REGULATION OF COLLAGEN IV GENE TRANSCRIPTION BY TGF-BETA

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DESCRIPTION (Adapted from Applicant's Abstract): Progression of renal disease is associated with the abnormal deposition of collagen IV and other matrix macromolecules. Humoral and cellular factors that regulate collagen IV synthesis in the normal and diseased kidney have not been well defined. TGF- beta1 is a potent cicatricial mediator that markedly stimulates fibrillar collagen (collagen I and collagen III) synthesis in many cell systems. Recent studies have demonstrated that TGF-beta1 is produced during experimental glomerulonephritis and may play a role in both glomerular and interstitial accumulation of collagen IV during disease progression.It is hypothesized that TGF-beta1 plays a dominant role in the progression of renal injury to renal failure by stimulating transcription of the collagen IV genes. The major objective of this grant application is to determine the mechanism by which TGF-beta1 increases transcription of the collagen IV genes. TGF-beta1 mediated induction of alpha1(IV) and alpha2(IV) collagen mRNA will be assessed in cell derived from rat glomeruli, tubular epithelium, and interstitium. Collagen IV gene transcription will be measured by nuclear run-on assays. Since two levels of transcriptional regulation of collagen IV, transcript initiation and transcript elongation have been identified, the role of TGF- beta1 in stimulation of transcript initiation and elongation will be characterized in NIH-3T3 cells, a readily manipulable cell line that responds to TGF-beta1 with increases in alpha1(IV) and alpha2(IV) collagen mRNA and collagen IV gene transcription. The alpha1(IV) and alpha2(IV) collagen genes share a 130 base pair bidirectional promoter; an enhancer element within the first intron of the alpha1(IV) gene is necessary for tissue-specific expression of the collagen IV genes. The role of these and other potential cis-acting factors in TGF-beta1 mediated induction of collagen IV transcription will be defined by transfecting chimeric collagen IV promoter/enhancer gene constructs into TGF-beta1 treated NIH-3T3 cells. Gel mobility shift assays and DNAse I footprinting studies will be used to identify putative trans-acting factors that interact with critical regulatory elements within the collagen IV genes. Elucidation of the cis- and trans- acting factors important in TGF-beta1 mediated induction of collagen IV gene transcription may provide the basis for development of better strategies for therapeutic interventions aimed at blocking the progression of renal injury to end stage renal disease.