Understanding the Role of Plasminogen Activator Inhibitor Type-2 (PAI-2, Serpinb2)

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2011 Understanding the role of plasminogen activator inhibitor type-2 (PAI-2, SerpinB2) in cancer: the relationship between biochemical properties and cellular function Blake J. Cochran University of Wollongong

Recommended Citation Cochran, Blake J., Understanding the role of plasminogen activator inhibitor type-2 (PAI-2, SerpinB2) in cancer: the relationship between biochemical properties and cellular function, Doctor of Philosophy thesis, University of Wollongong. School of Biological Sciences, University of Wollongong, 2011. http://ro.uow.edu.au/theses/3300

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UNDERSTANDING THE ROLE OF PLASMINOGEN

ACTIVATOR INHIBITOR TYPE-2 (PAI-2, SERPINB2) IN

CANCER: THE RELATIONSHIP BETWEEN

BIOCHEMICAL PROPERTIES AND CELLULAR

FUNCTION

A thesis submitted in fulfilment of the requirements for the award of the degree

Doctor of Philosophy from University of Wollongong

by Blake J. Cochran Bachelor of Biotechnology (Honours 1)(Advanced)

School of Biological Sciences University of Wollongong 2011

CERTIFICATION

I, Blake J. Cochran, declare that this thesis, submitted in fulfilment of the requirements for the award of Doctor of Philosophy, in the School of Biological

Sciences, University of Wollongong, is wholly my own work unless otherwise referenced or acknowledged. The document has not been submitted for qualifications at any other academic institution.

Blake J. Cochran

18 July 2011

I LIST OF PUBLICATIONS

Cochran, B.J., Gunawardhana, L.P., Vine, K.L., Lee, J.A., Lobov, S. and Ranson, M. 2009. The CD-loop of PAI-2 (SERPINB2) is redundant in the targeting, inhibition and clearance of cell surface uPA activity. BMC Biotechnology 9: 43.

Lee, J.A., Cochran, B.J. , Lobov, S. and Ranson, M. 2011. Forty years on and the role of Plasminogen Activator Inhibitor Type–2/SERPIN-B2 is still an enigma. Seminars in Thrombosis and Haemostasis 37 : 395.

Cochran, B.J. , Croucher, D.R., Lobov, S. and Ranson, M. 2011. Dependence on endocytic receptor binding via a minimal binding motif underlies the differential prognostic profiles of SerpinE1 and SerpinB2 in cancer. The Journal of Biological Chemistry 286 : 24467.

II LIST OF CONFERENCE PRESENTATIONS

CONFERENCE ORAL PRESENTATIONS Ranson M., Cochran B.J. , Croucher D.R., Lobov S., Saunders D.N. and Vine K.L. 2011. Selective targeting of urokinase-type plasminogen activator (uPA) malignant cells: biological rationale for the utility of PAI-2 versus the related uPA inhibitor PAI- 1. XIIIth International Workshop on Molecular & Cellular Biology of Plasminogen Activation, Clare College, Cambridge, UK.

Cochran, B.J. The molecular basis for the differential prognostic profiles of PAI- 1 and PAI-2 in cancer. Thompson Prize Final, Sydney Protein Group, Childrens Medical Research Institute, NSW, Australia

Cochran, B.J. , Croucher, D.R., Saunders, D.N., Lobov, S. and Ranson, M. 2010 A molecular basis for the differential prognostic profiles of Plasminogen Activator Inhibitors type-1 (PAI-1) and -2 (PAI-2) in cancer. Sydney Protein Group Young and Fresh Spring Meeting, University of Sydney, NSW, Australia

Cochran, B.J. , Croucher, D.R., Lobov, S. and Ranson, M. 2009. Endocytosis mechanisms and physiological functions of PAI-1 and PAI-2. XIIth International Workshop on Molecular & Cellular Biology of Plasminogen Activation, Cold Spring Harbor, NY, USA.

CONFERENCE POSTER PRESENTATIONS Cochran, B.J. , Croucher, D.R., Lobov, S. and Ranson, M. 2010. A molecular basis for the differential extracellular function of PAI-1 and PAI-2 in cancer. OzBio2010, Melbourne, VIC, Australia.

Cochran, B.J. , Croucher, D.R., Lobov, S. and Ranson, M. 2010. A potential molecular basis for understanding the PAI-1 versus PAI-2 paradox in cancer. 22 nd Lorne Cancer Conference, Lorne, VIC, Australia.

III Cochran, B.J. , Croucher, D.R., Lobov, S. and Ranson, M. 2010. A molecular basis for the differential endocytosis receptor binding mechanisms of PAI-1 and PAI-2. 30 th Lorne Conference on Protein Structure and Function, Lorne, VIC, Australia *Awarded Best Poster Prize

Cochran, B.J. , Lobov, S., Croucher, D.R. and Ranson, M 2007. Preliminary insights into the differential receptor mediated endocytosis mechanisms of PAI-1 and PAI-2. XIth International Workshop on Molecular & Cellular Biology of Plasminogen Activation, Saltsjöbaden, Sweden.

LIST OF FIGURES Page Figure 1.1: The metastatic process in cancer 4 Figure 1.2: The urokinase plasminogen activation system 7 Figure 1.3: Structures of uPA and uPAR 10 Figure 1.4: General conformations of serpins 16 Figure 1.5: The inhibitory mechanism of serpins 17 Figure 1.6: Structure of plasminogen activator inhibitor type-2 20 Figure 1.7: Structure of plasminogen activator inhibitor type-1 30 Figure 1.8: Structural representations of the LDLR members LDLR, VLDLR and LRP1 35 Figure 1.9: Structural comparison of the LDLR binding region of PAI- 1 and the corresponding region in PAI-2 39 Figure 2.1: Positions of oligonucleotide primers used to sequence PAI-2 cDNA 53 Figure 2.2: SDS-PAGE and western blot analysis of highly enriched recombinant PAI-2 proteins 61 Figure 2.3: Quantitative analysis of the purification of PAI-2 ∆CD-loop from pQE9 62 Figure 2.4: A positive ion ESI-MS of PAI-2 ∆CD-loop from pQE9 in 10 mM ammonium acetate (pH 6.8) containing 0.1% formic acid 64 Figure 2.5: PAI-2 ∆CD-loop efficiently inhibits both solution phase and cell bound uPA activity 65 Figure 2.6: Surface plasmon resonance analysis of the interaction between uPA:PAI-2 wild-type and uPA:PAI-2 ∆CD-loop and VLDLR 68 Figure 2.7: Cellular internalisation of uPA is equivalent between PAI-2 wild-type and PAI-2 ∆CD-loop as determined by flow cytometry 70 Figure 2.8: Hypothesised location of the CD-loop of PAI-2 based on intramolecular distance measurements 73 Figure 3.1: The LDLR minimal binding motif in PAI-1 is not conserved in PAI-2 80

V Figure 3.2: Schematic diagram of modified site directed mutagenesis used to produce PAI-2 mutants 88 Figure 3.3: Agarose gel electrophoresis indicating successful construction of TAGzyme pQE1/PAI-2 ∆CD-loop 91 Figure 3.4: Sequence analysis of TAGzyme pQE1/PAI-2 ∆CD-loop 92 Figure 3.5: Purification of PAI-2 ∆CD-loop by immobilised metal affinity chromatography 93 Figure 3.6: Small scale removal of 6 × his-tag from PAI-2 ∆CD-loop expressed from the TAGzyme pQE1 vector 94 Figure 3.7: Structural modelling of PAI-1 and PAI-2 96 Figure 3.8: Screening of PAI-2 mutants for VLDLR binding 99 Figure 3.9: Surface plasmon resonance analysis of the interaction between PAI-2 constructs and VLDLR 102 Figure 3.10: Surface plasmon resonance analysis of the interaction between uPA:PAI complexes and LRP1 104 Figure 3.11: PAI-2 forms maintained overall secondary structure 105 Figure 3.12: PAI-2 forms maintained inhibitory activity 106 Figure 3.13: The receptor binding surface of the uPA:PAI-1 complex 111 Figure 4.1: Cell surface antigen profiling of the HT1080 human fibrosarcoma cell line 121 Figure 4.2: Cell surface antigen profiling of the HeLa human cervical carcinoma cell line 128 Figure 4.3: Cell surface antigen profiling of the PC-3 human prostate carcinoma cell line 123 Figure 4.4: Cell surface antigen profiling of the MCF-7 human breast epithelial carcinoma cell line 124 Figure 4.5: uPA binding profile of cancer cell lines 126 Figure 4.6: Increased LDLR affinity leads to increased internalisation of uPA:PAI complexes by cancer cells 128 Figure 4.7: uPA:PAI-2YK induces promitogenic signalling pathways in MCF-7 cells similar to those of uPA:PAI-1 130 Figure 4.8: uPA:PAI-2YK increased cell proliferation in MCF-7 cells similar to uPA:PAI-1 131

VI Figure 5.1: Proposed models by which uPA:PAI complexes mediate ERK activation via VLDLR-mediated endocytosis 142 Figure 5.2: Physiological actions of PAI-1 and PAI-2 upon inhibition and clearance of uPA 147 Figure A.1: Standard curve constructed using protein markers 193 Figure A.2: Standard curve constructed using Eco RI + Hin III λ DNA markers 194 Figure A.3: Alignment of mutagenised regions of PAI-2 α-helix D and adjacent residues used in this study 195 Figure A.4: Construction of VLDLR chip for use in surface plasmon resonance 196 Figure A.5: Optimisation of initial seeding cell density for examination of proliferative effects in MCF-7 cells 197

VII LIST OF TABLES Page Table 1.1: The expression of PAI-2 mRNA and/or protein can be regulated by a range of biological agents in a diverse set of cell types 22 Table 1.2: Reported affinities of plasminogen activation system components for receptors of the low-density lipoprotein receptor family 37 Table 2.1: Oligonucleotides used for sequencing of pQE9/PAI-2 constructs 52 Table 2.2: Kinetic parameters of the interaction between PAI-2 ∆CD- loop or PAI-2 wild-type and VLDLR, both alone and in complex with uPA 69 Table 3.1: Oligonucleotides used for vector construction, sequencing and mutagenesis 85 Table 3.2: PAI-2 mutants used to investigate the differential VLDLR and LRP1 binding mechanisms of PAI-2 and PAI-1. 95 Table 3.3: Kinetic parameters of the interaction between PAI-2 forms used in this study and the endocytosis receptor VLDLR 100 Table 3.4: Kinetic parameters of the interaction between PAI forms used in this study and the endocytosis receptor LRP1 103 Table 3.5: Summary of PAI-2 binding interactions with VLDR and LRP1 109 Table A.1: Mobility of protein markers 193 Table A.2: Mobility of Eco RI + HindI III λ DNA markers 194

VIII LIST OF ABBREVIATIONS

AIIt Annexin II heterotetramer

α2-AP α2-antiplasmin AP-1 Activator protein 1 AP-2 Activator protein 2 BM Basement Membrane bp Base pairs CAM Cell adhesion molecule CD Circular dichroism CD-loop Polypeptide loop between α-helicies C and D present in some clade B serpins CI Cell index ECL Enhance chemiluminesence ECM Extra-cellular matrix EDTA Ethylenediaminetetraacetic acid ESI-MS Electrospray ionisation-mass spectrometry FAK Focal adhesion kinase FGF2 Basic fibroblast growth factor g Gravity G-CSF Granulocyte colony-stimulating factor HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HMW-uPA High molecular weight urokinase plasminogen activator HPLC High performance liquid chromatography IL Interleukin Jak/Stat Janus Kinase/Signal Transducer and Activator of Transcription

ka Association rate

KD Affinity constant

kd Dissociation rate LDL Low-dentisy lipoprotein LDLR Low-dentisy lipoprotein LMW-uPA Low molecular weight urokinase plasminogen activator LPS Lipopolysaccharide LRP1 Low-density lipoprotein receptor related protein-1 LRP2 Low-density lipoprotein receptor related protein-2 (megalin, gp330)

IX MAPK/ERK Mitogen-activated protein kinase/ Extracellular signal-regulated kinase MAZ Myc-associated zinc finger protein M-CSF Macrophage colony-stimulating factor MES 2-(N-morpholino)ethanesulfonic acid MMP Matrix metalloprotease NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells OD Optical density PA Plasminogen activator PAI-1 Plasminogen activator inhibitor type-1 PAI-2 Plasminogen activator inhibitor type-2 PAUSE-1 PAI-2 upstream silencer element-1 PC Primary culture PMA phorbol-12-myristate-13-acetate PVDF Polyvinylidene fluoride Q-TOF-MS Quantitative-time of flight-mass spectrometry RAP Receptor associated protein Rb Retinoblastoma protein RCL Reactive centre loop RPMI Roswell Park Memorial Institute medium RU response units sc-tPA Single chain tissue plasminogen activator SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis Serpin Serine protease inhibitor sorLA Sortilin-related receptor TBS(T) Tris buffered saline (with tween) tc-tPA Two chain tissue plasminogen activator TGF-β Transforming growth factor-β TNF-α Tumour necrosis factor-α tPA Tissue plasminogen activator U Units uPA Urokinase plasminogen activator uPAR Urokinase plasminogen activator receptor UTR Unstranslated region

X VLDL Very low-density lipoprotein VLDLR Very low-density lipoprotein receptor

XI ACKNOWLEDGEMENTS The completion of a PhD is a huge achievement and something that cannot be solely attributed to one person. Whilst the work contained herein is my own, there are a huge number of people who served as a support crew over the last four years that a thank you on a page can only begin to repay. First and foremost, my supervisor Marie Ranson for the opportunity to work on a project that was both challenging and rewarding. Working in a dynamic lab and gaining experience in a wide spectrum of techniques is something for which I am incredibly grateful. Also, thank you for your kind words and understanding during some of the darker days as well as the kick and motivation I sometimes required to produce nothing less than quality science. My co-supervisor David Croucher, for being the contact inside the Garvan and allowing me to have almost unlimited access to the BIAcore and also your role in the development and progression of this project, without it this project would never have moved forwards. Thanks also to Darren Saunders for your help in getting the manuscript up to standard for submission to a quality journal and your advice about my project and science in general. To the current and past members of the Ranson lab and those otherwise involved with the lab, thank you for making going to work an enjoyable experience: Kara (Ann, Egg, Tree) Perrow, Sergei Lobov, Jodi Lee, Nathan ‘Mildred’ Ralston-Bryce, Hayden Matthews, Lidia Matestic, Vineesh Indira Chandran, Samantha Roberts, Mel Desouza, Colin Corte, Lakshitha Gunawardhana, Sarah Vella (despite re-telling the same joke thousands of times and still thinking its funny), Bronwyn Spalding. Also, to the staff of Biology and IHMRI, thanks for answering questions when I needed clarification on some technique I had no clue about and making both buildings enjoyable places to work. Long suffering housemates (there has been 16 of you lucky people over the last 5 or so years) some of whom I will probably never see or talk to again, others who will be a fixture in my life for years to come (Harraposaurus, Nathan and Alison and others) – I am sure I was always a ‘delight and joy’ to live with. My friends, thank you all for what you bring to my life. To attempt to name everyone would only result in the omission of many, but special mention goes to Simon, Aleta, James, Rebecca (Burecca), Jim, Iman, Carola, Cork, Bodge, Dave/Ian, Zoe, the Trivia

XII Crew and the Wollongong Mustangs. You (and everyone else) were instrumental in my survival during the PhD. Perhaps my greatest thanks go to my family. Thank you for always believing in me and supporting me in everything I do and to my parents Garry and Helen for taking me back in when my scholarship dried up. This thesis is for you. Finally, on a lighter note, I would like to thank Kaldi, the Ethiopian goat hearder who discovered coffee in the 9 th century, and Saccharomyces cerevisiae for providing two substances critical to the writing process.

XIII ABSTRACT Overexpression of the plasminogen activation system has been found to play an important role in the progression of a number of forms of cancer. Plasminogen is activated to plasmin via the actions of the serine proteases urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA).Whilst cell surface bound plasminogen is protected from direct inhibition, uPA and tPA are inhibited by plasminogen activator inhibitor type-1 (PAI-1) and type-2 (PAI-2). PAI-1 and PAI-2 belong to the serpin protein superfamily (SerpinE1 and SerpinB2, respectively) which, despite significant variations in primary sequence fold into a well conserved structure consisting of three β–sheets and nine α-helices. PAI-2 is unique among the serpins in containing a 33-amino acid interhelical loop between α-helices C and D

(the CD-loop), which is commonly removed in recombinant PAI-2 production. Upon inhibition of cell bound uPA (or tPA), plasminogen activator:inhibitor complexes are endocytosed by members of the low-density lipoprotein receptor (LDLR) family.

Tumour overexpression of uPA and PAI-1 correlates with poor prognosis and increased metastatic potential. Conversely, tumour expression of PAI-2 (in combination with uPA) is associated with favourable outcome and relapse free survival. Previous work has suggested that the distinct binding interactions of PAI-1 and PAI-2, due to the presence of a high-affinity LDLR binding motif in PAI-1 that is absent in PAI-2, may underlie differential downstream cellular behaviours.

The aims of this thesis were to: (1) Determine the impact that removal of the CD-loop of PAI-2 had on uPA inhibition and clearance and confirm that this PAI-2 form (PAI-

2 ∆CD-loop) could be utilised in the further aims of this study; (2) characterise the molecular basis underlying the differential interactions of PAI-1 and PAI-2, both individually and in complex with uPA, with receptors of the LDLR family; and (3)

XIV examine the functional consequences resulting from the complementation of LDLR binding in PAI-2, specifically the initiation of promitogenic signalling and cell proliferation.

Removal of the CD-loop of PAI-2 was shown to have no impact on the inhibition of both solution phase and cell surface uPA or on the rate of clearance of uPA from the surface of MCF-7 cells. Additionally, uPA:PAI-2 ∆CD-loop had similar binding kinetics (K D ~ 5 nM) for the very low-density lipoprotein receptor (VLDLR) to that previous published for uPA:PAI-2 complexes. Furthermore, PAI-2 ∆CD-loop expressed and purified in the system described here gave both higher yield and purity than wild-type PAI-2 expressed and purified under identical conditions.

Introduction of the LDLR minimal binding motif present in the α-helix D of PAI-1 in

PAI-2 via site-directed mutagenesis enabled high affinity binding to VLDLR. The binding of this mutant, PAI-2YK in complex with uPA was remarkably similar to that of uPA:PAI-1 in both mechanism and affinity (heterogeneous analyte, K D values of

1.35 nM & 70.1 nM). Interestingly, mutation of an additional residue adjacent to the

LDLR binding motif was required for high affinity binding of PAI-2 to low-density lipoprotein receptor related protein-1 (LRP1).

Complementation of the LDLR binding motif in PAI-2 had profound impacts on the downstream functionality of PAI-2. Specifically, the LDLR-binding PAI-2 forms enhanced uPA endocytosis on both VLDLR and LRP1 expressing cell lines, demonstrating that LDLR affinity is the critical determinant in the rate of uPA internalisation. Furthermore, treatment of MCF-7 cells with PAI-2 YK sustained ERK activation, and increased proliferation in a manner indistinguishable from the effects of PAI-1.

XV The findings presented in this thesis clearly demonstrate that introduction of an otherwise absent LDLR minimal binding motif in PAI-2 increases affinity of uPA:PAI-2 complex for LDLRs to mimic that of uPA:PAI-1. This has a profound impact on the downstream function of PAI-2 via sustained activation of cell signalling pathways and subsequent proliferative effects. This has clear relevance to understanding the paradoxical disease outcomes associated with overexpression of these proteins in cancer.

XVI TABLE OF CONTENTS Page CERTIFICATION I PUBLICATIONS II LIST OF CONFERENCE PRESENTATIONS III Conference Oral Presentations III Conference Poster Presentations III LIST OF FIGURES V LIST OF TABLES VIII LIST OF ABBREVIATIONS IX ACKNOWLEGEMENTS XII ABSTRACT XIV TABLE OF CONTENTS XVII CHAPTER 1: LITERATURE REVIEW 1 1.1 INTRODUCTION TO CANCER 2 1.1.1 Invasion and metastasis 3 1.2 THE PLASMINOGEN ACTIVATION SYSTEM 5 1.2.1 General overview 5 1.2.2 tPA 8 1.2.3 uPA and uPAR 9 1.2.4 Inhibitors 13 1.3 PLASMINOGEN ACTIVATOR INHIBITORS 14 1.3.1 Overview of Serpins 14 1.3.2 Plasminogen Activator Inhibitor type-2 (PAI-1, SerpinB2) 18 1.3.2.1 Expression of PAI-2 21 1.3.2.2 Polymorphisms 24 1.3.2.3 Intracellular roles 26 1.3.2.4 Extracellular roles 27 1.3.3 Plasminogen Activator Inhibitor type-1 (PAI-1, SerpinE1) 29 1.3.4. Prognostic values of PAI-1 and PAI-2 in cancer 31 1.4 NON -PROTEOLYTIC FUNCTIONS OF THE PLASMINOGEN ACTIVATION SYSTEM 33 1.4.1 The LDLR superfamily 33 1.4.2 Endocytosis of uPA:PAI complexes 36 1.4.3 Signalling pathways 40 1.5 POTENTIAL THERAPEUTIC VALUE OF PAI-2 42 1.6 SUMMARY AND AIMS 43 CHAPTER 2: THE CD-LOOP OF PAI-2 (SERPINB2) IS REDUNDANT IN THE TARGETING, INHIBITION AND CLEARENCE OF CELL SURFACE uPA ACTIVITY 45 2.1 INTRODUCTION 46 2.2 MATERIALS AND METHODS 49 2.2.1 Materials 49 2.2.2 Preparation of electrocompetent E. coli for transformation 50 2.2.3 Generation of pQE9/PAI-2 constructs 50 2.2.4 DNA sequence analysis 51 2.2.5 Expression and purification of PAI-2 52 2.2.6 SDS-PAGE 54

XVII 2.2.7 Western blot analysis 54 2.2.8 Cation-exchange high performance liquid chromatography (HPLC) 55 2.2.9 Electrospray ionisation mass spectrometry (ESI-MS) 56 2.2.10 Tissue culture conditions 56 2.2.11 uPA inhibitory assays 57 2.2.11.1 Formation of SDS-stable complexes 57 2.2.11.2 Solution phase flurogenic uPA activity assay 57 2.2.11.3 Cell surface flurogenic uPA activity assay 57 2.2.12 Surface plasmon resonance 58 2.2.13 Internalisation assays 58 2.3 RESULTS 60 2.3.1 Expression and purification of PAI-2 60 2.3.2 Electrospray ionisation mass spectrometry (ESI-MS) 63 2.3.3 The absence of the CD-loop does not affect the solution phase or cell surface uPA activity inhibitory function of PAI-2 63 2.3.4 Removal of the CD-loop does not affect the clearance of uPA from the cell surface 67 2.4 DISCUSSION 71 CHAPTER 3: CHARACTERISING THE DIFFERENTIAL LDLR BINDING PROFILES OF PAI-1 AND PAI-2 77 3.1 INTRODUCTION 78 3.2 MATERIALS AND METHODS 83 3.2.1 Materials 83 3.2.2 Agarose gel electrophoresis 83 3.2.3 Construction and characterisation of TAGzyme pQE1/PAI-2 ∆CD loop 84 3.2.3.1 Amplification of PAI-2 cDNA 84 3.2.3.2 Colony PCR 86 3.2.4 Small-scale his-tag removal 86 3.2.5 Protein structure modelling 87 3.2.6 Site directed mutagenesis 87 3.2.7 Surface plasmon resonance 89 3.2.8 Far-UV circular dichroism spectrometry 89 3.2.9 uPA activity assay 90 3.3 RESULTS 90 3.3.1 Construction and characterisation of TAGzyme pQE1/PAI-2 ∆CD- loop 90 3.3.1.1 Construction of TAGzyme TAGzyme pQE1/PAI-2 ∆CD-loop 90 3.3.1.2 Purification of PAI-2 ∆CD-loop 93 3.3.1.3 Small scale removal of 6 × his-tag 94 3.3.2 Structural comparison indicates absence of a consensus LDLR- binding motif in PAI-2 95 3.3.3 LDLR binding can be introduced to PAI-2 by complementation of the PAI-1 LDLR binding motif 98 3.3.4 PAI-2 mutants maintain general secondary structure and inhibitory activity 105 3.4 DISCUSSION 107

XVIII CHAPTER 4: CELLULAR EFFECTS MEDIATED BY COMPLEMENTATION OF THE LDLR BINDING MOTIF IN PAI-2 112 4.1 INTRODUCTION 113 4.2 MATERIALS AND METHODS 116 4.2.1 Materials 116 4.2.2 Cell lines 116 4.2.3 Analysis of cell surface antigen expression by flow cytometry 116 4.2.4 Analysis of uPAR saturation at the cell surface 117 4.2.5 Endocytosis assay 117 4.2.6 Analysis of ERK activation 118 4.2.7 Cell proliferation assay 118 4.2.8 Statistical analyses 119 4.3 RESULTS 120 4.3.1 Components of the plasminogen activation system are differentially expressed on cancer cell lines 120 4.3.2 Characterisation of uPAR saturation 125 4.3.3 Introduction of LDLR binding to PAI-2 increased uPAR mediated endocytosis 127 4.3.4 Introduction of VLDLR binding to PAI-2 induces mitogenic signalling in MCF-7 cells 129 4.4 DISCUSSION 134 CHAPTER 5: CONCLUSIONS AND FUTURE DIRECTIONS 138 CHAPTER 6: REFERENCES 148 APPENDIX I: BUFFERS AND SOLUTIONS 183 APPENDIX II: PLASMID MAPS 187 APPENDIX III: CONSTRUCTION OF PROTEIN STANDARD CURVE 193 APPENDIX IV: CONSTRUCTION OF DNA STANDARD CURVE 194 APPENDIX V: SEQUENCING OF PAI-2 MUTANTS 195 APPENDIX VI: PREPARATION OF BIA CORE ANALYSIS SURFACE 196 APENDIX VII: OPTIMSATION OF CELL SEEDING DENSITY USING THE X CELL IGENGE SYSTEM 197 APPENDIX VIII: PUBLICATIONS 198

XIX

CHAPTER 1

LITERATURE REVIEW

1.1 INTRODUCTION TO CANCER

Cancer is a genetic disease caused by the sequential acquisition of chromosomal mutations leading to uncontrolled cell growth and proliferation. In Australia alone, cancer is responsible for approximately 40,000 deaths per annum (29% of total deaths) (Australian Institute of Health and Welfare & Australasian Association of

Cancer Registries, 2010). Tumour progression is categorised by an interrelated set of six key physiological events: evasion of apoptosis, self-sufficiency of growth signals, insensitivity to anti-growth signals, limitless replicative potential, sustained angiogenesis (the formation of blood vessels in the tumour) and tissue invasion and metastasis (the formation of secondary tumours) (Bertram 2000; Hanahan and

Weinberg 2000; Bergers and Benjamin 2003). A tumour is only classified as malignant upon acquisition of the final trait. The order in which these characteristics develop varies between the over one hundred distinct types of cancer that have been identified (Hanahan and Weinberg 2000). However, the ability of a cancer to invade and metastasise is the primary determinant of cancer prognosis (Duffy et al. 1999;

Brabletz et al. 2005), with mestastases accounting for 90% of cancer deaths (Sporn

1996).

This review will focus on the roles of the plasminogen activation system in invasion and metastasis with an emphasis on the inhibition of this system at the cell surface by plasminogen activator inhibitors type-1 (PAI-1, SerpinE1) and type-2 (PAI-2,

SerpinB2). The non-proteolytic roles of PAI-1 and PAI-2 in the plasminogen activation system will then be examined, in order to further the understanding on the apparent contrasting roles of PAI-1 and PAI-2 in tumour progression. Finally, the

2 current hypotheses and models believed to underlie this functional divergence will be discussed.

1.1.1 INVASION AND METASTASIS

Metastasis is the spread of cancer from its primary site to sites elsewhere in the body and occurs at some stage during the course of most forms of human cancer (Hanahan and Weinberg 2000). For metastasis to occur successfully, malignant cells have to detach from the biologically heterogeneous primary tumour (Fidler 2002), invade a blood or lymphatic vessel, travel via circulation to a site distant from the primary tumour and establish a new cellular colony (Rusoslahti 1996; Choong and

Nadesapillai 2003) (Figure 1.1). This colony may be within an organ or tissue, requiring escape from the blood or lymphatic vessel and subsequent invasion

(Tryggvason et al. 1987; Mignatti and Rifkin 1993), or within the vessel itself (Fidler

2002). There is increasing evidence that tumour associated cells, such as fibroblasts and immune cells, play an important role in invasion and metastasis, engaging in cellular cross talk via exchanging of proteases and cytokines with cancer cells (Wever and Mareel 2003; Kalluri and Zeisberg 2006; Mareel and Madani 2006; Steidl et al.

2010; Turner and Grose 2010). In order to develop into a secondary tumour, the invading cell must recruit and co-operate with the right combination of stromal cells in the new environment (Danø et al. 2005). There is also evidence that metastases are partly controlled by messages in the microenvironment of the organs to which they spread (Couzin 2003). Tumour invasion is facilitated by diminished expression of cellular adhesion molecules (CAMs) and the breakdown of the underlying basement membrane (BM) and extracellular matrix (ECM) (Liotta 1984; Andreasen et al. 1997).

Figure 1.1: The metastatic process in cancer . Metastatic cancer cells are able to colonise tissues distant to the primary tumour. (A) Mutagenesis leads to aberrant cell proliferation resulting in formation of a tumour mass. (B) Invasive cells must break away from the primary tumour, breakdown the extracellular matrix (ECM) and the basement membrane in order to reach blood or lymph vessels. (C) These cells then enter circulation in a process called intravasation. (D & E) Leaving circulation, extravasation requires breakdown of the basement membrane and ECM at the secondary site (F) for the formation of metastases. Adapted from Alberts et al. (2008).

CAMs provide not only a physical anchor between cells and the surrounding stroma

(Rusoslahti 1996), but adjacent cells that are connected by E-cadherin bridges also convey mutual anti-growth signals (Christofori and Semb 1999). Overcoming E- cadherin mediated cell adhesion has been demonstrated to be a rate-limiting step in the conversion of a benign to an invasive phenotype in epithelial cancer (Christofori and Semb 1999).

The ECM consists of collagens, glycoproteins (such as laminin, fibronectin, entactin and nidogen), proteoglycans and glycosaminoglycans as a mesh surrounding connective tissue (Mignatti and Rifkin 1993). The BM, which is comprised of layers of ECM, separates epithelial or endothelial cells from underlying connective tissue and blood vessels (Mignatti and Rifkin 1993). Penetration of the BM allows the entry of metastatic cancer cells into the circulatory system (intravasation), with their subsequent escape into tissues (extravastion) requiring BM degradation at the secondary site. This degradation is accomplished by the concerted action of several proteases, including the serine protease plasmin and a number of matrix metalloproteases (MMPs). Plasmin is generated from the zymogen precursor plasminogen via the actions of the plasminogen activation system. As such, this system is important in the context of tumour progression and metastasis.

1.2 THE PLASMINOGEN ACTIVATION SYSTEM

1.2.1 GENERAL OVERVIEW

The plasminogen activation system (Figure 1.2) is a multi-component regulatory system that, under normal conditions, functions in the clearance of blood clots and matrix degradation during tissue remodeling processes. However, unregulated

5 plasminogen activation is implicated in key events in malignant tumour progression, specifically solid tumour growth, invasion and metastatic spread (Andreasen et al.

1997; Ranson et al. 1998; Scherrer et al. 1999; Schmitt et al. 2000; Choong and

Nadesapillai 2003; Ranson and Andronicos 2003; Duffy 2004; Danø et al. 2005;

McMahon and Kwaan 2008; Kwaan and McMahon 2009). Initiation of the plasminogen activation system is facilitated by the serine proteases urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), resulting in the conversion of the zymogen plasminogen to the active, serine protease plasmin

(Figure 1.2). Activation of plasminogen involves proteolytic cleavage of the peptide bond Arg561 – Val 562 (Violand and Castellino 1976; Ponting et al. 1992; Syrovets and

Simmet 2004). Plasmin is able to digest the majority of proteins present in the ECM – laminin, fibronectin, fibrin, vitronectin, proteoglycans and collagen (Mignatti and

Rifkin 1993; Andreasen et al. 1997; Andreasen et al. 2000). Additionally, plasmin can activate MMPs and other proteases that can also degrade ECM proteins (Castellino and Ploplis 2005). Plasmin is also able to activate transforming growth factor-β (TGF-

β) and trigger the release of fibroblast growth factor-2 (FGF2) (Rifkin 1997) which have the potential to enhance tumour progression (Duffy 2004).

Generally, uPA is agreed to be the enzyme of most relevance to tumour biology

(Andreasen et al. 1997; Andreasen et al. 2000; Weigelt et al. 2005; McMahon and

Kwaan 2008) with the primary role of tPA in the generation of plasmin for fibrinolysis in blood vessels (Andreasen et al. 2000; McMahon and Kwaan 2008).

However, tPA has been found to play an important role in some forms of cancer such

Figure 1.2: The urokinase plasminogen activation system. The activation of plasminogen to plasmin is catalysed by the urokinase-type plasminogen activator (uPA) which is bound to the specific uPA receptor (uPAR). In turn, plasmin converts the zymogen pro-uPA to the active form of the enzyme. Additional enzymes such as cathespins can also activate pro-uPA. Plasmin mediates extracellular matrix (ECM) degradation by both direct proteolysis and indirectly via the activation of matrix metalloproteases (MMPs) and growth factor release. Whilst circulating plasmin can be inactivated by a number of inhibitors, such as α2-antiplasmin ( α2-AP), cell surface plasmin is protected from inhibition. Plasminogen activator inhibitors (PAI) can indirectly inhibit plasminogen activation by irreversibly inhibiting cell surface bound uPA. Adapted from Croucher et al. (2008).

7 as melanoma and neuroblastoma (Rijken and Collen 1981; Nielsen et al. 1982; Bizik et al. 1990). As a matter of completeness, tPA will be discussed briefly before an in- depth examination of the role of uPA in cancer.

1.2.2 tPA tPA is a 562 amino acid serine protease secreted from numerous cell types. Unlike uPA, the 72 kDa glycosylated single chain tPA (sc-tPA) form is active, with enhanced catalytic activity in the two chain form. Sc-tPA is converted to two chain tPA (tc-tPA) via cleavage of the Arg 310 – Ile 311 bond by plasmin, tissue kallikrein or factor Xa. The basic structure of tPA is comprised of an N-terminal finger domain, an EGF-like domain, two kringle domains and a serine protease domain. The primary biological function of tPA, thrombosis and fibrinolysis, is mediated via the ability of tPA to bind to fibrin with high affinity. Both the single and double chain forms of tPA display enhanced enzymatic activity for plasminogen (~ 2 fold) when bound to fibrin

(Hoylaerts et al. 1982). Similar to this, binding of tPA to heparin also results in an increase in plasminogen activation (Liang et al. 2000).

In addition to interacting with fibrin, tPA is also able to bind to monocyte, macrophage, endothelial and human tumour cell lines via lysine dependent interactions and activate cell-surface associated plasminogen (Ljungner et al. 1983;

Felez et al. 1991; Noorman et al. 1995; Lobov et al. 2008; Lee et al. 2010). tPA has been demonstrated to interact with annexin II (p36), the annexin II heterotetramer

(AIIt) (comprised of two subunits of p36 and two of p11), the low-density lipoprotein receptor-related protein-1 (LRP1), LRP-1B, the very low-density lipoprotein receptor

(VLDLR), laminin, cytokeratin-8 and the mannose receptor (Biessen et al. 1997;

Goldfinger et al. 2000; Gonias et al. 2001; Liu et al. 2001; Strickland et al. 2002; Ling et al. 2004; Lee et al. 2010). Expression of annexin II and tPA in breast cancer cells has been demonstrated to mediate cell motility and correlate to tumour neovascularisation (Sharma et al. 2010). The role of tPA in cancer may lay primarily in the effects mediated by tumour associated cells, with tPA activity upregulated in breast cancer associated fibroblasts (Sadlonova et al. 2009) and in normal tissues adjacent to gastrointestinal carcinomas (Scicolone et al. 2006). Additionally,

Borgfeldt et al. (2003) found that high levels of tPA in the plasma were associated with shorter patient survival in ovarian cancer.

1.2.3 uPA AND uPAR uPA is translated as a single chain 53 kDa precursor, pro-uPA (Duffy 2004) (Figure

1.3A). Plasmin-mediated cleavage of the Lys 158 – Ile 159 bond of pro-uPA generates active two-chain uPA. The two chains are covalently linked via the disulfide bridge

Cys 148 -Cys 279 (Duffy 2004). Receptor (uPAR, CD88) binding potentiates the activation of pro-uPA and results in the initiation of a pro-enzyme activation feedback loop, resulting in amplification of plasmin production (Behrendt 2004; Danø et al.

2005). Both pro-uPA and uPA are able to bind to uPAR, which is crucial for efficient cell-associated plasminogen activation (Ellis et al. 1999; Ranson and Andronicos

2003; Stillfried et al. 2007). In addition to plasmin, pro-uPA can also be activated by matriptase, hepsin, kallikreins and cathepsins (Kobayashi et al. 1991; List et al. 2000;

Kilpatrick et al. 2006; Moran et al. 2006) (Figure 1.2). The two chains of uPA consist of 4 distinct regions: an epidermal growth factor domain (residues 1-49); a kringle domain (50-131) and an interdomain linker region (132-158) in the A chain and the

A B

+ - NH 3 COO

Figure 1.3: Structures of uPA and uPAR. The A-chain of uPA contains an epidermal growth factor domain (yellow), a kringle domain (green) and an interdomain linker region (blue). The protease domain (red) is present in the B chain. Activation of pro-uPA to active two chain uPA (high molecular weight uPA, HMW-

uPA) occurs via cleavage if the Lys 158 -Ile 159 bond. Low molecular weight uPA (LMW-uPA) retains the proteolytic activity of uPA, but is unable to bind to uPAR. (B) Ribbon structure of the serine protease domain (residues 159 – 404) of uPA shown in detail. (C) The growth factor (residues 7 – 43) and kringle (residues 50 – 131) domains of uPA (grey) in complex with uPAR (domains are indicated). Structures are coloured from N-termius (blue) to C-terminus (red). Constructed using PDB coordinates 1GJ8 (Katz et al. 2001), 2FD6 (Huai et al. 2006) and the PyMOL molecular graphics system (DeLano 2002).

10 serine protease domain comprising the whole of the B-chain (159-411) (Figure 1.3A)

(Petersen et al. 2001). Low molecular weight uPA (LMW-uPA), which is N - terminally truncated at Lys 136 , retains the catalytic activity of native uPA but is unable to bind to uPAR (Bansal and Roychoudhury 2006) and is produced by autocatalytic conversion of active uPA. The high affinity binding (K D

~0.2 nM) (Lin et al. 2010) of (pro)uPA to uPAR consists of the growth factor domain of uPA inserting into a deep binding cavity (~ 14 Å) in uPAR (Huai et al. 2006)

(Figure 1.3C). This binding is mediated by residues in the epidermal growth factor domain of uPA (Lys 23 , Tyr 24 , Phe 25 and Ile 28 ) (Lin et al. 2010). The ligation of uPA to uPAR results in the cleavage of the Arg 83 - Ala84 and Arg 89 -Ser 90 bonds in the linker region between domains 1 and 2 of uPAR and is thought to iniate major conformational changes in uPAR which have a dramatic influence on the functionality of this receptor (Blasi 1999; Rasch et al. 2008; Smith and Marshall 2010).

In general, uPAR is expressed at relatively low levels in normal tissues (Behrendt

2004), but is overexpressed by many cancer cell lines (Schmitt et al. 2000). uPAR is also highly expressed on the surface of activated macrophages and various other leukocytes. The low concentration of uPA in plasma and the fact that the majority of circulating uPA is found bound to the inhibitor plasminogen activator inhibitor type-1

(PAI-1) suggests that autocrine and paracrine releases of uPA are mostly responsible for plasminogen activation (Ranson and Andronicos 2003).

As discussed, initiation of uPA facilitated activation of plasminogen at the cell surface requires binding of uPA to its receptor uPAR (Andreasen et al. 1997), with bound uPA regulated by plasminogen activator inhibitors (Figure 1.2). Acceleration of the

11 activation of plasmin is believed to occur via the co-localisation of plasminogen receptors and uPAR at the cell surface (Mignatti and Rifkin 1993; Félez 1998; Ranson and Andronicos 2003; Behrendt 2004) (Figure 1.2). Additionally, as mentioned previously, receptor bound plasmin/ogen is protected from inhibition by circulating plasmin inactivators (Hall et al. 1991).

Inappropriate activation of the plasminogen activation system in cancer, and hence increased metastatic potential, occurs via upregulated expression of components of the urokinase plasminogen activation system on the cell surface (Andreasen et al. 1997;

Duffy et al. 1999; Schmitt et al. 2000). As such, uPA/uPAR overexpression is a feature of a number of cancers including acute leukemias, breast, gastric, colorectal, esophageal, renal, lung, brain, urinary tract, endometrial, thyroid and ovarian cancers

(Duffy et al. 1999; Scherrer et al. 1999; Schmitt et al. 2000; Tecimer et al. 2001; Look et al. 2002; Weigelt et al. 2005; Ulisse et al. 2006; Mengele et al. 2010) and has also been linked to poor patient outcome (Knoop et al. 1998; Foekens et al. 2000; Choong and Nadesapillai 2003; Duffy 2004; Ohba et al. 2005; Schmitt et al. 2010).

uPAR plays a critical role in pericellular proteolysis but has also been identified as possessing a number of other important physiological roles: cell adhesion and motility; growth promoting activity; mitogenic activity in normal and tumour cells; chemotactic activity as demonstrated in neutrophils, endothelial cells and keratinocytes; signal transduction; ligand internalisation and functional interplay with integrins (Ehart et al. 1998; May et al. 1998; Mazar 2008; Blasi and Sidenius 2010). uPAR is also able to compete with integrins for vitronectin present in the ECM

(Behrendt and Stephens 1998), impacting on cell adhesion and motility (Kjoller

2002). It has been determined that vitronectin binding at the leading edge of the cell promotes cell migration, whilst binding at the trailing edge of the cell inhibits migration (Andreasen et al. 1997; Behrendt and Stephens 1998). Many studies using different cell lines have shown that uPA and uPAR are overexpressed at the leading cell edge of malignant cancer cells (Estreicher et al. 1990; Mazzieri and Blasi 2005;

Raghu et al. 2010). Thus, the uPA system plays a key role in tumour progression and metastasis through involvement in cellular adhesion and chemotaxis/migration (i.e., through non-proteolytic functions) in addition to proteolytic degradation (Ranson and

Andronicos 2003; Blasi and Sidenius 2010).

1.2.4 INHIBITORS

Due to the far-reaching and potentially damaging effects of plasmin, regulation of the plasminogen activation system is crucial for normal non-invasive cell function. The inhibition of inappropriate extracellular proteolysis is recognised as a valid treatment for cancer. Physiological regulation of the plasminogen activation system may be facilitated by either direct inhibitors of plasminogen/plasmin ( α2-antiplasmin and α2- macroglobulin) or specific plasminogen activator inhibitors [PAI-1, plasminogen activator inhibitor type-2 (PAI-2), protein C inhibitor and protease nexin-1] (Behrendt

2004).

Both PAI-1 and PAI-2 efficiently inhibit uPA in solution and at the cell surface

(Astedt et al. 1987; Ellis et al. 1990; Al-Ejeh et al. 2004) through the formation of covalent, irreversible enzyme:substrate complexes. As the focus of this review is the biological role of PAI-2 relative to that of PAI-1, the functional aspects of α2- antiplasmin, α2-macroglobulin, protease nexin-1 and protein C inhibitor will not be

13 discussed further. For reviews on these inhibitors the reader is referred to Kruithof

(1988), Geiger et al. (1996), Stassen et al. (2004) and Arnout et al. (2006), respectively.

1.3 PLASMINOGEN ACTIVATOR INHIBITORS

1.3.1 OVERVIEW OF SERPINS

PAI-1 and PAI-2 belong to a superfamily of proteins known as the serpins (ser ine protease in hibitors) (Silverman et al. 2001). The majority of serpins serve as regulators of serine or cystine protease activity in such diverse roles as complement activation, inflammation, coagulation, fibrinolysis, carcinogenesis, angiogenesis and apoptosis (Irving et al. 2000). The physiological importance of serpins is highlighted by their presence in all major forms of life (Bacteria , Archaea, Eukarya and Viridae )

(Irving et al. 2000; Roberts et al. 2004). Members of the serpin superfamily are organised into sixteen clades, designated A through P based on their structure, sequence and function (Silverman et al. 2001). Despite considerable variations in primary peptide sequence (Huber and Carrel 1989), members of the serpin family generally fold into a well-conserved tertiary structure consisting of 3 β-sheets, 8-9 α- helices and a reactive centre loop (RCL). Most serpins contain 350-500 amino acids

(Silverman and Lomas 2004), with variations due to amino- or carboxyl- terminal extensions or by glycosylation in the case of extracellular serpins (Potempa et al.

1994).

Whilst a number of the characterised serpins have no known inhibitory function, the remainder, including both PAI-1 and PAI-2, display an inhibitory activity that

Silverman and Lomas (2004) described as ‘a unique, irreversible suicide substrate-like

14 mechanism of inhibition’, occurring via a branched pathway (Silverman et al. 2001).

The serpin inhibitory mechanism is facilitated by the ability of serpins to undergo structural rearrangements. These differing conformations are largely dependent on the positioning of the RCL. In the active ‘native/stressed’ conformation (Figure 1.4A) the

RCL is exposed and intact. Cleavage of the RCL at the active site (denoted P1-P1’) and insertion into β-sheet A, forming a fully anti-parallel β-sheet, yields the cleaved

‘relaxed’ conformation (Figure 1.4B). The transition from the ‘stressed’ to ‘relaxed’ state represents a lowering in the thermodynamic state of the serpin molecule. The latent conformation (Figure 1.4C), which is inactive, is formed by the partial insertion of the RCL into β-sheet A without the cleavage of the P1-P1’ bond. The initial step of inhibition involves the reversible formation of a noncovalent Michaelis-like enzyme- substrate complex (Figure 1.5B) through interactions with residues flanking the P1-

P1’ bond (Silverman et al. 2001). The target protease attacks the P1-P1’ bond, triggering covalent bonding between the target protease and the backbone carbonyl of the P1 residue and cleavage of the P1-P1’ peptide bond to form an acyl-enzyme intermediate (Petersen et al. 2001). From this point, the complex can proceed via the non-inhibitory pathway, resulting in release of active protease and an RCL-cleaved serpin (Gettins 2002) (Figure 1.5D), or the inhibitory pathway (Figure 1.5C), depending on serpin identity (Stratikos and Gettins 1999). In the inhibitory pathway, the RCL fully inserts into β-sheet A, translocating the P1 covalently linked protease a distance of more than 70 Ǻ (Fa et al. 2000) to the opposite end of the serpin. The protease is thereby inactivated by deformation against the base of the serpin

(Huntington et al. 2000).

RCL A

hA hD

B C

Figure 1.4: General conformations of serpins. Serpins exist in three main ‘normal’ states. The native ‘stressed’ conformation (A) is required for inhibitory activity. Cleavage of the P1-P1’ bond within the RCL and full insertion of the reactive centre loop (RCL) into β-sheet A yields the cleaved ‘relaxed’ conformation (B) , the most thermodynamically stable state for inhibitory serpins. In the ‘latent’ confirmation (C) , the RCL inserts into β-sheet A without P1-P1’ cleavage. Adapted from Gettins (2002).

A B

Figure 1.5: The inhibitory mechanism of serpins. The ability to undergo major conformational changes is central to the inhibitory mechanism of serpins. The native conformation (A) is required for inhibitory activity. The first step in the inhibitory mechanism is the reversible formation of the noncovalent Michaelis-like complex (B) . From this point the reaction can traverse either the inhibitory or non- inhibitory pathway. The inhibitory pathway results in the formation of a covalent complex (C) with the active site of the protease inactivated via distortion against the base of the serpin. The non-inhibitory pathway results in cleavage of the serpin and release of the active protease (D). Adapted Law et al. (2006).

1.3.2 PLASMINOGEN ACTIVATOR INHIBITOR TYPE 2 (PAI-2, SERPIN B2)

PAI-2 is a 415 amino acid clade B serpin that is present in two forms in the human body, an extracellular 60 kDa glycosylated form and a non-glycosylated 47 kDa form found both intra- and extracellularly (Genton et al. 1987; Wohlwend et al. 1987). PAI-

2 is an efficient inhibitor of both uPA (second order rate constant = 2.1 × 10 6 M -1s-1) and tc-tPA (second order rate constant = 5.3 × 10 5 M -1s-1), but does not efficiently

inhibit sc-tPA (second order rate constant = 4.6 × 10 3 M -1s-1) (Thorsen et al. 1988).

Whilst PAI-2 levels in plasma are normally too low to be detected, in certain conditions, such as pregnancy and some myelomonocytic leukemias, PAI-2 is consistently detected in plasma (Kawano et al. 1970; Astedt et al. 1987; von Heijne et al. 1991; Brenner 2004). The main physiological producers of PAI-2 are activated monocytes and macrophages (Wohlwend et al. 1987; Hamilton et al. 1993; Ritchie et al. 1997), however additional cell types including eosinophils (Swartz et al. 2004), keratinocytes (Robinson et al. 1997), microglia (Akiyama et al. 1993), endothelial and epithelial cells (Kruithof et al. 1995) have also been demonstrated to produce PAI-2 in vivo .

The identification of PAI-2 as a clade B serpin was due to its significant amino acid homology with chicken ovalbumin (SerpinB14), the presence of an internal secretion signal, shorter N- and C- termini than a prototypical serpin and a penultimate serine residue (Kruithof et al. 1995). Whilst most clade B serpins have only an intracellular distribution, this is not the case for PAI-2. However, the majority of expressed PAI-2 is not secreted (Dickinson et al. 1995) due to two mildly hydrophobic internal regions that serve as an inefficient internal signal sequence (Belin et al. 1989; von Heijne et

al. 1991). The secretion efficiency of PAI-2 can be significantly increased by fusing

the signal sequence of PAI-1 to the N-terminus of the PAI-2 encoding gene (Mikus et

al. 1993; Mikus and Ny 1996).

The tertiary structure of PAI-2 is that of a typical serpin, with 9 α-helices (A-I) and 3

β-sheets (A-C) (Figure 1.6). PAI-2 does not readily convert into the latent serpin

conformation. The gene encoding PAI-2 possesses an exon encoding a polypeptide

loop (the CD-loop) between the α-helices C and D. At 33 amino acids, the CD-loop of PAI-2 is the longest among known serpins (Remold-O'Donnell 1993; Scott et al.

1999). The CD-loop is involved in transglutaminase mediated cross-linking to cellular and ECM proteins (Jensen et al. 1996). The CD-loop regulates the ability of PAI-2 to polymerise (Wilczynska et al. 2003), with PAI-2 the only known serpin to polymerise spontaneously and reversibly under physiological conditions, via the formation of a disulfide bond between Cys 79 of the CD-loop and Cys 161 at the bottom of helix F

(Mikus et al. 1993; Mikus and Ny 1996). Whilst mutations in other serpins, such as

α1-antitrypsin and neuroserpin result in protein polymerisation and are characteristic

of conformational diseases (Gooptu and Lomas 2009), no known pathology is

associated with PAI-2 polymerisation. The tertiary structure of PAI-2 has only been

resolved for a mutant lacking the CD-loop (Harrop et al. 1999) (Figure 1.6) and as

such, the precise conformation of the CD-loop is unknown. Using intramolecular

measurements, Lobov et al. (2004) triangulated the CD-loop to the side of the

molecule in monomeric PAI-2. In the polymerogenic form, the CD-loop folds under

the bottom of the molecule to facilitate formation of the Cys 79 -Cys 161 disulfide bond

(Lobov et al. 2004).

RCL

CD-loop

Figure 1.6: Structure of plasminogen activator inhibitor type-2. As a member of the serpin family of proteins (clade B), PAI-2 contains 9 α-helices, 3 β-sheets and a reactive centre loop (RCL). PAI-2 also possesses a CD-loop – a polypeptide chain between α-helices C and D (putative position shown as a dotted line). The conformation of the CD-loop has yet to be resolved and the structure shown is a CD- loop and RCL deleted mutant. Structure is coloured from N-terminus (blue) to C- terminus (red). Constructed using PDB coordinates 1BY7 (Harrop et al. 1999) and the PyMOL molecular graphics system (DeLano 2002).

1.3.2.1 Expression of PAI-2

PAI-2 expression is acutely up-regulated in pregnancy, inflammation, infection and

other pathophysiological conditions. Isolation and characterisation of the PAI-2

promoter region has identified mechanisms by which transcription of PAI-2 mRNA

and/or protein levels can be up-regulated by a wide range of activating molecules

(Table 1.1). The PAI-2 promoter region contains a putative MAZ (Myc-associated

zinc finger protein) binding site (Almeida-Vega et al. 2009), an ASC-1 (activating

signal cointegrator-1) site (Almeida-Vega et al. 2009), two AP1-like binding sites

(Cousin et al. 1991), an AP2-like binding site (Cousin et al. 1991), CRE-like element

(Cousin et al. 1991; Park et al. 2005) and a NF-κB binding site (Mahony et al. 1999;

Park et al. 2005). Response elements to glucocorticoids (Schuster et al. 1993;

Schuster et al. 1994), retinoic acid (Schuster et al. 1993; Schuster et al. 1994) and xenobiotics (Sutter et al. 1991) have also been localised to the 5’ promoter region of

PAI-2. In addition to the promoters described above, the PAI-2 gene contains at least two repressor regions in the 5’ region of the PAI-2 gene (positions,1859-1100 and

259-219) (Antalis et al. 1996). The latter site also contains a binding site for an as yet unidentified nuclear protein (Dear et al. 1996). A repressor motif termed PAUSE-1

(PAI-2 upstream silencer element-1, sequence: 5’-CTCTCTAGAGAG-3’) (Antalis et al. 1996) is located at position -1832 and binds a specific, ~ 67 kDa binding factor

(Ogbourne and Antalis 2001). This region overlaps with the repressor described by

Dear et al. (1996), suggesting that these studies described the same repressor.

Full length PAI-2 mRNA is unstable, with a half-life of approximately one hour

(Maurer et al. 1999). The best characterised mRNA degradation components are AU-

Table 1.1: The expression of PAI-2 mRNA and/or protein can be regulated by a range of biological agents in a diverse set of cell types. Expression Agent mRNA Protein Cell Type Cell Line Ref.

Pro-inflammatory Cytokines TNF-α ↑ ↑ Fibroblast HT-1080 (Medcalf et al. 1988) ↑ ND Fibroblast HT1080 (Tierney and Medcalf 2001) Fibroblast PC ↑ ↑ (Pytel et al. 1990) Melanocyte SK-MEL-109 ↑ ND Myeloid U-937, PC (Gyetko et al. 1992) ↑ ↑ Vascular SM PC (Jang et al. 2004) ↑ ↑ Keratinocyte PC (Wang and Jensen 1998) Interferon-γ ↑ ↑ Monocyte U-937, PC (Gyetko et al. 1992) Fibroblast RT4, RT112, DAG-1, ND ↑ (Champelovier et al. 2003) (Bladder) T24, J82S, TCCsup Fibroblast PC IL-1β ND ↑ (Hannocks et al. 1992) (BM) ↑ ND Monocyte PC (Gyetko et al. 1993) Fibroblast PC ↑ ↑ (Hamilton et al. 1993) (Synovial) IL-2 ↑ ND Monocyte PC (Gyetko et al. 1993) Epithelial PC IL-13 ↑ ND (Woodruff et al. 2007) (Bronchial) G-CSF ↑ ↑ Monocyte PC (Hamilton et al. 1993) M-CSF ↑ ↑ Monocyte PC (Hamilton et al. 1993) Steriods/Hormones Epithelial Gastrin ↑ ND AGS (Varro et al. 2002) (Gastric) Epidermal Growth Epithelial A-431 Factor (EGF) ↑ ↑ (George et al. 1990) (Epidermal) Cumulus PC ↑ ND Granulosa- PC (Piquette et al. 1993) luteal Glucocorticoids ↓ ↓ Fibroblast HT-1080 (Medcalf et al. 1988) Epithelial ↓ ND PC (Woodruff et al. 2007) (Bronchial) Pathogenic Stimuli LPS ↑ ↑ Monocyte PC (Schwartz and Bradshaw 1992) ↑ ND Macrophage RAW-267, PC (Costelloe et al. 1999) ↑ ND Monocyte PC (Suzuki et al. 2000) ↑ ↑ Fibroblast PC (Xiao and Bartold 2004)

Signalling mediators cAMP ↑ ND Myeloid PL-21 (Niiya et al. 1994) (Wang and Jen sen 1998; Seo et Calcium ↑ ↑ Keratinocytes PC, HaCaT al. 2002) ↑ ND Neurons PC (Zhang et al. 2009) Toxins Dioxin ↑ ND Keratinocyte SCC-12F (Sutter et al. 1991) Epithelial ↑ ND M13SV1 (Ahn et al. 2005) (Breast) Kainate ↑ ND Neuronal Whole tissue (Sharon et al. 2002) Okadaic acids ↑ ND Fibroblast HT-1080 (Medcalf 1992) Myeloid U-937 Oxamflatin ↑ ↑ Myeloid U-937 (Dear and Medcalf 2000) Tumour promoting agents PMA ↑ ND Macrophage RAW-267, PC (Costelloe et al. 1999) U-937, THP-1, HL 60, (Genton et al. 1987; Schl euning ↑ ↑ Myeloid K-562 et al. 1987; Schuster et al. 1993) Vasculature effectors and regulators Angiotensin II ↑ ND Aortic SM PC (Feener et al. 1995) D-Dimer ND ↑ Myeloid NOMO-1 (Hamaguchi et al. 1991) Factor VIIa ↑ ND Keratinocytes HaCaT (Camerer et al. 2000) Monocyte/ U-937, primary (Lundgren et al. 1994; Ritchie et Thrombin ↑ ↑ Macrophage cultures al. 1995) Other Lipoprotein-a ↑ ↑ Monocyte PC (Buechler et al. 2001) U-937, THP-1, HL 60, Retinoic acid ↑ ↑ Myeloid (Schuster et al. 1993) K562 ↑ ND Keratinocytes PC (Braungart et al. 2001) ↑ ↑ Monocyte PC (Montemurro et al. 1999) VLDL and LDL ND ↑ Monocyte PC (Wada et al. 1994)

ND: Not Determined PC: Primary Culture

rich regions in the 3’ region of mRNAs. The 3’-untranslated region of PAI-2 mRNA

contains an AU-rich region which plays an important role in the degradation of PAI-2

mRNA (Maurer and Medcalf 1996). Disruption of the nonameric motif 5’-

UUAUUUAUU-3’ via mutagenesis led to an increase in PAI-2 mRNA and protein

expression levels (Maurer et al. 1999). Additionally, this motif is a binding site for

HuR, which has been associated with mRNA decay (Maurer et al. 1999).

Tristetraprolin, a CCCH tandem zinc finger protein that binds to ARE-containing transcripts, also interacts with the AU-rich region of the 3’-UTR and mediates PAI-2 mRNA degradation (Yu et al. 2003). Exon 4 of PAI-2 also contains an mRNA instability region, sharing homology with instability determinants of other mRNAs

(Tierney and Medcalf 2001).

1.3.2.2 Polymorphisms

Whilst SERPINB2 -/- mice have been successfully generated and described (Dougherty et al. 1999), no naturally occurring PAI-2 deficient humans have been reported.

Additionally, PAI-2 has not, as yet, been linked to any ‘serpinopathies’, that is, diseases directly related to or caused by aberrant PAI-2 function. However, PAI-2 expression by trophoblasts during the very early stages of pregnancy (Astedt et al.

1998) suggests that mutations within the PAI-2 gene resulting in protein dysfunction could be embryonic lethal in humans. Successful development of the SERPINB2 -/-

mouse model can be explained by inter-species variability, whereby PAI-2 expression

is not detectable in littermates until late stage gestation (Ong et al. 2000) and therefore

does not appear to play a role in early murine fetal development or placental

homeostasis. Differences in PAI-2 expression between humans and mice during

24 pregnancy indicate the unsuitability of this mouse model for human fetal development studies.

Whilst no mutation within the PAI-2 gene resulting in dysfunction at the protein level has been described, there are numerous studies showing correlation between disease states and polymorphisms of the PAI-2 gene. Two isoforms of PAI-2 have been described, with variations in three amino acids (Antalis et al. 1988; van den Berg et al.

1990). These variations occur at positions 120, 404 and 413 with Asn, Asn and Ser in isoform A and Asp, Lys and Cys in isoform B. Each of these changes is the result of a single base change in each codon (120, Aat/Gat; 404, aaC/aaG; 413, tCc/tGc), however it appears that all three mutations are linked (ie. they are a haplotype) and do not exist in isolation. Whilst the functional consequences of these residue changes are unknown, a number of studies have associated PAI-2 genotype and disease. Form A has been associated with lupus erythematosus and thrombic pneumonia in patients with this condition (Palafox-Sanchez et al. 2009), anti-phospholipid syndrome

(Vazquez-Del Mercado et al. 2007) and mycocardial infarction (Buyru et al. 2003;

McCarthy et al. 2004), although Foy and Grant (1997) found no evidence of the link between PAI-2 variants and mycocardial infarction. These disease states all relate to disorders in haemostasis, highlighting a role for PAI-2 in this system. In addition,

Shioji et al. (2005) showed an association between the A form and prostate cancer.

PAI-2 B is linked to preterm birth in subjects later diagnosed with cerebral palsy

(Gibson et al. 2007) and poor overall survival in patients with non-small cell lung cancer (Di Bernardo et al. 2009). Recently, Byrol et al. (2010) found that isoform B is associated with dose-dependent proteasome inhibitor drug related peripheral neuropathy in multiple myeloma patients.

1.3.2.3 Intracellular roles

The distribution of PAI-2 within the nucleus, cytoplasm was well as the extracellular

environment suggests accessory functions independent of plasminogen activator

inhibitor activity as neither tPA or uPA are found in the intracellular environment and

no binding partner for intracellular PAI-2 as an inhibitory serpin has yet to be

identified. Intracellular PAI-2 has been associated with a variety of potential roles

such as signal transduction (Antalis et al. 1998), inhibition of apoptosis (Dickinson et

al. 1995), monocyte proliferation and differentiation (Yu et al. 2002). Zhou et al.

(2001) found that over expression of PAI-2 in keratinocytes resulted in sustained

cutaneous papilloma lesions compared to controls within a mouse model potentially

due to protection from apoptosis. Earlier studies reported that HT1080 fibrosarcoma

and HeLa epithelial cell lines transfected with PAI-2 were protected from TNF-α

induced apoptosis (Dickinson et al. 1995) with this function dependant on the CD-

loop (Dickinson et al. 1998). Additionally, PAI-2 was also reported to protect primary

culture human macrophages from Mycobacterium avium infection (Gan et al. 1995).

More recently, the hypothesis that PAI-2 protects against apoptosis by these

mechanisms has been questioned. Recently, Fish and Kruithof (2006) conducted a

thorough investigation of PAI-2 in the prevention of TNF-α induced apoptosis using

HeLa, Isreco-1 and HT1080 cell lines. Unlike earlier works, this study used a lenti- viral transfection system which did not require clonal selection and subsequently found that PAI-2 had no effect on apoptosis, concluding that previous transfection methodology had most likely resulted in a clonal bias effect.

The tumour suppressor retinoblastoma protein (Rb) was previously identified as a binding partner for PAI-2 (Darnell et al. 2003), stabilising Rb and thereby inhibiting

apoptosis. Subsequent experiments reported suppression of human papilloma virus

(HPV) type-18 oncogenes (E6/E7) by PAI-2 through the stabilisation of Rb (Darnell

et al. 2005). However, Fish and Kruithof (2006) were unable to detect any difference

in Rb levels when comparing IS-1 and HT1080 cell lines transfected with PAI-2.

Furthermore, Schroder et al. (2010) were unable to find a correlation between PAI-2

and Rb expression when comparing the SERPINB2 -/- mouse model to littermates and

Major et al. (2011) showed that PAI-2 did not bind to or regulate the expression of

Rb. As the studies describing the PAI-2 Rb interaction (Darnell et al. 2003; Darnell et al. 2005) used the same HeLa cell lines stably transfected with PAI-2 as those used in the early apoptosis studies (Dickinson et al. 1995), it appears likely that the reported stabilisation of Rb by PAI-2 is also a product of clonal bias. The role of intracellular

PAI-2 thus remains poorly defined.

1.3.2.4 Extracellular roles

Secreted or released PAI-2 (the previously described 60 kDa or 47 kDa forms, respectively) have been detected in various biological fluids including pregnancy plasma, amniotic fluids and umbilical cord blood (Astedt et al. 1985; Lecander and

Astedt 1987; Booth et al. 1988; Astedt et al. 1998), gingival fluid (Kinnby 2002), saliva (Virtanen et al. 2006), peritoneal fluid (Scott-Coombes et al. 1995), infectious pleural effusions (Aleman et al. 2003), as well as in human tears (Csutak et al. 2008).

PAI-2 has also been detected in the plasma of patients with monocytic leukaemia (up to 160 ng.mL -1) (Scherrer et al. 1991), severe sepsis (Robbie et al. 2000), in ascites fluid and tumour vessel blood from patients with advanced ovarian cancer (up to 108 ng.mL -1) (Lecander et al. 1990), and in the plasma of hypergastrinemic patients

(Varro et al. 2002).

Extracellular PAI-2 may regulate plasminogen activation through inhibition of uPA

(and tPA) via the inhibitory serpin pathway. Al-Ejeh et al. (2004) found that cell-

surface uPA:PAI-2 complex formation on breast and prostate cancer cells at 37ºC was

-1 both efficient and rapid (inactivation rate constant [k i] of 0.32 – 0.47 min and inhibition constant [K I] of < 81 pM). The ability of PAI-2 to inhibit two chain is tPA

dependant on the type of tPA binding interaction to either cell surfaces or proteins.

For example, PAI-2 has been demonstrated to inhibit two chain tPA bound to various

cell types in vitro (Lee et al. 2010), tPA bound to purified annexin II heterotetramer

(Lobov et al. 2008) and poly-D-lysine-bound-tPA (Leung et al. 1987). However, as is

the case for PA1-1, fibrin-bound tPA is protected from inhibition by PAI-2 (Leung et

al. 1987).

Direct evidence of PA:PAI-2 complexes as indicative of protease inhibition in vivo is

lacking, though PAI-2 in the plasma is able to form inhibitory complexes with added

PAs. Kiso et al. (1991) identified placental derived PAI-2 cleaved at the P1-P1’

position, indicating that it had reacted with a target protease. Additionally, PAI-2 and

uPA have been shown to co-localize within syncytiotrophoblasts of the placenta

(Nakashima et al. 1996). Furthermore, PAI-2 and tPA exist as high molecular weight

SDS-stable complexes in the gingival crevicular fluid, suggesting inhibitory complex

formation (Brown et al. 1995). Using a monoclonal antibody (2H5), that specifically

recognises the ‘relaxed’ conformation of PAI-2 (as adopted in a protease:serpin

inhibitory complex (Saunders et al. 1998)), the presence of relaxed PAI-2 in gingival

epithelium has been shown (Lindberg et al. 2001), again suggesting inhibition of

plasminogen activators.

1.3.3 PLASMINOGEN ACTIVATOR INHIBITOR TYPE -1 (PAI-1, SERPIN E1)

PAI-1, the primary regulator of tPA and potentially uPA activity in human blood

(Agirbasli 2005; Dellas and Loskutoff 2005), is a 379 amino acid clade E serpin secreted as a 60 kDa protein by platelets, monocytes, macrophages, endothelial cells and smooth muscle cells (Gettins 2002; De Taeye et al. 2004). PAI-1 effectively inhibits both uPA and tPA with second order rate constants of 2.5 × 10 7 M-1s-1 and 1 ×

10 7 M -1s-1, respectively (Thorsen et al. 1988). PAI-1 also inhibits pro-uPA, but with a

> 1000 fold reduction in the rate of inhibition compared to uPA (De Taeye et al.

2004). Unlike PAI-2, PAI-1 is able to efficiently inhibit sc-tPA (second order rate

constant = 5.5 × 10 6 M -1s-1) (Thorsen et al. 1988). The structure of PAI-1 conforms to

that of the general serpin architecture (Figure 1.7) and is remarkably similar to that of

PAI-2 despite significant variation (24.2% sequence homology) in peptide sequence

(Huber and Carrel 1989). The relationships between the structural domains of PAI-1

and functionality are well understood due to extensive characterisation (De Taeye et

al. 2004). Under physiological conditions, PAI-1 is relatively unstable and rapidly

converts to a latent form incapable of interacting with uPA (Lindahl et al. 1989;

Andreasen et al. 2000; Stout et al. 2000). However, the half-life of active PAI-1,

which is normally 1-2 hours, can be extended two to four-fold via binding to

vitronectin (Keijer et al. 1991; Padmanabhan and Sane 1995). This binding has been

shown, via site directed mutagenesis, to be dependent on residues Arg 101 and Met 110 ,

Leu 116 and Gln 123 , which reside in α-helix E and strand 1 of β-sheet A of PAI-1

(Lawrence et al. 1994; Arroyo De Prada et al. 2002; Jensen et al. 2002; Einholm et al.

2003; Jensen et al. 2004). As vitronectin is an ECM component, PAI-1 thus can

modulate cellular adhesion and migration (Dellas and Loskutoff 2005).

RCL

Figure 1.7: Structure of plasminogen activator inhibitor type-1. PAI-1 conforms to the general structure of a serpin with 9 α-helices, 3 β-sheets and a reactive centre loop (RCL). The structure shown is lacking the RCL and represents the cleaved form of PAI-1. Structure is coloured from N-terminus (blue) to C-terminus (red). Constructed using PDB coordinates 1A7C (Xue et al. 1998) and the PyMOL molecular graphics system (DeLano 2002).

As the primary physiological inhibitor of plasminogen activation (Agirbasli 2005;

Dellas and Loskutoff 2005), PAI-1 is an important regulator of the fibrinolytic

system, which serves to prevent intravascular thrombosis and is thought to participate

in the regulation of such processes as ovulation, embryogenesis and wound repair

(Duffy 2004). Whilst PAI-1 inhibits plasminogen activation at the cell surface, via

inactivation and clearance of uPA and tPA, it has also been found to play a critical

role in cancer development, being associated with tumor growth, invasion, metastasis

and angiogenesis (Webb et al. 2001; Degryse et al. 2004). The use of PAI-1 (along

with co-expression of uPA) has been suggested as a prognostic marker in cancer

diagnosis (Harris et al. 2007), with (somewhat paradoxically) high PAI-1 levels

associated with increased malignancy in gynaecological cancers, gliomas, sarcomas,

renal cell carcinoma and cancers of the breast, bladder and gastrointestinal tract

(Foekens et al. 2000; Stefansson et al. 2003; Bajou et al. 2004; Czekay and Loskutoff

2004; Degryse et al. 2004). This may be due to the effects of PAI-1 on proliferation

and migration which occur through involvement in non-proteolytic roles, the specifics

of which will be discussed in Section 1.4. It has been suggested that the cancer related

actions of PAI-1 appear to be dose-dependent, with physiological concentrations of

PAI-1 promoting tumour invasion and metastasis whilst supraphysiological levels of

PAI-1 inhibit tumour vascularisation (Bajou et al. 2004).

1.3.4 PROGNOSTIC VALUES OF PAI-1 AND PAI-2 IN CANCER

Whilst the inhibition of cell surface uPA activity by PAI-1 and PAI-2 in vitro serves

to decreases plasminogen activity, tumour overexpression of uPA, uPAR and PAI-1 is

associated with poor patient prognosis (Foekens et al. 2000; Choong and Nadesapillai

2003; Duffy 2004; Ohba et al. 2005) and increased metastatic potential (Duffy et al.

1999; Scherrer et al. 1999; Schmitt et al. 2000; Look et al. 2002; Weigelt et al. 2005).

The significance of this relationship in expression and disease outcomes is further

highlighted by the recommendation of uPA/PAI-1 expression as a tumour marker in

breast cancer by the American Society of Clinical Oncology (Harris et al. 2007). In

contrast, high levels of PAI-2 expression in breast carcinomas is correlated with

increased, relapse-free survival (Sumiyoshi et al. 1992; Bouchet et al. 1994; Foekens

et al. 1995; Ishikawa et al. 1996; Duggan et al. 1997; Umeda et al. 1997; Bouchet et

al. 1999; Foekens et al. 2000; Borstnar et al. 2002; Borstnar et al. 2002; Spyratos et al.

2002; Duffy and Duggan 2004; Meijer-van Gelder et al. 2004), whilst low expression

has the opposite effect. Whilst relatively few studies have assessed the prognostic

value of PAI-2 expression in other cancer forms, there is a general trend between

PAI-2 expression and neutral or positive outcomes (Croucher et al. 2008).

Whilst definitive identification of uPA:PAI-2 complexes within tumour masses is

absent, the relationship between positive patient prognosis and high PAI-2 levels in

node negative breast cancer is only statistically relevant if uPA levels are also

elevated (Bouchet et al. 1999; Foekens et al. 2000). This suggests that the inhibitory

relationship between uPA and PAI-2 in the tumour microenvironment mediates this

functionality. Previous studies have hypothesised that PAI-2 may be the bona fide uPA inhibitor in this context (Nakamura et al. 1992; Bouchet et al. 1994; Umeda et al.

1997; Nozaki et al. 1998; Bouchet et al. 1999; Zhao et al. 2003). Whilst it appears that the sole function of extracellular PAI-2 is to inhibit and clear uPA (and tPA) activity, the potential functions of PAI-1 are much more diverse as PAI-1 is also able to compete for binding to the ECM protein vitronectin, thereby moderating cell adhesion/deadhesion. Furthermore, PAI-1 has been demonstrated, upon binding to

LDLRs to activate pro-mitogenic signalling pathways, resulting in cell proliferation

and migration (discussed in section 1.4.3). These additional physiological roles may

explain the paradoxical prognostic profiles of PAI-1 verses PAI-2 expression in

cancer.

1.4 NON-PROTEOLYTIC FUNCTIONS OF THE PLASMINOGEN

ACTIVATION SYSTEM

Subsequent to formation of the enzyme:substrate complex, uPA:PAI-1 and uPA:PAI-

2 are cleared from the cell surface via receptor mediated endocytosis and subsequently degraded in lysosomes (Ploug et al. 1991; Al-Ejeh et al. 2004). This endocytosis is dependent on cell surface uPA to uPAR association (Hussain et al.

1999; Strickland et al. 2002; Strickland and Ranganathan 2003; Al-Ejeh et al. 2004).

As uPAR is anchored to the plasma membrane by a glycosylphosphadityl inositol

(GPI) moiety (Ploug et al. 1991), endocytosis requires the participation of an additional transmembrane receptor. Members of the LDLR superfamily have been demonstrated to mediate the internalisation of uPA:PAI complexes (Kounnas et al.

1993; Argraves et al. 1995; Kasza et al. 1997; Strickland et al. 2002; Croucher et al.

2006; Croucher et al. 2007).

1.4.1 THE LDLR SUPERFAMILY

The LDLR superfamily contains (among other receptors), LDLR itself (low density lipoporiten receptor), the low-density lipoprotein receptor-related protein-1 (LRP1), megalin (LRP2) and the very low-density lipoprotein receptor (VLDLR). Members

of the LDLR superfamily are implicated in diverse physiological roles such as

lipoprotein metabolism, cell signaling in a variety of tissue types and regulation of

extracellular proteolytic activity through an ability to bind to a variety of functionally

and sequentially diverse ligands (such as lipoproteins, MMPs, serpin:protease

complexes and cytotoxic species) (Strickland et al. 2002; Strickland and Ranganathan

2003; May et al. 2007).

LDLR-family members are composed of epidermal growth factor (EGF)-like repeats, clusters of YWTD-containing repeats (believed to be arranged in β-propeller

domains), clusters of ligand type binding repeats, a single transmembrane domain and

a cytoplasmic domain (Strickland et al. 2002) (Figure 1.8). Binding to target proteins

occurs via electrostatic interactions mediated by negatively charged regions in the

ligand type binding repeats (Lillis et al. 2005), with this supported by the resolved

crystal structure of an LDLR-RAP (receptor associated protein) complex revealing

interactions between acidic residues in the receptor and basic regions in the ligand

(Fisher et al. 2006). Further, Jensen et al. (2006) proposed an LDLR minimal binding

motif for ligands consisting of two basic residues separated by 2-5 residues and N-

terminal flanked by hydrophobic residues. The cytoplasmic domain of LDLR

members contains sequence motifs that are recognised by adaptor proteins, resulting

in clathrin coated pit-mediated internalisation (Chen et al. 1990). All LDLRs contain

at least one copy of the NPxY motif, but may contain other internalisation motifs. For

example, the YxxL in LRP1 serves as the dominant adapter protein recognition motif

rather than either of the two NPxY motifs (Li et al. 2000). Intracellular signal

transducing adapter proteins are also able to interact with the cytoplasmic domains of

LDLR members (Strickland and Ranganathan 2003; Lillis et al. 2005).

Figure 1.8: Structural representations of the LDLR members LDLR, VLDLR and LRP1 . All LDLR members comprise of a transmembrane domain and at least one ligand binding domain, EFG repat, YWTD β-propeller and cytoplasmic domain containing at least one NxPY motif. Adapted from Rebeck et al. (2006).

Subsequent to internalisation, the receptor and ligand are delivered to endosomes

where dissociation of the complex takes place. Typically, the receptor is returned to

the plasma membrane whilst the ligand is degraded (Hussain et al. 1999), with Brown

et al. (1997) suggesting that recycling can occur over 100 times before receptor

degradation. As LDLRs are localised in clatherin coats pits on the cell surface,

LDLRs are repetitively internalised and recycled to the cell surface regardless of

bound ligand. Release of the ligand from the receptor is moderated by the acidic pH

of the endosome. X-ray crystallography has revealed that, under acidic conditions, the

YWTD propeller domain of LDLR can act as an alternative ligand for the ligand

binding type repeats, displacing bound LDL (Innerarity 2002; Rudenko et al. 2002). It

is likely that a similar mechanism is involved in the release of other ligands from

LDLRs.

1.4.2 ENDOCYTOSIS OF uPA:PAI COMPLEXES

The internalisation of uPA:PAI-1 complexes by LDLRs is best characterised for the receptors VLDLR and LRP1, although LRP2 (also known as megalin and gp330) and sorLA (a member of the Vps10p-domian receptor family, but containing LDL ligand binding repeat domains (Hermey 2009)) have also been shown to bind (and presumable internalise) proteins of the plasminogen activation system (Table 1.2).

Whilst early studies seemed to suggest that PAI-2 is not internalised following inhibition of uPAR-bound uPA (Ragno et al. 1995; Andreasen et al. 1997; Andreasen et al. 2000; Schmitt et al. 2000), it is now clear that uPA:PAI-2 complexes are internalised via LRP1 (Croucher et al. 2006) and VLDLR (Croucher et al. 2007) and subsequently localised to endosomes and lysosomes (Al-Ejeh et al. 2004). Unlike

Table 1.2: Reported affinities of plasminogen activation system components for receptors of the low-density lipoprotein receptor family Ligand LRP1 K D (nM) LRP2 K D (nM) VLDLR K D (nM) sorLA K D (nM) 12 1# Pro-uPA 45 2* 18.4 8• - 5 16^ 5 16^ 50 1# 2* 60 3♦ ♦ • 100 uPA 100 3 10.3 8 12 16^ 209 & 31.2 14^ 200 13^ 7 16^ 55 1# 93 2* PAI-1 11 8• 51.8 14^ 23 16^ 35 5^ 24 16^ PAI-2 No interaction 13 - No interaction 14 - PN-1 - - - - 0.4 1# 0.8 6• 3 2* 1.5 3♦ ♦ • 0.5 3 0.8 9 14 11 • 0.48 15^ uPA:PAI-1 • • 1.4 6 1.1 8 15 12* 2.3 16^ 0.226 15^ 84.8 & 1.51 14^ 2.1 16^ 1.6 15^

uPA:PAI-2 36.2 13^ - 4.68 15^ -

uPA:PN-1 0.8 6• - 0.14 6• - 1130 1# 158 5^ tPA - 124 17^ - 323 7^ 66.9 17^ 3 1# 6.1 5^ tPA:PAI-1 - 23.0 & 139 17^ 23 16^ 12 16^ 0.95 & 21.2 17^

tPA:PAI-2 66.1 17^ - 131 17^

Plasminogen - ~225 10* - -

References 12: Argraves et al. 1995 Experiment type 1: Nykjaer et al. 1994 13: Croucher et al. 2006 # Competition binding (NB. As such

2: Kounnas et al. 1993 14: Croucher et al. 2007 these are EC 50 values and not K D values) 3: Heegaard et al. 1995 15: Skeldal et al. 2006 * ELISA 4: Degryse et al. 2004 16: Gliemann et al. 2004 ^ Surface plasmon resonance (NB.

5: Horn et al. 1997 17. Lee et al. 2010 Where more than one K D value is 6: Kasza et al. 1997 present, multiple binding sites are 7: Samson et al. 2008 present in the species) 8: Stefansson et al. 1995 • Solid phase binding assay – microtitre 9: Moestrup et al. 1993 plate ( 125 I labelled ligand) 10:Kanalas and Makker 1991 ♦ Solid phase binding assay 11: Mikhailenko et al. 1999 electrophoretically immobilised analyte (125 I labelled ligand)

PAI-1 (Nykjaer et al. 1994), PAI-2 is unable to bind directly to LRP1 or VLDLR

(Croucher et al. 2006; Croucher et al. 2007).

The affinity of the uPA:PAI-1 and uPA:PAI-2 complexes for LDLR members are

significantly higher than that for uPA alone (Table 1.2), with this increased affinity

resulting in an increase in the rate of uPA clearance from the cell surface (Nykjær et

al. 1994; Croucher et al. 2006; Croucher et al. 2007). The binding of uPA:PAI-1 to

LDLR members is dependent on sites in the A-chain and serine protease domain of

uPA (Andreasen et al. 1994; Nykjær et al. 1994; Skeldal et al. 2006) and in the PAI-1

moiety of the complex (Horn et al. 1998; Rodenburg et al. 1998; Stefansson et al.

1998; Skeldal et al. 2006). Previous studies have identified a number of basic residues

in and around the α-helix D of PAI-1 that mediate the high affinity binding of the

PAI-1 moiety of the uPA:PAI-1 complex to LDLRs (Figure 1.9). In addition, the α- helix D of PAI-1 contains a proposed common binding motif for high affinity LDLR family ligands consisting of two basic residues separated by 2-5 residues and N- terminally flanked by hydrophobic residues (Jensen et al. 2006) (Figure 1.9E). This motif is not conserved within the α-helix D of PAI-2 (Figure 1.9E), with the first

hydrophobic/basic amino acid couple present, but the second pair replaced with serine

residues (Ser 111 , Ser 112 ). Results obtained by Croucher et al. (2006) and Croucher et

al. (2007) suggest that the recognition sites in uPA:PAI-2 for both LRP1 and VLDLR

A C

B D

E 69 88 PAI-1 KGMAPALRHLYK ELMGPWN PAI-2 DKIHSSFRSLSS AINASTG 101 119

Figure 1.9: Structural comparison of the LDLR binding region of PAI-1 and the corresponding region in PAI-2. (A, B) Structure of PAI-1 and PAI-2 showing location of residues in α-helix D of PAI-1 implicated in the interaction with LDLRs and the corresponding residues in PAI-2. (C, D) Comparison of surface potential around the α-helix D region of PAI-1 and PAI-2, regions of positive electrostatic potential are in blue, regions of negative potential in red and neutral regions in white. (E) Alignment of α-helix D amino acid sequence from PAI-1 and PAI-2. The putative minimal binding motif (Jensen et al. 2006) in PAI-1 is underlined with necessary basic and hydrophobic residues highlighted in yellow and blue respectively. Adapted from Croucher et al. (2007).

lie solely within the uPA moiety of the complex, highlighting the importance of this

LDLR minimal binding motif. The increased affinity of uPA:PAI-2 relative to uPA

alone is thought to be due to changes to the structure of uPA upon complex formation.

Few crystal structures of serpin:protease complexes exist, and whilst Dementiev et al.

(2006) showed that no major structural changes occur in porcine pancreatic elastase

when in complex with α1-antitrypsin (SerpinA1), there are major structural differences in uPA and porcine pancreatic elastase. This lack of an LDLR binding site in the PAI-2 moiety of the uPA:PAI-2 complex results in reduced binding affinity for

LDLRs compared to uPA:PAI-1 (Table 1.2). Whilst these differences may seem somewhat trivial, their impact on downstream functional effects is remarkable.

1.4.3 SIGNALLING PATHWAYS

Initially, it was thought that the function of the plasminogen activation system components in cancer was restricted to roles in the degradation of tissue boundaries

(Duffy 2004). However, as previously mentioned, further studies have identified the participation of plasminogen activator system components in cancer-related activities such as cell adhesion and motility. Additionally, components of the plasminogen activation system have been linked to the activation of pro-oncogenic cell signaling and promotion of cell migration (Chapman 1997; Andreasen et al. 2000; Kjoller

2002).

Binding of uPA to uPAR at the cell surface of MCF-7 cells has been demonstrated to result in transient extracellular signal-regulated kinase (ERK) phosphorylation

(Nguyen et al. 1999; Webb et al. 2001). The MAPK/ERK signalling pathway regulates cell proliferation, differentiation, apoptosis and migration (Webb et al.

2001). The phosphorylation/activation of FAK, p130 Cas , Src and Rac have also been reported to occur by uPA:uPAR mediated signal transduction (Kjoller 2002). The activation of the above signalling pathways occur independent of LDLR-family member association and are instead mediated via interactions with cell-surface integrins (Resnati et al. 2002).

PAI-1 has been shown to promote cancer cell line development through the activation of signalling pathways (Chapman 1997; Foekens et al. 2000; Webb et al. 2001; Duffy and Duggan 2004; Ohba et al. 2005; Weigelt et al. 2005). Endocytosis of uPA:PAI-1 via VLDLR has been found to result in sustained ERK phosphorylation in MCF-7

(Webb et al. 2001) and SGC7901 (Di et al. 2010) cells, with this sustained activation dependant on the high affinity interaction between the PAI-1 moiety of the complex and VLDLR (Webb et al. 2001). The mechanism underlying this signalling response is yet to be fully elucidated, and is likely mediated by a combination of described models (Strickland et al. 2002). PAI-1 is also able to stimulate cell migration independent of uPA, via activation of the Jak/Stat pathway (Degryse et al. 2004)

Endocytosis of uPA:PAI-2 does not result in sustained activation of signalling pathways, as determined by decreased global tyrosine phosphorylation (Croucher et al. 2007), but rather transient activation of ERK similar to that of uPA/uPAR association (Croucher, unpublished observation). Furthermore, treatment of MCF-7 cells with uPA:PAI-2 did not result in increased cell proliferation (Croucher et al.

2007). The hypothesis that this difference in the moderation of mitogenic signalling activity is dependent on the high affinity interaction between the PAI-1 moiety of the complex and VLDLR is supported by the lack of ERK activation by uPA:PAI-1 R76E

(uPA:PAI-1 R76E binds to LDLRs with decreased affinity, discussed further in

Chapter 3) and ability of RAP (which competes for LDLR binding) to block ERK

mediated cell proliferation. This difference in the activation of cell signalling

pathways may play an important role in the difference in biological activity of PAI-1

and PAI-2 in cancer.

1.5 POTENTIAL THERAPEUTIC VALUE OF PAI-2

The ability of PAI-2 to efficiently inhibit and clear uPA (Al-Ejeh et al. 2004), without activating pro-invasive pathways in cancer cells like PAI-1 (Croucher et al. 2007) has led to the development of PAI-2 as a basis for novel, targeted cancer therapies. High plasma levels of PAI-2 are well tolerated, as in the third trimester of pregnancy PAI-2 levels can reach 250 ng.mL -1 in plasma (Kruithof et al. 1987; Hunt et al. 2009) with

no apparent associated health risks. In addition, PAI-2 cannot inhibit sc-tPA or fibrin

bound tPA (Leung et al. 1987), negating what could be problematic interplay in

fibrinolysis. Also, PAI-2 does not convert to the latent serpin conformation –

remaining active until reacting with uPA or being cleared from the bloodstream.

Conjugation of PAI-2 to bismuth-213 (an alpha-emitting radioisotope) has been used

to treat breast, prostate and melanoma cancer xenografts in animal models (Allen et

al. 2001; Li et al. 2002; Ranson et al. 2002; Allen et al. 2003; Stutchbury et al. 2007).

Suwa et al. (2008) found that treatment of mice bearing a human colon cancer

xenograft with an intraperationeal infusion of recombinant PAI-2 inhibited primary

tumour growth, decreased the frequency of metastasis and increased the apoptotic

index of tumours. Transfection with PAI-2 of uPA expressing cells in xenograft

models has also been demonstrated to inhibit or completely prevent metastasis (Laug

et al. 1993; Mueller et al. 1995; Praus et al. 1999). Vine et al. (2010) showed that 2’-

deoxy-5-fluorouridine (a pro-drug form of the common chemotherapeutic 5-

flurouracil) conjugated to PAI-2 was able to preferentially kill uPA/uPAR

overexpressing cell lines.

1.6 SUMMARY AND AIMS

PAI-1 and PAI-2 appear to play disparate roles in the progression of cancer. Whilst both are able to inhibit uPA at the cell surface, thereby decreasing pericellular plasminogen activation, PAI-1 is implicated in non-proteolytic roles stimulating cell proliferation and migration. The molecular basis of the strong relationship between breast cancer prognosis and expression of uPA and PAI-1 versus PAI-2 is unknown.

Croucher et al. (2008) proposed a model to explain the differential prognosis profiles of PAI-1 and PAI-2 based on differing interactions with receptors in an extracellular context. Whilst attempts have been made to relate the protective role of cancer in

PAI-2 to an intracellular role, as yet no clear mechanism has been described.

Characterisation of the interactions between uPA:PAI-2 and the endocytosis receptors

LRP1 and VLDLR has demonstrated clear differences in the way in which PAI-1 and

PAI-2 interact with these proteins (Croucher et al. 2006; Croucher et al. 2007). Whilst it could be argued that on a biochemical basis these interactions differ only slightly, the impact on downstream physiological function is striking. Further structural characterisation of the interactions potentially underlying the differences in LDLR- mediated cell signalling activities of PAI-1 and PAI-2 on carcinoma invasion and metastasis may help explain the apparently paradoxical biomarker data for PAI-

1/PAI-2 in cancer. In addition, increased characterisation of the mechanism of

43 internalisation of PAI-2 will help provide further rationale for the development of

PAI-2 based targeted therapies.

Given the lack of a definitive mechanism to explain the contrasting impact of PAI-1 and PAI-2 expression in cancer, the overall aim of this thesis was to elucidate the molecular basis underlying the differences in LDLR binding between PAI-1 and PAI-

2 and examine the functional consequences of manipulating this interaction. Initially, characterisation of an improved method for the purification and production of PAI-2 to be used further in this study will be described (Chapter 2). As this PAI-2 construct lacks the interhelical CD-loop, the redundancy of this domain in the uPA-dependent functionality and subsequent endocytosis of the uPA:PAI-2 complex will be confirmed (Chapter 2). By comparing the residues previously implicated in the interaction between PAI-1 and LDLRs with the corresponding residues in PAI-2, a series of mutants will be made to introduce LDLR binding to PAI-2 (Chapter 3). The impact of introducing LDLR binding to PAI-2 on downstream cellular functions related to uPA internalisation will be characterised to fully detail the molecular basis underlying the differential LDLR dependent differences of PAI-1 and PAI-2 in cancer in vitro (Chapter 4).

CHAPTER 2

THE CD-LOOP OF PAI-2 (SERPINB2) IS

REDUNDANT IN THE TARGETING,

INHIBITION AND CLEARANCE OF CELL

SURFACE uPA ACTIVITY

2.1 INTRODUCTION

As previously introduced in Chapter 1, PAI-2 is able to specifically target, inactivate and clear cell surface uPA without interacting with components of the ECM or modifying other cellular behaviours that may promote tumour cell behaviour (unlike

PAI-1) (Croucher et al. 2007; Croucher et al. 2008). This supports the use of exogenous PAI-2 for uPA targeted cancer treatments. As discussed previously in

Section 1.5, promising results using bismuth-213 labelled PAI-2 have been obtained in a number of in vitro , in vivo and preclinical evaluations which show clear cell

targeting specificity and tumour efficacy with minimal side effects in relevant animal

models (Li et al. 2002; Ranson et al. 2002; Allen et al. 2003; Allen et al. 2004; Qu et

al. 2005; Abbas Rizvi et al. 2006; Song et al. 2006; Song et al. 2007; Stutchbury et al.

2007).

To date, all work undertaken in developing PAI-2 based targeted therapies have used

full length, PAI-2 wild-type. However, it may be possible to utilise smaller, more

easily producible PAI-2 constructs without compromising the uPA inhibitory and

clearance functions of PAI-2. Previous studies have reported the purification of PAI-2

from placenta (Wun and Reich 1987), cultured human monocytes (Kruithof et al.

1986), transfected CHO cells (Mikus et al. 1993; Mikus and Ny 1996), baculovirus

infected insect larvae (Zhang et al. 1998) and from recombinant expression systems in

yeast (Steven et al. 1991; Gould et al. 2003) and Escherichia coli (Ye et al. 1987;

Antalis et al. 1988; Mikus et al. 1993; Jensen et al. 1994; Zhou et al. 1997;

Muehlenweg et al. 2000; Jankova et al. 2001; Wilczynska et al. 2003; Wilczynska et

al. 2003; Lobov et al. 2004; Di Giusto et al. 2005; Suwa et al. 2008). Methods of PAI-

2 expression in E. coli have generally utilised a one or two step purification

procedure, usually involving metal affinity chromatography and/or ion exchange

chromatography. The shift in the literature towards affinity tag based systems for the

production of recombinant PAI-2 constructs (Zhou et al. 1997; Muehlenweg et al.

2000; Wilczynska et al. 2003; Wilczynska et al. 2003; Lobov et al. 2004; Di Giusto et

al. 2005) allows for the purification of PAI-2 under milder, native conditions and

avoidance of denaturation/renaturation (Zhou et al. 1997) or extreme pH treatment

(Mikus et al. 1993) as used previously. The presence of an N-terminal 6 × his-tag has previously been shown to have no significant impact on the uPA inhibitory activity of

PAI-2 (Wilczynska et al. 2003). Generally, his-tags are believed to have no effect on overall protein structure (Carson et al. 2007).

An issue associated with the purification of recombinant PAI-2 wild-type is that the

CD-loop of PAI-2 is readily cleaved in both E. coli or mammalian expression systems

(Lobov et al. 2004). This results in two fractions of recombinant PAI-2 which retain inhibitory activity but require additional purification steps such as ion-exchange chromatography (Lobov et al. 2004) to achieve a homogenous protein population. Di

Giusto et al. (2005) showed that 6 × his-tagged PAI-2 lacking the CD-loop (termed

PAI-2 ∆CD-loop) can be purified with a one-step procedure and exhibited identical soluble phase uPA inhibitory activity to that previously reported for full length PAI-2.

As discussed in Chapter 1, the functionality of the CD-loop has been described primarily in an intracellular context (Croucher et al. 2008).

Altogether, this suggested that PAI-2 ∆CD-loop, being easy to produce and purify in

addition to retaining uPA inhibitory activity, could be used as an exogenous uPA

targeting substitute for PAI-2 wild-type in a therapeutic setting. Furthermore, the use

of PAI-2 ∆CD-loop may be suitable to further characterise the uPA-dependent roles of PAI-2 in vitro . This chapter compares the expression and purification of 6 × his- tagged wild type PAI-2 and PAI-2 ∆CD-loop and shows for the first time that the cell

bound uPA inhibitory activity and rapid clearance of uPA/uPAR by PAI-2 is not

compromised by the 6 × his-tagged form lacking the CD-loop.

2.2 MATERIALS AND METHODS

2.2.1 MATERIALS

The expression vectors pET15b/PAI-2 wild-type and pET15b/PAI-2 ∆CD-loop (both

human type A PAI-2 cDNA) (Appendix II) were a kind gift from Prof. T Ny (Umea

University, Sweden); pQE9 vector and M15[pREP4] E. coli obtained from QIAGEN

(Hilden, Germany); Oligonucleotides from Geneworks (Adelaide, Australia); phorbyl

myristate acetate (PMA) from Sigma-Aldrich (St. Louis, MO, USA);

Alexa 488 labelling kit, Alexa 488 polyclonal antibody and BL21 Star (DE3) E. coli from

Invitrogen (Carlsbad, CA, USA); IPTG from Applichem (Darmstadt, Germany);

Monoclonal antibodies against human uPA (394) and human PAI-2 (3750), and high

molecular weight (HMW)-uPA from American Diagnostica Inc. (Stamford, CT,

USA); Ampicillin and kanamycin from Amresco (Solon, OH, USA); TALON metal

affinity resin from Clontech (Mountain View, CA, USA); Bam HI restriction enzyme,

shrimp alkaline phosphatase and T4 DNA ligase from Fermentas (Vilnius, Lithuania);

CM5 BIAcore chip, PD-10 desalting columns and Hyperfilm ECL from GE

Healthcare (Princeton, NJ, USA); Recombinant VLDLR ligand binding region (V1-7)

was a gift from D Blaas (Medical University of Vienna, Austria); Z-Gly-Gly-Arg-

AMC from Merck (Darmstadt, Germany); Human recombinant non-histagged PAI-2

wild-type was from PAI-2 Pty Ltd (Sydney, Australia); Pfu HS Fusion II DNA

polymerase from Agilent Technologies (Santa Clara, CA, USA). Wizard SV Gel and

PCR Clean-Up System and Wizard Plus SV Minipreps DNA Purification kit were

from Promega (Madison, WI, USA). Precision Plus Protein Prestaind Standards and

PVDF membrane were from Bio-Rad (Hercules, CA, USA). SuperSignal West Pico

Chemiluminescence Substrate was from Pierce (Rockford, IL, USA).

2.2.2 PREPARATION OF ELECTROCOMPETENT E. COLI FOR

TRANSFORMATION

Electrocompetent Escherichia coli JM109, BL21 Star (DE3) and M15[pREP4] cells were prepared as described by Sharma and Schimke (1996). E. coli were grown overnight at 37 °C with shaking in YENB medium (Appendix I). Cells were diluted

1:200 into 1 L of fresh YENB and grown at 37 °C with shaking until mid-log phase

(OD 600 ~ 0.6). Cells were harvested by centrifugation (4000 g for 10 min), washed twice with 200 mL cold sterile MilliQ water, once with 50 mL of cold sterile 10% glycerol and finally resuspended in 2 mL of cold sterile 10% glycerol. Prepared cells were stored at -80 °C for up to one year. Transformation was performed by electroporation of competent cells. On ice, 1 µL of purified plasmid DNA (~100 ng) was added to 40 µL of electrocompetent cells and electropulsed at 2.5 V, 200 Ω and

25 µFD using 0.2 cm MicroPulser cuvettes and a MicroPulser (BioRad, Hercules, CA,

USA). After transformation, 400 µL of SOC medium (Appendix I) was added, the

culture grown at 37 °C with shaking for 1 h and then plated onto LB agar (Appendix I)

inoculated with the appropriate antibiotic(s). Where required, plasmids were extracted

from storage strains using a Wizard Plus SV Minipreps DNA Purification kit.

2.2.3 GENERATION OF pQE9/PAI-2 CONSTRUCTS

This work was previously conducted by Lakshitha Gunawardhana (B Biotech Hons,

University of Wollongong, Australia) and the methodology is included here, with his permission, for the sake of completeness. PAI-2 wild-type and PAI-2 ∆CD-loop

cDNAs lacking the 3' untranslated region were amplified from pET15b using Pfu HS

Fusion II DNA polymerase and the following primers: 5'-

GCGCGGATCCCTCGAGGATCTTTGTGTGGC-3' (forward) and 5'-

GCGCGGATCCTTAGGGTGAGGAAAATCTGCCG-3' (reverse). The PCR

parameters used were: initial denaturing step 95ºC for 2 min, 30 cycles of denaturing

at 95ºC for 10 s, annealing at 55ºC for 20 s, extension at 72ºC for 1 min, followed by

a final extension step of 72ºC for 3 min. The PCR product was purified using agarose

gel electrophoresis and the WizardSV Gel and PCR Clean-Up System. Primers

contained Bam HI sites to allow for ligation into pQE9. Both vector and insert were digested with Bam HI (37°C for 2 h), linear pQE9 de-phosphorylated using shrimp

alkaline phosphatise (as per manufacturer’s instructions) and the insert ligated into the

vector using T4 DNA ligase (as per manufacturer’s instructions). Successful insertion

and sequence integrity were confirmed using restriction digestion and DNA

sequencing.

2.2.4 DNA SEQUENCE ANALYSIS

DNA sequence analysis was used to confirm successful construction of of pQE9/PAI-

2 wild-type and pQE9/PAI-2 ∆CD-loop. Isolated plasmid DNA was sequenced using an ABI Prism BigDye Primer Cycle Sequencing Kit (Applied-Biosystems, Sydney,

Australia), electrophoresed in an ABI PRISM 377 DNA sequencer (Perkin-Elmer,

Waltham, MA, USA) and analysed using BioEdit (Hall 1999). For each sequencing reaction, 200 ng of DNA (in 1 µL), 0.5 µL BigDye, 2 µL 5 × sequencing buffer, 2

pmol of primer in 1 µL (Table 2.1, Figure 2.1) and 5.5 µL dH 2O were placed in a

PCR thermocycler and set to the following program: 25 cycles, 96ºC for 30 s, 52ºC for 15 s, 60ºC for 4 min. Excess dye terminators were removed from the sequencing reactions via ethanol precipitation (Sambrook et al. 1989) and sequencing undertaken.

Table 2.1: Oligonucleotides used for sequencing of pQE9/PAI-2 constructs. PAI-2 S4 is the reverse complement of PAI-2 S1. Location of primers on PAI-2 cDNA is indicated in Figure 2.1 Primer name Sequence Direction

PAI-2 S1 5’-CACCGAAGACCAGATGG-3’ Forward

PAI-2 S2 5’-GTCAAGACTCAAACCAAAGG-3’ Forward

PAI-2 S3 5’-TTGCCGATGTGTCCACTG-3’ Forward

PAI-2 S4 5’-GCCATCTGGTCTTCGGTGC-3’ Reverse

pQE promoter 5’-CGGATAACAATTTCACACAG-3’ Forward

2.2.5 EXPRESSION AND PURIFICATION OF PAI-2

Purified pET15b/PAI-2 ∆CD-loop, pQE9/PAI-2 ∆CD-loop and pQE9/PAI-2 wild- type vectors were transformed into electrocompetent BL21 Star (DE3) and M15

[pREP4] cells, respectively as described in section 2.2.2. Cells were cultured overnight at 37°C with shaking in LB containing 100 µg.mL -1 ampicillin and

25 µg.mL -1 kanamycin (only for M15[pREP4] cells). A 20 mL aliquot of this starter culture was added to 1 L Z-broth (Appendix I) and grown to an OD 600 of ~ 0.6.

Expression of PAI-2 ∆CD-loop was induced by the addition of 0.5 mM IPTG and the culture incubated for a further 4 h. Cells were collected by centrifugation at 10,000 g for 10 min. Pelleted cells were resuspended in 40 mL of ice-cold loading buffer

(Appendix I) and lysed using an EmulsiFlex-C5 homogeniser (Avestin, Ottawa,

Canada). Cell debris was then pelleted by centrifugation at 17,000 g for 30 min. The

supernatant was loaded onto equilibrated TALON metal affinity resin at a rate of 1

mL.min -1. Unbound proteins were removed using 10 column volumes of loading

buffer followed by 10 column volumes of wash buffer containing previously

10 20 30 40 50 60 70 80 90 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| PAI-2 ATGG AGG ATCTTT GTGTGG CAAA CACACTCTTT GCCC TCAA TTT ATT CAA GCATCTGG CAAAA GCAA GCCCC ACCC AGAA CC TCTT CC TC M E D L C V A N T L F A L N L F K H L A K A S P T Q N L F L

100 110 120 130 140 150 S1/S4 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| PAI-2 TCCCC ATGG AGCATCTCGTCC ACC ATGG CC ATGG TCTACATGGG CTCC AGGGG CAGCACC GAA GACC AGATGG CC AA GG TGCTT CAGTTT S P W S I S S T M A M V Y M G S R G S T E D Q M A K V L Q F

190 2 00 210 220 230 240 250 260 270 ....|....|....|....|....|....|....|....|.... |....|....|....|....|....|....|....|....|....| PAI-2 AA TGAA GTGGG AGCC AA TGCAGTT ACCCCC ATGACTCC AGAGAA CTTT ACC AGCTGTGGG TT CATGCAGCAGATCC AGAA GGG TAGTT AT N E V G A N A V T P M T P E N F T S C G F M Q Q I Q K G S Y

280 290 300 310 320 330 340 350 360 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| PAI-2 CC TGATGCGATTTT GCAGG CACAA GCTGCAGATAAAA TCC ATT CATCC TT CC GCTCTCTCAGCTCTGCAA TCAA TGCATCC ACAGGG AA T P D A I L Q A Q A A D K I H S S F R S L S S A I N A S T G N

370 380 390 400 410 420 430 440 450 ....|....|....|....|....|....|....|....|.... |....|....|....|....|....|....|....|....|....| PAI-2 TATTT ACTGG AAA GTGTCAA TAA GCTGTTT GG TGAGAA GTCTGCGAGCTT CC GGG AA GAA TATATT CGACTCTGTCAGAAA TATT ACTCC Y L L E S V N K L F G E K S A S F R E E Y I R L C Q K Y Y S

460 470 480 490 500 510 520 S2 530 54 0 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| PAI-2 TC AGAA CCCC AGG CAGTAGACTT CC TAGAA TGTGCAGAA GAA GCTAGAAAAAA GATT AA TT CC TGGGTCAA GACTCAAA CC AAA GG CAAA S E P Q A V D F L E C A E E A R K K I N S W V K T Q T K G K

550 560 570 580 590 600 610 620 630 ....|....|....|....|....|....|....|....|.... |....|....|....|....|....|....|....|....|....| PAI-2 ATCCC AAA CTT GTT ACC TGAA GG TT CTGTAGATGGGG ATACC AGG ATGG TCC TGG TGAA TGCTGTCTACTT CAAA GG AAA GTGG AAAA CT I P N L L P E G S V D G D T R M V L V N A V Y F K G K W K T

640 650 660 670 680 690 700 710 720 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| PAI-2 CC ATTT GAGAA GAAA CTAAA TGGG CTTT ATCC TTT CC GTGTAAA CTCGG CTCAGCGCACACC TGTACAGATGATGTACTT GCGTGAAAA G P F E K K L N G L Y P F R V N S A Q R T P V Q M M Y L R E K

730 740 750 760 770 780 790 800 810 ....|....|....|....|....|....|....|....|.... |....|....|....|....|....|....|....|....|....| PAI-2 CTAAA CATT GG ATACATAGAA GACC TAAA GG CTCAGATT CTAGAA CTCCC ATATGCTGG AGATGTT AGCATGTT CTT GTT GCTT CC AGAT L N I G Y I E D L K A Q I L E L P Y A G D V S M F L L L P D

820 S3 830 840 850 860 870 880 890 900 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| PAI-2 GAAA TT GCC GATGTGTCC ACTGGCTT GG AGCTGCTGG AAA GTGAAA TAA CC TATGACAAA CTCAA CAA GTGG ACC AGCAAA GACAAAA TG E I A D V S T G L E L L E S E I T Y D K L N K W T S K D K M

910 920 930 940 950 960 970 980 990 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| PAI-2 GCTGAA GATGAA GTT GAGG TATACATACCCC AGTT CAAA TT AGAA GAGCATT ATGAA CTCAGATCC ATT CTGAGAA GCATGGG CATGG AG A E D E V E V Y I P Q F K L E E H Y E L R S I L R S M G M E

1000 1010 1020 1030 1040 1050 1060 1070 1080 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| PAI-2 GA CGCC TT CAA CAA GGG ACGGG CC AA TTT CTCAGGG ATGTCGG AGAGG AA TGACC TGTTT CTTT CTGAA GTGTT CC ACC AA GCC ATGG TG D A F N K G R A N F S G M S E R N D L F L S E V F H Q A M V

1090 1100 1110 1120 1130 1140 1150 1160 1170 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| PAI-2 GATGTGAA TGAGG AGGG CACTGAA GCAGCC GCTGG CACAGG AGG TGTT ATGACAGGG AGAA CTGG ACATGG AGG CCC ACAGTTT GTGG CA D V N E E G T E A A A G T G G V M T G R T G H G G P Q F V A

1180 1190 1200 1210 1220 1230 1240 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|... PAI-2 GA TCATCC TTTT CTTTTTCTT ATT ATGCATAA GATAA CC AA CTGCATTTT ATTTTT CGG CAGATTTT CC TCACCC TAA D H P F L F L I M H K I T N C I L F F G R F S S P *

Figure 2.1: Positions of oligonucleotide primers used to sequence PAI-2 cDNA (isoform A) as described in Table 2.1. The ‘pQE promoter’ binds to a region in pQE9 as shown in Appendix I. The region of the PAI-2 cDNA encoding for the CD-loop is underlined. The positions of amino acids that differ between isoforms A and B are double underlined.

optimised imidazole concentrations (Appendix I; 5 mM imidazole for PAI-2

expressed from pQE9, 15 mM for PAI-2 expressed from pET15b). Bound protein was

eluted from the column using elution buffer (Appendix I). Purification was analysed

using SDS-PAGE under reducing conditions and the successful isolation of PAI-

2 ∆CD-loop confirmed by western blotting using a monoclonal antibody against PAI-

2 (3750). Samples were buffer exchanged into PBS (Appendix I) using a PD-10

column to remove imidazole for subsequent experiments.

2.2.6 SDS-PAGE

SDS-PAGE was used to assess the performance of protein purification and expression levels. Samples were diluted 1:4 with either 5 × reducing sample buffer (Appendix I) or 5 × non-reducing sample buffer (Appendix I) and boiled for 3-5 min. The gel was prepared using a Mini PROTEAN3 Cell system (BioRad, Hercules, CA, USA) using

4% stacking and 12% resolving acrylamide gels (Appendix I). Gels were electrophoresed at 140 V for 2 h in 1 × SDS running buffer (Appendix I). Gels were stained with coomassie rapid staining solution (Appendix I) and protein bands resolved using firstly rapid destain (Appendix I) and then final destain solutions

(Appendix I). Standard curves were constructed using Precision Plus Protein

Prestained Standards to allow for estimation of protein size (Appendix III).

2.2.7 WESTERN BLOT ANALYSIS

Western blots were conducted using a Mini Trans-Blot Cell system (BioRad,

Hercules, CA, USA) and PVDF membrane. Prior to use, the membrane was activated by soaking in methanol and equilibrated in transfer buffer (Appendix I). The blot was assembled as per the manufacturers instructions with all components soaked in

transfer buffer and run at 100 V for 1 h. Subsequent to protein transfer, the membrane

was blocked for 1 h at room temperature (RT) with 10% (w/v) powdered milk in

TBST (Appendix I). The membrane was incubated for 1 h at RT with a 1:500 dilution

of the primary antibody in 2% (w/v) powdered milk in TBST, re-blocked with 6%

(w/v) powdered milk in TBST and then incubated for 1 h at RT with a 1:5000 dilution

of the HRP-labelled secondary antibody in 2% (w/v) powdered milk in TBST.

Between each step, the membrane was washed with TBST and finally washed in TBS.

Labelled proteins were visualised using SuperSignal West Pico Chemiluminescence

Substrate and Hyperfilm ECL.

2.2.8 CATION -EXCHANGE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

(HPLC)

This work was performed by Dr Kara Perrow (University of Wollongong, Australia) and is included with her permission. The purity of PAI-2 ∆CD-loop expressed and purified from pQE9 was further determined by high performance liquid chromatography using a BioSuite SP 10 µm, C × C (7.5 cm × 75 mm) cation

exchange semi-preparative column (Waters, Milford, MA, USA). The column was pre-equilibrated with approximately 10 column volumes of 20 mM 2-(N- morpholino)ethanesulfonic acid (MES, pH 5.0). PAI-2 ∆CD-loop (100 µL, ~1 mg.mL -1) was then loaded and eluted with a linear (curve 6) 0.5 M NaCl salt gradient at 1 mL.min -1. The data was collected on a photodiode array at 280 nm and percent purity determined using Empower Pro V2 (Waters, Milford, MA, USA) software.

2.2.9 ELECTROSPRAY IONISATION MASS SPECTROMETRY (ESI-MS)

A positive ion mass spectrum of PAI-2 ∆CD-loop expressed and purified from pQE9 was acquired on a quadrupole time of flight spectrometer (Q-TOF-MS) (Micromass

Q-TOF Ultima, Waters, Milford, MA, USA) fitted with a Z-spray ionisation source. A sample of freshly prepared protein in PBS (pH 7.4) was exchanged into 10 mM ammonium acetate buffer (pH 6.8) containing 0.1% formic acid and made up to a final concentration of 10 µM (Sample prepared by Dr Kara Perrow, University of

Wollongong, Australia). The protein was then injected into the Q-TOF Ultima mass

spectrometer (20 µL) and the mass spectrum acquired with a capillary voltage of 2.6 kV, cone voltage of 50 V, source block temperature of 40°C, and a resolution power of 5000 Hz. Ceasium iodide was used for external calibration. The mass spectrum data is presented as raw data, on an m/z scale. Mass was calculated using MassLynx

MS software (Waters, Milford, MA, USA).

2.2.10 TISSUE CULTURE CONDITIONS

The human breast epithelial adenocarcinoma cell line MCF-7 and the leukemic monocyte lymphoma cell line U937 were routinely cultured in RPMI-1640 supplemented with 5% (v/v) heat-inactivated FCS (foetal calf serum). Cells were incubated at 37°C in a humidified incubator with a 5% carbon dioxide/air atmosphere and routinely passaged using trypsin/EDTA (MCF-7) or dilution (U937) as required.

Cells were routinely tested for absence of mycoplasma contamination.

2.2.11 uPA INHIBITORY ASSAYS

2.2.11.1 Formation of SDS-stable complexes

To ensure that PAI-2 ∆CD-loop retained uPA inhibitory activity, a 1:2 molar ratio of

HMW-uPA to PAI-2 ∆CD-loop was incubated at 37 oC for 1 h. Complex formation

was then qualified by SDS-PAGE as previously described (Saunders et al. 2001).

2.2.11.2 Solution phase fluorogenic uPA activity assay

Several substrate and uPA concentrations were used to find the optimum range and to

set the gain on a Fluorostar Optima fluorescence plate reader (BMG Labtech,

Germany). PAI-2 ∆CD-loop or PAI-2 wild-type were diluted in reaction buffer

(Appendix I) and mixed with fluorogenic substrate, Z-Gly-Gly-Arg-AMC in 180 µL reaction buffer. After a brief preincubation at 37°C, HMW-uPA (final concentration

0.675 nM) was added to start the reaction and fluorescence emission measured immediately at 37°C. All assays were performed in triplicate and values corrected by subtracting the background (reaction buffer plus substrate only).

2.2.11.3 Cell surface fluorogenic uPA activity assay

For cell based assays, PMA treated U937 cells (used to enhance the expression of unoccupied uPAR (Picone et al. 1989)) were washed and preincubated with 50 nM

HMW-uPA in binding buffer (Appendix I) for 10 min to saturate uPAR. After 2 washes the cells were resuspended in binding buffer to give a final concentration of 5

× 10 5 cells.mL -1. To initiate the reaction 100 µL aliquots of the cell suspension were transferred to a 96-well fluor plate containing 100 µL of 0.5 mM substrate +/- PAI-2

in binding buffer pre-equilibrated to 37°C (final concentrations, 0.25 mM substrate,

12.5 or 50 nM PAI-2) and fluorescence emission measured. All assays were

performed in triplicate and values corrected by subtracting background fluorescence

[binding buffer plus substrate +/- PAI-2 (the presence of PAI-2 made no difference to

background levels)]. Differences in uPA activity were tested for significance using an

unpaired Student's t-test with p < 0.05 considered statistically significant.

2.2.12 SURFACE PLASMON RESONANCE

Surface plasmon resonance was used to determine the impact of the removal of the

CD-loop and presence of the 6 × his-tag on the affinity of uPA:PAI-2 complexes for

VLDLR, essentially as described by Croucher et al. (2007). VLDLR was immobilised on a CM5 BIAcore chip, according to the manufacturer’s instructions (Appendix VI).

The chip was activated with a 1:1 mixture of 0.2 M N-ethyl-N'-(3-

dimethylaminopropyl)carbodi-imide and 0.05 M N-hydroxysuccimide. VLDLR was

coated onto the chip at 40 µg.mL -1 in 10 mM sodium acetate (pH 3) to a level of

~2,000 response units. Unoccupied binding sites were blocked using 1 M

ethanolamine (pH 8.5). Ligands were diluted into BIAcore running buffer (Appendix

I) before application to the BIAcore chip at 20 µL.min -1. Regeneration was achieved using 100 mM H 3PO 4. For kinetic assays, data was analysed using BIAevaluation

software (Version 4), using a blank cell as the reference cell. Binding models were

selected based on the lowest χ2 value and analysis of residual plots.

2.2.13 ENDOCYTOSIS ASSAYS

Endocytosis assays were conducted essentially as previously described (Al-Ejeh et al.

2004; Croucher et al. 2006; Croucher et al. 2007), using the MCF-7 cell line. Cells were grown for 48 h in 6 well plates to subconfluency, washed with phenol red free hanks buffered salt solution (Appendix I) then incubated in binding buffer for 1 h at

37°C to allow for receptor recycling. To confirm LDLR specificity, cells were

preincubated for 10 min with 200 nM RAP. Cells were incubated with 10 nM

uPA:Alexa 488 , both alone and previously complexed with either PAI-2 wild-type or

PAI-2 ∆CD-loop for 1 h at 37°C, to allow for internalisation. After this incubation period, the cells were washed twice with ice cold binding buffer, harvested using 5 mM EDTA, washed again and resuspended in ice cold binding buffer containing

-1 4 µg.mL Alexa 488 quenching polyclonal antibody. After 30 min the cells were washed twice with ice-cold PBS and analysed by dual colour flow cytometry with propidium iodide to exclude non-viable cells. Propidium iodide is membrane impermeant, but displays increased fluorescence intensity when bound to DNA, allowing cells fluorescing at 695 nm to be excluded from further analysis. Differences in the amount of uPA internalised between treatments were tested for significance using the unpaired Student's t-test with p < 0.05 considered statistically significant.

2.3 RESULTS

2.3.1 EXPRESSION AND PURIFICATION OF PAI-2

Expression and purification of PAI-2 ∆CD-loop from both the pET15b and pQE9 vector systems yielded a product migrating as a major band at approximately 45 kDa

(Figure 2.2A). This corresponds to the expected molecular weight of PAI-2 ∆CD-loop

and its identity was confirmed via western blotting with a monoclonal antibody to

PAI-2 (Figure 2.2B). The PAI-2 ∆CD-loop from the pQE9 system contained less impurities (seen as lower molecular weight bands; Figure 2.2A) compared to PAI-

2 ∆CD-loop from pET15b expressed and purified under similar conditions (Figure

2.2A, compare lanes 1 and 2). The abundance of PAI-2 ∆CD-loop expressed and purified from the pQE9 versus the pET15b system was found to be ~95% pure compared to ~74%, respectively, as measured by densitometry. The expression and purification of PAI-2 ∆CD-loop using the pQE9 system regularly achieved yields between 10 and 15 mg.L-1 of culture. PAI-2 wild-type expressed and purified using

the pQE9 system was less pure (Figure 2.2A, compare lanes 2 and 3) and of lower

yield (~5 mg.L-1) compared to PAI-2 ∆CD-loop. When visualised by SDS-PAGE

under reducing conditions, PAI-2 wild-type yielded two bands that were both

identified as PAI-2 via western blotting (Figure 2.2C). This agrees with the previous

finding that a portion of wild-type PAI-2 is cleaved in the CD-loop (Lobov et al.

2004), which would require further purification steps to obtain a homogenous protein

population. When analysed under non-reducing conditions, PAI-2 ∆CD-loop

displayed an additional band at ~74 kDa that was not present in the reduced sample

(Figure 2.3B). The purity of PAI-2 ∆CD-loop expressed in the pQE9 system was confirmed via HPLC, with integration of the elution profile giving PAI-2 ∆CD-loop

an abundance of ~95% (Figure 2.3).

A B C

1 2 3 B 1 2 3 A MW (kDa)

37 *

Figure 2.2: SDS-PAGE and western blot analysis of highly enriched recombinant PAI-2 proteins . A total protein amount of 10 µg from PAI-2 ∆CD-loop purified from either pET15b (lane 1) or pQE9 (lane 2), or PAI-2 wild-type from pQE9 (lane 3) were fractionated by a 12% SDS-PAGE under reducing conditions and stained with coomassie blue to detect total protein (A) . A sample of PAI-2 ∆CD-loop from pQE9 was also analysed under non-reducing conditions (B) . Samples transferred to PVDF membrane and probed with monoclonal PAI-2 antibody (3750) (C) . The slight difference in size between PAI-2∆CD-loop from pET15b and pQE9 is due to differences in tag/linker length. * marks cleaved PAI-2 in wild-type population as mentioned in text.

Figure 2.3: Quantitative analysis of the purification of PAI-2 ∆CD-loop from pQE9 . Protein (100 µL) was injected onto a cation exchange column and eluted using a linear NaCl gradient (0–1 M) at 1 mL.min -1. PAI-2 ∆CD-loop was detected at 280 nm with a RT of 42.606 min. Integration of the peak corresponding to PAI-2 ∆CD-loop measured the purity at 94.5%. The green line indicates the relative amount of buffer B (MES pH 5.0, 1 M NaCl) to buffer A (MES pH 5.0). Insert: Relative retention times and purity as calculated from AUC using Empower Pro V2 (Waters, UK) software. RT = retention time.

2.3.2 ELECTROSPRAY IONISATION MASS SPECTROMETRY (ESI-MS)

The ESI-MS spectrum of PAI-2 ∆CD-loop (Figure 2.4) shows a pattern of multiply charged ions (13 + to 16 +) in the m/z range of 2700–3500 consistent for the 45 kDa monomeric species. The calculated molecular weight of 44496.72 Da is in agreement with the theoretical monoisotopic molecular weight of 44480.16 Da. In addition, five higher charge states (20 + to 24 +) from m/z 3,700 – 4,500 representing an 89 kDa dimeric species were also observed, this may correspond to the high molecular weight bands observed when PAI-2 ∆CD-loop was analysed under non-reducing conditions by SDS-PAGE (Figure 2.2B).

2.3.3 THE ABSENCE OF THE CD-LOOP DOES NOT AFFECT THE SOLUTION

PHASE OR CELL SURFACE U PA ACTIVITY INHIBITORY FUNCTION OF PAI-2

To ensure that PAI-2 ∆CD-loop retained uPA inhibitory activity, a 2-fold molar excess of PAI-2 ∆CD-loop was incubated with uPA and analysed by SDS-PAGE under reducing conditions (Figure 2.5A). A band at ~70 kDa was observed corresponding to covalently complexed uPA:PAI-2 ∆CD-loop (whilst the expected size of the uPA:PAI-2 complex is ~95 kDa, under reducing conditions the A-chain of uPA is not present in the complex. Additionally, western blotting of this complex formation using a monoclonal antibody to uPA showed no free uPA remained after incubation (Figure 2.5B). PAI-2 ∆CD-loop and PAI-2 wild-type gave super- imposable kinetic inhibition curves for HMW-uPA in solution (Figure 2.5C), confirming that the absence of the CD-loop does not affect uPA inhibitory activity of

PAI-2. Di Giusto et al. (2005) showed that his-tagged PAI-2 ∆CD-loop had the same second order rate constant for uPA inhibition in solution (2.40 × 10 6 M-1 s -1) as the

Figure 2.4: A positive ion ESI-MS of PAI-2 ∆CD-loop from pQE9 in 10 mM ammonium acetate (pH 6.8) containing 0.1% formic acid . The m/z spectrum shows a Gaussian-type distribution of multiply charged ions ranging from m/z 2700 – 4500. Charge states are indicated. The monomeric ( A) and dimeric ( B) forms of the protein gave a measured molecular mass of 44496 Da (± 0.89) and 88961 Da (± 0.84), respectively. Mass was calculated using MassLynx MS software (Waters) and caesium iodide was used for external calibration.

A C uPA only 100 80

MW (kDa) 60 uPA:PAI-2 ratio 9570 uPA:PAI-2 ∆CD-loop 40 1:1 20 45 PAI-2 ∆CD- Units Fluoresence loop control) (% uninhibited 1:5 0 0 500 1000 1500 2000 2500 3000 Time (s)

B D uPA:PAI-2 MW (kDa) uPA ** ∆CD-loop 100

7095 80

anti-uPA 60

3035 40

Units Fluoresence 20 (% uninhibited control) (% uninhibited 0 0 12.5 50 0 12.5 50 PAI-2 (nM)

Figure 2.5: PAI-2 ∆CD-loop efficiently inhibits both solution phase and cell bound uPA activity . (A) PAI-2 ∆CD-loop is able to form SDS stable complexes with uPA. PAI-2 ∆CD- loop was incubated in a 2:1 molar ratio with HMW-uPA for 30 min and analysed SDS-PAGE under reducing conditions. (B) Western blot analysis of complex formation between uPA and PAI-2 ∆CD-loop using a monoclonal antibody to the B-chain of uPA (~30 kDa), indicating that no residual uPA remains in the sample. (C) Kinetic inhibition curves for PAI-2 wild-type (dark red) versus PAI-2 ∆CD-loop (bright pink) against HMW-uPA in solution. The uPA fluorogenic substrate was briefly pre-incubated with the PAI-2 forms or buffer alone (open circles) and the assays initiated by the addition of HMW-uPA. Fluorescence units were converted to a percentage of the maximal (uninhibited) uPA activity. Values shown are the means of triplicate determinations. Errors (< 10%) are not shown for clarity of presentation. (D) U937 cells were incubated with uPA fluorogenic substrate in the absence or presence of PAI-2 wild-type (filled bars) or PAI-2 ∆CD-loop (open bars) for 1 h at 37°C. Fluorescence units were converted to a percentage of the maximal (uninhibited) uPA activity. Values shown are means ± SEM (n = 3). *p < 0.001 compared to inhibited samples for both PAI-2 forms.

published wild-type non-recombinant form. Moreover, we show that the inhibitory activity of

both variants was also identical towards cell surface receptor bound uPA as there were no

significant differences seen between the two PAI-2 forms (Figure 2.5D).

2.3.4 REMOVAL OF THE CD-LOOP DOES NOT AFFECT THE CLEARANCE OF U PA

FROM THE CELL SURFACE

Preliminary dot-blot analysis suggested that removal of the CD-loop of PAI-2 has no impact on the interaction between the uPA:PAI-2 complex and members of the LDLR family

(Croucher et al. 2006). Surface plasmon resonance was thus used to measure the affinity of uPA complexed with PAI-2∆CD-loop or PAI-2 wild-type for the endocytosis receptor

VLDLR (Figure 2.6). Both gave similar on (k a) and off (k d) rates and the affinity constant

(KD) of both uPA:PAI-2 ∆CD-loop and uPA:PAI-2 wild-type for VLDLR was found to be ~5 nM (Table 2.2) with these interactions both best fitting a 1:1 binding model. No interaction was observed between VLDLR and either PAI-2 ∆CD-loop or PAI-2 wild-type alone

(Table 2.2).

Additionally, when complexed with uPA, both PAI-2 wild-type and PAI-2 ∆CD-loop lead to

an almost two-fold increase in the amount of uPA endocytosed by MCF-7 cells relative those

treated with uPA alone (Figure 2.7). Importantly, there was no significant difference between

cells treated with uPA:PAI-2 wild-type and uPA:PAI-2 ∆CD-loop. Pre-treatment of cells

with the LDLR antagonist RAP significantly reduced the amount of uPA internalised in all

treatments (Figure 2.7).

uPA:PAI-2 wild-type 250

200 100 nM 150

100

Response (RU) Response 50

6.25 nM 0 0 200 400 600 Time (s)

uPA:PAI-2 ∆∆∆CD-loop 250

200 100 nM 150

100

Response (RU) Response 50

6.25 nM 0 0 200 400 600 Time (s)

Figure 2.6: Surface plasmon resonance analysis of the interaction between uPA:PAI-2 wild-type and uPA:PAI-2 ∆CD-loop and VLDLR. Samples were injected at a rate of 20 µL.min -1 at concentrations of 100 nM, 50 nM, 25 nM, 12,5 nM and 2.35 nM. Data is representative of at least three experiments.

Table 2.2: Kinetic parameters of the interaction between PAI-2 ∆CD-loop or PAI-2 wild-type and VLDLR, both alone and in complex with uPA.

Binding ka kd KD Analyte χ2 Model (M -1 s-1) (s -1) (nM)

PAI-2 wild-type No binding - - - -

3.75 × 10 5 1.44 × 10 -3 4.81 uPA:PAI-2 wild-type 1:1 4.71 (± 1.1 × 10 5) (± 0.39 × 10 -3) (± 0.64)

PAI-2 ∆CD-loop No binding - - - -

4.16 × 10 5 1.36 × 10 -3 4.34 uPA:PAI-2 ∆CD-loop 1:1 3.28 (± 1.8 × 10 5) (± 0.23 × 10 -3) (± 0.56) Values were determined using surface plasmon resonance. Binding data was fitted using the BIAevaluation 4.0 software. The binding model chosen represents that with the lowest χ2 value. (Values are the average ± SEM, n = 3).

-RAP 200 +RAP

150 * * 100 * (% control) 50 uPA Internalisation uPA

0 p o PA o u type -l d- D ∆∆∆C

2 I- PAI-2 wil A : :P A P uPA u

Figure 2.7: Cellular internalisation of uPA is equivalent between PAI-2 wild-type and PAI-2 ∆CD-loop as determined by flow cytometry . MCF-7 cells were incubated with 10 nM uPA:Alexa 488 , either alone or in complex with PAI-2 wild-type or PAI-2 ∆CD-loop for 1 h at 37°C. Any surface bound uPA:Alexa 488 remaining was quenched by incubation with -1 4 µg.mL anti-Alexa 488 polyclonal antibody for 30 min prior to analysis by dual colour flow cytometry. Values shown are means ± SEM (n = 3) as a percentage of uPA only treatment. * p < 0.005 compared to internalisation in the absence of RAP for each treatment.

2.4 DISCUSSION

As introduced in Section 1, the 33 amino acid CD-loop of PAI-2 is the largest amongst known serpins. Whilst seven other members of the clade B serpin family contain a CD-loop

(SerpinB3, SerpinB4, SerpinB7, SerpinB10, SerpinB11, SerpinB12 and SerpinB13), the only described function of this domain (other than those proposed for PAI-2) is in SerpinB10, where the CD-loop contains a nuclear import sequence (Chuang and Schleef 1999). Much of the previous work describing the role of the CD-loop of PAI-2 in an intracellular context is controversial, as discussed in Section 1.3.2.3. The CD-loop is involved in transglutaminase mediated cross-linking to cellular and ECM proteins (Jensen et al. 1994; Jensen et al. 1996), although the functional significance of this crosslinking is unknown. Interestingly, cross- linked PAI-2 maintains uPA inhibitory activity.

Lobov et al . (2004) and Wilczynska et al . (2003) found that, when purified, a fraction of his- tagged PAI-2 was always cleaved, with this cleavage occurring in the CD-loop at positions

Phe 76 -Thr 77 , Gly 80 - Phe 81 , Phe 81 -Met 82 and Ala 93 -Ile 94 . This CD-loop cleaved conformation accounts for the band observed at approximately 36 kDa in the purified sample (Figure 2.2A, lane 3). Furthermore, Lobov et al . (2004) and Wilczynska et al . (2003) reported that this cleavage of the CD-loop did not result in a loss of uPA inhibitory activity. Di Giusto et al.

(2005) previously showed that his-tagged PAI-2 ∆CD-loop had the same second order rate

constant for uPA (2.40 × 10 6 M-1 s-1) as the published wild-type non-tagged PAI-2 providing

further evidence that the CD-loop of PAI-2 plays no role in the inhibition of uPA in vitro .

The results presented here go further than previous studies, comparing not only the solution- phase uPA inhibitory properties of PAI-2 wild-type and PAI-2 ∆CD-loop directly, but

showing that the CD-loop of PAI-2 plays no role in uPA inhibition in the more

physiologically relevant setting of the cell surface. This is to be expected as the majority of

71 serpin sequence deviation occurs in the loops joining secondary structures (Irving et al.

2000). Moreover, the intrahelical CD-loop is absent and hence not required for function in inhibitory serpins of all other clades (Silverman et al. 2004). Intramolecular distance measurements indicate that the CD-loop of PAI-2 in the monomeric form is folded to the side of the molecule (Lobov et al. 2004) (Figure 2.8), away from the domains of the protein involved in protease inhibition (namely the RCL, β-sheet A and other residues previously deemed as important as reviewed by Gettins (2002)), further suggesting that the CD-loop plays no role in substrate recognition or translocation of the protease with the RCL across the helix-F face of PAI-2 to the base of the molecule.

Additionally, the findings presented here indicate that the use of PAI-2 ∆CD-loop is advantageous compared to PAI-2 wild-type due to superior purity and yield of the recombinant protein under identical conditions. A lower amount of contaminating proteins were co-purified in the PAI-2 ∆CD-loop purification than PAI-2 wild-type. As western blotting with a monoclonal antibody to PAI-2 (and previous experiments using a PAI-2 polyclonal antibody, data not shown ) indicated that the additional protein bands co-purified with PAI-2 wild-type did not contain breakdown products of PAI-2, they may be of bacterial origin. Moreover, as these proteins are negligible in the PAI-2 ∆CD-loop sample, it is feasible that these proteins may co-purify with PAI-2 wild-type via interactions with the CD- loop.

RCL

β-sheet A CD-loop

Figure 2.8: Hypothesised location of the CD-loop of PAI-2 based on intramolecular distance measurements. In the stable monomeric form of PAI-2, the CD-loop is believed to fold to the side of the molecule. The precise structure of the CD-loop has not been determine by X-ray crystallography and the conformation shown here is a prediction. Numbers and green spheres show locations of cysteine residues used in triangulation measurements. Figure from Lobov et al. (2004).

Previous studies expressing PAI-2 ∆CD-loop from E. coli have generally used the pET15b plasmid in the BL21 Star (DE3) E. coli expression strain. Recloning of the cDNA encoding for PAI-2 ∆CD-loop lacking the 3’ untranslated region, which has been shown to contain mRNA instability regions (Maurer and Medcalf 1996), into pQE9 is one of the potential reasons for the increased yield and purity of PAI-2 ∆CD-loop when purified under optimised conditions from the pQE9 verses the pET15b vector system. A lower concentration of imidizole was used in the binding and wash buffers during the purification of PAI-2 ∆CD-

loop from pQE9 relative to pET15 as pET15b/PAI-2 ∆CD-loop encodes for a thrombin

recognition sequence between the six histadine residues and the first amino acid of PAI-2

∆CD-loop which serves as a ‘linker’, extending the his-tag away from α- helix A PAI-2

∆CD-loop and leading to a slightly increased affinity for the TALON resin.

SDS-PAGE under non-reducing conditions and mass spectrometry of purified PAI-2 ∆CD-

loop indicated the presence of a dimeric form of PAI-2 ∆CD-loop. Whilst the CD-loop of

PAI-2 controls the ability of PAI-2 to polymerise, Lobov et al. (2004) showed that whilst less polymergenic than PAI-2 wild-type, PAI-2 ∆CD-loop was still able to form dimers and higher order aggregates. Serpins have been shown to polymerise via the insertion of a portion of the RCL into β-sheet A or into β-sheet C of another serpin molecule (Chang et al. 1997).

As insertion of the RCL into β-sheet C requires the ‘recipient’ serpin to convert to the latent

conformation and PAI-2 is known not to convert to this state, formation of PAI-2 ∆CD-loop dimmers cannot be explained via this mechanism. However, it is possible that PAI-2 may form polymers via a CD-loop independent mechanism involving insertion of the RCL into β-

sheet A.

Using a non-tagged, PAI-2 wild-type produced and purified in an alternative, mass

production scale procedure, Croucher et al. (2007) showed that PAI-2 wild-type did not

interact directly with VLDLR and that the interaction between uPA:PAI-2 wild-type and

VLDLR was mediated solely by the uPA moiety of the complex. As introduced in Section

1.4.1, LDLRs generally bind regions of basic charge on ligands. Whilst the CD-loop is

largely electroneutral, containing only one lysine and two glutamic acid residues, it may have

been possible that the CD-loop was able to block potential LDLR binding residues in PAI-2,

that would be revealed upon removal of the CD-loop. However, kinetic analysis of PAI-2

∆CD-loop both alone and in complex with uPA gave identical values to PAI-2 wild-type, indicating that removal of the CD-loop had no impact on VLDLR binding. Furthermore, removal of the CD-loop of PAI-2 had no impact on the internalisation of uPA:PAI-2 ∆CD-

loop relative to uPA:PAI-2 wild-type, suggesting that the CD-loop of PAI-2 plays no role in

the VLDLR mediated internalisation of these complexes.

PAI-2 ∆CD-loop represents a desirable starting material for the development of PAI-2 based uPA targeted cancer therapies. Tumour uptake increases as protein size is decreased (Thomas et al. 1989), suggesting that this shortened but fully active form of PAI-2 may not only be simpler to express and purify than PAI-2 wild-type, but also exhibit a favourable pharmacokinetic profile. Pharmacokinetic and bio-distribution studies have suggested that the half life of PAI-2 wild-type in mice is quite short (< 2 min) (Hang et al. 1998) and this is not significantly changed by removal of the CD-loop (Dr Kara Perrow, personal communication ), indicating that additional modifications are required to both PAI-2 species to increase protein half life and increase tumour uptake. Nevertheless, the ease of purification of PAI-2 ∆CD-loop makes it the preferential target upon which to optimise this property. The

ability of PAI-2 ∆CD-loop to target and clear uPA from the cell surface in a manner

75 indistinguishable from PAI-2 wild-type combined with improved purification simplicity and protein yield makes the use of PAI-2 ∆CD-loop favourable when studying the uPA- dependent functions of PAI-2. Additionally, as the VLDLR binding profile of PAI-2 ∆CD- loop is identical to PAI-2 wild-type, this protein form may also prove useful in the further characterisation of the LDLR-dependent functionality of PAI-2.

CHAPTER 3

CHARACTERATION OF THE DIFFERENTIAL

LDLR BINDING PROFILES OF PAI-1 AND PAI-2

3.1 INTRODUCTION

PAI-1 and PAI-2 both efficiently inhibit uPA (Thorsen et al. 1988). As inhibitory members of the serpin protein family, PAI-1 and PAI-2 exhibit an irreversible inhibitory mechanism and conform to a strongly conserved structure (as discussed in Section 1.3.1) despite significant variation in peptide sequence. Upon inhibition of uPAR-bound uPA, uPA:PAI complexes are rapidly cleared via receptor mediated endocytosis mediated by LDLRs and delivered to endosomes and lysosomes (Argraves et al. 1995; Al-Ejeh et al. 2004; Croucher et al. 2006).

Whilst PAI-1 has been shown to bind directly to LRP1, VLDLR, LRP2 and sorLA (Kounnas et al. 1993; Nykjaer et al. 1994; Stefansson et al. 1995; Horn et al. 1997; Gliemann et al.

2004; Croucher et al. 2007), previous studies have shown that PAI-2 does not bind directly to

LDLRs, namely LRP1 (Croucher et al. 2006) and VLDLR (Croucher et al. 2007).

Additionally, the interaction between uPA:PAI-2 and these LDLRs is markedly different to that of uPA:PAI-1 in terms of both affinity and mechanism (Croucher et al. 2006; Skeldal et al. 2006; Croucher et al. 2007). Croucher et al. (2007) previously modelled the electrostatic surface potential of both PAI-1 and PAI-2 in and around α-helix D, noting differences in the alkalinity of the region surrounding R 76 of PAI-1 and the corresponding residue in PAI-2

(R 108 ). As LDLRs bind to target proteins via electrostatic interactions mediated by negatively

charged regions receptor and positive regions in the ligand (Lillis et al. 2005; Fisher et al.

2006), this may explain the differential LDLR binding of PAI-1 and PAI-2 to LDLRs.

Numerous studies have identified several positively charged residues within (R 76 , K 80 ) and

nearby (K 88 , R 118 , K 122 ) the α-helix D of PAI-1 that contribute to the high-affinity binding of uPA:PAI-1 with LDLRs (Horn et al. 1998; Rodenburg et al. 1998; Stefansson et al. 1998;

Skeldal et al. 2006). In addition, the α-helix D of PAI-1 contains a proposed LDLR minimal binding motif of two basic residues separated by two to five residues and N-terminally

flanked by hydrophobic residues (Jensen et al. 2006) (Figure 3.1A). This motif facilitates the

high affinity interaction of a number of LDLR binding proteins, such as RAP (Figure 3.1B).

Previous analyses suggest that the decreased affinity of uPA:PAI-2 for LDLRs is likely due

to an incomplete LDLR binding motif within α-helix D of PAI-2, in which the first hydrophobic/basic amino acid pair is conserved, whilst the second pair is replaced by serine residues (Croucher et al. 2007) (Figure 3.1C, D and E). Indeed, previous studies using a mutant form of PAI-1 with an incomplete LDLR binding motif (R76E) observed decreased

LRP1 and VLDLR binding affinity (Stefansson et al. 1998; Webb et al. 2001; Croucher et al.

2007). Whilst these differences in receptor binding may appear trivial, they underpin stark differences in the biological actions of PAI-1 and PAI-2.

The lack of an LDLR binding site in PAI-2 facilitates the removal of cell surface uPA and hence proteolytic activity, without activation of the pro-mitogenic/motogenic signalling pathways associated with PAI-1 (Croucher et al. 2007; Croucher et al. 2008). As introduced in Section 1.4.3, the binding of uPA:PAI-1 to VLDLR results in increased cell growth in

MFC-7 cells (Webb et al. 2001) and increased expression of MMP-2 and MMP-9 in

SGC7901 cells (Di et al. 2010) via sustained activation of the MAPK/ERK signalling pathway. Webb et al. (2001) demonstrated that this activity was dependent on the interaction between the PAI-1 moiety of the complex and VLDLR as both ERK signalling and cell proliferation were reduced to levels similar to controls when treated with uPA:PAI-1 R76E.

Croucher et al. (2007) found that the affinity of uPA:PAI-1 R76E for VLDLR (9.95 nM) was almost 10-fold lower than uPA:PAI-1 and was best described by a 1:1 binding model similar to uPA:PAI-2.

K63 L62

W59 K60

C D

Figure 3.1: The LDLR minimal binding motif in PAI-1 is not conserved in PAI-2. The proposed LDLR minimal binding motif consists of two basic amino acids N-terminal flanked by hydrophobic residues and separated by 2-5 residues. These residues bind to acid residues coordinated with tryptophan on the receptor. (B) The LDLR-binding interface of RAP showing two lysine residues and the flanking hydrophobic residues. (C) Alignment of α-helix D and adjacent residues of PAI-1 and PAI-2. The LDLR minimal binding motif in PAI-1 is underlined and residues previously reported to mediate binding of PAI-1 to LDLRs coloured. (D) Location of the LDLR binding motif in α-helix D of PAI-1 and (E) the corresponding residues in PAI-2. Structure analysis performed using PyMol (DeLano 2002), and structural coordinates for RAP (1LRE (Nielsen et al. 1997)), PAI-1 (9PAI (Aertgeerts et al. 1995)) and PAI-2 (1JRR (Jankova et al. 2001)).

The results described in this chapter show that by replacing residues of PAI-2 with those

corresponding to LDLR binding sites in PAI-1, LDLR binding can be introduced to PAI-2.

Interestingly, the mutations required to introduce binding differ for VLDLR and LRP1. This

provides important insights into the differences in the mechanisms of uPA:PAI-1 and

uPA:PAI-2 endocytosis (and downstream functionality, which will be discussed in Chapter

4), the proposed LDLR minimal binding motif and the differing binding specificities of LRP1

and VLDLR.

Whilst a robust, reproducible methodology for the production and purification of PAI-2

∆CD-loop was described in Chapter 2, the 6 × his-tag present at the N-terminus of PAI-2

∆CD-loop produced in this system cannot be removed. Whilst the presence of this tag has no

impact on the uPA and LDLR dependent function of PAI-2, the use of PAI-2 ∆CD-loop in future applications (such as animal testing or crystallography) may require removal of the 6 × his-tag. To this end, PAI-2 ∆CD-loop cDNA was ligated into the TAGzyme pQE1 vector

(QIAGEN, Hilden, Germany). According to the manufacturer, this vector is optimised for the

removal of 6 × his-tags by a novel dipeptidase. The vector and expression system do not

differ significantly from pQE9, and as such the already optimised expression and purification

protocols used in the production of PAI-2 ∆CD-loop from this system (Section 2.2.5) may be

utilised.

3.2 MATERIALS AND METHODS

3.2.1 MATERIALS

PCR MasterMix was from Promega (Madison, WI, USA). TAGzyme pQE1 vector,

TAGzyme enzyme kit and anti-pentahis antibody were from QIAGEN (Hilden, Germany).

Oligonucleotide primers were from Geneworks (Adelaide, Australia). Lambda

DNA/ Eco RI+ Hind III molecular weight markers, Hind III and Pvu II restriction enzymes were

from Fermentas (Vilnius, Lithuania). QuikChange site-directed mutagenesis kit was from

Stratagene. PAI-1 14-1B stable mutant was from Molecular Innovations (Novi, MI, USA).

The PAI-1 14-1B stable mutant contains four mutations (N150H/K154T/Q319L/M354I) that

increase the functional stability of PAI-1 without significantly altering the rate of inhibition

of uPA or tPA (Berkenpas et al. 1995). Human purified placental LRP1 was a kind gift from

Prof. Phillip Hogg (University of New South Wales, Sydney, Australia). SnakeSkin dialysis tubing with a nominal 3.5 kDa molecular weight cut off was from Pierce (Rockford, IL,

USA).

3.2.2 AGAROSE GEL ELECTROPHORESIS

Agarose gel electrophoresis was conducted using a 1% agarose gel (Appendix I) in 1 × TAE buffer (Appendix I) at 100 V for 1 h in a MiniSub Cell GT electrophoresis tank (BioRad,

Hercules, CA, USA). DNA in the gel was visualised using a UVP BioImaging System (UVP,

Upland, CA, USA) after staining with 0.5 mg.mL -1 ethidium bromide for 30 min. To allow

for size estimation of observed DNA bands, standard curves for each gel were constructed

using Lambda DNA/ Eco RI+ Hind III molecular weight markers (Appendix IV).

3.2.3 CONSTRUCTION AND CHARACTERISATION OF TAG ZYME pQE1/PAI-2 ∆CD-

LOOP

pQE9 does not encode a protease cleavage site for the removal of the his-tag from the N- terminus of PAI-2 ∆CD-loop. Whilst the presence of the 6 × his-tag has no impact on the uPA-dependent and LDLR-dependent functionalities of PAI-2, future work may require removal of this tag. To this end, PAI-2 ∆CD-loop cDNA was recloned into the vector

TAGzyme pQE1, which is similar to pQE9 but allows for the removal of the 6 × his-tag

using the TAGzyme system.

3.2.3.1 Amplification of PAI-2 cDNA

PAI-2 ∆CD-loop cDNA (type A) was amplified from pQE9/PAI-2 ∆CD-loop using Pfu HS

Fusion II DNA polymerase and the oligonucleotide primers ‘pQE1 PAI-2 Up’ and ‘pQE1

PAI-2 Down’ (Table 3.1). The PCR parameters used were: initial denaturing step 95ºC for 2

min, 30 cycles of denaturing at 95ºC for 10 s, annealing at 55ºC for 20 s, extension at 72ºC

for 1 min, followed by a final extension step of 72ºC for 3 min. Successful amplification was

assessed by agarose gel electrophoresis. To allow for ligation into TAGzyme pQE1, the

‘pQE1 PAI-2 Down’ primer contained a Hind III recognition sequence and the ‘pQE1 PAI-2

Up’ primer was 5’ phosphorylated. Purified PCR product was digested with Hind III (37°C for 2 h) and TAGzyme pQE1 digested with Hind III and Pvu II (37°C for 2 h), as advised by the manufacturer. Ligation with T4 DNA ligase was conducted as per manufacturer’s instructions and 1 µL from the ligation reaction used to transform electrocompletent E coli.

JM109.

Table 3.1: Oligonucleotides used for vector construction, sequencing and mutagenesis. P = phosphate group. Mutating codons are underlined. Primer name Sequence Direction

PAI-2 cDNA amplification primers

pQE1 PAI-2 Up 5’-P-ATGGAGGATCTTTGTGTGGC-3’ Forward pQE1 PAI-2 Down 5’-GCGCAAGCTTTTAGGGTGAGGAAAATCTGC-3’ Reverse

Colony PCR Screening Primers

pQE promoter 5’-CCCGAAAAGTGCCACCTG-3’ Forward

S2 reverse 5’-CCTTTGGTTTGAGTCTTGAC-3’ Reverse

Site-directed Mutagenesis Primers

N101K sense 5’-GAAGTGGGAGCCGCTGCAAAG AAAATCCATTCATCCTTCC-3’ Forward

N101K antisense 5’-GGAAGGATGAATGGATTTTCTT TGCAGCGGCTCCCACTTC-3’ Reverse

S112K sense 5’-CATCCTTCCGCTCTCTCAGCAAG GCAATCAATGCATCCACAGG-3’ Forward

S112K antisense 5’-CCTGTGGATGCATTGATTGCCTT GCTGAGAGAGCGGAAGGATG-3’ Reverse

S111Y/S112K 5’-CATTCATCCTTCCGCTCTCTCTATAAG GCAATCAATGCATCCAC-3’ Forward sense

S111Y/S112K 5’-GTGGATGCATTGATTGCCTTATA GAGAGAGCGGAAGGATGAATG-3’ Reverse antisense

N120K sense 5’-CAATCAATGCATCCACAGGGAAG TATTTACTGGAAAGTGTCAATAAG-3’ Forward

N120K antisense 5’-CTTATTGACACTTTCCAGTAAATACTT CCCTGTGGATGCATTGATTG-3’ Reverse

3.2.3.2 Colony PCR

To determine if the PAI-2 ∆CD-loop cDNA had successfully ligated into TAGzyme pQE1, bacterial colonies were screened via colony PCR. The PCR reaction was made as follows: 3.5

µL PCR Master Mix, 1 µM each of ‘pQE promoter’ primer and ‘S2 reverse’ primer (Table

3.1) and dH 2O to a final volume of 7 µL. A single colony from the culture plate was added to

the reaction tube, resuspended and run at the following PCR parameters: intial denaturing

step, 95°C 2 min; 30 cycles, 95°C 30 s, 55°C 30 s, 72°C 1 min and a final extension step,

72°C 5 min. Reaction product was observed via agarose gel electrophoresis, with positive

vector construction indicated by amplification of a ~ 700 base pair band, corresponding to the

distance between the ‘pQE promoter’ and ‘S2 reverse’ primers. Plasmid DNA from these

colonies was extracted and the plasmid sequenced (as described in Section 2.2.3) to confirm

successful construction. PAI-2 ∆CD-loop was expressed and purified as described in Section

2.2.4.

3.2.4 SMALL -SCALE HIS -TAG REMOVAL

Optimisation of tag removal was carried out using 50 µg of PAI-2 ∆CD loop. As PAI-2

∆CD-loop does not contain an intrinsic DAPase stop point, Qcyclase was used so that the

DAPase would stop digestion at the glutamine residue directly before the PAI-2 ∆CD loop

sequence. PAI-2 ∆CD loop was buffer exchanged into 1 × TAGZyme buffer (20 mM sodium

phosphate, 150 mM NaCl, pH 7.0) using a PD-10 column immediately before the reaction.

50 µg of PAI-2 ∆CD loop was incubated with 2.5 mU DAPase, 150 mU Qcyclase and 80 µM

Cysteamine.HCl in a final volume of 50 µL. The reaction carried out at 37° C for 60 min.

Samples were taken from the reaction at 10 min time points and fractionated by SDS-PAGE,

western blotted to PVDF membrane and probed for the presence of histadine tagged protein

using a pentahis antibody.

3.2.5 PROTEIN STRUCTURE MODELLING

Molecular modelling and figure preparation was performed using structural co-ordinates obtained from X-ray crystal structures of the relaxed conformations of PAI-1 (Aertgeerts et al. 1995) (PDB file 9PAI) and PAI-2 (Jankova et al. 2001) (PDB file 1JRR) and the PyMol molecular graphics program.

3.2.6 SITE DIRECTED MUTAGENESIS

Residues of PAI-2 were replaced with the corresponding residues of PAI-1 on the pQE9/PAI-

2 ∆CD-loop backbone using the primers described in Table 3.1. The PAI-2 ∆CD-loop backbone was used as removal of the CD-loop of PAI-2 allows for easier purification of protein without compromising the inhibitory activity or LDLR binding affinity of PAI-2

(Chapter 2). Control PAI-2 ∆CD-loop protein will hence be referred to as PAI-2 in this study.

Initially, site directed mutagenesis was attempted using the Quickchange II kit to replace

specific residues of PAI-2 with the corresponding residues from PAI-1. However, after

numerous attempts, no mutagenesis product was produced in any of the reactions carried out.

As such, an alternative mutagenesis protocol was employed, based on a protocol described by

Higuchi et al. (1988) (Figure 3.2). This methodology involved the use of the primers

described in Table 3.1, but PCR of only the cDNA to be mutagenised, rather than

amplification of the entire plasmid. This methodology consisted of an initial step producing

two, overlapping DNA fragments, both containing the desired mutation. These fragments

were generated using separate reactions consisting of 100 ng template DNA, 0.2 µM of each

primer (one amplification primer and one mutagenesis primer), 25 mM dNTPs, 5 µL 10 ×

PfuUltra II reaction buffer, 1 µL PfuUltra II Fusion HS DNA polymerase in a 50 µL reaction.

Reactions were conducted in a PCR thermocycler (Eppendorf, Hamburg, Germany) using the

pQE1 PAI-2 Up primer Sense primer cDNA to be mutated

Anti-sense primer pQE1 PAI-2 Down primer

PCR

5’ fragment 3’ fragment

PCR with pQE1 PAI-2 Up and pQE1 PAI-2 Down primers

Complete cDNA including mutations

Figure 3.2: Schematic diagram of modified site directed mutagenesis used to produce PAI-2 mutants , based on the method of Higuchi et al. (1988). TAGzyme pQE1/PAI-2 ∆CD- loop was used as the cDNA template, and the UP and DOWN primers were ‘pQE1 PAI-2 Up’ and ‘pQE1 PAI-2 Down’, respectively, as described in Table 3.1. Red sections denote codon/s to be mutagenised.

following parameters: initial denaturing step 95ºC for 2 min, then 40 cycles of denaturing at

95ºC for 10 s, annealing at 55ºC for 20 s, extension at 72ºC for 1 min, followed by a final extension step of 72ºC for 3 min. The overlapping PCR products were purified by agarose gel electrophoresis using a WizardSV Gel and PCR Clean-Up System. In order to produce the full length PAI-2 cDNA including the desired mutation, 1 µL of each of the purified PCR products was added to a new reaction containing only the ‘pQE1 PAI-2 Up’ and ‘pQE1 PAI-

2 Down’ primers and the PCR protocol repeated as previous. The resulting PCR product was

purified via agarose gel electrophoresis, digested with Hind III (5 U, 1 h at 37 °C) and ligated into TAGzyme pQE1 as described in section 3.2.3.1. Constructs were screened using colony

PCR (Section 3.2.3.2) and sequenced (Section 2.2.3) to confirm successful residue mutation.

As for PAI-2 ∆CD-loop, PAI-2 mutants were expressed and purified as described in Section

2.2.4.

3.2.7 SURFACE PLASMON RESONANCE

Surface plasmon resonance was performed essentially as previously described (Croucher et al. 2006; Croucher et al. 2007). VLDLR or LRP1 were loaded onto CM5 BIAcore sensor chips to a level of ~2000 response units (Appendix VI). Analytes were diluted into BIAcore running buffer (Appendix I) and ran over the chip at 20 µL/min. Between samples, the sensor surface was regenerated with 100 mM H 3PO 4. Data was analysed using BIAevaluation

software (Version 4), using a blank cell as the reference cell. Where multiple CM5 chips

were used, results were validated against control chips and samples to ensure reproducibility.

3.2.8 FAR -UV CIRCULAR DICHROISM SPECTROMETRY

The structural integrity of PAI-2 constructs was assessed using far UV circular dichroism

(CD) spectroscopy to determine if the replacement of residues of PAI-2 with those of PAI-1 resulted in significant secondary structural changes. Proteins were buffer exchanged into 10 mM sodium phosphate by dialysis and diluted to a concentration of 0.4 mg.mL -1. CD

spectroscopy was performed using a J-810 spectropolarimeter (Jasco, Tokyo, Japan)

equipped with a thermoelectric temperature control at 25°C. Spectra were collected from 190

nm to 240 nm at 1 nm intervals, with each spectrum representing the average of ten scans,

with a sample of 10 mM sodium phosphate serving as a reference.

3.2.9 uPA ACTIVITY ASSAY

To ensure that mutagenesis of PAI-2 did not result in any changes in uPA inhibition, PAI-2 mutants were tested as described in Section 2.2.11.

3.3 RESULTS

3.3.1 CONSTRUCTION AND CHARACTERISATION OF TAG ZYME P QE1/PAI-2 ∆CD-

LOOP

3.3.1.1 Construction of TAGzyme pQE1/PAI-2 ∆CD-loop

In order to generate TAGzyme pQE1/PAI-2 ∆CD-loop, PAI-2 ∆CD-loop cDNA was

amplified from pQE9/PAI-2 ∆CD-loop using the ‘pQE1 PAI-2 Up’ and ‘pQE1 PAI-2 Down’ primers as described in section 3.2.3.1. The reaction product was analysed by agarose gel electrophoresis (Figure 3.3A) to determine if amplification was successful. A single band at approximately 1100 base pairs corresponding to the expected size of PAI-2 ∆CD-loop cDNA

was observed. The band was purified from the gel and ligated into TAGzyme pQE1. The

ligation reaction was transformed into E coli. JM109. Colonies were screened for correct

ligation of PAI-2 ∆CD-loop cDNA into TAGzyme pQE1 by colony PCR using the ‘pQE promoter’ and ‘PAI-2 S2R primers’. Potential successful construction of TAGzyme pQE1/PAI-2 ∆CD-loop was observed in 10 of the 12 colonies analysed as indicated by the

presence of a ~ 700 base pair DNA band in these samples (Figure 3.3B). Sequence analysis

indicated that PAI-2 ∆CD-loop cDNA was correctly inserted in TAGzyme pQE1, contained

no mutations and was confirmed as type A (ie. with N1 20 , N 404 and S 413 ) (Figure 3.4).

A M PCR product Size (bp)

21226

5148 4268

2027 1904 1584 1375

947 831

B M Colonies 1 - 12 Size (bp) 21226

5148 4268

2027 1904 1584 1375

947 831

564

Figure 3.3: Agarose gel electrophoresis indicating successful construction of TAGzyme pQE1/PAI-2 ∆CD-loop. (A) PCR amplification of PAI-2 ∆CD-loop cDNA yielded a band of ~ 1100 base pairs, corresponding to the molecular weight of PAI-2 ∆CD-loop cDNA. (B) Colony PCR screen of E. coli colonies subsequent to transformation with TAGzyme pQE1/PAI-2 ∆CD-loop ligation mix. Succesful insertion of PAI-2 ∆CD-loop cDNA into TAGzyme pQE1 was observed in most colonies screened, as indicated by the presence of an ~700 base pair band. M = Lambda DNA/ Eco RI+ Hind III molecular weight markers.

10 20 30 40 50 60 70 80 90 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| ATGAAA CATCACC ATCACC ATCACGAGG ATCTTT GTGTGG CAAA CACACTCTTT GCCC TCAA TTT ATT CAA GCATCTGG CAAAA GCAA GC M K H H H H H H E D L C V A N T L F A L N L F K H L A K A S

100 110 120 130 140 150 160 170 180 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| CCC ACCC AGAA CC TCTT CC TCTCCCC ATGG AGCATCTCGTCC ACC ATGG CC ATGG TCTACATGGG CTCC AGGGG CAGCACC GAA GACC AG P T Q N L F L S P W S I S S T M A M V Y M G S R G S T E D Q

190 200 210 220 230 240 250 260 270 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| ATGG CC AA GG TGCTT CAGTTT AA TGAA GTGGG AGCC GCTGCAGATAAAA TCC ATT CATCC TT CC GCTCTCTCAGCTCTGCAA TCAA TGCA M A K V L Q F N E V G A A A D K I H S S F R S L S S A I N A

280 290 300 310 320 330 340 350 360 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| TCCACAGGG AA TT ATTT ACTGG AAA GTGTCAA TAA GCTGTTT GG TGAGAA GTCTGCGAGCTT CC GGG AA GAA TATATT CGACTCTGTCAG S T G N Y L L E S V N K L F G E K S A S F R E E Y I R L C Q

370 380 390 400 410 420 430 440 450 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| AAA TATT ACTCC TCAGAA CCCC AGG CAGTAGACTT CC TAGAA TGTGCAGAA GAA GCTAGAAAAAA GATT AA TT CC TGGG TCAA GACTCAA K Y Y S S E P Q A V D F L E C A E E A R K K I N S W V K T Q

460 470 480 490 500 510 520 530 540 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| ACC AAA GG CAAAA TCCC AAA CTT GTT ACC TGAA GG TT CTGTAGATGGGG ATACC AGG ATGG TCC TGG TGAA TGCTGTCTACTT CAAA GG A T K G K I P N L L P E G S V D G D T R M V L V N A V Y F K G

550 560 570 580 590 600 610 620 630 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| AA GTGG AAAA CTCC ATTT GAGAA GAAA CTAAA TGGG CTTT ATCC TTT CC GTGTAAA CTCGG CTCAGCGCACACC TGTACAGATGATGTAC K W K T P F E K K L N G L Y P F R V N S A Q R T P V Q M M Y

640 650 660 670 680 690 700 710 720 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| TT GCGTGAAAA GCTAAA CATT GG ATACATAGAA GACC TAAA GG CTCAGATT CTAGAA CTCCC ATATGCTGG AGATGTT AGCATGTT CTT G L R E K L N I G Y I E D L K A Q I L E L P Y A G D V S M F L

730 740 750 760 770 780 790 800 810 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| TT GCTT CC AGATGAAA TT GCC GATGTGTCC ACTGG CTT GG AGCTGCTGG AAA GTGAAA TAA CC TATGACAAA CTCAA CAA GTGG ACC AGC L L P D E I A D V S T G L E L L E S E I T Y D K L N K W T S

820 830 840 850 860 870 880 890 900 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| AAA GACAAAA TGG CTGAA GATGAA GTT GAGG TATACATACCCC AGTT CAAA TT AGAA GAGCATT ATGAA CTCAGATCC ATT CTGAGAA GC K D K M A E D E V E V Y I P Q F K L E E H Y E L R S I L R S

910 920 930 940 950 960 970 980 990 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| ATGGG CATGG AGG ACGCC TT CAA CAA GGG ACGGG CC AA TTT CTCAGGG ATGTCGG AGAGG AA TGACC TGTTT CTTT CTGAA GTGTT CC AC M G M E D A F N K G R A N F S G M S E R N D L F L S E V F H

1000 1010 1020 1030 1040 1050 1060 1070 1080 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| CAA GCC ATGG TGG ATGTGAA TGAGG AGGG CACTGAA GCAGCC GCTGG CACAGG AGG TGTT ATGACAGGG AGAA CTGG ACATGG AGG CCC A Q A M V D V N E E G T E A A A G T G G V M T G R T G H G G P

1090 1100 1110 1120 1130 1140 1150 1160 1170 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....| CAGTTT GTGG CAGATCATCC GTTT CTTTTT CTT ATT ATGCATAA GATAA CC AA CTGCATTTT ATTTTT CGG CAGATTTT CC TCACCC TAA Q F V A D H P F L F L I M H K I T N C I L F F G R F S S P *

Figure 3.4: Sequence analysis of TAGzyme pQE1/PAI-2 ∆CD-loop (type A). Plasmid sample was extracted from transformed E. coli and sequenced as described in section 2.2.3. Sequence analysis performed using the BioEdit analysis program (Hall 1999). The predicted translated product is shown beneath the DNA sequence.

3.3.1.2 Purification of PAI-2 ∆CD-loop

As the general features of the pQE9 and TAGzyme pQE1 vectors are the same, PAI-2 ∆CD-

loop from TAGzyme pQE1/PAI-2 ∆CD-loop was expressed and purified as described in

Section 2.2.4. Throughout the purification process, samples were taken for subsequent

analysis by SDS-PAGE (Figure 3.5). All eluted fractions exhibited a major band at ~ 45 kDa,

correlating to the molecular weight of PAI-2 ∆CD-loop. Additional protein bands were also

present in the first elution fraction from the column, however the intensity of these bands was

significantly diminished in subsequent elution fractions whilst the total amount of protein

Size (kDa) 1 2 3 4 5 6 7 8 150

100 75 50

Figure 3.5: Purification of PAI-2 ∆CD-loop by immobilised metal affinity chromatography. PAI-2 ∆CD-loop was expressed in E. coli M15 [pREP4] and PAI-2 ∆CD- loop purified using TALON resin. Samples from each stage of the purification process were analysed by SDS-PAGE under reducing conditions to assess purification performance and purity of eluted PAI-2 ∆CD-loop samples. Lane 1, crude cell lysate; lane 2, column loading flow through; lane 3, wash buffer elution; lane 4-8, elution fractions.

93 eluted remained relatively constant over the five fractions analysed by SDS-PAGE. Elution fractions 2-5 (lanes 5-8 in Figure 3.5) were combined and buffer exchanged against PBS to remove imidazole that may have impacted on further experiments. Yields of 10-15 mg of protein per litre of culture were routinely achieved.

3.3.1.3 Small scale removal of 6 × his-tag

The vector TAGzyme pQE1 is designed to allow for the removal of the 6 × his-tag. Tag removal was trialled on a small scale as suggested by the manufacturer over a period of 60 min. Western blot analysis of samples taken at 10 min time points indicated that the majority of his-tag cleavage occurred in the first 10 min, as determined by a lack of reactivity to an anti-pentahis antibody (Figure 3.6). After 60 min no reactivity to the anti-pentahis antibody was observed, indicating that tag removal from PAI-2 ∆CD-loop was complete. Probing of these same fractions with a monoclonal antibody for PAI-2 (3570, as described in section

2.2.6) indicated that no loss of protein from the fractions had occurred. However, attempts to scale up this process did not result in successful tag removal.

Min 0 10 20 30 40 50 60 Antibody pentahis

PAI-2

Figure 3.6: Small scale removal of the 6 × his-tag from PAI-2 ∆CD-loop expressed from the TAGzyme pQE1 vector. PAI-2 ∆CD-loop was incubated with DAPase and Qcyclase for the indicated time and probed with either an anti-pentahis or a PAI-2 antibody.

As the presence of an N-terminus his-tag does not impact on the uPA and LDLR dependent

functions of PAI-2 (as demonstrated in Chapter 2 as PAI-2 proteins used retained N-terminal his-tags), PAI-2 mutants generated to investigate the impact of complementing LDLR binding to PAI-2 retained the 6 × his-tag. However, the option to subsequently remove the his-tag was kept as future work may require generation of tag-less PAI-2.

3.3.2 STRUCTURAL COMPARISON INDICATES ABSENCE OF A CONSENSUS LDLR-

BINDING MOTIF IN PAI-2

The surface charge and topology of the region defining the consensus LDLR binding motif on α-helix D of PAI-1 are clearly different to the corresponding region of PAI-2. Hence, the differential binding to VLDLR and subsequent functional effects of PAI-1 and PAI-2 may be due to an incomplete LDLR binding motif within PAI-2 (Croucher et al. 2008). To directly investigate this residues of PAI-2 were substituted with those corresponding to the LDLR binding motif within α-helix D of PAI-1, (Ehrlich et al. 1992; Horn et al. 1998; Stefansson et

al. 1998; Skeldal et al. 2006) (Figure 3.7A-C, Table 3.2). Specifically, S 111 and S112 of PAI-2

were replaced with tyrosine and lysine respectively (corresponding to Y 79 and K 80 in PAI-1)

Table 3.2: PAI-2 mutants used to investigate the differential VLDLR and LRP1 binding mechanisms of PAI-2 and PAI-1. Protein Description PAI-2 ∆CD-loop Amino acid residues 66-98 (CD-loop) deleted 1 K1 PAI-2 D101 in PAI-2 corresponds with K 69 in PAI-1 K2 PAI-2 S112 in PAI-2 corresponds with K 80 in PAI-1 KK PAI-2 D101 /S 112 in PAI-2 corresponds with K 69 /K 80 in PAI-1 YK PAI-2 S111 /S 112 in PAI-2 corresponds with Y 79 /K 80 in PAI-1 KYK PAI-2 D101 /S 111 /S 112 in PAI-2 corresponds with K 69 /Y 79 /K 80 in PAI-1 YKK PAI-2 S111 /S 112 /N 120 in PAI-2 corresponds with Y79 /K 80 /K 88 in PAI-1 PAI-1 Full length PAI-1 1 All PAI-2 mutants constructed on this backbone.

Figure 3.7: Structural modelling of PAI-1 and PAI-2. (A) Amino acid sequence alignment of the area in and adjacent to α-helix D of PAI-1 and PAI-2. Residues reported to be important for the interaction between PAI-1 and LDLRs (Horn et al. 1998; Rodenburg et al. 1998; Stefansson et al. 1998; Skeldal et al. 2006) and the corresponding residues in PAI-2 are coloured. The amino acid residues comprising the proposed LDLR minimal binding motif in PAI-1 are underlined. (B,C) Ribbon representation and (D,E) surface representations of the relative locations of PAI-1 residues implicated in LDLR binding and the corresponding residues in PAI-2. Analysis performed using PyMol (DeLano 2002), PAI-1 (PDB file 9PAI (Aertgeerts et al. 1995)) and PAI-2 (PDB file 1JRR (Jankova et al. 2001)).

to generate PAI-2YK , completing an LDLR minimal binding motif (Figure 3.7A). To further

dissect the role of this LDLR minimal binding motif as well as other residues implicated in

the binding of PAI-1 to LDLRs, an panel of PAI-2 mutants were generated (Table 3.2, Figure

K1 K2 3.7A): PAI-2 (D101 of PAI-2 corresponds to K 69 in PAI-1); PAI-2 (S 112 of PAI-2

KK KYK corresponds to K 80 in PAI-1), PAI-2 (D101K, S112K); PAI-2 (D101K, S111Y, S112K)

YKK and PAI-2 (S111Y, S112K, N120K (N120 of PAI-2 corresponds to K88 of PAI-1). All PAI-

2 mutants were generated on the TAGzyme pQE1/PAI-2 ∆CD-loop backbone, sequenced to ensure successful mutagenesis (Appendix V) and purified as described in Section 2.2.4 and gave similar purification profiles to PAI-2 ∆CD-loop (Section 3.3.1.2).

3.3.3 LDLR BINDING CAN BE INTRODUCED TO PAI-2 BY COMPLEMENTATION OF

THE PAI-1 LDLR BINDING MOTIF

To investigate the molecular basis of the differential binding of PAI-1 and PAI-2 to LDLRs residues of PAI-2 were substituted with those corresponding to PAI-1 previously identified as important for LDLR binding. PAI-2 mutants (100 nM) were tested for the ability to bind to

VLDLR using surface plasmon resonance (Figure 3.8). Previous studies have demonstrated that PAI-2 not in complex with uPA does not interact with either LRP1 of VLDLR (Croucher et al. 2006; Croucher et al. 2007). Replacement of PAI-2 residues with the basic residues in the α-helix D of PAI-1 important for LDLR binding, either in isolation (PAI-2K1 , PAI-2K2 ) or

together (PAI-2KK ) (Figure 3.7, Table 3.2) did not impact on binding to VLDLR (Figure 3.8,

Table 3.3). uPA-independent binding to VLDLR was only observed by PAI-2 mutants containing a complete LDLR minimal binding motif (i.e. PAI-2YK and PAI-2KYK ) (Figure

150

100 PAI-2 KYK PAI-2 YK 50

Response (RU) Response PAI-2 (control) PAI-2 K1 0 K2 0 100 200 300 400 PAI-2 KK Time (s) PAI-2

Figure 3.8: Screening of PAI-2 mutants for VLDLR binding. Surface plasmon resonance sensograms showing the interaction between 100 nM PAI-2 mutants and immobilised VLDLR. Data shown is representative of at least three independent experiments.

3.8). Dose- dependent kinetic analysis gave affinity constants (K D) of 37.3 nM and 50.1 nM

for PAI-2YK and PAI-2KYK , respectively (Table 3.3). Kinetic analyses indicated that

interactions of uPA:PAI-2YK and uPA:PAI-2KYK with VLDLR were best described by a heterogeneous analyte model with two independent, competing binding sites on the complex

YK with affinity constants (K D) of 1.35 nM and 70.1 nM for uPA:PAI-2 ; and 1.53 nM and 51.2

nM for uPA:PAI-2KYK (Figure 3.9C,D; Table 3.3). These values were similar to those previously published for uPA:PAI-1 when analysed under comparable conditions (Skeldal et al. 2006; Croucher et al. 2007). Kinetic analyses of uPA:PAI-2 binding to VLDLR confirmed the single site binding model interaction (K D = 4.34 nM) (Figure 3.9B; Table 3.3). Croucher et al. (2006) previously compared the differential binding interactions of uPA:PAI-1 and uPA:PAI-2 with a related LDLR family member, LRP1. Interestingly, the interaction between uPA:PAI-2YK complex and LRP1 displayed different kinetics to those observed for

Table 3.3: Kinetic parameters of the interaction between PAI-2 forms used in this study and the endocytosis receptor VLDLR. Values were determined using surface plasmon resonance. Binding data was fitted using BIAevaluation 4.0 software. The binding model chosen represents that with the lowest χ2 value. (Values are the average ± SEM, n ≥ 3).

ka kd KD 2 Analyte Binding Model χχχ (M -1 s -1) (s -1) (nM)

PAI-2 No binding - - - -

PAI-2K1 No binding - - - -

PAI-2K2 No binding - - - -

PAI-2KK No binding - - - -

PAI-2YK 1:1 6.77 × 10 4 (± 2.68 × 10 4) 1.99 × 10 -3 (± 0.76 × 10 -3) 37.3 ( ± 7.0) 0.97

PAI-2KYK 1:1 6.68 × 10 4 (± 2.91 × 10 4) 4.31 × 10 -3 (± 2.22 × 10 -3) 50.1 ( ± 10.2) 1.02

uPA:PAI-2 1:1 4.16 × 10 5 (± 1.80 × 10 5) 1.36 × 10 -3 (± 0.23 × 10 -3) 4.3 ( ± 0.56) 3.28

4.78 × 10 4 (± 0.38 × 10 4) 3.15 × 10 -5 ( ± 1.07 × 10 -5) 1.35 ( ± 0.41) uPA: PAI-2YK Heterogeneous analyte 7.6 1.09 × 10 5 ( ± 0.51 × 10 5) 5.69 × 10 -3 ( ± 1.35 × 10 -3) 70.1 ( ± 24.9)

2.01 × 10 4 ( ± 0.22 × 10 4) 3.7 × 10 -5 ( ± 2.11 × 10 -5) 1.53 ( ± 0.43) uPA: PAI-2KYK Heterogeneous analyte 4.3 1.66 × 10 5 (± 0.33 × 10 5) 3.35 × 10 -3 (± 1.05 × 10 -3) 51.2 ( ± 12.9)

100

YK uPA:PAI-1 (uPA:PAI-1 K D = 0.252 nM and 2.13 nM; uPA:PAI-2 K D = 0.808 nM and 57.8 nM) (Figure 3.10; Table 3.3). Since Skeldal et al. (2006) found that Lys 88 of

PAI-1 (referred to as Lys 90 in their study) is critical for the interaction between uPA:PAI-1 and LRP1 (but not VLDLR), the PAI-2YKK mutant was generated by replacing the asparagine at position 120 (corresponding to K 88 of PAI-1) with lysine

(Figure 3.7A). uPA:PAI-2YKK complex displayed strikingly similar LRP1-binding affinities to uPA:PAI-1, with the high and lower affinity interactions (K D = 0.128 nM and 2.72 nM) not significantly different to those of uPA:PAI-1. However, it should be noted that individual k a and k d values were significantly different (Figure 3.10C,D;

Table 3.3), indicating subtle differences in interaction mechanics that may reflect variations in local surface potential and/or steric effects.

101

uPA:PAI-2 300

200

100 Response (RU) Response

0 0 100 200 300 400 500 600 Time (s)

uPA:PAI-2 YK 300

200

100 Response (RU) Response

0 0 100 200 300 400 500 600 Time (s)

uPA:PAI-2 KYK 300

200

100 Response (RU) Response

0 0 100 200 300 400 500 600 Time (s)

Figure 3.9: Surface plasmon resonance analysis of the interaction between PAI-2 constructs and VLDLR. Surface plasmon resonance analysis of the dose dependant interaction between uPA complexed PAI-2 mutants and VLDLR . The data presented here is representative of at least three experiments with analyte concentrations from 100 to 6.25 nM.

102

Table 3.4: Kinetic parameters of the interaction between PAI forms used in this study and the endocytosis receptor LRP1. Values were determined using surface plasmon resonance. Binding data was fitted using BIAevaluation 4.0 software. The binding model chosen represents that with the lowest χ2 value. (Values are the average ± SEM, n ≥ 3).

ka kd KD Analyte Binding Model χχχ2 (M -1 s -1) (s -1) (nM)

9.52 × 10 4 ( ± 2.89 × 10 4) 2.68 × 10 -5 ( ± 0.84 × 10 -5) 0.252 ( ± 0.087) uPA:PAI-1 Heterogeneous analyte 8.31 6.09 × 10 5 ( ± 1.56 × 10 5) 1.30 × 10 -3 ( ± 0.33 × 10 -3) 2.13 ( ± 0.56 )

uPA:PAI-2 1:1 6.74 × 10 4 (± 4.53 × 10 4) 6.71 × 10 -4 (± 1.32 × 10 -4) 1.02 ( ± 0.54) 3.41

1.38 × 10 5 (± 0.48 × 10 5) 1.9 × 10 -5 (± 0.87 × 10 -5) 0.808 ( ± 0.246) uPA:PAI-2YK Heterogeneous analyte 3.35 1.24 × 10 5 (± 0.44 × 10 5) 8.06 × 10 -3 ( ± 7.41 × 10 -3) 57.8 ( ± 39.0)

3.43 × 10 5 (± 1.34 × 10 5) 3.68 × 10 -5 (± 1.67 × 10 -5) 0.128 ( ± 0.081) uPA:PAI-2YKK Heterogeneous analyte 7.32 1.97 × 10 5 (± 1.39 × 10 5) 1.10 × 10 -2 (± 1.55 × 10 -2) 2.72 ( ± 1.51)

103

uPA:PAI-2 uPA:PAI-2 YK 300 300

200 200

100 100 Response (RU) Response (RU) Response

0 0 0 250 500 750 1000 0 250 500 750 1000 Time (s) Time (s)

uPA:PAI-2 YKK uPA:PAI-1 300 300

200 200

100 100 Response (RU) Response Response (RU) Response

0 0 0 250 500 750 1000 0 250 500 750 1000 Time (s) Time (s)

Figure 3.10: Surface plasmon resonance analysis of the interaction between uPA:PAI complexes and LRP1. Surface plasmon resonance analysis of the dose dependant interaction between LRP1 and uPA complexed PAI-2 mutants as indicated and uPA:PAI-1. The data presented here is representative of at least three experiments with analyte concentrations from 100 to 6.25 nM.

104

3.3.4 PAI-2 MUTANTS MAINTAIN GENERAL SECONDARY STRUCTURE AND

INHIBITORY ACTIVITY

To ensure that any observed changed in LDLR binding were a function of residue replacement and not due to protein misfolding and exposure of alternative receptor binding determinants, LDLR-binding PAI-2 mutants were tested for relative secondary structure using far-UV circular dichroism spectroscopy. All PAI-2 species tested were found to have similar ellipticity values to the control PAI-2 across the full range of wavelengths measured

(Figure 3.11). Additionally, PAI-2 mutants retained inhibitory activity indistinguishable from control PAI-2, as assessed by the formation of SDS stable complexes with uPA (Figure

3.12A) and solution phase inhibitory assays (Figure 3.12B).

40000 PAI-2 PAI-2 YK PAI-2 KYK PAI-2 YKK 20000 Elipticity

0 200 220 240

-20000 Wavelength (nm)

Figure 3.11: PAI-2 forms maintained overall secondary structure. Far-UV circular dichroism spectroscopy indicated that the relative secondary structure did not vary between PAI-2, PAI-2YK , PAI-2KYK and PAI-2YKK . Each spectrum is the average of ten scans.

105

YK KYK YKK PAI-2 PAI-2 PAI-2 PAI-2

uPA - + - + - + - +

uPA:PAI-2

PAI-2

100 PAI-2 uPA only PAI-2 YK PAI-2 KYK PAI-2 YKK 75

uPA:PAI-2 ratio

50 1:1 (Fluorescence units) (Fluorescence % Uninhibited control % Uninhibited 25

0 0 200 400 600 800 1000 1200 1400 1600 1800 Time (s)

Figure 3.12: PAI-2 forms maintained inhibitory activity. (A) LDLR-binding PAI-2 mutants were able to form SDS-stable complexes with uPA. PAI-2 mutants were incubated at a 1:1 molar ratio with uPA for 30 min at 37 °C and analysed by SDS-PAGE. (B) PAI-2 proteins showed similar solution phase uPA inhibitory activity (assessed as described in Section 2.2.11.2). HMW-uPA was added at a concentration of 0.675 nM and fluorescence emission measured at 37°C.

106

3.4 DISCUSSION

Whilst numerous studies have utilised site directed mutagenesis of both PAI-1 and uPA to characterise the LDLR binding of these proteins both alone and in complex, substantially less is known about the interaction between uPA:PAI-2 and LDLRs, with the biological importance of PAI-2 often underplayed in the literature. However, there is growing evidence that PAI-2 is present in pericellular and extracellular environments and is clearly linked to anti-tumourogenic activity suggesting that PAI-2 has an important role as a bona fide

inhibitor of uPA in vivo (Croucher et al. 2008).

As recently discussed by Dupont et al. (2009), understanding the receptor-ligand interaction

between uPA:PAI-1 and LDLRs is complicated by the increased affinity of the complex

compared to each moiety of the complex separately. This is also true for PAI-2 as, like PAI-

1, the uPA:PAI-2 complex has increased affinity for LDLRs compared to uPA alone

(Croucher et al. 2006; Croucher et al. 2007), despite no apparent interaction between the PAI-

2 moiety of the complex and the receptor. As such, it is reasonable to infer that this increase

in affinity is due to conformational changes in the protease. Whilst Dementiev et al. (2006)

showed that no major structural changes occur in porcine pancreatic elastase when in

complex with α1-proteinase inhibitor, there are major structural differences between uPA and

porcine pancreatic elastase and as such, the inferences that can be drawn between the

elastase: α1-proteinase inhibitor and uPA:PAI complexes is limited.

As previously reported, there are distinct differences in the interactions of uPA:PAI-2 and

uPA:PAI-1 with LDLRs (Croucher et al. 2006; Skeldal et al. 2006; Croucher et al. 2007). The

findings here further confirm this. The kinetic data for the interactions between uPA:PAI-1

and both VLDLR and LRP1 best fit a heterogeneous analyte model, whereby there are two

107 competing binding sites in the complex, suggesting that the interaction between is dependant on interactions in both the serpin and protease moieties of the complex. Interestingly, reported affinity constants for uPA:PAI-1 with mutations in PAI-1 to reduce LRP1 and

VLRLR binding (Skeldal et al. 2006; Croucher et al. 2007) are similar to those of uPA:PAI-2 reported both here and previously (Croucher et al. 2007). In addition, the removal of these basic residues in and around α-helix D of PAI-1 significantly reduced or eliminated the direct

binding of PAI-1 to LDLRs (Skeldal et al. 2006; Croucher et al. 2007).

As introduced previously, α-helix D of PAI-1 contains a proposed LDLR minimal binding motif of two basic residues N-terminally flanked by hydrophobic residues and separated by two to five residues (Jensen et al. 2006). This motif is not conserved in PAI-2 (Croucher et al.

2007; Croucher et al. 2008). This study demonstrates the robustness of the LDLR minimal binding motif, as LDLR binding was introduced to PAI-2 via the mutation of PAI-2 residues to corresponding residues in PAI-1 identified previously as important in the interaction of

PAI-1 and uPA:PAI-1 with LDLRs. As this motif is further validated, new, high affinity

LDLR ligands may be identified. Whilst the region in and around α-helix D of PAI-1 is mostly positively charged (basic), the same region in PAI-2 is more neutral and surrounded by multiple electronegative (acidic) regions (Croucher et al. 2007; Croucher et al. 2008).

Despite the generally accepted notion that LDLRs bind via interactions between acidic residues in the receptor and basic regions in the ligand (Fisher et al. 2006), replacement of

D101 and S 112 with lysine(s), PAI-2 still did not interact with VLDLR. However, VLDLR

binding could be introduced to PAI-2 through mutagenesis of S 111 , which corresponds to Y 79

in PAI-1. Interestingly, despite uPA:PAI-2YK binding to VLDLR in an almost identical

manner to uPA:PAI-1 (reported affinity constants of 1.51 nM (Croucher et al. 2007) and 1.6

108

Table 3.5: Summary of PAI-2 binding interactions with VLDR and LRP1. uPA activity 1/VLDLR K (nM) (for uPA:PAI complexes) Protein D binding kinetics 2 VLDLR LRP1 PAI-2 ∆CD-loop - 4.34 3 1.02 PAI-2K1 Unchanged/Unchanged ND ND PAI-2K2 Unchanged/Unchanged ND ND PAI-2KK Unchanged/Unchanged ND ND PAI-2YK Unchanged/Similar to PAI-1 1.35 ND PAI-2KYK Unchanged/Similar to PAI-1 1.53 0.808 PAI-2YKK Unchanged/ND ND 0.128 PAI-1 - 1.51 4 0.252 1 & 2 Compared to PAI-2 ∆CD-loop. Binding kinetics determined by SPR analysis of free PAI-2 (Section 3.3.3). 3 Section 2.3.4 5 Croucher et al. (2007). ND: Not determined. As these constructs show no direct binding to VLDLR, the affinity of the uPA:PAI-2 construct complex for the receptor was not measured.

nM (Skeldal et al. 2006)) measured using surface plasmon resonance analysis), this was not the case for LRP1. Skeldal et al. (2006) found that K 88 (referred to as K 90 due to differences in residue numbering systems), whilst somewhat important in the interaction of PAI-1 with

VLDLR, was more critically implicated in the interaction of PAI-1 with LRP1 (Figure 3.13).

YKK As such, an additional mutation was introduced in PAI-2, generating PAI-2 (N 120

corresponds to K 88 in PAI-1) which gave similar kinetics and mechanism of interaction with

LRP1 as uPA:PAI-1. This difference in the interaction surfaces highlights the distinct, whilst overlapping, specificities of LDLRs (Strickland et al. 2002). Additionally, the binding of uPA:PAI complexes to LRP1 is complicated by the involvement of both CTR (C-terminal repeat) clusters II and IV (Strickland et al. 2002; Croucher 2006).

The mutations made to complement LDLR binding in PAI-2 were found to have no impact on the general level of secondary structure or inhibitory activity of these proteins. Despite significant differences in primary sequence, there are 51 core residues conserved in approximately 70% of serpins (Irving et al. 2000). However, none of the mutations made here

109 correspond to these residues. As no difference in inhibitory activity was detected between the control PAI-2 and the PAI-2 mutants used in this study, any downstream functional differences observed are not a function of uPA inhibitory activity, suggesting that the differential extracellular functionalities in terms of protease clearance and degradation ascribed to PAI-1 and PAI-2 lies primarily in differential interactions with LDLRs.

The results presented here provides evidence that the high affinity interaction between uPA:PAI-1 and LDLRs is mediated by an avidity effect due to the combination of two binding sites on the same ligand. Furthermore, as mutations can easily modify the LDLR binding properties of PAI-1 and PAI-2 to act essentially like one another, it can be inferred that upon inhibition, conformational changes occur in uPA which, in turn, leads to an increase in affinity for LDLRs. Modification of the LDLR binding affinity/mechanism of uPA:PAI complexes may be further implicated in the contrasting non-proteolytic activities of

PAI-1 and PAI-2, namely cell growth, adhesion and activation of pro-mitogenic cell signalling pathways.

110

Figure 3.13: The receptor binding surface of the uPA:PAI-1 complex showing the difference in the importance of K 90 (equivalent to K 88 in this study due to differences in the numbering system used) of PAI-1 in the interaction of uPA:PAI-1 with VLDLR and LRP1 as determined by Skeldal et al. (2006). Alanine substitution of residues coloured red led to a greater than 5-fold reduction in binding of the uPA:PAI-1 complex to the appropriate receptor, residues coloured pink led to a 1.5 to 5-fold reduction. Alanine substitution of residues coloured blue led to a less than 1.5-fold reduction in binding. Reproduced from Skeldal et al. (2006).

111

CHAPTER 4

CELLULAR EFFECTS MEDIATED BY

COMPLEMENTATION OF THE LDLR

BINDING MOTIF IN PAI-2

112

4.1 INTRODUCTION

PAI-1 (SerpinE1) and PAI-2 (SerpinB2) are both efficient inhibitors of the urokinase- type plasminogen activator (uPA), a key enzyme in the tissue remodelling process.

Combined tumour overexpression of uPA, its receptor (uPAR) and PAI-1 is strongly correlated with poor patient prognosis (Foekens et al. 2000; Choong and Nadesapillai

2003; Duffy 2004) and metastatic potential (Duffy et al. 1999; Scherrer et al. 1999;

Schmitt et al. 2000; Look et al. 2002; Weigelt et al. 2005) and uPA/PAI-1 expression has been recommended as an independent prognostic indicator in node-negative breast cancer (Harris et al. 2007). On the contrary, tumour expression of PAI-2 is correlated with increased relapse-free survival in breast cancer (and possibly other cancer types) (Croucher et al. 2008). Further, the relationship between outcome and high PAI-2 levels in node negative breast cancer is only statistically relevant if uPA levels are also elevated (Bouchet et al. 1999; Foekens et al. 2000), suggesting that the inhibitory relationship between uPA and PAI-2 mediates this functionality.

Binding of uPA to uPAR induces the activation of numerous cell-type specific signalling pathways, such as MAPK/ERK, Jak/Stat, Src, FAK and Rac, thereby affecting cell adhesion, migration, differentiation and apoptosis (D'Alessio and Blasi

2009). As uPAR is not a transmembrane protein, but is rather linked to the cell surface by a glycosylphosphatidylinositol-anchor, uPA/uPAR signalling is mediated by integrins and several other adaptor molecules, including members of the low- density lipoprotein receptor (LDLR) superfamily (Preissner et al. 2000; Resnati et al.

2002; Strickland and Ranganathan 2003; D'Alessio and Blasi 2009). Both PAI-1 and

PAI-2 efficiently inhibit uPA in solution and at the cell surface (Astedt et al. 1987;

Ellis et al. 1990; Al-Ejeh et al. 2004) through the formation of covalent, irreversible

113 enzyme:substrate complexes. Following the formation of an inhibitory complex at the cell surface, uPA:PAI complexes are rapidly cleared via LDLR mediated endocytosis

(Argraves et al. 1995; Al-Ejeh et al. 2004; Croucher et al. 2006; Croucher et al. 2007).

Inhibition of uPA by PAI-1 has previously been shown to modulate uPA/uPAR signalling, resulting in sustained activation of the MAPK/ERK signalling pathway in both MCF-7 and SGC7901 cells (Webb et al. 2001; Di et al. 2010) leading to increased cell growth (Webb et al. 2001) and expression of MMP-2 and MMP-9 (Di et al. 2010) in these cell lines, respectively. Webb et al. (2001) demonstrated that this pro-mitogenic activity was dependent on the direct, high affinity interaction between the PAI-1 moiety of the uPA:PAI-1 complex and the very low-density lipoprotein receptor (VLDLR). Further, previous work has demonstrated that PAI-2 does not initiate these signalling events following uPA inhibition and VLDLR mediated endocytosis (Croucher et al. 2007) and PAI-2 (unlike PAI-1) does not bind directly to

LDLRs (Croucher et al. 2006; Croucher et al. 2007). Additionally, the interaction between the uPA:PAI-2 complex and these LDLRs is markedly different to that of uPA:PAI-1 in terms of both affinity and mechanism (Croucher et al. 2006; Skeldal et al. 2006; Croucher et al. 2007). As detailed in Chapter 3, numerous studies have identified several positively charged residues within (Lys 69 , Arg 76 , Lys 80 ) and nearby

(Lys88 , Arg 118 , Lys 122 ) the α-helix D of PAI-1 that contribute to the high-affinity binding of uPA:PAI-1 with LDLRs (see Figure 3.7) (Horn et al. 1998; Rodenburg et al. 1998; Stefansson et al. 1998; Skeldal et al. 2006). In addition, the α-helix D of

PAI-1 contains a proposed LDLR minimal binding motif (75-LRHLYK-80), consisting of two basic residues (Arg 76 and Lys 80 ) separated by two to five residues and N-terminally flanked by hydrophobic residues (Leu 75 and Tyr 79 ) (Jensen et al.

2006). Analysis by Croucher et al. (2007) suggested that the decreased affinity of

114 uPA:PAI-2 is likely due to an incomplete LDLR binding motif within α-helix D of

PAI-2 (107-FRSLSS-112), in which the first hydrophobic/basic amino acid pair is conserved, whilst the second pair is replaced by serine residues (Figure 3.7). Indeed, previous studies using a mutant form of PAI-1 with an incomplete LDLR binding motif (R76E) observed decreased LRP1 and VLDLR binding affinity and hence functional outcomes (Stefansson et al. 1998; Webb et al. 2001; Croucher et al. 2007).

PAI-2 may inhibit and clear cell surface uPA (and hence proteolytic activity) without influencing pro-mitogenic signalling pathways that are activated via PAI-1 and this may account for the differential prognosis seen between these two serpins (Croucher et al. 2007; Croucher et al. 2008).

Using the LDLR-binding PAI-2 mutants generated in Chapter 3 (see Table 3.2), the results presented here show that introduction of LDLR binding to PAI-2 has a profound impact on the functionality of PAI-2 in the clearance of uPA from the cell surface and initiates sustained cell signalling events similar to PAI-1 resulting in increased cell proliferation. These findings agree with the hypothesis of Croucher et al. (2007) that LDLR binding may be the biochemical basis underlying the functional disparity between PAI-1 and PAI-2 in cancer prognosis.

115

4.2 MATERIALS AND METHODS

4.2.1 MATERIALS

Protease inhibitor cocktail, glutamine, transferrin, selenium, rabbit IgG and peroxidise conjugated anti-rabbit IgG were from Sigma-Aldrich (St Louis, MO, USA). Purified human receptor associated protein (RAP) were from Molecular Innovations (Novi,

MI, USA). Rabbit antibodies to ERK and phosphorylated ERK from Cell Signaling

Technology (Danvers, MA, USA). VLDLR polyclonal antibody from Santa Cruz

Biotechnology (Santa Cruz, CA, USA). Monoclonal antibodies to human LRP1

(3402), uPA (390) and uPAR (3936) were from American Diagnostica Inc. (Stamford,

CT, USA). Matched isotype control antibodies IgG2a and IgG1 were from Millipore

(Billerica, MA, USA). Goat anti-rabbit FITC- IgG and sheep antimouse FITC-IgG were from BioRad (Hercules, CA, USA).

4.2.2 CELL LINES

The human breast adenocarcinoma (MCF-7), human prostate carcinoma (PC-3), human fibrosarcoma (HT1080) and human cervical cancer carcinoma (HeLa) cell lines were obtained from American Type Culture Collection distributed by Cryosite

(Sydney, Australia). All cell lines were routinely cultured as described in Section

2.2.9.

4.2.3 ANALYSIS OF CELL SURFACE ANTIGEN EXPRESSION BY FLOW

CYTOMETRY

Prior to analysis, cells were grown to ~ 80 % confluency over a period of at least 48 h.

Cells were detached using PBS with 5 mM EDTA, collected by centrifugation at 200 g for 5 min at 4°C and washed twice with ice cold binding buffer (Appendix I). The

116 cells were resuspended at 1 × 10 6 cells.mL -1 in ice cold binding buffer containing primary antibody or matched isotype control (5 µg.mL -1) and incubated on ice for 45 min. Cells were washed three times with ice cold binding buffer and incubated with

FITC-labelled secondary antibody (1:50 dilution) for 45 min on ice. After three further washes with ice cold binding buffer, cells were analysed by flow cytometry, with non-viable cells excluded from measurements by gating for positive propidum idiode flourecence as described previously in Section 2.2.13

4.2.4 ANALYSIS OF U PAR SATURATION AT THE CELL SURFACE

The level of uPAR saturation in cell lines expressing significant levels of uPA was analysed to determine if these cell lines were suitable for downstream characterisation of uPA:PAI complexes. Cells, grown to ~ 80 % confluency were prepared as described in section 4.2.2. Cells were resuspended in either binding buffer (Appendix

I), or Cell Acid Elution buffer (Appendix I) for 3 min at room temperature to remove surface bound uPA. Cells were neutralised with 1/5 volumes of Cell Neutralisation buffer (Appendix I) and immediately collected by centrifugation at 200 g for 5 min at

4°C and washed twice in ice cold binding buffer. Cells were incubated with 0, 5, 10,

25, 50, 75 or 100 nM Alexa 488 labelled uPA for 30 min on ice. Cells were again washed twice to remove unbound uPA and analysed by flow cytometry as described above.

4.2.5 ENDOCYTOSIS ASSAY

Cell internalisation of Alexa 488 -labelled uPA alone or in complex with either PAI-1 or

PAI-2 forms was conducted in the absence or presence of the LDLR antagonist RAP

(to confirm the involvement of LDLRs in this process) essentially as described in

Section 2.2.13.

117

4.2.6 ANALYSIS OF ERK ACTIVATION

Activation of ERK was analysed as described previously (Webb et al. 2001). Briefly,

MCF-7 cells were grown in 12 well plates to ~ 60% confluency and then serum starved (in RPMI) for 12 h. Cells were then incubated with 10 nM uPA or uPA:PAI complexes (in RPMI) for the time periods indicated. After washing twice in ice cold

RIPA buffer (Appendix I), wells were incubated for 5 min on ice in RIPA buffer, collected and insoluble inclusions removed by centrigufation at 14,000 g for 5 min at

4ºC. Supernatants were electrophoresed on 12% SDS-PAGE gels under reducing conditions, transferred to PVDF membranes and probed with antibodies that detect phosphorylated and total ERK. Bound phosphorylated and total ERK were detected using peroxidise conjugated anti-rabbit IgG secondary antibodies and visualised via

ECL.

4.2.7 CELL PROLIFERATION ASSAY

Cell proliferation was examined in real-time using the xCELLigence RTCA DP

System (Roche, Basel, Switzerland). The xCELLigence system allows continuous quantitative monitoring of cellular behaviour including proliferation by measuring electrical impedance. MCF-7 cells were seeded at 5000 cells per well into E-Plate 16 wells (cell number per well was previously optimised for this cell line, Appendix VII) and cultured for 24 h. The media was replaced with 100 µL of serum free RPMI containing 300 µg.mL -1 glutamine, 5 µg.mL -1 transferrin and 38 nM selenium in the presence of 10 nM uPA, uPA:PAI complexes or media alone. Proliferation was continuously monitored every 15 min over a time period of 72 h. Data analysis was carried out using RTCA Software 1.2.1 supplied with the instrument.

118

4.2.8 STATISTICAL ANALYSES

Statistical significance of treatment groups as compared to control groups was determined using an unpaired Student’s t-test or one-way ANOVA with a Tukey post- test (GraphPad Prism V 5.1). p values < 0.05 were considered statistically significant.

119

4.3 RESULTS

4.3.1 COMPONENTS OF THE PLASMINOGEN ACTIVATION SYSTEM ARE

DIFFERENTIALLY EXPRESSED ON CANCER CELL LINES

Prior to analysing the cellular functional consequences of complementing LDLR binding in PAI-2, a panel of cancer cells lines were screened for expression of receptors involved in the endocytosis of uPA:PAI complexes. As there are differences in the determinants required to increase the affinity of uPA:PAI-2 for LRP1 and

VLDLR to a level similar to that of uPA:PAI-1 binding (Chapter 3), cell lines that express either LRP1 or VLDLR and not both receptors were desirable to allow for non-confounding characterisation. HT1080 cells have previously been shown to express LRP1 (Webb et al. 2000; Degryse et al. 2004) and this was confirmed (Figure

4.1). These cells also expressed considerable amounts of uPA and uPAR, but did not express any detectable VLDLR (Figure 4.1). The HeLa human cervical cancer cell line was found to express uPA, uPAR and both LRP1 and VLDLR (Figure 4.2). As such, HeLa cells were deemed unsuitable for further use due to the dual expression of

LRP1 and VLDLR. Al-Ejeh et al. (2004) and Croucher et al. (2006) previously characterised the internalisation of PAI-2 on PC-3 cells, noting that these cells expressed uPA, uPAR and LRP1. Analysis here confirms expression of these antigens, and very low to negligible levels of VLDLR (Figure 4.3). The level of uPA expressed by PC-3 cells was significantly less than that of HeLa cells (p < 0.05).

MCF-7 cells were used in Chapter 2 and previously (Webb et al. 2001; Croucher et al.

2007) to characterise uPA:PAI internalisation in a VLDLR dependent manner. As anticipated, these cells expressed significant levels of uPAR, VLDLR, a small but significant level of uPA and negligible levels of LRP1 (Figure 4.4).

120

uPA 90.8 ± 6.7 uPAR 68.6 ± 9.6

LRP1 52.1 ± 4.1 VLDLR 2.1 ± 1.8

Figure 4.1: Cell surface antigen profiling of the HT1080 human fibrosarcoma cell line. HT1080 cells were incubated with 5 µg.mL -1 primary antibody against either uPA, uPAR, LRP or VLDLR, washed, incubated with a secondary FITC labelled antibody and cell surface fluorescence on intact cells measured by flow cytometry. Representative histograms are shown. Values in top right corner of each panel are the difference between means of treatment and matched isotype control ± standard deviations (n = 3).

121

uPA 11.0 ± 0.3 uPAR 107.8 ± 13.1

LRP1 23.4 ± 3.6 VLDLR 6.1 ± 1.8

Figure 4.2: Cell surface antigen profiling of the HeLa human cervical carcinoma cell line. HeLa cells were incubated with 5 µg.mL -1 primary antibody against either uPA, uPAR, LRP or VLDLR, washed, incubated with a secondary FITC labelled antibody and cell surface fluorescence on intact cells measured by flow cytometry. Representative histograms are shown. Values in top right corner of each panel are the difference between means of treatment and matched isotype control ± standard deviations (n = 3).

122

uPA 7.2 ± 1.8 uPAR 49.2 ± 4.5

LRP1 18.1 ± 2.9 VLDLR 1.2 ± 0.9

Figure 4.3: Cell surface antigen profiling of the PC-3 human prostate carcinoma cell line. PC-3 cells were incubated with 5 µg.mL -1 primary antibody against either uPA, uPAR, LRP or VLDLR, washed, incubated with a secondary FITC labelled antibody and cell surface fluorescence on intact cells measured by flow cytometry. Representative histograms are shown. Values in top right corner of each panel are the difference between means of treatment and matched isotype control ± standard deviations (n = 3).

123

uPA 5.1 ± 2.8 uPAR 69.2 ± 8.3

LRP1 2.9 ± 2.0 VLDLR 52 ± 5.9

Figure 4.4: Cell surface antigen profiling of the MCF-7 human breast epithelial carcinoma cell line. MCF-7 were incubated with 5 µg.mL -1 primary antibody against either uPA, uPAR, LRP or VLDLR, washed, incubated with a secondary FITC labelled antibody and cell surface fluorescence on intact cells measured by flow cytometry. Representative histograms are shown. Values in top right corner of each panel are the difference between means of treatment and matched isotype control ± standard deviations (n = 3).

124

4.3.2 CHARACTERISATION OF U PAR SATURATION

As the focus of this study was on the role of uPA:PAI complexes (as explained in

Section 4.1, the prognostic value of PAI-1 and PAI-2 in cancer is only significant when co-expressed with uPA), internalisation and downstream functional consequences of introducing LDLR binding to PAI-2 was examined using pre- prepared uPA:PAI complexes. This allows for the assumption that any changes in cell behaviour upon treatment with uPA:PAI complexes is due to differences in complex affinity and not dependent on the differing inhibition rates of PAI-1 and PAI-2.

Additionally, whilst PAI-1 can moderate cell adhesion/deadhesion via interactions with vitronectin, the uPA:PAI-1 complex does not readily bind vitronectin. This is particularly relevant given that PAI-1 alone, but not PAI-2, interacts with vitronectin and causes considerable cell detachment (Lobov and Ranson, Cancer Letters, in press ), which confounds endocytosis analysis. Further, cell lines used to examine the link between LDLR affinity and uPA:PAI complex endocytosis were required to bind exogenous uPA. The level of uPAR saturation of MCF-7 cells was not examined as these cells have previously been demonstrated to internalise uPA:PAI complexes in an

LDLR-affinity dependent manner represent a suitable model (Section 2.3.4 and

Croucher et al. (2007)). HT1080 cells did not readily bind exogenous uPA (at concentrations up to 100 nM) (Figure 4.5A). However, after acid stripping to elute non-covalently associated surface proteins, HT1080 cells showed a dose dependent uPA binding response, suggesting that under native conditions, uPAR is fully saturated with uPA. As such, these cells do not present an ideal model to test uPA:PAI internalisation as endogenous uPA would first need to be stripped from the cell surface and this may cause other confounding effects. In contrast, PC-3 cells readily bound exogenous uPA (Figure 4.5B). As such,

125

A HT1080

200 uPA binding Acid Stripped 150 uPA Binding

100

0 Mean Fluorescence (515 nm) (515 Fluorescence Mean 0 20 40 60 80 100 uPA Concentration (nM)

B PC-3

200 uPA Binding Acid Stripped 150 uPA Binding

100

0 Mean Fluorescence (515 nm) (515 Fluorescence Mean 0 20 40 60 80 100 uPA Concentration (nM)

Figure 4.5: uPA binding profile of cancer cell lines. Any endogenous uPAR bound uPA was either removed by acid stripping (red lines) or not (black lines) from (A) HT1080 and (B) PC-3 cells. Cells were then incubated with increasing concentrations of Alexa 488 labelled uPA. Excess uPA was removed via centrifugation and cell surface associated fluorescence measured by flow cytometry. Values are means ± standard deviation (n = 3).

126

PC-3 cells were further used to characterise the internalisation of uPA:PAI complexes in an LRP1 dependent system.

4.3.3 INTRODUCTION OF LDLR BINDING TO PAI-2 INCREASED U PAR-

MEDIATED ENDOCYTOSIS

Croucher et al. (2007) previously demonstrated that the affinity of uPA or uPA:PAI complex for LDLRs is a key determinant of its rate of endocytosis. This was confirmed here with significantly more uPA:PAI-2 endocytosis (~4 fold, relative to uPA alone) by MCF-7 cells in an LDLR antagonist (RAP)-sensitive manner over a 30 min time period (Figure 4.6A). Given that VLDLR is the only LDLR family member expressed by MCF-7 cells (Figure 4.4), these data reflect the increased affinity of the complex for VLDLR (see Table 3.3). Accordingly, the RAP-sensitive endocytosis of uPA:PAI-2YK was increased to a level comparable with uPA:PAI-1 (~7 fold compared to uPA alone) (Figure 4.6A). The uPA:PAI-2KYK complex had no significant impact on endocytosis over and above that of uPA:PAI-2YK (Figure 4.6A). This result reflected the similar VLDLR binding affinities of the PAI-2 mutants and PAI-1 (Table

3.2) and further confirms that completion of the LDLR binding motif is sufficient for enhanced endocytosis via VLDLR.

Further investigation of endocytosis in an LRP1-dependent cell model (PC-3) showed that the LDLR-binding motif alone was not sufficient for enhanced endocytosis via

LRP1. Although a small but significant increase in RAP-sensitive uPA:PAI-2 endocytosis was observed compared to uPA alone, no significant difference in internalisation was observed between uPA:PAI-2 and uPA:PAI-2YK (Figure 4.6B).

127

A n.s.

† † † 800 **** - RAP 700 + RAP 600 500 400 300 200 100 % uPA internalisation 0

A YK P I-2 KYK I-1 u A I-2 2 A I- :PA A:P :P A :P uP PA A uPA u uP

B n.s. n.s.

† † **** 300 - RAP + RAP 200

100 % uPA internalisation 0

A -2 YK KK 1 2 Y uP AI :P AI- :PAI- A P uP PA: A:PAI-2 uPA u uP

Figure 4.6: Increased LDLR affinity leads to increased internalisation of uPA:PAI complexes by cancer cells. (A) MCF-7 cells or (B) PC-3 cells were incubated in the presence or absence of RAP (200 nM) for 15 min at 37°C, prior to * analysis of endocytosis of Alexa 488 -labelled uPA. Values are means ± SEM (n = 3). p < 0.005 compared to relative uPA treatment, † p < 0.005 compared to relative uPA:PAI-2 treatment, n.s., no significant difference between these values.

128

However, the level of uPA:PAI-2YKK and uPA:PAI-1 endocytosis were similarly significantly enhanced ~2.5 fold compared with uPA alone; and ~1.6 fold compared with uPA:PAI-2 (Figure 4.6B). These functional data are entirely consistent with the biochemical data describing the differential interactions of uPA:PAI-2YK versus uPA:PAI-2YKK with LRP1 (Table 3.3).

4.3.4 INTRODUCTION OF VLDLR BINDING TO PAI-2 INDUCES MITOGENIC

SIGNALLING IN MCF-7 CELLS

The sustained ERK phosphorylation that is initiated in breast cancer cells via a high- affinity LDLR minimal binding motif in PAI-1 is not observed in cells treated with uPA:PAI-2 (Webb et al. 2001; Croucher et al. 2007). This was confirmed in this study as the effect of uPA:PAI-2 being indistinguishable from uPA (Figure 4.7). The importance of an intact LDLR minimal binding motif in facilitating this activity was confirmed, as stimulation of MCF-7 cells with uPA:PAI-2YK initiated a significant increase in both acute and sustained ERK phosphorylation (~3.5 fold within 3 min, relative to uPA alone) (Figure 4.7B). The kinetics of ERK phosphorylation by uPA:PAI-2 was similar to that observed for uPA:PAI-1.

A downstream functional outcome of sustained ERK phosphorylation in MCF-7 cells stimulated by uPA:PAI-1 is induction of proliferation (Webb et al. 2001; Croucher et al. 2007). Thus, the effect of uPA:PAI-2YK on MCF-7 cell proliferation was measured in real time using the xCELLigence system (Figure 4.8A). Cell indices for uPA:PAI-

2YK and uPA:PAI-1 treatments were distinct from those observed for cells incubated with uPA or uPA:PAI-2 (Figure 4.8A). Clear proliferative effects were evident over

129

Figure 4.7: uPA:PAI-2YK induces promitogenic signalling pathways in MCF-7 cells similar to those of uPA:PAI-1. MCF-7 cells were cultured in serum-free medium for 12 h and then exposed to 5 nM uPA, uPA:PAI-2, uPA:PAI-2YK or uPA:PAI-1 for the indicated times. (A) Cell lysates were analysed for phosphorylated and total ERK by western blotting. (B) Analysis of phoshorylated ERK levels using densitometry was performed using the Quantitiy One software package (BioRad).

130

2.0 uPA:PAI-1 uPA:PAI-2 YK 1.5 uPA:PAI-2 uPA Media only 1.0

0.5

Normalised Cell Index (CI) Index Cell Normalised 0.0 0 20 40 60 80 100 Time (h) B

24 h 48 h

150 n.s. 150 n.s.

** 100 100

50 50

0 0 K y A Y control) (% Index Cell Relative I-1 control) (% Index Cell Relative I-2 2 onl uP A only uPA A - a :P :PAI-2 dia A A e Medi uPA:PAI-2 uPA:P M uP uP uPA:PAI

72 h n.s.

200

Relative Cell Index (% control) (% Index Cell Relative

131

Figure 4.8: uPA:PAI-2YK increased cell proliferation in MCF-7 cells similar to uPA:PAI-1. (A) MCF-7 cells were cultivated in E-plates with RPMI/5% FCS for 24 h, before changing to serum free media containing 10 nM uPA, uPA:PAI-2, uPA:PAI- 2YK , uPA:PAI-1 or media only and cell proliferation monitored over 72 h in real-time using an xCELLigence RTCA DP System. The electronic readout of cell sensor impedance is displayed continuously as the Cell Index (CI) normalised to the compound addition timepoint (indicated by vertical dotted line). The CI value at each time point is defined as (R n–Rb)/R b, where R n is the electrode impedance of wells with cells and R b is the background of wells with media alone. Curves are means of 4 replicates. (B) Bar graphs showing CI values 24, 48 and 72 h after addition of proteins as percentage of media only treatment (time points are indicated on electronic readout by arrows). Values are means ± SEM (n = 4). * p < 0.005 as determined by one-way ANOVA with a Tukey post-test; n.s., no significant difference between treatments.

132 an extended time-frame following addition of either uPA:PAI-2YK or uPA:PAI-1, as reflected in an apparent logarithmic phase growth pattern (Figure 4.8A). This resulted in a significant (~1.6 fold) increase in cell index relative to controls (media, uPA or uPA:PAI-2) after 72 h (Figure 4.8B). Interestingly, cell indices following uPA:PAI-

2YK or uPA:PAI-1 treatment were initially slightly lower than those obtained following treatment with uPA or uPA:PAI-2. The reason for this is unclear. No difference was observed between cells incubated with uPA or uPA:PAI-2 (Figure

4.8B), with both reaching a plateau between 12 and 36 h post-treatment before declining, suggesting a decrease in cell number.

133

4.4 DISCUSSION

Together with the results presented in Chapter 3, this study provides the first definitive molecular characterisation of the key structural elements that underlie the differential interactions of PAI-1 and PAI-2 with LDLR receptors and how these are associated with VLDLR-mediated downstream mitogenic signalling and cell proliferation. By completing the LDLR binding motif in PAI-2 with residues corresponding to those in PAI-1, PAI-2 displayed the pro-mitogenic functionalities of

PAI-1 when complexed with uPA. This suggests that the high LDLR binding affinity and endocytosis rate of uPA:PAI-1 are part of the repertoire of effects attributable to the pro-mitogenic function of PAI-1. This helps to explain why, despite a shared serpin inhibitory function, high tumour levels of uPA and PAI-1 promote tumour progression, whereas high levels of PAI-2 are protective (Croucher et al. 2008).

The existence of a strong correlation between the affinity of uPA:PAI complexes for

VLDLR and uPA:PAI endocytosis has been previously reported (Webb et al. 2001;

Croucher et al. 2007), suggesting that VLDLR affinity may be the rate limiting determinant of uPA/uPAR clearance from the cell surface (Croucher et al. 2007). By comparison to uPA:PAI-1, the uPA:PAI-2YK complex displayed similar binding affinity to VLDLR (Section 3.3.3), and was internalised via this endocytosis receptor to the same extent. The importance of the LDLR binding motif for high affinity

VLDLR binding and enhanced endocytosis was confirmed by the lack of a significant impact of uPA:PAI-2KYK over and above that of uPA:PAI-2YK . As members of the

LDLR family have overlapping, yet distinct ligand specificities (Strickland et al.

2002), presence of the LDLR minimal binding motif alone was not sufficient to introduce binding to LRP1 (as discussed in Chapter 3). Skeldal et al. (2006) reported

134 that Lys 88 was critically implicated in the interaction of PAI-1 with LRP1. In agreement with this, modification of the residue Asn 120 in PAI-2 (Asn 120 corresponds to Lys 88 in PAI-1) was also required in conjunction with a complete LDLR minimal binding motif (PAI-2YKK ) to result in LRP1 binding kinetics and LRP1-mediated endocytosis similar to that of uPA:PAI-1. This thus confirms that LDLR affinity is the rate-limiting step of uPA:PAI endocytosis.

Webb et al. (2001) demonstrated that affinity of uPA:PAI-1 complexes for VLDLR correlates to the sustained activation of ERK and increased cell proliferation in MCF-

7 cells. The VLDLR binding and downstream functional activity associated with PAI-

1 was also introduced into PAI-2 via completion of the LDLR minimal binding motif within the α-helix D of PAI-2. As such, it is clear that VLDLR affinity is the key property underlying the differences in sustained ERK activation and subsequent effects on cell proliferation between uPA:PAI-1 and uPA:PAI-2. Given this, it was unnecessary to investigate the impact of uPA:PAI-2KYK (or other PAI-2 mutants generated with incomplete LDLR binding motifs) on mitogenic signalling as the

VLDLR binding affinity of this complex was indistinguishable from that of uPA:PAI-

2YK . Direct binding of PAI-1 to LRP1 has also been demonstrated to activate the

Jak/Stat pathway, leading to increased cell motility (Degryse et al. 2004). However, as the prognostic data for PAI-1 is only significant in tumours also expressing uPA

(Foekens et al. 2000; Choong and Nadesapillai 2003; Duffy 2004; Ohba et al. 2005), the impact of introducing PAI-2 LRP1 binding on the activation of this pathway was also not investigated in this study. Furthermore, PAI-1 alone causes significant cellular detachment (Lobov and Ranson, Cancer Letters in press ) which complicates analysis of endocytosis and cell signalling.

135

Whilst high affinity binding of uPA:PAI complexes to VLDLR is clearly vital for the induction of downstream ERK activation, it is important to note that high affinity binding of any ligand to VLDLR will not necessarily induce the same response. This is particularly highlighted by the high affinity interaction of RAP with VLDLR (K D =

0.7 nM) (Battey et al. 1994), which fails to induce downstream ERK activation and cell proliferation (Webb et al. 2001; Croucher et al. 2007). Therefore, the rapid formation of a multi-molecular complex consisting of VLDLR, a uPA:PAI complex, uPAR and any associated integrins or co-receptors is likely implicated in the induction of this signal, not merely the recognition of a high affinity ligand by

VLDLR

Other factors that would impact on the prognostic differences between overexpression of PAI-1 versus PAI-2 in cancer include the complex interplay of interactions between uPA/uPAR, PAI-1 and other ligands and co-receptors, by which

PAI-1 modulates adhesion and migration. For example, PAI-1 competes with uPAR and integrins for vitronectin binding which has further implications on cell adhesion/deadhesion (Deng et al. 1996; Stefansson et al. 2003; Croucher et al. 2008).

PAI-2 does not bind vitronectin (Mikus et al. 1993). Furthermore, the vitronectin binding site/s in PAI-1 are distinct from the LDLR minimal binding motif (Jensen et al. 2002) and in agreement with this, the PAI-2 constructs used in this study did not bind to vitronectin or impact on cell adhesion (Dr Sergei Lobov, personal communication ). This indicated that the proliferative effects seen with PAI-2YK with a

VLDLR-dependent cell line (Figure 4.8) were not due to modulation of cellular adhesion/deadhesion, but solely reliant on the increased affinity for VLDLR.

Regardless, the absence of an LDLR minimal binding motif in the PAI-2 moiety of

136 the uPA:PAI-2 complex indicates how, unlike PAI-1, extracellular PAI-2 would inhibit and clear uPA from the cell surface, thereby preventing plasmin-mediated

ECM degradation, without impacting on cell proliferation.

In conclusion, this study clearly demonstrates that VLDLR affinity is the critical determinant underlying the disparate roles of PAI-1 and PAI-2 in mitogenic signalling and cell proliferation. Understanding the molecular basis of such physiological roles increases the understanding of the association of these proteins with their respective roles in predicting disease severity and outcome.

137

CHAPTER 5

CONCLUSIONS AND FUTURE

DIRECTIONS

138

The findings described in this thesis define the relationship between LDLR affinity and the physiological roles of PAI-2 verses PAI-1. Generally, as PAI-1 is considered the primary physiological mediator of thrombosis and fibrinolysis, PAI-1 has attracted significantly more attention than PAI-2. Numerous studies have analysed the links between the molecular biology and physiological function in PAI-1 (De Taeye et al.

2004) whilst irrefutable proof of PAI-2 internalisation and the mechanisms facilitating this endocytosis, at least in vitro , have only been described more recently (Al-Ejeh et al. 2004; Croucher et al. 2006; Croucher et al. 2007). Significant resistance exists in the acceptance of the role of PAI-2 in inhibiting plasminogen activation due to the majority of expressed PAI-2 being unsecreted. Whilst there has been no definitive identification of uPA:PAI-2 complexes in vivo , a number of studies have detected

PAI-2 co-localised with a plasminogen activator(s) (Nakashima et al. 1996; Kinnby et al. 1999) or in forms likely to have reacted with uPA or tPA (Kiso et al. 1991; Brown et al. 1995). Furthermore, expression of PAI-2 in predicting outcomes in node negative breast cancer is only significant when combined with increased uPA expression (Foekens et al. 2000), suggesting that this relationship is due to the inhibitory action of PAI-2. Future studies will aim to isolate intact PA:PAI-2 complexes to definitivly confirm the role of PAI-2 as a protease inhibitor in vivo . The

2H5 monoclonal antibody has been been demonstrated to recognise PAI-2 in the

‘relaxed’ conformation (Saunders et al. 1998) and could be used to isolate PAI-2 in complex with plasminogen activators from PAI-2 rich tissues such as placenta, gingivia or plasma from women in the third trimester of pregnancy. Further validation of the role of PAI-2 in the inhibition of cancer progression should involve an animal model. For example, SERPINB2 -/- mice would be engineered to express human PAI-2 using the Cre/ lox system (Sauer and Henderson 1988) with Cre under the control of a

139 fibroblast specific promoter. These mice, which would express human PAI-2 only in fibroblast cells, would then be utilised in xenograft models using human cancer cell lines. The rationale behind the transfection of only fibroblast cells is that in doing so, the ECM surrounding the xenograft would contain PAI-2, with the PAI-2 inhibiting the intravasation, presenting a ‘barrier’ which the invading cells must pass. The impact of enhancing the secretion of PAI-2 from the fibroblasts could also be assessed with the use of a PAI-2 construct fused to the signal peptide of PAI-1 which has previously been reported to increase the secretion efficiency of PAI-2 (von Heijne et al. 1991).

Understanding the interactions between PAI-2 and members of the LDLR family is important in providing rationale for the continued development of PAI-2 as a targeted cancer therapeutic. The ability of PAI-2 to target, inhibit and clear uPA without influencing additional biochemical pathways is an important characteristic in the choice to develop PAI-2 rather than PAI-1 as a therapeutic. Previous work defining the interactions of PAI-2 with VLDLR and LRP1 could not definitively rule out the

CD-loop blocking an interaction between the PAI-2 moiety of the uPA:PAI-2 complex and LDLRs (Croucher et al. 2006; Croucher et al. 2007) and as such, it could not be assumed that removal of the CD-loop would not alter the binding of uPA:PAI-

2 to LDLRs and thereby activate promitogenic signalling pathways. The findings described in this thesis showed that removal of the CD-loop had no impact on the uPA targeting function of PAI-2, nor does the CD-loop block an LDLR binding site in

PAI-2. As removal of the CD-loop enabled easier purification from a prokaryotic system, PAI-2 ∆CD-loop is currently being utilised as the starting material to further

140 the development of PAI-2 as a targeted cancer therapeutic (Vine et al. 2010 and Dr

Kara Perrow, University of Wollongong, manuscript submitted ).

The strong prognostic value of tumour expression of uPA and PAI-1 in predicting poor patient outcome is well established and is gaining traction as a diagnostic clinical tool in node negative breast cancer. In contrast, there is a growing body of evidence linking tumour expression of PAI-2 and favourable patient outcome. The biochemical basis underlying this disparate role in disease outcome is poorly understood and represents a fundamental shortfall in basic tumour pathobiology. This thesis directly addresses this shortfall and provides a definitive mechanistic basis for the apparently paradoxical relationship of PAI-1 and PAI-2 with disease outcome.

Previous work demonstrated clear differences in the interactions of PAI-2 and PAI-1 with LDLR endocytosis receptors (Croucher et al. 2006; Croucher et al. 2007). By introducing the LDLR minimal binding motif in PAI-2, the findings presented here demonstrate that these biochemical differences define the disparate cellular functions of PAI-1 and PAI-2 in cancer. The sustained activation of ERK initiated in MCF-7 and SGC7901 cells by uPA:PAI-1 is clearly mediated via the high affinity interaction between the complex and VLDLR. However, the precise mechanism by which this occurs remains undefined. Strickland et al. (2002) outlined three possible, non- mutually exclusive mechanisms by which uPA:PAI-1 may initiate this signalling

(Figure 5.1). Transient phosporylation of ERK due to the binding of uPA to uPAR occurs via interactions of the uPA:uPAR complex with integrins (Ossowski and

Aguirre-Ghiso 2000) (Figure 5.1A). All of the proposed mechanisms by which

141

A Integrin or other uPA uPAR co-receptor uPAR

Transient ERK phosphorylation B C D PAI-1 PAI-1 VLDLR PAI-1 VLDLR VLDLR

uPAR uPA uPAR uPA uPAR uPA

Unidentified adaptor protein?

•Complex internalisation •Complex internalisation •Bridging of intracellular domains •uPAR recycling •Delivery to endosomes of integrins and VLDLR •Sustained ERK phosphorylation •Sustained ERK phosphorylation •Sustained ERK phosphorylation as sum of transient events from endsome via uPAR machinery

E F G PAI-2 PAI-2 VLDLR PAI-2 VLDLR VLDLR

uPAR uPA uPAR uPA uPAR uPA

•Complex internalisation •Complex internalisation •Insufficent uPA:PAI-2 complex •uPAR recycling •Delivery to endosomes affinity to bridge intracellular •Transient ERK phosphorylation •Transient ERK phosphorylation domains of integrins and VLDLR •Transient ERK phosphorylation

PAI-2YK H VLDLR

uPAR uPA

•Increased uPA:PAI-2YK affinity sufficient to bridge intracellular domains of integrins and VLDLR? •Receptor recycling? •Sustained ERK phosphorylation •Promitogenic effects similar to uPA:PAI-1 142

Figure 5.1: Proposed models by which uPA:PAI complexes mediate ERK activation via VLDLR-mediated endocytosis. (A) The binding of uPA to uPAR induces a transient phosphorylation of ERK which is mediated via adapter proteins such as integrins . Strickland et al. (2002) proposed three models (B-D) by which uPA:PAI-1 may sustain ERK phosphorylation. (B) After internalisation of the uPAR:uPA:PAI-1:VLDLR complex, uPAR is recycled to the cell surface where it can potentiate further cell signalling resulting in a sustained ERK phosphorylation that is the sum of many transient responses. (C) Subsequent to internalisation, the complex is delivered to endosomes where pH changes result in structural changes in uPAR which activates signalling proteins associated with uPAR and thereby sustaining activation of ERK. (D) In the final model, the high affinity of the uPA:PAI-1 complex results in the colocalisation of integrins and VLDLR resulting in bridging of proteins associated with the intracellular domains of integrins and signalling motifs in VLDLR. This thesis further characterises the internalisation of uPA:PAI-2, building upon previous studies (Al-Ejeh et al. 2004; Croucher et al. 2006; Croucher et al. 2007) and shows that the differential mechanisms by which uPA:PAI-1 and uPA:PAI-2 differentially modulate ERK activity in MCF-7 cells upon complex internalisation is due to differences in the affinity of the complex for the receptor. (E) Internalisation of uPA:PAI-2 results in uPAR recycling (Al-Ejeh et al. 2004), but only transient phosphorylation of ERK. (F) Like PAI-1, upon internalisation the uPAR:uPA:PAI-2 complex is delivered to endosomes. As such, it appears unlikely that the difference in ERK phosphorylation mediated by uPA:PAI-1 and uPA:PAI-2 is due to signalling through uPAR associated changes that occur in the endosome. (G) The lower affinity of the uPA:PAI-2 complex for VLDLR is not sufficient to bridge the intracellular domains of VLDLR and integrins/uPAR coreceptors and as such, the proteins associated with these intracellular domains are not able to sustain ERK phosphorylation. (H) Introducing VLDLR binding to PAI-2 in uPA:PAI-2YK results in sustained phosphorylation of ERK similar to uPA:PAI-1 with this likely occurring via the increased receptor affinity allowing for interactions between the cytoplasmic domains of integrins and VLDLR or by altering the uPAR recycling profile. A-D adapted from Strickland et al. (2002).

143 uPA:PAI-1 require interplay between the uPA:uPAR:integrin complex and VLDLR

(Figure 5.1B-D), as high affinity binding to VLDLR is not, in itself, able to induce sustained ERK activation – as evidenced by the lack of signal transduction induced upon treatment of cells with RAP (K D = 0.7 nM). Additionally, the general co- localisation of VLDLR and integrins mediated by uPA:PAI-2 does not sustain phosphorylation of ERK (Figure 5.1E-G), suggesting that there may be some threshold in the affinity of the integrin:uPAR:uPA:PAI complex for VLDLR at which point ERK phosphorylation is sustained. If the sustained ERK phosphorylation observed in cells treated with uPA:PAI-1 was solely due to receptor recycling of uPAR (Figure 5.1B) a similar signalling response would be expected in cells treated with uPA:PAI-2, rather than just a transient phosphorylation of ERK (Figure 5.1E).

Furthermore, as both uPA:PAI-1 and uPA:PAI-2 mediated endocytosis of the integrin:uPAR:uPA:PAI complex results in delivery of the complex to endosomes

(Figure 5.1C,F), both uPA:PAI-1 and uPA:PAI-2 would be expected to sustain ERK phosphorylation if this was mediated via structural changes in uPAR initiated by a change in pH. As such, it appears most likely that it is the bridging of the intracellular domains of integrins and VLDLR facilitated by uPA:PAI-1 that results in sustained phosphorylation of ERK (Figure 5.1D). The affinity of uPA:PAI-2 for VLDLR is insufficient to bridge the intracellular domains of integrins and VLDLR and results only in the transient activation mediated by the integrin:uPAR interaction (Figure

5.1G). The results presented in this thesis lend weight to this hypothesis as increasing the affinity of PAI-2 for VLDLR (in PAI-2YK ) to a level indistinguishable from that of

PAI-1 led to sustained ERK phosphorylation in MFC-7 cells (Figure 5.1H). As the rate of co-localisation of uPAR and VLDLR following ligand stimulation may underpin the activation of signalling events, the kinetics of uPAR and VLDLR co-

144 localisation could be measured in real time following ligand stimulation via bimolecular fluorescence complementation (Kerppola 2006). Identification of the protein/s that associate with the intracellular domain of VLDLR and mediate sustained ERK phosphorylation could also be analysed by combining bimolecular fluorescence complementation with a high through-put screen using a human

ORFeome set. Previous studies have shown that reelin, which is involved in brain development and neuroplasticity, contains two LDLR binding sites, is able to initiate cell signalling events via clustering of VLDLR and Dab-1, a tyrosine kinase associated with the interacellular domain of VLDLR but expressed only in neural cells (Strasser et al. 2004). Whilst uPA:PAI-1 and uPA:PAI-2YK do contain two independent binding sites for VLDLR, the low affinity interaction (K D values of 84.8

YK nM and 70.1 nM for uPA:PAI-1 and uPA:PAI-2 ) is less than that of uPA alone (K D

31.2 nM) and would likely not result in clustering of VLDLR.

A recent study by Di et al. (2010) found that treatment of SGC7901 cells with uPA:PAI-1 led to an increased ratio of type II VLDLR to type I VLDLR. These forms of VLDLR differ in the presence (type I) or absence (type II) of the O-linked sugar domain (Iijima et al. 1998) with type I VLDLR primarily found in heart and skeletal muscle whilst type II VLDLR is predominant in non-muscle tissue. Previous studies have found that the ratio of type II to type I VLDLR in breast cancer, gastroinintestinal adenocarcinoma and hepatocellular carcinoma tissues and cell lines in significantly higher compared to control tissues or cells (Chen et al. 2005; He et al.

2010; Luo et al. 2010). Luo et al. (2010) found that silencing expression of type II

VLDLR in hepatocellular carcinoma cell lines decreased cell proliferation suggesting perhaps the expression of type II VLDLR is not merely an indicator of cell proliferation but facilitates proliferation by an as yet identified mechanism. The

145 impact of incubating cells with uPA or uPA:PAI-2 on the expression pattern of

VLDLR has not been investigated. Upregulation of VLDLR expression via the ERK signalling pathway in cells treated with VLDL and β-VLDL has been reported (Liu et al. 2009); the increased expression of type II VLDLR could be a function of the sustained ERK activation mediated by uPA:PAI-1.

The results of this thesis further the understanding of the relationship between the biochemical properties and cellular function of PAI-2. Whilst both PAI-1 and PAI-2 efficiently inhibit and clear uPA from the cell surface, the high affinity interaction between uPA:PAI-1 and VLDLR mediates promitogenic signalling effects, resulting in increased metastatic potential (Figure 5.2). The lack of an LDLR minimal binding motif in PAI-2 lowers the affinity of uPA:PAI-2 for VLDLR to a level below that required to initiate promitogenic signalling. Furthermore, differences in adhesion/deadhesion effects further underlie the disparate roles of PAI-1 and PAI-2 in cancer pathobiology. The relationships between these biochemical function and physiological roles provides rationale for the use of PAI-1 and PAI-2 as markers of disease progression and the continued development of PAI-2 as a targeted cancer therapeutic. More generally, these findings also further validate the importance of the

LDLR minimal binding motif in endocytosis and downstream function.

146

Integrin/ Integrin/ co-receptor co-receptor PAI-1 VLDLR PAI-2 VLDLR

uPAR uPA uPAR uPA

•Enhanced affinity for LDLRs •Enhanced affinity for LDLRs relative to uPA alone relative to uPA alone •Enhanced internalisation •Enhanced internalisation and •Sustained activation of clearance of uPA without promitogenic signaling pathways sustained signaling •Increased proliferation (and •No impact on proliferation or migration in other settings) migration •Increased metastasis •Inhibition of metastasis

Figure 5.2: Physiological actions of PAI-1 and PAI-2 upon inhibition and clearance of uPA. PAI-1 is able to promote metastasis via the activation of promitogenic signalling pathways that are dependent on the high affinity uPA:PAI- 1:VLDLR interaction. Additionally, PAI-1 can influence cell adhesion/deadhesion via completion for vitronectin. Whilst PAI-2 also rapidly clears uPA from the cell surface, the uPA:PAI-2 complex lacks sufficient affinity for VLDLR to activate promitogenic signalling and PAI-2 does not compete for vitronectin (Lobov and Ranson, Cancer Letters, in press ) defining the protective role of PAI-2 in cancer.

147

CHAPTER 6

REFERENCES

148

Abbas Rizvi, S. M., Li, Y., Song, E. Y., Qu, C. F., Raja, C., Morgenstern, A., Apostolidis, C. and Allen, B. J. (2006). "Preclinical studies of bismuth-213 labeled plasminogen activator inhibitor type 2 (PAI2) in a prostate cancer nude mouse xenograft model." Cancer Biology & Therapy 5: 386-393.

Aertgeerts, K., De Bondt, H. L., De Ranter, C. J. and Declerck, P. J. (1995). "Mechanisms contributing to the conformational and functional flexibility of plasminogen activator inhibitor-1." Nature Structural Biology 2: 891-897.

Agirbasli, M. (2005). "Pivotal role of plasminogen-activator inhibitor 1 in vascular disease." International Journal of Clinical Practice 59 : 102-106.

Ahn, N. S., Hu, H., Park, J. S., Kim, J. S., An, S., Kong, G., Aruoma, O. I., Lee, Y. S. and Kang, K. S. (2005). "Molecular mechanisms of the 2,3,7,8- tetrachlorodibenzo-p-dioxin-induced inverted U-shaped dose responsiveness in anchorage independent growth and cell proliferation of human breast epithelial cells with stem cell characteristics." Mutat Research 579 : 189-199.

Akiyama, H., Ikeda, K., Kondo, H., Kato, M. and McGeer, P. L. (1993). "Microglia express the type 2 plasminogen activator inhibitor in the brain of control subjects and patients with Alzheimer's disease." Neuroscience Letters 164 : 233-235.

Al-Ejeh, F., Croucher, D. and Ranson, M. (2004). "Kinetic analysis of plasminogen activation inhibitor type-2: urokinase complex formation and subsequent internalisation by carcinoma cell lines." Experimental Cell Research 297 : 259- 271.

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. and Walter, P. (2008). Molecular Biology of the Cell . Garland Science, New York, USA.

Aleman, C., Alegre, J., Monasterio, J., Segura, R. M., Armadans, L., Angles, A., Varela, E., Ruiz, E. and Fernandez de Sevilla, T. (2003). "Association between inflammatory mediators and the fibrinolysis system in infectious pleural effusions." Clinical Science 105 : 601-607.

Allen, B. J., Raja, C., Rizvi, S., Li, Y., Tsui, W., Zhang, D., Song, E., Qu, C. F., Kearsley, J., Graham, P. and Thompson, J. (2004). "Targeted alpha therapy for cancer." Physics in Medecine and Biology 49 : 3703-3712.

Allen, B. J., Rizvi, S., Li, Y., Tian, Z. and Ranson, M. (2001). "In vitro and preclinical targeted alpha therapy for melanoma, breast, prostate and colorectal cancers." Critical Reviews in Oncology/Hematology 39 : 139-146.

Allen, B. J., Tian, Z., Rizvi, S. M. A., Li, Y. and Ranson, M. (2003). "Preclinical studies of targeted α therapy for breast cancer using 213 Bi-labelled- plasminogen activator inhibitor type 2." British Journal of Cancer 88 : 944– 950.

149

Almeida-Vega, S., Catlow, K., Kenny, S., Dimaline, R. and Varro, A. (2009). "Gastrin activates paracrine networks leading to induction of PAI-2 via MAZ and ASC-1." American Journal of Physiology Gastrointestinal and Liver Physiology 296 : G414-423.

Andreasen, P. A., Egelund, R. and Petersen, H. H. (2000). "The plasminogen activation system in tumor growth, invasion, and metastasis." Cellular and Molecular Life Sciences 57 : 25-40.

Andreasen, P. A., Kjoller, L., Christensen, L. and Duffy, M. J. (1997). "The urokinase-type plasminogen activator system in cancer metastasis: a review." International Journal of Cancer 72 : 1-22.

Andreasen, P. A., Sottrup-Jensen, L., Kjoller, L., Nykjaer, A., Moestrup, S. K., Petersen, C. M. and Gliemann, J. (1994). "Receptor-mediated endocytosis of plasminogen activators and activator/inhibitor complexes." FEBS Letters 338 : 239-245.

Antalis, T. M., Clark, M. A., Barnes, T., Lehrbach, P. R., Devine, P. L., Schevzov, G., Goss, N. H., Stephens, R. W. and Tolstoshev, P. (1988). "Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor." Proceedings of the National Academy of Sciences U S A 85 : 985-989.

Antalis, T. M., Costelloe, E., Muddiman, J., Ogbourne, S. and Donnan, K. (1996). "Regulation of the plasminogen activator inhibitor type-2 gene in monocytes: localization of an upstream transcriptional silencer." Blood 88 : 3686-3697.

Antalis, T. M., La Linn, M., Donnan, K., Mateo, L., Gardner, J., Dickinson, J. L., Buttigieg, K. and Suhrbier, A. (1998). "The serine proteinase inhibitor (serpin) plasminogen activation inhibitor type 2 protects against viral cytopathic effects by constitutive interferon alpha/beta priming." The Journal of Experimental Medicine 187 : 1799-1811.

Argraves, K. M., Battey, F. D., MacCalman, C. D., McCrae, K. R., Gafvels, M., Kozarsky, K. F., Chappell, D. A., Strauss, J. F. and Strickland, D. K. (1995). "The very low density lipoprotein receptor mediates the cellular catabolism of lipoprotein lipase and urokinase-plasminogen activator inhibitor type 1 complexes." The Journal of Biological Chemistry 270 : 26550-26557.

Arnout, J., Hoylaerts, M. F. and Lijnen, H. R. (2006). "Haemostasis." Handbook of Experimental Pharmacology 176 : 1-41.

150

Arroyo De Prada, N., Schroeck, F., Sinner, E. K., Muehlenweg, B., Twellmeyer, J., Sperl, S., Wilhelm, O. G., Schmitt, M. and Magdolen, V. (2002). "Interaction of plasminogen activator inhibitor type-1 (PAI-1) with vitronectin." European Journal of Biochemistry 269 : 184-192.

Astedt, B., Bladh, B., Christensen, U. and Lecander, I. (1985). "Different inhibition of one and two chain tissue plasminogen activator by a placental inhibitor studied with two tripeptide-p-nitroanilide substrates." Scandinavian Journal of Clinical and Laboratory Investigation 45 : 429-435.

Astedt, B., Lecander, I. and Ny, T. (1987). "The placental type of plasminogen activator inhibitor, PAI-2." Fibrinolysis 1: 203-208.

Astedt, B., Lindoff, C. and Lecander, I. (1998). "Significance of the plasminogen activator inhibitor of placental type (PAI-2) in pregnancy." Seminars in Thrombosis and Hemostasis 24 : 431-435.

Australian Institute of Health and Welfare & Australasian Association of Cancer Registries 2010. Cancer in Australia: an overview, 2010. Cancer series no. 60. Cat. no. CAN 56. Canberra: AIHW.

Bajou, K., Maillard, C., Jost, M., Lijnen, R., Gills, A., Declerck, P., Carmeliet, P., Foidart, J.-M. and Noel, A. (2004). "Host-derived plasminogen activator inhibitor-1 (PAI-1) concentration is critcal for in vivo tumoral angiogenesis and growth." Oncogene 23 : 6986-6990.

Bansal, V. and Roychoudhury, P. K. (2006). "Production and purification of urokinase: A comprehensive review." Protein Expression and Purification 45 : 1-14.

Battey, F. D., Gafvels, M. E., FitzGerald, D. J., Argraves, W. S., Chappell, D. A., Strauss, J. F., 3rd and Strickland, D. K. (1994). "The 39-kDa receptor- associated protein regulates ligand binding by the very low density lipoprotein receptor." The Journal of Biological Chemistry 269 : 23268-23273.

Behrendt, N. (2004). "The urokinase receptor (uPAR) and the uPAR-associated protein (uPARAP/Endo180): membrane proteins engaged in matrix turnover during tissue remodelling." Biological Chemistry 385 : 103-136.

Behrendt, N. and Stephens, R. W. (1998). "The urokinase receptor." Fibrinolysis & Proteolysis 12 : 191-204.

Belin, D., Wohlwend, A., Schleuning, W. D., Kruithof, E. K. O. and Vassalli, J. D. (1989). "Facultative polypeptide translocation allows a single mRNA to encode the secreted and cytosolic forms of plasminogen activators inhibitor 2." The EMBO Journal 8: 3287-3294.

Bergers, G. and Benjamin, L. E. (2003). "Tumorigenesis and the angiogenic switch." Nature Reviews Cancer 3: 401-410.

151

Berkenpas, M. B., Lawrence, D. A. and Ginsburg, D. (1995). "Molecular evolution of plasminogen activator inhibitor-1 functional stability." The EMBO Journal 14 : 2969-2977.

Bertram, J. S. (2000). "The molecular biology of cancer." Molecular Aspects of Medicine 21 : 167-223.

Biessen, E. A., van Teijlingen, M., Vietsch, H., Barrett-Bergshoeff, M. M., Bijsterbosch, M. K., Rijken, D. C., van Berkel, T. J. and Kuiper, J. (1997). "Antagonists of the mannose receptor and the LDL receptor-related protein dramatically delay the clearance of tissue plasminogen activator." Circulation 95 : 46-52.

Bizik, J., Lizonová, A., Stephens, R. W., Grófová, M. and Vaheri, A. (1990). "Plasminogen activation by t-PA on the surface of human melanoma cells in the presence of alpha2-macroglobulin secretion." Cell Regulation 1: 895-905.

Blasi, F. (1999). "The urokinase receptor. A cell surface, regulated chemokine." Acta Pathologica, Microbiologica et Immunologica Scandinavica 107 : 96-101.

Blasi, F. and Sidenius, N. (2010). "The urokinase receptor: focused cell surface proteolysis, cell adhesion and signaling." FEBS Letters 584 : 1923-1930.

Booth, N. A., Reith, A. and Bennett, B. (1988). "A plasminogen activator inhibitor (PAI-2) circulates in two molecular forms during pregnancy." Journal of Thrombosis and Haemostasis 59 : 77-79.

Borgfeldt, C., Bendahl, P. O., Ferno, M. and Casslen, B. (2003). "High preoperative plasma concentration of tissue plasminogen activator (tPA) is an independent marker for shorter overall survival in patients with ovarian cancer." Gynecologic Oncology 91 : 112-117.

Borstnar, S., Vrhovec, I. and Cufer, T. (2002). "Prognostic value of plasminogen activator inhibitors in breast cancer." The International Journal of Biological Markers 17 : 96-103.

Borstnar, S., Vrhovec, I., Svetic, B. and Cufer, T. (2002). "Prognostic value of the urokinase-type plasminogen activator, and its inhibitors and receptor in breast cancer patients." Clinical Breast Cancer 3: 138-146.

Bouchet, C., Hacene, K., Martin, P. M., Becette, V., Tubiana-Hulin, M., Lasry, S., Oglobine, J. and Spyratos, F. (1999). "Dissemination risk index based on plasminogen activator system components in primary breast cancer." Journal of Clinical Oncology 17 : 3048-3057.

Bouchet, C., Spyratos, F., Martin, P. M., Hacene, K., Gentile, A. and Oglobine, J. (1994). "Prognostic value of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitors PAI-1 and PAI-2 in breast carcinomas." British Journal of Cancer 69 : 398-405.

152

Brabletz, T., Jung, A., Spaderna, S., Hlubek, F. and Kirchner, T. (2005). "Migrating cancer stem cells - an integrated concept of malignant tumor progression." Nature Reviews Cancer 5: 744-749.

Braungart, E., Magdolen, V. and Degitz, K. (2001). "Retinoic acid upregulates the plasminogen activator system in human epidermal keratinocytes." Journal of Investigative Dermatology 116 : 778-784.

Brenner, B. (2004). "Haemostatic changes in pregnancy." Thrombosis Research 114 : 409-414.

Brown, J. M., Watanabe, K., Cohen, R. L. and Chambers, D. A. (1995). "Molecular characterization of plasminogen activators in human gingival crevicular fluid." Archives of Oral Biology 40 : 839-845.

Brown, M. S., Herz, J. and Goldstein, J. L. (1997). "Calcium cages, acid baths and recycling receptors." Nature 388 : 629-630.

Broyl, A., Corthals, S. L., Jongen, J. L., van der Holt, B., Kuiper, R., de Knegt, Y., van Duin, M., el Jarari, L., Bertsch, U., Lokhorst, H. M., Durie, B. G., Goldschmidt, H. and Sonneveld, P. (2010). "Mechanisms of peripheral neuropathy associated with bortezomib and vincristine in patients with newly diagnosed multiple myeloma: a prospective analysis of data from the HOVON-65/GMMG-HD4 trial." Lancet Oncology 11 : 1057-1065.

Buechler, C., Ullrich, H., Ritter, M., Porsch-Oezcueruemez, M., Lackner, K. J., Barlage, S., Friedrich, S. O., Kostner, G. M. and Schmitz, G. (2001). "Lipoprotein (a) up-regulates the expression of the plasminogen activator inhibitor 2 in human blood monocytes." Blood 97 : 981-986.

Buyru, N., Altinisik, J., Gurel, C. B. and Ulutin, T. (2003). "PCR-RFLP detection of PAI-2 variants in myocardial infarction." Clinical and Applied Thrombosis Hemostasis 9: 333-336.

Camerer, E., Gjernes, E., Wiiger, M., Pringle, S. and Prydz, H. (2000). "Binding of factor VIIa to tissue factor on keratinocytes induces gene expression." The Journal of Biological Chemistry 275 : 6580-6585.

Carson, M., Johnson, D. H., McDonald, H., Brouillette, C. and DeLucas, L. J. (2007). "His-tag impact on structure." Acta Crystallographica Section D 63 : 295-301.

Castellino, F. J. and Ploplis, V. A. (2005). "Structure and function of the plasminogen/plasmin system." Thrombosis and Haemostasis 93 : 647-654.

Champelovier, P., Simon, A., Garrel, C., Levacher, G., Praloran, V. and Seigneurin, D. (2003). "Is interferon gamma one key of metastatic potential increase in human bladder carcinoma?" Clinical Cancer Research 9: 4562-4569.

153

Chang, W. S., Whisstock, J., Hopkins, P. C., Lesk, A. M., Carrell, R. W. and Wardell, M. R. (1997). "Importance of the release of strand 1C to the polymerization mechanism of inhibitory serpins." Protein Science 6: 89-98.

Chapman, H. A. (1997). "Plasminogen activators, integrins, and the coordinated regulation of cell adhesion and migration." Current Opinion in Cell Biology 9: 714-724.

Chen, T., Wu, F., Chen, F. M., Tian, J. and Qu, S. (2005). "Variations of very low- density lipoprotein receptor subtype expression in gastrointestinal adenocarcinoma cells with various differentiations." World Journal of Gastroenterology 11 : 2817-2821.

Chen, W.-J., Goldstein, J. L. and Brown, M. S. (1990). "NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor." The Journal of Biological Chemistry 265 : 3116-3123.

Choong, P. F. M. and Nadesapillai, A. P. W. (2003). "Urokinase plasminogen activator system: A multifunction role in tumor progression and metastasis." Cancer Orthopadedics and Related Research 415S : S46-S58.

Christofori, G. and Semb, H. (1999). "The role of the cell-adhesion molecule E- cadherin as a tumour-suppressor gene." Trends in Biochemical Sciences 24 : 73-76.

Chuang, T. L. and Schleef, R. R. (1999). "Identification of a nuclear targeting domain in the insertion between helices C and D in protease inhibitor-10." The Journal of Biological Chemistry 274 : 11194-11198.

Costelloe, E. O., Stacey, K. J., Antalis, T. M. and Hume, D. A. (1999). "Regulation of the plasminogen activator inhibitor-2 (PAI-2) gene in murine macrophages. Demonstration of a novel pattern of responsiveness to bacterial endotoxin." Journal of Leukocyte Biology 66 : 172-182.

Cousin, E., Medcalf, R. L., Bergonzelli, G. E. and Kruithof, E. K. (1991). "Regulatory elements involved in constitutive and phorbol ester-inducible expression of the plasminogen activator inhibitor type 2 gene promoter." Nucleic Acids Research 19 : 3881-3886.

Couzin, J. (2003). "Tracing the steps of metastasis, cancer’s menacing ballet." Science 299 : 1002-1006.

Croucher, D. R. (2006). Defining the mechanism and functional consequences of PAI- 2-mediated uPA/uPAR endocytosis. School of Biological Sciences . Wollongong, University of Wollongong. PhD thesis.

Croucher, D. R., Saunders, D. N., Lobov, S. and Ranson, M. (2008). "Revisiting the biological roles of PAI2 (SERPINB2) in cancer." Nature Reviews Cancer 8: 535-545.

154

Croucher, D. R., Saunders, D. N. and Ranson, M. (2006). "The urokinase/PAI-2 Complex: A new high affinity ligand for the endocytosis receptor low density lipoprotein receptor-related protein." The Journal of Biological Chemistry 281 : 10206-10213.

Croucher, D. R., Saunders, D. N., Stillfried, G. E. and Ranson, M. (2007). "A structural basis of differential signaling by PAI-1 and PAI-2 in breast cancer: Implications for metastatic potential." Biochemical Journal 408 : 203-210.

Csutak, A., Silver, D. M., Tozser, J., Steiber, Z., Bagossi, P., Hassan, Z. and Berta, A. (2008). "Plasminogen activator inhibitor in human tears after laser refractive surgery." Journal of Cataract & Refractive Surgery 34 : 897-901.

Czekay, R.-P. and Loskutoff, D. J. (2004). "Unexpected role of plasminogen activator inhibitor 1 in cell adhesion and detachment." Experimental Biology and Medicine 229 : 1090–1096.

D'Alessio, S. and Blasi, F. (2009). "The urokinase receptor as an entertainer of signal transduction." Frontiers in Bioscience 14 : 4575-4587.

Danø, K., Behrendt, N., Hoyer-Hansen, G., Johnsen, M., Lund, R. F., Ploug, M. and Romer, J. (2005). "Plasminogen activation and cancer." Thrombosis and Haemostasis 93 : 676-681.

Darnell, G. A., Antalis, T. M., Johnstone, R. W., Stringer, B. W., Ogbourne, S. M., Harrich, D. and Suhrbier, A. (2003). "Inhibition of retinoblastoma protein degradation by interaction with the serpin plasminogen activator inhibitor 2 via a novel consensus motif." Molecular and Cellular Biology 23 : 6520-6532.

Darnell, G. A., Antalis, T. M., Rose, B. R. and Suhrbier, A. (2005). "Silencing of integrated human papillomavirus type 18 oncogene transcription in cells expressing SerpinB2." The Journal of Virology 79 : 4246-4256.

De Taeye, B., Gils, A. and Declerck, P. J. (2004). "The story of the serpin plasminogen activator inhibitor 1: is there any need for another mutant?" Thrombosis and Haemostasis 92 : 898-924.

Dear, A. E. and Medcalf, R. L. (2000). "The novel anti-tumour agent oxamflatin differentially regulates urokinase and plasminogen activator inhibitor type 2 expression and inhibits urokinase-mediated proteolytic activity." Biochimica et Biophysica Acta 1492 : 15-22.

Dear, A. E., Shen, Y., Ruegg, M. and Medcalf, R. L. (1996). "Molecular mechanisms governing tumor-necrosis-factor-mediated regulation of plasminogen-activator inhibitor type-2 gene expression." European Journal of Biochemistry 241 : 93- 100.

155

Degryse, B., Neels, J. G., Czekay, R., Aertgeerts, K., Kamikubo, Y. and Loskutoff, D. J. (2004). "The low density lipoprotein receptor-related protein is a motogenic receptor for plasminogen activation inhibitor-1." The Journal of Biological Chemistry 279 : 22595-22604.

DeLano, W. L. (2002). The PyMOL molecular graphics system. San Carlos, CA, USA, DeLano Scientific.

Dellas, C. and Loskutoff, D. J. (2005). "Historical analysis of PAI-1 from its discovery to its potential role in cell motility and disease." Thrombosis and Haemostasis 93 : 631-640.

Dementiev, A., Dobo, J. and Gettins, P. G. W. (2006). "Active site distortion is sufficient for proteinase inhibition by serpins: Structure of the covalent complex of α1-proteinase inhibitor with porcine pancreatic elastase." The Journal of Biological Chemistry 281 : 3452-3457.

Deng, G., Royle, G., Wang, S., Crain, K. and Loskutoff, D. J. (1996). "Structural and functional analysis of the plasminogen activator inhibitor-1 binding motif in the somatomedin B domain of vitronectin." The Journal of Biological Chemistry 271 : 12716-12723.

Di Bernardo, M. C., Matakidou, A., Eisen, T. and Houlston, R. S. (2009). "Plasminogen activator inhibitor variants PAI-1 A15T and PAI-2 S413C influence lung cancer prognosis." Lung Cancer 65 : 237-241.

Di Giusto, D. A., Sutherland, A. P. R., Jankova, L., Harrop, S. J., Curmi, P. M. G. and King, G. C. (2005). "Plasminogen activator inhibitor-2 is highly tolerant to P8 residue substitution—Implications for serpin mechanistic model and prediction of nsSNP activities." Journal of Molecular Biology 353 : 1069– 1080.

Di, Y., Liu, Z., Tian, J., Zong, Y., Yang, P. and Qu, S. (2010). "TFPI or uPA-PAI-1 complex affect cell function through expression variation of type II very low density lipoprotein receptor." FEBS Letters 584 : 3469-3473.

Dickinson, J. L., Bates, E. J., Ferrante, A. and Antalis, T. M. (1995). "Plasminogen activator inhibitor type 2 inhibits tumor necrosis factor alpha-induced apoptosis: evidencefor an alternate biological function." The Journal of Biological Chemistry 270 : 27894-27904.

Dickinson, J. L., Norris, B. J., Jensen, P. H. and Antalis, T. M. (1998). "The C-D interhelical domain of the serpin plasminogen activator inhibitor-type 2 is required for protection from TNF-α induced apoptosis." Cell Death and Differentiation 5: 163-171.

Dougherty, K. M., Pearson, J. M., Yang, A. Y., Westrick, R. J., Baker, M. S. and Ginsburg, D. (1999). "The plasminogen activator inhibitor-2 gene is not required for normal murine development or survival." Proceedings of the National Academy of Sciences U S A 96 : 686-691.

156

Duffy, M. J. (2004). "The urokinase plasminogen activator system: role in malignancy." Current Pharmaceutical Design 10 : 39-49.

Duffy, M. J. and Duggan, C. (2004). "The urokinase plasminogen activator system: a rich source of tumor markers for the individualised management of patients with cancer." Clinical Biochemistry 37 : 541-548.

Duffy, M. J., Maguire, T. M., McDermott, E. W. and O'Higgins, N. (1999). "Urokinase plasminogen activator: A prognostic marker in multiple types of cancer." Journal of Surgical Oncology 71 : 130-135.

Duggan, C., Kennedy, S., Kramer, M. D., Barnes, C., Elvin, P., McDermott, E., O'Higgins, N. and Duffy, M. J. (1997). "Plasminogen activator inhibitor type 2 in breast cancer." British Journal of Cancer 76 : 622-627.

Dupont, D. M., Madsen, J. B., Kristensen, T., Bodker, J. S., Blouse, G. E., Wind, T. and Andreasen, P. A. (2009). "Biochemical properties of plasminogen activator-1." Frontiers in Bioscience 14 : 1337-1361.

Ehart, M., Koshelnick, Y., Stockinger, H. and Binder, B. R. (1998). "Interactions of uPAR: impact on receptor regulation and signal transduction." Fibrinolysis & Proteolysis 12 : 211-217.

Ehrlich, H. J., Gebbink, R. K., Keijer, J. and Pannekoek, H. (1992). "Elucidation of structural requirements on plasminogen activator inhibitor 1 for binding to heparin." The Journal of Biological Chemistry 267 : 11606-11611.

Einholm, A. P., Pedersen, K. E., Wind, T., Kulig, P., Overgaard, M. T., Jensen, J. K., Bodker, J. S., Christensen, A., Charlton, P. and Andreasen, P. A. (2003). "Biochemical mechanism of action of a diketopiperazine inactivator of plasminogen activator inhibitor-1." The Biochemical Journal 373 : 723-732.

Ellis, V., Whawell, S. A., Werner, F. and Deadman, J. J. (1999). "Assembly of urokinase receptor-mediated plasminogen activation complexes involves direct, non-active-site interactions between urokinase and plasminogen." Biochemistry 38 : 651-659.

Ellis, V., Wun, T. C., Behrendt, N., Ronne, E. and Dano, K. (1990). "Inhibition of receptor-bound urokinase by plasminogen-activator inhibitors." The Journal of Biological Chemistry 265 : 9904-9908.

Estreicher, A., Muhlhauser, J., Carpentier, J. L., Orci, L. and Vassalli, J. D. (1990). "The receptor for urokinase type plasminogen activator polarizes expression of the protease to the leading edge of migrating monocytes and promotes degradation of enzyme inhibitor complexes." The Journal of Cell Biology 111 : 783-792.

157

Fa, M., Bergström, F., Hägglöf, P., Wilczynska, M., Johansson, L. B. A. and Ny, T. (2000). "The structure of a serpin–protease complex revealed by intramolecular distance measurements using donor–donor energy migration and mapping of interaction sites." Structure 8: 397-405.

Feener, E. P., Northrup, J. M., Aiello, L. P. and King, G. L. (1995). "Angiotensin II induces plasminogen activator inhibitor-1 and -2 expression in vascular endothelial and smooth muscle cells." The Journal of Clinical Investigation 95 : 1353-1362.

Felez, J. (1998). "Plasminogen binding to cell surfaces." Fibrinolysis & Proteolysis 12 : 183-189.

Felez, J., Chanquia, C. J., Levin, E. G., Miles, L. A. and Plow, E. F. (1991). "Binding of tissue plasminogen activator to human monocytes and monocytoid cells." Blood 78 : 2318-2327.

Fidler, I. J. (2002). "Critical determinants of metastasis." Seminars in Cancer Biology 12 : 89-96.

Fish, R. J. and Kruithof, E. K. (2006). "Evidence for serpinB2-independent protection from TNF-alpha-induced apoptosis." Experimental Cell Research 312 : 350- 361.

Fisher, C., Beglova, N. and Blacklow, S. C. (2006). "Structure of an LDLR-RAP complex reveals a general mode for ligand recognition by lipoprotein receptors." Molecular Cell 22 : 277–283.

Foekens, J. A., Buessecker, F., Peters, H. A., Krainick, U., van Putten, W. L., Look, M. P., Klijn, J. G. M. and Kramer, M. D. (1995). "Plasminogen activator inhibitor-2: prognostic relevance in 1012 patients with primary breast cancer." Cancer Research 55 : 1423–1427.

Foekens, J. A., Peters, H. A., Look, M. P., Portengen, H., Schmitt, M., Kramer, M. D., Brunner, N., Janicke, F., Meijer-van Gelder, M. E., Henzen-Logmans, S. C., van Putten, W. L. and Klijn, J. G. (2000). "The urokinase system of plasminogen activation and prognosis in 2780 breast cancer patients." Cancer Research 60 : 636-643.

Foy, C. A. and Grant, P. J. (1997). "PCR-RFLP detection of PAI-2 gene variants: prevalence in ethnic groups and disease relationship in patients undergoing coronary angiography." Thrombosis and Haemostasis 77 : 955-958.

Gan, H., Newman, G. W. and Remold, H. G. (1995). "Plasminogen activator inhibitor type 2 prevents programmed cell death of human macrophages infected with Mycobacterium avium, serovar 4." The Journal of Immunology 155 : 1304- 1315.

158

Geiger, M., Zechmeister-Machhart, M., Uhrin, P., Hufnagl, P., Ecke, S., Priglinger, U., Xu, J., Zheng, X. and Binder, B. R. (1996). "Protein C inhibitor (PCI)." Immunopharmacology 32 : 53-56.

Genton, C., Kruithof, E. K. and Schleuning, W. D. (1987). "Phorbol ester induces the biosynthesis of glycosylated and nonglycosylated plasminogen activator inhibitor 2 in high excess over urokinase-type plasminogen activator in human U-937 lymphoma cells." The Journal of Cell Biology 104 : 705-712.

George, F., Pourreau-Schneider, N., Arnoux, D., Boutiere, B., Dussault, N., Roux- Dosseto, M., Alessi, M. C., Martin, P. M. and Sampol, J. (1990). "Modulation of tPA, PAI-1 and PAI-2 antigen and mRNA levels by EGF in the A431 cell line." Blood Coagulation & Fibrinolysis 1: 689-693.

Gettins, P. G. W. (2002). "Serpin structure, mechanism, and function." Chemical Reviews 102 : 4751-4803.

Gibson, C. S., MacLennan, A. H., Dekker, G. A., Goldwater, P. N., Dambrosia, J. M., Munroe, D. J., Tsang, S., Stewart, C. and Nelson, K. B. (2007). "Genetic polymorphisms and spontaneous preterm birth." Obstetrics & Gynecology 109 : 384-391.

Gliemann, J., Hermey, G., Nykjaer, A., Petersen, C. M., Jacobsen, C. and Andreasen, P. A. (2004). "The mosaic receptor sorLA/LR11 binds components of the plasminogen-activating system and platelet-derived growth factor-BB similarly to LRP1 (low-density lipoprotein receptor-related protein), but mediates slow internalization of bound ligand." Biochemical Journal 381 : 203- 212.

Goldfinger, L. E., Jiang, L., Hopkinson, S. B., Stack, M. S. and Jones, J. C. (2000). "Spatial regulation and activity modulation of plasmin by high affinity binding to the G domain of the alpha 3 subunit of laminin-5." The Journal of Biological Chemistry 275 : 34887-34893.

Gonias, S. L., Hembrough, T. A. and Sankovic, M. (2001). "Cytokeratin 8 functions as a major plasminogen receptor in select epithelial and carcinoma cells." Frontiers in Bioscience 6: D1403-1411.

Gooptu, B. and Lomas, D. A. (2009). "Conformational pathology of the serpins: themes, variations, and therapeutic strategies." Annual Review of Biochemistry 78 : 147-176.

Gould, A. R., Sharp, P. J., Smith, D. R., Stegink, A. J., Chase, C. J., Kovacs, J. C., Penglis, S., Chatterton, B. E. and Bunn, C. L. (2003). "Increased permeability of psoriatic skin to the protein, plasminogen activator inhibitor 2." Archives of Dermatological Research 295 : 249-254.

Gyetko, M. R., Shollenberger, S. B. and Sitrin, R. G. (1992). "Urokinase expression in mononuclear phagocytes: cytokine-specific modulation by interferon-gamma and tumor necrosis factor-alpha." Journal of Leukocyte Biology 51 : 256-263.

159

Gyetko, M. R., Wilkinson, C. C. and Sitrin, R. G. (1993). "Monocyte urokinase expression: modulation by interleukins." Journal of Leukocyte Biology 53 : 598-601.

Hall, S. W., Humphries, J. E. and Gonias, S. L. (1991). "Inhibition of cell surface receptor-bound plasmin by α2-antiplasmin and α2-macroglobulin." The Journal of Biological Chemistry 266 : 12329-12336.

Hall, T. A. (1999). "BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT." Nucleic Acids Symposium Series 41 : 95-98.

Hamaguchi, M., Morishita, Y., Takahashi, I., Ogura, M., Takamatsu, J. and Saito, H. (1991). "FDP D-dimer induces the secretion of interleukin-1, urokinase-type plasminogen activator, and plasminogen activator inhibitor-2 in a human promonocytic leukemia cell line." Blood 77 : 94-100.

Hamilton, J. A., Whitty, G. A., Stanton, H., Wojta, J., Gallichio, M., McGrath, K. and Ianches, G. (1993). "Macrophage colony-stimulating factor and granulocyte- macrophage colony-stimulating factor stimulate the synthesis of plasminogen- activator inhibitors by human monocytes." Blood 82 : 3616-3621.

Hamilton, J. A., Wojta, J., Gallichio, M., McGrath, K. and Filonzi, E. L. (1993). "Contrasting effects of transforming growth factor-beta and IL-1 on the regulation of plasminogen activator inhibitors in human synovial fibroblasts." The Journal of Immunology 151 : 5154-5161.

Hanahan, D. and Weinberg, R. A. (2000). "The hallmarks of cancer." Cell 100 : 57-70.

Hang, M. T. N., Ranson, M., Saunders, D. N., Liang, X.-M., Bunn, C. L. and Baker, M. S. (1998). "Pharmacokinetics and biodistribution of recombinant human plasminogen activator inhibitor type 2 (PAI-2) in control and tumour xenograft-bearing mice." Fibrinolysis & Proteolysis 12 : 145-154.

Hannocks, M. J., Oliver, L., Gabrilove, J. L. and Wilson, E. L. (1992). "Regulation of proteolytic activity in human bone marrow stromal cells by basic fibroblast growth factor, interleukin-1, and transforming growth factor beta." Blood 79 : 1178-1184.

Harris, L., Fritsche, H., Mennel, R., Norton, L., Ravdin, P., Taube, S., Somerfield, M. R., Hayes, D. F. and Bast, R. C. (2007). "American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer." Journal of Clinical Oncology 25 : 5287-5312.

Harrop, S. J., Jankova, L., Coles, M., Jardine, D., Whittaker, J. S., Gould, A. R., Meister, A., King, G. C., Mabbutt, B. C. and Curmi, P. G. M. (1999). "The crystal structure of plasminogen activator inhibitor 2 at 2.0 Å resolution: implications for serpin function." Structure 7: 43-54.

160

He, L., Lu, Y., Wang, P., Zhang, J., Yin, C. and Qu, S. (2010). "Up-regulated expression of type II very low density lipoprotein receptor correlates with cancer metastasis and has a potential link to beta-catenin in different cancers." BMC Cancer 10 : 601.

Heegaard, C. W., Simonsen, A. C. W., Oka, K., Kjdler, L., Christensen, A., Madsen, B., Ellgaard, L., Chan, L. and Andreasen, P. A. (1995). "Very low density lipoprotein receptor binds and mediates endocytosis of urokinase-type plasminogen activator-type-1 plasminogen activator inhibitor complex." The Journal of Biological Chemistry 270 : 20855-20861.

Hermey, G. (2009). "The Vps10p-domain receptor family." Cellular and Molecular Life Sciences 66 : 2677-2689.

Higuchi, R., Krummel, B. and Saiki, R. K. (1988). "A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions." Nucleic Acids Research 16 : 7351-7367.

Horn, I. R., van den Berg, B. M. M., Moestrup, S. K., Pannekoek, H. and van Zonneveld, A.-J. (1998). "Plasminogen activation inhibitor 1 contains a cryptic high affinity receptor binding site that is exposed upon complex formation with tissue-type plasminogen activator." Thrombosis and Haemostasis 80 : 822-828.

Horn, I. R., van den Berg, B. M. M., van der Meijden, P. Z., Pannekoek, H. and van Zonneveld, A.-J. (1997). "Molecular analysis of ligand binding to the second cluster of complement-type repeats of the low density lipoprotein receptor- related protein: Evidence for an allosteric component in receptor-associated protein-mediated inhibition of ligand binding." The Journal of Biological Chemistry 272 : 13608-13613.

Hoylaerts, M., Rijken, D. C., Lijnen, H. R. and Collen, D. (1982). "Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin." The Journal of Biological Chemistry 257 : 2912-2919.

Huai, Q., Mazar, A. P., Kuo, A., Parry, G. C., Shaw, D. E., Callahan, J., Li, Y., Yuan, C., Bian, C., Chen, L., Furie, B., Furie, B. C., Cines, D. B. and Huang, M. (2006). "Structure of human urokinase plasminogen activator in complex with its receptor." Science 311 : 656-659.

Huber, R. and Carrel, R. W. (1989). "Implications of the three-dimensional structure of alpha1-antitrypsin for structure and function of serpins." Perspectives in Biochemistry 28 : 8951-8966.

Hunt, B. J., Missfelder-Lobos, H., Parra-Cordero, M., Fletcher, O., Parmar, K., Lefkou, E. and Lees, C. C. (2009). "Pregnancy outcome and fibrinolytic, endothelial and coagulation markers in women undergoing uterine artery Doppler screening at 23 weeks." Journal of Thrombosis and Haemostasis 7: 955-961.

161

Huntington, J. A., Read, R. J. and Carrell, R. W. (2000). "Structure of a serpin- protease complex shows inhibition by deformation." Nature 407 : 923-926.

Hussain, M. M., Strickland, D. K. and Bakillah, A. (1999). "The mammalian low- density lipoprotein receptor family." Annual Review of Nutrition 19 : 141-172. Iijima, H., Miyazawa, M., Sakai, J., Magoori, K., Ito, M. R., Suzuki, H., Nose, M., Kawarabayasi, Y. and Yamamoto, T. T. (1998). "Expression and characterization of a very low density lipoprotein receptor variant lacking the O-linked sugar region generated by alternative splicing." The Journal of Biochemistry 124 : 747-755.

Innerarity, T. L. (2002). "LDL receptor's β-propeller displaces LDL." Science 298 : 2337-2338.

Irving, J. A., Pike, R. N., Lesk, A. M. and Whisstock, J. C. (2000). "Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function." Genome Research 10 : 1845–1864.

Ishikawa, N., Endo, Y. and Sasaki, T. (1996). "Inverse correlation between mRNA expression of plasminogen activator inhibitor-2 and lymph node metastasis in human breast cancer." Japanese Journal of Cancer Research 87 : 480-487.

Jang, W. G., Kim, H. S., Park, K. G., Park, Y. B., Yoon, K. H., Han, S. W., Hur, S. H., Park, K. S. and Lee, I. K. (2004). "Analysis of proteome and transcriptome of tumor necrosis factor alpha stimulated vascular smooth muscle cells with or without alpha lipoic acid." Proteomics 4: 3383-3393.

Jankova, L., Harrop, S. J., Saunders, D. N., Andrews, J. L., Bertram, K. C., Gould, A. R., Baker, M. S. and Curmi, P. M. (2001). "Crystal structure of the complex of plasminogen activator inhibitor 2 with a peptide mimicking the reactive center loop." The Journal of Biological Chemistry 276 : 43374-43382.

Jensen, G. A., Andersen, O. M., Bonvin, A. M. J. J., Bjerrum-Bohr, I., Etzerodt, M., Thøgersen, H. C., O'Shea, C., Poulsen, F. M. and Kragelund, B. B. (2006). "Binding site structure of one LRP–RAP complex: Implications for a common ligand–receptor binding motif." Journal of Molecular Biology 362 : 700-716.

Jensen, J. K., Durand, K. V., Skeldal, S., Dupont, D. M., Bødker, J. S., Wind, T. and Andreasen, P. A. (2004). "Construction of a plasminogen activator inhibitor-1 variant without measurable affinity to vitronection but otherwise normal." FEBS Letters 556 : 175-179.

Jensen, J. K., Wind, T. and Andreasen, P. A. (2002). "The vitronectin binding area of plasminogen activator inhibitor-1, mapped by mutagenesis and protection against an inactivating organochemical ligand." FEBS Letters 521 : 91-94.

Jensen, P. H., Jensen, T. G., Laug, W. E., Hager, H., Gliemann, J. and Pepinsky, B. (1996). "The exon 3 encoded sequence of the intracellular serine proteinase inhibitor plasminogen activator inhibitor 2 is a protein binding domain." Journal of Biological Chemistry 271 : 26892-26899.

162

Jensen, P. H., Schuler, E., Woodrow, G. C., Richardson, M., Goss, N. H., Hojrup, P., Petersen, T. E. and Ranmussen, L. K. (1994). "A unique interhelical insertion in plasminogen activator inhibitor-2 contains three glutamines, Gln83, Gln84, Gln86, essential for transglutaminase-mediated cross-linking." Journal of Biological Chemistry 269 : 15394-15398.

Kalluri, R. and Zeisberg, M. (2006). "Fibroblasts in cancer." Nature Reviews Cancer 6: 392-401.

Kanalas, J. J. and Makker, S. P. (1991). "Identification of the rat Heymann nephritis autoantigen (GP330) as a receptor site for plasminogen." The Journal of Biological Chemistry 266 : 10825-10829.

Kasza, A., Petersen, H. H., Heegaard, C. W., Oka, K., Christensen, A., Dubin, A., Chan, L. and Andreasen, P. A. (1997). "Specificity of serine proteinase/serpin complex binding to very-low-density lipoprotein receptor and aplha2- macroglobulin receptor/low-density-lipoprotein-receptor-related protein." European Journal of Biochemistry 248 : 270-281.

Katz, B. A., Sprengeler, P. A., Luong, C., Verner, E., Elrod, K., Kirtley, M., Janc, J., Spencer, J. R., Breitenbucher, J. G., Hui, H., McGee, D., Allen, D., Martelli, A. and Mackman, R. L. (2001). "Engineering inhibitors highly selective for the S1 sites of Ser190 trypsin-like serine protease drug targets." Chemistry & Biology 8: 1107-1121.

Kawano, T., Morimoto, K. and Uemura, Y. (1970). "Partial purification and properties of urokinase inhibitor from human placenta." Journal of Biological Chemistry (Tokyo) 67 : 333-342.

Keijer, J., Linders, M., Wegman, J. J., Ehrlich, H. J., Mertens, K. and Pannekoek, H. (1991). "On the target specificity of plasminogen activator inhibitor 1: The role of heparin, vitronectin, and the reactive site." Blood 78 : 1254-1261.

Kerppola, T. K. (2006). "Visualization of molecular interactions by fluorescence complementation." Nature Reviews Molecular Cell Biology 7: 449-456.

Kilpatrick, L. M., Harris, R. L., Owen, K. A., Bass, R., Ghorayeb, C., Bar-Or, A. and Ellis, V. (2006). "Initiation of plasminogen activation on the surface of monocytes expressing the type II transmembrane serine protease matriptase." Blood 108 : 2616 - 2623.

Kinnby, B. (2002). "The plasminogen activating system in periodontal health and disease." Biological Chemistry 383 : 85-92.

Kinnby, B., Lindberg, P., Lecander, I. and Matsson, L. (1999). "Localization of plasminogen activators and plasminogen-activator inhibitors in human gingival tissues demonstrated by immunohistochemistry and in situ hybridization." Archives of Oral Biology 44 : 1027-1034.

163

Kiso, U., Henschen, A., Bohn, H., Heimburger, N., Radtke, K. P., Lecander, I. and Astedt, B. (1991). "Identity between the placental protein PP10 and the specific plasminogen activator inhibitor of placental type PAI-2." Biochimica et Biophysica acta 1074 : 74-78.

Kjoller, L. (2002). "The urokinase plasminogen activator receptor in the regulation of the actin cytoskeleton and cell motility." Biological Chemistry 383 : 5-19.

Knoop, A., Andreasen, P. A., Andersen, J. A., Hansen, S., Laenkholm, A.-V., Simonsen, A. C. W., Andersen, J., Overgaard, J. and Rose, C. (1998). "Prognostic significance of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 in primary breast cancer." British Journal of Cancer 77 : 932-940.

Kobayashi, H., Schmitt, M., Goretzki, L., Chucholowski, N., Calvete, J., Kramer, M., Gunzler, W. A., Janicke, F. and Graeff, H. (1991). "Cathepsin B efficiently activates the soluble and the tumor cell receptor-bound form of the proenzyme urokinase-type plasminogen activator (Pro-uPA)." The Journal of Biological Chemistry 266 : 5147-5152.

Kounnas, M. Z., Henkin, J., Argraves, W. S. and Strickland, D. K. (1993). "Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor mediates cellular uptake of pro-urokinase." The Journal of Biological Chemistry 268 : 21862-21867.

Kruithof, E. K., Baker, M. S. and Bunn, C. L. (1995). "Biological and clinical aspects of plasminogen activator inhibitor type 2." Blood 86 : 4007-4024.

Kruithof, E. K., Tran-Thang, C., Gudinchet, A., Hauert, J., Nicoloso, G., Genton, C., Welti, H. and Bachmann, F. (1987). "Fibrinolysis in pregnancy: a study of plasminogen activator inhibitors." Blood 69 : 460-466.

Kruithof, E. K., Vassalli, J.-D., Schleuning, W.-D., Mattaliano, R. J. and Bachman, F. (1986). "Purification and characterization of a plasminogen activator inhibitor from the histiocytic lymphoma cell line U-937." The Journal of Biological Chemistry 261 : 11207-11213.

Kruithof, E. K. (1988). "Plasminogen activator inhibitors--a review." Enzyme 40 : 113-121.

Kruithof, E. K., Baker, M. S. and Bunn, C. L. (1995). "Biological and clinical aspects of plasminogen activator inhibitor type 2." Blood 86 : 4007-4024.

Kwaan, H. C. and McMahon, B. (2009). "The role of plasminogen-plasmin system in cancer." Cancer Treatment and Research 148 : 43-66.

Laug, W. E., Cao, X. R., Yu, Y. B., Shimada, H. and Kruithof, E. K. (1993). "Inhibition of invasion of HT1080 sarcoma cells expressing recombinant plasminogen activator inhibitor 2." Cancer Research 53 : 6051-6057.

164

Law, R. H. P., Zhang, Q., McGowan, S., Buckle, A. M., Silverman, G. A., Wong, W., Rosado, C. J., Langendorf, C. G., Pike, R. N., Bird, P. I. and Whisstock, J. C. (2006). "An overview of the serpin superfamily." Genome Biology 7: 216.

Lawrence, D. A., Berkenpas, M. B., Palaniappan, S. and Ginsburg, D. (1994). "Localization of vitronectin binding domain in plasminogen activator inhibitor-1." The Journal of Biological Chemistry 269 : 15223-15228.

Lecander, I. and Astedt, B. (1987). "Specific plasminogen activator inhibitor of placental type PAI 2 occurring in amniotic fluid and cord blood." Journal of Laboratory and Clinical Medicine 110 : 602-605.

Lecander, I., Martinsson, G., Casslén, B., Andreasen, P. A. and Åstedt, B. (1990). "Occurrence of the specific plasminogen activator inhibitor of placental type, PAI-2 in ascitic fluid and tumour vessel blood from patients with ovarian carcinoma." Fibrinolysis 4: 221-224.

Lee, J. A., Croucher, D. R. and Ranson, M. (2010). "Differential endocytosis of tissue plasminogen activator by serpins PAI-1 and PAI-2 on human peripheral blood monocytes." Journal of Thrombosis and Haemostasis 104 .

Leung, K. C., Byatt, J. A. and Stephens, R. W. (1987). "Poly-D-lysine dependent inactivation of tissue plasminogen activator by a class PAI-2 inhibitor (minactivin)." Thrombosis Research 46 : 767-777.

Leung, K. C., Byatt, J. A. and Stephens, R. W. (1987). "The resistance of fibrin- stimulated tissue plasminogen activator to inactivation by a class PAI-2 inhibitor (minactivin)." Thrombosis Research 46 : 755-766.

Li, Y., Marzolo, M. P., van Kerkhof, P., Strous, G. J. and Bu, G. (2000). "The YXXL motif, but not the two NPXY motifs, serves as the dominant endocytosis signal for low density lipoprotein receptor-related protein." The Journal of Biological Chemistry 275 : 17187-17194.

Li, Y., Rizvi, S. M., Ranson, M. and Allen, B. J. (2002). " 213 Bi-PAI2 conjugate selectively induces apoptosis in PC3 metastatic prostate cancer cell line and shows anti-cancer activity in a xenograft animal model." British Journal of Cancer 86 : 1197-1203.

Liang, J. F., Li, Y. and Yang, V. C. (2000). "The potential mechanism for the effect of heparin on tissue plasminogen activator-mediated plasminogen activation." Thrombosis Research 97 : 349-358.

Lillis, A. P., Mikhailenko, I. and Strickland, K. D. (2005). "Beyond endocytosis: LRP function in cell migration, proliferation and vascual permeability." Journal of Thrombosis and Haemostasis 3: 1884-1893.

165

Lin, L., Gardsvoll, H., Huai, Q., Huang, M. and Ploug, M. (2010). "Structure-based engineering of species selectivity in the interaction between urokinase and its receptor: implication for preclinical cancer therapy." The Journal of Biological Chemistry 285 : 10982-10992.

Lindahl, T. L., Sigurdardottir, O. and Wiman, B. (1989). "Stability of plasminogen activator inhibitor 1 (PAI-1)." Thrombosis and Haemostasis 62 : 748-751.

Lindberg, P., Baker, M. S. and Kinnby, B. (2001). "The localization of the relaxed form of plasminogen activator inhibitor type 2 in human gingival tissues." Histochemistry and Cell Biology 116 : 447-452. Ling, Q., Jacovina, A. T., Deora, A., Febbraio, M., Simantov, R., Silverstein, R. L., Hempstead, B., Mark, W. H. and Hajjar, K. A. (2004). "Annexin II regulates fibrin homeostasis and neoangiogenesis in vivo." The Journal of Clinical Investigation 113 : 38-48.

Liotta, L. A. (1984). "Tumor invasion and metastases: role of the basement membrane. Warner-Lambert Parke-Davis Award lecture." The American Journal of Pathology 117 : 339-348.

List, K., Jensen, O. N., Bugge, T. H., Lund, L. R., Ploug, M., Dano, K. and Behrendt, N. (2000). "Plasminogen-independent initiation of the pro-urokinase activation cascade in vivo. Activation of pro-urokinase by glandular kallikrein (mGK-6) in plasminogen-deficient mice." Biochemistry 39 : 508-515.

Liu, C. X., Li, Y., Obermoeller-McCormick, L. M., Schwartz, A. L. and Bu, G. (2001). "The putative tumor suppressor LRP1B, a novel member of the low density lipoprotein (LDL) receptor family, exhibits both overlapping and distinct properties with the LDL receptor-related protein." The Journal of Biological Chemistry 276 : 28889-28896.

Liu, Z., Li, H., Li, Y., Wang, Y., Zong, Y., Feng, Y., Feng, Z., Deng, Y. and Qu, S. (2009). "Up-regulation of VLDL receptor expression and its signaling pathway induced by VLDL and beta-VLDL." Journal of Huazhong University of Science and Technology [Med Sci] 29 : 1-7.

Ljungner, H., Holmberg, L., Kjeldgaard, A., Nilsson, I. M. and Astedt, B. (1983). "Immunological characterisation of plasminogen activators in the human vessel wall." Journal of Clinical Pathology 36 : 1046-1049.

Lobov, S., Croucher, D. R., Saunders, D. N. and Ranson, M. (2008). "Plasminogen activator inhibitor type 2 inhibits cell surface associated tissue plasminogen activator in vitro: potential receptor interactions." Thrombosis and Haemostasis 100 : 319-329.

Lobov, S., Wilczynska, M., Bergstro, F., Johansson, L. B. A. and Ny, T. (2004). "Structural bases of the redox-dependent conformational switch in the serpin PAI-2." Journal of Molecular Biology 344 : 1359–1368.

166

Look, M. P., van Putten, W. L. J., Duffy, M. J., Harbeck, N., Christensen, I. J., Thomssen, C., Kates, R., Spyratos, F., Fernö, M., Eppenberger-Castori, S., Sweep, C. G. J. F., Ulm, K., Peyrat, J.-P., Martin, P.-M., Magdelenat, H., Brünner, N., Duggan, C., Lisboa, B. W., Bendahl, P.-O., Quillien, V., Daver, A., Ricolleau, G., Meijer-van Gelder, M. E., Manders, P., Fiets, W. E., Blankenstein, M. A., Broët, P., Romain, S., Daxenbichler, G., Windbichler, G., Cufer, T., Borstnar, S., Kueng, W., Beex, L. V. A. M., Klijn, J. G. M., O’Higgins, N., Eppenberger, U., Jänicke, F., Schmitt, M. and Foekens, J. A. (2002). "Pooled analysis of prognostic impact of urokinase-type plasminogen activator and its inhibitor PAI-1 in 8377 breast cancer patients." Journal of the National Cancer Institute 94 : 116-128.

Lundgren, C. H., Sawa, H., Sobel, B. E. and Fujii, S. (1994). "Modulation of expression of monocyte/macrophage plasminogen activator activity and its implications for attenuation of vasculopathy." Circulation 90 : 1927-1934.

Luo, M., Liu, Y. J., Xia, L. M., Yan, W., Zhu, Q. and Tian, D. A. (2010). "Very low density lipoprotein receptor subtype II silencing by RNA interference inhibits cell proliferation in hepatoma cell lines." Hepatogastroenterology 57 : 882-890.

Mahony, D., Kalionis, B. and Antalis, T. M. (1999). "Plasminogen activator inhibitor type-2 (PAI-2) gene transcription requires a novel NF-kappaB-like transcriptional regulatory motif." European Journal of Biochemistry 263 : 765- 772.

Major, L., Schroder, W. A., Gardner, J., Fish, R. J. and Suhrbier, A. (2011). "Human papilloma virus transformed CaSki cells constitutively express high levels of functional SerpinB2." Experimental Cell Research 317 : 338-347.

Mareel, M. and Madani, I. (2006). "Tumour-associated host cells participating at invasion and metastasis: Targets for therapy?" Acta Chirurgica Belgica 106 : 635-640.

Maurer, F. and Medcalf, R. L. (1996). "Plasminogen activator inhibitor type 2 gene induction by tumor necrosis factor and phorbol ester involves transcriptional and post-transcriptional events. Identification of a functional nonameric AU- rich motif in the 3'-untranslated region." The Journal of Biological Chemistry 271 : 26074-26080.

Maurer, F., Tierney, M. and Medcalf, R. L. (1999). "An AU-rich sequence in the 3'- UTR of plasminogen activator inhibitor type 2 (PAI-2) mRNA promotes PAI- 2 mRNA decay and provides a binding site for nuclear HuR." Nucleic Acids Research 27 : 1664-1673.

May, A. E., Kanse, S. M., Chavakis, T. and Preissner, K. T. (1998). "Molecular interactions between the urokinase receptor and integrins in the vasculature." Fibrinolysis & Proteolysis 12 : 205-210.

167

May, P., Woldt, E., Matz, R. L. and Boucher, P. (2007). "The LDL receptor-related protein (LRP) family: An old family of proteins with new physiological functions." Annals of Medicine 39 : 219–228.

Mazar, A. P. (2008). "Urokinase plasminogen activator receptor choreographs multiple ligand interactions: implications for tumor progression and therapy." Clinical Cancer Research 14 : 5649-5655.

Mazzieri, R. and Blasi, F. (2005). "The urokinase receptor and the regulation of cell proliferation." Thrombosis and Haemostasis 93 : 641-646.

McCarthy, J. J., Parker, A., Salem, R., Moliterno, D. J., Wang, Q., Plow, E. F., Rao, S., Shen, G., Rogers, W. J., Newby, L. K., Cannata, R., Glatt, K. and Topol, E. J. (2004). "Large scale association analysis for identification of genes underlying premature coronary heart disease: cumulative perspective from analysis of 111 candidate genes." Journal of Medical Genetics 41 : 334-341.

McMahon, B. and Kwaan, H. C. (2008). "The plasminogen activator system and cancer." Pathophysiology of Haemostasis and Thrombosis 36 : 184-194.

Medcalf, R. L. (1992). "Cell- and gene-specific interactions between signal transduction pathways revealed by okadaic acid. Studies on the plasminogen activating system." The Journal of Biological Chemistry 267 : 12220-12226.

Medcalf, R. L., Kruithof, E. K. and Schleuning, W. D. (1988). "Plasminogen activator inhibitor 1 and 2 are tumor necrosis factor/cachectin-responsive genes." The Journal of Experimental Medicine 168 : 751-759.

Medcalf, R. L., Van den Berg, E. and Schleuning, W. D. (1988). "Glucocorticoid- modulated gene expression of tissue- and urinary-type plasminogen activator and plasminogen activator inhibitor 1 and 2." The Journal of Biological Chemistry 106 : 971-978.

Meijer-van Gelder, M. E., Look, M. P., Peters, H. A., Schmitt, M., Brunner, N., Harbeck, N., Klijn, J. G. and Foekens, J. A. (2004). "Urokinase-type plasminogen activator system in breast cancer: association with tamoxifen therapy in recurrent disease." Cancer Research 64 : 4563-4568.

Mengele, K., Napieralski, R., Magdolen, V., Reuning, U., Gkazepis, A., Sweep, F., Brunner, N., Foekens, J., Harbeck, N. and Schmitt, M. (2010). "Characteristics of the level-of-evidence-1 disease forecast cancer biomarkers uPA and its inhibitor PAI-1." Expert Review of Molecular Diagnostics 10 : 947-962.

Mignatti, P. and Rifkin, D. B. (1993). "Biology and biochemistry of proteinases in tumor invasion." Physiological Reviews 73 : 161-195.

Mikhailenko, I., Considine, W., Argraves, K. M., Loukinov, D., Hyman, B. T. and Strickland, D. K. (1999). "Functional domains of the very low density lipoprotein receptor: molecular analysis of ligand binding and acid-dependent ligand dissociation mechanisms." Journal of Cell Science 112 : 3269-2381.

168

Mikus, P. and Ny, T. (1996). "Intracellular polymerization of the serpin plasminogen activator inhibitor type 2." The Journal of Biological Chemistry 271 : 10048- 10053.

Mikus, P., Urano, T., Liljestrom, P. and Ny, T. (1993). "Plasminogen-activator inhibitor type 2 (PAI-2) is a spontaneously polymerising SERPIN. Biochemical characterisation of the recombinant intracellular and extracellular forms." European Journal of Biochemistry 218 : 1071-1082.

Moestrup, S. K., Nielsen, S., Andreasen, P., Jorgensen, K. E., Nykjaer, A., Roigaard, H., Gliemann, J. and Christensen, E. I. (1993). "Epithelial glycoprotein-330 mediates endocytosis of plasminogen activator-plasminogen activator inhibitor type-1 complexes." The Journal of Biological Chemistry 268 : 16564-16570.

Montemurro, P., Barbuti, G., Conese, M., Gabriele, S., Petio, M., Colucci, M. and Semeraro, N. (1999). "Retinoic acid stimulates plasminogen activator inhibitor 2 production by blood mononuclear cells and inhibits urokinase-induced extracellular proteolysis." British Journal of Haematology 107 : 294-299.

Moran, P., Li, W., Fan, B., Vij, R., Eigenbrot, C. and Kirchhofer, D. (2006). "Pro- urokinase-type plasminogen activator is a substrate for hepsin." The Journal of Biological Chemistry 281 : 30439-30446.

Muehlenweg, B., Guthaus, E., Arroyo De Prada, N., Schmitt, M., Schmiedeberg, N., Kotzsch, M., Creutzburg, S., Kramer, M. D., Kessler, H., Wilhelm, O. G. and Magdolen, V. (2000). "Epitope mapping of monoclonal antibodies directed to PAI-1 using PAI-1/PAI-2 chimera and PAI-1-derived synthetic peptides." Thrombosis Research 98 : 73-81.

Mueller, B. M., Yu, Y. B. and Laug, W. E. (1995). "Overexpression of plasminogen activator inhibitor 2 in human melanoma cells inhibits spontaneous metastasis in scid/scid mice." Proceedings of the National Academy of Sciences U S A 92 : 205-209.

Nakamura, M., Konno, H., Tanaka, T., Maruo, Y., Nishino, N., Aoki, K., Baba, S., Sakaguchi, S., Takada, Y. and Takada, A. (1992). "Possible role of plasminogen activator inhibitor 2 in the prevention of the metastasis of gastric cancer tissues." Thromb Research 65 : 709-719.

Nakashima, A., Kobayashi, T. and Terao, T. (1996). "Fibrinolysis during normal pregnancy and severe preeclampsia relationships between plasma levels of plasminogen activators and inhibitors." Gynecologic and Obstetric Investigation 42 : 95-101.

Nguyen, D. H., Catling, A. D., Webb, D. J., Sankovic, M., Walker, L. A., Somlyo, A. V., Weber, M. J. and Gonias, S. L. (1999). "Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner." Journal of Cell Biology 146 : 149-164.

169

Nielsen, L. S., Hansen, J. G., Skriver, L., Wilson, E. L., Kaltoft, K., Zeuthen, J. and Dano, K. (1982). "Purification of zymogen to plasminogen activator from human glioblastoma cells by affinity chromatography with monoclonal antibody." Biochemistry 21 : 6410-6415.

Nielsen, P. R., Ellgaard, L., Etzerodt, M., Thogersen, H. C. and Poulsen, F. M. (1997). "The solution structure of the N-terminal domain of alpha2-macroglobulin receptor-associated protein." Proceedings of the National Academy of Sciences U S A 94 : 7521-7525.

Niiya, K., Taniguchi, T., Shinbo, M., Ishikawa, T., Tazawa, S., Hayakawa, Y. and Sakuragawa, N. (1994). "Different regulation of plasminogen activator inhibitor 2 gene expression by phorbol ester and cAMP in human myeloid leukemia cell line PL-21." Thrombosis and Haemostasis 72 : 92-97. Noorman, F., Braat, E. A. and Rijken, D. C. (1995). "Degradation of tissue-type plasminogen activator by human monocyte-derived macrophages is mediated by the mannose receptor and by the low-density lipoprotein receptor-related protein." Blood 86 : 3421-3427.

Nozaki, S., Endo, Y., Kawashiri, S., Nakagawa, K., Yamamoto, E., Yonemura, Y. and Sasaki, T. (1998). "Immunohistochemical localization of a urokinase-type plasminogen activator system in squamous cell carcinoma of the oral cavity: association with mode of invasion and lymph node metastasis." Oral Oncology 34 : 58-62.

Nykjær, A., Kjoller, L., Cohen, R. L., Lawrence, D. A., Garni-Wagner, B. A., Todd, R. F., van Zonneveld, A. J., Gliemann, J. and Andreasen, P. A. (1994). "Regions involved in binding of urokinase-type-1 inhibitor complex and pro- urokinase to the endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Evidence that the urokinase receptor protects pro-urokinase against binding to the endocytic receptor." Journal of Biological Chemistry 269 : 25668-25676.

Ogbourne, S. M. and Antalis, T. M. (2001). "Characterisation of PAUSE-1, a powerful silencer in the human plasminogen activator inhibitor type 2 gene promoter." Nucleic Acids Research 29 : 3919-3927.

Ohba, K., Miyata, Y., Kanda, S., Koga, S., Hayashi, T. and Kanetake, H. (2005). "Expression of urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor and plasminogen activator inhibitors in patients with renal cell carcinoma: correlation with tumour associated macrophage and prognosis." Journal of Urology 174 : 461-465.

Ong, K., Horsfall, W., Conway, E. M. and Schuh, A. C. (2000). "Early embryonic expression of murine coagulation system components." Thrombosis and Haemostasis 84 : 1023-1030.

Ossowski, L. and Aguirre-Ghiso, J. A. (2000). "Urokinase receptor and integrin partnership: coordination of signaling for cell adhesion, migration and growth." Current Opinion in Cell Biology 12 : 613-620.

170

Padmanabhan, J. and Sane, D. C. (1995). "Localisation of a vitronectin binding region of plasminogen activation inhibitor-1." Thrombosis and Haemostasis 73 : 829- 834.

Palafox-Sanchez, C. A., Vazquez-Del Mercado, M., Orozco-Barocio, G., Garcia-De la Torre, I., Torres-Carrillo, N., Torres-Carrillo, N. M., Illades-Aguiar, B. and Munoz-Valle, J. F. (2009). "A functional Ser(413)/Ser(413) PAI-2 polymorphism is associated with susceptibility and damage index score in systemic lupus erythematosus." Clinical and Applied Thrombosis and Hemostasis 15 : 233-238.

Park, J. M., Greten, F. R., Wong, A., Westrick, R. J., Arthur, J. S., Otsu, K., Hoffmann, A., Montminy, M. and Karin, M. (2005). "Signaling pathways and genes that inhibit pathogen-induced macrophage apoptosis--CREB and NF- kappaB as key regulators." Immunity 23 : 319-329.

Petersen, H. H., Hansen, M., Schousboe, S. L. and Andreasen, P. A. (2001). "Localization of epitopes for monoclonal antibodies to urokinase-type plasminogen activator: Relationship between epitope localization and effects of antibodies on molecular interactions of the enzyme." European Journal of Biochemistry 268 : 4430-4439.

Picone, R., Kajtaniak, L. S., Nielsen, L. S., Behrendt, N., Mastronicola, M. R., Cubellis, M. V., Stoppelli, M. P., Pedersen, S., Dano, K. and Blasi, F. (1989). "Regulation of urokinase receptors in monocytelike U937 cells by phorbol ester myristate acetate." Journal of Cell Biology 108 : 693-702.

Piquette, G. N., Crabtree, M. E., el-Danasouri, I., Milki, A. and Polan, M. L. (1993). "Regulation of plasminogen activator inhibitor-1 and -2 messenger ribonucleic acid levels in human cumulus and granulosa-luteal cells." Journal of Clinical Endocrinology & Metabolism 76 : 518-523.

Ploug, M., Rønne, E., Behrendt, N., Picone, R. and Danø, K. (1991). "Cellular receptor for urokinase plasminogen activator." The Journal of Biological Chemistry 266 : 1926-1933.

Ponting, C. P., Marshall, J. M. and Cederholm-Williams, S. A. (1992). "Plasminogen: a structural review." Blood Coagulation and Fibrinolysis 3: 605-614.

Potempa, J., Korzus, E. and Travis, J. (1994). "The serpin superfamily of proteinase inhibitors: Structure, function, and regulation." The Journal of Biological Chemistry 269 : 15957-15960.

Praus, M., Wauterickx, K., Collen, D. and Gerard, R. D. (1999). "Reduction of tumor cell migration and metastasis by adenoviral gene transfer of plasminogen activator inhibitors." Gene Therapy 6: 227-236.

Preissner, K. T., Kanse, S. M. and May, A. E. (2000). "Urokinase receptor: a molecular organizer in cellular communication." Current Opinion in Cell Biology 12 : 621-628.

171

Pytel, B. A., Peppel, K. and Baglioni, C. (1990). "Plasminogen activator inhibitor type-2 is a major protein induced in human fibroblasts and SK-MEL-109 melanoma cells by tumor necrosis factor." Journal of Cellular Physiology 144 : 416-422.

Qu, C. F., Song, E. Y., Li, Y., Rizvi, S. M., Raja, C., Smith, R., Morgenstern, A., Apostolidis, C. and Allen, B. J. (2005). "Pre-clinical study of 213 Bi labeled PAI2 for the control of micrometastatic pancreatic cancer." Clinical & Experimental Metastasis 22 : 575-586.

Raghu, H., Sodadasu, P. K., Malla, R. R., Gondi, C. S., Estes, N. and Rao, J. S. (2010). "Localization of uPAR and MMP-9 in lipid rafts is critical for migration, invasion and angiogenesis in human breast cancer cells." BMC Cancer 10 : 647.

Ragno, P., Montuori, N. and Rossi, G. (1995). "Urokinase-type plasminogen activator/type-2 plasminogen-activator inhibitor complexes are not internalized upon binding to the urokinase-type plasminogen-activator receptor in THP-1 cells. Interaction of urokinase-type plasminogen activator/type-2 plasminogen activator inhibitor complexes with the cell surface." European Journal of Biochemistry 233 : 514-519.

Ranson, M. and Andronicos, N. M. (2003). "Plasminogen binding and cancer: Promises and pitfalls." Frontiers in Biosciences 8: s294-304.

Ranson, M., Andronicos, N. M., O'Mullane, M. J. and Baker, M. S. (1998). "Increased plasminogen binding is associated with metastatic breast cancer cells: differential expression of plasminogen binding proteins." British Journal of Cancer 77 : 1586-1597.

Ranson, M., Tian, Z., N.M., A., Rizvi, S. and Allen, B. J. (2002). " In vitro cytotoxicity of bismuth-213 ( 213 Bi)-labeled-plasminogen activator inhibitor type 2 (alpha-PAI-2) on human breast cancer cells." Breast Cancer Research and Treatment 71 : 149-159.

Rasch, M. G., Lund, I. K., Almasi, C. E. and Hoyer-Hansen, G. (2008). "Intact and cleaved uPAR forms: diagnostic and prognostic value in cancer." Frontiers in Bioscience 13 : 6752-6762.

Rebeck, G. W., LaDu, M. J., Estus, S., Bu, G. and Weeber, E. J. (2006). "The generation and function of soluble apoE receptors in the CNS." Molecular Neurodegeneration 1: 15.

Remold-O'Donnell, E. (1993). "The ovalbumin family of serpin proteins." FEBS Letters 315 : 105-108.

Resnati, M., Pallavicinim, I., Wang, J. M., Oppenheim, J., Serhan, C. N., Romano, M. and Blasi, F. (2002). "The fibrinolytic receptor for urokinase activates the G protein-coupled chemotactic receptor FPRL1/LXA4R." Proceedings of the National Academy of Sciences USA 99 : 1359-1364.

172

Rifkin, D. B. (1997). "Cross-talk among proteases and matrix in the control of growth factor action." Fibrinolysis & Proteolysis 11 : 3-9.

Rijken, D. C. and Collen, D. (1981). "Purification and characterization of the plasminogen activator secreted by human melanoma cells in culture." The Journal of Biological Chemistry 256 : 7035-7042.

Ritchie, H., Jamieson, A. and Booth, N. A. (1995). "Thrombin modulates synthesis of plasminogen activator inhibitor type 2 by human peripheral blood monocytes." Blood 86 : 3428-3435.

Ritchie, H., Jamieson, A. and Booth, N. A. (1997). "Regulation, location and activity of plasminogen activator inhibitor 2 (PAI-2) in peripheral blood monocytes, macrophages and foam cells." Thrombosis and Haemostasis 77 : 1168-1173.

Robbie, L. A., Dummer, S., Booth, N. A., Adey, G. D. and Bennett, B. (2000). "Plasminogen activator inhibitor 2 and urokinase-type plasminogen activator in plasma and leucocytes in patients with severe sepsis." British Journal of Haematology 109 : 342-348.

Roberts, T. H., Hejgaard, J., Saunders, N. F., Cavicchioli, R. and Curmi, P. M. (2004). "Serpins in unicellular Eukarya, Archaea, and Bacteria: sequence analysis and evolution." Journal of Molecular Evolution 59 : 437-447.

Robinson, N., Lapic, S., Welter, J. and Eckert, R. L. (1997). "S100A11, S100A10, Annexin I, Desmosomal Proteins, Small Proline-rich Proteins, Plasminogen Activator Inhibitor-2, and Involucrin Are Components of the Cornified Envelope of Cultured Human Epidermal Keratinocytes." The Journal of Biological Chemistry 272 : 12035-12046.

Rodenburg, K. W., Kjoller, L., Petersen, H. H. and Andreasen, P. A. (1998). "Binding of urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex to the endocytosis receptors alpha2 - macroglobulin receptor/low- density lipoprotein receptor-related and very-low-density lipoprotein receptor invloves basic residues in the inhibitor." Biochemical Journal 329 : 55-63.

Rudenko, G., Henry, L., Henderson, K., Ichtchenko, K., Brown, M. S., Goldstein, J. L. and Deisenhofer, J. (2002). "Structure of the LDL receptor extracellular domain at endosomal pH." Science 298 : 2353-2358.

Rusoslahti, E. (1996). "How cancer spreads." Scientific American 275 : 42-47.

173

Sadlonova, A., Bowe, D. B., Novak, Z., Mukherjee, S., Duncan, V. E., Page, G. P. and Frost, A. R. (2009). "Identification of molecular distinctions between normal breast-associated fibroblasts and breast cancer-associated fibroblasts." Cancer Microenvironment 2: 9-21.

Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning : a Laboratory Manual . Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press.

Samson, A. L., Nevin, S. T., Croucher, D., Niego, B., Daniel, P. B., Weiss, T. W., Moreno, E., Monard, D., Lawrence, D. A. and Medcalf, R. L. (2008). "Tissue- type plasminogen activator requires a co-receptor to enhance NMDA receptor function." Journal of Neurochemistry 107 : 1091-1101.

Sauer, B. and Henderson, N. (1988). "Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1." Proceedings of the National Academy of Sciences U S A 85 : 5166-5170.

Saunders, D. N., Buttigieg, K. M., Gould, A., McPhun, V. and Baker, M. S. (1998). "Immunological detection of conformational neoepitopes associated with the serpin activity of plasminogen activator inhibitor type-2." The Journal of Biological Chemistry 273 : 10965-10971.

Saunders, D. N., Jankova, L., Harrop, S. J., Curmi, P. M. G., Gould, A. R., Ranson, M. and Baker, M. S. (2001). "Interaction between the P14 residue and strand 2 of the beta-sheet B is critical for the reactive center loop insertion in plasminogen activator inhibitor-2." The Journal of Biological Chemistry 276 : 43383-43389.

Scherrer, A., Kruithof, E. K. and Grob, J. (1991). "Plasminogen Activator Inhibitor-2 in Patients with Monocytic Leukemia." Leukemia 5: 470-486.

Scherrer, A., Wohlwend, A., Kruithof, E. K. O., Vassalli, J. D. and Sappino, A. P. (1999). "Plasminogen activation in human acute leukaemias." British Journal of Haematology 105 : 920-927.

Schleuning, W. D., Medcalf, R. L., Hession, C., Rothenbuhler, R., Shaw, A. and Kruithof, E. K. (1987). "Plasminogen activator inhibitor 2: regulation of gene transcription during phorbol ester-mediated differentiation of U-937 human histiocytic lymphoma cells." Molecular Cell Biology 7: 4564-4567.

Schmitt, M., Wilhelm, O. G., Reuning, U., Krüger, A., Harbeck, N., Lengyel, E., Graeff, H., Gänsbacher, B., Kessler, H., Bürgle, M., Stürzebecher, J., Sperl, S. and Magdolen, V. (2000). "The urokinase plasminogen activator system as a novel target for tumour therapy." Fibrinolysis & Proteolysis 14 : 114-132.

Schmitt, M., Mengele, K., Napieralski, R., Magdolen, V., Reuning, U., Gkazepis, A., Sweep, F., Brunner, N., Foekens, J. and Harbeck, N. (2010). "Clinical utility of level-of-evidence-1 disease forecast cancer biomarkers uPA and its inhibitor PAI-1." Expert Review of Molecular Diagnostics 10 : 1051-1067.

174

Schroder, W. A., Le, T. T., Major, L., Street, S., Gardner, J., Lambley, E., Markey, K., Macdonald, K. P., Fish, R. J., Thomas, R. and Suhrbier, A. (2010). "A Physiological Function of Inflammation-Associated SerpinB2 Is Regulation of Adaptive Immunity." The Journal of Immunology 184 : 2663-2670.

Schuster, W. A., Medcalf, R. L. and Kruithof, E. K. (1993). "Retinoic acid potentiates phorbol ester-mediated induction of urokinase and plasminogen activator inhibitor type 2 in human myeloid leukemic cell lines." Endocrinology 133 : 1724-1730.

Schuster, W. A., Medcalf, R. L. and Kruithof, E. K. O. (1994). "Localization and characterisation of a retinoic acid response-like element in the plasminogen activator inhibitor-2 gene promoter." Fibrinolysis 8: 113-119.

Schwartz, B. S. and Bradshaw, J. D. (1992). "Regulation of plasminogen activator inhibitor mRNA levels in lipopolysaccharide-stimulated human monocytes. Correlation with production of the protein." The Journal of Biological Chemistry 267 : 7089-7094. Scicolone, G., Sanchez, V., Vauthay, L., Fuentes, F., Scicolone, A., Scicolone, L., Rapacioli, M. and Flores, V. (2006). "Tissue-type plasminogen activator activity in morphologically normal tissues adjacent to gastrointestinal carcinomas is associated with the degree of tumor progression." Journal of Cancer Research and Clinical Oncology 132 : 309-319.

Scott-Coombes, D., Whawell, S., Vipond, M. N. and Thompson, J. (1995). "Human intraperitoneal fibrinolytic response to elective surgery." British Journal of Surgery 82 : 414-417.

Scott, F. L., Eyre, H. J., Lioumi, M., Ragoussis, J., Irving, J. A., Sutherland, G. A. and Bird, P. I. (1999). "Human ovalbumin serpin evolution: phylogenic analysis, gene organization, and identification of new PI8-related genes suggest that two interchromosomal and several intrachromosomal duplications generated the gene clusters at 18q21-q23 and 6p25." Genomics 62 : 490-499.

Seo, E. Y., Piao, Y. J., Kim, J. S., Suhr, K. B., Park, J. K. and Lee, J. H. (2002). "Identification of calcium-induced genes in HaCaT keratinocytes by polymerase chain reaction-based subtractive hybridization." Archives of Dermatology 294 : 411-418.

Sharma, M., Ownbey, R. T. and Sharma, M. C. (2010). "Breast cancer cell surface annexin II induces cell migration and neoangiogenesis via tPA dependent plasmin generation." Experimental and Molecular Pathology 88 : 278-286.

Sharma, R. C. and Schimke, R. (1996). "Preparation of electro-competent E. coli using salt-free growth medium." Biotechniques 20 : 42-44.

Sharon, R., Abramovitz, R. and Miskin, R. (2002). "Plasminogen mRNA induction in the mouse brain after kainate excitation: codistribution with plasminogen activator inhibitor-2 (PAI-2) mRNA." Molecular Brain Research 104 : 170- 175.

175

Shioji, G., Ezura, Y., Nakajima, T., Ohgaki, K., Fujiwara, H., Kubota, Y., Ichikawa, T., Inoue, K., Shuin, T., Habuchi, T., Ogawa, O., Nishimura, T. and Emi, M. (2005). "Nucleotide variations in genes encoding plasminogen activator inhibitor-2 and serine proteinase inhibitor B10 associated with prostate cancer." Journal of Human Genetics 50 : 507-515.

Silverman, G. A., Bird, P. I., Carrell, R. W., Church, F. C., Coughlin, P. B., Gettins, P. G. W., Irving, J. A., Lomas, D. A., Luke, C. J., Moyer, R. W., Pemberton, P. A., Remold-O'Donnell, E., Salvesen, G. S., Travis, J. and Whisstock, J. C. (2001). "The serpins are an expanding superfamily of structurally similar but functionally diverse proteins." The Journal of Biological Chemistry 276 : 33293-33296.

Silverman, G. A. and Lomas, D. A. (2004). "Serpin identification, production, and characterization." Methods 32 : 71-72.

Silverman, G. A., Whisstock, J. C., Askew, D. J., Pak, S. C., Luke, C. J., Cataltepe, S., Irving, J. A. and Bird, P. I. (2004). "Human clade B serpins (ov-serpins) belong to a cohort of evolutionarily dispersed intracellular proteinase inhibitor clades that protect cells from promiscuous proteolysis." Cellular and Molecular Life Sciences 61 : 301-325.

Skeldal, S., Larsen, J. V., Pedersen, K. E., Petersen, H. H., Egelund, R., Christensen, A., Jensen, J. K., Gliemann, J. and Andreasen, P. A. (2006). "Binding areas of urokinase-type plasminogen activator–plasminogen activator inhibitor-1 complex for endocytosis receptors of the low-density lipoprotein receptor family, determined by site-directed mutagenesis." FEBS Journal 273 : 5143- 5159.

Smith, H. W. and Marshall, C. J. (2010). "Regulation of cell signalling by uPAR." Nature Reviews Molecular Cell Biolology 11 : 23-36.

Song, E. Y., Abbas Rizvi, S. M., Qu, C. F., Raja, C., Brechbiel, M. W., Morgenstern, A., Apostolidis, C. and Allen, B. J. (2007). "Pharmacokinetics and toxicity of (213)Bi-labeled PAI2 in preclinical targeted alpha therapy for cancer." Cancer Biology & Therapy 6: 898-904.

Song, Y. J., Qu, C. F., Rizvi, S. M., Li, Y., Robertson, G., Raja, C., Morgenstern, A., Apostolidis, C., Perkins, A. C. and Allen, B. J. (2006). "Cytotoxicity of PAI2, C595 and Herceptin vectors labeled with the alpha-emitting radioisotope Bismuth-213 for ovarian cancer cell monolayers and clusters." Cancer Letters 234 : 176-183.

Sporn, M. B. (1996). "The war on cancer." Lancet 347 : 1377–1381.

Spyratos, F., Bouchet, C., Tozlu, S., Labroquere, M., Vignaud, S., Becette, V., Lidereau, R. and Bieche, I. (2002). "Prognostic value of uPA, PAI-1 and PAI- 2 mRNA expression in primary breast cancer." Anticancer Research 22 : 2997- 3003.

176

Stassen, J. M., Arnout, J. and Deckmyn, H. (2004). "The hemostatic system." Current Medicinal Chemistry 11 : 2245-2260.

Stefansson, S., Kounnas, M. Z., Henkin, J., Mallampalli, R. K., Chappell, D. A., Strickland, D. K. and Argraves, W. S. (1995). "gp330 on type II pneumocytes mediates endocytosis leading to degradation of pro-urokinase, plasminogen activator inhibitor-1 and urokinase-plasminogen activator inhibitor-1 complex." Journal of Cell Science 108 : 2361-2368.

Stefansson, S., McMahon, G. A., Petitclerc, C. and Lawrence, D. A. (2003). "Plasminogen activator inhibitor-1 in tumor growth, angiogenesis and vascular remodeling." Current Pharmaceutical Design 9: 1545-1564.

Stefansson, S., Muhammad, S., Cheng, X., Battey, F. D., Strickland, D. K. and Lawrence, D. A. (1998). "Plasminogen activator inhibitor-1 contains a cryptic high affinity binding site for the low density lipoprotein receptor-related protein." The Journal of Biological Chemistry 273 : 6358-6366.

Steidl, C., Lee, T., Shah, S. P., Farinha, P., Han, G., Nayar, T., Delaney, A., Jones, S. J., Iqbal, J., Weisenburger, D. D., Bast, M. A., Rosenwald, A., Muller- Hermelink, H. K., Rimsza, L. M., Campo, E., Delabie, J., Braziel, R. M., Cook, J. R., Tubbs, R. R., Jaffe, E. S., Lenz, G., Connors, J. M., Staudt, L. M., Chan, W. C. and Gascoyne, R. D. (2010). "Tumor-associated macrophages and survival in classic Hodgkin's lymphoma." New England Journal of Medicine 362 : 875-885.

Steven, J., Cottingham, I. R., Berry, S. J., Chinery, S. A., Goodey, A. R., Courtney, M. and Ballance, D. J. (1991). "Purification and characterisation of plasminogen activator inhibitor 2 produced in Saccharomyces cerevisiae ." European Journal of Biochemistry 196 : 431-438.

Stillfried, G. E., Saunders, D. N. and Ranson, M. (2007). "Plasminogen binding and activation at the breast cancer cell surface: the integral role of urokinase activity." Breast Cancer Research 9: R14.

Stout, T. J., Graham, H., Buckley, D. I. and Matthews, D. J. (2000). "Structures of active and latent PAI-1: a possible stabilizing role for chloride ions." Biochemistry 39 : 8460-8469.

Strasser, V., Fasching, D., Hauser, C., Mayer, H., Bock, H. H., Hiesberger, T., Herz, J., Weeber, E. J., Sweatt, J. D., Pramatarova, A., Howell, B., Schneider, W. J. and Nimpf, J. (2004). "Receptor clustering is involved in Reelin signaling." Molecular and Cellular Biology 24 : 1378-1386.

Stratikos, E. and Gettins, P. G. W. (1999). "Formation of the covalent serpin- proteinase complex involves translocation of the proteinase by more than 70 Å and full insertion of the reactive center loop into beta-sheet A." Proceedings of the National Academy of Sciences U S A 96 : 4808-4813.

177

Strickland, D. K., Gonias, S. L. and Argraves, W. S. (2002). "Diverse roles for the LDL receptor family." Trends in Endocrinology & Metabolism 13 : 66-74.

Strickland, D. K. and Ranganathan, S. (2003). "Diverse roles of the LDL receptor- related protein in the clearance of proteases and in signalling." Journal of Thrombosis and Haemostasis 1: 1663–1670.

Stutchbury, T. K., Al-Ejeh, F., Stillfried, G. E., Croucher, D. R., Andrews, J., Irving, D., Links, M. and Ranson, M. (2007). "Preclinical evaluation of 213 Bi-labeled plasminogen activator inhibitor type 2 in an orthotopic murine xenogenic model of human breast carcinoma." Molecular Cancer Therapeutics 6: 203- 212.

Sumiyoshi, K., Serizawa, K., Urano, T., Takada, Y., Takada, A. and Baba, S. (1992). "Plasminogen activator system in human breast cancer." International Journal of Cancer 50 : 345-348.

Sutter, T. R., Guzman, K., Dold, K. M. and Greenlee, W. F. (1991). "Targets for dioxin: genes for plasminogen activator inhibitor-2 and interleukin-1 beta." Science 254 : 415-418.

Suwa, D., Konno, H., Tanaka, T. and Urano, T. (2008). "Intraperitoneal infusion of recombinant plasminogen activator inhibitor type 2 induced apoptosis in implanted human colon cancer and inhibited its growth and liver metastasis." Anticancer Res 28 : 693-698.

Suzuki, T., Hashimoto, S., Toyoda, N., Nagai, S., Yamazaki, N., Dong, H. Y., Sakai, J., Yamashita, T., Nukiwa, T. and Matsushima, K. (2000). "Comprehensive gene expression profile of LPS-stimulated human monocytes by SAGE." Blood 96 : 2584-2591.

Swartz, J. M., Bystrom, J., Dyer, K. D., Nitto, T., Wynn, T. A. and Rosenberg, H. F. (2004). "Plasminogen activator inhibitor-2 (PAI-2) in eosinophilic leukocytes." Journal of Leukocyte Biology 76 : 812-819.

Syrovets, T. and Simmet, T. (2004). "Novel aspects and new roles for the serine protease plasmin." Cellular and Molecular Life Sciences 61 : 873-885.

Tecimer, C., Doering, D. L., Goldsmith, L. J., Meyer, J. S., Abdulhay, G. and Wittliff, J. L. (2001). "Clinical relevance of urokinase-type plasminogen activator, its receptor, and its inhibitor type 1 in endometrial cancer." Gynecologic Oncology 80 : 48–55.

Thomas, G. D., Chappell, M. J., Dykes, P. W., Ramsden, D. B., Godfrey, K. R., Ellis, J. R. M. and Bradwell, A. R. (1989). "Effect of dose, molecular size, affinity, and protein binding on tumor uptake of antibody or ligand: A biomathematical model." Cancer Research 49 : 3290-3296.

178

Thorsen, S., Philips, M., Selmer, J., Lecander, I. and Astedt, B. (1988). "Kinetics of inhibition of tissue-type and urokinase-type plasminogen activator by plasminogen-activator inhibitor type 1 and type 2." European Journal of Biochemistry 175 : 33-39.

Tierney, M. J. and Medcalf, R. L. (2001). "Plasminogen activator inhibitor type 2 contains mRNA instability elements within exon 4 of the coding region. Sequence homology to coding region instability determinants in other mRNAs." The Journal of Biological Chemistry 276 : 13675-13684.

Tryggvason, K., Hoyhtya, M. and Salo, T. (1987). "Proteolytic degradation of extracellular matrix in tumor invasion." Biochimica et Biophysica acta 907 : 191-217.

Turner, N. and Grose, R. (2010). "Fibroblast growth factor signalling: from development to cancer." Nature Reviews Cancer 10 : 116-129.

Ulisse, S., Baldini, M., Toller, M., Marchioni, E., Giacomelli, L., De Antoni, E., Ferretti, E., Marzullo, A., Graziano, F. M., Trimboli, P., Biordi, L., Curcio, F., Gulino, A., Ambesi-Impiombato, F. S. and D'Armiento, M. (2006). "Differential expression of the components of the plasminogen activating system in human thyroid tumour derived cell lines and papillary carcinomas." European Journal of Cancer 42 : 2631-2638.

Umeda, T., Eguchi, Y., Okino, K., Kodama, M. and Hattori, T. (1997). "Cellular localization of urokinase-type plasminogen activator, its inhibitors, and their mRNAs in breast cancer tissues." The Journal of Pathol 183 : 388-397. van den Berg, E. A., le Clercq, E., Kooistra, T., Frants, R. R. and Bakker, E. (1990). "The human gene for plasminogen activator inhibitor 2 (PAI2) exhibits an EcoRI RFLP." Nucleic Acids Research 18 : 2837.

Varro, A., Hemers, E., Archer, D., Pagliocca, A., Haigh, C., Ahmed, S., Dimaline, R. and Dockray, G. J. (2002). "Identification of plasminogen activator inhibitor-2 as a gastrin-regulated gene: Role of Rho GTPase and menin." Gastroenterology 123 : 271-280.

Vazquez-Del Mercado, M., Garcia-Cobian, T. A., Munoz Valle, J. F., Torres-Carrillo, N., Martin-Marquez, B. T., Arana-Argaez, V. E., Best-Aguilera, C. R., Martinez-Garcia, E. A., Petri, M. H., Nunez-Atahualpa, L. and Delgado-Rizo, V. (2007). "Genotype Ser413/Ser of PAI-2 polymorphism Ser413/Cys is associated with anti-phospholipid syndrome and systemic lupus erythematosus in a familial case: comparison with healthy controls." Scandinavian Journal of Rheumatology 36 : 206-210.

Vine, K. L., Locke, J. M., Bremner, J. B., Pyne, S. G. and Ranson, M. (2010). "Selective targeting of 2'-deoxy-5-fluorouridine to urokinase positive malignant cells in vitro." Bioorganic & Medicinal Chemistry Letters 20 : 2908- 2911.

179

Violand, B. N. and Castellino, F. J. (1976). "Mechanism of the urokinase-catalyzed activation of human plasminogen." The Journal of Biological Chemistry 251 : 3906-3912.

Virtanen, O. J., Siren, V., Multanen, J., Farkkila, M., Leivo, I., Vaheri, A. and Koskiniemi, M. (2006). "Plasminogen activators and their inhibitors in human saliva and salivary gland tissue." European Journal of Oral Sciences 114 : 22- 26. von Heijne, G., Liljeström, P., Mikus, P., Andersson, H. and Ny, T. (1991). "The efficiency of the uncleaved secretion signal in the plasminogen activator inhibitor type 2 protein can be enhanced by point mutations that increased its hydrophobicity." The Journal of Biological Chemistry 266 : 15240-15243.

Wada, H., Kaneko, T., Wakita, Y., Minamikawa, K., Nagaya, S., Tamaki, S., Deguchi, K. and Shirakawa, S. (1994). "Effect of lipoproteins on tissue factor activity and PAI-II antigen in human monocytes and macrophages." International Journal of Cardiology 47 : S21-25.

Wang, Y. and Jensen, P. J. (1998). "Regulation of the level and glycosylation state of plasminogen activator inhibitor type 2 during human keratinocyte differentiation." Differentiation 63 : 93-99.

Webb, D. J., Nguyen, D. H. and Gonias, S. L. (2000). "Extracellular signal-regulated kinase functions in the urokinase receptor dependent pathway by which neutralization of low density lipoprotein receptor-related protein promotes fibrosarcoma cell migration and matrigel invasion." Journal of Cell Science 113 : 123-134.

Webb, D. J., Thomas, K. S. and Gonias, S. L. (2001). "Plasminogen activator inhibitor 1 functions as a urokinase response modifier at the level of cell signalling and thereby promotes MCF-7 cell growth." The Journal of Cell Biology 152 : 741- 752.

Weigelt, B., Peterse, J. L. and van't Veer, L. J. (2005). "Breast cancer metastasis: Markers and Models." Nature Reviews Cancer 5: 591-602.

Wever, O. D. and Mareel, M. (2003). "Role of tissue stroma in cancer cell invasion." Journal of Pathology 200 : 429–447.

Wilczynska, M., Lobov, S. and Ny, T. (2003). "The spontaneous polymerization of plasminogen activator inhibitor type-2 and Z-antitrypsin are due to different molecular aberrations." FEBS Letters 537 : 11-16.

Wilczynska, M., Lobov, S., Ohlsson, P.-I. and Ny, T. (2003). "A redox-sensitive loop regulates plasminogen activator inhibitor type 2 (PAI-2) polymerization." The EMBO Journal 22 : 1753-1761.

180

Wohlwend, A., Belin, D. and Vassalli, J. D. (1987). "Plasminogen activator-specific inhibitors produced by human monocytes/macrophages." Journal of Expermental Medicine 165 : 320-339.

Woodruff, P. G., Boushey, H. A., Dolganov, G. M., Barker, C. S., Yang, Y. H., Donnelly, S., Ellwanger, A., Sidhu, S. S., Dao-Pick, T. P., Pantoja, C., Erle, D. J., Yamamoto, K. R. and Fahy, J. V. (2007). "Genome-wide profiling identifies epithelial cell genes associated with asthma and with treatment response to corticosteroids." Proceedings of the National Academy of Sciences U S A 104 : 15858-15863.

Wun, T. C. and Reich, E. (1987). "An inhibitor of plasminogen activation from human placenta." The Journal of Biological Chemistry 262 : 3646-3653.

Xiao, Y. and Bartold, P. M. (2004). "Modulating effect of serum on the stimulation of plasminogen activator inhibitor 2 production in human gingival fibroblasts by lipopolysaccharide and interleukin-1beta." Journal of the International Academy of Periodontology 6: 81-88.

Xue, Y., Bjorquist, P., Inghardt, T., Linschoten, M., Musil, D., Sjolin, L. and Deinum, J. (1998). "Interfering with the inhibitory mechanism of serpins: crystal structure of a complex formed between cleaved plasminogen activator inhibitor type 1 and a reactive-centre loop peptide." Structure 6: 627-636.

Ye, R. D., Wun, T.-C. and Sadler, J. E. (1987). "cDNA cloning and expression in Escherichia coli of a plasminogen activator inhibitor from human placenta." The Journal of Biological Chemistry 262 : 3718-3752.

Yu, H., Maurer, F. and Medcalf, R. L. (2002). "Plasminogen activator inhibitor type 2: a regulator of monocyte proliferation and differentiation." Blood 99 : 2810- 2818.

Yu, H., Stasinopoulos, S., Leedman, P. and Medcalf, R. L. (2003). "Inherent instability of plasminogen activator inhibitor type 2 mRNA is regulated by tristetraprolin." The Journal of Biological Chemistry 278 : 13912-13918.

Zhang, S. J., Zou, M., Lu, L., Lau, D., Ditzel, D. A., Delucinge-Vivier, C., Aso, Y., Descombes, P. and Bading, H. (2009). "Nuclear calcium signaling controls expression of a large gene pool: identification of a gene program for acquired neuroprotection induced by synaptic activity." PLoS Genetics 5: e1000604.

Zhang, X. W., Sun, T., Zhou, X. F., Zeng, X. Y., Liu, X. and Gu, D. X. (1998). "Expression of human PAI-2 in the baculovirus expression system." Journal of Industrial Microbiology & Biotechnology 21 : 175-177.

Zhao, E. M., Han, D. M., Yu, Z. K., Fan, E. Z. and Li, Y. (2003). "[Expression and significance of the urokinase-type plasminogen activator and its inhibitors in squamous cell carcinoma of human larynx]." Zhonghua Er Bi Yan Hou Ke Za Zhi 38 : 39-42.

181

Zhou, A., Jiang, X., Dou, F., Zhu, D. and Xu, X. (1997). "Renaturation, purification, and characterization of human plasminogen activator inhibitor type 2 (PAI-2) accumulated at high level in Escherichia coli ." The Journal of Biochemistry 121 : 930-934.

Zhou, H. M., Bolon, I., Nichols, A., Wohlwend, A. and Vassalli, J. D. (2001). "Overexpression of plasminogen activator inhibitor type 2 in basal keratinocytes enhances papilloma formation in transgenic mice." Cancer Research 61 : 970-976.

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Appendix I: Buffers and solutions

TAE buffer , pH 8 40 mM Tris 1 mM EDTA 0.142% (v/v) glacial acetic acid

1% TAE agarose gel 1% (w/v) agarose 40 mM Tris 1 mM EDTA 0.142% (v/v) glacial acetic acid

Phosphate buffered saline (PBS), pH 7.4 12 mM Na 2HPO 4 2 mM NaH 2PO 4 2.7 mM KCl 137 mM NaCl

BIAcore running buffer , pH 7.4 10 mM HEPES 140 mM NaCl 1 mM CaCl 2 0.05% (v/v) Tween-20

Binding buffer , pH 7.4 0.98% (w/v) Hanks balanced salts 20 mM HEPES 1 mM CaCl 2 1 mM MgCl 2 0.1% (w/v) BSA

RIPA buffer , pH 7.4 50 mM HEPES 100 mM NaCl 1% (v/v) Nonidet P-40 2 mM EDTA 0.4 mg.mL -1 sodium orthovanadate 5 mg.mL -1 dithiothreitol 1 mL.L-1 protease inhibitor cocktail

BIAcore running buffer, pH 7.4 10 mM HEPES 150 mM NaCl 1 mM CaCl 2 0.005% Tween-20

Cell Acid Elution buffer , pH 3.0 50 mM glycine 0.15 M NaCl

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Cell Neutralising buffer , pH 7.5 0.5 M HEPES 0.1 M NaCl uPA activity assay reaction buffer , pH 7.6 20 mM HEPES 100 mM NaCl 0.5 mM EDTA 0.01% (v/v) Tween 20

Bacterial Culture Media Lauria-Bertani (LB) broth 1% (w/v) tryptone 0.5% (w/v) yeast extract 170 mM NaCl

LB agar 1% (w/v) tryptone 0.5% (w/v) yeast extract 170 mM NaCl 1.5% (w/v) agar

Z-broth 1% (w/v) tryptone 0.5% (w/v) yeast extract 170 mM NaCl 5.5 mM glucose 4.5 mM CaCl 2 YENB medium 0.75% (w/v) yeast extract 0.8% (w/v) tryptone

SOC medium 0.5% (w/v) yeast extract 2% (w/v) tryptone 10 mM NaCl 2.5 mM KCl 10 mM MgCl 2 10 mM MgSO 4 20 mM glucose

Wizard Plus SV Minipreps DNA Purification Cell resuspension solution 50 mM Tris-HCl (pH 7.5) 10 mM EDTA 100 µg.mL -1 RNase A

Cell lysis solution 0.2 M NaOH 1% (w/v) SDS

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Neutralisation solution 4.09 M guanidine hydrochloride 0.759 M potassium acetate 2.12 M glacial acetic acid Column wash solution 60 mM potassium acetate 8.3 mM Tris-HCl (pH 7.5) 0.04 mM EDTA (pH 8.0) 60% (v/v) ethanol

SDS-PAGE 5 ××× non-reducing sample buffer 225 mM Tris-Cl 20% (v/v) glycerol 2 mM SDS 0.02% (w/v) bromophenol blue

5 ××× reducing sample buffer 225 mM Tris-Cl 20% (v/v) glycerol 2 mM SDS 0.02% (w/v) bromophenol blue 250 mM DTT

4% stacking acrylamide gel 9% (v/v) 37.5:1 Bis:acrylamide 24.3% (v/v) 0.5 M tris-HCl (pH 6.8) 0.097% (w/v) SDS 0.0485% (w/v) APS 0.485% (v/v) TEMED

12% resolving acrylamide gel 30% (v/v) 37.5:1 Bis:acrylamide 24.3% (v/v) 1.5 M tris-HCl (pH 8.8) 0.097% (w/v) SDS 0.0485% (w/v) APS 0.485% (v/v) TEMED

SDS-PAGE running buffer 25 mM Tris 120 mM glycine 2 mM SDS pH 8.3

Coomassie staining solution 0.2% (w/v) coomassie blue R-250 40% (v/v) methanol 10% (v/v) glacial acetic acid

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Rapid destain 40% (v/v) methanol 10% (v/v) glacial acetic acid

Final destain 10% (v/v) glacial acetic acid 4% (v/v) glycerol

Western blotting Transfer buffer 25 mM Tris base 200 mM glycine 20% (v/v) methanol

TBS 50 mM Tris base 150 mM NaCl

TBST 50 mM Tris base 150 mM NaCl 0.05% (v/v) Tween 20

Immobilised metal affinity chromatography Loading buffer , pH 7.0 50 mM sodium phosphate 0.3 M NaCl 5 mM imidazole

Washing buffer , pH 7.0 50 mM sodium phosphate, 0.3 mM NaCl, 10 mM imidazole

Elution buffer , pH 7.0 50 mM sodium phosphate 0.3 mM NaCl 150 mM imidazole

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Appendix II: Plasmid maps pET15b/PAI-2 wild-type and pET15b/PAI-2 ∆CD-loop

Source: Novagen

187

Bam HI - 319 - G'GATC_C

ApR

PAI-2 wild-type (1808 bp) pET15b/PAI-2 wild-type 7556 bp Xho I - 2172 - C'TCGA_G

lacI

ApR

PAI-2 ∆CD-loop (1754 bp)

∆

lacI

188

pQE9/PAI-2 wildtype and pQE9/PAI-2 ∆CD-loop

Source: Qiagen

189

ApR PAI-2 wild-type (1251 bp)

Col E1

ApR PAI-2 ∆CD-loop (1152 bp)

∆

Col E1

190

TAGzyme pQE1/PAI-2 ∆CD-loop

Source: Qiagen

191

PvuII site lost

PAI-2 ∆CD-loop (1152 bp) ApR

∆

Col E1

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Appendix III: Construction of protein standard curve

Using molecular weight markers, standard curves were constructed to allow for the estimation of the size of proteins observed on SDS-PAGE gels. The standard curve constructed using the markers in Figure 3.5 is shown below (Table A.1 and Figure A.1).

Table A.1: Mobility of protein markers used for construction of a standard curve (Figure A.1). The data here is based on the markers present on the gel in Figure 3.5

Size (kDa) log 10 size Mobility (mm) 150 2.176091 11 100 2 19 75 1.875061 26 50 1.69897 32 37 1.568202 44 25 1.39794 56 20 1.30103 70 15 1.176091 84

2.5 y = -0.0135x + 2.2249 2.0 R2 = 0.9581

1.5

MW (kDa) 1.0 10

log 0.5

0.0 0 20 40 60 80 100 Mobility (mm)

Figure A.1: Standard curve constructed using protein markers from the SDS- PAGE gel in Figure 3.5

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Appendix IV: Construction of DNA standard curve

Using molecular weight markers, standard curves were constructed to allow for the estimation of the size of DNA bands observed on agarose gels. The standard curve constructed based on the markers in Figure 3.3 is shown below (Table A.2 and Figure A.2).

Table A.2: Mobility of Eco RI + HindI III λ DNA markers used for construction of a standard curve (Figure A.2). The data here is based on the markers present on the gel in Figure 3.3

Size (bp) log 10 size Mobility 5148 0.711639 31 4268 0.630224 34 2027 0.306854 50 1904 0.279667 52 1584 0.199755 56 1375 0.138303 59 947 -0.02365 68 831 -0.0804 74 564 -0.24872 81

4.0 y = -0.0188x + 4.2272 R2 = 0.9982 3.5 MW (kDa)

10 3.0 log

2.5 0 20 40 60 80 100 Mobility (mm)

Figure A.2: Standard curve constructed using Eco RI + Hin III λ DNA markers from the gel in Figure 3.3

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Appendix V: Sequencing of PAI-2 mutants To ensure successful mutagenesis of PAI-2 cDNA to generate the intended PAI-2 mutants, the full cDNA of all inserts generated was sequenced. The α-helix D and adjacent area of PAI-2 mutants is shown in Figure A.3.

295 360 ....|....|....|....|....|....|....|....|....|....|....|....|....|. PAI-2 GCTGCAGATAAAA TCC ATT CATCC TT CC GCTCTCTCAGCTCTGCAA TCAA TGCATCC ACAGGG AA T A A D K I H S S F R S L S S A I N A S T G N PAI-2K1 ...... A. G...... A A K K I H S S F R S L S S A I N A S T G N PAI-2K2 ...... AA G...... A A D K I H S S F R S L S K A I N A S T G N PAI-2KK ...... A. G...... AA G...... A A K K I H S S F R S L S K A I N A S T G N PAI-2YK ...... TATAA G...... A A D K I H S S F R S L Y K A I N A S T G N PAI-2KYK ...... A. G...... TATAA G...... A A K K I H S S F R S L Y K A I N A S T G N PAI-2YKK ...... TATAA G...... G A A D K I H S S F R S L Y K A I N A S T G K 99 120 Figure A.3: Alignment of mutagenised regions of PAI-2 α-helix D and adjacent residues used in this study. The full cDNAs of all mutants were sequenced to ensure that only the desired mutations were present. Region underlined denotes complete LDLR minimal binding motif.

195

Appendix VI: Preparation of BIAcore analysis surface In order to analyse the kinetics of the interactions between proteins used in this study, VLDLR or LRP1 were immobilised to BIAcore CM5 chips. The example shown here is for a VLDLR chip. The chip was activated with a 1:1 mixture of 0.2 M N-ethyl-N’ - (3-dimethylaminopropyl)carbodi-imide and 0.05 M N-hydroxysuccimide. For kinetic analysis a blank cell (Figure A.4A) and a VLDLR cell (Figure A.4B) were created. VLDLR was coated onto the chip at 40 µg/mL in 10 mM sodium acetate (pH 3) for 7 min at 5 µg/min. Un-occupied binding sites on both cells were blocked using 1 M ethanolamine, pH 8.5. Chips containing LRP1 were generated in a smiliar manner. A 30000 Ethanolamine blocking

20000 NHS Activation

10000 Response (RU) Response

0 0 500 1000 1500 Time (s)

B 40000

Ethanolamine blocking 30000

NHS Activation 20000

10000 VLDLR test VLDLR binding Response (RU) Response binding

0 0 500 1000 1500 2000 2500 Time (s)

Figure A.4: Construction of VLDLR chip for use in surface plasmon resonance . A blank flow cell to serve as a control (A) and a flow cell containing immobilised VLDLR (B) were generated for kinetic analysis.

196

Apendix VII: Optimsation of cell seeding density using the xCELLigenge system

Cells/well 8 5000 2500 6 1250 625 4 312 156 2 78 Relative Cell Index (CI) Index Cell Relative 0 0 10 20 30 40 Time (h)

Figure A.5: Optimisation of initial seeding cell density for examination of proliferative effects in MCF-7 cells. MCF-7 cells were seeded in E-plates at 5000, 2500, 1250, 625, 312, 156 or 78 cells per well and cultivated with RPMI/5% FCS over a 48 h period. Cell proliferation was monitored in real-time using an xCELLigence RTCA DP System. The electronic readout of cell sensor impedance is displayed continuously as the Cell Index (CI) normalised to the compound addition timepoint (indicated by vertical dotted line). The CI value at each time point is defined as (R n–Rb)/R b, where R n is the electrode impedance of wells with cells and R b is the background of wells with media alone. Curves are means of 4 replicates. Cell seeding at 5000 cells/well gave optimal logarithmic growth curves over an extended time frame and was chosen for further experiments.

197

Appendix VIII: Publications

198