Role of the von Hippel-Lindau tumour suppressor protein in the regulation of HIF-1alpha and its oxygen-regulated transactivation domains at high cell density.
Hypoxia-inducible factor-1alpha (HIF-1alpha) induction and associated transcription were investigated during high cell density, focusing on the negative regulator of HIF-1alpha expression, the von Hippel-Lindau (VHL) protein. In 293T and HeLa cells, HIF-1alpha protein levels and associated transcription were induced as cells approached confluence. To determine whether these changes were due to a deficit in nuclear VHL-mediated ubiquitination of HIF-1alpha at confluence, cells were stably transfected with VHL. Overexpression of VHL in 293T cells had no demonstrable effect on the induction and nuclear accumulation of HIF-1alpha during high cell density or associated transcription. Moreover, RCC cells stably transfected with full-length VHL failed to exhibit the cell-density-dependent induction of HIF-1alpha noted in other cell lines. Investigation of both N-terminal and C-terminal (aa 727-826) oxygen-regulated proline and asparagine hydroxylation of HIF-1alpha revealed that both are inhibited during high cell density, as determined by impaired capture of HIF-1alpha by VHL and enhanced C-terminal transactivation. Finally, cell-density-mediated induction of HIF-1alpha and GLUT1 in RCC cells could be completely reconstituted by mutations in VHL binding affinity, suggesting that cell-density dependent induction of HIF-1alpha and transactivation may underpin some of the deregulated gene expression observed in VHL disease.
Paltoglou
Steve M
SM
School of Pharmaceutical, Molecular and Biomedical Sciences, University of South Australia, Reid Building, Frome Rd., Adelaide 5000, Australia.
Roberts
Ben J
BJ
D002452:Cell Count; D002460:Cell Line; D051272:Glucose Transporter Type 1; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis; D020218:Response Elements / Q000502:physiology; D014157:Transcription Factors / Q000096:biosynthesis*; D015533:Transcriptional Activation; D025521:Tumor Suppressor Proteins / Q000502:physiology*; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*; D051794:Von Hippel-Lindau Tumor Suppressor Protein
D051272:Glucose Transporter Type 1; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009004:Monosaccharide Transport Proteins; C495677:SLC2A1 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human