1. ¹School of Pharmaceutical, Molecular and Biomedical Sciences, University of South Australia, Reid Building, Frome Rd., Adelaide 5000, Australia

Correspondence: BJ Roberts, School of Pharmaceutical, Molecular and Biomedical Sciences, University of South Australia, Reid Building, Frome Rd, Adelaide, Australia, 5000. E-mail: [email protected]

Received 28 October 2004; Revised 07 January 2005; Accepted 14 January 2005; Published online 07 March 2005.

Abstract

Hypoxia-inducible factor-1 (HIF-1) induction and associated transcription were investigated during high cell density, focusing on the negative regulator of HIF-1 expression, the von Hippel-Lindau (VHL) protein. In 293T and HeLa cells, HIF-1 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-1 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-1 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-1 noted in other cell lines. Investigation of both N-terminal and C-terminal (aa 727−826) oxygen-regulated proline and asparagine hydroxylation of HIF-1 revealed that both are inhibited during high cell density, as determined by impaired capture of HIF-1 by VHL and enhanced C-terminal transactivation. Finally, cell-density-mediated induction of HIF-1 and GLUT1 in RCC cells could be completely reconstituted by mutations in VHL binding affinity, suggesting that cell-density dependent induction of HIF-1 and transactivation may underpin some of the deregulated gene expression observed in VHL disease.