This is a new application by a new investigator with the long-term objectives to study intracellular networks of cytokine signaling which mediate renal fibrogenesis, a major process in the pathogenesis of end stage renal disease. The overall hypothesis is that members of the Smad family of intracellular proteins integrate opposing pro-fibrogenic and anti-fibrogenic signaling pathways into signaling thresholds that regulate fibrogenesis. This hypothesis will be tested by studying the function, downstream targets and regulation of the positive mediator Smad2, and the putative negative regulator Smad7, in the opposing signaling pathways of pro-fibrogenic TGFbeta and anti-fibrogenic TNFalpha. Preliminary studies indicate that Smad2 and Smad7 are promising candidate signaling molecules in renal fibrogenesis, with opposite activities. The first Specific aim will test the hypothesis that Smad2 promotes fibrosis by activation of fibrogenic genes in response to TGFbeta. The effects of Smad2 deficiency in Smad2 null mouse embryonic fibroblasts on the expression of selected genes encoding matrix proteins, and on fibroblast proliferation will be determined. The second Specific Aim will test the hypothesis that Smad2 function as a positive regulator and Smad7 functions as a negative regulator of an overlapping pool of fibrogenic genes. To do this the PI will identify and compare gene pools which are differentially-regulated in wild type mouse embryonic fibroblasts (MEFwt), Smad2 null MEFs (MEF2-/-) and MEFs with constitutive over expression of Smad7 (MEF7+), using high-throughput microarray methods for comparative analysis of gene expression. Genes with reduced expression in both MEF2-/- and MEF7+ cells relative to MEFwt cells will be identified and characterized. The third Specific Aim will test whether Smad7 functions by inhibition of Smad2 activation, or transcriptional repression of Smad2-activated target gene promoters, or both. The last Specific Aim will examine whether TGFbeta and TNFalpha signaling pathways converge to regulate transcription of Smad7. To do this, the PI has cloned the functional human Smad7 promoter region. Promoter deletion analyses and electrophoretic mobility shift assays (EMSAs) will be utilized to identify the functional cis-acting elements in the Smad7 gene and their cognate transcription factors.
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