Abstract
Hypoxia-inducible factor-1α (HIF-1α) induction and associated transcription were investigated during high cell density, focusing on the negative regulator of HIF-1α expression, the von Hippel-Lindau (VHL) protein. In 293T and HeLa cells, HIF-1α 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-1α 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-1α 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-1α noted in other cell lines. Investigation of both N-terminal and C-terminal (aa 727–826) oxygen-regulated proline and asparagine hydroxylation of HIF-1α revealed that both are inhibited during high cell density, as determined by impaired capture of HIF-1α by VHL and enhanced C-terminal transactivation. Finally, cell-density-mediated induction of HIF-1α and GLUT1 in RCC cells could be completely reconstituted by mutations in VHL binding affinity, suggesting that cell-density dependent induction of HIF-1α and transactivation may underpin some of the deregulated gene expression observed in VHL disease.
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This work was supported in part by the National Health and Medical Research Council of Australia, project grant no. 10365, awarded to BJ Roberts.
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Paltoglou, S., Roberts, B. Role of the von Hippel-Lindau tumour suppressor protein in the regulation of HIF-1α and its oxygen-regulated transactivation domains at high cell density. Oncogene 24, 3830–3835 (2005). https://doi.org/10.1038/sj.onc.1208531
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DOI: https://doi.org/10.1038/sj.onc.1208531
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