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Title: Epithelial-mesenchymal signalling : role of matrix metalloproteinase-7
Author: Hemers, Elaine Marie
ISNI:       0000 0001 2451 8646
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2004
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The gastric hormone gastrin not only regulates acid secretion, but also gene expression and the organisation of the gastric epithelium. Matrix metalloproteinases (MMPs) are involved in extracellular matrix (ECM) degradation, an essential mechanism in maintaining the organisation of the gastric epithelium, and in cancer. Stromal proliferation is a commonfeature of many carcinomas, with myofibroblasts the predominantstromalcell type present. The aim of this study was to investigate the role of gastrin in regulating MMP-7 expressionin gastric epithelial cells, and to examine the consequences for gastric myofibroblast function. Using Western blot analysis and enzyme-linked-immunosorbent assay (ELISA) it was found that hypergastrinaemic patients with pernicious anaemia (PA) and multiple endocrine neoplasia type 1 syndrome (MEN-1) had elevated gastric and plasma MMP-7. Immunohistochemistry demonstrated that the MMP-7 waslocalised to the epithelial cells within gastrin-dependent enterochromaffin-like (ECL) carcinoid tumour andnot to the abundantstromal cells. However myofibroblasts were plentiful, and distributed throughout the carcinoid tumour. Using co-culture assays of AGS cells with cholecystokinin (CCK)-2 receptor-expressing gastric epithelial cells, it was found that gastrin increased the expression of 2.3kb of the human MMP-7 promoter via a signal transduction pathway that involved NF«B, activation of cyclooxygenase (COX)-2 and the insulin-like growth factor I receptor (IGF-IR). Using Boyden chamber migration assays, primary gastric epithelial glands and culture medium significantly increased myofibroblast migration and this was inhibited by MMP-7 antisense oligonucleotide. Recombinant MMP-7 (rMMP-7)stimulated myofibroblast migration through activation of the mitogen-activated protein kinase (MAPkinase) and phosphatidylinositol 3-kinase (PI3-kinase) signalling pathways and partly through MMP-3 and -8. The proteomic techniques of two-dimensional gel electrophoresis (2DE) and mass spectrometry identified a number of MMP-7-, MMP-3- and -8-regulated proteins in myofibroblasts. Using these techniques IGFBP5 was identified in the culture medium of colonic myofibroblasts stimulated with these MMPs. Western blot analysis demonstrated that MMP-7 stimulated the secretion of IGFBP-5 from myofibroblasts and its subsequent cleavage. In addition, MMP-7increased the abundance of IGF-II in the myofibroblast culture medium and inhibiting the tyrosine kinase activity of the IGF-IR significantly inhibited myofibroblast migration. In contrast, cleavage of IGFBP-5 by MMP-3 and -8 produced different cleavage products compared to those obtained after cleavage by MMP-7. In addition, MMP-3 and -8 did not stimulate secretion of IGFBP-5 nor increase the abundance of fully processed IGF-II in the myofibroblast culture medium. In summary, gastrin regulates the expression of MMP-7 through the activation of NFkB, COX-2 and IGF-IR. Matrix metalloproteinase-7 stimulates migration of myofibroblasts, through activation of the MAPkinase and PI3-kinase signalling pathways and partly through activation of MMP-3 and -8. Matrix metalloproteinase-7 cleaves IGFBP-5, increases IGF-II in culture medium, and activates the IGF-IR resulting in the stimulation of myofibroblast migration. In conclusion,gastrin indirectly stimulates the migration of myofibroblasts through the increased expression and secretion of MMP-7.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral