Induction of erythropoietin (hypoxia marker) in Hep3B cells by hypoxia depends on NADPH-dependent enzyme

Abstract Regulation of gene expression in hypoxia is of fundamental importance in developmental, physiological, and pathophysiological processes. Erythropoietin (EPO) is a typical protein induced by hypoxia, and its induction appears to be dependent on signal transduction by hypoxia inducible factor 1 (HIF-1). The activation of HIF-1 by hypoxia depends on a sensing and signaling process that is poorly understood. In this study, we used several enzyme inhibitors to investigate the oxygen sensing mechanism of Hep3B cells. Hydrogen peroxide, Fe 2+ , and vitamin C were found to inhibit EPO induction by hypoxia; however; inhibitors of xanthine oxidase and nitric oxide synthase did not inhibit the induction of EPO. Diphenyleneiodonium chloride (DPIC), an inhibitor of NADPH oxidase, efficiently inhibited EPO induction; however, NADPH oxidase mRNA was not detected although NADPH– P 450 reductase was expressed abundantly. In addition, several metal ions mimic the hypoxic response of the cells. Cobalt and nickel induced EPO under normoxic conditions but zinc and cadmium did not. Zinc mediated the accumulation of HIF-1α in Hep3B cells but did not induce EPO, and cadmium promoted the degradation of HIF-1α. These results suggest that reactive oxygen species produced by the NADPH-dependent enzyme or transition metals are important in the stabilization of HIF-1α. Finally, cellular redox changes are likely to be involved in the oxygen-sensing mechanism of Hep3B cells.