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 u pregulation of several genes, including the hematopoietic hormone eryt hropoietin, the angiogenic vascular endothelial growth factor (VEGF), and glycolytic enzymes such as aldolase. Recently, the heme protein cy tochrome b558 of the nicotinamide adenine dinucleotide phosphate (NADP H) 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 phagocyte s and B lymphocytes. Mutations in these subunits result in cytochrome b558-negative chronic granulomatous disease (cytb(-) CGD), an inherite d disorder in humans characterized by reduced microbicidal activity du e 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 compar ed the mRNA levels of VEGF and aldolase with the untreated controls. N orthern blot analysis revealed unimpaired basal and inducible expressi on of VEGF and aldolase mRMA in all four cytb(-) CGD-derived B-cell li nes compared with wild-type cells. Furthermore, reconstitution of cyto chrome b558 expression in cytb(-) CGD-derived B cells by transfection with p22(phox) Or gp91(phox) expression vectors did not modify VEGF an d aldolase mRNA expression. Thus, cytochrome b558 of the NADPH oxidase complex appears not to be essential for hypoxia-activated gene expres sion and can be excluded as a candidate for the putative universal oxy gen sensor. (C) 1996 by The American Society of Hematology.