Cutting edge: infection by the agent of human granulocytic ehrlichiosis prevents the respiratory burst by down-regulating gp91phox

The agent of human granulocytic ehrlichiosis (HGE) is an emerging tick-borne pathogen that resides in neutrophils and can be cultured in a promyelocytic (HL-60) cell line. In response to microbes, polymorphonuclear leukocytes normally activate the NADPH oxidase enzyme complex and generate superoxide anion (O2-). However, HL-60 cells infected with HGE bacteria did not produce O2- upon activation with PMA. RT-PCR demonstrated that HGE organisms inhibited mRNA expression of a single component of NADPH oxidase, gp91phox, and FACS analysis showed that plasma membrane-associated gp91phox protein was reduced on the infected cells. Infection with HGE organisms also decreased gp91phox mRNA levels in splenic neutrophils in a murine model of HGE, demonstrating this phenomenon in vivo. Therefore, HGE bacteria repress the respiratory burst by down-regulating gp91phox, the first direct inhibition of NADPH oxidase by a pathogen.     

Gp91phox contributes to NADPH oxidase activity in aortic fibroblasts but not smooth muscle cells

Reactive oxygen species (ROS) derived from vascular NADPH oxidase are important in normal and pathological regulation of vessel growth and function. Cell-specific differences in expression and function of the catalytic subunit of NADPH oxidase may contribute to differences in vascular cell response to NADPH oxidase activation. We examined the functional expression of gp91phox on NADPH oxidase activity in vascular smooth muscle cells (SMC) and fibroblasts (FB). As measured by dihydroethidium fluorescence in situ, superoxide (O2-*) levels were greater in adventitial cells compared with medial SMC in wild-type aorta. In contrast, there was no difference in O2-* levels between adventitial cells and medial SMC in aorta from gp91phox-deficient (gp91phox KO) mice. Adventitial-derived FB and medial SMC were isolated from the aorta of wild-type and gp91phox KO mice and grown in culture. Consistent with the observations in situ, basal and stimulated ROS levels were reduced in FB isolated from aorta of gp91phox KO compared with FB from wild-type aorta, whereas ROS levels were similar in SMC derived from gp91phox KO and wild-type aorta. There were no differences in expression of superoxide dismutase between gp91phox KO and wild-type FB to account for these observations. Because gp91phox is associated with membranes, we examined NADPH-stimulated O2-. production in membrane-enriched fractions of cell lysate. As measured by chemiluminescence, NADPH oxidase activity was markedly greater in wild-type FB compared with gp91phox KO FB but did not differ among the SMCs. Confirming functional expression of gp91phox in FB, antisense to gp91phox decreased ROS levels in wild-type FB. Finally, deficiency of gp91phox did not alter expression of the gp91phox homolog NOX4 in isolated FB. We conclude that the neutrophil subunit gp91phox contributes to NADPH oxidase function in vascular FB, but not SMC.     

Molecular mechanism for activation of superoxide-producing NADPH oxidases

The membrane-integrated protein gp91phox, existing as a heterodimer with p22phox, functions as the catalytic core of the phagocyte NADPH oxidase, which plays a crucial role in host defence. The oxidase, dormant in resting cells, becomes activated to produce superoxide, a precursor of microbicidal oxidants, by interacting with the adaptor proteins p47phox and p67phox as well as the small GTPase Rac. In the past few years, several proteins homologous to gp91phox were discovered as superoxide-producing NAD(P)H oxidases (Nox's) in non-phagocytic cells; however, regulatory mechanisms for the novel oxidases have been largely unknown. Current identification of proteins highly related to p47phox and p67phox, designated Noxol (Nox organizer 1) and Noxal (Nox activator 1), respectively, has shed lights on common and distinct mechanisms underlying activations of Nox family oxidases.     

Nox3 regulation by NOXO1, p47phox, and p67phox

gp91(phox) (Nox2), the catalytic subunit of the superoxide-generating respiratory burst oxidase, is regulated by subunits p47(phox) and p67(phox). Nox1, a homolog of gp91(phox), is regulated by NOXO1 and NOXA1, homologs of p47(phox) and p67(phox), respectively. For both Nox1 and gp91(phox), an organizer protein (NOXO1 or p47(phox)) cooperates with an activator protein (NOXA1 or p67(phox)) to regulate the catalytic subunit. Herein, we investigate the subunit regulation of Nox3 compared with that of other Nox enzymes. Nox3, like gp91(phox), was activated by p47(phox) plus p67(phox). Whereas gp91(phox) activity required the protein kinase C activator phorbol myristate acetate (PMA), Nox3 activity was already high without PMA, but was further stimulated approximately 30% by PMA. gp91(phox) was also activated by NOXO1/NOXA1 and required PMA for high activity. gp91(phox) regulation required an intact activation domain in the activator protein, as neither p67(phox)(V204A) nor NOXA1(V205A) were effective. In contrast, p67(phox)(V204A) was effective (along with p47(phox)) in activating Nox3. Unexpectedly, Nox3 was strongly activated by NOXO1 in the absence of NOXA1 or p67(phox). Nox3 activity was regulated by PMA only when p47(phox) but not NOXO1 was present, consistent with the phosphorylation-regulated autoinhibitory region in p47(phox) but not in NOXO1. Deletion of the autoinhibitory region from p47(phox) rendered this subunit highly active in the absence of PMA toward both gp91(phox) and Nox3, and high activity required an activator subunit. The unique regulation of Nox3 supports a model in which multiple interactions with regulatory subunits stabilize an active conformation of the catalytic subunit.     

Cloning and Characterization of High-CO2-Specific cDNAs from a Marine Microalga, Chlorococcum littorale, and Effect of CO2 Concentration and Iron Deficiency on the Gene Expression

Two cDNA clones exclusively induced under an extremely high-CO₂concentration (20%) were isolated from Chlorococcum littoraleby differential screening and named HCR (high-CO₂ response)1 and 2, respectively. The amino acid sequence of the proteinencoded by HCR2 exhibited homology to the gp91-phox protein,a critical component of a human phagocyte oxidoreductase, andto the yeast ferric reductases, Saccharomyces cerevisiae FRE1and FRE2 and Schizosaccharomyces pombe Frpl. The induction ofboth HCR mRNAs required extremely high-CO₂ conditions and irondeficiency, being suppressed under air conditions and by ironsufficiency, suggesting that the expression of these two HCRgenes required extremely high-CO₂ conditions and iron deficiencyin combination. The HCR2 protein was detected in the membranefractions of cells grown under conditions which would favorthe induction of HCR2-mRNA and the protein level was loweredwhen the cells were transferred from iron deficient to 10 µMFeSO₄ conditions (with 20% CO²). (Received September 10, 1997; Accepted November 14, 1997)     

Rotenone activates phagocyte NADPH oxidase by binding to its membrane subunit gp91phox

Rotenone, a widely used pesticide, reproduces parkinsonism in rodents and associates with increased risk for Parkinson disease. We previously reported that rotenone increased superoxide production by stimulating the microglial phagocyte NADPH oxidase (PHOX). This study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91(phox), the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91(phox). Functional studies showed that both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91(phox)/p22(phox)) and cytosolic subunits (p67(phox) and p47(phox)). Rotenone-elicited extracellular superoxide release in p47(phox)-deficient macrophages suggested that rotenone enabled activation of PHOX through a p47(phox)-independent mechanism. Increased membrane translocation of p67(phox), elevated binding of p67(phox) to rotenone-treated membrane fractions, and coimmunoprecipitation of p67(phox) and gp91(phox) in rotenone-treated wild-type and p47(phox)-deficient macrophages indicated that p67(phox) played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91(phox). Rac1, a Rho-like small GTPase, enhanced p67(phox)-gp91(phox) interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91(phox); such an interaction triggered membrane translocation of p67(phox), leading to PHOX activation and superoxide production.     

Mutagenesis of an arginine- and lysine-rich domain in the gp91(phox) subunit of the phagocyte NADPH-oxidase flavocytochrome b558

Site-directed mutagenesis was used to generate a series of mutants harboring point or multiple substitutions within the hydrophilic, polybasic domain of gp91(phox) encompassed by residues 86-102, which was previously identified as a site of interaction with p47(phox) during phagocyte NADPH oxidase assembly. Recombinant wild-type or mutant gp91(phox) was expressed in a human myeloid leukemia cell line in which the endogenous gp91(phox) gene was disrupted by gene targeting. NADPH oxidase activity was measured in a cytochrome c reduction assay following granulocytic differentiation of cells that expressed recombinant gp91(phox). Expression of a gp91(phox) mutant in which amino acids 89-97 were replaced with nine alternate amino acids abolished NADPH oxidase activity. Expression of gp91(phox) mutants R89T, D95A, D95R, R96A, R96E, or K102T did not significantly affect NADPH oxidase activity. However, mutations of individual or paired arginine residues at positions 91 and 92 had substantial effects on superoxide generation. The R91E/R92E mutation completely abolished both NADPH oxidase activity and membrane-translocation of the cytosolic oxidase proteins p47(phox), p67(phox), Rac1, and Rac2. The phorbol 12-myristate 13-acetate-induced rate of superoxide production was reduced by approximately 75% in cells expressing R91T/R92A, R91E, or R92E gp91(phox) along with an increased lag time to the maximal rates of superoxide production relative to cells expressing wild-type gp91(phox). Taken together, these results demonstrate that Arg91 and Arg92 of gp91(phox) are essential for flavocytochrome b558 function in granulocytes and suggest that these residues participate in the interaction of gp91(phox) with the cytosolic oxidase proteins.     

Processing and maturation of flavocytochrome b558 include incorporation of heme as a prerequisite for heterodimer assembly

The phagocyte NADPH-dependent oxidase generates superoxide by reducing molecular oxygen through a transmembrane heterodimer known as flavocytochrome b(558) (flavocytochrome b). We investigated the biosynthesis of flavocytochrome b subunits gp91(phox) and p22(phox) to elucidate features of flavocytochrome b processing in myeloid cells. Although the gp91(phox) precursor, gp65, was processed to gp91(phox) within 4-8 h of chase, unassembled gp65 and p22(phox) monomers were degraded by the cytosolic proteasome. gp65 associated with p22(phox) post-translationally, within 1-4 h of chase, but prior to its modification in the Golgi complex. Moreover, p22(phox) coprecipitated with unglycosylated gp91(phox) primary translation product made in the presence of tunicamycin, suggesting that heterodimer formation does not require glycosylation. Blocking heme synthesis with succinyl acetone completely inhibited heterodimer formation, although biogenesis of gp65 and p22(phox) was unaffected. In succinyl acetone-treated cells, p22(phox) and gp65 were degraded completely by 8 h of chase, a process mediated by the cytosolic proteasome. Taken together, these data suggest that the formation of the gp65-p22(phox) heterodimer is relatively inefficient and that acquisition of heme by gp65 precedes and is required for its association with p22(phox), a process that requires neither the addition of N-linked oligosaccharides nor modification in the Golgi complex.     

NAD(P)H oxidase-derived reactive oxygen species regulate angiotensin-II induced adventitial fibroblast phenotypic differentiation

Phenotypic differentiation of adventitial fibroblasts into myofibroblasts is an essential feature of vascular remodeling. The present study was undertaken to test the hypothesis that reactive oxygen species (ROS) are involved in rat adventitial fibroblast differentiation to myofibroblast. Activation of alpha-smooth muscle actin (alpha-SMA) was used as a marker of myofibroblast. Angiotensin II increased intracellular ROS in adventitial fibroblasts that was completely inhibited by the free radical scavenger NAC, the NAD(P)H oxidase inhibitor DPI, and transfection of antisense gp91phox oligonucleotides. Myofibroblast differentiation was prevented by inhibition of ROS generation with DPI, NAC, and antisense gp91phox as shown by decreased expression of alpha-SMA. Angiotensin II rapidly induced phosphorylation of p38 MAPK and JNK, both of which were inhibited by DPI, NAC, antisense gp91phox, and the selective AT1 receptor antagonist, losartan. Inhibiting p38MAPK with SB202190 or JNK with SP600125 also reduced angiotensin II-induced alpha-SMA expression. These findings demonstrate that angiotensin II induces adventitial fibroblast differentiation to myofibroblast via a pathway that involves NADPH oxidase generation of ROS and activation of p38MAPK and JNK pathways.     

The Nox/Duox family of ROS-generating NADPH oxidases

Reactive oxygen species (ROS) generated by the NADPH oxidases are conventionally thought to be cytotoxic and mutagenic and at high levels induce an oxidative stress response. The phagocyte NADPH oxidase catalyzes the NADPH-dependent reduction of molecular oxygen to generate superoxide O2-., which can dismute to generate ROS species. Together, these ROS participate in host defence by killing or damaging invading microbes. Flavocytochrome b558 is the catalytic core of the phagocyte NADPH oxidase and consists of a large glycoprotein gp91phox or Nox-2 and a small protein p22phox. The other components of the NADPH oxidase are cytosolic proteins, namely p67phox, p47phox, p40phox and Rac. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections. Evidence is rapidly accumulating that low level of ROS were produced by NADPH oxidase homologs in non-phagocytic cells. To date, six human homologs (Nox-1, Nox-3, Nox-4, Nox-5, Duox-1 and Duox-2) have been recently identified in a variety of non-phagocytic cells. The identification of Nox-1 was quickly followed by the cloning of Nox-3, Nox-4, and Nox-5. In parallel, two very large members of the Nox family were discovered, namely Duox-1 and Duox-2, initially also referred to as thyroid oxidases. The physiological functions of Nox-dependent ROS generation are in progress and still require detailed characterization. Activation mechanisms and tissue distribution of the different members of the Nox family are very different, suggesting distinct physiological functions. Nox family enzymes are likely to be involved in a variety of physiological events including cell proliferation, host defence, differentiation, apoptosis, senescence and activation of growth-related signaling pathways. An increase and a decrease in the function of Nox enzymes can contribute to a wide range of pathological processes.     

清开灵对小胶质细胞BV2缺氧再复氧损伤gp~(91phox)表达的影响

目的:建立小胶质细胞缺氧再复氧损伤模型,观察产生ROS的NADPH氧化酶的重要功能亚基gp91phox的表达变化及清开灵的干预作用,丰富清开灵基于解毒通络法以祛除内毒恢复脉络的作用内涵。方法:体外培养小鼠胶质细胞BV2,细胞分为正常组、模型组和清开灵高、中、低剂量组,在1%O2三气培养箱中缺氧12小时再复氧12小时模拟缺血再灌注损伤,正常对照组在培养箱中培养24小时,实时荧光定量PCR法检测gp91phoxmRNA的转录水平,Western blot法检测gp91phox蛋白表达。结果:缺氧再复氧损伤后,模型组gp91phox基因转录水平和蛋白表达提高(P0.05);与模型组比较,清开灵低、中、高剂量组都有明显改善作用,其中低剂量(0.0625%)对基因转录降低更明显,高剂量组(0.25%)对gp91phox蛋白表达的抑制更显著,具有统计学意义(P0.05)。结论:清开灵可通过降低缺氧再复氧后小胶质细胞gp91phox的表达,减少活性氧的产生而抑制脑缺血损伤氧化应激反应。     

Gene therapy for inherited diseases using heamatopoietic stem cells--gene therapy for patients with chronic granulomatous disease

The possibility of gene therapy for inherited diseases with a single gene mutation in Figure 1 had been verified by the successful treatment with bone marrow transplantation. As the gene therapy method and theory has been progressing rapidly, it is expected that gene therapy will overcome the complications of bone marrow transplantation. Of these inherited diseases, chronic granulomatous disease (CGD) is the one of the most expected disease for gene therapy. CGD is an inherited immune deficiency caused by mutations in any of the following four phox genes encoding subunits of the superoxide generating phagocyte NADPH oxidase. It consists of membranous cytochrom b558 composed of gp91 phox and p22 phox, and four cytosolic components, p47 phox, p67 phox, rac p21 and p40 phox, which translocate to the membrane upon activation. In our group study, more than 220 CGD patients has been enrolled. The incidence of CGD patients was estimated as 1 out of 250,000 births. The expected life span of the CGD patients is 25 to 30 years old by the Kaplan Meier analysis. Comparing with the ratio of CGD subtype in US and Europe, that with p47phox deficiency is lower (less than 10%/o vs. 23%) and that of gp91 phox deficiency is higher (more than 75% vs. 60%). Prophylactic administration of ST antibiotics and IFN-gamma and bone marrow transplantation have been successfully employed in our therapeutic strategy. However, it is necessary to develop the gene therapy technology for CGD patients as more promising treatment. In the current study we constructed two retrovirus vectors; MFGS-gp91/293 SPA which contains only the therapeutic gp91 phox gene, a bicistronic retrovirus pHa-MDR-IRES-gp91/PA317 which carries a multi drug resistant gene (MDR1) and the gp91phox gene connected with an internal ribosome entry site (IRES). We demonstrate high efficiency transduction of gp 91 phox to CGD EB virus established cell line with high levels of functional correction of the oxidase by MFGS-gp91 and by pHa-MDR-IRES-gp91, respectively. We also demonstrate sufficient transduction of gp91 phox to CD34+ hematopoietic stem cell from the patients with gp91 phox deficiency by MFGS-gp91/293 SPA. Our current studies suggest that the combination of the 293-SPA packaging system and the bicistronic retrovirus system inserted MDR1 gene make our CGD gene therapy more feasible for clinical application.     

gp91phox-containing NAD(P)H oxidase mediates attenuation of nitric oxide-dependent control of myocardial oxygen consumption by ANG II

We have previously reported that ANG II stimulation increased superoxide anion (O2-) through the activation of NAD(P)H oxidase and inhibited nitric oxide (NO)-dependent control of myocardial oxygen consumption (MVo2) by scavenging NO. Our objective was to investigate the role of NAD(P)H oxidase, especially the gp91phox subunit, in the NO-dependent control of MVo2. MVo2 in mice with defects in the expression of gp91phox [gp91(phox)(-/-)] was measured with a Clark-type oxygen electrode. Baseline MVo2 was not significantly different between wild-type (WT) and gp91(phox)(-/-) mice. Stimulation of NO production by bradykinin (BK) induced significant decreases in MVo2 in WT mice. BK-induced reduction in MVo2 was enhanced in gp91(phox)(-/-) mice. BK-induced reduction in MVo2 in WT mice was attenuated by 10(-8) mol/l ANG II, which was restored by coincubation with Tiron or apocynin. In contrast to WT mice, BK-induced reduction in MVo2 in gp91(phox)(-/-) mice was not altered by ANG II. There was a decrease in lucigenin (5 x 10(-6) mol/l)-detectable O2- in gp91(phox)(-/-) mice compared with WT mice. ANG II resulted in significant increases in O2- production in WT mice, which was inhibited by coincubation with Tiron or apocynin. However, ANG II had no effect on O2- production in gp91(phox)(-/-) mice. Histological examination showed that the development of abscesses and/or the invasion of inflammatory cells occurred in lungs and livers but not in hearts and kidneys from gp91(phox)(-/-) mice. These results indicate that the gp91(phox) subunit of NAD(P)H oxidase mediates O2- production through the activation of NAD(P)H oxidase and attenuation of NO-dependent control of MVo2 by ANG II.