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The Pennsylvania State University

The Graduate School

College of Medicine

MEPRIN METALLOPROTEASES MODULATE

INTESTINAL HOST RESPONSE

A Dissertation in

Biochemistry and Molecular Biology

Sanjita Banerjee

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

May 2008

The thesis of Sanjita Banerjee was reviewed and approved* by the following:

Judith S Bond Professor and Chair of Department of Biochemistry and Molecular Biology Thesis Adviser Chair of Committee

Kristin A. Eckert Professor of Biochemistry and Molecular Biology

Sergei A. Grigoryev Associate Professor of Biochemistry and Molecular Biology

Harriet C. Isom Distinguished Professor of Microbiology and Immunology

Cara-Lynne Schengrund Professor of Biochemistry and Molecular Biology

*Signatures are on file in the Graduate School

ii ABSTRACT Meprins, zinc metalloproteases of the astacin family, are composed of two independently expressed subunits meprin α and β which associate to form homo and heter-oligomers, termed meprin A and B. Meprin isoforms are enriched in human as well as rodent intestine. They have also been reported in kidney, leukocytes, lung and skin. Considerable research has been conducted regarding the in vitro functions of these proteases, however the physiological roles of these proteases are unknown. Generation of meprin β, α and αβ knock-out mice were steps towards filling this void. This work reports the general characterization of the meprin α knock-out mouse and investigates the role of meprin proteases in the mouse intestine using a model of colitis. Several polymorphisms have been identified in the human meprin α gene that significantly correlate with ulcerative colitis and Crohn’s disease, which make this mouse model relevant to the human pathology. Human and rodent intestines have high but non-uniform meprin expression and hence allow one to study the contributions of different meprin isoforms. To understand the role of meprin A, colitis was induced in wild-type and meprin α knock-out mice by dextran sulfate sodium administration. Meprin α knock-out mice showed greater susceptibility to injury and had a phenotype of heightened inflammation as evidenced by different markers of inflammation at macroscopic as well as molecular level. Further investigations showed a role for mucosal meprin A in the process of tissue repair. To understand the phenotype of increased inflammation, meprin interaction with immune-mediators was studied at greater detail. This led to the first known documentation of an in vivo interaction of meprins with an immune-mediator, interleukin–18. Subsequent experiments using meprin αβ knock-out mice elucidated the contribution of meprin B in the phenotype in this model of inflammatory bowel disease. These experiments collectively demonstrated a novel function of meprins in immuno-modulation. This thesis furthers our knowledge about the roles of meprin metalloproteases in the rodent intestine and also elucidates the importance of proper distribution of meprin isoforms.

iii TABLE OF CONTENTS List of Figures ix List of Tables xii List of Abbreviations xiii Acknowledgements xvi Chapter 1: Introduction 1 1.1 Overview 1 1.1.1 Proteases: In Life and Death 1 1.1.2 The story of Meprin – Metalloendopeptidase from Renal Tissue 3 1.1.2.1 Discovery of Meprin 3 1.1.2.2 Characterization of Meprin 4 1.1.2.3 Meprins’ Unique Oligomerization 4 1.1.2.4 A first attempt at elucidating the function 5 1.1.2.5 Meprin Classification 7 1.1.2.6 Meprin localization 9 1.1.2.7 Domains of Meprins 10

1.1.2.7.1 The NH2 & COOH Domains 10 1.1.2.7.2 The I Domain 10 1.1.2.7.3 The Interaction Domains 12 1.1.2.8 The higher order structure of meprins 14 1.1.2.9 Meprin Substrates 16 1.1.2.10 Meprins in Disease 18 1.1.2.11 In vivo studies 18 1.1.2.12 Meprin distribution in the intestine 19 1.1.2.13 Inflammatory Bowel Disease 20 1.1.2.14 Rationale for this thesis work 24

Chapter 2: Characterization of Meprin α Knockout Mice 26 2.1 Overview 26 2.2 Experimental Procedures 28 2.2.1 Generation and validation of αKO mice 28 2.2.2 Urine and serum analyses 30 2.2.3 End-Point PCR 30

iv 2.2.4 Immunoblotting of urinary samples 32 2.2.5 Immunohistochemistry 32 2.2.6 Ileum brush-border membrane preparation 33 2.2.7 Meprin β activity assay 33 2.2.8 Statistical Analysis 34 2.3 Results 34 2.3.1 Meprin αKO mice did not express meprin α mRNA or protein 34 2.3.2 General Parameters 34 2.3.3 Litter size 37 2.3.4 Meprin β mRNA and protein is unaffected in αKO mice 37 2.4 Discussion 42

Chapter 3: Delineating the role of meprin A in a Model of 44 Inflammatory Bowel Disease 3.1 Overview 44 3.2 Experimental Procedures 47 3.2.1 Induction of Experimental Colitis by DSS 47 3.2.2 Histological Scoring 48 3.2.3 Myeloperoxidase Assay 48 3.2.4 Collection of blood samples 49 3.2.5 Measurement of Serum Nitrite levels 49 3.2.6 FITC-Dextran Oral Gavage 50 3.2.7 Measurement of Colon and Serum Cytokines 50 3.2.8 Statistical Analysis 51 3.3 Results 51 3.3.1 Meprin αKO mice show greater susceptibility to 51 DSS-induced colitis 3.3.2 DSS-treatment causes greater colon injury in meprin αKO mice 55 3.3.3 Meprin αKO colons show heightened inflammation 58 3.3.4 Meprin A is not involved in maintaining the epithelial barrier 62 function 3.3.5 Mucosal meprin A plays a role in tissue repair and remodeling 62 3.3.6 DSS-induced colitis elicits greater systemic inflammation in 66

v αKO mice 3.4 Discussion 72

Chapter 4: Delving into the interaction of Meprins with Interleukin-18 78 4.1 Overview 78 4.2 Experimental Procedures 81 4.2.1 Construction of proIL-18 expression vector 81

4.2.1.1 Generation of pET30b::His6-proIL-18 81

4.2.1.2 Generation of pET28a::His6-proIL-18 82 4.2.2 Preparation of BL21(DE3)-RIL Competent Cells 84

4.2.3 Induction and purification of His6-proIL-18 84 4.2.4 Thrombin cleavage and proIL-18 purification 87 4.2.5 Activation of recombinant meprins 88 4.2.6 Meprin – proIL-18 reaction 88 4.2.7 Identification of IL-18 site of cleavage 89 4.2.8 Kinetic measurements 90 4.2.9 Meprin B and IL-18 interaction in MDCK cells 90 4.2.10 NF-κB activation in EL-4 cells by IL-18 91 4.2.11 ELISA of serum IL-18 91 4.2.12 Statistical Analysis 92 4.3 Results 92 4.3.1 Generation of recombinant murine proIL-18 92 4.3.2 Cleavage of proIL-18 by meprins 93 4.3.3 Biochemical characterization of proIL-18 cleavage by meprins 99 4.3.4 Identification of IL-18 cleavage site 99 4.3.5 Meprin B and proIL-18 interaction in a cell-culture system 102 4.3.6 Meprin B cleavage results in active IL-18 105 4.3.7 Corroboration of Meprin B – IL-18 interaction in vivo 105 4.4 Discussion 109

Chapter 5: Elucidating a role for meprin B in the DSS-induced 114 colitis model 5.1 Overview 114

vi 5.2 Experimental Procedures 115 5.2.1 Induction of Experimental Colitis by DSS 115 5.2.2 Colon myeloperoxidase assay 115 5.2.3 Collection of blood samples for serum nitrite measurement 115 5.2.4 Statistical Analysis 116 5.3 Results 116 5.3.1 Meprin αβKO mice show greater susceptibility to 116 DSS-induced colitis 5.3.2 Meprin αβKO do not exhibit a higher degree of inflammation 120 than WT 5.4 Discussion 124

Chapter 6: Conclusions and Discussion 128 6.1 Overview 128 6.1.1 Meprins: similar yet different from MMPs 128 6.1.2 Advancement of meprin knowledge 129 6.1.2.1 Meprin Redundancy 129 6.1.2.2 Meprins in the intestine 130 6.1.2.3 Meprins expressed by immune-mediators 133 6.1.3 Possible roles for meprins 137 6.1.3.1 Meprin distribution in different leukocytic populations 137 6.1.3.2 Meprin involvement in MΦ - T cell cross talk 138 6.1.3.3 Meprin signalling in innate immune response 139 6.2 Closing remarks 142

Appendix: Characterization of meprin α expression in a human 145 hepatocellular carcinoma cell-line, HepG2 A.1 Overview 145 A.2 Experimental Procedures 146 A.2.1 End-point PCR 146 A.2.2 Immunoblotting of HepG2 culture medium 146 A.2.3 HepG2 growth assay 147 A.2.4 Matrigel assay 147

vii A.3 Results 147 A.3.1 HepG2 cells over-express meprin α mRNA and protein 147 A.3.2 Meprin α inhibition does not affect HepG2 growth 148 A.3.3 HepG2 invasiveness unaffected by the loss of meprin α activity 148 A.4 Discussion 148

Bibliography 154

viii List of Figures Figure 1.1 Meprin metalloproteases show unique oligomerization 6 Figure 1.2 Schematic view of the catalytic centre of the metzincins 8 Figure 1.3 Domain structure of meprin subunits 11 Figure 1.4 Importance of different domains in the transport of meprin 13 subunits Figure 1.5 Multimeric structures of meprin metalloproteases 15 Figure 1.6 Differences in substrate bond specificities between the two 17 subunits Figure 1.7 Different mouse models of colitis and their utilities 22 Figure 2.1 Strategy for Mep1a gene disruption 31 Figure 2.2 Validation of meprin αKO mice at mRNA and protein level 35 Figure 2.3 The αKO and WT mice show comparable growth rates 38 Figure 2.4 Meprin αKO mice have smaller litters 40 Figure 2.5 Meprin β is unaffected in the αKO mice 41 Figure 3.1A Meprin αKO mice severely affected by four day DSS 52 treatment Figure 3.1B Meprin αKO mice have higher occult blood scores at day 7 53 Figure 3.1C Meprin αKO mice have higher DAI scores 54 Figure 3.2A Significant colon shortening seen in meprin αKO mice 56 on day 7 Figure 3.2B Meprin αKO mice show greater colon injury than the 57 WT mice Figure 3.2C Meprin αKO mice have higher colon injury scores 59 Figure 3.3 Higher inflammation and leukocyte infiltration seen in 60 αKO mice Figure 3.4 Colon cytokines in αKO mice significantly more elevated 64 than WT mice Figure 3.5 Lack of meprin A does not affect intestinal barrier function 65 Figure 3.6A Meprin αKO mice show slower recovery than WT mice 67 Figure 3.6B Absence of epithelial meprin A impedes tissue repair and 68 remodeling

ix Figure 3.7 Meprin αKO mice show higher systemic inflammation than 70 WT mice Figure 3.8 Meprin αKO mice show significant elevation of a few 74 cytokines after DSS-treatment Figure 4.1 IL-1β and IL-18 share structural similarities 80 Figure 4.2A Construction of pET30b::IL-18 83 Figure 4.2B Strategy for pET28a::IL-18 construction 85 Figure 4.2C pET28a::IL-18 clone confirmation by KpnI – XbaI digestion 86

Figure 4.3 Induction and purification of His6-proIL-18 from 94 pET30b::IL-18 Figure 4.4 Non-specific cleavage seen upon enterokinase treatment 95

Figure 4.5 Induction and purification of His6-proIL-18 from 96 pET28a::IL-18 Figure 4.6 Purification of proIL-18 97 Figure 4.7 Different isoforms of meprin process proIL-18 differently 98 Figure 4.8 Presence of actinonin can inhibit meprin B interaction with 100 proIL-18 Figure 4.9 Identification of proIL-18 cleavage site 103 Figure 4.10 Meprin B expressed in MDCK cells can process proIL-18 104 Figure 4.11 Meprin B cleavage results in IL-18 activation as measured 106 by NF-κB activation Figure 4.12A Active IL-18 levels in meprin βKO mice sera are significantly 107 lower Figure 4.12B Meprin αKO mice sera show significant elevation of IL-18 108 upon DSS treatment Figure 4.13 Scheme elucidating the differences in levels of active 112 IL-18 in the serum of WT and meprin KO mice Figure 5.1A Meprin αβKO mice show greater susceptibility to 117 DSS-induced colitis than WT mice Figure 5.1B Meprin αβKO have higher DAI scores 118 Figure 5.2 Significant colon shortening seen in meprin αβKO mice 119 on day 7 Figure 5.3 WT and meprin αβKO mice show comparable inflammation 121

x Figure 5.4 IL-6, the sole cytokine that shows significant elevation in 123 the αβKO mice Figure 5.5 Systemic inflammation in WT mice higher than that seen 125 in αβKO mice Figure 6.1 Absence of meprin A results in greater tissue damage 132 upon DSS treatment Figure 6.2 Meprin αKO mice colons show higher degree of damage 135 Figure 6.3 Meprins in the leukocyte modulate the inflammatory 136 environment by interacting with the immune mediators Figure 6.4 Modulation of inflammatory response by MΦ – T cell 140 cross-talk Figure 6.5 Possibilities of meprin involvement in the different steps of 141 MΦ – T cell cross-talk to modulate inflammation Figure 6.6 MyD88 involvement in TLR signalling 143 Figure A.1 HepG2 cells express increased levels of meprin α mRNA 149 Figure A.2 Meprin α protein detectable only in HepG2 culture medium 150 Figure A.3 Meprin α inhibition does not affect the growth rate of HepG2 151 Figure A.4 Lack of meprin α activity does not affect HepG2 invasiveness 152

xi List of Tables Table 1.1 Different Mouse Colitis Model Systems 23 Table 1.2 Comparison between DSS-colitis model and human UC 25 and CD Table 2.1 Mus musculus Vital Statistics 27 Table 2.2 Serum chemistry of WT and meprin αKO mice 36 Table 2.3 Genotypic distribution of meprin α+/- mating 39 Table 3.1 Major IBD loci and their position 45 Table 3.2 Colon cytokines are elevated upon DSS treatment, with 61 greater inflammation in αKO mice Table 3.3 A few serum cytokines show significant elevation in the αKO 71 mice upon DSS administration Table 4.1 Kinetic constant determination of proIL-18 cleavage 101 Table 5.1 Colon cytokines are elevated upon DSS treatment in both the 122 genotypes

xii List of Abbreviations AM : After MATH BK+ : 2-aminobenzoyl-R-P-P-G-F-S-P-F-R-K-(dinitrophenyl)-G-OH BUN : Blood Urea Nitrogen C : Cytosolic tail CD : Crohn’s disease CID : Collision Induced Dissociation CUB : Complement C1r/C1s, Uegf and Bone morphogenetic protein-1 DAI : Disease Activity Index DC : Dendritic Cells DD : Death Domain DMEM : Dublecco’s Modified Eagle’s Medium DMF : Dimethyl formamide DMSO : Dimethyl Sulfoxide DSS : Dextran Sulfate Sodium E : EGF-like domain ES cells : Embryonic Stem cells ECM : Extra-Cellular Matrix EDTA : Ethylenediamine Tetraacetic Acid EGF : Epidermal Growth Factor EGFR : Epithelial growth factor receptor ELISA : Enzyme Linked Immunosorbent Assay ER : Endoplasmic Reticulum FBS : Fetal Bovine Serum FITC : Fluorescein Isothiocyanate Fmr : Fragile X mental retardation GAPDH : Glyceraldehyde-3-phosphate Dehydrogenase GM-CSF : Granulocyte Macrophage Colony Stimulating Factor GRP : Gastrin Releasing Peptide HGF : Hepatocyte Growth Factor HLA : Human Leukocyte Antigen HRP : Horseradish Peroxidase HTAB : Hexadecyltrimethyl-Ammonium Bromide

xiii I : Inserted domain IBD : Inflammatory Bowel Disease ICAM : Intercellular Adhesion Molecule ICE : IL-1β Converting Enzyme IEC : Intestinal Epithelial Cells IFNγ : Interferon IGIF : IFN-γ-Inducing Factor IL : Interleukin IL-18BP : IL-18 Binding Protein IL-18R : IL-18 Receptor IL-18Rβ : IL-18 Receptor β IL-18Rα : IL-18 Receptor α IRAK : IL-1 Receptor Associated Kinase KO : Knockout MAM : Meprin, A5 protein and protein Tyrosine Phosphatase μ MATH : Meprin and TRAF-Homology MBP : Mannan-Binding Protein MCP-1 : Monocyte Chemotactic Protein-1 MDCK : Madin-Darby Canine Kidney cells MEM : Minimum Essential Medium MHC : Major Histocompatibility Complex MIP-1α : Macrophage Inflammatory Protein-1α MMP : Matrix Metalloprotease MPO : Myeloperoxidase MyD88 : Myeloid Differentiation Primary Response Protein 88 MΦ : Macrophage NF-κB : Nuclear factor-kappa B NK : Natural Killer NMR : Nuclear Magnetic Resonance NO : Nitric Oxide NOS : Nitric Oxide Synthase nSPF : Nonspecific Pathogen Free OCK+ : 2-aminobenzoic acid-MGWM-DEIDK-2,4-dinitrophenyl-SG-OH

xiv PCR : Polymerase Chain Reaction PKC : Protein Kinase-C PMA : Phorbol 12-Myristate 13-Actetate PMN : Polymorphonuclear Leukocytes

PPH : N-Benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase PR-3 : Proteinase-3 PTH : Parathyroid Hormone RANTES : Regulated on Activation Normal T Cell Expressed and Secreted S : Signal sequence SNP : Single Nucleotide Polymorphism STI : Soybean Trypsin Inhibitor T : Transmembrane domain TAPI : Tumor necrosis factor A Proteinase Inhibitor TBS : Tris Buffered Saline TFA : Trifluoroacetic acid TGF-β : Transforming Growth Factor – β Th : T helper cell TIMP : Tissue Inhibitor of Metalloproteinase TIR : Toll/IL-1 Receptor TL1A : TNF-like Ligand 1A TLR : Toll-Like Receptors TNBS : Trinitrobenzene Sulfonic Acid TNF-α : Tumor Necrosis Factor-α TRAF : Tumor Necrosis Factor Receptor-Associated Factor Treg : Regulatory T cells UC : Ulcerative Colitis

xv Acknowledgements

Writing a doctoral thesis is a daunting task but effectively puts many things in perspective. Not only has this thesis reminded me of my many failed endeavours, it has also taken me along a nostalgic trip to my first experiments in this laboratory. This log book of six years makes me realise the enormous help and input of so many along the way without whom this would never have been possible. An expression of appreciation and thanks is necessary for the completion of my thesis. I am truly indebted to Judith Bond, my thesis adviser. Her encouragement and her confidence in me have been instrumental in building my confidence as a scientist. I am grateful for the scientific freedom that she gave me and letting me try out my ideas. I have had many stimulating and interesting discussions with Dr Bradley, science as well as non-science. While talking politics or philosophy has certainly been more invigorating, the many ideas that came out of our scientific conversations have taken this thesis miles ahead. I am thankful to my fellow lab members, past and present, for making the Bond lab a wonderful place to work in. Susan, Ryan, Christine, Xiaoli, Lourdes, Gail, Renee, Ge, John, Moige, Tim, Jialing…predecessors, colleagues and successors. Many thanks for their help, suggestions and inputs. Jennifer, for her help during her two under-graduation rotations. I wish to extend my thanks to Dr Leo Fitzpatrick from the Department of Pharmacology for his help with histology, Dr Jason Kim from the Department of Cellular and Molecular Physiology for letting me use his Bio-Imaging instrument, Bruce Stanley from the Penn State Mass-Spectrometry Core Facility for his help with mass-spectrometry and Kathy Griffin from the Flanagan lab for giving me the various vectors. I am also thankful to the Penn State College of Medicine Molecular Genetics Core for DNA sequencing and the Clawson lab in the Department of Pathology for the use of their microscope. My thanks go to the Biochemistry Office and the Graduate Student Office as well as Kathy Simon.

xvi To Dr Erwin Sterchi from the University of Berne and Dr Mike Henry from GlaxoSmithKline go my heartfelt appreciation and thanks for their valuable inputs in my thesis research. Ending on a personal note, my family without whom I would not have been where I am today. Ma and Baba…they tried to solve so many of my research problems without really understanding what I was talking about. Rinku…from being my tag-along sister to my very best friend. And Renjith, my greatest critic and truest friend.

xvii

Where the mind is without fear and the head is held high Where knowledge is free Where the world has not been broken up into fragments By narrow domestic walls Where words come out from the depth of truth Where tireless striving stretches its arms towards perfection Where the clear stream of reason has not lost its way Into the dreary desert sand of dead habit Where the mind is relentlessly led forward Into ever-widening thought and action Into that land of freedom, let humanity awake….

Adapted from “Where the Mind is Without Fear”, Rabindranath Tagore.

xviii Chapter 1: Introduction 1.1 Overview 1.1.1 Proteases: In Life and Death Enzymes that break peptide bonds between amino acids in a protein or a peptide are termed proteases. The genes encoding various proteases constitute about 2% of the genome in the case of human beings and 1-5% of the gene content of all organisms. Even though the proteases participate in several diverse processes, their catalytic mechanisms of action can be broadly categorized into 6 major classes which form the basis for their classification. To date, there are six major classes of proteases: serine, cysteine, aspartic acid, metallo, threonine and glutamic acid, the latter two included as late as in 1995 (Fenteany et al., 1995; Seemuller et al., 1995) and 2004 (Yabuki et al., 2004) respectively. Proteases are expressed within and at the surface of, as well as secreted from nearly all cell types; thereby functioning at a wide range of cellular and sub-cellular locations. Proteases are intimately involved in nearly every life process, from simple digestion to complicated regulatory networks. Life, from the beginning till its end, depends on proper functioning of proteases, and dysregulation of these enzymes leads to a myriad of diseases. Herein lies the importance of protease research. Proteases come into play right from the process of conception of life. Union of sperm and egg by fertilization initiates the process of new life. The mammalian egg is encased by a glycoprotein called the zona pellucida. For successful fertilization to occur, the sperm cell head, or acrosome, adheres to the zona pellucida. Subsequently the acrosomal reaction, which is an exocytotic event, causes the release of acrosomal components, which then interact with the zona pellucida ultimately resulting in fusion of the membranes followed by sperm entry (Bedford, 1998; Primakoff and Myles, 2002; Raven and Johnson, 1992; Wassarman, 1999). This penetration event is thought to occur via limited proteolysis with sperm proteases playing a major role. A collagenase-like peptidase, thought to play a role in this process, was identified in the mammaliam sperm as early as in 1973 (Koren and Milkovic, 1973). Since then several other proteases such as testase 1 and TESP5 have also been implicated in this process (Honda et al., 2002b; Zhu et al., 2001). Acrosin, an endopeptidase with trypsin-like cleavage specificity found in the acrosome, is one of the most well studied proteases (Klemm et al., 1990). The

1 collective and synchronized activity of these proteases is of paramount importance in successful embryo formation (Honda et al., 2002a; Primakoff and Myles, 2002). Proteases are also intricately involved in the processes of cell death, be it apoptosis or oncosis. Necrosis signifies the post-mortem changes associated with both types of cell-death even though it has been commonly used to denote non-apoptotic cell- death pathways including oncosis. First described by Kerr et al., apoptosis is one of the main types of programmed cell death, which involves a series of orchestrated biochemical events leading to characteristic cell morphology and death (Kerr et al., 1972). Apoptosis is characterized by activation of specific proteases, membrane- blebbing, nuclear and chromatin condensation followed by extensive nuclear fragmentation (Hockenbery, 1995; Steller, 1995). Caspases, a family of cysteine proteases, act as death messengers and play a major role in the apoptotic pathway (Danial and Korsmeyer, 2004). Lysosomes play an important role in this process by releasing an array of cystein cathepsins as well as aspartic protease cathepsin D (Stoka et al., 2007). Proteases of other families like proteasome (a threonine protease), granzyme B, thrombin and Omi (serine proteases) are also involved in the apoptotic pathway (Johnson, 2000; Moffitt et al., 2007; O'Connell et al., 2006). Oncosis, coined by von Recklinghausen more than a century back, includes “accidental” death processes and is seen when the cells are subjected to extreme insults. This results in chaotic breakdown of cellular integrity and the process is characterized by organelle and cytoplasmic swelling followed by blebbing and increased permeability (Boise and Collins, 2001; Majno and Joris, 1995). There is random nuclear fragmentation and significant plasma membrane damage (Chakrabarti et al., 2003; Matsubara et al., 1994). Cell death, either by apoptosis or oncosis, finally leads to necrosis which is signaled by irreversible changes in the nucleus as well as cytoplasm (Majno and Joris, 1995). Although caspase involvement is not thought to be an integral part of oncosis, caspase-1 dependant oncosis, known as pyrotosis, has been identified (Brennan and Cookson, 2000; Cookson and Brennan, 2001). Extensive studies have shown that activated calpains (cysteine proteases) are capable of mediating the process of oncosis (Chakrabarti and McClane, 2005; Denecker et al., 2001; Liu et al., 2004). Thus protease involvement is well-documented in the process of cellular death.

2 The above examples of involvement of proteases at two ends of the spectrum of life - origin of life and cell death - are intended to demonstrate the breadth of the gamut of functions carried out by proteases. Understanding the physiology of these proteins is not only interesting from the point of view of mere intellectual curiosity, but also has a practical importance. Research on proteases on the one hand gives us a better understanding of the functioning of our body, and on the other helps us in amending the system during dysfunction.

1.1.2 The story of Meprin – Metalloendopeptidase from Renal Tissue 1.1.2.1 Discovery of Meprin As is often the case in science, many of the discoveries and important advances come in the form of serendipities. During an analysis of proteolytic activity of diabetic mouse tissues, Bond et al. (1980) found that kidney samples showed elevated proteolytic activity towards azocasein (Bond, 1980). Further investigations led to the discovery of a novel renal endopeptidase which had a molecular mass of approximately 81,000 Da (Beynon et al., 1981). The active enzyme, when subjected to gel filtration, displayed a molecular mass of about 300,000Da and was assumed to be a tetramer. This glycoprotein showed activity at a basic pH range from 9 to 10, and was further classified as a metalloprotease as it was inhibited by metal chelators. Distinctive features such as lack of inhibition by phosphoramidon, an inhibitor of several metalloproteases, and a broad range of specifity excluded the probability of this being one of the already known renal metalloproteases (Beynon et al., 1981). In 1983, the renal protease, named meprin, was shown to be mouse strain-dependent. A cross between “meprin-sufficient” (later termed “high-meprin”) and “meprin-deficient” (“low-meprin”) strains of mice gave progeny which were meprin- sufficient indicating the trait for deficiency to be recessive (Beynon and Bond, 1983; Bond et al., 1983; Shannon et al., 1981). Very soon the location of meprin gene, Mep-1, was determined to be on chromosome 17 in mice, and linked to the major histocompatibility complex (MHC) (Bond et al., 1984; Reckelhoff et al., 1985; Reckelhoff et al., 1988).

3 1.1.2.2 Characterization of Meprin The following few years of meprin research was characterized by several interesting “meprin-facts” coming to light. Meprin specificity was studied in greater detail and suitable subtrate assays were generated in an effort to understand its ability to cleave a broad range of proteins (Butler et al., 1987; Wolz and Bond, 1990). In depth studies mapped the active site of meprin A. It was demonstrated that meprin A did not have strict requirements regarding residues adjacent to the cleavage site and multiple subsite interactions were involved in the cleavage reaction (Wolz et al., 1991). In concurrent studies in humans, N-Benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (PPH) was isolated from particulate fractions of intestine and it showed a high degree of similarity to meprin with regards to its size and presence of intersubunit disulphide bridges. Sterchi et al (1988) found that dimerization of PPH occurred in the rough endoplasmic reticulum giving rise to the possibility that this might affect its subsequent transport to the cell surface (Sterchi et al., 1988a; Sterchi et al., 1988b). In rats too, a closely related kidney brush-border membrane enzyme, endopeptidase-2, was discovered around this time; this enzyme showed close similarities not only to the mouse meprin, but also to the human PPH (Barnes et al., 1989; Stephenson and Kenny, 1988). This period of discovery was not without minor hitches though. Due to the similarities that endopeptidase-2 shared with P-substance degrading enzyme, there were arguments that these two were the same enzyme. This claim was strongly disputed and later refuted when meprin and endopeptidase-2 were found to be one and the same (Johnson and Hersh, 1992; Probert and Hanley, 1989).

1.1.2.3 Meprins’ Unique Oligomerization Meanwhile work on meprin-deficient strains of mice kidneys showed the existence of an altered gene product named meprin b, that was recognized by anti-meprin IgG, but had a different mobility from the then known 85 kDa meprin subunit (McKay et al., 1985). Meprin b was latent in vivo, and showed ‘meprin a like’ activity after trypsin activation. It was initially postulated that a mutation in the meprin gene or differential post- translational modification of the nascent protein gave rise to this altered gene product in the animals of low-meprin strain (Butler and Bond, 1988; Macadam et al., 1990). Very

4 soon though, meprin b was shown to be present in all strains of mice, irrespective of the levels of meprin a, and had activity against azocasein that was similar to meprin a. Therefore the possibility of meprin b being a novel gene product was suggested and was very soon confirmed (Macadam et al., 1990). Soon after this the individual subunits were named meprin α and β and the enzyme isoforms called meprin A and B. While meprin A was found in the urine, meprin B was not detected in rodent urine indicating differences in their processing (Flannery et al., 1991). To establish the connection between the relatively obscure meprin B and the by then extensively studied meprin A, the former was purified to homogeneity from mouse strains that did not express meprin A (C3H/He) and compared with meprin A (isolated from ICR mice kidney) using an array of biochemical techniques. While the two shared many similarities, distinct differences, including those in substrate specificity, confirmed the fact that meprin B was indeed a distinct enzyme (Kounnas et al., 1991). A major thrust then was elucidation of the subunit organization of these proteases. It was shown that meprin A and B form three distinct oligomeric complexes linked by disulphide bridges (Figure 1.1). Probing the oligomers with distinct antibodies to α and β subunits, it was found that the oligomers corresponded to α2 and β2 homooligomers and α2β2 heterotertramers (Gorbea et al., 1991). This finding made meprins novel in being endopeptidases with distinctive multimerization patterns.

1.1.2.4 A first attempt at elucidating meprin function A pertinent question facing meprin research was the function of this protease. Meprins were found in the urine of “high-meprin” mice. Based on this observation it was speculated that meprin action in the urine caused fragmentation of urinary proteins (Flannery et al., 1990a). At that point of research, presence of meprin was clearly established only in mice and rats, and hence it was suspected to have a role in sensory communication processes in rodents (Beynon et al., 1991; Beynon et al., 1996; Flannery et al., 1990b). Subsequent discovery of PPH in human intestine and the recognition that it was identical to meprin indicated a function that had to be broader in nature. Interestingly, differences were observed in meprin levels between the two sexes of mice. While the kidney meprin activity was similar in both the sexes, female mice showed

5 Meprin Subunits Meprin Oligomers Meprin A

Meprin B meprin β - - meprin α

secreted multimers membrane bound hetero-oligomers

Figure 1.1 Meprin metalloproteases show unique oligomerization Meprin metalloproteases are oligomers made up of two independently expressed subunits, meprin α and β. Homomeric meprin A is composed of multimers of meprin α subunits that are secreted into the lumen. Heteromeric meprin A is a membrane-bound tetramer comprised of meprin α and β subunits. Meprin B is entirely made up of meprin β dimers that are membrane-bound.

6 lower levels of meprin A than males by immunohistochemical techniques. Further differences were seen between the sexes with respect to meprin glycosylation (Stroupe et al., 1991).

1.1.2.5 Meprin Classification Soon after the different oligomeric structures of meprins were identified, the mouse and human enzymes were cloned and the amino acid sequences of the protease domains of meprins, PPH and astacin, a crayfish digestive protease were found to be markedly similar. While the mouse meprin A and human PPH shared 82% identity, 30% identity was found between meprin A and astacin as well as BMP-1. This led them to be grouped together as the “astacin family” (Dumermuth et al., 1991). Today meprins are classified as metalloproteases belonging to the clan MA of metalloproteases. By definition, a clan is a set of families in which all the peptidases have evolved from a single ancestor (Beynon and Bond, 2000). Clan MA is characterized by an HEXXH motif in which the two His residues are zinc ligands and the Glu has a catalytic function. This clan is further subdivided into two subclans depending on the nature of its third zinc ligand. Subclan MA(E) has Glu as the third ligand and is hence also known as “Glu-Zincins”. MA(M), or metzincins, have a His or Asp within the extended motif HEXXHXXGXXH/D. The conserved Gly is important for the β turn that brings all the three ligands together (Bode et al., 1993). Another difference between the two subclans is that while MA(E) is comprised of both exo- and endopeptidases, MA(M) is constituted only of endopeptidases. The metzincins also have a conserved β turn that underlies the active-site with a conserved Met residue, hence the structurally important “Met-turn” (Stocker et al., 1995). This turn provides the zinc ion and the three His residues at the catalytic centre with a hydrophobic environment (Bode et al., 1993) (Figure 1.2). Meprins, along with other endopeptidases of the MA(M) subclan are synthesized as inactive prescursors. In many of the members, the proenzyme remains inactive due to an interaction between a Cys residue in the propeptide and the catalytic zinc ion, thereby preventing binding of a water molecule (Van Wart and Birkedal-Hansen, 1990). However, this cysteine switch mechanism is not present in meprins. Instead, they have a catalytically important Tyr,

Figure 1.2 Schematic view of the catalytic centre of the metzincins Meprins, members of the metzincin superfamily, have Zn at their catalytic site. The conserved H-E-X-X-H-X-X-G-X-X-H motif is important for holding the Zn ion. Gly residue is involved in the providing β turn thereby bringing the third H ligand together with the other two. The characteristic “met-turn” provides the Zn ion and the three ligands at the catalytic centre a hydrophobic environment. The water molecule and the Tyr residue which, along with the three His residues, hold the active Zn ion in the trigonal bipyramidal coordination is also shown (Stocker and Bode, 1995).

8 two residues beyond the Met of the characteristic “Met-turn”. The Tyr residue along with the three His residues and a water molecule hold the catalytically active Zn in a trigonal bipyramidal coordination sphere (Bond and Beynon, 1995; Stocker et al., 1993). All the peptidases of the M12 family including meprins have either the cysteine switch (subfamily M12B or adamalysin) or the conserved Tyr (subfamily M12A or astacin) but not both. The M12 family is the second largest family in the superfamily MA(M). Crayfish astacin, the simplest enzyme of the M12A subfamily was the first protein from this family to be sequenced and since then its crystal structure has also been solved (Bode et al., 1992; Gomis-Ruth et al., 1993; Grams et al., 1996; Stocker et al., 1993; Titani et al., 1987). The smallest members of the astacin family, namely the crayfish astacin, teleostean choriolysin L and choriolysin H, have no domain C-terminal to the protease domain. Nearly all the other members, meprin included, contain one or more epidermal growth factor (EGF) and/or Complement C1r/C1s, Uegf and Bone morphogenetic protein-1 (CUB) domains. These interaction domains promote protein-protein interactions (Bond and Beynon, 1995).

1.1.2.6 Meprin localization In mouse the meprin α gene was localized to chromosome-17, indicating that the Mep-1 gene identified earlier corresponded to Mep-1a (Bond et al., 1984; Jiang and Beatty, 1997; Jiang et al., 1993). The meprin β gene in mouse was cloned next and this was localized to chromosome 18 (Gorbea et al., 1993). The study showed that even though α and β might have evolved from a common ancestor, they had moved apart significantly during the process of evolution as illustrated by localization on different chromosomes and differences in post-translational processing (Gorbea et al., 1993). In humans the MEP1A and MEP1B were found to be located on chromosomes 6p and 18q respectively (Bond et al., 1995; Jiang et al., 1995). Genomic structure analysis of these two proteins showed α to be composed of 14 exons, spanning 35kb, while β had 15 exons and a size of 27kb (Hahn et al., 2000; Jiang and Le, 2000).

1.1.2.7 Domains of Meprins

1.1.2.7.1 The NH2 & COOH Domains Continuing along the timeline of meprin research, the next milestone achieved was the cloning and sequencing of the entire mouse meprin α subunit and identification of the different domains of the protein. Presence of a signal peptide N-terminal to the protease domain was identified at this point. On the COOH end, there was an intervening region of unknown functional domain followed by an EGF-like domain and a putative transmembrane domain (Jiang et al., 1992). These discoveries marked the beginning of identification of various domains that made up the proteases called meprins (Figure 1.3). Probing for the meprin α gene in other organisms showed meprin α to be present in humans, primates and other mammals but not in yeast.

1.1.2.7.2 The I Domain Extensive research shed more light on the secretion of the α subunit. In rats as well as in humans, it was seen that the meprin α subunit was secreted into the cell culture medium as an inactive protein in spite of the presence of a transmembrane domain in the protein (Corbeil et al., 1993; Dumermuth et al., 1993; Grunberg et al., 1993). It was then found that when expressed individually, meprin α was secreted into the culture medium and meprin β was anchored at the membrane as a type-I transmembrane protein. Nevertheless when expressed together, α was found at the membrane bound to the β subunit via disulphide linkage (Johnson and Hersh, 1994; Marchand et al., 1994; Milhiet et al., 1994). Investigations into the reason behind these interesting differential expression patterns of the two subunits resulted in identification of the “I” or inserted domain in α. Meprin α was found to encode a 56-amino acid sequence N-terminal to the EGF-like domain that was not found in β. This domain was proteolytically processed in the pre- Golgi compartment that subsequently led to the secretion of the α subunit. The C- terminal tail of the α subunit was essential for mediating association with ER-machinery, helping in further processing of the C-terminal region (Hahn et al., 1997). A weak signal

10 A

S Pro Protease MAM TRAF I E T C

S Pro Protease MAM TRAF E T C

Figure 1.3 Domain structure of meprin subunits (A) Meprin α subunit is made up of a signal (S) sequence followed by the pro-domain. Protease domain has the catalytic centre followed by two interaction domains MAM and TRAF. The inserted (I) domain undergoes proteolytic processing that results in the loss of the subsequent domains: the EGF-like (E) domain, transmembrane (T) domain and cytosolic (C) tail. The mature meprin α therefore comprises only till its TRAF domain. (B) Meprin β subunit has the same domain composition as the α subunit with the lack of I domain. This ensures its retention of all the domains due to lack of any proteolytic processing.

11 for retention, that became strengthened upon oligomerisation led to its retention in the ER/cis-Golgi compartment for processing (Hengst and Bond, 2004). While a study suggested the enzyme responsible for this processing to be furin, an enzyme localized in Golgi apparatus, later studies confirmed that the processing was preGolgi and it was the I domain that enabled the processing without the requirement of furin or furin-like enzymes (Milhiet et al., 1995; Tang and Bond, 1998). Meprin β lacking the I domain retained its transmembrane domain and hence was expressed as a type I membrane protein (Marchand et al., 1995). The transmembrane domain and C-terminal tail in β also had an essential albeit different function. Retention of the tail was essential for proper transport of the protein from ER to Golgi suggesting interaction with a cargo protein (Litovchick et al., 1998). As clearly elucidated by Hengst et al (2004), the I domain of meprin α was sufficient of its ER/cis-Golgi processing, while the meprin β transmembrane domain resulted in rapid transport of the subunit from ER to Golgi (Figure 1.4). In humans though, instances of meprin β being released into the medium were detected. A 13-amino acid region was identified in the human meprin β that resulted in its processing. This shedding took place in trans-Golgi after complex glycosylation of the subunit (Lottaz et al., 1999a; Pischitzis et al., 1999). While O-linked glycosylation in this 13-amino acid region diminished meprin β cleavage, phorbol 12-myristate 13- actetate (PMA) mediated by activated protein kinase-C (PKC) enhances the ectodomain shedding (Hahn et al., 2003; Leuenberger et al., 2003).

1.1.2.7.3 The Interaction Domains Soon after, the remaining meprin domains were identified and named. The domain C- terminal to the protease domain was found to be conserved among functionally diverse group of receptors and was thought to act as an adhesion domain important for protein- protein interaction. It was named MAM (after meprin, A5 protein and protein tyrosine phosphatase μ) (Beckmann and Bork, 1993). The next domain to be identified was the tumour necrosis factor receptor-associated factor (TRAF). TRAFs were originally identified as intracellular proteins binding to the cytoplasmic domains of the TNF receptor family. The TRAFC domain of these proteins was found to share strong resemblance with the meprin domain C-terminal to the MAM domain and was hence

12 A B

C D

Figure 1.4 Importance of different domains in the transport of meprin subunits (A) WT meprin α is processed at its “I” or inserted domain which enables its subsequent secretion. (B) WT meprin β lacks I domain and is retained at the membrane. Meprin β requires its transmembrane domain and cytosolic tail (TC) for proper transportation along the secretory pathway. (C) The importance of the “I” domain is clearly elucidated using the meprin αΔI construct (meprin α subunit lacking the I domain) which shows retention in the ER/cis-Golgi compartment. (D) Meprin α subunit lacking the I domain but having merpin β transmembrane domain and cytosolic tail (meprin αΔI/βTC) mimics meprin β transportation. This shows the importance of the transmembrane domain and cytosolic tail (TC) of meprin β (adapted from Hengst and Bond, 2004).

13 named as MATH (meprin and TRAF-homology) domain. The MATH domain in the TRAF proteins functions in promoting interactions. Identification of yet another interaction domain shed more light on the meprin structure (Uren and Vaux, 1996). The remaining region between the MATH and EGF domains was named AM (after MATH). Later MATH and AM domains were referred to together as TRAF domain. Both the interaction domains, MAM, and TRAF were deemed essential for efficient transportation as well as proper folding so as to give rise to an active protein. Deletion of the MAM domain in recombinant meprins caused degradation of the protein via a proteasomal route (Tsukuba and Bond, 1998). The meprins were also shown to require carbohydrate residues for optimal enzymatic activity and processing (Kadowaki et al., 2000). Calnexin and calreticulin were shown to be intimately involved in the correct folding and transport of meprin to the surface and were also involved in the retrograde transport of the protein (Tsukuba et al., 2002).

1.1.2.8 The higher order structures of meprins After the overall structure of meprins was demonstrated, the next task was gaining further detailed insight into the structural organization. The first study done in this field was identification of the cysteine residues involved in α2, β2 and α/β dimerization in rat as well as mouse. Cysteine residues in the MAM domain were found to be involved in the inter-subunit bridging, lending credibility to the earlier hypothesis of MAM domains being involved in oligomerization (Chevallier et al., 1996; Ishmael et al., 2005; Marchand et al., 1996). Electron microscopy studies showed that the functional unit was a covalently linked dimer and higher associations were of a non-covalent nature (Ishmael et al., 2001). While meprin B could only form dimers, heteromeric meprin A was restricted to tetramers. Homomeric meprin A, on the other hand, formed crescent shaped or ring structures composed of as high 100 subunits, making them the largest known secreted protease (Figure 1.5) (Bertenshaw et al., 2003; Ishmael et al., 2001). However, it is worth mentioning here that a lack of availability of high resolution details of the meprin structure, either by X-ray crystallography or by nuclear magnetic resonance (NMR), is a lacuna in the field of meprin structural biology research.

A B

Figure 1.5 Multimeric structures of meprin metalloproteases (A) Latent homomeric meprin A forms higher order oligomers and is present as rings, spirals and crescents. (B) The heteromeric meprin A differs markedly from the homomeric form and shows a barrel structure with symmetrical arrangement of the two subunits. (C) Meprin B forms dimers made up of meprin β subunits which is seen as distinct areas of high density around the central cavity (adapted from Bertenshaw et al., 2003).

15 1.1.2.9 Meprin Substrates Substrate specificities and inhibitors are part of the defining characteristics of any protease. Initial characterization of meprin A had shown a broad specificity of substrates. Substrate specificity of meprin B was studied soon after and more substrates were predicted for this meprin isoform compared to meprin A (Chestukhin et al., 1997; Chestukhin et al., 1996). In-depth analyses of substrate specificities showed that in spite of the high degree of identity that the two meprin subunits shared at the level of their primary structures, the different enzyme isoforms formed by their association had marked differences in their cleavage preferences. While meprin A preferred small and hydrophobic amino acids at the P1’ and proline at P2’ position, meprin B was more discriminating in its choice for acidic amino acids at P1’ (Figure 1.6). While GRP-(14- 27) (gastrin releasing peptide fragment 14-27) was cleaved by both the isoforms, albeit with differing degrees of efficiencies, other substrates were exclusively cleaved by one isoform or the other. For example, while substance P is cleaved only by meprin A; gastrin 17, the best known meprin B substrate, is not cleaved by meprin A. Differences in their active site residues could explain the differences observed in substrate specificities. Meprin B has three basic residues in the active site cleft that have the potential to form salt bridges with acidic residues in the substrate. In contrast, meprin A has aromatic residues at the equivalent positions along with a tyrosine that protrudes into the active site cleft. This explains the preference for proline close to the scissile bonds (Bertenshaw et al., 2001; Villa et al., 2003). The marked differences in the substrate specificities raise the possibility of diverse, even opposing, functions for the different meprin isoforms. The physiological significance of this difference is dealt with in the later chapters of this thesis. Inhibition studies established actinonin, a naturally occurring hydroxamate, as a potent competitive inhibitor of meprins. Actinonin inhibits meprin A in nanomolar range (Ki = 20 nM) and meprin B in micromolar range (Ki = 1.7 μM). Other hydroxamate- based inhibitors such as tumor necrosis factor α proteinase inhibitor (TAPI) -0 and TAPI- 2 could also inhibit meprins but none were found to be as efficient as actinonin (Kruse et al., 2004; Wolz, 1994). Recently, mannan-binding protein (MBP), a serum lectin, was found to be a natural inhibitor of meprins (Hirano et al., 2005).

16 A

Figure 1.6 Differences in substrate bond specificities between the two subunits (A) Meprin α shows a preference for small and aromatic residues in its P1’ position with a marked selection against proline. In its P2’ position proline was most favored. (B) In contrast meprin β showed very different substrate specificity with a strong preference for acidic residues in its P1’ position. Aspartic acid strongly favored followed by glutamic acid. Like meprin α, proline was strongly selected against. There was no clear preference for meprin β in its P2’ position (adapted from Bertenshaw et al., 2001).

17 1.1.2.10 Meprins in Disease While structural and biochemical information about these metalloproteases was progressively being gleaned, meprins were for the first time implicated in a disease condition. Trachtman et al (1993) reported that meprin activity decreased in puromycin- induced glomerulopathy, which might contribute to progressive renal injury (Trachtman et al., 1993). Meprin was also shown to be a major enzyme in renal cortex with the ability to degrade components of the extracellular matrix (Kaushal et al., 1994). In rats as well as humans, meprins were shown to degrade parathyroid hormone (PTH) in the kidney (Yamaguchi et al., 1994a; Yamaguchi et al., 1994b). Meprins were also implicated in acute renal failure as “high-meprin” mice suffered greater damage than meprin-deficient mice upon renal clamping. It was hypothesized that upon injury, meprins are dislocated to the basolateral membrane where they cause damage by degrading the extracellular matrix (Trachtman et al., 1995; Walker et al., 1998). In 2005, a genetic association between meprins and diabetic nephropathy was established; polymorphisms in the meprin β gene (MEP1B) in humans were found to be significantly associated with diabetic nephropathy in the Pima Indian population (Red Eagle et al., 2005). The role of meprins in disease in the case of intestine also came into focus when a novel β’ mRNA was detected in human and mouse colon cancer cell-lines. This transcript differed from the normal β mRNA in its 5’ untranslated region and the translated protein had differences in the signal sequence. This gave rise to the possibility of the protein being directed to different cellular compartment in cancerous cells (Dietrich et al., 1996; Matters and Bond, 1999b). In colorectal cancer cells, Caco-2, redistribution and increased secretion of meprin α was observed, further linking these proteases to cancer pathogenesis. This redistribution resulted in increased proteolytic potential in the tumour stroma, which in turn facilitated tumour progression by increased migration and metastasis (Kohler et al., 2000; Lottaz et al., 1999b).

1.1.2.11 In vivo Studies As can be seen from the research material discussed so far, meprin has traveled a long way, from its discovery in early 1980’s into the 21st century now when significant details are known about its structure along with glimpses of its functions. Recently, meprins

18 have been found in lungs [personal communication; D. Bergin] and skin (Becker-Pauly et al., 2007), tissues at the interface of the host and environment, suggesting a broader role in interactions of the host immune system with extraneous factors such as pathogens. While biochemical as well as cell-culture studies give us meaningful insights into the mechanism and the possible functions of these complex proteases, obtaining physiological significance necessitates the use of an animal model. This led to the generation of the first meprin KO mouse, the meprin βKO mouse. The meprin βKO mice though born normal and fertile, were underrepresented in a heterozygote cross (Norman et al., 2003a). As expected, the mouse only had homomeric meprin A. It was shown in vitro that leukocytes isolated from βKO had impaired ability to migrate as compared to the WT mice (Crisman et al., 2004). This observation led to speculation that the βKO mice might show differences in immune response as leukocyte migration is a key phenomenon in an immune process. As a natural progression, the next step was generation and characterization of the αKO mice. General characterization of the αKO mice and detailed investigation of meprin function using intestine as a model organ constitutes the major portion of the work described in this thesis.

1.1.2.12 Meprin distribution in the intestine Meprin expression has been detencted in various tissues. While meprins were initially identified in kidney and intestine, its presence in other sites like skin, lung and leukocytes is relatively recent. Meprin function in the intestine was chosen for this investigation due to a multitude of reasons. Even though meprins are highly expressed in the kidney, meprin B is inactive and meprin A is active in vivo in the rodents. In humans though, both the meprin isoforms are active in vivo. Thus extrapolation of functions of mice meprins to the human proteins is likely to be cumbersome in the renal system. Intestinal meprins, in contrast, are active in mice as well as humans. In addition, the intestinal distribution of meprins is non-uniform, characterized by differential expression of the two subunits along the intestinal tract. In mice, meprin distribution varies along the small and large intestine. Meprin concentration increases along the small intestine, with ileum having the highest concentration of both the subunits. In the colon, meprin β expression is very low

19 and even though the meprin α expression levels also drop, it is the main subunit that is expressed. So while ileum has all the three meprin isoforms, namely homomeric and heteromeric meprin A and meprin B; in colon, homomeric meprin A appears to be the major isoform. In humans, the meprin distribution also follows a similar pattern, with ileum enjoying the highest levels of both the subunits and meprin β levels dropping to near negligible amounts throughout the entire large intestine (Banerjee et al., unpublished) (Bankus and Bond, 1996; Lottaz et al., 1999a). This provides one with an advantage while trying to distinguish functionally between the different isoforms of meprins. Hence, in order to look at intestinal pathophysiology, an experimental model of inflammatory bowel disease (IBD) was used.

1.1.2.13 Inflammatory Bowel Disease Inflammatory Bowel Diseases are multi-factorial disorders whose etiologies are not clearly understood, but are thought to be brought about by an ungoverned immune response to the normal gut flora (Elson et al., 1995; Strober et al., 2007; Xavier and Podolsky, 2007). It is being increasingly understood that IBD is the manifest outcome of three major interactive factors: mucosal immunity, genetic susceptibility of the host and the enteric flora. Mucosal immunity is essential for proper recognition and interpretation of the local microenvironment. It should be tolerant to the enteric flora and yet be capable of mounting a response to pathogenic challenges (Shanahan, 2000). Individuals with a predisposition towards IBD show a breakdown in regulation of mucosal immunity, consequently mounting a response to the enteric microflora. In humans, IBD encompasses the two distinct clinical entities of Crohn’s disease (CD) and ulcerative colitis (UC). Both share various common clinical features such as rectal bleeding, diarrhea, abdominal pain and weight loss (Elrod et al., 2005). However, significant differences exist between the two conditions. While UC was reported as early as 1875, CD is a recent disease, being first detailed as late as 1932 (Crohn et al., 1932; Wilks and Moxon, 1875). Even then, it was not until the 1960s that differences between the two were clearly identified (Lockhart-Mummery and Morson, 1960). In CD, inflammation can occur anywhere along the entire gastrointestinal tract, from mouth to anus whereas the large intestine is the main target for UC. While inflammation is mucosal and more

20 uniform in case of UC, it is transmural and discontinuous in CD. Granulomas and fistulae formation are classic features associated with CD (Boismenu and Chen, 2000). Even at the cellular and molecular levels differences have been observed; CD is generally characterized by a Th-1 response (involving IL-12, IFN-γ, TNF-α), while UC shows a predominantly Th-2 mediated response (IL-4, IL-5) (Ishii et al., 2004; Shanahan, 2001). Elucidating the mechanisms underlying these two clinical conditions was impeded for a long time by the lack of suitable experimental model systems. It was not till 1990s that a number of model systems, mainly mouse, became available which enabled scientists to mimic IBD in mice in order to study the etiology, pathogenesis and prognosis of the these multifactorial diseases (Elson et al., 1998; Elson et al., 1995). In today’s parlance, several models of colitis are available, each of which allows one to look at one or more aspects of this condition (Figure 1.7). As very aptly put by Elson et al. (1995), while there is no ideal model for IBD, different models have their own advantages as well as handicaps. Broadly, these models can be classified into four main categories: chemically-induced models, spontaneous models, transgenic/gene deletion models and adoptive cell transfer models (Table 1.1). While the models belonging to the latter two categories are more recently developed, chemically-induced models were established in late 1980s to early 1990s. The two most commonly used chemically induced models are the dextran sulfate sodium (DSS) model and the trinitrobenzene sulfonic acid (TNBS) model. While administration of DSS leads to UC-like colitis development, TNBS generates a CD-like condition (Boismenu and Chen, 2000). For the study described in this thesis, colitis was induced using the DSS-model. First described by Okayasu et al (1990), in this model DSS is administered ad libitum in the drinking water to generate colitis-like features. DSS is a sulfated glucose molecule, containing up to 3 sulfate groups per sugar molecule (Ricketts, 1952). The exact mechanism by which DSS provokes intestinal irritation has not been clearly established. Nevertheless, DSS is thought to cause increased epithelial cell toxicity, increased intestinal membrane permeability as well as macrophage activation, all of which can be factors leading to colitis (Cooper et al., 1993; Dieleman et al., 1994). DSS-induced colitis is thought to be a result of direct cytotoxicity which results in inflammation as a secondary phenomenon (Egger et al., 2000; Ni et al., 1996). In line with DSS eliciting a UC-like condition, DSS uptake has

21 : probable usefulness : definite usefulness Figure 1.7 Different mouse models of colitis and their utilities The different experimental models of colitis can be used to study the various contributing factors of IBD (adapted from Elson et al., 1998).

22 Category Affected areas Inflammation Similarity

Chemically induced Acetic acid Descending colon/rectum Acute UC DSS Colon Acute/chronic UC Oxazolone Descending colon Acute/chronic UC TNBS/ethanol Colon Acute/chronic CD

Spontaneous C3H/HeBir Descending colon/rectum Acute/chronic SAMP1/Yit Ileum/caecum Acute/chronic CD

Transgenic/gene deletion -/- Gαi Colon Chronic > acute UC IL-2-/- Colon Acute/chronic UC IL-10-/- Small intestine/colon Chronic UC Keratin 8-/- Colon Chronic N-cadherin-/- Small intestine Chronic CD TCR-α-/- Colon Chronic UC

Adoptive cell transfer CD4+ Tcells/SCID Colon > ileum Chronic > acute CD CD3ε (Tgε26) Colon Acute/chronic CD

Table 1.1 Different Mouse Colitis Model Systems Mouse models of colitis have been broadly classified into four categories. The first category includes the chemically-induced models like DSS and TNBS models of colitis. The spontaneous models of colitis make up the next category. The last two categories, namely the transgenic models and the adoptive cell transfer models have been established more recently (Boismenu and Chen, 2000).

23 been reported early on in the colon, mesenteric lymph nodes and liver. DSS accumulation in the ileum was not seen till the very end of the DSS study, which reaffirms the disease to be restricted to the colon (Table 1.2) (Kitajima et al., 1999; Okayasu et al., 1990). There were several factors favoring the use of the DSS-model of colitis for understanding the role of meprins in the intestinal pathophysiology. Yap et al (2006) have shown polymorphisms in the human meprin α (MEP1A) gene with significant correlations with both UC and CD. Interestingly, the correlation is much stronger in the case of UC (Yap et al., 2006). As already mentioned, DSS administration induces colitis in mice that resembles human UC. In addition, ease of use compared to the TNBS model, and the fact that it has been previously utilized and characterized in the laboratory (Crisman et al., 2004) were the other advantages of using the DSS model.

1.1.2.14 Rationale for this thesis work The work conducted for this thesis includes the general characterization of the αKO mouse along with a study of its phenotype in an experimental model of intestinal stress. Meprin metalloproteases are highly expressed in the mammalian intestine and have been implicated in IBD. In addition, meprins are capable of degrading many proteins and peptides including ECM proteins and cytokines in vitro. In order to gain insight into the function of meprin metalloproteases in the intestine, meprin αKO mice were generated. WT and meprin αKO mice were subjected to a model of intestinal stress and then monitored for differences. For this purpose, the DSS model of IBD was chosen. The insights gained from this experimental model of IBD indicated a role for meprins in immuno-modulation by direct interaction with cytokines. This lead was followed by studying meprin interaction with one of the immune mediators, IL-18, which is one of the central players in the pathogenesis of IBD. As the meprin αKO mice have a skewed distribution of all the meprin isoforms, the contribution of meprin B towards any phenotype observed in the αKO needed additional investigation. For this purpose, meprin αβKO mice were generated and subjected to the DSS-model of colitis. This set of experiments helped in further defining the roles of different meprin isoforms in the intestinal patho-physiology.

Features CD UC DSS-colitis

Location Small intestine/colon Colon Colon Depth Transmural Mucosal Mucosal Extent Discontinuous Continuous Continuous Symptoms Non-bloody diarrhea, Bloody diarrhea, no Bloody diarrhea, no fistula fistula fistula Granuloma Yes No No Genetic Yes Yes Yes Microflora Yes Yes Yes Immune system Yes Yes Yes Inflammation Transmural Epithelium Epithelium TNF-α Elevated Elevated Elevated

Table 1.2 Comparison between DSS-colitis model and human UC and CD The DSS-model of colitis resembles the human UC condition. The pathology is restricted to the colon and is continuous and mucosal in nature. (Boismenu and Chen, 2000; Okayasu et al., 1990).

25 Chapter 2: Characterization of Meprin α Knockout Mice 2.1 Overview One of the recent landmarks in the field of science was the sequencing of the human genome, which was expected to lay open many of the mysteries of life. As is the case with every scientific discovery, it succeeded in raising many more questions reiterating the complexities of life. Deciphering protein sequences and their subsequent structures and functions has proved to be one of the major challenges facing science inspite of the many advances that have been achieved so far. Over time, it has become clear that a protein in the test-tube may behave in quite different a manner than it does in the organism. It is increasingly being realized that in vivo studies are necessary tools to gain insight into the physiological significance of a protein. Thus, the significance of animal models came to be realized. Of the various model systems available, mice are one of the most common models used for research. Not only does this mammalian model resemble humans in many physiological aspects, their life statistics make them an ideal model to study (Table 2.1) (Hogan et al., 1994). Not only do mice have large litters, they also reach maturity as early as 4-5 weeks, making for a good model system. Additionally, the field of mouse genomics has seen many rapid advances. Following human genome, mouse genome was the next to be sequenced. As of today, partial or complete sequences of many strains such as C57BL/6, BALB/c, C3H, NOD and several 129 substrains are available. This further increases the attractiveness of the mouse as a model organism. Another major advantage of this model system is the availability of knockout mice for different genes. The rapid evolution of molecular biology techniques in 80’s and 90’s made it possible to manipulate the mouse genome and thus transgenic and knockout mice were created as model systems to study genes and their functional products (Capecchi, 1989a; Capecchi, 1989b; Evans, 1989; Koller et al., 1989; Koller and Smithies, 1989). The technology of creating knockout mice, which earned Mario Capecchi, Martin Evans and Oliver Smithies the 2007 Nobel Prize in Medicine and Physiology, allowed scientists to study the effect of a gene in the animal physiology by inactivating and thereby removing its function. This allowed the study of in vivo functions of a gene in its physiological setting.

Genome Information Chromosome number (diploid) : 40 Diploid DNA content : ~ 6pg/cell Approximate number of genes: 0.5-1.0 x 105

Reproductive Biology Gestation time: 19-21 days Age at weaning: 3 weeks Age at sexual maturity: 6-8 weeks Lifespan in laboratory: 1.5-2.5 years Average litter size: 6-8 pups Total number of litters per breeding female: 4-8 Useful breeding life of females: 6-8 months Useful breeding life of males: 18-24 months

Table 2.1 Mus musculus Vital Statistics Table adapted from Hogan, et al. 1994; (page-3).

27 Another important factor to be appreciated when using mouse as a model organism is the strain of the mouse. An inbred strain is defined as one that has been maintained for more than 20 generations of brother-to-sister mating and is essentially considered to be homozygous for all genetic loci barring spontaneous mutations (Hogan et al., 1994). Several instances are present when phenotype resulting from mutations change depending on the background of the carrier. Epithelial Growth Factor Receptor (EGFR) knockout mice show varied phenotypes from lethality to hair fiber abnormalities which are dependent on the genetic background (Sibilia and Wagner, 1995). A Transforming Growth Factor (TGF)-β1 knockout mouse provides another such example, where the cause, time and penetrance of embryonic lethality depend on the strain background of the Tgf-β-/- mouse (Kallapur et al., 1999). Fragile X mental retardation (Fmr)1-/- mice, which serve as a model for human Fragile X mental retardation syndrome, show mild to pronounced deficiency in learning the Morris water maze task depending upon their strain (Dobkin et al., 2000). The phenotype of Noggin-/- mouse is also strain dependent (Tylzanowski et al., 2006). Hence, it is imperative that while conducting experiments the different groups have the same genetic background for proper comparisons to be made. In order to investigate the function of meprin metalloproteases in their in vivo setting, meprin knockout mice were generated. Meprin βKO mice were first generated in our laboratory to study the effect of loss of meprin β gene (Norman et al., 2003a). The next step was the generation of meprin αKO mice by inactivation of meprin α (Mep1a) gene. This chapter deals with the general characterization of the meprin αKO mouse on a mixed C57BL/6 x 129/Sv background.

2.2 Experimental Procedures 2.2.1 Generation and validation of αKO mice The Mep1a gene on chromosome 17 was inactivated by homologous recombination with a targeted vector that inserted a neomycin resistant cassette into the catalytic centre, encoded by exon 7, of the Mep1a gene and deleting 140-bp at the targeted site. Primers 5’-CAGGTGAGTATGACTCGAGCAGAGTAGG-3’ (containing an XhoI restriction site, corresponding to exon7/intron7 junction) and 5’-

28 GATGAGGCAGCTAGCATACTGGGATTC-3’ (encoding an NheI site and corresponding to intron 8) were used to generate a 4.9 kb amplicon from the 129 x 1/SvJ genomic DNA (Jackson Stock No. 000691). The XhoI-NheI digested amplicon was ligated into XhoI-NheI cut Osdupdel (a kind gift from Prof. Oliver Smithies, University of North Carolina, Chapel Hill) to generate Osdupdel- Mep1a-4.9. A second amplicon was made using primers 5’- GTCAGAAGAAATCTAGACAGTAGATCAGTG-3’ (containing an XbaI restriction site and corresponding to intron 6) and 5’- CCCACCTGTGGATCCCCAATCATAGAC-3’ (encoding a BamHI restriction site and corresponding to exon 7). This 2.5 kb fragment was XbaI-BamHI digested and ligated into XbaI-BamHI cut Osdupdel-Mep1a-4.9. The final vector, Osdupdel-Mep1a, had exon 7 disrupted by a 1.2 kb neomycin cassette with an in-frame stop codon (Figure 2.1A). (Restriction enzyme sites of corresponding enzymes underlined in the primers mentioned). The construct was designed and developed by Dr Gail Matters. The targeting vector, Osdupdel-Mep1a, was electroporated into R1 mouse ES cells (Nagy et al., 1993). A total of 390 clones were screened by PCR. PCR product from the WT allele corresponded to 3.1 kb and that of the targeted allele corresponded to 4.3 kb. Screening of ES cells showed three clones to be carrying the targeted allele. The clones were microinjected into blastocysts of C57BL/6 mice. Of the 38 ES cell-mouse chimeras obtained, 4 were germline, transmitting the disrupted Mep1a allele to their offspring. Electroporation and selection of targeted ES cells and subsequent blastocyst injections were performed at the University of Michigan Transgenic Animal Facility. Chimeric animals were crossed with C57BL/6 mice at Pennsylvania State University College of Medicine in full compliance with animal use and care regulations. Tail biopsies were digested overnight with agitation at 42 0C with 6 U of proteinase K in 0.4 ml of buffer (50 mM Tris [pH 7.5], 100 mM EDTA, 125 mM NaCl, and 1% sodium dodecyl sulfate). A saturated NaCl solution (200 μl) was added to the digested samples. Samples were vortexed and then centrifuged for 15 min at 16,000 x g. The DNA was then ethanol precipitated from the resulting supernatant fraction. Southern blot as well as PCR analyses were used to determine genotype. PCR primers 5’- CCCCTGGAGTCTGTCTAGTAGCCATCATC-3’ and 5’- GCGAAGGACCTCCCATG ATAAACTTAG-3’ gave 3.1 kb and 4.3 kb products for WT and KO allele respectively.

29 For southern analysis, HindIII was used to digest 10 μg of DNA. A 740 bp probe, corresponding to intron 8, was generated using primers 5’- GCTCTGGTGAAACTGCCCCACTCGAAGCTG-3’ and 5’-AGTATGGGAGGCTCTC AGCA -3’ to detect WT (4.7 kb) and disrupted (3.5 kb) alleles (Figure 2.1B).

2.2.2 Urine and serum analyses Urine was collected from WT and meprin αKO mice by bladder massage. Chemstrip 10 SG dip sticks (Roche) were used for semiquantitative analysis of urinary pH, protein, nitrite, glucose, ketones, urobilinogen, bilirubin, blood, leukocytes, and specific gravity. The mice were anesthetized by isofluorane and blood was withdrawn by cardiac puncture and collected in heparin coated microvette tubes (Sarstedt). Blood cells were removed by centrifugation at 10,000 x g for 10 min and serum was used for analysis of blood urea nitrogen (BUN), total serum protein, serum albumin, serum creatinine, serum Na+ and serum K+ levels. Urine and serum creatinine levels were measured by using the Infinity Creatinine Assay (Sigma Diagnostics). The rate of change of color caused by the addition of 1:10 diluted urine sample to an alkaline picrate is followed at 500 nm. Total urinary protein was measured by using the Bio-Rad Bradford dye standard assay. Serum albumin was determined by using the Sigma Diagnostics albumin reagent. BUN was measured by using the BUN rate reagent (Sigma Diagnostics), based on the Talke and Schubert method (Dobbelstein et al., 1971a; Dobbelstein et al., 1971b).

2.2.3 End-Point PCR Total RNA isolated from mouse kidney using TRIzol (Invitrogen) was subjected to RT- PCR with the Superscript One-Step RT-PCR kit (Invitrogen). The amount of amplicon at various cycles was analyzed by using integrated band density data obtained with the Stratagene Eagle-Eye II system equipped with Eagle-Sight software. Glyceraldehyde-3- phosphate dehydrogenase (GAPDH) was amplified as a control. Meprin α was amplified using primers 5’-CGCCTCAAGTCTTGTGTGGATT-3’ and 5’-ATTTCATGTTCAAT GGTGGCCT T-3’, which gave a 164-bp product. For meprin β, the primers 5’- AGGATTCAGCCAGGCAAGGA-3’ and 5’-CGTGACGATGGTAGACTCTGTCC-3’ were used to obtain a 145-bp product.

30 A 3.5 kb

WT allele 6 HindIII probe 7 HindIII 8 9

HEXXHXXGFXHEXXRXDRD Catalytic Centre

4.7 kb

αKO allele 6 7 7 8 9 HindIII probe HindIII

Disrupted Catalytic Center 1.2 kb PGK-Neo Insert B -/- +/+ +/-

4.7 kb (αKO allele) 3.5 kb (WT allele)

Figure 2.1 Strategy for Mep1a gene disruption (A) Schematic diagram of a portion of the exon-intron structure of the WT and meprin αKO alleles. Exons (6 to 9) are represented as black boxes. The neomycin cassette derived from the targeting vector is depicted as a gray box in exon 7 of the KO allele. The 19 amino acid consensus sequence for the catalytic center of astacin family metalloproteases is also shown. (B) Southern blot analysis of tail-derived genomic DNA. The probe used for southern blotting detects two HindIII-generated fragments: 3.5 kb corresponding to the WT allele and 4.7 kb from the KO allele. Lanes: -/-, meprin αKO DNA; +/+, WT DNA; +/-, meprin α heterozygous DNA.

31 2.2.4 Immunoblotting of urinary samples Presence of meprin α was tested by western analysis of urine from WT and αKO mice (Norman et al., 2003a). Equal urinary creatinine samples were loaded for proper comparison. Loading equal urine creatinine enables comparative analyses across animals, since urine creatinine secretion is constant over 24 h. Normalization of urine is important as samples obtained will have variable volume and/or concentration (Chen et al., 1995; Ginsberg et al., 1983). The assumption made in this case is that urine creatinine levels of the mice used are constant over 24 h. As serum creatinine levels of WT and αKO mice are not statistically different, the assumption, is considered valid. Urine samples were subjected to 7.5% polyacrylamide gel electrophoresis under reducing and denaturing conditions and transferred to nitrocellulose membranes. The membranes were blocked with 10% non-fat milk in Tris-buffered saline (TBS) supplemented with 0.1% Tween-20 and antibodies incubated in 5% non-fat milk in TBS/0.1% Tween-20. Polyclonal rabbit anti-recombinant meprin α primary antibodies were used. Horseradish peroxidase- conjugated anti-rabbit secondary antibodies (GE HealthCare) were detected by chemiluminescence using the SuperSignal Dura substrate (Pierce).

2.2.5 Immunohistochemistry Tissues were fixed in methyl Carnoy's solution (60% methanol, 30% chloroform, 10% acetic acid), dehydrated in ethanol, embedded in paraffin wax, thin sectioned (5 µm) and fixed onto SuperFrost Plus microscope slides (Fisher HealthCare). Tissues were deparaffinized with xylene and re-hydrated with decreasing concentrations of ethanol and submerged in a 4:1 methanol/hydrogen peroxide wash to quench endogenous peroxidases. An avidin-biotin blocking kit (Vector Laboratories) was used to block background contributed by endogenous biotin or biotin-binding proteins. Background Buster (Accurate Chemical & Scientific Corp.) was then used as a general blocking agent. Polyclonal rabbit anti-recombinant meprin α or β served as the primary antibody. A pre-immune rabbit serum was used as negative control. Biotinylated goat anti-rabbit immunoglobulin G (heavy plus light chains) at a final concentration of 7.5 µg/ml (Vector Laboratories) was used as the secondary antibody. Sections were washed and then incubated with Vectastain ABC Elite reagents (Vector Laboratories) following

32 manufacturer’s directions. 3,3'-Diaminobenzidine (1 mg/ml) in Tris (pH 7.6) with 8.5% hydrogen peroxide was applied to all sections. Sections were counterstained with hematoxylin-eosin, rinsed in 100% ethanol, submerged in xylene, and mounted with Permount (Fisher). Digital photographs were taken with a Nikon Eclipse E600 microscope.

2.2.6 Ileum brush-border membrane preparation Mouse ileum was dissected; samples weighed and homogenized in 10 volumes of cold homogenization buffer (50 mM Mannitol, 2 mM Tris-Cl, pH 7.0) in the presence of

Complete Mini Protease Cocktail Inhibitor (Roche). CaCl2 was added to a final concentration of 10 mM and the homogenate stirred slowly at 4 0C for 15 min. This was followed by centrifugation at 3,000 x g for 15 min and the pellet was discarded. The supernatant fraction was spun at 27,000 x g for 30 min and the pellet was suspended in 0.5 volumes homogenization buffer. After a final spin at 27,000 x g for 30 min, the pellet suspended in 0.5 volumes of homogenization buffer comprised of the brush-border membrane enriched fraction.

2.2.7 Meprin β activity assay Meprin β activity was assayed using the intramolecularly quenched fluorogenic substrate, 2-aminobenzoic acid-MGWM-DEIDK-2,4-dinitrophenyl-SG-OH (OCK+). The peptide sequence of OCK+ was designed based on the cleavage site within two peptides, orcokinin and cholecystokinin, which are specific substrates for meprin B (Bertenshaw et al., 2002). Equal amounts of protein were activated by addition of trypsin (1 mg/ml in 20 mM Tris buffer, pH 7.5) in 20:1, followed by incubation at 37 0C for 1 h. The trypsin was then inactivated by addition of 3X soybean trypsin inhibitor (STI) (3 mg/ml in 20 mM Tris buffer, pH 7.5) and incubated for 20 min at 25 0C. The volume of the reaction was made up to 298 μl with 20 mM Tris/100 mM NaCl, pH 6.7. OCK+ was prepared as a 1.5 mM solution in 100% DMF and used in a final concentration of 10 μM in the final assay to detect meprin β activity. Substrate hydrolysis was followed by monitoring the increase in fluorescence in a Hitachi F-2000 fluorescence spectrophotometer using excitation and emission wavelengths of 326 and 418 nm respectively. Actinonin, dissolved in 50%

33 dimethyl sulfoxide (DMSO), was added to a final concentration of 33 μM for inhibiting meprin β activity. Vehicle control samples had equal amounts of 50% DMSO.

2.2.8 Statistical Analysis 2 Genotypic distribution of F2 populations was determined using a chi-square (χ ) test. For all other analyses, unpaired, two-tailed t-test was used. P-values < 0.05 were considered significant.

2.3 Results 2.3.1 Meprin αKO mice did not express meprin α mRNA or protein The genotypes of 21 day old pups were determined by both Southern analysis as well as PCR from tail DNA biopsies (Figure 2.1B). The absence of meprin α mRNA and protein was also verified. Kidney mRNA from WT and αKO mice was isolated and tested for meprin α by end-point PCR (Figure 2.2A). Meprin α was present only in the WT kidney. To test the presence of meprin α protein in urine, samples from WT and αKO mice, normalized for creatinine, were subjected to immunoblotting with meprin α antibody (Figure 2.2B). Only the WT urine samples had detectable meprin α protein, as expected. Immunohistochemistry for meprin α was performed on ileal sections from WT and αKO mice. In line with the earlier observations, only sections from WT mice showed the presence of meprin α along the ileal villi (Figure 2.2C).

2.3.2 General Parameters Deletion of meprin α gene was not lethal and the αKO mice did not show any overt physical abnormalities. Serum was analyzed for BUN, serum creatinine, total protein, serum albumin and serum Na+ and K+ levels. All the parameters tested for αKO were within the normal range and did not vary from their WT counterparts (Table 2.2). The meprin αKO mice were fertile and did not show any obvious birth or developmental defects. Body weights of both the genotypes were measured at different time points to determine difference in growth rate if any. Males and females of both the genotypes had

34 A B 198 kDa 131 kDa 164 bp

WT αKO 82 kDa

1 2 3 4

WT αKO C-i C-ii C-iii

Figure 2.2 Validation of meprin αKO mice at mRNA and protein level (A) Semi-quantitative end-point PCR showing meprin α mRNA levels in kidney of WT (first lane) and meprin αKO (second lane) mice. The lanes contain an equal amount of RNA. (B) Western blot for meprin α in the urine of WT (lanes 1 & 2) and αKO (lanes 3 & 4) mice. Samples were normalized for equal creatinine (3 μg). (C) Immunohistochemistry showing meprin α distribution in villi of the ileum of WT (i) and meprin αKO (ii) mice. WT non-immune sera control (iii). Arrows point to the anti-meprin α antibody immunostaining.

WT (α+/+) αKO (α-/-) BUN (mg/dl) 20.13 ± 5.89 21.25 ± 2.12 Serum Creatinine (mg/dl) 0.17± 0.05 0.18 ± 0.08 Total Serum Protein (mg/dl) 4.73 ± 0.91 4.38± 1.11 Serum Albumin (mg/dl) 2.53 ± 0.39 2.43 ± 0.26 Serum Na+ (mmol/L) 151.00 ± 3.74 152.33 ± 2.07 Serum K+ (mmol/L) 5.46 ± 0.89 6.20 ± 0.98 Table 2.2 Serum chemistry of WT and meprin αKO mice Serum was collected from adult male WT and αKO mice (n = 5) and analyzed for the above mentioned parameters. All values were within the normal range.

36 comparable body weights at birth (~1.6 g), during weaning (~9.3 g) and at 44 days (males ~ 21.5 g, females ~ 17.8 g) as adults (Figure 2.3).

2.3.3 Litter size +/- -/- F1 heterozygote matings (α x α ) gave the expected Mendelian genotypic distribution; with approximately 25% of the F2 progeny being meprin αKO (Table 2.3). The WT (α+/+) and αKO (α-/-) mice obtained from heterozygous mating were mated further to generate WT and αKO litters. Interestingly, the litter size of WT (α+/+ x α+/+) mating was significantly larger than that of an αKO (α-/- x α-/-) mating. When the average litter size of WT was calculated from a total of 59 litters and 444 pups, a value of 7.5 ± 0.25 pups per litter was obtained. The corresponding value for the αKO litter was 6.1 ± 0.29, obtained from 63 litters and a total of 388 pups, (*,P < 0.004) (Figure 2.4).

2.3.4 Meprin β mRNA and protein is unaffected in αKO mice Even though the expression of two meprin subunits is independent of each other, their spatial distribution is not. The two subunits are encoded on different choromosomes and expressed independently. But, at the same time, the subunits combine as homo- and hetero-oligomers to form their functional units. Their dependence stems from the fact that absence of one subunit results in a change in the distribution of meprin isoforms. Hence, effect of meprin α deletion on meprin β distribution, if any, was investigated at mRNA as well as protein levels. Kidney mRNA obtained from WT and αKO animals showed equivalent amounts of meprin β as seen by end-point PCR (Figure 2.5A). Immunohistochemistry for meprin β was performed on ileum sections from both the genotypes. Both WT and αKO sections showed similar pictures of meprin β staining along the villi of the ileum (Figure 2.5B). Finally, meprin β activity was measured, using the fluorogenic substrate OCK+, in the ileum brush-border fraction of WT and αKO mice. Meprin β activity was seen in both WT and αKO mice, which was inhibited by actinonin (Figure 2.5C).

37 A

30 WT 20 αKO 10

Weight (gms)Weight 0 Day 2 Day 10 Day 21 Day 44

Days

25 WT 20 αKO 15

5 Weight (gms) Weight

0 Day 2 Day 10 Day 21 Day 44 Days

Figure 2.3 The αKO and WT mice show comparable growth rates Weight progression of male (A) and female (B) WT and αKO mice from birth to adulthood. Both the genotypes show comparable weights at birth (Day 2), during weaning (Day 21) and as adults (Day 44).

Observed % Predicted % Observed Total 87 WT (α+/+) 25 25% 28.7% Hetero (α+/-) 40 50% 46.0% αKO (α-/-) 22 25% 25.3% Male 36 WT (α+/+) 8 25% 22.2% Hetero (α+/-) 19 50% 52.8% αKO (α-/-) 9 25% 25.0% Female 51 WT (α+/+) 17 25% 33.3% Hetero (α+/-) 21 50% 41.2% αKO (α-/-) 13 25% 25.5% Table 2.3 Genotypic distribution of meprin α+/- mating

Genotypic frequencies are for progeny of F1 heterozygous crosses. The meprin αKO mice show the expected Mendelian distribution of 1:2:1. There is an equal distribution of both the sexes.

9 8 *

7 *

6 5 4 3

# of pups/litter of # 2

0 WT αKO (444 pups; 59 litters) (388 pups; 63 litters)

Parental Genotype

Figure 2.4 Meprin αKO mice have smaller litters Meprin αKO mice have significantly smaller litters than their WT counterparts (*, P < 0.004). WT litters had an average size of 7.5 ± 0.25 pups, while the αKO litter size averaged to 6.1 ± 0.29 pups.

40 B-i B-ii A

Meprin β 145 bp

345 bp GAPDH

WT αKO B-iii C - Inhibitor + Inhibitor

0.4 *

0.3 *

0.2

0.1 Activity Rate/ug Activity

T O W K

Figure 2.5 Meprin β is unaffected in the αKO mice (A) Semi-quantitative end-point PCR showing meprin β mRNA levels in kidney of WT (first lane) and meprin αKO (second lane) mice. GAPDH served as a control for equal RNA loading. (B) Immunohistochemistry showing meprin β distribution in villi of the ileum of WT (i) and meprin αKO (ii) mice; meprin αKO non-immune sera control (iii). (C) Meprin β enzyme activity using OCK+ (2-aminobenzoic acid-MGWMDEIDK-2,4-dinitrophenyl-SG-OH) as substrate. WT and meprin αKO ileum brush border membrane samples were assayed in the presence and absence of actinonin (33 µM), an inhibitor for meprins.

41 2.4 Discussion The work reported in this chapter describes the construction and the general characterization of meprin αKO mouse. The disrupted allele can be transcribed till exon 6 which translates to the signal and the pro sequence on meprin. Though no meprin α mRNA was detected, presence of truncated mRNAs can not be ruled out. But the fact that polyclonal meprin α antibodies failed to detect any protein as well as the inability to express truncated meprin α subunit consisting of signal, pro and protease domain in our laboratory definitively rule out the presence of meprin α subunit in the αKO mice (Tsukuba and Bond, 1998). From the results, it can be concluded that meprin β expression is unaffected by the absence of meprin α. There was also no change in the spatial distribution or activity of meprin β along the ileal villi in the αKO mouse. However, it must be noted that the meprin β present in the WT mouse is distributed into two isoforms; heteromeric membrane-bound meprin A and meprin B. In contrast, in the αKO mouse, meprin β can only form meprin B. So similar levels of meprin β in effect, translates to higher amounts of meprin B in the αKO animal as there is no heteromeric meprin A. Hence, any difference in phenotype in the αKO can therefore be attributed not only to the loss of meprin A, but also to the possible increase in meprin B. As discussed earlier, the genetic background can play a role in the animal’s physiology. It is important that the mice are not only age and sex-matched, but also share the same genetic background (Sanford et al., 2001). All experiments reported in the subsequent chapters were carried out with WT and αKO mice on C57BL/6 x 129/Sv background. Congenic αKO mice are currently being generated by back-crossing with C57BL/6 mice obtained from Jackson Laboratory. Mice homozygous for the disrupted meprin α gene are not negatively selected as can be seen from the Mendelian distribution from a heterozygous cross. However, the αKO does show a reduction in litter size. Studies with meprin βKO mice had shown a similar result with the βKO mice being underrepresented (Norman et al., 2003a). Taken together, these observations are indicative of a possible novel role for these proteases in embryogenesis.

42 Embryo implantation is a tightly controlled process, and is a critical step in the establishment of pregnancy. For successful implantation, synchronization between invasiveness of the embryo and receptivity of endometrium is required. This process involves complex remodeling of ECM, where MMPs play a pivotal role (Fata et al., 2000; Salamonsen, 1999; Waterhouse et al., 1993). Recent studies indicate a role for E- cadherin via the regulation of MMP-2 and MMP-9 expression (Liu et al., 2006). Interstingly, meprins interact with E-cadherin on the cell-surface [personal communication; D. Lottaz]. Another interesting line of support stems from the fact that hatching enzymes across major eukaryotic groups belong to the evolutionary family that meprins belong to, “the astacin family of metalloproteinases”. These enzymes are involved in the breakdown of the egg membrane, chorion, allowing the embryo to emerge during hatching. Recently Quesada et al identified human and mouse ovastacin (Quesada et al., 2004), which shares similarities with hatching enzymes from Astacus (AEA) (Geier and Zwilling, 1998), eel (EHE-7) (Hiroi et al., 2004), medaka fish (HC and LCE) (Yasumasu et al., 1992), Xenopus (XHE) (Katagiri et al., 1997) and quail (CAM-1) (Elaroussi and DeLuca, 1994). Hence, it is plausible that meprin α, another member of the same family may have an as yet unsuspected role in the process of mouse embryogenesis. Previous work from our laboratory, using in situ hybridization, detected both the meprin subunits in the mouse embryo, as early as day 11 of gestation (Kumar and Bond, 2001). All these data, taken together with the observation of reduced litter size in the αKO mouse, makes it tempting to suggest a role for these proteases in the developmental process. As the loss of meprin A did not seem to affect the mouse under normal conditions, I decided to study the phenotype of these mice under a condition of physiological stress. A murine model of IBD was chosen as a model for intestinal stress. Meprins are highly expressed in human as well as rodent gut but the two isoforms enjoy non-uniform spatial distribution. Consequently, a phenotype observed using this model may allow one to understand as well as distinguish between the functions of the different meprin isoforms. Hence to understand the role of meprins in intestinal patho-physiology, a DSS model of colitis was chosen, which is described in the next chapter.

43 Chapter 3: Delineating the role of meprin A in a Model of Inflammatory Bowel Disease 3.1 Overview Inflammatory Bowel Diseases are characterized by chronic illness of unknown etiology with genetic, environmental and immunological factors influencing its outcome (Rodriguez-Bores et al., 2007). Epidemiological studies increasingly suggest genetic susceptibility being a major driving force behind the development of this condition. Not unexpectedly, IBD does not follow a simple Medelian model of inheritance, but are considered complex polygenic diseases and multiple putative or candidate IBD loci have been identified (Table 3.1) (Rodriguez-Bores et al., 2007). Existence of genetic susceptibility towards IBD is documented by familial clustering of the disease (Colombel et al., 1996; Mayberry, 1989; Orholm et al., 1991; Satsangi et al., 1996a) as well as increased concordance in monozygotic twins (Thompson et al., 1996; Tysk et al., 1988). Studies by Hugot et al (1996) and Satsangi et al (1996) revealed significant susceptibility loci on chromosomes 16 and 12, now known as IBD1 and IBD2 respectively (Hugot et al., 1996; Satsangi et al., 1996b). Since then, many candidate genes have been identified that predispose an individual towards IBDs, the most prominent among them being NOD- 2, on chromosome 16, showing a strong linkage to CD (Hugot et al., 2001; Ogura et al., 2001). Subsequent studies by Rioux et al (2000) identified an additional locus for CD susceptibility on chromosome 5q21-q33 (Rioux et al., 2000). The same study also identified a susceptibility locus on chromosome 19p13 for both UC and CD. Another important region linked with both UC and CD is the human leukocyte antigen (HLA) loci on chromosome 6p (Hampe et al., 1999a; Hampe et al., 1999b), which also harbors the meprin α gene, MEP1A (Jiang et al., 1995). While several groups have shown evidence for HLA linkage with UC (Bouma et al., 1997; Perri et al., 1998; Roussomoustakaki et al., 1997; Uyar et al., 1998), Forcione et al (1996) have linked it with the occurrence of CD (Forcione et al., 1996). An inflammatory cell infiltrate associated with changes in proliferation and migration of epithelial cells characterizes the chronic mucosal inflammation in IBD. This is accompanied by extensive remodeling of sub-epithelial connective tissue resulting in

Region Localization Genes involved

IBD1 Chromosome 16 NOD2/CARD15, IL-4R, CD11B

IBD2 Chromosome 12 Vitamin D receptor (VDR), STAT6, Interferon γ, β7 integrine

IBD3 Chromosome 6 Major Histocompatibility complex (MHC): Class I, II, III

IBD4 Chromosome 14 T-Lymphocyte Receptor (TCR), Leukotriene B4

IBD5 Chromosome 5 Organic cations transporter (OCTN), Drosophila long disc homologue gene 5 (DLG5), Multidrug resistant gene (MDR), IL-6, CD14

IBD6 Chromosome 19 Thrombaxane A2, Leukotriene B4, ICAM-1

IBD7 Chromosome 1 Transforming growth factor (TGF-β, TNFα receptors

IBD8 Chromosome 16 Unknown

IBD9 Chromosome 16 CCR5, CCR9, IL-12

Table 3.1 Major IBD loci and their position IBD is a polygenic condition and several susceptibility loci spread across different chromosomes have been identified (Rodriguez-Bores, et al., 2007).

45 increased turnover of ECM components (Kovacs and DiPietro, 1994; von Lampe et al., 2000). ECM turnover is a highly regulated event with different classes of proteinases such as cysteine, serine and metalloproteinases being involved in the process. Several metzincins, the superfamily which includes MMPs as well as meprins, are thought to have an active role in this process. An important player in normal tissue remodeling, MMPs have been shown to be upregulated in several animal models of colitis as well as in patients with active colitis (Bailey et al., 1994; Baugh et al., 1999; Castaneda et al., 2005; Ravi et al., 2007). Though predicted to play a role, no connection between meprins and IBD has been shown so far. Investigations in mice and humans have reported the differential expression patterns of meprin α and β subunits in the intestinal epithelial cells. While meprin β is abundant in the ileum, meprin α is found in the small as well as the large intestines. Thus meprin in the intestine is found in different oligomeric forms and in varying amounts (Bankus and Bond, 1996). In the small intestine, membrane- bound as well as secreted forms of meprin A are found, while in colon it is mainly present in its secreted homo-oligomeric form (Bankus and Bond, 1996; Lottaz et al., 1999a) (Banerjee et al., unpublished). Of further interest is the fact that in pathological conditions, for example in colorectal cancer, increased meprin α activity is observed along with a redistribution from apical to basolateral region of the cell (Lottaz et al., 1999b). This observation has prompted speculations about this protease being involved in cell-migration, a possibility further supported by the fact that different ECM proteins like collagen-IV, nidogen-1, fibrinogen and laminins-1 and 5 are meprin α substrates (Kohler et al., 2000; Kruse et al., 2004). In addition to its abundance in the intestinal epithelium, meprins have also been detected in human and mouse leukocytes under normal conditions as well as during inflammation (Crisman et al., 2004; Lottaz et al., 2007; Lottaz et al., 1999a). Proteolysis of intestinal mucosa is a characteristic feature of IBD and recently several SNPs have been identified in the MEP1A gene which have significant associations with both UC and CD, with a stronger association with the former (Yap et al., 2006). These observations imply a role for meprin A in intestinal homeostasis. Therefore, an experimental model of colitis was used to study the role meprins in the intestinal patho-physiology employing WT and αKO mice. The DSS model of colitis,

46 which recapitulates the symptoms of UC, was chosen for the purpose of this study (Boismenu and Chen, 2000; Okayasu et al., 1990).

3.2 Experimental Procedures 3.2.1 Induction of Experimental Colitis by DSS All the experiments were performed with 8-9 week old male WT and αKO mice on C57BL/6 x 129/Sv background. The mice were housed under conventional nonspecific pathogen free (nSPF) conditions. The experimental and control groups of each genotype were caged separately, with no more than five mice per cage. Colitis was induced in the experimental groups by 3.5% (w/v) DSS (mol. wt 44,000; TDB Consultancy, Uppsala, Sweden) administration in the drinking water for 4 days, followed by normal drinking water. The controls were given normal drinking water throughout the experiment. For recovery studies, a lower dosage of DSS was used: the experimental groups were given 2.5% DSS for four days and the study was carried on for 10 days. The volume of water consumed was monitored. The mice were weighed daily starting from day (-)1 to day 7. An average of day (-)1 and day 0 was calculated which was used to compare the subsequent loss of weight over the remaining period of the study. Weight loss was plotted as a percentage of the weight lost from the start of the experiment. For the recovery study, weight change with respect to the previous day was calculated. Stool formation and rectal bleeding was monitored over the period of study. Rectal bleeding was followed by visualization as well as collecting rectal swabs and testing for blood using hemoccult blood slides (Beckman Coulter). The principle of the test is based on the oxidation of guaiac by hydrogen peroxide to give a blue colored quinone compound. If blood is present in the fecal matter, the heme portion of hemoglobin shows peroxidase activity thereby catalyzing the oxidation of alpha- guaiaconic acid (present in the blood slide) by hydrogen peroxidase (active component of the developer) and forming a highly conjugated quinone compound that is blue in color (Kratochvil et al., 1971). Disease activity index (DAI), giving equal weight to all the three parameters of weight loss, rectal bleeding and stool formation, was calculated for each mouse. The scoring was expressed on a scale of 0 to 4; 0 indicated no disease and 4 corresponding to

47 maximal disease activity (Cooper et al., 1993). Mice were necropsied on day 7 by inhalation of isofluorane followed by cervical dislocation. The entire colon was removed using standard surgical procedures and measured for length before being dissected for sample preparation. A 7-day DSS experiment occasionally led to mortality of αKO mice. Thus, for the time-course experiments, the period was shortened to 5 days. Subsets of WT and αKO mice from both control and experimental groups were killed on days 1 to 5. Blood was collected by cardiac puncture and processed for further studies. Various organs were harvested and flash-frozen for further characterization of inflammation.

3.2.2 Histological Scoring A histological examination was performed on samples of proximal colon from each animal. The samples were fixed as described in the previous chapter, stained with hematoxylin-eosin and evaluated for tissue damage by an investigator blinded to the treatment groups using the validated scoring system of Williams et al (2001) (Williams et al., 2001). The colon sections were scored by Dr. Leo Fitzpatrick, Department of Pharmacology. The scoring system takes into account the severity and extent of inflammation, crypt damage and percent involvement. Based on all four criteria, each section is given an injury score, with higher scores reflecting greater damage.

3.2.3 Myeloperoxidase Assay Colon samples were weighed and assayed for myeloperoxidase (MPO) activity (Bradley et al., 1982; Medina et al., 2003). Myeloperoxidase is essential for oxygen-dependent bactericidal system of polymorphonuclear leukocytes (PMNs) and is hence used as a marker to assess PMN accumulation, which in turn is a hallmark of the early stages of an acute inflammation (Krawisz et al., 1984; Schierwagen et al., 1990). Colon samples were longitudinally opened and washed gently with PBS to remove the fecal matter, weighed and homogenized in 10 volumes of phosphate buffer (50 mM, pH 7.4) and centrifuged at 20,000 x g for 20 min. The resulting sediment was suspended in an equivalent volume of phosphate buffer (50 mM, pH 6.0) containing 0.5% hexadecyltrimethyl-ammonium bromide (HTAB) and 10 mM EDTA, sonicated thrice for 5 sec each, freeze-thawed thrice and incubated at 4 0C for 20 min. Addition of HTAB to the reaction buffer serves two

48 purposes; it solubilises MPO bound to granular membranes and totally inhibits the pseudo peroxidase activity of haemoglobin and myoglobin that may remain associated with the membranous pellet. HTAB also selectively inhibits heme nucleus of hemoglobin and myolgobin without affecting MPO activity (Grisham et al., 1990). The samples were centrifuged at 4000 x g for 15 min and 0.1 μl of the supernatant fluid was added to 2.9 ml of phosphate buffer (50 mM, pH 6.0) containing o-dianisidine dihydrochloride (0.167 mg/ml) and H2O2 (0.0005%) and absorbance was measured at 460 nm for 5 min. One unit of enzyme activity is defined as the amount of MPO that degrades 1 mmol of H2O2 per min at 25 0C.

3.2.4 Collection of blood samples 5 Blood was collected by cardiac puncture using a 25G” /8 needle fitted to a 1 ml syringe. After collection, the blood was transferred to EDTA coated microvette (Sarstedt) and centrifuged at 10,000 x g for 10 min. This resulted in separation of blood into erythrocytes and leukocytes with plasma at the top. The clear plasma was removed into a fresh microcentrifuge tube and stored at -80 0C. Part of it (~100 μl) was filtered using a 10,000 MW cut-off microcon (Millipore) and stored at -80 0C for measurement of serum nitrite levels.

3.2.5 Measurement of Serum Nitrite levels Serum, ultra-filtered with a microcon, was used to measure total nitrite levels using Greiss assay (Cayman Chemicals) following the manufacturer’s instructions. The assay relies on a diazotization reaction first described by Greiss in 1879 (Greiss, 1879), which has since then undergone several modifications. The method involves formation of a deep purple colored azo dye which can be measured spectrophometrically at 540 nm. It is a two step reaction, the first step being conversion of nitrate into nitrite utilizing nitrate reductase. The second step involves nitrosation of sulfanilamide (Greiss Reagent I) by NOx intermediates followed by coupling of the Greiss reagents, sulfanilamide and N-(1- Naphthyl)ethylenediamine (Greiss Reagent II) which gives the colored azo dye product with a λmax at 540 nm (Fiddler, 1977; Nims et al., 1995). Serum samples were assayed in a 96-well format. A nitrate standard curve was generated using the nitrate standard stock

49 provided. Serum samples (25 μl) were added to the sample wells and volume made up to 40 μl using the Assay buffer. Enzyme cofactor (5 μl) followed by Nitrate Reductase (5 μl) was added and the plate was incubated at 25 0C for 3 h. At the end of the incubation period, 25 μl of Greiss Reagents I and II were added, making the total reaction volume 100 μl. The color was developed for 10 min at 25 0C, and the plate was read using a plate reader at A540nm. The standard curve was generated and the nitrite concentration in the serum samples was calculated.

3.2.6 FITC-Dextran Oral Gavage In order to measure intestinal barrier permeability, the mice were gavaged with a permeability tracer, fluorescein isothiocyanate (FITC)-labeled dextran (60 mg/100 g body weight of FITC-labeled dextran, mol wt. 4000; Sigma-Aldrich) (Furuta et al., 2001; Garg et al., 2006; Napolitano et al., 1996). Food and water was withdrawn four hours before the gavage. After four more hours, blood was withdrawn by cardiac puncture as described. Fluorescence intensity of the serum samples was measured using Hitachi F- 2000 spectrophotometer (excitation 490 nm; emission 525 nm). FITC-dextran concentrations were determined from a FITC-dextran standard curve.

3.2.7 Measurement of Colon and Serum Cytokines Colon and serum cytokines in control and DSS-treated mice were measured using mouse Inflammation Cytokine Array (Biolegend), following the manufacturer’s instructions. Colons were homogenized in DMEM containing 10% FBS (0.1 gm/ml). Colon and serum samples were diluted 1:5 in the Dilution Buffer provided with the kit. The assay was carried out in a 96-well format. Diluted colon and serum samples (30 µl) were added to the plate and incubated for 1 h on a shaker. The plates were washed with wash buffer and 30 µl of Detection mix was added and incubated for 1 h. After further washes, the wells were incubated for 15 min with Streptavidin-HRP Buffer and finally 40 µl of Chemiluminescent substrate was added and the plate developed using Biochemi EC3 imaging system (UVP). Intensity of the spots were analysed using the software provided (Quansys Biosciences).

50 3.2.8 Statistical analysis The unpaired, two-tailed t test was used for all statistical analysis; P values of <0.05 at a confidence interval of 95% were considered significant.

3.3 Results 3.3.1 Meprin αKO mice show greater susceptibility to DSS-induced colitis Throughout the 7-day period of the study both the control groups receiving plain water maintained normal progression of their body weights (Figure 3.1A). Weight loss, starting from day 4, was noted in both the experimental groups, which were given 3.5% DSS in their drinking water. The WT mice, by day 7 had lost around 15% of their initial body weight (*,P < 0.03). In contrast, meprin αKO mice showed a loss of more than 25% of their body weight (*,P < 0.03). While discernable weight loss in both the genotypes started on day 4, the slope of weight loss for the αKO mice was significantly steeper than the WT mice (†,P < 0.01, **,P < 0.005). These results indicated that meprin αKO mice were more susceptible to the 4 day DSS treatment than WT mice. When fecal matter of DSS-treated and control mice was monitored for occult blood to assess rectal bleeding, the control groups had occult scores less than 1 (WT values, 0.86 ± 0.14; meprin αKO values, 0.71 ± 0.29) which indicated no bleeding (Figure 3.1B). There was a significant increase in fecal occult blood by 7 days in the mice treated with DSS (*,P < 0.002), with αKO (mean values 2.8 ± 0.37) tending to bleed more than the WT mice (mean values 2.0 ± 0.22). The rectal bleeding in the αKO mice also started as early as day 4, compared to day 6 in WT mice. The DAI, comprising of weight loss, occult blood and stool formation, is often used as a quantitative measure of the degree of injury brought about by inflammation (Cooper et al., 1993). This index was used for the WT and meprin αKO mice in order to quantify the injury suffered (Figure 3.1C). The DAI values were greater for the meprin αKO mice than for WT mice from days 4 to 7. While the DAI score for the DSS-treated meprin αKO mice (1.86 ± 0.3) was significantly higher than controls (0.1 ± 0.1) at day 4 (*,P < 0.0001), the DAI values for the DSS-treated WT mice started

51 Day s 1 2 3 4 5 6 7

5 * * * * * * * * 0 -5 -10 † -15

% Weight Change Weight % -20 ** -25 WT Control WT DSS aKO Control aKO DSS -30 ** ** 3.5% DSS Water

Figure 3.1A Meprin αKO mice severely affected by four day DSS treatment Body weight loss in WT and meprin αKO mice was monitored over a 7-day period. DSS (3.5%) was administered in the drinking water for four days; then replaced with water. Controls for both genotypes received water only. Both WT and meprin αKO DSS groups showed significant weight loss by day 4 (n = 7 mice per group; WT DSS-treated vs control, *,P < 0.03; αKO DSS-treated vs control, *,P < 0.03). The meprin αKO DSS group lost a greater percent of body weight than the WT DSS group (†,P < 0.01; **,P < 0.005).

DSS No DSS

WT * Genotype aKO *

0 0.5 1 1.5 2 2.5 3 3.5 Occult Blood Score

Figure 3.1B Meprin αKO mice have higher occult blood scores at day 7 Upon necropsy on day 7, fecal matter was tested for occult blood. Both WT and meprin αKO DSS groups showed significantly higher bleeding than their corresponding control groups (n = 7 mice per group; *,P < 0.002).

53 4.5 ** ** * 4.0 * 3.5 ** 3.0 WT Con * † ** 2.5 ** WT DSS * 2.0 † ** aKO Con DAI Score DAI 1.5 † ** aKO DSS 1.0 ** 0.5 0.0 1234567 3.5% DSS Water Day s

Figure 3.1C Meprin αKO mice have higher DAI scores WT and αKO controls maintained DAI scores of 0-0.5 over the 7-day period. The DAI for the meprin αKO DSS group was significantly increased starting on day 4 (αKO DSS- treated vs control, *,P < 0.0001), while the WT DSS DAI increased on day 5 (WT DSS- treated vs control, †,P < 0.002). There was a significant difference in the DAI scores of the two DSS-treated groups day 4 onward (**,P < 0.002).

54 increasing only by day 5 (control, 0.3 ± 0.2; DSS, 1.0 ± 0) (†,P < 0.002). On day 5, the DAI for the DSS-treated meprin αKO mice (2.43 ± 0.3) was more than double the score for the WT group (**,P < 0.002). By day 7, the DAI for the meprin αKO group reached a value of 4.0 ± 0.01, accompanied by drastic weight loss, diarrhea and extensive bleeding, while the WT group value was a modest 2.29 ± 0.36. All these parameters together pointed towards the greater susceptibility that the αKO mice have towards intestinal colitis.

3.3.2 DSS-treatment causes greater colon injury in meprin αKO mice To assess the tissue damage caused by DSS-induced colitis, colon lengths were measured (Figure 3.2A). Colons from both the DSS-treated groups showed prominent shortening compared to their respective control populations (WT, *,P < 0.02; meprin αKO, †,P < 0.0001). Nevertheless, the decrease in colon length was significantly greater in the αKO mice than WT (**,P < 0.04). The severity of damage directly correlated to colon shortening, a hallmark of IBD. To obtain a picture of the damage at histological level, sections of proximal colon of all the groups were examined after hematoxylin-eosin staining (Figure 3.2B). Both the WT (i) and meprin αKO (ii) controls showed normal colon morphology; the crypts were straight and the base of the crypt was sitting on muscularis mucosae. After DSS- treatment, marked changes in colon structure were observed on day 7. The DSS-treated WT mice showed some crypt destruction and infiltrating leukocytes (iii). In comparison, meprin αKO mice showed greater damage as evidenced by massive crypt destruction and ulceration (iv). In several of the experimental meprin αKO mice the entire crypt architecture was lost though the surface epithelium was retained. Extensive leukocytic infiltration was seen both in lamina propria and submucosal regions. The colon sections were then scored for injury using a multifactorial scoring system by Dr Leo Fitzpatrick (Williams et al., 2001). As described earlier, the scoring system takes into account the severity and extent of inflammation, crypt damage and percent involvement, based on which an injury score is assigned to each section. The DSS-treated mice had a greater injury score than the controls of both the genotypes (WT = 14.5 ± 2.3

55 No DSS DSS ** † 8 * * 6 † 4

2 Colon length (cms) length Colon 0 WT aKO Genotype

Figure 3.2A Significant colon shortening seen in meprin αKO mice on day 7 The average colon length for both WT and meprin αKO mice administered 3.5% DSS was shorter than their respective controls at day 7 (WT DSS-treated vs control, *,P < 0.02; αKO DSS-treated vs control, †,P < 0.0001). The meprin αKO DSS group had shorter colons than WT DSS group (n = 7 mice per group; **, P < 0.04).

56 i ii

iv iii

Figure 3.2B Meprin αKO mice show greater colon injury than the WT mice Proximal colons from WT and meprin αKO mice were fixed in methyl Carnoy’s, stained with hematoxylin and eosin, and scored for injury. Representative histological sections from (i) WT control, (ii) meprin αKO control, (iii) WT DSS treated, (iv) meprin αKO DSS-treated are shown. Colon sections from both the control groups have normal appearance (i and ii). DSS-treated colon of WT mouse (iii) shows crypt destruction (red arrow) along with leukocytic infiltration in the lamina propria (top black arrow) as well as in the submucosa (bottom black arrow). Greater damage is evident in the DSS-treated αKO section (iv) where massive crypt destruction is seen in an area of ulceration (red arrow). Heavy leukocyte infiltration is evident in the lamina propria and submucosal regions (black arrows).

57 versus 12.6 ± 1.8; αKO = 21.1 ± 1.1 versus 11.9 ± 1.3) (P < 0.01). Moreover the DSS- treated αKO mice but had higher colon injury scores than the DSS-treated WT mice. The injury scores of the DSS-treated groups were normalized to their corresponding control populations in order to assess the degree of colon injury brought about by DSS-treatment (Figure 3.2C). While the WT experimental sections showed 1.14-fold higher scores, the fold-increase for meprin αKO sections was 1.77. This difference was significant between the two groups (*,P < 0.04).

3.3.3 Meprin αKO colons show heightened inflammation In order to assess further the phenotype of greater injury in αKO mice, inflammation of colon was investigated. Inflammation of colonic mucosa was evaluated by MPO assay to assess the infiltration of neutrophils (Figure 3.3). Colons from both the control groups showed negligible MPO activity (WT, 0.37 ± 0.03; αKO, 0.39 ± 0.03) over the 5 day study period. MPO activity of the DSS-treated groups showed marked elevation on days 4 and 5 (*, P < 0.01; #, P < 3x10-8). The DSS-treated meprin αKO mice had significantly greater MPO activity than their WT counterparts (**, P < 0.007). As MPO activity indicates leukocyte infiltration, which in turn is an early event of inflammation, the data reflect a more severe inflammation in the meprin αKO mice. To elucidate further the differences between the WT and αKO mice, as well as understand the inflammatory environment upon IBD induction, colon cytokines were measured using an inflammation cytokine array that quantifies the levels of a panel of 16 cytokines and chemokines. On day 5 of DSS-treatment regime both WT and αKO mice showed greater than 5-fold elevation of IL-1α, IL-1β and IL-6 levels compared to their untreated controls. Comparisons of DSS-treated WT and αKO mice showed that the colon tissues from αKO mice had higher levels of most of the cytokines tested (Table 3.2). Levels of IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-9, GM-CSF, MIP-1α and TNF-α were not significantly different between WT and αKO DSS-treated groups. The other seven cytokines: IL-5, IL-6, IL-10, IL-12, MCP-1, RANTES and IFN-γ were all significantly elevated in the αKO mice compared to the WT mice (**,P < 0.05) (Figure 3.4). Upon DSS treatment, WT mice showed a 4-fold elevation in IL-5 levels, which nearly doubled

WT Genotype aKO *

0 0.5 1 1.5 2 Colon Injury

Figure 3.2C Meprin αKO mice have higher colon injury scores The injury scores of WT and meprin αKO DSS-treated groups were normalized to the corresponding control populations. Colons from meprin αKO mice show significantly greater damage than from WT mice (n = 7 mice per group; *,P < 0.04).

59 6 **† # 5 WT Con WT DSS aKO Con aKO DSS

4 m

3 † MPO U/g 2 * * 1

0 Day 1 Day 2 Day 3 Day 4 Day 5 3.5% DSS Water

# Days

Figure 3.3 Higher inflammation and leukocyte infiltration seen in αKO mice MPO activities of WT and meprin αKO colons from DSS-treated and untreated animals were measured from days 1 to 5. The control mice of both the genotypes showed negligible MPO activity throughout the period of study. The DSS-treated mice had increased MPO activity relative to their controls at day 4 and 5 (n = 6 per group; *,P < 0.00002; †,P < 0.00005). The colon MPO activity of DSS-treated meprin αKO mice was significantly greater than that of the DSS-treated WT mice (**,P < 0.007).

60 Cytokine WT αKO P-value (pg/ml) Control DSS Elevation P-value Control DSS Elevation P-value (DSS grps)

IL-1α 190.78 ± 22.42 3038.67 ± 313.90 15.9 0.001 164.89 ± 25.89 1601.78 ± 419.32 9.7 0.04 0.07 IL-1β 207.22 ± 22.22 1407.22 ± 38.49 6.8 0.00002 207.22 ± 44.44 2518.33 ± 974.49 12.1 0.008 0.38 IL-2 33.34 ± 0.00 51.56 ± 3.64 1.5 0.01 29.70 ± 4.21 58.84 ± 9.17 2.0 0.05 0.55 IL-3 54.04 ± 0.00 102.50 ± 18.56 1.9 0.08 60.50 ± 6.46 117.04 ± 25.49 1.9 0.1 0.67 IL-4 32.61 ± 0.00 69.12 ± 5.75 2.1 0.005 42.57 ± 0.00 92.35 ± 12.85 2.2 0.02 0.21 IL-5 18.28 ± 0.00 78.36 ± 22.94 4.3 0.05 20.99 ± 1.36 162.20 ± 20.40 7.7 0.004 0.05 IL-6 52.00 ± 8.37 1470.71 ± 29.67 28.3 0.00002 60.37 ± 4.18 3056.81 ± 89.00 50.6 0.000008 0.0001 2656.90 ± IL-9 2518.97 ± 0.00 3484.48 ± 158.02 1.4 0.006 5260.34 ± 611.28 2.0 0.02 0.06 137.93 IL-10 254.04 ± 0.0 441.94 ± 52.71 1.7 0.03 233.17 ± 10.44 629.83 ± 0.00 2.7 0.000003 0.03 IL-12 19.64 ± 0.65 39.65 ± 7.68 2.0 0.05 15.74 ± 3.06 86.51 ± 8.15 5.5 0.002 0.01 TNF-α 142.31 ± 6.99 182.52 ± 13.20 1.3 0.05 142.31 ± 6.99 245.45 ± 31.32 1.7 0.04 0.11 GM-CSF 32.63 ± 8.42 58.31 ± 7.56 1.8 0.07 34.96 ± 9.34 75.81 ± 6.07 2.2 0.01 0.12 IFN-γ 216.39 ± 19.34 467.84 ± 52.97 2.2 0.01 332.45 ± 69.74 719.29 ± 73.02 2.2 0.02 0.04 MIP-1α 377.73 ± 87.34 465.07 ± 65.50 1.2 0.5 159.39 ± 43.67 2604.80 ± 842.24 16.3 0.05 0.09 MCP-1 424.70 ± 8.67 992.54 ± 75.45 2.3 0.0014 450.71 ± 8.67 2158.55 ± 39.73 4.8 0.000003 0.00007 RANTES 30.89 ± 0.00 68.73 ± 24.38 2.2 0.2 61.10 ± 46.74 224.39 ± 42.92 3.7 0.05 0.02 Table 3.2 Colon cytokines are elevated upon DSS treatment, with greater inflammation in αKO mice An array of 16 cytokines were tested in WT and αKO mice (n = 5). Cytokines from both the DSS-treated groups show significant elevation. Comparing the WT and αKO mice treated with DSS (last column), 7 cytokines (shown in bold) were significantly elevated in the αKO colon. The rest of the cytokines, with the exception of IL-1α, showed an upward trend in the αKO colon.

61 for αKO mice with a 7-fold increase (Figure 3.4A). While the IL-6 levels in WT were elevated over 25-fold after DSS treatment, the fold increase for αKO mice was 50-fold higher (Figure 3.4B). The other pro-inflammatory molecules, namely IL-12, MCP-1 and RANTES, showed 2 to 3 fold increases in WT mice, and 4 to 5-fold increases in αKO mice (Figure 3.4C-E). Interestingly, both the genotypes upon DSS treatment showed significantly elevated levels of IL-10, an anti-inflammatory cytokine, in the colons (Figure 3.4F). While MIP-1α levels in DSS-treated WT mice showed negligible increase, it was significantly elevated in the αKO mice with a robust 16-fold elevation (Figure 3.4G). Thus it can be concluded that DSS-induced colitis elicits greater inflammation in the αKO mice, thereby making it more susceptible to IBD. As meprin A is expressed in the mouse colon as well as leukocytes, contributions of mucosal versus leukocytic meprins have to be understood before the role of meprins in IBD pathogenesis can be clarified. Therefore the next set of experiments was addressed towards understanding the role of epithelial meprin A towards maintenance of epithelial barrier permeability.

3.3.4 Meprin A is not involved in maintaining the epithelial barrier function To evaluate the role of epithelial meprin A, barrier function was tested on days 5, 6 and 7, when the mice were on water following DSS for 4 days. Mice were administered FITC- labeled dextran by oral gavage and FITC levels in the serum were quantitated after 4 h. As can be seen from Figure 3.5, compared to water-treated controls, both the DSS- treated groups show increased levels of FITC-labeled dextran in their serum suggesting decreased barrier function (WT DSS-treated vs control, *,P < 0.006; αKO DSS-treated vs control, #,P < 0.005). There is no difference in the barrier function between the two DSS- treated genotypes, indicating that lack of meprin A does not affect the integrity of the epithelial membrane.

3.3.5 Mucosal meprin A plays a role in tissue repair and remodeling The previous experiment addressed the role of meprin A in maintenance of epithelial barrier function, but it could not shed light on the possible involvement of this protease in tissue repair. Treating the mice with 3.5% DSS for four days led to an irreversible

62 Figure 3.4 Colon cytokines in αKO mice significantly more elevated than WT mice Colon cytokine and chemokine levels in WT and αKO mice treated with or without DSS were measured on day 5 (n = 5 per group; WT DSS-treated vs control, *,P < 0.03; αKO DSS-treated vs control, #,P < 0.05). (A) Upon DSS-treatment, αKO mice showed greater elevation than the WT mice (**,P, < 0.05). (B) IL-6 levels were markedly elevated upon DSS treatment in both WT and αKO mice; significantly higher values were found in the αKO mice (**,P < 0.0001). (C-E) Higher levels of IL-12 (**,P < 0.01) (C), MCP-1 (**,P < 0.00007) (D) and RANTES **,P < 0.02) (E), were observed in the DSS-treated αKO colons when compared to the DSS-treated WT group. (F) IL-10, an anti-inflammatory cytokine, was also elevated upon DSS-treatment; meprin αKO colons showed significantly higher levels of IL-10 (**,P < 0.03). (G) MIP-1α levels in αKO mice showed a marked increase upon DSS-treatment.

64 100 * 80 † WT-Con WT-DSS 60 * aKO-Con 40 aKO-DSS FITC (ug/ml) * †

20 † † * * * 0 567 # Days

Figure 3.5 Lack of meprin A does not affect intestinal barrier function Mice were given FITC-labeled dextran by oral gavage and FITC levels were monitored in their serum to test epithelial barrier function. The water-treated mice showed low levels of FITC indicating intact epithelial membrane. Compared to the control groups, both the DSS-treated groups showed increased FITC levels in their serum indicating a leaky membrane (n = 7 per group; WT DSS-treated vs control, *,P < 0.006; αKO DSS- treated vs control, †,P < 0.005). There was no difference between the two DSS-treated genotypes.

65 damage from which the mice did not recover. As tissue remodeling can only be addressed during a recovery phase, the experimental set-up had to be modified accordingly. A lower dosage (2.5%) of DSS was used for four days and the period of study extended to 10 days. The lower dosage of DSS caused intestinal damage sufficient enough to generate a visible pathology, but at the same time mild enough for the mice to recover following withdrawal of DSS. When daily weight change was monitored, it was seen that the WT mice given 2.5% DSS lost weight steadily from day 4 to day 6, after which the weight loss slowed down and finally the mice gained weight on days 9 and 10 (*,P < 0.016). The meprin αKO also followed a similar trend but the recovery process was slower and they gained weight only by day 10 (†,P < 0.02) (Figure 3.6A). On day 9 there was a significant difference between the two DSS-treated genotypes with the WT mice showing a weight gain as opposed to the αKO mice which still had not recovered from the DSS-treatment (**,P < 0.004). When barrier function was tested, compared to the controls, both the DSS-treated groups showed significantly higher FITC-dextran values in their serum on day 8 indicating a leaky membrane (n = 7 per group; WT DSS-treated vs control, *,P < 0.02; αKO DSS-treated vs control, †,P < 0.01) (Figure 3.6B). The FITC-dextran values in the WT decreased to levels of control groups by day 9. On day 9, the DSS-treated αKO mice, compared to their control group as well as DSS-treated WT mice, had significantly higher FITC-dextran values (**,P < 0.05). The FITC-dextran levels in αKO mice returned to control levels on day 10. These data indicate that loss of epithelial meprin A impedes the recovery process and hence the αKO mice are slower to recover from tissue damage after colitis. The other aspect to be studied in order to understand the phenotype of meprin αKO mice is the part played by immune cell-derived meprins. As differences in systemic inflammation in the mice will point towards a role for meprins in the leukocytes, experiments were designed to address that question.

3.3.6 DSS-induced colitis elicits greater systemic inflammation in αKO mice Total nitrite levels in the serum of DSS-treated and control mice were measured to gain insight into the degree of systemic inflammation. Nitric Oxide (NO) is synthesized by the

66 # Days 6

4 1 2 3 4 5 6 7 8 9 10 2 ** † WT-Con * † WT-DSS 0 * † * * aKO-Con -2 * aKO-DSS eight Change **

% W * -4 * † † † * 2.5% DSS * Water -6

Figure 3.6A Meprin αKO mice show slower recovery than WT mice The experimental groups were given 2.5% DSS for four days and then replaced with water for the remainder of the study which was carried out till day 10. The control groups were given water throughout the period of study. Weight progression showed while both the DSS-treated groups suffered sufficient damage and elicited a visible phenotype; they were also able to recover by the end of the study (n = 7 per group; WT DSS-treated vs control, *,P < 0.016; αKO DSS-treated vs control, †,P < 0.02). The meprin αKO mice recovered more slowly than the WT mice. On day 9, while the WT group showed a weight gain, the αKO group still showed weight loss (**,P < 0.004).

67 70 * 60

50 WT-Con ** ) † WT-DSS 40 † aKO-Con

30 aKO-DSS FITC (ng/ul 20 ** 10

0 78910 # Days

Figure 3.6B Absence of epithelial meprin A impedes tissue repair and remodeling Mice were given FITC-labeled dextran by oral gavage and FITC levels were monitored in their serum to test epithelial barrier function. The control groups had low FITC values indicating intact epithelial membrane. The DSS-treated groups showed a significant elevation in the FITC levels, which indicated loss of epithelial barrier function. (n = 7 per group; WT DSS-treated vs control, *, P < 0.02; αKO DSS-treated vs control, †,P < 0.01). On day 9, the WT mice given DSS-treatment showed recovery, as seen by return of FITC values to control levels. The αKO mice in contrast had significantly elevated levels of FITC in their serum (**,P < 0.05).

68 enzyme Nitric Oxide Synthase (NOS) by the L-arginine-nitric oxide pathway (Moncada, 1992; Moncada and Higgs, 1993; Moncada et al., 1989). Studies have shown that while under physiological conditions, NO is anti-inflammatory (Miller et al., 1993), however in active colitis, NO is generated in excess and leads to mucosal damage and generates further pro-inflammatory reaction (Ialenti et al., 1992; Levine et al., 1998; Middleton et al., 1993; Miller et al., 1993). The total nitrite levels in control mice of both the genotypes were low (WT, 3.7 ± 0.6 μM; αKO, 2.4 ± 0.3 μM) (Figure 3.7). No significant elevation in nitrite levels were seen in the WT DSS-treated mice till day 5 (*,P < 0.01). By contrast, the nitrite concentration in the DSS-treated meprin αKO group markedly increased by day 1 and remained significantly elevated (†,P < 0.02). The NO values were significantly different between the two DSS-treated groups day 4 onwards (**,P < 0.02). This indicated that the αKO mice showed greater systemic inflammation as a response to the damage caused by DSS. This intuitively leads one to suspect a role for meprins in the immune cells in this process. To further this line of investigation, cytokine levels in the serum of WT and αKO were measured using the previously mentioned inflammation cytokine array as well as IL-18 ELISA. In contrast to the colon picture, both the DSS-treated groups showed significant elevation of only a few of the cytokines compared to their respective control populations (WT DSS-treated vs control, *,P < 0.03; αKO DSS-treated vs control, #,P < 0.05) (Table 3.3). As can be expected, greater elevation of cytokines was observed in the serum of αKO mice treated with DSS compared to their WT counterparts. Comparison between the two DSS-treated groups showed that significant elevation was limited to a set of five cytokines, which included IL-1β, IL-6, MCP-1, RANTES, and IL-18 (**,P < 0.05) (Figure 3.8). IL-1β levels in the DSS-treated WT mice showed a 9-fold increase, while in the αKO mice, the elevation was over 200-fold (Figure 3.8A). Serum IL-6 levels in the WT mice showed over 4-fold increase after DSS-treatment. But in the αKO counterparts, the fold increase was over 16-fold (Figure 3.8B). Likewise, serum IL-18 and MCP-1 showed greater increase in αKO DSS-treated group (Figure 3.8C, D). Surprisingly, while RANTES levels in the DSS-treated WT mice dropped, it showed

69 ** 50 † WT Con WT DSS aKO Con aKO DSS 40

20 † Nitrate (uM) ** † † * † 10 † † † *

0 12345 3.5% DSS Water # Days

Figure 3.7 Meprin αKO mice show higher systemic inflammation than WT mice Serum nitric oxide levels of all the four groups were measured throughout the course of study from days 1 to 5. Both the control groups showed lower levels of nitric oxide in their serum. Compared to its control group, DSS-treated meprin αKO mice showed significantly higher levels as early as day 1, whereas the WT DSS-treated mice showed no significant elevation till day 5 (n = 6 per group; WT DSS-treated vs control, *,P <0.01; αKO DSS-treated vs control, †,P < 0.035). The meprin αKO levels were significantly higher than the WT DSS levels by day 4 (**,P < 0.02).

70 Cytokine WT αKO P-value (pg/ml) Control (n = 7) DSS (n = 13) Elevation P-value Control (n = 7) DSS (n = 15) Elevation P-value (DSS grps) IL-1α 28.3 ± 6.5 32.0 ± 5.2 1.1 0.69 17.8 ± 3. 6 36. 7 ± 4.9 2.1 0.02 0.54 IL-1β 1.3 ± 1.2 12.7 ± 6.7 9.8 0.71 1.15 ± 0.15 261.5 ± 109.2 227.4 0.13 0.04 IL-2 0.04 ± 0.00 0.32 ± 0.00 8.0 0.53 0.8 ± 0.0 4.5 ± 3.7 5.6 0.42 0.30 IL-3 156.3 ± 26.6 199.0 ± 26.9 1.3 0.35 85.2 ± 19.3 160.3 ± 31.5 1.9 0.14 0.37 IL-4 36.0 ± 7.9 49.8 ± 10.2 1.4 0.37 23.8 ± 2.3 41.2 ± 3.9 1.7 0.01 0.43 IL-5 39.5 ± 15.2 46.3 ± 10.1 1.2 0.71 18.6 ± 4.9 58.7 ± 13.6 3.2 0.06 0.48 IL-6 14.4 ± 7.9 66.0 ± 13.6 4.6 0.02 0.0 ± 0.0 260.4 ± 47.5 18.1 0.001 0.001 IL-9 4693.7 ± 713.0 5290.4 ± 268.5 1.1 0.57 4834.2 ± 563.7 5488.1 ± 407.2 1.1 0.37 0.79 IL-10 215.1 ± 49.9 211.5 ± 22.7 1.0 0.94 175.5 ± 13.0 259.0 ± 28.6 1.5 0.07 0.23 IL-12 66.0 ± 7.3 78.7 ± 4.3 1.2 0.13 53.0 ± 6.7 76.3 ± 5.2 1.4 0.02 0.73 IL-18 432.8 ± 21.6 617.2 ± 34.9 1.4 0.0002 404.2 ± 19.9 903.8 ± 52.3 2.2 4.6 x 10-12 0.0007 TNF-α 0.0 ± 0.0 0.0 ± 0.0 -- -- 0.0 ± 0.0 0.0 ± 0.0 ------GM-CSF 9.6 ± 4.1 24.2 ± 6.9 2.5 0.19 3.9 ± 2.6 34.9 ± 9.2 8.9 0.05 0.39 IFN-γ 4968.1 ± 350.2 5445.4 ± 268.5 1.1 0.33 4983.3 ± 525.7 5112.3 ± 378.9 1.0 0.85 0.49 MIP-1α 1836.0 ± 312.2 1730.9 ± 355.7 0.9 0.84 1775.2 ± 343.9 1775.2 ± 244.4 1.0 0.94 0.92 MCP-1 392.6 ± 9.3 438.9 ± 6.7 1.1 0.0008 386.4 ± 7.8 580.7 ± 70.0 1.5 0.08 0.05 RANTES 6.5 ±3.2 2.6 ± 1.2 0.4 0.20 7.9 ± 5.3 17. 9 ± 6.5 2.3 0.37 0.05 Table 3.3 A few serum cytokines show significant elevation in the αKO mice upon DSS administration Sera of WT and αKO mice were tested for 17 cytokines. Comparing WT and αKO mice treated with DSS (last column), 5 cytokines (shown in bold) were significantly elevated in the αKO serum.

71 more than 2-fold elevation in the αKO group (Figure 3.8E). IL-10 levels in the serum remained unaltered upon DSS-treatment (Figure 3.8F). 3.4 Discussion This chapter describes the phenotype of meprin αKO in a DSS-model of colitis and thereby furthers the knowledge about the role of these proteases in intestinal pathophysiology. The studies illustrate that the αKO mice are more susceptible to the DSS model of colitis as indicated by several criteria. The meprin αKO mice showed greater weight loss and had higher DAI scores. When colon damage was scored by histology, the αKO mice had greater injury scores. The colonic tissues of αKO mice also displayed greater inflammation and leukocyte infiltration with elevation of pro-inflammatory cytokine molecules compared to the WT mice. The baseline levels of the 16 cytokines tested were not statistically different between the untreated control groups. While IL-1α and IL-6 showed more than 10-fold elevation in the WT mice upon DSS treatment, in the αKO mice, IL-1β and MIP-1α, in addition to IL-1α and IL-6, were more than 10-fold elevated. The significantly higher IL-6 levels in the αKO directly correlate with the damage to the intestinal mucosa (Atreya and Neurath, 2005). Furthermore, the drastic and differential rise of MIP-1α, a chemokine that boosts the Th1 responses, can explain the early development of the IBD symptomology in the αKO mice (Pender et al., 2005). In humans, Banks et al. have shown a positive correlation for MCP-1 increase in both the epithelium and inflammatory cells of IBD patients (Banks et al., 2003). The doubling of MCP-1 levels in the αKO mice parallels with the increased MPO activity as well as higher tissue damage. In summary, the exaggerated response of meprin αKO mice to DSS indicates that meprin A has a role in the intestinal homeostatis and that the lack of meprin α activity aggravates mucosal inflammation. The genetic background makes a major contribution to the aetiology of IBD. Several instances of strain differences leading to differences in susceptibility to IBD have been observed in mice. Among those, it is of particular interest to note that the C3H/He mice, which are “low-meprin” mice, show greater susceptibility to IBD. This is in contrast to C57Bl/6J and DBA/2J strains, which apart from being “high-meprin α" mice,

72 Figure 3.8 Meprin αKO mice show significant elevation of a few cytokines after DSS-treatment The DSS-treated groups showed increases in their serum cytokine levels compared to their respective water control (n = 7 to 15 mice per group; WT DSS-treated vs control, *,P < 0.03; αKO DSS-treated vs control, #,P < 0.05). (A) IL-1β levels were markedly elevated upon DSS treatment in both WT and αKO mice, with significantly higher values in the latter (**,P < 0.04). (B-D) Significant increases in the levels of IL-6 (**,P < 0.001) (B), IL-18 (**,P < 0.0007) (C) and MCP-1 (**,P < 0.05) (D), were observed in the DSS- treated αKO colons when compared to the DSS-treated WT group. (E) RANTES levels decreased in WT mice upon DSS-treatment, but showed more than 2-fold elevation in the corresponding αKO group (**,P < 0.05). (F) Serum IL-10 levels remained unchanged by DSS treatment.

73 74 are also more resistant to DSS-induced colitis (Mahler et al., 1998). These correlations are not definitive however because these inbred strains differ in many other alleles which may modulate pathogenicity and immune responses. In addition, microarray analysis by Lawrance et al (2001) reported a 3-fold decrease of meprin α mRNA levels in UC patients (Lawrance et al., 2001). It can be seen that the phenotype of increased susceptibility exhibited by the meprin αKO mice in the DSS-model of colitis reported here is in concordance with these known observations. Both the meprin subunits are expressed in the intestine and distributed non- uniformly along its length. While both the meprin subunits are highly expressed in the ileum, their expression reduces in the large intestine with meprin β present at only negligibly low levels in human as well as mouse colon (Bankus and Bond, 1996; Lottaz et al., 1999a). In the ileum therefore, both soluble and membrane-bound meprin A as well as meprin B are present. In the colon though, negligible meprin β expression results in soluble meprin A being the major meprin isoform. The other source of meprins that need to be taken into account while trying to arrive at an explanation for the phenotype reported in this model of colitis is the meprins expressed in the leukocytes. As leukocytes express both the subunits of meprin, all the different isoforms of meprins are present (Crisman et al., 2004). So in the αKO mouse, while there is a lack of epithelial meprin A in the colon, in the leukocytes there is an increase in meprin B levels along with the absence of meprin A. In order to understand the mechanism behind the observed phenotype, comprehension of the contributions of meprins from both these sources is necessary. One of the characteristic features of chronic inflammation is a breakdown of barrier function along with extensive tissue remodeling through increased turnover of ECM components (Bailey et al., 1994). Intestinal membrane integrity was investigated in order to shed light on the role of mucosal meprin A in this phenomenon. The results showed that while lack of meprin A in the αKO mice did not compromise their barrier function, it did impede recovery subsequent to the challenge. This assigns a function for epithelial meprin A in the process of tissue repair and remodeling. Indeed, quite a few proteases of the metzincin superfamily are known to be involved in maintenance of tissue homeostasis. Several MMPs like MMP-9 and MMP-14 have been shown to play major

75 roles in bone-remodeling (Page-McCaw et al., 2007). Of particular interest are the MMP- 14 knockout mice, which show gross defects in connective tissue remodeling. The phenotype of this knockout demonstrates how loss of a matrix metalloprotease leads to decreased ECM remodeling, a phenotype previously thought to be brought about by increased proteolysis (Holmbeck et al., 1999). Other examples include MMP-3 and MMP-7, both of which are involved in wound healing (Bullard et al., 1999; McGuire et al., 2003). MMP-7 aids wound-healing by processing E-cadherin which results in increased epithelial migration (McGuire et al., 2003). von Lampe et al (2000) report upregulation of MMP-1 and MMP-3 leading to ECM remodeling in the inflamed colonic mucosa of patients with IBD (von Lampe et al., 2000). Meprin A itself possesses a known ability to process various ECM proteins like laminin, type IV collagen, fibronectin and nidogen (Kaushal et al., 1994; Walker et al., 1998). This in turn indicates a possible route by which meprin A might carry out a physiological function in tissue-remodeling. IBD initiates as an intestinal inflammation, but as the disease advances there is progressive systemic inflammation. The measurable characteristics of systemic inflammation include NO levels and changes in the cytokine levels in the serum. Since these changes are mediated through leukocytes, a difference in these parameters between the two genotypes will help in explaining the role of meprins in the leukocytes. Hence NO levels as well as serum cytokine and chemokine levels were compared between the WT and αKO mice. Compared to the DSS-treated WT mice, meprin αKO showed increased NO levels. In a panel of 17 cytokines tested five cytokines, namely IL-1β, IL-6, IL-18, MCP-1 and RANTES, were significantly elevated in the meprin αKO mice serum. Interestingly, among these IL-1β, MCP-1 and RANTES are known meprin substrates in vitro. Meprins are known to process various cytokines and chemokines. Meprin A can inactivate MIP-1α and RANTES, both of which are upregulated in IBD (Danese and Gasbarrini, 2005; Norman et al., 2003b; Uguccioni et al., 1999). MCP-1 is also truncated by meprin A, which reduces its ability to activate a major receptor CCR2 (Gong and Clark-Lewis, 1995). On the other hand, meprin B has been shown to activate IL-1β (Herzog et al., 2005). Given this background, the altered serum cytokine profile of the αKO suggests that leukocytic meprins are involved in immune modulation by interacting with cytokines.

76 Meprins have not been reported to interact with either IL-6 or IL-18. Nevertheless, not only does IL-18 share high structural similarity with IL-1β but it also belongs to the same family. Furthermore, both the cytokines are activated by caspase-1 cleavage (Fantuzzi and Dinarello, 1999). This led to the hypothesis that meprins might be able to interact with IL-18. Hence, meprin – IL-18 interaction was studied to test this hypothesis that is dealt with in the next chapter.

77 Chapter 4: The interaction of Meprins with Interleukin-18 4.1 Overview The discovery of interleukin (IL)-18 is relatively recent and elucidation of its role is still in its infancy, unlike other illustrious members of the cytokine family. Okamura et al., in 1982 first reported induction of IFN-γ in the serum of LPS challenged mice treated with Propionibacterium acnes. However, the authors could not explain the mechanism behind their observation (Okamura et al., 1982). Over the next few years, the same group identified and characterized the serum factor responsible and named it IFN-γ-inducing factor (IGIF) (Nakamura et al., 1993; Nakamura et al., 1989; Okamura et al., 1995a). Thereafter investigations gained speed, the protein was cloned and its biological function was studied in greater detail. Bazan et al. (1996) gave the first indication of its relationship with the interleukin family, when it was found that the predicted structure of the protein had striking similarities with the IL-1 family of cytokines (Bazan et al., 1996). In addition to its IFN-γ induction abilities, IGIF was shown to have pleiotropic effects and was later renamed as IL-18 (Ushio et al., 1996). Due to its ability to induce IFN-γ, IL-18 is, by default, a member of the T helper type 1 cell (Th1)-inducing family of cytokines, some of the other members of the family being IL-1β, IL-2, IL-12 and IL-15 (Dinarello, 1999). IL-18 is capable of inducing both CXC and CC chemokines like IL-8, MIP-1α and MCP-1 (Puren et al., 1998). IL-18 can activate the translocation of NFκB in Th1 cells (Matsumoto et al., 1997) as well as enhance proliferation and cytotoxicity of natural killer (NK) cells (Dao et al., 1998; Okamura et al., 1995b; Son et al., 2001). In addition to inducing IFN-γ, IL-18 also induces GM-CSF and suppresses the levels of IL-10 production (Ushio et al., 1996). Enhancement of Fas-ligand expression (Ohtsuki et al., 1997) along with increased intercellular adhesion molecule (ICAM)-1 expression in KG- 1 (Kohka et al., 1998), a macrophagic cell line, as well as in T cells and NK cells is brought about by this cytokine (Dao et al., 1996). Micallef et al (1997) have reported IL- 18 exhibiting anti-tumour activity associated with induction of immunological memory and generation of cytotoxic CD4+ cells in mice (Micallef et al., 1997).

78 While the pleiotropic functions of this cytokine were being increasingly understood, its structure and activation mechanism were also being addressed. IL-18 was found to be very closely related to IL-1β than to any other cytokine (Figure 4.1). Not only do the precursor forms of these two cytokines lack signal sequence, but also they are synthesized in biologically inactive pro-forms (Okamura et al., 1995b). Furthermore, both these cytokines are cleaved by IL-1β-converting enzyme (ICE) at an Asp-X site generating their biologically active mature forms (Fantuzzi and Dinarello, 1999; Ghayur et al., 1997; Gu et al., 1997). Significant similarities have been observed at a structural level as well between these two interleukins. Both IL-1β and IL-18 are primarily composed of β-sheets, a feature that is very uncommon among other cytokine molecules. IL-18 consists of 12 strands, forming three twisted four-stranded β-sheets packed against each other into a β-trefoil fold (Kato et al., 2003). Mature IL-18 binds to IL-18 receptor α (IL-18Rα), followed by binding of IL-18 receptor β (IL-18Rβ). Formation of this IL-18 receptor (IL-18R) complex is essential for IL-18 activity (Dinarello, 1999; Kato et al., 2003). The mature form of IL-18 is stored within the cell, to be released only upon activation. The activity of the mature IL-18 released into the serum is further regulated by IL-18 binding protein (IL-18BP). IL-18BP binds to IL-18 thereby blocking its bioavailability and therefore its function (Novick et al., 1999). As mentioned in the earlier chapter, IBDs are characterized by chronic inflammation of the gastrointestinal tract. It is being increasingly realized that cytokine secretion patterns play a pivotal role in the pathogenesis of the disease (Badolato and Oppenheim, 1996; Boismenu and Chen, 2000). A broad range of cytokines have been implicated in the progression of IBD, including the proinflammatory cytokines like IL-1, IL-2, IL-12, IL-18, IL-23, IFN-γ, TNF and TL1A, as well as the anti-inflammatory IL-4, IL-10 and IL-13 (Bamias et al., 2003; Becker et al., 2003; Li and He, 2004; Reuter and Pizarro, 2004). Several groups have reported increased expression of IL-18 mRNA as well as protein in patients with active IBD (Kanai et al., 2000; Monteleone et al., 1999; Pizarro et al., 1999). The physiological implications of this observation were supported when intestinal damage could be attenuated by blocking IL-18 in various murine models of experimental IBD (Kanai et al., 2001; Sivakumar et al., 2002). The paradigm that IBD evolves in two distinct phases is gaining credence through emerging

79 Murine IL-1β Human IL-18

Figure 4.1 IL-1β and IL-18 share structural similarities IL-1β (PDB ID - 2MIB) and IL-18 (PDB ID – 1J0S) share significant structural similarity (Kato et al., 2003; van Oostrum et al., 1991). Both the members of the IL-1 family of cytokines are primarily composed of β sheets.

80 evidence that shows distinct inflammatory mechanisms mediating early versus late phases of colitis in mice. While the early phase is characterized by increased levels of IL- 12 and IFN-γ, the later stage shows upregulation of Th2 cytokines, IL-4 and IL-13 (Spencer et al., 2002). Though historically IL-18 has been classified as a member of the Th1 family, IL-18 has also been shown to polarize naïve CD4+ cells to produce IL-4 and IL-13 (Yoshimoto et al., 2000). This raises the possibility of IL-18 playing differential roles in the two phases of IBD. Such a possibility can be explained by the fact that a dramatic shift occurs in the cellular source of IL-18 production over the course of the disease. It is produced in intestinal epithelial cells (IEC) in the early phase, and in macrophages (MΦ) and dendritic cells (DC) in the later chronic phase (Pizarro et al., 1999). This type of evidence increasingly supports the contention that IL-18 is a major player in the pathogenesis of IBD. Recently, IL-18 promoter region has been shown to be closely related to the etiology of UC (Rodriguez-Bores et al., 2007; Takagawa et al., 2005). Serum IL-18 levels were significantly elevated in meprin αKO mice compared to the WT mice after colitis induction as mentioned in the Chapter 3. Furthermore, IL-1β, with which IL-18 shares significant similarities, was recently discovered to be a meprin B substrate (Herzog et al., 2005). These observations led to the working hypothesis of this chapter, that meprins interact with IL-18, thereby carrying out a role in modulation of the inflammatory environment.

4.2 Experimental Procedures 4.2.1 Construction of proIL-18 expression vector pCR3.1::IL-18, a eukaryotic expression system for murine proIL-18 was obtained as a gift from Dr. Camille Locht, Institute Pasteur de Lille, France. The proIL-18 gene was subcloned into an Escherichia coli (E.coli) expression system for ease of purification.

4.2.1.1 Generation of pET30b::His6-proIL-18 proIL-18 was inserted into pET30b vector in such a way as to express the protein with an N-terminus histidine tag joined by a linker region containing an enterokinase cleavage site. The IL-18 in pCR3.1::IL-18 had an NcoI site coinciding with its start codon (Kremer

81 et al., 1999). Due to the presence of multiple NcoI sites in pCR3.1 vector, the IL-18 fragment was excised using NheI and EcoRI restriction enzymes. The 630bp NheI-EcoRI fragment was further digested with NcoI. The NcoI-EcoRI fragment was ligated into the NcoI-EcoRI cut pET30b to generate pET30b::IL-18. The resulting plasmid had full- length IL-18 with an N-terminal histidine tag (Figure 4.2A).

4.2.1.2 Generation of pET28a::His6-proIL-18

pET30b::IL-18 expresses His6-proIL-18 with an enterokinase cleavage site in the linker region. However, use of enterokinase treatment to remove the histidine tag resulted in non-specific cleavage of the protein. Hence it became necessary to adopt a different strategy for proIL-18 purification. For this purpose, proIL-18 was further subcloned from pET30b::IL-18 into pET28a which has a thrombin cleavage site between an N-terminal histidine tag and the multiple cloning sequence. To make use of the NdeI restriction site that is in-frame with the histidine linker sequence in pET28a, the NcoI site of pET30b::IL-18 was converted to an NdeI site by site-directed mutagenesis (pET30b::IL- 18-NdeI). This sequence change does not affect the IL-18 protein. Primers 5’-CGA CGA CGA CGA CAA CAT|ATG GCT GCC ATG TCA G-3’ (sense) and 5’-C TGA CAT GGC AGC CAT|ATG TTG TCG TCG TCG TCG-3’ (antisense) were used for this purpose. The PCR conditions were as follows: Buffer - 1X Pfu Turbo DNA Polymerase (Stratagene) pET30b::IL-18 – 5 ng Primers – 0.2 μM each dNTPs – 0.2 mM Pfu DNA Polymerase – 2.5 U/50 μl reaction. The thermal cycling parameters consisted of an initial denaturation step of 95 0C for 2 min followed by 17 cycles of 95 0C, 55 0C and 68 0C for 30 sec, 1 min and 7 min respectively with a final extension at 68 0C for 10 min. An extension period of 7 min was used to ensure proper amplification of the 6 kb pET30b::IL-18 plasmid. In order to digest the parental, methylated and non-mutated DNA, 10 U of DpnI was added to the 50 μl reaction and incubated at 37 0C for 1 h. The plasmid was finally transformed in XL1-Blue supercompetent cells (Stratagene) and plated on LB agar in the presence of Kanamycin

82 NheI NcoI NcoI 6XHis tag NcoI EcoRI proIL-18 enterokinase cleavage site EcoRI pCR3.1-Uni::IL-18 pET30b

NcoI

NcoI fragment release by NheI-EcoRI digestion; NcoI digestion NcoI-EcoRI digestion; ligation NcoI 6XHis tag proIL-18 enterokinase cleavage site EcoRI

pET30b::IL-18

Figure 4.2A Construction of pET30b::IL-18 proIL-18 gene was subloned from pCR3.1-Uni::IL-18, (gift from Dr Camille Locht, Institute Pasteur de Lille, France), into pET30b. proIL-18 fragment was excised by NheI- EcoRI digestion. Following NcoI digestion, the fragment was ligated with the NcoI- EcoRI digested pET30b vector to obtain the final pET30b::IL-18 plasmid. The resulting plasmid had an N-terminal hexa-histidine tag containing an enterokinase cleavage site in the linker region.

83 (50 μg/ml). The clones thus obtained were sequenced in the Core Sequencing Facility to confirm the NcoI to NdeI change and absence of any other sequence variation in the proIL-18 gene. Since the resultant pET30b::IL-18-NdeI plasmid contained two NdeI restriction sites, the proIL-18 fragment was excised using BglII-EcoRI restriction enzymes. The 650 bp fragment thus obtained was further digested with NdeI. The resulting NdeI-EcoRI fragment was ligated into the NdeI-EcoRI cut pET28a to generate the final pET28a::IL-18 plasmid (Figure 4.2B). To reduce the vector self-ligation background, the NdeI-EcoRI cut pET28a was treated with Calf Intestinal Alkaline Phosphatase (NEB) for 40 min at 37 0C and then solution purified. The ligation reaction was transformed into XL-1 Blue supercompetent cells and plated on LB-KanR plate. The colonies on the positive (vector + insert) plate were further screened for confirmation of the correct clone using restriction enzymes KpnI and XbaI. The double digestion will result in linearization of the negative (vector only) clones as the pET28a vector lacks KpnI site, but will cause a release of a 650 bp insert for positive (pET28a::IL-18) clones (Figure 4.2C). The positive clones were further verified by sequencing reaction.

4.2.2 Preparation of BL21(DE3)-RIL Competent Cells A 5 ml primary inoculum of BL21(DE3)-RIL was grown O/N at 37 0C in LB-CamR medium. The O/N culture was inoculated into 200 ml SOB-CamR medium and grown for 0 2 h at 37 C till an O.D.600 of 0.4-0.6 was reached. The cells were centrifuged at 4000 rpm for 10 min at 4 0C. The resulting sediment was suspended in 125 ml of cold 50 mM

CaCl2 and incubated on ice for 30 min. The suspension was centrifuged at 4000 rpm for 0 10 min at 4 C and the pellet was re-suspended in 5 ml of cold 50 mM CaCl2-15% glycerol. Aliquots of 100 μl were flash-frozen in liquid nitrogen and stored at -70 0C.

4.2.3 Induction and purification of His6-proIL-18 In the initial experiments, vector pET30b::IL-18 was transformed into E.coli strains BL21(DE3) and BL21(DE3)-RIL and the transformants were selected in the presence of Kanamycin (50 μg/ml) and Kanamycin-Chloramphenicol (25 μg/ml) respectively. The vector pET28a::IL-18 was transformed into the E.coli strain BL21(DE3)-RIL and the transformants were selected in the presence of Kanamycin-Chloramphenicol.

84 6XHis tag 6XHis tag NdeI proIL-18 EcoRI NdeI NcoI BglII thrombin EcoRI cleavage site pET30b::IL-18 pET28a

NcoI to NdeI mutagenesis NdeI-EcoRI digestion; ligation

NdeI fragment release by BglII-EcoRI digestion; 6XHis tag NdeI proIL-18 NdeI digestion NdeI 6XHis tag proIL-18 BglII thrombin EcoRI cleavage site EcoRI pET30b::IL-18-NdeI pET28a::IL-18

Figure 4.2B Strategy for pET28a::IL-18 construction The NcoI site of pET30b::IL-18 was coverted to an NdeI restriction site to generate pET30b::IL-18-NdeI. Full-length IL-18 fragment was excised by BglII-EcoRI digestion. Following NdeI digestion, the fragment was ligated with the NdeI-EcoRI digested pET28a vector to obtain the final pET28a::IL-18 plasmid. The final construct had an N-terminal hexa-histidine tag with a thrombin cleavage site in the linker region.

1 2 3 4 5

insert release

Figure 4.2C pET28a::IL-18 clone confirmation by KpnI – XbaI digestion Confirmation of pET28a::IL-18 was done by double digestion using KpnI and XbaI. The negative clones (pET28a vector alone) lack a KpnI restriction enzyme site and hence only linearization of the DNA is seen (lanes 1 and 2). The pET28a::IL-18 positive clones released a 650 bp insert (lanes 4 and 5). Lane 3 – 1 kb MW marker.

86 The transformants were inoculated into LB broth with suitable antibiotics and

grown to an O.D.600 of 0.4-0.5, at which point expression of His6-proIL-18 was induced by adding IPTG to a final concentration of 1 mM. Aliquots were taken out 2 h and 4 h post-induction to monitor the induction of the protein. For a large scale purification of the protein, the culture was centrifuged at 5000 rpm for 15 min at 4 0C. Purification of the hexa histidine tagged proIL-18 was carried out using Ni-NTA matrix (Qiagen). The purification was carried out following the manufacturer’s protocol for purification of recombinant proteins under native conditions with slight modifications. An extra wash step of 150 mM imidazole was introduced as a more stringent wash step. All the fractions were analysed on a 12% SDS PAGE. The pure fractions were pooled and dialysed

against a buffer containing 50 mM NaH2PO4, 150 mM NaCl and 10 mM imidazole (pH 8.0) for further processing. Protein concentrations of the dialyzed fractions were determined using Micro-BCA Assay (Pierce).

4.2.4 Thrombin cleavage and proIL-18 purification

His6-proIL-18 was treated with thrombin-agarose, from the Recomt Thrombin CleanCleave Kit (Sigma), to remove the N-terminal histidine tag. The thrombin is immobilized to an agarose resin which circumvents the subsequent need for thrombin removal. The slurry was prepared following the manufacturer’s guidelines, CaCl2 was added to a final concentration of 10mM followed by addition of 1 μg of His6-proIL-18. The reaction was incubated at 25 0C for 8-9 h with gentle agitation, at the end of which, the thrombin resin was spun down and the supernatant removed as two 1 ml fractions. The beads were washed and stored at -20 0C for re-use. The cleavage specificity and efficiency were checked by analyzing the eluates on a 15% SDS PAGE.

The N-terminal linker region and remant His6-proIL-18 were removed by Ni- NTA purification. The eluates were loaded on the Ni-NTA matrix and washed with a buffer containing 50 mM NaH2PO4, 300 mM NaCl and 10 mM imidazole (pH 8.0). The collected fractions were checked for the presence of proIL-18 as well as the loss of full- length His6-proIL-18 by separation on 15% SDS PAGE. Protein concentrations of the fractions of interest were quantitated using Micro-BCA Assay.

87 4.2.5 Activation of recombinant meprins Recombinant histidine-tagged mouse meprin α (homomeric meprin A), rat meprin αβ (heteromeric meprin A) and rat meprin β (meprin B) were used to study the interaction between meprins and proIL-18 (Bertenshaw et al., 2002; Villa et al., 2003). Subsequent to protein concentration calculation, using Micro-BCA Assay, trypsin (1 mg/ml in 20 mM Tris buffer, pH 7.5) was added in 20:1 ratio, and the samples incubated at 37 0C for 1 h. The trypsin activated samples were loaded onto a charged Sephadex G-25 (Sigma) column and spun briefly at 6000 rpm for 6 min. The trypsin was thus removed from the reaction and activated meprins were obtained. The eluate was collected and 2 μl was used to check for meprin α and/or β activity by BK+ or OCK+ assay respectively. BK+ (2-aminobenzoyl-R-P-P-G-F-S-P-F- R-K-(dinitrophenyl)-G-OH), a fluorogenic analogue of bradykinin was used to measure meprin α activity. The volume of the reaction was made upto 298 μl with 50 mM Ethanolamine/HCl (pH 8.7). BK+ was prepared as a 1.5 mM solution in 50% DMSO and used in a final concentration of 10 μM in the final assay mix. Substrate hydrolysis was followed by monitoring the increase in fluorescence in a Hitachi F-2000 fluorescence spectrophotometer using excitation and emission wavelengths of 320 and 417 nm respectively. Meprin β activity was measured by OCK+ assay as described earlier. While one unit of homomeric meprin A and meprin B was regarded as an α-α and β-β dimer respectively, αβ-βα tetramer was taken as a unit for heteromeric meprin A. This ensured equimolar amounts of different isoforms having identical amounts of the individual subunits.

4.2.6 Meprin – proIL-18 reaction Meprin (0.11 μM) and proIL-18 (2.2 μM) were incubated in 1:20 molar ratio in a total reaction volume of 50 μl made up with 20 mM Tris, 50 mM NaCl (pH 7.0) buffer. The reaction was incubated at 37 0C for 0 min, 30 min and 45 min. For actinonin inhibition studies, proIL-18 and meprin B were incubated at 37 0C in 20:1 molar ratio for 10 min, 20 min and 30 min in the presence or absence of actinonin (35 μM). The samples without actinonin had equal volume of 50% DMSO (vehicle control). The reaction samples were

88 subjected to two 15% SDS polyacrylamide gels under reducing conditions. One gel was processed for silver-staining and the other for immunoblotting with mouse IL-18 antibody (Rockland Immunochemicals). For silver-staining, the gel was soaked in a fixative solution (50% (v/v) Ethanol, 12% (v/v) Glacial acetic acid, 0.0185% (v/v) Formaldehyde) for 1 h followed by three 20

min washes in 50% ethanol. The gel was sensitized with 0.02% (w/v) Na2S2O3.5H2O for

exactly 1 min followed by 20 min treatment of 0.2% (w/v) AgNO3 solution containing 0.028% (v/v) formaldehyde. The gel was incubated in the developing solution (3% (w/v)

K2CO3, 0.0004% (w/v) Na2S2O3.5H2O, 0.0185% (v/v) Formaldehyde) till bands became visible and the reaction was stopped by adding the stop solution (1% (w/v) Glycine). Immunoblotting was carried out, as described earlier, using commercially available polyclonal rabbit anti-mouse IL-18 antibodies (1:4000).

4.2.7 Identification of IL-18 site of cleavage For mass-spectrometry analysis of the cleavage product, full-length IL-18 and IL-18 after incubation with meprin B were subjected to 15% SDS PAGE in duplicates. One half was silver stained and the corresponding positions from the unstained lanes were excised and processed further. This circumvented the need for destaining the bands and in-gel tryptic digestions could be done directly. The gel pieces were minced up thoroughly, transferred to a microcentrifuge tube and incubated at 60 0C for 30 min after addition of 30 μl of 5

mM (NH4)HCO3. After removing the solution, the sample was alkylated by addition of 0 30 μl of 55 mM iodoacetamide/20 mM (NH4)HCO3 and incubated at 25 C for 30 min in the dark. This was followed by two 20 min incubations with 100 μl of 50 mM

(NH4)HCO3/50% acetonitrile. Another 20 min wash step was carried out in 75% acetonitrile and the gel pieces were then dried completely. The dried pieces were then 0 incubated in 20 μg/ml trypsin in 20 mM (NH4)HCO3 solution overnight at 37 C. Trypsin was added so as to moisten all the gel pieces. The gel pieces were then treated with 100 μl 50% acetonitrile/0.1% TFA for 20 min to extract the protein into the solution and the solution was transferred to a fresh microcentrifuge tube. For complete extraction, the process was repeated with 80 μl of the extraction solution. The solution was dried completely followed by re-suspension in 10 μl 0.5% TFA.

4.2.8 Kinetic measurements Kinetic measurements of proIL-18 cleavage by meprin isoforms were carried out using quantitative western analysis. Substrate (proIL-18) concentrations ranging from 0.5 μM to 8.0 μM were incubated with 0.1 μM of meprin B or heteromeric meprin A for varying times, separated over a 15% polyacrylamide gel and product formation was quantified using Quantity One software (Bio-Rad). Different enzyme concentrations, at a saturating proIL-18 concentration, were first tested in order to determine catalytic concentration of enzyme. The rates of product formation were plotted against substrate concentration using GraphPad Prism to calculate KM and Vmax. Catalytic constant (kcat) was calculated using the equation:

kcat = Vmax/[Etotal]; and specificity constant (kcat/KM) was then calculated.

4.2.9 Meprin Β and IL-18 interaction in MDCK cells Madin-Darby Canine Kidney (MDCK) cells were grown in minimum essential medium (MEM) with Earle’s salts supplemented with HEPES (0.049 gm/lit), and 10% (v/v) FBS. Full-length rat meprin β cDNA was transfected into MDCK cells grown to 30% confluence in a 24-well plate using Lipofectamine 2000 (Invitrogen). Meprin β was activated by limited trypsin digestion 36 h after transfection. MDCK cells were washed twice with serum-free medium and 10 μl of trypsin (1 mg/ml in 50 mM Tris-HCl, pH 7.5), diluted in 1 ml of serum-free medium, was added and incubated at 37 0C for 30 min. After trypsin removal, cells were washed with serum-free medium, followed by addition of 10 μl of soyabean trypsin inhibitor (STI) (2 mg/ml in water) in 1 ml serum-free medium and incubated for 30 min at 37 0C. MDCK cells were washed again before addition of proIL-18 (1 μg in 1 ml serum-free MEM) and incubated at 37 0C for 22 h. Negative controls underwent the same treatment without trypsin. Culture medium was collected and separated over a 15% polyacrylamide gel and probed with anti-IL-18 antibody. The cells were washed with PBS, sonicated and subjected to electrophoresis using a 7.5% polyacrylamide gel and probed with anti-meprin β antibody.

90 4.2.10 NF-κB activation in EL-4 cells by IL-18 IL-18 bioactivity was tested as a function of NF-κB activation in EL-4 cells. EL-4 cells were maintained in Dublecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) donor horse serum. The cells were washed with PBS and suspended in serum- free medium to a concentration of 1 million cells/ml. proIL-18 and meprin B pre- incubated for 10 min at 37 0C in 20:1 molar ratio was added to final amount of 200 ng/ml proIL-18 and incubated for 30 min. Meprin B alone and proIL-18 alone controls had equivalent amount of respective proteins. Cell-lysates were prepared and total protein quantified using Micro-BCA kit (Pierce). Fifty micrograms of total protein was used for NF-κB activation assay using p65 TransAM kit following the manufacturer’s suggestion. Wells were pre-incubated with 30 μl Complete Binding Buffer to which 50 μg of cell- lysates, made upto 20 μl with Complete Lysis Buffer, was added. After 60 to 90 min incubation, the wells were thoroughly washed and 100 μl of primary antibody (NF-κB p65 1:1000 dilution) was added and incubated for 1 h. After further washes, 100 μl of secondary antibody (Anti-rabbit HRP-conjugated IgG, 1:1000 dilution) was added and incubated for an hour. Developing solution (100 μl) was added and incubated for 4 min in dark and the reaction was stopped with 100 μl stop solution. Absorbance was read at 450 nm using a plate reader. NF-κB activation was calculated as fold-increase over unstimulated EL-4 cells.

4.2.11 ELISA of serum IL-18 Serum samples from meprin αKO and meprin βKO mice along with their corresponding WT mice treated with or without DSS were collected over days 1 to 5, as described earlier. Levels of active IL-18 in the serum were measured using mouse IL-18 ELISA Kit (MBL International) following the manufacturer’s instructions. An IL-18 standard curve was generated. The serum samples were diluted 1:5 with the assay diluent buffer. Standards and samples (100 μl) were added to the 96-well plate provided and incubated at 25 0C for 1 h. At the end of the incubation, the plate was washed with wash buffer thoroughly before adding 100 μl of the Conjugate solution. After 1 h incubation, followed by washes, 100 μl of the substrate was added and incubated for 30 min. The

91 reaction was stopped by adding 100 μl of the stop solution and absorbance read at 450 nm using a plate reader, within 30 min. The serum IL-18 values were calculated from the standard curve multiplied by the dilution factor of 5 to determine the final IL-18 concentration (pg/ml).

4.2.12 Statistical Analysis The unpaired, two-tailed t test was used for all statistical analysis; P values of <0.05 at a confidence interval of 95% were considered significant.

4.3 Results 4.3.1 Generation of recombinant murine proIL-18 In order to study meprin interaction with proIL-18, large amounts of proIL-18 were required. To facilitate the production and purification of proIL-18, the gene was subcloned into a bacterial expression system with a histidine tag as described in the Experimental Procedures of this chapter. Full-length IL-18 DNA was cloned into pET30b to generate pET30b::IL-18. This expression system has an N-terminal hexa histidine tag attached to the gene of interest via a linker containing an enterokinase cleavage sequence for subsequent removal of the fusion tag. The clone was sequence verified and checked to see that the IL-18 start codon was in-frame with the histidine start codon, for proper translation. When proIL-18 induction with IPTG was checked after transformation in E.coli BL21(DE3) strain, no induction was visible (Figure 4.3A, lanes 1-3). One of the drawbacks of recombinant protein expression using the E.coli system is the occurrence of rare codons. Certain tRNAs, frequently occurring in the heterologous protein might be limited in E.coli, resulting in translation arrest. To circumvent such an occurrence, BL21-CodonPlus strains have been engineered that contain extra copies of genes that encode the tRNAs rare in E.coli but frequently seen in the heterologous proteins. BL21-CodonPlus(DE3)-RIL strain contains extra copies of the argU, ileY, leuW tRNA genes which encode for tRNAs that recognize AGA and AGG arginine codons, AUA isoleucine codon and CUA leucine codon respectively. As murine proIL-18 had rare codons, pET30b::IL-18 was transformed into BL21-CodonPlus(DE3)-RIL strain for

IPTG induction. Induction of the His6-proIL-18 was clearly visible using this strategy

92 (Figure 4.3A, lanes 5-6), and the protein was purified using Ni-NTA matrix (Figure

4.3B). Treatment of His6-proIL-18 with enterokinase to remove the histidine tag, led to the generation of multiple bands as visualized by silver staining (Figure 4.4, lanes 1 and 2) as well as immunoblotting with anti-IL-18 antibodies (Figure 4.4, lanes 3 and 4). This indicated that enterokinase cleavage was non-specific and caused further fragmentation of proIL-18. To circumvent this problem, proIL-18 was subcloned from pET30b into pET28a, which joined the histidine tag and the protein of interest via a thrombin cleavage site. After sequence verification, the plasmid was transformed in BL21-CodonPlus(DE3)-RIL strain, induced with 1 mM IPTG (Figure 4.5A), and the protein was purified over Ni- NTA matrix (Figure 4.5B). The pure fractions were pooled and subjected to thrombin cleavage to remove the N-terminal linker region. After thrombin digestion, the fractions were checked for specificity and completion of cleavage by analyzing on 15% SDS PAGE and silver staining (Figure 4.6A). As seen in Figure 4.6A, thrombin cleavage generated a 24 kDa protein, the expected size of proIL-18. The reaction was further

purified over a Ni-NTA column to separate any remaining uncut His6-proIL-18 and the linker region from thrombin cleaved proIL-18 fraction, which would not bind to the Nickel column. The eluates were pooled together and analyzed by SDS PAGE and silver stained to verify removal of the unwanted fragments (Figure 4.6B). Protein concentration of the different proIL-18 fractions were calculated using Micro-BCA protein assay.

4.3.2 Cleavage of proIL-18 by meprins Recombinant homomeric meprin A, heteromeric meprin A and meprin B were activated by limited trypsin digestion. Subsequent to each activation reaction, protease activities of the proteins were measured against their respective fluorescent substrates, and specificity confirmed using actinonin, a meprin selective inhibitor. Activated meprins were incubated with proIL-18 and the products were analyzed by electrophoresis and visualized by silver-staining (Figure 4.7A) as well as immunoblotting with polyclonal anti-mouse IL-18 antibody (Figure 4.7B). It can be seen from Figure 4.7 that while homomeric meprin A (soluble oligomers of meprin α) was unable to cleave proIL-18, both heteromeric meprin A (membrane-bound tetrameric meprin αβ) and meprin B

93 A 1 2 3 4 5 6

His6-proIL-18

0h 2h 3h 0h 2h 3h BL21(DE3) BL-21(DE3)-RIL

B 1 2 3 4 5 6 7 8 9 10 11 12 13

His6- proIL-18

Figure 4.3 Induction and purification of His6-proIL-18 from pET30b::IL-18

(A) Induction of His6-proIL-18 in E.coli Codon Plus strain. There was no induction at the

beginning of IPTG induction in both the strains (lanes 1 and 4). Over-expression of his6- proIL-18 by IPTG induction was not seen even after 2h and 3h in E.coli BL21(DE3) strain (lanes 2 and 3). A strong IPTG induction was seen when pET30b::IL-18 was transformed into E.coli BL-21(DE3)-RIL Codon Plus strain (lanes 5 and 6). (B)

Purification of His6-proIL-18 over Ni-NTA matrix. 1- load; 2- flow-through; 3- 50mM immidazole wash; 4- 150mM immidazole wash; 5 to 13 – eluates in 250mM immidazole.

94 1 2 3 4

His6-proIL-18

fragments

Figure 4.4 Non-specific cleavage seen upon enterokinase treatment

His6-proIL-18 was treated with enterokinase (rEK) to remove the N-terminal histidine tag. As can be seen from silver stain (lanes 1 and 2) and anti-IL-18 immunoblotting

(lanes 3 and 4), fragmentation of his6-proIL-18 is seen after rEK treatment (lanes 2 and 4).

95 A 1 2 3

His6-proIL-18

0h 2h 4h

B 1 2 3 4 5 6 7 8 9 10 11 12 13 14

His6-proIL-18

Figure 4.5 Induction and purification of His6-proIL-18 from pET28a::IL-18

(A) Induction of His6-proIL-18 in E.coli Codon Plus strain. At 0 h (lane 1) there was no endogenous his6-proIL-18. After IPTG induction, his6-proIL-18 over-expression was seen at 2 h and 4 h (lanes 2 and 3). (B) Purification of His6-proIL-18 over Ni-NTA matrix. 1- flow-through; 2- 150 mM immidazole wash; 3 to 14- eluates in 150 mM immidazole.

96 A 1 2 3

His6-proIL-18 proIL-18

- thrombin + thrombin

B 1 2 3 4 5 6 7 8

His6-proIL-18 proIL-18

uncut cut Ni2+ eluates

Figure 4.6 Purification of proIL-18

(A) The His6-proIL-18 obtained was treated with thrombin in order to remove the histidine tag. As seen above, thrombin cleavage resulted in generation of a slightly smaller fragment (24 kDa) that corresponded to proIL-18 (lanes 2 and 3). (B) In order to remove the linker region as well as the uncleaved his6-proIL-18, the fractions were separated over a Ni-NTA matrix. The eluates collected comprised of proIL-18 (lanes 3 to 8).

1 2 3 4 5 6 7 8 9 A

0m 30m 45m 0m 30m 45m 0m 30m 45m homomeric meprin A (mep α) heteromeric meprin A (mep αβ) meprin B (mep β) Figure 4.7 Different isoforms of meprin process proIL-18 differently Homomeric meprin A (0.11 μM) (lanes 1-3), heteromeric meprin A (0.11 μM) (lanes 4- 6) and meprin B (0.11 μM) (lanes 7-9) were incubated with proIL-18 (2.2 μM) in 1:20 molar ratio, incubated for indicated time-points and visualized by silver-staining (A) as well as by immunoblotting using anti-IL-18 antibody (B). Homomeric meprin A does not cleave proIL-18, but both heteromeric meprin A and meprin B can cleave proIL-18 in a time-dependent fashion (lanes 5, 6 and 8, 9 respectively).

98 (membrane-associated dimers of meprin β) showed pro-IL18 cleavage in a time- dependent manner, generating a 17 kDa fragment. Meprin B displayed a greater propensity to cleave proIL-18 than heteromeric meprin A (Figure 4.7A & B, compare lanes 5-6 with 8-9). Specificity of this cleavage was confirmed by carrying the reaction out in the presence or absence of actinonin, an inhibitor for meprins. As can be seen from Figure 4.8, addition of actinonin clearly inhibits the cleavage of IL-18 (lanes 4-6). It is worth mentioning here that with longer incubation a proportional increase in intensity of the 17 kDa band was not seen. This indicated the possibility of meprin B degrading the smaller fragment on prolonged incubation.

4.3.3 Biochemical characterization of proIL-18 cleavage by meprins The kinetics of cleavage of proIL-18 by heteromeric meprin A and meprin B was examined. Rate of product formation (generation of the 17 kDa fragment) over time was

determined by densitometric analysis. The kinetic constants KM and kcat as well as the

specificity constant kcat/KM for the reactions were calculated by directly plotting the rates of product formation against substrate concentration to the Michaelis-Menten equation by

non-linear regression analysis. Meprin B had a significantly lower KM (1.31 ± 0.001 μM) -1 -1 (*,P = 0.004) and higher kcat/KM (5.19 ± 0.47 μM sec ) (**,P = 0.05) for cleaving proIL- -1 -1 18 than heteromeric meprin A (KM = 5.49 ± 0.27 μM; kcat/KM = 3.0 ± 0.19 μM sec ) indicating greater efficiency (Table 4.1).

4.3.4 Identification of IL-18 cleavage site In order to identify the site of proIL-18 cleavage, the products of proIL-18 cleavage by meprin B were separated by a 15% SDS-PAGE. Subsequently, the 17 kDa band was excised, tryptic digested and subjected to analysis by C18 nanoflow followed by MS/MS. Mass-spectrometric analysis was done by Dr. Bruce Stanley. A peak corresponding to the C-terminus of IL-18 was found in the spectrum, indicating that the cleavage removed a fragment from the N-terminal region of the protein. Comparison of the peaks generated against the theoretical peaks expected from tryptic digestion of proIL-18 revealed a 1119 Da fragment which could arise from a semi-tryptic digestion (i.e., a trypsin cleavage on the C-terminus and a non-trypsin cleavage on the N-terminus). The amino acid sequence

99 1 2 3 4 5 6 A

10m 20m 30m 10m 20m 30m

- Actinonin + Actinonin (35μM)

Figure 4.8 Presence of actinonin can inhibit meprin B interaction with proIL-18 Meprin B (0.11 μM) and proIL-18 (2.2 μM) were incubated in 1:20 molar ratio for indicated time-points in the presence or absence of actinonin (35 μM), a meprin selective inhibitor. Addition of actinonin inhibits the cleavage (lanes 4 to 6) indicating specificity as visualized by (A) silver staining as well as (B) immunoblotting using anti-IL-18 anitbody.

100 -1 -1 -1 KM (μM) kcat (sec ) kcat/KM (μM sec )

Meprin B (meprin β) 1.31 ± 0.0013* 6.79 ± 0.55 5.19 ± 0.47**

Heteromeric meprin A 5.49 ± 0.275 16.44 ± 0.24 3.0 ± 0.19 (meprin αβ)

Table 4.1 Kinetic constant determination of proIL-18 cleavage Measurement of kinetic parameters of proIL-18 cleavage by meprin B and heteromeric meprin A were calculated by quantitative western analysis by directly fitting the rate of product formation against substrate concentration to Michaelis-Menten equation. ProIL- 18 (0.5 μM to 8.0 μM) was incubated with meprin B or heteromeric meprin A (0.1 μM) in 20 mM Tris.Cl 50 mM NaCl (pH 7.5) for varying times (0 min to 5 min). Meprin B had significantly lower KM (*,P = 0.004) and higher kcat/KM (**,P = 0.05) than heteromeric meprin A.

101 of the fragment was identified by further subjecting it to CID (collision induced dissociation) followed by MS/MS. Fragmentation in this process is induced by collision and the resulting ions are further analyzed. The masses of ions thus formed are determined and compared with the theoretical masses of ions predicted. This helps to reveal the identities of consecutive ions and thereby the peptide fragment. From the set of y-ions thus generated, the sequence was deduced to be (N) DQVLFVDKR (Q) (Figure 4.9). The cleavage site thus identified is consistent with previously established substrate preferences for meprin B, which prefers acidic residue in its P1’ position (Bertenshaw et al., 2001).

4.3.5 Meprin B and proIL-18 interaction in a cell-culture system The next question addressed was whether meprin B expressed in cells could similarly process proIL-18. Full-length rat meprin β was transiently expressed in MDCK cells and trypsin activated. Subsequent to trypsin activation, proIL-18 was added to the cell culture medium. The cell culture medium was collected after 22 h and the presence of IL-18 was assessed by immunoblotting (Figure 4.10). As can be seen from lanes 1 and 2, no endogenous IL-18 was detected in the cell-culture medium of MDCK cells. Full-length IL-18 was detected intact after 22 h in medium from un-transfected cells, irrespective of trypsin treatment (lanes 3 and 4). Medium from MDCK cells expressing latent meprin B also contained comparable levels of full-length IL-18 (lane 5). However, MDCK cells expressing active meprin B contained considerably less IL-18 in the culture medium, which is clearly apparent in lane 6. It was interesting to note that the disappearance of proIL-18 band was not accompanied with appearance of the 17 kDa cleaved product. This is reminiscent of the recombinant meprin B – proIL-18 reaction where with longer incubation disappearance of the cleaved product was evident. While full-length IL-18 is stable (Figure 4.10 lanes 3 and 4), the 17 kDa product appears to be more unstable. The lack of the smaller fragment may be a result of either meprin B degrading the cleaved product over time, and/or an increased susceptibility of the fragment to the various proteases secreted by the MDCK cells during the period of incubation.

102

caspase-1 putative PR-3 meprin B 10 20 30 40 50 60 MAAMSEDSCV NFKEMMFIDN TLYFIPEENG DLESDNFGRL HCTTAVIRNI NDQVLFVDKR

caspase-3 70 80 90 100 110 120 QPVFEDMTDI DQSASEPQTR LIIYMYKDSE VRGLAVTLSV KDSKMSTLSC KNKIISFEEM

Figure 4.9 Identification of proIL-18 cleavage site Meprin B cleavage site is marked and the nine amino acid sequence identified by MS/MS is shown in bold. Caspase-1 and putative PR-3 sites are also shown in bold and underlined. Region of caspase-3 cleavage is italicized and underlined by dotted line.

103 1 2 3 4 5 6

proIL-18 culture medium

meprin B cell-lysate (latent) meprin B (active)

mep B - - - - + +

trp act - + - + - +

proIL-18 - - + + + +

Figure 4.10 Meprin B expressed in MDCK cells can process proIL-18 Full length rat meprin β cDNA was transfected into MDCK cells and proIL-18 added to the culture medium. Upper panel shows proIL-18 in the culture medium while the lower panel detects meprin B in the cell-lysate fraction. As can be seen in lanes 1 and 2, there is no endogenous proIL-18 detected in the MDCK culture medium. Full-length IL-18 was unaffected by trypsin treatment in MDCK cells un-transfected with meprin β (trypsin untreated – lane 3; trypsin treated – lane 4). The culture medium from cells containing latent meprin B also showed comparable amounts of proIL-18 (lane 5). Considerable loss of proIL-18 is seen in the culture medium from MDCK cells expressing active meprin B (lane 6). Lanes 1 to 4 show no endogenous expression of rat meprin B. Meprin B is seen in both lanes 5 and 6, with the latter showing two meprin B bands corresponding to active and latent forms.

104 4.3.6 Meprin B cleavage results in active IL-18 The crucial issue to address at this juncture to understand the relevance of this interaction was the effect of meprin B cleavage on the function of IL-18. For this purpose NF-κB activity was monitored using the p65 TransAM kit. EL-4 cells were treated with proIL-18 alone, meprin B alone or proIL-18 pre-incubated with meprin B, for 30 min and the cell- lysates were assayed for NF-κB activity. As mature IL-18 is capable of enhancing NF-κB (Kojima et al., 1998), NF-κB activation in this system reflects the activity of IL-18. EL-4 cells treated with proIL-18 alone showed a 1.5-fold increase in NF-κB activity over untreated EL-4 cells. There was a 2.6-fold increase in NF-κB activity in EL-4 cells when proIL-18 pre-incubated with meprin B was added to the cells (Figure 4.11A). The difference between these two values was statistically significant (*,P < 0.02). Comparing the two treatments, meprin B cleaved IL-18 has 1.8-fold higher NF-κB activity than proIL-18 (Figure 4.11B). This clearly indicated that meprin B cleavage of proIL-18 leads to its activation.

4.3.7 Corroboration of meprin B – IL-18 interaction in vivo An in vivo recapitulation of the meprin – IL-18 interaction was essential before trying to assign a physiological function to the interaction. If indeed meprin B were capable of activating serum IL-18, the meprin αKO and βKO mice would be expected to show contrasting serum IL-18 levels. For this purpose, colitis was induced in the meprin βKO and WT mice and serum IL-18 levels were measured. IL-18 levels were also measured in the serum of DSS-treated and untreated meprin αKO and their corresponding WT mice (IL-18 levels on day 5 discussed in chapter 3). While both the WT and meprin βKO mice show elevated levels of active IL-18 upon DSS-treatment (n = 5 per group, WT DSS- treated vs control, *,P < 0.02; βKO DSS-treated vs control, †,P < 0.0002), the elevation is indeed significantly lower in the βKO mice (**,P<0.05) (Figure 4.12A). This is in contrast to what was observed in the αKO mice, which showed significantly higher serum IL-18 than WT mice when subjected to DSS-induced colitis (n = 7 per group, WT DSS-treated vs control, *,P < 0.0002; αKO DSS-treated vs control, #,P < 5 x 10-11; DSS- treated WT vs αKO, **,P < 0.0015) (Figure 4.12B).

105 A NF-κB Activation

4 3.5 3 e * 2.5 2 * 1.5 Fold Increas 1 0.5 0 Jurkat EL-4 proIL mepb proIL + mepb Treatments

B NF-κB Activation

2.5

2 s a e r 1.5 c n I d l 1 Fo 0.5

0 proIL mepb proIL + mepb Treatments

Figure 4.11 Meprin B cleavage results in IL-18 activation as measured by NF-κB activation EL-4 cells were treated with proIL-18 pre-incubated with meprin B, proIL-18 or meprin B alone for 30 min and cells lysates were assayed for NF-κB activation using p65 TransAM kit. Jurkat cells act as a positive control for the NF-κB activation assay. (A) While proIL-18 treament shows 1.5 fold NF-κB activation; a significantly higher NF-κB activation (2.6 fold) is seen with proIL-18 pre-incubated with meprin B shown as fold increase over unstimulated EL-4 cells (*,P < 0.02). (B) NF-κB activation when calculated as fold increase over proIL-18 alone treatment, shows a value of 1.18.

106 A

800 * ** * 600 † † ** WT-Con * 400 WT-DSS † † * * * bKO-Con IL-18 [pg/ml] 200 bKO-DSS

0 345 # Days

Figure 4.12A Active IL-18 levels in meprin βKO mice sera are significantly lower WT and βKO mice treated with 3.5% DSS for four days had increased levels of active IL-18 compared to their respective control groups (n = 5 per group, WT DSS-treated vs control, *,P < 0.02; βKO DSS-treated vs control, †,P < 0.0002). Meprin βKO mice, treated with DSS, showed significantly lower levels of serum IL-18 than WT mice given DSS-treatment (**,P < 0.05).

107 B

1500 ** † 1200 ** † † 900 * WT Con * * WT DSS 600 aKO Con IL-18 (pg/ml) * † * † * † aKO DSS 300

0 345 # Days

Figure 4.12B Meprin αKO mice sera show significant elevation of IL-18 upon DSS treatment In contrast to βKO, meprin αKO mice show significantly elevated levels of IL-18 compared to the WT mice, after the same DSS treatment (n = 7 per group, **,P < 0.0015). Both the DSS-treated groups showed significant elevation in their serum IL-18 levels compared to the respective control populations (WT DSS-treated vs control, *,P < 0.0002; αKO DSS-treated vs control, †,P < 5 x 10-11).

108 4.4 Discussion The work described in this chapter identifies proIL-18 as a novel substrate of meprin B, biochemically characterizes this enzyme substrate interaction and elucidates the functional relevance of this interaction. This also reports the first instance known where different meprin A isoforms exhibit varying degrees of specificities towards the same substrate. While meprin B cleavage results in activation of this immune mediator, it is interesting to note that over time, the mature IL-18 fragment generated also shows progressive degradation. IL-18 is a pleiotropic cytokine with an ability to carry out a multitude of functions. Hence, it is imperative that its activity be tightly regulated. Thus the possibility of meprins activating the pro-form as well as degrading the mature form as a regulatory mechanism is attractive but not surprising. Furthermore, IL-18 appears to be one of the best known meprin B substrates to

date when the kinetic parameters of this interaction are compared with the KM and kcat/KM values for the previously known substrates. Gastrin 17, a regulatory molecule of the gastrointestinal tract, is the best known meprin B substrate with a KM of 7.1 μM and specificity constant of 1.75 μM-1s-1 (Bertenshaw et al., 2001). Not only does meprin B show a greater affinity for proIL-18, but the reaction is also catalytically more efficient, -1 -1 with a lower KM (1.31 μM) and a higher kcat/KM (5.19 μM s ). However, several differences that exist between the previous kinetic analyses and the present study need to be taken into account. The kinetic constants determined by Bertenshaw et al., (2001) were measured using various peptide substrates of meprins as opposed to the larger protein substrate that has been used in the present study. As the peptides have less complicated structures than proIL-18, reaction of the latter with meprins can be expected to involve more protein-protein interactions. On the one hand, absence of a tertiary structure in the case of peptides may increase the accessibility of the substrate to the enzyme catalytic centre, which could favor the interaction. But on the other hand, the more complex set of protein-protein interactions involved in the proIL-18 – meprin interaction may also be involved in improving the cleavage reaction, either directly or indirectly. The second factor that needs to be considered is that while recombinant meprins expressed from human cells (HEK 293) were used to for the present study; earlier studies were done with meprins purified from rat kidney. The two sources of

109 meprins may have differences in their glycan composition. Variations in glycosylation patterns can have an impact on meprin activity, as MBP has been shown to inhibit meprin activity by interacting with the carbohydrate moieties (Hirano et al., 2005). In addition, Bertenshaw et al., (2001) used quantitative HPLC analysis to measure the kinetic constants, whereas meprin – IL-18 interaction kinetics have been investigated using quantitative immunoblotting which is a very different technique. All the above mentioned factors make a direct comparison between the current study and the previous one difficult. Nevertheless, given the remarkably high efficiency observed for meprin B – proIL-18 in this work, proIL-18 indeed appears to be the best meprin B substrate known so far. Although both the members of the IL-1 family, IL-1β and IL-18, are synthesized as inactive molecules and are activated by caspase-1 before being secreted, inactive forms of these cytokines are also found outside the cell indicating the existence of a caspase-1 independent pathway (Fantuzzi and Dinarello, 1999). While many proteases have been identified that can cleave and activate IL-1β (Black et al., 1988; Schonbeck et al., 1998), only a few are known to act upon proIL-18. Caspase-1 and PR-3 cleavage can activate proIL-18, while caspase-3 cleaves both the precursor and mature form of IL-18 to inactivate it (Akita et al., 1997; Ikawa et al., 2005; Sugawara et al., 2001). Here, for the first time meprin B is shown to cleave and activate proIL-18. While the soluble homomeric meprin A showed no detectable activity towards proIL-18, the other two forms could cleave proIL-18. Interestingly, even between the two isoforms differences could be identified in the kinetics of the cleavage. Equimolar amounts of both the isoforms have identical amounts of meprin β and hence the decreased cleavage efficiency of heteromeric meprin A is an inherent catalytic property of this isoform. Therefore, it is conceivable that differences in proportions of various meprin isoforms distributed in the tissue can significantly modulate the IL-18 activity, especially in a meprin knockout mouse where this distribution is skewed and aberrant. The importance of proper distribution of meprin isoforms can be very well illustrated taking the meprin - IL-18 interaction as an example (Figure 4.13). In the WT mice (upper panel), the different meprin isoforms can modulate the IL-18 levels during inflammation. In contrast, in the meprin deficient mice, there is an abnormal distribution

110 Figure 4.13 Model illustrating the differences in levels of active IL-18 in the serum of WT and meprin KO mice. WT mice (upper panel) have all the three meprin isoforms, homomeric meprin A, heteromeric meprin A and meprin B. While homomeric meprin A does not process proIL-18, heteromeric meprin A as well as meprin B cleaves proIL-18 resulting in the release of mature IL-18. In the βKO mice (lower panel; left) only homomeric meprin A is present which is unable to generate IL-18 that results in a decrease in IL-18 levels. In the αKO mice (lower panel; right) there is a possible excess of meprin B. This is reflected as higher turnover of IL-18. Caspase-1 is present and releases a baseline level of active IL- 18.

111 Homomeric meprin A Heteromeric meprin A Meprin B IL-18

P P P P P P

P P P P P cell surface P

cytosol

caspase-1

proIL-18

Homomeric meprin A Meprin B IL-18

P P P P P P P P P P P P P P P cell P P

cytosol

caspase-1

proIL-18

proIL-18 βKO αKO

112 of meprin isoforms which results in differences in IL-18 levels that were observed in the serum of the mice upon IBD induction. For example, in the meprin βKO mice, lack of both heteromeric meprin A and meprin B results in meprins being present exclusively as homomeric meprin A, the isoform incapable of cleaving proIL-18. This is reflected as a drop in the serum IL-18 levels in the βKO mice (lower panel; left). Conversely, the meprin αKO mice are not only bereft of meprin A, but also show an aberrant meprin B distribution with all the meprin β subunits constituting meprin B which can cleave proIL- 18 with a greater efficiency than the heteromeric meprin A. The consequent imbalance can explain the elevation of active IL-18 levels in the meprin αKO mice compared to the WT (lower panel; right). This piece of work, when analyzed with the picture of differences in serum cytokine profile seen between the WT and αKO mice upon colitis induction, lends credence to the earlier hypothesis of leukocytic meprins interacting with cytokines and modulating the immune environment. Furthermore, it clearly demonstrates that a change in the levels in the cytokines, seen in the αKO mice, is a reflection of the distribution of all the meprin isoforms as opposed to the contribution of any single isoform. In turn, this implies that meprin B also contributes to the phenotype of heightened inflammation that is seen in the αKO mice upon colitis induction. To investigate the involvement of meprin B, if any, colitis was studied in WT and αβKO mice, that is described in the following chapter.

113 Chapter 5: The role of Meprin B in the murine model of DSS- induced colitis 5.1 Overview Studying the role of meprins in the intestinal system provides an opportunity to differentiate between the functional contributions of the various meprin isoforms. Expression and distribution of meprins along the intestinal tract is variable. In the colon of the WT mice, due to negligibly low meprin β expression, only homomeric meprin A protease is present. Thus on first inspection, any difference observed between the WT and αKO mice after colitis induction may appear to be a simple situation where the phenotype can be attributed to the absence of meprin A protease in the latter. However, the presence of infiltrating leukocytes, which potentially express both the meprin subunits and hence have all the different isoforms of meprins, confounds such a simplistic conclusion. Therefore it becomes imperative to first differentiate the roles of meprins from these two sources. Experiments discussed in Chapter 3 addressed these concerns in part and showed that while epithelial meprin A definitely had a role in the progression of IBD, meprins in the leukocytes also contributed to the phenotype. The role of leukocytic meprins was investigated by studying the ability of meprins to interact with immune molecules in vitro as well as in vivo. In Chapter 4, the increased bioactivity of IL-18 observed in the serum of the αKO led to the speculation that it was the result of a cumulative effect of the presence of meprin B by itself and the absence of meprin A. This implied a role for meprin B also in the phenotype of heightened susceptibility that the αKO mice exhibit. The natural progression, therefore, was to define the contribution of meprin B in this phenotype. Using meprin βKO mice to determine the role of meprin B in the IBD phenotype would be complicated by the presence of homomeric meprin A in excess. To circumvent this problem, meprin αβKO mice were used, which lacked all the isoforms of meprin. As meprin αβKO mice lacked meprin β in addition to meprin α, comparing the phenotype of the αβKO with the known phenotype of αKO would illuminate the contribution of meprin B in the latter. DSS-induced colitis was induced in WT and αβKO mice, and the

114 phenotype of αβKO was compared with that of αKO mice in order to dissect out the role of meprin B.

5.2 Experimental Procedures 5.2.1 Induction of Experimental Colitis by DSS All the experiments were performed with 8-9 week old male WT and αβKO mice on C57BL/6 x 129/Sv background. The mice were housed under conventional nSPF conditions. The experimental and control groups of each genotype were caged separately, with no more than five mice per cage. Colitis was induced as described in Chapter 3. In brief, the experimental groups were given 3.5% (w/v) DSS (mol. wt 44,000; TDB Consultancy, Uppsala, Sweden) in their drinking water for 4 days, followed by normal drinking water for 3 days. The controls were given normal drinking water during the 7 day period. Stool formation and rectal bleeding was monitored to calculate the DAI scores. The mice were necropsied on different days by inhalation of isofluorane followed by cervical dislocation. The entire colon was removed using standard surgical procedures and measured for length before being dissected for sample preparation. Blood was collected by cardiac puncture and processed for further studies.

5.2.2 Colon myeloperoxidase assay Colon samples were weighed and assayed for MPO activity as described earlier in Chapter 3 (Bradley et al., 1982; Medina et al., 2003).

5.2.3 Collection of blood samples for serum nitrite measurement Blood was collected by cardiac puncture, transferred to EDTA coated microvette (Sarstedt) and centrifuged at 10,000 x g for 10 min to obtain the serum. The serum, ultra- filtered with a 10,000 MW cut-off microcon, was used to measure total nitrite levels using Greiss assay (Cayman Chemicals) following the manufacturer’s instructions as described earlier.

115 5.2.4 Statistical Analysis The unpaired, two-tailed t test was used for all statistical analysis; P values of <0.05 at a confidence interval of 95% were considered significant.

5.3 Results 5.3.1 Meprin αβKO mice show greater susceptibility to DSS-induced colitis Colitis was induced in WT and meprin αβKO mice by DSS administration to examine the contributions of all meprin isoforms in the pathogenesis of IBD. By day 4, both the groups drinking DSS-water lost comparable amounts of body weight (n = 17 mice per group; WT DSS-treated vs control, *,P < 0.0002; αβKO DSS-treated vs control, †,P < 0.002). On day 7, the αβKO mice showed greater weight loss than the corresponding WT group (**,P < 0.024) (Figure 5.1A). DAI scores were calculated giving equal importance to weight loss, stool formation and rectal bleeding. Both the WT and αβKO DSS-treated groups had significantly higher DAI scores than their control groups by day 4 (n = 17 mice per group; WT DSS-treated vs control, *,P < 0.0002; αβKO DSS-treated vs control, †,P < 0.003). The DSS-treated αβKO mice showed significantly higher scores than WT mice on days 6 and 7 (**,P < 0.0008) (Figure 5.1B). Tissue damage caused by DSS-induced colitis was also assessed by measuring colon lengths (Figure 5.2). Both the DSS-treated groups showed significant shortening of colon (n = 6 mice per group; WT DSS-treated vs control, *,P < 6.5 x 10-7; αβKO DSS-treated vs control, †,P < 1.4 x 10-4). Comparing the two DSS-treated groups, the αβKO mice showed greater colon shortening compared to the WT (**,P < 0.03). Taken together, the data indicate that the αβKO mice are more susceptible to colitis than WT mice. In order to assess the damage in αβKO with respect to the αKO mice, the DSS- treated WT groups corresponding to the two KO genotypes were compared. Damage and weight loss were found to be similar in these two WT subsets, enabling comparison of αKO mice with the αβKO mice upon IBD induction. Interestingly, when weight loss and DAI scores are employed for a comparison with the αKO mice, αβKO appear to be more resistant to colitis. By day 7, αKO mice had lost more than 25% of their body weight

116 # Days

1 2 3 4 5 6 7 † † 5 † † † † * * * * 0

-5 * †

-10 * † -15 * * † % Weight Loss % Weight -20 WT-Con WT-DSS abKO-Con abKO-DSS aKO-DSS ** -25 †

-30 3.5% DSS Water

Figure 5.1A Meprin αβKO mice show greater susceptibility to DSS-induced colitis than WT mice Body weight loss in WT and meprin αβKO mice was monitored over a 7-day period. DSS (3.5%) was administered in the drinking water for four days; then replaced with water. Controls for both genotypes received water only. Both WT and meprin αβKO DSS groups showed significant weight loss by day 4 (n = 17 mice per group; WT DSS- treated vs control, *,P < 0.0002; αβKO DSS-treated vs control, †,P < 0.002). The meprin αβKO DSS group lost a greater percent of body weight than the WT DSS group (**,P < 0.024).

117 4.5 4 3.5 † 3 † ** WT-Con ** 2.5 WT-DSS † DAI 2 abKO-Con 1.5 † ** abKO-DSS * 1 aKO-DSS * 0.5 0 1234567 # Days 3.5% DSS Water

Figure 5.1B Meprin αβKO mice have higher DAI scores WT and αβKO controls maintained DAI scores of 0-0.5 over the 7-day period. The DAI for the WT and meprin αβKO DSS group significantly increased day 4 onwards (n = 17 mice per group; WT DSS-treated vs control, *,P < 0.0002; αβKO DSS-treated vs control, †,P < 0.003). There was a significant difference in the DAI scores of the two DSS-treated groups on days 6 and 7 (**,P < 0.0008). DAI score for αKO mice treated with DSS was higher than αβKO mice throughout the period of study.

118 ** † 8 *

* 6 † No DSS 4 DSS

Length [cms] 2

0 WT abKO Genotype

Figure 5.2 Significant colon shortening seen in meprin αβKO mice on day 7 The average colon length for both WT and meprin αβKO mice administered 3.5% DSS was shorter than their respective controls at day 7 (WT DSS-treated vs control, *,P < 6.5 x 10-7; αβKO DSS-treated vs control, †,P < 1.4 x 10-4). The meprin αβKO DSS group had shorter colons than WT DSS group (n = 6 mice per group; **,P < 0.03).

119 compared to about 20% weight loss seen in the αβKO mice (Figure 5.1A). When DAI scores were compared, αKO had plateaued at the score of 4.0, the maximum possible in this scoring system, while the αβKO was still lower at 3.6 (Figure 5.1B). This mild, albeit significant attenuation of the colitis phenotype in the αβKO compared to the αKO indicated a hitherto undefined role for meprin B in the IBD phenotype. As meprin β expression in mouse colon is negligibly low, it is possible that meprin β expressed in the leukocyte is involved in the mediation of this function. With this question in mind, inflammation and leukocytic infiltration were compared between the WT and αβKO mice.

5.3.2 Meprin αβKO do not exhibit a higher degree of inflammation than WT Inflammation and leukocytic infiltration were investigated by measuring colon myeloperoxidase activity (Figure 5.3). The colons of both the control groups had negligible MPO activity. On day 3, there was no detectable elevation of MPO activity in the DSS-treated groups. By day 5 both the groups had significantly higher MPO activities than their corresponding control populations (n = 6 mice per group; WT DSS-treated vs control, *,P < 0.0003, αβKO DSS-treated vs control, †,P < 0.0005). However, there was no difference in the degree of inflammation between the WT and the αβKO mice as seen by this assay. This was in contrast to the αKO mice, which showed significantly higher MPO activity than WT on day 5 (see Figure 3.3). The inflammatory environment was further characterized by measuring the cytokine levels in the WT and αβKO colons (Table 5.1). The majority of cytokines in DSS-treated mice showed significant elevation over their water treated controls (n = 10 mice per group; WT DSS-treated vs control, *,P < 0.03, αβKO DSS-treated vs control, #,P < 0.05). When differences between the two DSS-treated genotypes were examined, IL-6 was the sole cytokine that appeared to be significantly elevated in the αβKO mice (**,P < 0.03) (Figure 5.4). This indicates that upon DSS-treatment, while the colons of both the genotypes are inflamed, there is not much difference in the inflammatory environment between the two genotypes.

120 16 14 †

m * WT-Con 10 WT-DSS 8 abKO-Con MPO U/g 6 abKO-DSS 4 * 2 † 0 35 # Days

Figure 5.3 WT and meprin αβKO mice show comparable inflammation Inflammation and leukocytic infiltration was measured by MPO activity in the colons of the WT and αβKO mice treated with or without DSS. While both the control groups had negligible MPO activity, significant elevation was seen in both the DSS-treated groups on day 5 (n = 6 per group, WT DSS-treated vs control, *,P < 0.0003, αβKO DSS-treated vs control, †,P < 0.0005). The MPO activity between WT and αβKO mice on DSS- treatment was comparable on day 5.

121 Cytokine WT αβKO P-value (pg/ml) Con (n = 5) DSS (n = 10) P-value Con (n = 5) DSS (n = 10) P-value (DSS grps) IL-1α 164.94 ± 5.43 1165.91 ± 213.44 0.03 157.77 ± 0.00 1472.59 ± 259.13 0.03 0.46 IL-1β 43.28 ± 3.85 186.87 ± 29.30 0.02 31.40 ± 4.44 273.54 ± 64.25 0.13 0.33 IL-2 0.00 ± 0.00 0.00 ± 0.00 -- 0.00 ± 0.00 0.00 ± 0.00 -- -- IL-3 12.56 ± 6.06 5.58 ± 2.32 0.31 0.00 ± 0.00 65.27 ± 27.02 0.25 0.11 IL-4 103.55 ± 12.22 154.53 ± 19.99 0.21 103.55 ± 7.46 154.53 ± 18.23 0.19 0.10 IL-5 46.39 ± 5.66 61.95 ± 3.00 0.05 39.90 ± 4.11 65.84 ± 3.53 0.01 0.51 IL-6 0.00 ± 0.00 735.80 ± 139.80 0.02 0.00 ± 0.00 2649.08 ± 611.40 0.05 0.03 IL-9 2584.54 ± 245.50 3730.37 ± 221.31 0.03 2678.29 ± 178.35 7105.37 ± 1265.32 0.11 0.06 IL-10 0.00 ± 0.00 139.67 ± 6.19 0.0000001 102.47 ± 14.61 172.23 ± 12.86 0.05 0.1 IL-12 17.57 ± 1.84 87.98 ± 16.12 0.04 32.82 ± 7.90 55.56 ± 4.68 0.07 0.13 TNF-α 0.00 ± 0.00 0.00 ± 0.00 -- 0.00 ± 0.00 0.00 ± 0.00 -- -- GM-CSF 38.19 ± 0.00 50.41 ± 4.76 0.26 23.55 ± 6.96 65.34 ± 6.44 0.01 0.14 IFN-γ 406.95 ± 138.75 664.65 ± 72.15 0.16 358.33 ± 62.77 971.51 ± 141.54 0.06 0.13 MIP-1α 0.00 ± 0.00 66.25 ± 31.96 0.28 0.00 ± 0.00 236.32 ± 125.90 0.36 0.3 MCP-1 449.09 ± 0.00 1029.45 ± 179.81 0.16 449.09 ± 0.00 1208.02 ± 222.15 0.17 0.62 RANTES 0.00 ± 0.00 372.64 ± 56.15 0.004 0.00 ± 0.00 218.59 ± 34.07 0.01 0.16 Table 5.1 Colon cytokines are elevated upon DSS treatment in both the genotypes An array of 16 cytokines were tested in WT and αβKO mice. Cytokines from both the DSS-treated groups show significant elevation (shown in bold). Comparing between the WT and αβKO mice treated with DSS (last column), only IL-6 (shown in bold) level is heightened in the αβKO mice.

122

** 3000 #

2000 WT-Con WT-DSS # 1000 * abKO-Con abKO-DSS * # Conc [pg/ml] 400

# 200 # * # * # * * * # 0 * 3 6 0 2 1 α S -γ L- L- -1 -1 P- -1 E N I I IL IL C IP T IF M M AN R Cytokines

Figure 5.4 IL-6, the sole cytokine that shows significant elevation in the αβKO mice The DSS-treated groups showed increase in their colon cytokine levels compared to their respective water control on day 5 (WT DSS-treated vs control, *,P < 0.03; αKO DSS- treated vs control, #,P < 0.05). Only IL-6 levels in the DSS-treated αβKO mice showed significant elevation compared to the corresponding WT group (**,P < 0.03).

123 Systemic inflammation, measured in terms of total nitrite levels in the serum, reflects the leukocytic involvement in the processes of colitis development and progression. While both the genotypes on water showed no elevation in their NOx levels, WT mice on DSS-treatment showed elevated nitrite values on day 5 (n = 12 per group, WT DSS-treated vs control, *,P < 0.0006) (Figure 5.5). Unexpectedly, even by day 5, the αβKO mice on DSS-water showed no increase in their NOx levels. Not only was this difference significant between the two groups (**,P < 0.03), the trend was in direct contrast to the phenotype that was observed in the αKO mice, which showed higher systemic inflammation, as seen by significantly higher NOx values (see Figure 3.7).

5.4 Discussion The purpose of this study was to dissect out the role of meprin B, if any, in the phenotype of increased susceptibility that is observed in the αKO mice upon DSS-treatment. The working hypothesis for this set of experiments was that if meprin B had a minimal contribution to the phenotype of colitis, the αβKO mice would show a phenotype that was similar to the αKO mice. While αβKO mice did show a phenotype that was similar to the αKO mice in that they were more susceptible to colitis than their WT counterparts, significant and interesting differences were observed. Even when the αβKO mice resembled the αKO mice in showing greater weight loss, they were less vulnerable than the latter. Further differences became evident when this phenotype was investigated at the molecular level. One of the most conspicuous features of the phenotype exhibited by the αKO mice was exacerbated inflammation at the tissue as well as systemic levels in comparison to the WT counterparts. Interestingly, the αβKO mice, while being more susceptible to DSS-induced colitis, did not show significantly higher inflammation than the WT mice. The observation that there was less inflammation in the αβKO mice evinced a role for meprin B in modulating the host response seen in the αKO mice. Negligible levels of meprin B in the rodent colon combined with the fact that a difference in the phenotype of inflammation indicates involvement of leukocytes; strongly implicated leukocytic meprin B in this process. Previous studies in our laboratory have reported the presence of meprin

124 120 ** * 100

80 ** WT-Con 60 * WT-DSS abKO-Con Nitrite (uM) Nitrite 40 abKO-DSS

0 35 # Days

Figure 5.5 Systemic inflammation in WT mice higher than that seen in αβKO mice Serum nitric oxide levels of all the four groups were measured during the course of study on days 3 and 5. Both the control groups showed lower levels of nitric oxide in their serum. Compared to its control group, DSS-treated WT mice showed significant elevation only on day 5 (n = 12 per group, WT DSS-treated vs control, *,P < 0.0006). Surprisingly, the αβKO mice did not show any serum nitrite elevation even on day 5, a difference that was significant (**,P < 0.03).

125 B in the leukocytes (Crisman et al., 2004). Similar levels of myeloperoxidase activity observed in the WT as well as αβKO colons suggested that neutrophil and/or macrophage infiltration, in response to DSS treatment, in both these genotypes was comparable. Thus, increased infiltration that was evident in the αKO mice can be explained by the presence of higher levels of meprin B in some of the leukocytic populations. Further differences became apparent when the cytokine profiles between WT and αβKO mice were compared with that seen between αKO and their corresponding WT populations. Most of the cytokine levels in the colons of the WT and αβKO mice were similar with the exception of IL-6 which was significantly elevated in the latter. In contrast, most of the cytokines in the αKO colons showed elevation over the WT levels, in further support of the phenotype of heightened inflammation. IL-6, originally identified as a B-cell differentiation factor, is now known as a multifunctional cytokine with pleiotropic functions (Hirano, 1998; Hirano et al., 1986; Teranishi et al., 1982). Involvement of IL-6 in immune response, hematopoeisis, acute phase response and inflammation has been well-documented (Hirano, 1998). As IL-6 is expressed not only by the infiltrating T cells, macrophages and B cells but also by the endothelial cells, it plays an important role in the pathogenesis of IBD apart from its role in promoting inflammation (Jirik et al., 1989; Jones et al., 1993; Stevens et al., 1992). Patients with active IBD have been reported to have increased levels of this cytokine (Ishihara and Hirano, 2002). Furthermore, mice deficient in IL-6 are resistant to the DSS- induced colitis (Suzuki et al., 2001). IL-6 was the only cytokine that showed consistent elevation in the DSS-treated colons of both the αβKO and αKO mice. This may reflect greater tissue damage and inflammation that sets the stage for development of the chronic phase of IBD. Unexpectedly, while the WT mice on DSS-water showed elevated NOx levels on day 5, the nitrite levels in the serum of αβKO mice were significantly lower, perhaps reflecting the lower levels of IL-1β and TNF-α. Nitric oxide is synthesized by the L- arginine nitric oxide pathway (Moncada, 1992; Moncada and Higgs, 1993). Nitrate and nitrite is formed from L-arginine by macrophages (MΦ) upon activation (Marletta et al., 1988; Palmer et al., 1988; Stuehr and Marletta, 1985). Hence, lower nitrite levels in the αβKO mice reflect a decrease in the degree of MΦ activation. Meprin B is present in MΦ

126 and the lack of meprin B impairs the ability of leukocytes to migrate in vitro (Crisman et al., 2004). Taken together, this opens up the possibility of a role for meprin B in the pathway of MΦ activation. MΦ activation plays an important role in the quality, duration as well as the magnitude of an inflammatory reaction. Classical activation pathway in MΦ leads to production of a milieu of pro-inflammatory cytokines along with secretion of nitric oxide molecules that in turn cause tissue damage and microbicidal activity. On the other hand, the alternative activation pathway does not result in NO production (Rutschman et al., 2001). MΦ activated thus cannot kill intracellular microbes; these MΦ are involved in ECM repair and tissue homeostasis (Duffield, 2003; Gordon, 2003; Mosser, 2003). A decrease in NO production therefore indicates impairment in MΦ activation by the classical pathway, which in turn can explain the phenotype of diminished inflammation in the αβKO mice. In conclusion, the phenotype exhibited by the αβKO mice showed that while absence of meprin B attenuated the phenotype of injury and inflammation, it was unable to compensate for the lack of meprin A. Furthermore, the most striking differences between the αβKO and αKO mice were seen in the degree of inflammation in these two genotypes. These observations indicate a role for leukocytic meprin B, in the process of modulating injury and inflammation in the DSS-induced colitis model of IBD.

127 Chapter 6: Conclusions and Discussion 6.1 Overview 6.1.1 Meprins: similar yet different from MMPs Meprin research has travelled a long distance, from merely being a metalloendopeptidase present in the renal tissue, to now being found in multiple tissues including the intestinal villi, leukocytes, lung and skin. Significantly, associations with various pathologies are coming to light for both the meprin subunits. While significant SNPs have been identified in the meprin β (MEP1B) gene in association with diabetic nephropathy in the Pima Indian population (Red Eagle et al., 2005), more recently meprin α (MEP1A) has been implicated in both UC and CD (Banerjee et al, unpublished). Meprins are closely related to MMPs, in that both are members of the metzincin superfamily. The MMP family of proteases is among the most extensively researched families and while meprins, their less-studied cousins, share numerous similarities, significant differences also exist. MMP levels are low in normal healthy tissues, with upregulation observed during repair and inflammatory processes. Meprins, conversely, are highly expressed in physiological conditions and meprin downregulation has been associated with several disease conditions such as acute renal injury (Ricardo et al., 1996). This is highly suggestive of a role for meprins in tissue homeostasis. The other marked difference is inability of endogenous MMP inhibitors, TIMPs, to inhibit meprin activity (Kruse et al., 2004). However, both MMPs and meprins are capable of degrading ECM proteins in vitro and have been traditionally thought to play a role in ECM turnover, degradation as well as destruction. It has also been shown in vivo that some MMPs do indeed cleave ECM proteins (Asahi et al., 2001; Lochter et al., 1997; Zhou et al., 2000). Interestingly, the majority of MMPs have also been implicated in a myriad of, often unanticipated, functions related to immune response and host defense. Almost all the MMP-knockout mice, except MMP-14, show no phenotype under normal physiological conditions (Parks et al., 2004). However, upon challenge the scenario changes drastically. The MMP-deficient mice reveal a multitude of phenotypes that implicate them in a variety of processes such as tissue repair, angiogenesis, host defense and inflammation (Balbin et al., 2003; Itoh et al., 1998; Warner et al., 2001). This is reminiscent of the meprin-knockout mice, which under normal physiological conditions

128 show only minor phenotypes, but when challenged respond in ways different from their WT counterparts. While the striking similarities between meprins and MMPs imply overlapping functions for these proteases, differences in their expression patterns during health and disease speak of a unique role for meprins in the human and rodent physiology. This thesis attempts to investigate the function of meprin proteases in the intestinal physiology utilizing meprin KO mice. DSS-induced colitis model was used as a tool to elicit intestinal inflammation in order to probe the role of these proteases in intestinal pathophysiology.

6.1.2 Advancement of meprin knowledge 6.1.2.1 Meprin redundancy Meprin αKO mice are fertile and show minimal, if any, developmental or growth defects. This establishes that meprin A is not essential and lack of meprin A is not lethal. Nevertheless, a requirement during development is suggested by a significant reduction in litter size. Meprins belong to the astacin family of proteases that also includes the various eukaryotic hatching enzymes. Thus it is conceivable that meprins might also have a role in embryogenesis. The fact that meprin αKO mice do not show an overt phenotype under normal conditions indicates redundancy of meprin proteases in the normal functioning of the animal. It is clear that various proteases have overlapping substrate preferences. Thus compensation may take place seamlessly in the case of critical functions. Redundancy of function is very important from the point of view of evolution of life. It is a safeguard by which stability and viability of life is maintained in face of mutations or abnormalities alternating protein functions. In such scenarios, the KO mice may appear to be normal when unchallenged, but a phenotype is often discernable when subjected to situations of stress. Proteins perform multiple functions, and under a condition of stress failure of an otherwise compensated for function may surface. At the beginning of this study, a role for meprins in the intestinal system was considered quite plausible owing to its high expression profile in both human and mouse intestines (Bankus and Bond, 1996; Lottaz et al., 1999a).

129 6.1.2.2 Meprins in the intestine The foremost conclusion that can be drawn from the experimental colitis model used here is that meprin αKO mice are more susceptible to colitis than their WT counterparts. The αKO mice showed greater damage and mortality when subjected to the DSS treatment. This phenotype was characterized by massive tissue damage accompanied with exacerbated inflammation. As has become apparent from the results discussed in the previous sections, the phenotype observed is in fact an amalgam of different meprins participating in multiple ways from more than one location. The etiology of IBD is complicated as it involves multiple players not only from the gut epithelium but also from the immune system. Meprins, with their diverse distribution in the intestinal epithelial cells as well as in the infiltrating leukocytes can be envisaged to affect the progress of IBD pathogenesis via multiple pathways. While meprins are abundant in the intestine, mucosal meprin A, surprisingly was found not to have a role in maintaining the intestinal barrier integrity. However, the lack of meprin A does impede the recovery process. This translates to greater tissue damage that is seen in both meprin αKO and meprin αβKO mice and appears to be the mode by which lack of mucosal meprin A contributes to the heightened susceptibility of these mice to DSS induced colitis (Figure 6.1). Such a simplistic explanation fails to adequately describe the phenotype exhibited by the αβKO mice, which, though more susceptible than WT mice in terms of weight loss and DAI, is less vulnerable than the αKO mice. To explain this interesting difference between phenotypes, one needs to take into account the tissue resident macrophages and the epithelial cells secreting immune mediators that make up the inflammatory environment of the colon. Meprin B is present in the macrophage population (Crisman et al., 2004), and has the potential to interact with the cytokine and chemokine molecules, thereby modulating the immune environment. When the inflammatory environment of the colon was tested using an array of cytokine molecules, meprin αKO mice showed a clear phenotype of exacerbated inflammation. The αβKO mice colons exhibited a tissue inflammation profile with striking similarity to the WT profile; notwithstanding the higher susceptibility the former had to DSS induced colitis. Hence, mucosal repair and tissue damage due to inflammation taken together translates to the

130 Figure 6.1 Absence of meprin A results in greater tissue damage upon DSS treatment The epithelial membrane of WT, αKO and αβKO mice colon are intact under normal physiological conditions (upper panel). Only WT colon secretes homomeric meprin A ( ). DSS administration results in injury to the intestinal epithelial membrane (lower panel). This insult causes inflammation and influx of leukocytes along with release of cytokines ( ) and chemokines ( ). Cytokine release is greater in DSS-treated meprin αKO mice than WT or meprin αβKO mice. In WT colon, presence of mucosal meprin A aids in tissue repair and remodeling thereby containing the damage. In the meprin αKO and the αβKO, lack of mucosal meprin impedes the recovery process leading to greater injury.

131 lumen epithelial mep A (α) lumen lumen

mep A mep B(β) mep (αβ) leukocytes leukocyte leukocyte blood vessel blood vessel blood vessel

colitis induction by DSS administration lumen lumen lumen

immune molecules

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132 final phenotype that is observed in the three genotypes. The αKO mice have slower recovery, coupled with higher inflammation that results in massive tissue destruction and crypt damage. The αβKO mice show impaired tissue remodeling, but also have reduced inflammatory reaction, which is reflected as a more subdued phenotype (Figure 6.2). Though the studies carried out implicate meprin A in tissue repair, additional experiments are required in order to understand the mechanism(s) involved. Meprins can interact with E-cadherin (personal communication, D. Lottaz) which is a central molecule in facilitating epithelial cell migration during wound repair (Dunsmore et al., 1998). MMPs have been shown to modulate re-epithelization and tissue repair by direct interaction with E-cadherin (Dunsmore et al., 1998; McGuire et al., 2003). Investigating the ability of meprin A to promote wound-healing in vitro and in vivo may provide meaningful insights. Cell-lines, such as MDCK, stably expressing meprin A can be used for in vitro experiments. Colon explants from WT and meprin αKO mice can be another in vitro tool. Experimental wound healing models with the various meprin KO mice will be an interesting in vivo system to address similar questions.

6.1.2.3 Meprins expressed by immune-mediators Another aspect that merits further attention is the meprin distribution in different leukocytic populations. Results of experiments presented in Chapter 4 of this thesis have led to the suggestion that meprins interact with cytokine molecules. The meprin – IL-18 interaction was studied in great detail, and serves as a proof of principle in this case. Meprins can be envisioned to act in an autocrine manner, thereby modulating the immune response. The interaction of meprins with IL-18 can be expanded to explain the observation of heightened cytokines in the αKO serum. Meprin proteases expressed in the leukocyte can lead to either activation or inactivation of different immune mediators. These processes in concert contribute to shape the inflammatory environment upon injury (Figure 6.3). As can be seen from the upper panel, WT leukocytes express all the different meprin isoforms. As examples for two extremes of the spectrum of actions possible, upon colitis induction, meprin A inactivates RANTES, and meprin B activates IL-1β and IL-18. The immune environment is therefore modified as a net result of

133 Figure 6.2 Meprin αKO mice colons show higher degree of damage The colons of WT, meprin αKO and meprin αβKO show injury as a result of DSS administration which leads to an inflammatory response (upper panel). Meprin B, present in the tissue resident MΦ of WT and αKO colons, also attribute to the progression of inflammation. When DSS is withdrawn (lower panel), the presence of mucosal meprin A aids the WT mice to recover faster. The αKO, which suffer from slower recovery coupled with higher inflammation, is most susceptible to colitis. Though the αβKO mice also have impaired recovery, it also has reduced inflammation. So it enjoys slightly better protection than the αKO mice against colitis, but remains more vulnerable than WT mice due to lack of epithelial meprin A.

134 lumen lumen lumen

immune molecules

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WT colon αKO colon αβKO colon

135 α α α β β α β β β β β β α α α β β α β β

WT leukocyte αKO leukocyte αβKO leukocyte

colitis induction after treatment with DSS

IL-1β IL-12 IL-12 IL-1β IL-12 IL-1β

RANTES RANTES RANTES

IL-18 IL-18

IL-18

inflammation inflammation inflammation

Figure 6.3 Meprins in the leukocyte modulate the inflammatory environment by interacting with the immune mediators Leukocytes in the WT mice (first panel) express all the different meprin isoforms. Upon colitis induction, cytokine activation as well as inactivation is brought about by a combined action of meprin A and B. For example, RANTES is inactivated by meprin A and IL-1β and IL-18 are activated by heteromeric meprin A as well as meprin B. This is manifested as systemic inflammation. In the leukocytes of the meprin αKO mice (second panel), only meprin B is present. This leads to anomaly in cytokine activation or degradation. For example, lack of meprin A results in RANTES remaining active. IL-1β and IL-18 activities are also enhanced by the presence of greater amounts of meprin B, resulting in heightened inflammation that is characteristic of the αKO. In the αβKO leukocytes (third panel), none of the meprin isoforms are present. Thus while RANTES activity is enhanced, IL-1β and IL-18 activities are diminished. This causes subdued inflammation akin to the WT phenotype. IL-12 serves as an example of a cytokine which gets activated upon intestinal insult, but is not affected directly by meprin isoforms.

136 activities of all the meprin isoforms. As can be seen in the αKO mice in the middle panel, due to lack of meprin A, all the meprin β subunits form meprin B. This is reflected as RANTES retaining its activity and greater degree of IL-1β and IL-18 activation, which together result in greater inflammation. The meprin αβKO mice, as shown in the panel to the right, lack all meprin isoforms. In this case, while RANTES is active, IL-1β and IL- 18 activities are decreased. Considering these cytokines as examples, it is evident how meprin expression can be critical in defining the immune environment of the mice. Nevertheless, to show unequivocally the role of immune-cell derived meprins in the observed phenotypes, reciprocal transfer experiments between WT and the αKO mice using bone marrow transplantation is ideally required.

6.1.3 Possible roles for meprins 6.1.3.1 Meprin distribution in different leukocytic populations Continuing along the lines of the immuno-modulatory roles of meprins via their expression in the leukocytes, it is evident that leukocyte recruitment and their further interaction with the immune mediators form a major component of the phenotype that is observed in IBD. While meprins have been detected in the lamina propria leukocytes in humans and in the mesenteric lymph nodes in mice, little is known about which sub- populations express meprins (Crisman et al., 2004; Lottaz et al., 1999a). Meprin B is + - present in CD11b as well as in CD11b populations. Meprin A, on the other hand, was + shown to be absent in the CD11b population of leukocytes (Crisman et al., 2004). CD11b is a cell-surface marker expressed in most natural killer (NK) cells, as well as mature neutrophils and monocytes/macrophages (Landay et al., 1983; Sanchez-Madrid et al., 1983; Springer et al., 1978; Springer et al., 1979). Meprin B was further shown to be present in the MΦ populations (Crisman et al., 2004). Hence it follows that meprin A is - present along with meprin B in the CD11b population, which consists mostly of T-cells. Thus it becomes evident that while certain leukocytic sub-populations will not be affected by the loss of meprin A (as it was absent in the first place); other sub-populations will see a change in the distribution of the meprin isoforms in the meprin αKO mouse. The process of inflammation involves substantial cross-talk between the different leukocytic

137 populations, raising the possibility of meprins modulating the immune environment in a paracrine fashion in addition to the autocrine manner.

6.1.3.2 Meprin involvement in MΦ - T cell cross talk In this context, the total nitrite levels in the serum of DSS-treated mice present an intriguing picture and draw further attention. While the αKO mice show a significant rise in NO production after DSS treatment in line with the observation of heightened injury and inflammation, αβKO mice had NO levels which were surprisingly lower than even the WT levels. As mentioned earlier, NO production is mainly brought about by MΦ, activated in response to IL-1β and/or TNF-α stimuli. One possibility, that has already been touched upon in Chapter 5, is that meprin B is involved in activating MΦ, and hence its lack results in decreased NO production in the αβKO mice. But this phenomenon alone is not able to explain the increased levels of NO that are seen in the αKO mice, in particular since MΦ lack meprin A. There are reports of extensive cross-talk between T cells and MΦ during inflammation. The presence of meprin B in the MΦ and predicted localization of both the meprins in the T cell population encourage interesting and novel speculations. The CD4+ Th1 cells characteristically produce IFN-γ, which in turn activates the MΦ to produce NO (Deng et al., 1993; Ding et al., 1988; Tian et al., 1995). NO can also influence T cell differentiation into various sub-populations. At low doses, NO enhances the differentiation of precursor T cells into Th1 cells which are major players in the inflammatory cascade (Niedbala et al., 1999). Thus there exists a NO – Th1 amplification cycle that plays a major role in promoting inflammation. But, mice lacking iNOS show an enhancement in their Th1 response, indicating the existence of a negative feedback mechanism (Huang et al., 1998; Wei et al., 1995). Thus NO fine tunes the immune homeostatis by upregulating Th1 production at low doses to mount an immune response, and subsequently at high doses dampens the very same response to protect the self against injury. This negative feedback is partly brought about by inhibition of IL-12 synthesis. As IL-12 is a major driving force in Th1 differentiation, a decrease in IL-12 results in a decrease in Th1 population (Trinchieri, 1995). In addition, NO also induces a population of regulatory T cells (Treg), by a mechanism still unknown, that are CD4+

138 CD25+ and are suppressive in nature (Niedbala et al., 2006). These immuno-suppresive cells have been shown to play a role in inhibition of murine colitis (Read et al., 2000; Siegmund et al., 2001). Figure 6.4 shows a simplified schematic explaining the MΦ – T cell interaction that modulates the inflammatory response. Inspecting the molecular parameters in the αKO mice after DSS-induced colitis, one can see significantly elevated levels of NO. This phenotype of greater inflammation has two implications. On the one hand, there maybe a stronger Th1 amplification signal, or on the other hand, the negative feedback mechanism might be impaired in the αKO - mice. As mentioned earlier, CD11b cells, which mainly comprise the lymphocytic population, are meprin A as well as meprin B positive. Thus absence of meprin A as well as the presence of meprin B alone can affect this loop. One possibility maybe impaired Th1 inhibition by Treg cells in the αKO mice during inflammation. This would result in a constitutively active positive loop resulting in heightened inflammation (Figure 6.5-1). Inhibition of IL-12 synthesis is one of the ways in which this inhibition is brought about. Although a defect in this pathway is another mechanism, it is unlikely as MΦ is the site of synthesis of IL-12 and they lack meprin A. But an inbihition by some indirect signaling can not be ruled out (Figure 6.5–2). Yet another plausible pathway where a change in meprin distribution may have an effect is in the conversion of resting T cells (CD4+ CD25-) to Treg (CD4+ CD25+) cells (Figure 6.5–3). A first step towards addressing these sets of questions would be a detailed and thorough investigation of distribution meprin subunits in the different leukocyte sub- populations. This can be done by immuno-staining different leukocytic populations with specific markers and identifying meprin co-localization. A clearer picture of meprin distribution in the leukocyte populations will help in designing further experiments aimed at dissecting meprin involvement in the cross-talks involved.

6.1.3.3 Meprin signaling in innate immune response Meprins present in the different leukocytic populations may be involved in cleaving different immune or second messengers which then bring about further changes in the signaling pathways. Meprin involvement in intracellular cell signaling via the cytosolic tail of meprin β is another interesting possibility. In this context, the phenotypes

139 4 pro-inflammatory IFNγ IL-12 Th1 2

MΦ 1

NOlow 6

anti-inflammatory NOhigh 3 Treg 5

T cell

CD4+ CD25+ + - CD4 CD25 Figure 6.4 Modulation of inflammatory response by MΦ – T cell cross-talk MΦ and T cells are major players in contributing to the inflammatory response. (1) MΦ produce IL-12 and NO, both of which induce T cell differentiation into Th1 cell-type which in turn produce IFNγ, thus promoting inflammation (2). IFNγ production in turn activates MΦ to produce higher levels NO, thereby establishing a positive amplification cycle (3). High levels of NO inhibit IL-12 synthesis, thereby dampening Th1 differentiation (4). It also induces CD4+ CD25- resting T cells to differentiate into CD4+ CD25+ regulatory T cell (Treg) by an unknown mechanism (5). Treg cells are immuno-suppressive in nature and dampen the immune response (6).

140 IFNγ IL-12 Th1

MΦ

NOlow 1

2 NOhigh 3 Treg

T cell

CD4+ CD25+ + - CD4 CD25 Figure 6.5 Possibilities of meprin involvement in the different steps of MΦ – T cell cross-talk to modulate inflammation Inhibition of Th1 cells by Treg might involve meprin A. In the meprin αKO absence of this protein may lead to constitutive activation of this loop resulting in increased inflammation. (2) Increased levels of NO inhibit IL-12 synthesis by MΦ. Meprin A involvement in this step, though improbable, cannot be ruled out. As meprin A is not present in the MΦ population to begin with, a direct involvement seems unlikely. (3) Another route by which NO inhibits the pro-inflammatory cycle is by converting resting T cells to regulatory T cells which are immuno-suppressive in nature. The mechanism of this conversion is still not known and meprins being involved in this process cannot be ruled out.

141 exhibited by the myeloid differentiation primary response protein 88 (MyD88)-KO mice are of utmost interest. The MyD88-deficient mice show a phenotype that mimics the αKO mice not only in the DSS-induced colitis model, but also the endotoxin model that is currently being studied in the laboratory (personal communication, R.E. Yura) (Araki et al., 2005; Kawai et al., 1999). MyD88 is a cytoplasmic adaptor molecule with an N- terminal death domain (DD) and a C-terminal Toll/IL-1 receptor (TIR) domain. Through its TIR domain, it interacts with the TIR domains of toll-like receptors (TLR) as well as IL-1 and IL-18 receptors. MyD88 recruits IL-1 receptor associated kinase (IRAK) via homophilic DD-DD interaction which leads to further cell-signaling that ends with NF- κB activation (Figure 6.6) (Akira and Takeda, 2004). MyD88 is an important molecule in the TLR signaling pathways which are hallmarks of innate responses. Another point of interest that must be noted here is that while the meprin αKO mice showed increased susceptibility at a dosage of 3.5%, the MyD88-deficient mice had a more severe susceptibility at a dosage as low as 1.2%. This implies that MyD88 involvement lies further downstream to meprin involvement in the signaling cascade. Meprin β lacks a TIR domain, which is necessary for a direct interaction with MyD88. Thus it points most likely to an indirect interaction. In addition, soluble meprin A may bind or activate an, as yet unknown receptor triggering one of the many MyD88-dependant signaling pathways. Existence of any of the above mentioned mechanisms would imply meprin involvement in the innate immune system in a paracrine fashion. Although on first inspection a direct meprin B – MyD88 interaction does not seem to be possible due the lack of a TIR domain, in vitro experiments to test MyD88 interaction with the cytoplasmic tail of meprin β will be valuable in validating the hypothesis.

6.2 Closing remarks The aetiology of IBD is complicated as it involves multiple players not only from the gut epithelium but also in the immune system. Meprins, with their diverse distribution in the intestinal epithelial cells as well as in the infiltrating leukocytes can affect the progress of IBD via multiple pathways. While epithelial meprin A affects the outcome of IBD by impeding tissue repair, mucosal as well as leukocytic meprins might be capable of modulating the immune environment, thereby regulating inflammation and injury. Thus

142 Figure 6.6 MyD88 involvement in TLR signaling TLR stimulation leads to MyD88 association via the TIR domains. MyD88, in turn recruits IRAK via DD interaction. IRAK activation leads to further signaling that ultimately results in NF-κB activation (adapted from Akira and Takeda, 2004).

143 the phenotype that is seen in the meprin KO mice is a result of a combination of inputs from all these players. The work reported in this thesis achieves identification of two novel physiological functions for the meprin metalloproteases: a role in tissue repair and in immuno-modulation. One of the most interesting features of meprins is their unique patterns of oligomerization. Thus meprin α and β subunits, in spite of being independently expressed are intricately connected as far as protein distribution and eventual function are concerned. Instances of extremely complicated signaling networks with innumerable checks and cross-checks to fine-tune the proper functioning are not unknown in human or rodent physiology. Meprin balance may be another example of how the two subunits, α and β form, through intricate associations, isoforms that counter-balance each other and ultimately modulate the immune environment. In conclusion, meprins are a novel example of how inactivation of one subunit results not only in loss-of-function of one isoform, but also a gain-of-function of other isoforms giving rise to phenotypes which are a combination of both.

144 Appendix: Characterization of meprin α expression in a human hepatocellular carcinoma cell-line, HepG2 A.1 Overview The role of proteases in cancer development and progression has been long documented. Porteases function in various stages of the disease including initiation, neovascularization, intravasation as well as extravasation (Keppler and Sloane, 1996; Rochefort et al., 1996; Wu et al., 1997). There have been several reports about involvement of MMPs and meprins, members of the metzincin superfamily, in cancer (Lin et al., 1997; Matters et al., 2005). Meprin expression has been detected in various colon and breast cancer cell-lines (Dietrich et al., 1996; Matters and Bond, 1999a). It has been shown that meprins cleave ECM proteins like fibronectin and collagen-IV, and thus may contribute to the ability of cancer cells to metastasize (Kruse et al., 2004). Meprin α is normally secreted from the apical surface of polarized colon cells. However in Caco-2 cells, a colon carcinoma cell-line, meprin α was found to be secreted from both apical and basolateral plasma membrane along with increased enzymatic activity. A consequential increase in proteolytic activity was observed in the stromal compartment (Lottaz et al., 1999b; Rosmann et al., 2002). There is some evidence that points to enhanced expression of meprins in the metastatic phase of cancer. For example, the pattern of meprin α expression in the two colon cancer cell-lines, SW480 and SW620, that have been extensively used as a model for colon cancer progression (Hewitt et al., 2000). The SW480 cell-line was established from a primary colon tumor, while SW620 represents a metastatic tumor cell-line derived from the same individual (Leibovitz et al., 1976). When meprin α expression patterns were investigated in detail in this pair, the metastatic and invasive SW620 cell-line showed significantly higher meprin α expression than the non- metastatic SW420 cell-line (Bond et al., 2005). Hepatocellular carcinoma is a malignancy of the liver and is one of the causes of cancer related death. The prognosis for this cancer is very poor with a 5 year survival rate of less than 5 percent (El-Serag and Mason, 1999). This is largely due to late detection of the tumor and lack of reliable early markers. Elevated expression of meprin α has been detected in different kinds of cancers (Matters et al., 2005). The

145 present study was initiated with the aims of identifying the expression levels of meprin α in hepatocellular carcinoma and investigating any role of the protease in cancer growth and invasiveness using the two hepatocellular carcinoma cell lines HepG2 and HuH-7. HepG2 cell-line was established from liver tumor biopsies of a 15-year old Caucasian male with primary hepatoblastoma from Argentina in 1975 (Aden et al., 1979). HuH-7 cell-line was established from a 57-year old Japanese man with hepatocellular carcinoma (Nakabayashi et al., 1982). Both of these cell-lines do not express hepatitis B virus surface antigen and are non-metastatic in nature (Knowles et al., 1980; Nakabayashi et al., 1982).

A.2 Experimental Procedures A.2.1 End-point PCR Total RNA was extracted from HepG2 cells grown to confluence using TRIzol (Invitrogen) and subsequently subjected to RT-PCR with the Superscript One-Step RT-PCR kit (Invitrogen). The amount of amplicon at various cycles was analyzed by using integrated band density data obtained with the Stratagene Eagle-Eye II system equipped with Eagle-Sight software. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as a control. Meprin α was amplified using primers 5’- ATCGGAGGCACGGCTGGCGT-3’ and 5’-GCCTGCCCTCATGGAGCTTACAG- 3’, which gave a 380-bp product.

A.2.2 Immunoblotting of HepG2 culture medium Levels of meprin α protein in the HepG2 and Huh7 culture medium were tested using quantitative western analysis. Huh7 culture medium was concentrated nearly 10-fold using Centricon 50,000 (Millipore). HepG2 and Huh7 culture medium was subjected to 5% polyacylamide gel electrophoresis under non-reducing and denaturing conditions, transferred to nitrocellulose membrane and probed with polyclonal rabbit anti-recombinant meprin α antibody. Horseradish peroxidase-conjugated anti-rabbit secondary antibodies (GE HealthCare) were detected by chemiluminescence using the SuperSignal Dura substrate (Pierce).

146 A.2.3 HepG2 growth assay HepG2 growth was monitored over a period of 6 days in the presence and absence of actinonin, a meprin inhibitor. Each well was seeded with 0.06 x 106 cells at day 0 and the total numbers of cells were counted at every time-point in triplicates. The cells were treated with trypsin and cells were counted using a hemocytometer. Two concentrations of actinonin (25 nM and 50 nM), dissolved in 50% DMSO, were used for the inhibition study. Vehicle control corresponding to the higher concentration of actinonin was also used to rule out the effect of DMSO on cell-growth, if any.

A.2.4 Matrigel assay Invasiveness of HepG2 cells was investigated using the matrigel invasion assay following the manufacturer’s instructions (BD Biosciences). The control and matrigel inserts and wells were equilibrated with serum-free DMEM for 2 h in humidified 0 tissue culture chamber at 37 C, 5% CO2. After medium removal serum-free DMEM, with or without chemoattractant and/or inhibitor, was added to the wells. Hepatocyte growth factor (HGF) at a final concentration of 100 ng/ml was used as a chemoattractant. Actinonin (5 μM) served as meprin α inhibitor. HepG2 cells (0.05 x 106), suspended in serum-free DMEM, in the presence or absence of actinonin, were added to the pre-equilibrated inserts and incubated in the humidified tissue culture 0 chamber at 37 C, 5% CO2 for 48 h. After incubation, the non-invading cells were removed from the upper surface of the insert by scrubbing and the lower surface stained using Hema 3 stain and 10 randomly selected fields /filter were counted. Percent invasion was calculated as follows: % Invasion = (mean # of cells invading through the matrigel insert) x 100 (mean # cells migrating through the control insert)

A.3 Results A.3.1 HepG2 cells over-express meprin α mRNA and protein To assess the level of meprin α expression in hepatocellular carcinoma cells, meprin α mRNA and protein levels were compared in two hepatocellular carcinoma cell- lines, HepG2 and Huh7. End-point PCR showed that, meprin α mRNA levels in HepG2 was several fold higher than Huh7 (Figure A.1). Equal amounts of amplicons were observed for GAPDH, the loading control. When protein levels were determined for these two cell-lines, the same trend was observed. Meprin α protein was readily

147 detected from HepG2 culture medium, but Huh7 media, even after 10-fold concentration did not show detectable amounts of meprin α protein (Figure A.2). Thus, it was concluded that HepG2 cells, but not Huh7 cells over-expressed meprin α mRNA as well as protein. The next set of experiments was designed to understand the relevance of the observed meprin α over-expression.

A.3.2 Meprin A inhibition does not affect HepG2 growth Functional relevance of the high levels of meprin α expression was investigated by monitoring HepG2 growth after inhibition of meprin A activity. Equal density of HepG2 cells were seeded in the presence and absence of increasing concentrations of actinonin, a meprin selective inhibitor, and progression in cell densities was assessed by estimating the cell counts every 24 h over a period of 6-day. Addition of actinonin did not affect the growth rate of the HepG2 cells (Figure A.3).

A.3.3 HepG2 cell invasiveness is unaffected by the loss of meprin A activity A role for meprin A in determining the invasiveness of HepG2 was studied using the matrigel invasion assay. HepG2 cells showed increased invasiveness in the presence to HGF, a hepatocyte chemoattractant, irrespective of the state of meprin activity. Even in the presence of actinonin, i.e. inhibition of meprin A activity, no discernible inhibition of invasion was seen (Figure A.4). This indicated that meprin activity was not required for HepG2 invasion in vitro.

A.4 Discussion While extracellular proteases are known to play a role in different stages of cancer cell progression, the extent of their importance as well as the exact functions are still unclear (Egeblad and Werb, 2002; McCawley and Matrisian, 2000). Various MMPs have been implicated in tumor invasion and metastasis (Woessner, 1991). In addition, MMP-2 and MMP-9 levels have been shown to directly correlate with the metastatic potential of many malignant cells (Liotta et al., 1991; Sato et al., 1992). Meprin expression has been detected in various cancer cells (Lottaz et al., 1999b; Matters et al., 2005). Furthermore, increased meprin A activity has been associated with the metastatic state of the cancer cells (Bond et al., 2005).

148 HepG2 Huh7 Standards HepG2 Huh7

GAPDH Meprin α (580bp) (380 bp)

Figure A.1 HepG2 cells express high levels of meprin α mRNA Meprin α mRNA of two human hepatoma cell-lines, HepG2 and Huh7, were compared. HepG2 expressed higher levels of meprin α mRNA than the Huh7 cells. Similar levels of GAPDH show equal loading control.

149 H meprin α std 10x conc Huh7 Huh7 HepG2 2ng 1ng 0.5ng

Figure A.2 Meprin α protein detectable only in HepG2 culture medium Meprin α protein levels were assessed in HepG2 and Huh7 culture media. While meprin α was seen in unconcentrated HepG2 medium, Huh7 culture medium did not show the presence meprin α protein even after 10-fold concentration.

150 4.00E+05 3.50E+05

r 3.00E+05 2.50E+05 2.00E+05 Control Vehicle Control Cell Numbe Cell 1.50E+05 1.00E+05 25nM Actinonin 5.00E+04 50nM Actinonin 0.00E+00 123456 Time [days]

Figure A.3 Meprin A inhibition does not affect the growth rate of HepG2 cells HepG2 growth rate was compared in the presence and absence of two concentrations of actinonin, meprin inhibitor. Inhibition of meprin A activity did not affect the growth of HepG2 cells.

151 HepG2 Invasion

100 90 80 70 60 50 40 % Invasion % 30 20 10 0 Control + HGF + HGF + Inh Conditions

Figure A.4 Lack of meprin A activity does not affect HepG2 invasiveness HepG2 cells (0.5 x 106) were seeded on control and matrigel inserts in the presence or absence of HGF and/or actinonin (5 μM) for 48 h. The number of invading cells in 10 random fields was counted.

152 Investigations into the expression pattern of meprins in hepatocellular carcinoma cell-lines showed constitutive over-expression of meprin α mRNA as well as protein in the HepG2 cell-line, but not in HuH-7 cells. Instances of constitutive expression of MMP-9 in leukemic cell-line HL60 have been reported. These cells express an autocatalytically truncated form of MMP-9 that is insensitive to inhibition by TIMPs, and thereby escapes regulation (Ries et al., 1996; Ries and Petrides, 1995). Meprin A does indeed play a role in the invasiveness of MDA-MB-435 metastatic breast cancer cell in vitro (Matters et al., 2005). These suggested the possibility of a functional relevance for the meprin α over-expression observed in HepG2. However, from the experiments reported here, growth and invasiveness of HepG2 cells do not appear to be dependent on meprin A activity. Nevertheless, it needs to be mentioned that the experiments conducted were of a limited scope, and do not rule out a significant role for meprin α. Further functional characterization of meprin α expression is necessary to determine the role of meprin α in this hepatocellular carcinoma cell-line. It is also worth noting that given the highly elevated expression of meprin α observed in HepG2 cells, investigating its potential as a diagnostic tool appears promising.

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181 VITA Sanjita Banerjee Education Ph.D. Biochemistry and Molecular Biology 2002 - 2008 The Pennsylvania State University

M.Sc. Biotechnology 2000 - 2002 Indian Institute of Technology, Bombay, India

B.Sc. Zoology Honours 1997 - 2000 University of Calcutta, India

Awards Metzincin Metalloproteases in Health and Disease, Monte Verita, 2006 Switzerland Travel Award and Best Poster Prize

ASBMB Travel Award, Washington DC 2007

Selected to participate in 2nd NIH National Graduate Student 2007 Research Festival, NIH Bethesda MD,USA

Publications • Bond, J.S., Matters, G.L., Banerjee, S., Dusheck, R.E. (2005). Meprin metalloprotease expression and regulation in kidney, intestine, urinary tract infections and cancer. FEBS Letters, 579, 3317-3322.

• Banerjee, S., Oneda, B., Yap, L.M., Jewell, D., Matters, G. L., Fitzpatrick, L. R., Sterchi, E., Ahmad, T., Lottaz, D., Bond, J.S. MEP1A encodes a meprin metalloprotease subunit and is a susceptibility gene for inflammatory bowel disease (in preparation).

• Banerjee, S., Bond, J.S. ProInterleukin-18 is activated by meprin β in vitro and in vivo in intestinal inflammation (in preparation).

• Banerjee, S., Bond, J.S. Mice lacking both meprin A and B show altered response to DSS-induced colitis (in preparation).