Copper Is Required for Cobalt-Induced Transcriptional Activity of Hypoxia-Inducible Factor-1

Cobalt inhibits prolyl hydroxylases, leading to the accumulation of hypoxia-inducible factor-1 alpha (HIF-1 alpha) and a concomitant increase in the transcriptional activity of HIF-1. Therefore, cobalt has been under development as a drug for activating HIF-1 under some disease conditions. However, it has been shown that ischemic conditions resulted in the loss of copper, and the activation of HIF-1 would not occur unless copper was supplemented. The present study was undertaken to test the hypothesis that copper is also required for the cobalt activation of HIF-1 transcriptional activity. Human umbilical vein endothelial cells subjected to treatment with cobalt chloride (CoCl2) at concentrations above 25 mu M for 2 h resulted in an accumulation of HIF-1 alpha, which was determined by Western blot analysis, and an increase in the expression of vascular endothelial growth factor (VEGF), which was determined by real-time reverse transcription-polymerase chain reaction analysis for mRNA levels and enzyme-linked immunosorbent assay analysis for protein levels. The copper chelator tetraethylenepentamine at 25 mu M did not significantly affect the accumulation of HIF-1 alpha but blocked increases in VEGF mRNA and protein levels, an effect that could be reversed by the addition of 25 mu M copper sulfate (CuSO4). In addition, gene silencing of the copper chaperone for Cu,Zn-superoxide dismutase blocked VEGF expression with little effect on cobalt-induced HIF-1 alpha accumulation. The present study thus demonstrates that copper was required for cobalt-activated transcriptional activity of HIF-1, although copper did not affect cobalt-induced accumulation of HIF-1 alpha in the cells.