Abstract
Previous studies have demonstrated that copper up-regulates hypoxia-inducible factor 1 (HIF-1). The present study was undertaken to test the hypothesis that copper is required for HIF-1 activation. Treatment of HepG2 cells with a copper chelator tetraethylenepentamine (TEPA) or short interfering RNA targeting copper chaperone for superoxide dismutase 1 (CCS) suppressed hypoxia-induced activation of HIF-1. Addition of excess copper relieved the suppression by TEPA, but not that by CCS gene silencing, indicating the requirement of copper for activation of HIF-1, which is CCS-dependent. Copper deprivation did not affect production or stability of HIF-1α but reduced HIF-1α binding to the hypoxia-responsive element (HRE) of target genes and to p300, a component of HIF-1 transcriptional complex. Copper probably inhibits the factor inhibiting HIF-1 to ensure the formation of HIF-1 transcriptional complex. This study thus defines that copper is required for HIF-1 activation through the regulation of HIF-1α binding to the HRE and the formation of the HIF-1 transcriptional complex.
Footnotes
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Support for this study was provided in part by National Institutes of Health grants HL59225 and HL63760 (to Y.J.K.), Kentucky Science and Engineering Foundation grant KSEF-888-RDE-008 (to W.F. and Y.J.K.), and American Diabetes Association grant 7-07-JF-23 (to W.F.).
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Y.J.K. is a Distinguished University Scholar at the University of Louisville.
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ABBREVIATIONS: VEGF, vascular endothelial growth factor; TEPA, tetraethylenepentamine; HIF, hypoxia-inducible factor; HRE, hypoxia-responsive element; DFO, deferoxamine; FIH-1, factor inhibiting hypoxia-inducible factor 1; CCS, copper chaperone for superoxide dismutase 1; IGF, insulin-like growth factor; PHD, hypoxia-inducible factor prolyl hydroxylase; EMSA, electrophoretic mobility shift assay; siRNA, short interfering RNA; PBS, phosphate-buffered saline; DTT, dithiothreitol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
- Received August 22, 2008.
- Accepted October 8, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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