Influence of the RNA-Binding Protein HuR in pVHL-Regulated p53 Expression in Renal Carcinoma Cells

Abstract

A recent analysis of gene expression in renal cell carcinoma cells led to the identification of mRNAs whose translation was dependent on the presence of the von Hippel-Lindau (VHL) tumor suppressor gene product, pVHL. Here, we investigate the finding that pVHL-expressing RCC cells (VHL⁺) exhibited elevated levels of polysome-associated p53 mRNA and increased p53 protein levels compared with VHL-defective (VHL⁻) cells. Our findings indicate that p53 translation is specifically heightened in VHL⁺ cells, given that (i) p53 mRNA abundance in VHL⁺ and VHL⁻ cells was comparable, (ii) p53 degradation did not significantly influence p53 expression, and (iii) p53 synthesis was markedly induced in VHL⁺ cells. Electrophoretic mobility shift and immunoprecipitation assays to detect endogenous and radiolabeled p53 transcripts revealed that the RNA-binding protein HuR, previously shown to regulate mRNA turnover and translation, was capable of binding to the 3′ untranslated region of the p53 mRNA in a VHL-dependent fashion. Interestingly, while whole-cell levels of HuR in VHL⁺ and VHL⁻ cells were comparable, HuR was markedly more abundant in the cytoplasmic and polysome-associated fractions of VHL⁺ cells. In keeping with earlier reports, the elevated cytoplasmic HuR in VHL⁺ cells was likely due to the reduced AMP-activated kinase activity in these cells. Demonstration that HuR indeed contributed to the increased expression of p53 in VHL⁺ cells was obtained through use of RNA interference, which effectively reduced HuR expression and in turn caused marked decreases in p53 translation and p53 abundance. Taken together, our findings support a role for pVHL in elevating p53 expression, implicate HuR in enhancing VHL-mediated p53 translation, and suggest that VHL-mediated p53 upregulation may contribute to pVHL's tumor suppressive functions in renal cell carcinoma.

ACKNOWLEDGMENTS

We are grateful to J. Gnarra for providing the UOK121 cells, B. Zbar and M. Lerman for the UMRC6 cells, and O. Iliopoulos and W. G. Kaelin for the 786-O cells. We thank K. Becker and C. Cheadle (DNA Array Unit, NIA-IRP) for helpful discussions and for providing cDNA arrays for analysis.

S.G. was sponsored by a German Academic Exchange Service (DAAD) grant through the Johannes Gutenberg University in Mainz, Germany.