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 p22phox and gp91phox 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 p22phox or gp91phox, to hypoxia (1% oxygen) or CoCl2 (100 mumol/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 mRNA 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 p22phox or gp91phox 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.
Wenger
R H
RH
Institute of Physiology, University of Zürich, Switzerland.
Marti
H H
HH
Schuerer-Maly
C C
CC
Kvietikova
I
I
Bauer
C
C
Gassmann
M
M
Maly
F E
FE
D001402:B-Lymphocytes / Q000378:metabolism*; D001483:Base Sequence; D015415:Biomarkers; D015687:Cell Hypoxia; D002461:Cell Line, Transformed; D003035:Cobalt / Q000494:pharmacology; D003573:Cytochrome b Group / Q000502:physiology*; D016228:Endothelial Growth Factors / Q000096:biosynthesis* / Q000235:genetics; D005634:Fructose-Bisphosphate Aldolase / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation; D006105:Granulomatous Disease, Chronic / Q000201:enzymology / Q000235:genetics / Q000473:pathology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008222:Lymphokines / Q000096:biosynthesis* / Q000235:genetics; D008562:Membrane Glycoproteins / Q000172:deficiency* / Q000502:physiology; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000502:physiology*; D009247:NADH, NADPH Oxidoreductases / Q000502:physiology*; D009252:NADPH Dehydrogenase / Q000172:deficiency* / Q000502:physiology; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D010100:Oxygen / Q000378:metabolism; D010313:Partial Pressure; D010750:Phosphoproteins / Q000172:deficiency* / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D015415:Biomarkers; D003573:Cytochrome b Group; D016228:Endothelial Growth Factors; D008222:Lymphokines; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009097:Multienzyme Complexes; D010750:Phosphoproteins; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D003035:Cobalt; C036888:cytochrome b558; D006861:Hydrogen Peroxide; D009247:NADH, NADPH Oxidoreductases; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human; D009252:NADPH Dehydrogenase; D005634:Fructose-Bisphosphate Aldolase; C018021:cobaltous chloride; D010100:Oxygen