Increased biosynthesis and accumulation of extracellular matrix macromolecules, particularly type I collagen, are hallmarks of keloid, a prototype of skin fibrosis. Recently, endothelial cell growth factor, its constituent basic (bFGF) and acidic (aFGF) fibroblast growth factors (FGFs), in the presence of heparin have been shown to effectively down- regulate type I collagen gene expression by keloid fibroblasts. The objectives of the proposed studies are to elucidate, at the transcriptional and post-transcriptional level, the molecular mechanisms of action of the FGFs in the regulation of genes encoding the collagens and other matrix macromolecules in keloid and normal skin fibroblasts. Specifically, (i) Nuclear transcriptional activities will be examined by in vitro run-on analysis to compare the relative transcriptional activities of the matrix genes altered by aFGF or bFGF in the absence and presence plus/minus of heparin; (ii) Collagen promotor activity will be assayed in transient transfections with collagen promotor reporter gene constructs, that presently include the pro-alpha 2(I) promotor-CAT construct; (iii) Elucidation of trans-acting regulatory factors involved in the control of gene expression will be conducted by gel mobility shift and foot-printing analyses to determine whether the FGFs are capable of regulating such factors; (iv) The identification of responsive cis-acting regulatory elements by deletion analysis; (v) Stability of the mRNAs (post- transcriptional) will be evaluated to determine possible alteration of mRNA turnover by the FGFs. Proposed experiments will deal with human recombinant bFGF, aFGF plus/minus heparin. The studies will focus on, but will not be limited to, the collagen genes, such as type I collagen gene expression. Fibroblasts from age- and site-matched normal controls will be included for comparative purposes with keloid fibroblasts. The significance of the proposal is to pin-point the precise mechanisms involved in regulatory control of collagen matrix gene expression by FGFs, that could lead to alternative modalities that would ameliorate the over- abundant expression of matrix components, such as collagen, by cells in fibrosis.
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