View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Helsingin yliopiston digitaalinen arkisto Helsinki University Biomedical Dissertations No. 98 Molecular biology and functions of epilysin (MMP-28) Sara A. Illman Departments of Pathology and Virology, Haartman Institute and Helsinki University Hospital, Biomedicum Helsinki University of Helsinki Finland Academic Dissertation To be presented, with the permission of the Faculty of Medicine, University of Helsinki, for public criticism, in the Lecture Hall 2 in Biomedicum Helsinki, Haartmaninkatu 8, Helsinki, on December 13th, at 1 o’clock PM. Helsinki 2007 Supervised by: Professor Jorma Keski-Oja and Docent Jouko Lohi Departments of Pathology and Virology Biomedicum and Haartman Institute University of Helsinki Helsinki, Finland Reviewed by: Professor Johanna Myllyharju Department of Medical Biochemistry and Molecular Biology Biocenter Oulu University of Oulu Oulu, Finland and Docent Panu Jaakkola Center for Biotechnology University of Turku and Åbo Akademi University Turku, Finland Opponent: Docent Jukka Westermarck Institute of Medical Technology University of Tampere Tampere, Finland ISSN 1457-8433 ISBN 978-952-10-4338-3 (paperback) ISBN 978-952-10-4339-0 (PDF) http://ethesis.helsinki.fi Yliopistopaino Helsinki 2007 2 Till mamma och pappa 3 4 Original publications............................................................................................................7 Abbreviations .......................................................................................................................8 Abstract ..............................................................................................................................10 1 Introduction ................................................................................................................11 1.1 Extracellular matrix............................................................................................11 1.1.1 Interstitial matrix...........................................................................................11 1.1.2 Basement membrane .....................................................................................12 1.1.3 Interactions between cells and the ECM ........................................................13 1.2 Matrix Metalloproteinases..................................................................................13 1.2.1 Structures and substrates of the different MMP subfamilies ....................14 1.2.1.1 Collagenases..........................................................................................16 1.2.1.2 Gelatinases ............................................................................................16 1.2.1.3 Stromelysins..........................................................................................17 1.2.1.4 Matrilysins.............................................................................................17 1.2.1.5 Membrane-type MMPs ..........................................................................17 1.2.1.6 Other MMPs..........................................................................................18 1.2.2 Targeted disruption of Mmp genes in the mouse........................................19 1.2.3 Epilysin (MMP-28)......................................................................................21 1.2.3.1 Epilysin in wound healing......................................................................22 1.2.3.2 Epilysin in cancer ..................................................................................23 1.2.3.3 Expression of epilysin in specialized tissues ..........................................23 1.2.4 Regulation of MMP activity........................................................................25 1.2.4.1 Transcriptional regulation......................................................................25 1.2.4.2 Activation of latent MMPs.....................................................................26 1.2.4.3 Pericellular localization .........................................................................27 1.2.4.4 Inhibition of MMP activity ....................................................................29 1.2.5 Modulation of bioactive molecules by MMPs.............................................29 1.2.5.1 Cryptic fragments of ECM molecules ....................................................29 1.2.5.2 Growth factors and cytokines.................................................................30 1.2.5.3 Cell adhesion molecules ........................................................................30 1.3 Epithelial to mesenchymal transition .................................................................31 1.3.1 Role of MMPs in growth factor mediated EMT.............................................32 1.3.2 MMPs are induced by Snail and Sip1 ............................................................33 1.3.3 MMPs induce Snail .......................................................................................34 1.4 MMPs in cancer ..................................................................................................34 1.5 MMPs and other proteolytic pathways ..............................................................35 2 Outline of the present study .......................................................................................36 3 Materials and Methods...............................................................................................37 3.1 Cell culture and reagents ....................................................................................37 3.1.1 Antibodies.....................................................................................................37 3.1.2 Enzymes, chemicals and growth factors.........................................................37 3.2 DNA and protein/DNA interaction analysis.......................................................38 3.2.1 Construction of expression vectors ................................................................38 3.2.2 Transfections.................................................................................................39 3.2.3 Promoter activity assays ................................................................................39 5 3.2.4 Gel mobility shift assay .................................................................................39 3.3 RNA analysis .......................................................................................................40 3.3.1 Extraction of RNA from mouse tissues..........................................................40 3.3.2 RNase protection analysis..............................................................................40 3.3.3 Northern hybridization ..................................................................................40 3.4 Protein analysis ...................................................................................................40 3.4.1 Immunofluorescence .....................................................................................40 3.4.2 SDS-PAGE and immunoblotting ...................................................................41 3.4.3 Gelatin zymography ......................................................................................41 3.4.4 TGF-b activity assays....................................................................................41 3.5 Transwell migration assays and collagen invasion ............................................42 4 Results .........................................................................................................................43 4.1 Characterization of human and mouse epilysin promoters (I)..........................43 4.1.1 Identification of potential regulatory sites......................................................43 4.1.2 Analysis of transcriptional activity ................................................................43 4.1.3 The epilysin promoter interacts with Sp1 and Sp3 .........................................44 4.2 Cloning of mouse epilysin cDNA (II)..................................................................44 4.2.1 Mouse and human epilysin proteins are highly similar...................................44 4.2.2 Epilysin is processed by a furin-like pro-protein convertase...........................45 4.2.3 Epilysin is widely expressed in different tissues in the mouse........................45 4.3 Epilysin induces EMT in lung carcinoma cells (III) ..........................................46 4.3.1 TGF-b mediates the epilysin-induced EMT ...................................................46 4.3.2 Induction of gelatinase B and MT1-MMP in epilysin-induced EMT..............47 4.4 Epilysin is associated with the surface of epithelial cells (III) ...........................47 4.4.1 Cell surface association of epilysin is lost upon EMT ....................................48 4.5 Epilysin increases the motility of lung carcinoma cells (III)..............................48 4.5.1 Epilysin-induced EMT results in a collagen invasive phenotype ....................49 4.6 Central findings of the work...............................................................................49 5 Discussion....................................................................................................................50 5.1 Regulation of epilysin gene expression (I) ..........................................................50 5.1.1 Specific regulation of gene expression by Sp1