PU.1 is dominant and HAF-1 supplementary for activation of the gp91(phox) promoter in human monocytic PLB-985 cells

Gp91(phox) is a key component of the phagocyte NADPH oxidase. Mutations of its promoter found in patients with chronic granulomatous disease cause deficient binding of PU.1 and HAF-1. Because the two factors bind to the same site (Pu box) of the promoter, we attempted to clarify their relative in vivo contributions to activation of the gp91(phox) promoter in monocytically differentiated PLB-985 cells using a dual luciferase reporter assay and a gel shift competition assay. We found that the activity of a series of single-point-mutated promoters increases or decreases according to an increase or decrease, respectively, in the affinity of the promoters to PU.1 but not to HAF-1. Two of 7 mutants showing weak binding affinity to PU.1 exhibited moderate promoter activity and normal binding affinity for HAF-1. These results suggest PU.1 is the dominant activator and HAF-1 is supplementary. The increased promoter activity of single-, double-, and triple-point-mutated constructs with sequences closer to that of the Ets-binding element correlates with their binding affinity to PU.1 but not to HAF-1, supporting that PU.1 is a more efficient activator than HAF-1. In contrast to co-expressed wild-type PU.1, dominant-negative PU.1 significantly inhibited the activity of a PU.1-optimised gp91(phox) promoter construct. Therefore, we conclude that PU.1 and HAF-1 binding to the Pu box is dominant and supplementary, respectively, for activation of the gp91(phox) promoter in human monocytic cells.     

Upregulation of p67(phox) and gp91(phox) in aortas from angiotensin II-infused mice

Although NAD(P)H oxidase-derived superoxide (O(2)(-)) is increased during the development of angiotensin II (ANG II)-dependent hypertension, vascular regulation at the protein level has not been reported. We have shown that four major components of NAD(P)H oxidase are located primarily in the vascular adventitia as a primary source of vascular O(2)(-). Here we compare vascular levels of O(2)(-) and NAD(P)H oxidase in normotensive and ANG II-infused hypertensive mice and show that, after 7 days of ANG II infusion (750 microg. kg(-1). day(-1) ip) in C57B1/6 mice, systolic blood pressure was increased compared with that after sham infusion, concomitant with increased O(2)(-) in the thoracic aorta as measured using lucigenin (25 microM)-enhanced chemiluminescence. Both p67(phox) and gp91(phox) were detectable by Western blotting in aortic homogenates, and we observed increased protein levels of NAD(P)H oxidase subunits. These ANG II-induced increases were normalized by simultaneous treatment with the AT(1) receptor antagonist losartan. Moreover, the primary location of these subunits was the adventitia as detected immunohistochemically. Our results suggest that ANG II-induced increases in O(2)(-) are due to increased adventitial NAD(P)H oxidase activity, brought about by the heightened expression and interaction of its components.     

Activation of the flavoprotein domain of gp91phox upon interaction with N-terminal p67phox (1-210) and the Rac complex

A series of truncated forms of His(6)-tagged gp91phox were expressed, solubilized, and purified in the presence of 30 microM FAD. The truncated gp91phox with the longest sequence in the C-terminal region (221-570) (gp91C) showed the highest activity (turnover rate, 0.92) for NADPH diaphorase in the presence of either 0.3% Triton X-100 or 0.5% Genapol X-80. Activity was not inhibited by superoxide dismutase but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium. The flavinated gp91C contained approximately 0.9 mol of FAD/mol of protein (MW 46 kDa) and 12% alpha-helix content. In the absence of p47phox, p67phox showed considerable activation of gp91C in the presence of Rac. Carboxyl-terminal truncated p67phox (1-210) (p67N), which is the minimal active fragment, was fused with Rac or Q61LRac. The fusion protein p67N-Rac (or p67N-Q61LRac) showed a 2-fold higher stimulatory effect on NBT reductase activity of gp91C than the combination of the individual cytosolic p67N and Rac proteins. In contrast, Rac-p67N, a fusion with the opposite orientation, showed a smaller significant effect on the enzyme activity. The EC(50) values for p67phox, p67N, p67N-Rac, and Rac-p67N were 8.00. 4.35, 2.56, and 15.2 microM, respectively, while the K(m) value for NADPH in the presence and absence of the cytosolic components was almost the same (40-55 microM). In the presence of Rac, p67N or p67phox bound to gp91C with a molar ratio of approximately 1:1 but neither p67N nor Rac alone showed significant binding.     

Hypoxic pulmonary hypertension: role of superoxide and NADPH oxidase (gp91phox)

Chronic exposure to low-O2 tension induces pulmonary arterial hypertension (PAH), which is characterized by vascular remodeling and enhanced vasoreactivity. Recent evidence suggests that reactive oxygen species (ROS) may be involved in both processes. In this study, we critically examine the role superoxide and NADPH oxidase plays in the development of chronic hypoxic PAH. Chronic hypoxia (CH; 10% O2 for 3 wk) caused a significant increase in superoxide production in intrapulmonary arteries (IPA) of wild-type (WT) mice as measured by lucigenin-enhanced chemiluminescence. The CH-induced increase in the generation of ROS was obliterated in NADPH oxidase (gp91phox) knockout (KO) mice, suggesting that NADPH oxidase was the major source of ROS. Importantly, pathological changes associated with CH-induced PAH (mean right ventricular pressure, medial wall thickening of small pulmonary arteries, and right heart hypertrophy) were completely abolished in NADPH oxidase (gp91phox) KO mice. CH potentiated vasoconstrictor responses of isolated IPAs to both 5-hydroxytryptamine (5-HT) and the thromboxane mimetic U-46619. Administration of CuZn superoxide dismutase to isolated IPA significantly reduced CH-enhanced superoxide levels and reduced the CH-enhanced vasoconstriction to 5-HT and U-46619. Additionally, CH-enhanced superoxide production and vasoconstrictor activity seen in WT IPAs were markedly reduced in IPAs isolated from NADPH oxidase (gp91phox) KO mice. These results demonstrate a pivotal role for gp91phox-dependent superoxide production in the pathogenesis of CH-induced PAH.     

Heme histidine ligands within gp91(phox) modulate proton conduction by the phagocyte NADPH oxidase

The membrane subunit of the phagocyte NADPH oxidase, gp91(phox), possesses a H(+) channel motif formed by membrane-spanning histidines postulated to coordinate the two heme groups forming the redox center of the flavocytochrome. To study the role of heme-binding histidines on proton conduction, we stably expressed the gp91(phox) cytochrome in human embryonic kidney 293 cells and measured proton currents with the patch clamp technique. Similar to its shorter homologue, NADPH oxidase homologue 1, which is predicted not to bind heme, gp91(phox) generated voltage-activated, pH-dependent, H(+)-selective currents that were reversibly blocked by Zn(2+). The gp91(phox) currents, however, activated faster, deactivated more slowly, and were markedly affected by the inhibition of heme synthesis. Upon heme removal, the currents had larger amplitude, activated faster and at lower voltages, and became sensitive to the histidine reagent diethylpyrocarbonate. Mutation of the His-115 residue to leucine abolished both the gp91(phox) characteristic 558-nm absorbance peak and voltage-activated currents, indicating that His-115 is involved in both heme ligation and proton conduction. These results indicate that the gp91(phox) proton channel is activated upon release of heme from its His-115 ligand. During activation of the oxidase complex, changes in heme coordination within the cytochrome might increase the mobility of histidine ligands, thereby coupling electron and proton transport.     

Sequence-specific binding of HMG-I(Y) to the proximal promoter of the gp91-phox gene.

Screening of a cDNA expression library with a CCAAT-box element derived from the myelomonocyte-specific gp91-phox promoter resulted in the isolation of three independent HMG-I(Y) cDNA clones. Filter binding competition studies reveal that HMG-Y binds to this promoter element in a sequence-specific manner and exhibits a gradient of binding affinities for various A/T-rich sequences. Two adjacent A/T-rich regions within the gp91-phox promoter CCAAT-box element are required for maximal binding. In addition, competition experiments demonstrate that the binding affinity of HMG-Y is influenced by sequences that flank A/T-rich core binding sites.     

Expression of NOX-I, gp91phox, p47phox and P67phox in the aorta segments above and below coarctation

Aorta coarctation results in hypertension (HTN) in the arterial tree proximal to stenosis and, as such, provides an ideal model to discern the effects of different levels of blood pressure on the vascular tissue in the same animal. Compelling evidence has emerged supporting the role of oxidative stress as a cause of HTN. However, whether or not HTN (independent of the circulating humoral factors) can cause oxidative stress is less certain. NAD(P)H oxidase isoforms are the main source of reactive oxygen species (ROS) in the vascular tissues. We therefore compared the expressions of NOX-I, gp91phox and the regulatory subunits of the enzyme in the aorta segments residing above and below coarctation in rats with abdominal aorta banding. Rats were studied 4 weeks after aorta banding above the renal arteries or sham operation. Subunits of NAD(P)H oxidase and its NOX-I isoform as well as endothelial NO synthase (eNOS) and nitrotyrosine (footprint of NO oxidation by superoxide) were measured in the aorta segments above and below coarctation. The gp91phox, p47phox, and p67phox subunits of NAD(P)H oxidase, NOX-I isoform, eNOS and nitrotyrosine were markedly increased in the aorta segment above coarctation (hypertensive zone), but were virtually unchanged in the segment below coarctation. Since, excepting blood pressure, all other conditions were constant, the upregulation of NAD(P)H oxidase isoforms and the increased NO oxidation in the aorta segment above, but not below, coarctation prove that HTN, per se, independent of circulating mediators can cause oxidative/nitrosative stress in the arterial wall. These observations suggest that HTN control may represent a specific form of antioxidant therapy for hypertensive disorders.     

Differential effects of diabetes on the expression of the gp91phox homologues nox1 and nox4

The nox2-dependent NADPH oxidase was shown to be a major superoxide source in vascular disease, including diabetes. Smooth muscle cells of large arteries lack the phagocytic gp91phox subunit of the enzyme; however, two homologues have been identified in these cells, nox1 and nox4. It remained to be established whether also increases in protein levels of the nonphagocytic NADPH oxidase contribute to increased superoxide formation in diabetic vessels. To investigate changes in the expression of these homologues, we measured their expression in aortic vessels of type I diabetic rats. Eight weeks after streptozotocin treatment, we found a doubling in nox1 protein expression, while the expression of nox4 remained unchanged. This was associated with a significant increase in the NADPH oxidase activity in membrane fractions of diabetic heart and aortic tissue. Furthermore, we observed a decreased sensitivity of diabetic vessels to acetylcholine and nitroglycerin and a decrease in both acetylcholine-stimulated NO production and phosphorylation of VASP, despite an increase in endothelial NO synthase (NOSIII) expression. In addition, xanthine oxidase activity was markedly increased in plasma and 100,000 g supernatant of cardiac tissue of diabetic rats, while myocardial mitochondrial superoxide formation was only weakly enhanced. We conclude that in addition to phagocytic NADPH oxidase, also nonphagocytic, vascular NADPH oxidase subunit nox1, uncoupled NOSIII, and plasma xanthine oxidase contribute to endothelial dysfunction in the setting of diabetes mellitus.     

Heme-ligating histidines in flavocytochrome b(558): identification of specific histidines in gp91(phox).

The phagocyte NADPH-dependent oxidase generates superoxide (O(2)) by reducing molecular oxygen through flavocytochrome b(558) (flavocytochrome b), a heterodimeric oxidoreductase composed of gp91(phox) and p22(phox) subunits. Although each flavocytochrome b molecule contains two heme groups, their precise distribution within the heterodimer is unknown. Among functionally and/or structurally related oxidoreductases, histidines at codons 101, 111, 115, 119, 209, 210, and 222 of gp91(phox) are conserved and potential candidates to ligate heme. We compared biochemical and functional features of normal flavocytochrome b with those in cells expressing gp91(phox) harboring amino acid substitutions at each of these histidines. Surface expression of flavocytochrome b and heterodimer formation were relatively unaffected in cells expressing gp91(phox) H111L, H119L, or H210L. These mutations also had no effect on the flavocytochrome b heme spectrum, although NADPH oxidase activity was decreased in cells expressing gp91(phox) H119L or H210L. In contrast, gp65 was not processed to gp91(phox), heterodimers did not form, and flavocytochrome b was not expressed on the surface of cells expressing gp91(phox) H101L, H115L, H115D, H209C, H209Y, H222L, H222C, or H222R. Similarly, this subset of mutants lacked detectable O(2)-generating activity, and flavocytochrome b purified from these cells contained little or no heme. These findings demonstrate that His(101), His(115), His(209), and His(222) of gp91(phox) are critical for heme binding and biosynthetic maturation of flavocytochrome b.     

Identification of a spectrally stable proteolytic fragment of human neutrophil flavocytochrome b composed of the NH2-terminal regions of gp91(phox) and p22(phox)

A heme-bearing polypeptide core of human neutrophil flavocytochrome b(558) was isolated by applying high performance, size exclusion, liquid chromatography to partially purified Triton X-100-solubilized flavocytochrome b that had been exposed to endoproteinase Glu-C for 1 h. The fragment was composed of two polypeptides of 60-66 and 17 kDa by SDS-polyacrylamide gel electrophoresis and retained a native heme absorbance spectrum that was stable for several days when stored at 4 degrees C in detergent-containing buffer. These properties suggested that the majority of the flavocytochrome b heme environment remained intact. Continued digestion up to 4.5 h yielded several heme-associated fragments that were variable in composition between experiments. Digestion beyond 4.5 h resulted in a gradual loss of recoverable heme. N-Linked deglycosylation and reduction and alkylation of the 1-h digestion fragment did not affect the electrophoretic mobility of the 17-kDa fragment but reduced the 60-66-kDa fragment to 39 kDa. Sequence and immunoblot analyses identified the fragments as the NH(2)-terminal 320-363 amino acid residues of gp91(phox) and the NH(2)-terminal 169-171 amino acid residues of p22(phox). These findings provide direct evidence that the primarily hydrophobic NH(2)-terminal regions of flavocytochrome b are responsible for heme ligation.