The VHL tumor suppressor gene product (pVHL) is an E3 ubiquitin ligase, and it is believed that pVHL inactivation results in the stabilization of specific target proteins, leading to tumorigenesis. VHL-inactivated tumors are highly vascular, and over-expression of vascular endothelial growth factor (VEGF) and other hypoxia-responsive products have been implicated in the etiology of these tumors. Under hypoxic conditions (e.g., 1% O2), VEGF is induced at the transcriptional level through the binding of hypoxia-inducible factor (HIF) to enhancer elements in the VEGF promoter, and at the post-transcriptional level through the binding of stability factors to AU-rich elements (AREs) in the 3' UTR of the VEGF transcript. Under normal oxygen conditions, pVHL plays a central role in regulation of VEGF expression. pVHL controls VEGF transcription by targeting HIF alpha subunits for ubiquitylation and subsequent proteasomal degradation. pVHL also regulates VEGF mRNA stability through a mechanism involving the association of trans-acting factors with the VEGF mRNA 3' UTR ARE sequences. Our preliminary results show that pVHL levels were down- regulated under hypoxic conditions in kidney cells and we hypothesize that pVHL turnover may play a central role in determining stability or turnover of target proteins such as HIF alpha as well as decay of VEGF or other hypoxia-responsive mRNAs. pVHL and oxygen levels controlled expression of a VEGF ARE- containing reporter construct in RCC cells. The RNA binding proteins AUF1 and HuR bound to the VEGF 3' UTR under both normoxic and hypoxic conditions. pVHL binds directly to these hnRNPs, and under normoxic conditions where VEGF expression is low, we detected pVHL-hnRNP complexes in association with VEGF ARE RNA. We hypothesize that pVHL, through association with AUF1 and HuR, targets VEGF mRNA for decay. Under hypoxic conditions where VEGF is expressed at high levels, AUF1 and HuR bound to VEGF ARE RNA in the absence of pVHL.
The Specific Aims of this project are: (1) to determine mechanisms of regulation of pVHL protein levels by hypoxia; (2) to characterize pVHL-hnRNP interaction domains and pVHL-dependent hnRNP turnover; and (3) to determine post-transcriptional control of VEGF expression through pVHL-hnRNP interactions. These studies will lead to a greater understanding of pVHL regulation of hypoxia-regulated gene expression, as well as mechanisms of pVHL tumor suppression. ? ? ?
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