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Role of Transglutaminases in Signalling That Regulates Epithelial Responses in Wound Healing

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Role of Transglutaminases in Signalling That Regulates Epithelial Responses in Wound Healing Role of Transglutaminases in Signalling that Regulates Epithelial Responses in Wound Healing. Thesis submitted in fulfilment of the requirements of the degree of Doctor of Philosophy, Cardiff University September 2010 Jessica Edwards, B.Sc. (Hons), M.Sc. Tissue Engineering and Reparative Dentistry. Dental School, Cardiff University, Cardiff i UMI Number: U518502 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U518502 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 DECLARATION This work has not previously been accepted in substance for any degree and is not concurrently submitted in candidature for any degree. Signed . ...................... ...........(candidate) Date ~lO[& STATEMENT 1 This thesis is being submitted in partial fulfillment of the requirements for the degree o f ........................................(insert MCh, MD, MPhil, PhD etc, as appropriate) Signed .... (candidate) Date STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. Signed (candidate) Tolo Date STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed (candidate) . Date II “Science never solved a problem without creating ten more” George Bernard Shaw. 1856 - 1950. Ill Role o f Transglutaminases in signalling that regulates epithelial responses in wound healing. Contents Pages Acknowledgments I Summary II Abbreviations III Chapter 1 General Introduction 1 Aims of the Investigation 102 Chapter 2 Materials and Methods 103 Chapter 3 TG2 and its Role in Re-Epithelialisation. 133 Chapter 4 Metalloproteinase Activity and Subsequent 161 Downstream Effects in Re-Epithelialisation. Chapter 5 TG2 Mediates Transactivation of the EGFR pathway in 195 Keratinocytes. Chapter 6 GPR56: a Potential Receptor linking Extracellular TG2 234 to Cell Migration and Proliferation. Chapter 7 General Discussion 262 References 281 IV Acknowledgements First and foremost, I have to thanks my supervisors Professor Daniel Aeschlimann and Dr Vera Knauper. Secondly, I would like to thanks my friends Dr Loma Fiedler, and Dr Sally Rosser-Davies for your proof reading and patience! Dr Mathew Locke, Mr Martin Langley, Mrs Pascale Aeschlimann and anyone else in the Dental School who has answered my questions about techniques and equipment, swapped ideas, sympathy and motivation when things have not worked out quite right! Thank you to my family, for their support both financially and otherwise through what has been a lengthy process increased by illness! I would also like to thank my fiance Chris, for listening and supporting me every day. Finally, this study would not have been possible without the funding from the Dental School. V Summary Wound healing is a complex process broken down into five main stages fibrin clot formation, re-epithelialisation, granulation tissue formation and wound contraction, and finally angiogenesis. In the second stage, re-epithelialisation over fibroblast- derived matrix occurs which is necessary for wound closure. During this process, TG2, a multi-functional enzyme with both cross linking and GTP signalling ability is involved in both stabilisation of the extracellular matrix by cross linking and by allowing downstream signalling events to occur which lead to the wound closing. This thesis has investigated the mechanisms by which re-epithelialisation occurs with regard to TG2. In the first stage of wound healing, an influx of growth factors and metalloproteinases occurs, that through the initial interaction of TG2 are able to stabilise the matrix and stimulate keratinocyte cells to migrate and proliferate to close the wound. Experiments have indicated that TG2 is able to stimulate proliferation and migration of keratinocyte cells both directly and indirectly by modulating metalloproteinase signalling, leading to the activation of the EGFR by EGF ligands liberated from the ECM. Furthermore, through the course of altering TG2’s conformation and activity experiments have determined that TG2 must be in an open and active conformation in order for it to affect keratinocyte signalling leading to proliferation and migration. Finally, a G protein coupled receptor has been investigated as to whether it may be involved in TG2 driven proliferation and migration in keratinocytes. Previous work by Xu et al., 2006 had shown TG2 to be a binding partner of GPR56 and this GPCR has been shown to be involved in proliferation and migration of cells located in the brain and heart. Although investigations of GPR56 are at this time inconclusive regarding keratinocyte proliferation and migration this thesis confirms the presence of GPR56 in keratinocytes. Therefore GPR56 may be involved in keratinocyte migration and proliferation. VI Abbreviations ADAM A Disintegrin And Metalloproteinase ADAM-TS A disintegrin and metalloproteinase with thrombospondin motifs AREG Amphiregullin AS (ECM) Antisense/ TG2 null extracellular matrix BCA Bicinchoninic acid BMP Bone morphogenic Protein BSA Bovine Serum albumin BTC Betacellulin CBL Covalently bound lipid CE Comified Envelope CTGF Connective tissue growth factor DEJ Dermal Epidermal Junction DMEM Dulbecco's Modified Eagle Medium DSFM Defined serum free medium-keratinocytes DTT Dithiothretol DMSO Dimethyl sulfoxide ECM Extracellular Matrix EDTA Ethylenediaminetetraacetic acid EGFR Epidermal Growth factor Receptor ErBB Epidermal growth factor receptor 1 EbBl Alternative name for epidermal growth factor receptor 1 ErbB2 -4 Alternative name for epidermal growth factor receptor 2-4 EPGN Epigen EREG Epiregulin ERK MAPK/ERK pathway Activated Ras activates the protein kinase activity of RAF kinase. (MAPK). FAK Focal Adhesion Kinase FAM Fluorencin FCS Foetal Calf Serum FGF Fibroblast Growth factor receptor VII FITC Fluorescein FPLC Fast protein liquid chromatography FN Fibronectin GM-CSF Granulocyte macrophage colony stimulating factor GPCR G protein coupled receptor GPCR56 G protein coupled receptor 56 GPS GPCR protein cleavage site GST Glutathione S-transferase HEPES N-2-hydroxyethlpiperazine-N’-2-ethane sulphonic acid monosodium salt Her 1-4 Epidermal growth factor receptors (alternative names 1-4) HB-EGF Heparin Bound-Epidermal Growth Factor IGFR Insulin like Growth factor receptor IL- Interleukin IPTG Isopropyl P D-thiogalactoside K Keratin LI Lamellar ichthyosis LPA Lysophosphalidic acid M/Mk (ECM) Mock (endogenous) extracellular matrix MDC Monodansyl cadaverine MMP Matrix Metalloproteinase (numbered) MT-MMP Membrane Type Matrix Metalloproteinases(numbered) MAPK Mitogen-activated protein kinase NRG 1-4 Neuregulin PBS Phosphate buffered saline PDGFR Platelet derived growth factor receptor PMSF Phenylmethyl sulphonylfluoride PNGase Peptide N-Glycosidase PSG Penicillin/Streptomycin/Glutamine P13K Phosphatidylinositol 3 Kinase QPCR Quantitative real time polymerase chain reaction RGD Arginine Glycine Aspartate VIII RT Room Temperature SDS Sodium dodecyl sulphate SE Skin Equivalent SiRNA Small interfering RNA. S (ECM) Sense (overexpressed) extracellular matrix. SPR Small proline rich proteins SVMP Snake venom metalloproteinases TAE buffer Tris/acetate/EDTA buffer TAMRA T etramethy lrhodamine TBS Tris buffered saline TBE Tris-Borate EDTA TCA Trichloroacetic acid TG Transglutaminase (1-7) TGF Transforming Growth Factor (alpha/beta) TIG3 Tarazotene induced Gene 3 TIMP Tissue inhibitors of metalloproteinases TNF Tumour necrosis factor VEGF Vascular Endothelial Growth Factor Receptor UV Ultra Violet IX Role of Transglutaminases in Signalling that Regulates Epithelial responses in Wound Healing. Chapter 1 introduction Role of Transglutaminases in Signalling that Regulates Epithelial Responses in Wound Healing. Contents. Page 1.0 Transglutaminases General Introduction. 3 1.1 Transglutaminase Family Structure. 7 1.1.1 Transglutaminase Regulation by Nucleotide Binding. 9 1.2 Summary of Nine Transglutaminase Isoforms. 12 1.2.1 FXIII. 18 1.2.2 Band 4.2. 20 1.2.3 Transglutaminase 1. 21 1.2.4 Transglutaminase 2. 25 1.2.4.1 Intracellular Transglutaminase 2 function. 27 Cytosol. 27 Membrane. 28 Nucleus. 28 Extracellular TG2. 29 1.2.4.2 TG2 Knock Out Murine Model. 33 1.2.5 Transglutaminase 3. 34 1.2.6 Transglutaminase 4. 37 1.2.7 Transglutaminase 5. 37 1.2.8 Transglutaminase 6. 38 1.2.9 Transglutaminase 7. 39 1.3 Metalloproteinase General Introduction. 40 1.3.1 Structure of Metalloproteinase Super Family. 41 1.3.2Metalloproteinase Family and Domain Structure. 42 1.3.3 Control of Metalloproteinases. 47 1.3.4 Endogenous Regulation of Metalloproteinases (TIMPS). 48 1.3.5 ADAMS Subfamily. 50 1.3.6 ADAMS Family Structure. 50 1.3.7 ADAMS Family Function. 56 1.3.8 ADAMS as Regulators of EGFR Signalling. 57 1.4 Wound healing General Introduction. 64 1.4.1 The Fibrin Clot and Inflammatory Cells. 64 1.4.2 Re-epithelialisation. 66 1 Role of Transglutaminases in Signalling that Regulates Epithelial Responses in Wound Healing. 1.4.2 Granulation Tissue and Wound Contraction. 69 1.4.3 Angiogenesis. 72 1.4.4 Mesenchymal Epidermal Interactions. 72 1.4.5 Transglutaminase Role in Wound Healing. 75 1.4.5.1 FXIII. 75 1.4.5.2 TGI. 75 1.4.5.3 TG2. 76 1.4.5.4 TG2 and Tissue Fibrosis. 78 1.5 Metalloproteinase Activity in Wound Healing. 78 1.6 EGF and its Role in Wound Healing. 79 1.7 TGFp and its Role in Wound Healing. 83 1.8 Skin General Introduction. 85 1.8.1 The Dermis. 87 1.8.2 The Epidermis. 87 1.8.2.1 Stratum Basale. 8 8 1.8.2.2 Stratum Spinosum. 88 1.8.2.3 Stratum Granulosum. 89 1.8.2.4 Transition Zone. 89 1.8.2.5 Stratum Comeum. 90 1.9 The Comified Envelope.
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