Influence of the RNA-binding protein HuR in pVHL-regulated p53 expression in renal carcinoma cells

Mol Cell Biol. 2003 Oct;23(20):7083-95. doi: 10.1128/MCB.23.20.7083-7095.2003.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions
  • Actins / metabolism
  • Adenylate Kinase / metabolism
  • Antigens, Surface*
  • Base Sequence
  • Blotting, Northern
  • Blotting, Western
  • Carcinoma, Renal Cell / metabolism*
  • Cell Line, Tumor
  • Cytoplasm / metabolism
  • DNA, Complementary / metabolism
  • ELAV Proteins
  • ELAV-Like Protein 1
  • Gene Expression Regulation, Neoplastic*
  • Humans
  • Kidney Neoplasms / metabolism*
  • Microscopy, Fluorescence
  • Molecular Sequence Data
  • Oligonucleotide Array Sequence Analysis
  • Plasmids / metabolism
  • Polyribosomes / metabolism
  • Precipitin Tests
  • Protein Binding
  • Protein Biosynthesis
  • RNA / metabolism
  • RNA Interference
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / metabolism
  • RNA-Binding Proteins / metabolism
  • RNA-Binding Proteins / physiology*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Time Factors
  • Transfection
  • Tumor Suppressor Protein p53 / metabolism*
  • Tumor Suppressor Proteins / metabolism*
  • Ubiquitin-Protein Ligases / metabolism*
  • Up-Regulation
  • Von Hippel-Lindau Tumor Suppressor Protein

Substances

  • 3' Untranslated Regions
  • Actins
  • Antigens, Surface
  • DNA, Complementary
  • ELAV Proteins
  • ELAV-Like Protein 1
  • ELAVL1 protein, human
  • RNA, Messenger
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • Tumor Suppressor Protein p53
  • Tumor Suppressor Proteins
  • RNA
  • Ubiquitin-Protein Ligases
  • Von Hippel-Lindau Tumor Suppressor Protein
  • Adenylate Kinase
  • VHL protein, human