Increased endothelial expression of the potent connective tissue mitogen platelet-derived growth factor B-chain (PDGF B-chain) has been implicated in the pathogenesis of hypoxia-associated pulmonary vascular remodeling. We have identified a novel regulatory sequence in the promoter of the PDGF-B chain gene that is necessary for basal transcription in endothelial cells and may be an hypoxia-response element. Transversion mutation of this region results in a 48% reduction in basal transcription based on transient transfection analysis width reporter constructs. Evidence suggests that at least five-nuclear proteins bind specifically to this regulatory sequence based on electromobility shift assays (EMSA). EMSA with many antibodies capable of binding known transcription factors failed to supershift bands suggesting that these factors may be unique. These 5 proteins have been purified in small quantities and partially characterized. Molecular weights range from 35 to 42 kDa, and they appear to be ubiquitous, being found in the lung and several other tissues and organs of different species. At least one of these DNA binding proteins appear to be up-regulated under hypoxic conditions and may play a role in mediating pulmonary vascular remodeling by increasing PDGF B-chain transcription under chronic hypoxic conditions. We propose to further characterize this putative hypoxia-response-element, and molecularly clone and structurally analyze the transcription factors binding to this regulatory sequence. The functional significance of these regulatory elements in the context of hypoxia and other pathophysiologically relevant inducers of PDGF B-chain will be examined.
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