PDGF GENE TRANSCRIPTION IN RENAL EPITHELIAL CELLS

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Platelet-derived growth factor (PDGF) plays a critical role in the regulation of cell proliferation during embryonic development, cellular differentiation and tissue repair. PDGF is also synthesized and secreted at sites of abnormal cellular growth, and may act to stimulate autonomous cellular proliferation by an autocrine or paracrine mechanism. In particular, PDGF may play a pathological role in proliferative lesions of the kidney, where mesangial, endothelial and epithelial cells express significant quantities of the PDGF A- and B-chains. We have identified a non-transformed renal epithelial cell line (BSC-1; African green monkey) which expresses measurable quantities of the mRNA encoding the cognate PDGF chains, as well as significant levels of A-chain promoter activity. We have localized regions within the upstream regulatory region of this gene which mediate positive and negative transcriptional activity of the gene. Three specific aims are proposed to determine the molecular mechanisms which mediate the constrained but measurable rate of PDGF A-chain gene transcription in these cells. These aims are focused upon the localization and characterization of a negative regulatory element identified upstream (-1029 to -879) of the start site of PDGF A-chain transcription. First, the negative regulatory element (NRE) will be examined to determine whether it exerts a dominant repressing effect on two adjacent transcriptional enhancers (-879 to-634 and -84 to-62) within the PDGF A-chain promoter. NRE influence upon these enhancers will be evaluated in a panel of cell lines which express PDGF A-chain at low versus high levels to determine if transcriptional repression/derepression plays an important role in mediating relative basal rates of A-chain gene transcription. Second, the DNA response element which mediates transcriptional silencer activity will be finely localized by site-directed mutagenesis of the -1029 to-879 region. Third, nuclear factors which bind with high affinity and specificity to the minimal NRE will be identified and characterized by electrophoretic mobility shift assay (EMSA) and DNA footprinting. DNA sequence requirements for NRE function and factor binding will be compared to determine the functional importance of any identified silencer elements binding proteins for NRE activity. The proposed experiments offer the potential for identifying molecular mechanism which control the physiological and pathophysiological expression of PDGF in the kidney and possibly other cell types.