NADPH-cytochrome P-450 reductase in the plasma membrane modulates the activation of hypoxia-inducible factor 1.

Hypoxia induces a group of physiologically important genes that include erythropoietin (EPO) and vascular endothelial growth factor (VEGF). Hypoxia-inducible factor 1 (HIF-1) was identified as a hypoxia-activated transcription factor; however, the molecular mechanisms that underlie hypoxia signal transduction in mammalian cells remain undefined. In this study, we found that a flavoprotein, NADPH-P450 reductase (NPR), could regulate the induction of EPO mRNA under hypoxic conditions. Hypoxic EPO mRNA induction in Hep3B cells was inhibited by diphenyleneiodonium chloride, which is an inhibitor of NADPH-dependent enzymes. NPR antisense cDNA was transfected into Hep3B cells, and NPR-deficient hepatocyte cells (NPR(-) cells) were established. NPR(-) cells lacked EPO induction under hypoxia, and HIF-1alpha in NPR(-) cells did not respond to either transcriptional activation or translocation to the nucleus based on electrophoretic mobility shift assays and reporter gene assay including hypoxia response element. In contrast, NPR overexpression in Hep3B cells enhanced the DNA binding activity of HIF-1alpha by luciferase reporter gene assay. A study with HeLa S3 cells produced the same results. Furthermore, anti-NPR IgG inhibited EPO induction. EPO induction inhibited by diphenyleneiodonium chloride was recovered by bovine serum albumin-NADPH (a covalent binding complex of bovine serum albumin and NADPH) as well as NADPH. These results suggested that NPR located at the plasma membrane regulates EPO expression in hypoxia, including HIF-1 activation and translocation. We further studied the expression of NPR and VEGF mRNAs in human tumor tissues and found that the NPR mRNA levels were correlated with the VEGF mRNA levels, suggesting that NPR might be an important factor in the hypoxic induction of genes such as VEGF in vivo.