Hypoxic induction of gene expression in chronic granulomatous disease-derived B-cell lines: Oxygen sensing is independent of the cytochrome b558-containing nicotinamide adenine dinucleotide phosphate oxidase

Reduced oxygenation of a variety of cells results in transcriptional upregulation of several genes, including the hematopoietic hormone erythropoietin, the angiogenic vascular endothelial growth factor (VEGF), and glycolytic enzymes such as aldolase. Recently, the heme protein cytochrome b558 of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex has been proposed as a key component of the oxygen-sensing mechanism. Cytochrome b558 consists of the p22(phox) and gp91(phox) subunits and is essential for superoxide generation in phagocytes and B lymphocytes. Mutations in these subunits result in cytochrome b558-negative chronic granulomatous disease (cytb(-) CGD), an inherited disorder in humans characterized by reduced microbicidal activity due to deficient superoxide generation. To test whether NADPH oxidase is involved in oxygen sensing, we exposed wild-type B-cell lines as well as cytb(-) CGD-derived B cell lines, deficient in either p22(phox) or gp91(phox), to hypoxia (1% oxygen) or CoCl2 (100 mu mol/L) and compared the mRNA levels of VEGF and aldolase with the untreated controls. Northern blot analysis revealed unimpaired basal and inducible expression of VEGF and aldolase mRMA in all four cytb(-) CGD-derived B-cell lines compared with wild-type cells. Furthermore, reconstitution of cytochrome b558 expression in cytb(-) CGD-derived B cells by transfection with p22(phox) Or gp91(phox) expression vectors did not modify VEGF and aldolase mRNA expression. Thus, cytochrome b558 of the NADPH oxidase complex appears not to be essential for hypoxia-activated gene expression and can be excluded as a candidate for the putative universal oxygen sensor. (C) 1996 by The American Society of Hematology.