Connective tissue growth factor (CTGF) is a 38 kD protein induced by TGF_beta in fibroblasts. In asthma, the activation of fibroblasts by inflammatory cells is associated with the development of subepithelial fibrosis. CTGF production is increased in fibrotic diseases, however, the role of CTGF in the development of subepithelial fibrosis in asthma is not known. Mast cells elaborate cytokines and proteases that activate fibroblasts. Preliminary data indicate that the cell products TGF-beta and IL-4, and trypsin (a protease related to mast cell tryptase) induce CTGF and type I collagen in fibroblasts. The overall goal of this proposal is to establish the role of mast cell products in the development of subepithelial fibrosis in asthma. It is hypothesized that the mast cell products TGF-beta, IL-4, and tryptase induce fibroblast CTGF production with subsequent deposition of collagen and other matrix elements in the subepithelial layer.
The specific aims are: (1) To characterize TGF-beta, IL-4, and tryptase mediated regulation of CTGF expression in cultured human lung fibroblasts using northern and western blot analyses. The signal transduction mechanisms of these effectors will also be studied. Using a murine model of subepithelial fibrosis in asthma, histopathologic and physiologic parameters will be assessed. (2) To analyze the molecular regulation of CTGF expression and to localize and characterize the TGF-beta, IL-4, and tryptase response elements of the human CTGF promoter. Transcriptional run-on assays and mRNA half-life determinations will be performed. The TGF-beta, IL-4, and tryptase response elements of the human CTGF promoter will be localized using deletion analysis and the nuclear proteins binding to these elements will be identified using gel mobility shift assays. Clinically, subepithelial fibrosis may contribute to airway obstruction and hyperresponsiveness in asthma. A better understanding of the development of subepithelial fibrosis may lead to novel therapeutic approaches based on the inhibition of mast cell cytokine and protease production. The proposed research training plan will include participation in molecular biology and ethics seminars and regular review by the advisory committee. This award will prepare the investigator for an independent research career.
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