TGF BETA--TRANSCRIPTIONAL CONTROL

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DESCRIPTION (Taken directly from applicant's abstract) In children with progressive glomerular disease, glomerulosclerosis results from a shift in the balance between matrix synthesis and degradation to favor extracellular matrix (ECM) accumulation. Data increasingly support a mediating role for TGF-B1 in this process. However, the intracellular mechanism(s) by which TGF-B1 stimulates cells to accumulate matrix are not well understood. Mesangial cells treated with TGF-B1 assume a matrix-accumulating phenotype defined by changes in cell structure and activity as well as in ECM turnover. Changes in expression of mRNAs for ECM proteins, proteases and protease inhibitors are preceded by changes in expression of c-Ets2 and c-jun mRNA. Binding sites for these transcription factors (Ets and AP-1, respectively), as well as for another transcription factor, Sp1, have been identified in the promoters of genes related to ECM turnover. We hypothesize that changes in expression or activity of Ets, AP-1 and Sp1 transcription factors mediate TGF-B1 induced changes in mesangial cell ECM turnover related to sclerosis. To address this hypothesis, we propose two specific aims. First, to determine whether TGF-B1 modulates expression or activity of AP-1, Ets or Sp1 transcription factors in mesangial cells, they will be treated with TGF-B1 and examined for the following: specific transcription factor mRNA and protein expression; nuclear extract binding to DNA binding sites for AP-1, Ets or Sp1 transcription factors; phosphorylation of AP-1 and Ets transcription factors; and transcription factor activity, evaluated in cells transiently expressing specific binding site-thymidine kinase promoter-luciferase reporter constructs. Timing of changes induced by TGF-B1 will be related to timing of changes in ECM turnover. The second Specific Aim addresses whether activation of specific transcription factors may mediate TGF-B1 induced changes in gene expression related to ECM turnover. Studies will identify signaling pathways that could mediate TGF-B1 modulation of transcription factor activity, such as cell membrane protein tyrosine kineses, cellular MAP kineses, or the TGF-B specific Smad transducer family. Inhibitors of these pathways will be examined for ability to inhibit TGF-B1 induced transcription factor activity in mesangial cells. Finally, inhibitors of TGF-B1 induced changes in transcriptional activity will be evaluated for ability to block TGF-B1 induced changes in mesangial cell matrix turnover. These studies will elucidate cellular mechanisms of progressive glomerulosclerosis, and may suggest therapeutic approaches to these mechanisms in progressive childhood renal disease.