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Metalloproteinase Cleavage of Versican at The METALLOPROTEINASE CLEAVAGE OF VERSICAN AT THE FIBROBLAST CELL SURFACE by Sean Bertram Maurice B.Kin., University of Calgary, 1999 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Dental Science) THE UNIVERSITY OF BRITISH COLUMBIA October, 2009 © Sean Bertram Maurice, 2009 ABSTRACT Versican is a large aggregating proteoglycan expressed in the pericellular matrix of fibroblast cells. It is highly expressed during development and remodeling. The regulated synthesis and degradation of versican are associated with physiological remodeling. Versican is expressed in fibroproliferative lesions of human pulmonary fibrosis and atherosclerosis. Stromal expression of versican is associated with many forms of cancer and may be predictive of poor prognosis. Abnormal persistence of the versican-rich matrix may contribute to fibroproliferative and oncogenic processes. The process of versican degradation is not understood, but as versican is a pericellular molecule, physiological degradation likely involves cell surface-associated proteolysis. As such, the overarching hypothesis for this work is that regulated versican turnover involves the cell surface-associated metalloproteinases ADAMTS-2, MMP-2 and MT1-MMP, that are expressed in versican-rich remodeling lesions. ADAMTS-2 is a procollagen N-propeptidase involved in collagen fibrillogenesis. As procollagen is synthesized in a versican-rich matrix, it was hypothesized that ADAMTS- 2 might bind and process versican. MMP-2 and MT1-MMP in complex with TIMP-2, are activated at the cell surface during wound healing, pulmonary fibrosis and cancer. Versican was purified from human fetal lung fibroblast cultures for in vitro proteolysis experiments. The purified versican preparation was characterized by electrophoresis, chromatography, spectrophotometry and mass spectrometry. ADAMTS-2 and versican localization in normal and fibrotic human lungs were investigated. ADAMTS-2 was shown to co-purify with versican from human fetal lung fibroblasts. Bovine ADAMTS-2 was purified from fetal calf skin and shown to cleave purified human versican. The plant lectin concanavalin-A (ConA) induces a matrix degradative phenotype and is used here to investigate the process of versican degradation relative to apoptotic ii events in human fetal lung fibroblast cultures. Con-A induced increased expression of MMP-2 and MT1-MMP in human fetal lung fibroblasts and a concomitant loss of versican from the matrix. Microarray analysis was used to investigate expression of possible versican-degrading enzymes and their inhibitors, expressed in response to ConA. Recombinant MMP-2 and MT1-MMP were shown to process purified versican in vitro. This work expands upon the body of knowledge of versican turnover and should help in the search for therapeutic avenues to treat fibroproliferative and oncogenic processes. iii TABLE OF CONTENTS Abstract ....................................................................................................................ii Table of Contents ....................................................................................................iv List of Tables ...........................................................................................................ix List of Figures.......................................................................................................... x Abbreviations......................................................................................................... xiii Dedication..............................................................................................................xiv Acknowledgements ................................................................................................xv Co-authorship Statement.......................................................................................xvi CHAPTER 1 – BACKGROUND 1.1. Versican in normal physiology .................................................................... 1 1.1.1. Development ............................................................................................ 2 1.1.2. Reproduction............................................................................................ 4 1.2. Versican in wound healing disorders.......................................................... 5 1.2.1. Wound healing and fibrosis...................................................................... 5 1.2.2. Pulmonary fibrosis.................................................................................. 10 1.2.3. Atherosclerosis....................................................................................... 13 1.2.4. Cancer stroma........................................................................................ 15 1.2.5. Arthritis ................................................................................................... 19 1.2.6. Tendinopathies....................................................................................... 19 1.3. Versican structure....................................................................................... 20 1.3.1. Hyalectans ............................................................................................. 21 1.3.2. Gene organization and regulation .......................................................... 21 1.3.3. Splice variants........................................................................................ 24 iv 1.3.4. Glycosaminoglycans .............................................................................. 27 1.3.5. N-terminus/G1........................................................................................ 30 1.3.6. C-terminus/G3........................................................................................ 30 1.4. Versican turnover in tissue remodeling.................................................... 31 1.4.1. Matrix metalloproteinases ...................................................................... 32 1.4.2. ADAMTS ................................................................................................ 37 1.4.3. Known and unknown versican proteolytic events .................................. 40 1.4.4. Versican proteolysis ............................................................................... 41 1.4.5. Glial hyaluronate binding protein............................................................ 41 1.4.6. Hyaluronectin ......................................................................................... 43 1.4.7. Matrikines............................................................................................... 44 1.4.8. Potential mechanisms of altered proteolysis in aberrant tissue remodeling ........................................................................................................ 45 1.5. Rationale ....................................................................................................... 45 1.5.1. Overarching hypothesis .......................................................................... 48 1.5.2. Specific aims ........................................................................................... 48 1.6. References ................................................................................................... 50 CHAPTER 2 – PURIFICATION AND CHARACTERIZATION OF VERSICAN 2.1. Summary ...................................................................................................... 78 2.2. Introduction.................................................................................................. 78 2.3. Experimental procedures ........................................................................... 80 2.3.1. Cell culture ............................................................................................. 80 2.3.2. Immunofluorescence staining and microscopy ...................................... 81 2.3.3. Isolation of versican ............................................................................... 82 v 2.3.4. Electrophoretic techniques..................................................................... 82 2.3.5. Western blotting ..................................................................................... 83 2.3.6. Quantification of chondroitin sulfate concentration with the DMMB assay................................................................................................................. 84 2.3.7. Gel filtration chromatography ................................................................. 84 2.3.8. Characterization of proteolytic fragments............................................... 84 2.3.9. Enzyme incubation................................................................................. 85 2.3.10. Proteomic identification ........................................................................ 85 2.4. Results.......................................................................................................... 86 2.4.1. Versican purification from human fetal lung fibroblast cells ................... 86 2.4.2. Optimization of versican purification ...................................................... 89 2.4.3. Gel filtration analysis of versican............................................................ 91 2.4.4. Characterization of versican degradation............................................... 91 2.4.5. Mass spectrometric characterization of versican ................................... 93 2.4.6. Versican glycosylation and characterization .........................................
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