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Structural Interactions Between the Α3β1 Integrin and Mmp-2: a Potential Functional Role in Cell Adhesion

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Structural Interactions Between the Α3β1 Integrin and Mmp-2: a Potential Functional Role in Cell Adhesion Virginia Commonwealth University VCU Scholars Compass Theses and Dissertations Graduate School 2009 STRUCTURAL INTERACTIONS BETWEEN THE Α3Β1 INTEGRIN AND MMP-2: A POTENTIAL FUNCTIONAL ROLE IN CELL ADHESION James Bowman Virginia Commonwealth University Follow this and additional works at: https://scholarscompass.vcu.edu/etd Part of the Nervous System Commons © The Author Downloaded from https://scholarscompass.vcu.edu/etd/1861 This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. School of Medicine Virginia Commonwealth University This is to certify that the dissertation prepared by James Russell Bowman, III entitled STRUCTURAL INTERACTIONS BETWEEN THE Α3Β1 INTEGRIN AND MMP-2: A POTENTIAL FUNCTIONAL ROLE IN CELL ADHESION has been approved by his or her committee as satisfactory completion of the dissertation requirement for the degree of Doctor of Philosophy David G. Simpson, Ph.D., School of Medicine John W. Bigbee, Ph.D., School of Medicine Robert F. Diegelmann, Ph.D School of Medicine Helen Fillmore, Ph.D., School of Medicine Babette Fuss, Ph.D., School of Medicine John T. Povlishock, Ph.D., Department Chair, Department of Anatomy and Neurobiology Jerome Strauss, M.D. Ph.D., Dean, School of Medicine Dr. F. Douglas Boudinot, Dean of the Graduate School July 16, 2009 James Russell Bowman, III, 2009 All Rights Reserved STRUCTURAL INTERACTIONS BETWEEN THE Α3Β1 INTEGRIN AND MMP-2: A POTENTIAL FUNCTIONAL ROLE IN CELL ADHESION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. by JAMES RUSSELL BOWMAN, III B.S., Hampden-Sydney College, 1998 M.S., Virginia Commonwealth University, DIRECTOR: DAVID G. SIMPSON, PH.D. Associate Professor, Department of Anatomy and Neurobiology Virginia Commonwealth University Richmond, Virginia July, 2009 Acknowledgements What we’ve got here is failure to communicate, Some men you just can’t reach, So you get what we have here last week, Which is the way he wants it, Well, he gets it, And I don’t like it anymore than you men. Cool Hand Luke, 1967 With that said, I want to thank my committee members, John Bigbee, Bob Diegelmann, Helen Fillmore, and Babette Fuss for their utmost patience and understanding as the years we have spent together have been, at times, arduous. Your consistent motivation and direction have shaped this project and my abilities. David, the words “thank you” fall short of describing the mentorship and guidance that you provided as “thank you” does not encompass the friendship or represent the selflessness by which have been there for me for nearly a decade. Lastly, I thank my family for their never ending love and support. ii Table of Contents Page Acknowledgements............................................................................................................. ii List of Tables ..................................................................................................................viii List of Figures .................................................................................................................... ix Chapter 1 Introduction and Overview of Project .............................................................. 1 2 Cell Adhesion Molecules..................................................................................... 6 3 Integrin Biology................................................................................................. 10 Integrin fine structure ................................................................................. 10 Integrin activation....................................................................................... 12 Integrin binding properties ......................................................................... 14 4 Cell Adhesion..................................................................................................... 19 Conventional adhesion assays .................................................................... 24 ECIS assays ................................................................................................ 25 Centrifugal adhesion assays........................................................................ 26 Molecular adhesion..................................................................................... 27 Morphological assessment of cell adhesion ............................................... 28 5 The Matrix Metalloproteinases........................................................................ 30 The Matrix Metalloproteinases................................................................... 33 The Gelatinases: MMP-2 and MMP-9 ....................................................... 35 Functional Consequences of MMP-2 and MMP-9..................................... 39 MMPs in the Heart in Development........................................................... 42 MMPs in the Heart in Adult Life................................................................ 45 MMP Interactions with Integrins................................................................ 46 6 Methods.............................................................................................................. 51 iii Cell Culture................................................................................................. 51 Cell Isolation ........................................................................................ 51 Culture Maintenance ............................................................................ 53 Culture Media Formulation.................................................................. 54 Adhesion Assays......................................................................................... 55 Conventional Adhesion Assays............................................................. 55 Centrifugal Adhesion Assays................................................................ 56 Enzyme Inhibition ...................................................................................... 58 GM-6001............................................................................................... 58 SB-3CT.................................................................................................. 59 Adhesion experimentation with protease inhibitors ............................. 60 Viability Assays..................................................................................... 62 Microscopy ................................................................................................. 64 Hoffman Modulation Contrast.............................................................. 64 Colocalization experiments: Quantitative Scanning confocal microscopy............................................................................. 66 Interference Reflection Microscopy (IRM ............................................ 69 Gelatinase Labeling .................................................................................... 71 Titration of Gelatinase label................................................................. 71 Evaluation of SB-3CT effect on the cell surface associated MMPs.................................................................................. 73 Immunoprecipitation .................................................................................. 75 Routine immunoprecipitation .............................................................. 75 Crosslinking of cell surface .................................................................. 77 Seize IP ................................................................................................. 78 Immunoprecipitation and gelatin zymography..................................... 80 Zymography.......................................................................................... 81 Western Blotting ................................................................................... 82 iv 7 Identification of a MMP-2/Integrin/TIMP-2 Complex in the Cultured Neonatal Cardiac Fibroblast........................................................................ 85 Introduction ................................................................................................ 85 Results ........................................................................................................ 85 Immunoprecipitation from cell suspensions ........................................ 85 Summary..................................................................................................... 97 8 Functional Implications of MMP-2 mediated Protease Activity in Cell Adhesion Processes in the Cultured Neonatal Cardiac Fibroblast .......... 99 Introduction ................................................................................................ 99 Results ...................................................................................................... 100 Conventional Adhesion Assays .......................................................... 100 Centrifugal Adhesion Assays ............................................................. 108 Cell Viability in the face of protease inhibition ................................. 115 Summary................................................................................................... 119 9 In Situ Relationships between MMP-2, Specific Integrin
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