Membrane Translocation Of P47phox Research Articles

Homocysteine stimulates phosphorylation of NADPH oxidase p47phoxand p67phoxsubunits in monocytes via protein kinase Cβ activation

Hyperhomocysteinaemia is an independent risk factor for cardiovascular diseases due to atherosclerosis. The development of atherosclerosis involves reactive oxygen species-induced oxidative stress in vascular cells. Our previous study [Wang and O (2001) Biochem. J. 357, 233-240] demonstrated that Hcy (homocysteine) treatment caused a significant elevation of intracellular superoxide anion, leading to increased expression of chemokine receptor in monocytes. NADPH oxidase is primarily responsible for superoxide anion production in monocytes. In the present study, we investigated the molecular mechanism of Hcy-induced superoxide anion production in monocytes. Hcy treatment (20-100 microM) caused an activation of NADPH oxidase and an increase in the superoxide anion level in monocytes (THP-1, a human monocytic cell line). Transfection of cells with p47phox siRNA (small interfering RNA) abolished Hcy-induced superoxide anion production, indicating the involvement of NADPH oxidase. Hcy treatment resulted in phosphorylation and subsequently membrane translocation of p47phox and p67phox subunits leading to NADPH oxidase activation. Pretreatment of cells with PKC (protein kinase C) inhibitors Ro-32-0432 (bisindolylmaleimide XI hydrochloride) (selective for PKCalpha, PKCbeta and PKCgamma) abolished Hcy-induced phosphorylation of p47phox and p67phox subunits in monocytes. Transfection of cells with antisense PKCbeta oligonucleotide, but not antisense PKCalpha oligonucleotide, completely blocked Hcy-induced phosphorylation of p47phox and p67phox subunits as well as superoxide anion production. Pretreatment of cells with LY333531, a PKCbeta inhibitor, abolished Hcy-induced superoxide anion production. Taken together, these results indicate that Hcy-stimulated superoxide anion production in monocytes is regulated through PKC-dependent phosphorylation of p47phox and p67phox subunits of NADPH oxidase. Increased superoxide anion production via NADPH oxidase may play an important role in Hcy-induced inflammatory response during atherogenesis.

G-CSF induced reactive oxygen species involves Lyn-PI3-kinase-Akt and contributes to myeloid cell growth

Granulocyte colony-stimulating factor (G-CSF) drives the production, survival, differentiation, and inflammatory functions of granulocytes. Reactive oxygen species (ROSs) provide a major thrust of the inflammatory response, though excessive ROSs may be deleterious. G-CSF stimulation showed a time- and dose-dependent increase in ROS production, correlating with activation of Lyn and Akt. Inhibition of Lyn, PI3-kinase, and Akt abrogated G-CSF-induced ROS production. This was also blocked by DPI, a specific inhibitor of NADPH oxidase. Following G-CSF stimulation, neutrophils from Lyn-/- mice produced less ROSs than wild-type littermates. G-CSF induced both serine phosphorylation and membrane translocation of p47phox, a subunit of NADPH oxidase. Because patients with a truncated G-CSF receptor have a high risk of developing acute myeloid leukemia (AML), we hypothesized that dysregulation of ROSs contributes to leukemogenesis. Cells expressing the truncated G-CSF receptor produced more ROSs than those with the full-length receptor. G-CSF-induced ROS production was enhanced in bone marrow-derived neutrophils expressing G-CSFRdelta715, a truncated receptor. The antioxidant N-acetyl-L-cysteine diminished G-CSF-induced ROS production and cell proliferation by inhibiting Akt activation. These data suggest that the G-CSF-induced Lyn-PI3K-Akt pathway drives ROS production. One beneficial effect of therapeutic targeting of Lyn-PI3K-kinase-Akt cascade is abrogating ROS production.

Endothelin-1-induced oxidative stress in DOCA-salt hypertension involves NADPH-oxidase-independent mechanisms

We have demonstrated recently [Callera, Touyz, Teixeira, Muscara, Carvalho, Fortes, Schiffrin and Tostes (2003) Hypertension 42, 811-817] that increased vascular oxidative stress in DOCA (deoxycorticosterone acetate)-salt rats is associated with activation of the ET (endothelin) system via ETA receptors. The exact source of ET-1-mediated oxidative stress remains unclear. The aim of the present study was to investigate whether ET-1 increases generation of ROS (reactive oxygen species) in DOCA-salt hypertension through NADPH-oxidase-dependent mechanisms. Xanthine oxidase, eNOS (endothelial nitric oxide synthase) and COX-2 (cyclo-oxygenase-2) were also examined as potential ET-1 sources of ROS as well as mitochondrial respiration. DOCA-salt and control UniNX (uninephrectomized) rats were treated with the ETA antagonist BMS182874 (40 mg.day(-1).kg(-1) of body weight) or vehicle. Plasma TBARS (thiobarbituric acid-reacting substances) were increased in DOCA-salt compared with UniNX rats. Activity of NADPH and xanthine oxidases in aorta, mesenteric arteries and heart was increased in DOCA-salt rats. BMS182874 decreased plasma TBARS levels without influencing NADPH and xanthine oxidase activities in DOCA-salt rats. Increased p22(phox) protein expression and increased p47(phox) membrane translocation in arteries from DOCA-salt by rats were not affected by BMS182874 treatment. Increased eNOS and COX-2 expression, also observed in aortas from DOCA-salt rats, was unaltered by BMS182874. Increased mitochondrial generation of ROS in DOCA-salt rats was normalized by BMS182874. ETA antagonism also increased the expression of mitochondrial MnSOD (manganese superoxide dismutase) in DOCA-salt rats. In conclusion, activation of NADPH oxidase does not seem to be the major source of oxidative stress induced by ET-1/ETA in DOCA-salt hypertension, which also appears to be independent of increased activation of xanthine oxidase or eNOS/COX-2 overexpression. Mitochondria may play a role in ET-1-driven oxidative stress, as evidenced by increased mitochondrial-derived ROS in this model of hypertension.

NAD(P)H oxidase-stimulating activity of serum from type 2 diabetic patients with retinopathy mediates enhanced endothelial expression of E-selectin

Endothelial expression of E-selectin is enhanced in diabetic patients with retinopathy, however, the underlying mechanisms are unclear. Therefore, this study was aimed to determine if endothelial expression of E-selectin is stimulated with serum from type 2 diabetic patients with retinopathy, and whether this process is related to NAD(P)H oxidase-derived oxidative stress. Serum was obtained from type 2 diabetic patients with (T2DR) or without (T2DM) retinopathy, and age-matched non-diabetic healthy person (Control). Serum was added to in vitro-grown human coronary artery endothelial cells (HCAEC), after which E-selectin expression, reactive oxygen species (ROS) production, and NAD(P)H oxidase activity were measured. Serum from T2DR induced a significantly higher expression of E-selectin than serum from T2DM and control in association with an enhanced production of ROS in HCAEC. T2DR serum enhanced E-selectin expression in a ROS-dependent manner since this process was significantly attenuated not only by tiron (1 mM), a superoxide scavenger, but also by DPI (10 μmol/L) and apocynin (100 μmol/L), inhibitors of NAD(P)H oxidase. Furthermore, the activity of NADH oxidase was markedly increased by T2DR serum, and this was accompanied by the enhanced membrane translocation of p47phox, a cytosolic subunit of NAD(P)H oxidase. These findings suggest that serum from T2DR induced up-regulation of E-selectin expression in HCAEC, and this process might be dependent on activation of endothelial NADH oxidase via an enhanced membrane translocation of p47phox.

Impaired NADPH oxidase activity in Rac2-deficient murine neutrophils does not result from defective translocation of p47phox and p67phox and can be rescued by exogenous arachidonic acid

Rac2 is a hematopoietic-specific Rho-GTPase that plays a stimulus-specific role in regulating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and other functional responses in neutrophils. In this study, rac2-/- neutrophils were shown to have significantly decreased NADPH oxidase activity and actin remodeling in response to exogenous arachidonic acid (AA), as previously observed for phorbol 12-myristate 13-acetate (PMA) or formyl-Met-Leu-Phe (fMLP) as agonists. PMA-, fMLP-, or AA-induced translocation of p47phox and p67phox to the plasma membrane was not impaired in rac2-/- neutrophils. Combined stimulation of rac2-/- neutrophils with exogenous AA and PMA had a synergistic effect on NADPH oxidase activity, and superoxide production increased to a level that was at least as high as wild-type cells and had no effect on fMLP-elicited enzyme activity. Membrane translocation of p47phox and p67phox as well as Rac1 activation was not increased further by combined PMA and AA stimulation. Inhibitor studies were consistent with important roles for phorbol ester-activated protein kinase C (PKC) isoforms and an atypical isoform, PKCzeta, in superoxide production by wild-type and rac2-/- neutrophils stimulated with AA and PMA. In addition, PMA-stimulated release of AA and cytoplasmic phospholipase A2 expression in rac2-/- neutrophils were similar to wild-type, suggesting that deficient AA production by PMA-stimulated rac2-/- neutrophils does not explain the effect of exogenous AA on oxidase activity. Although not required for translocation of p47phox and p67phox, Rac2 is necessary for optimal activity of the assembled oxidase complex, an effect that can be replaced by exogenous AA, which may act directly or via an exogenous AA-induced mediator.

Perturbation of actin dynamics induces NF-κB activation in myelomonocytic cells through an NADPH oxidase-dependent pathway

Although several reports showed the effect of compounds disrupting microtubules on NF-kappaB (nuclear factor kappaB) activation, nothing is known about agents perturbing actin dynamics. In the present study, we have shown that actin cytoskeleton disruption induced by actin-depolymerizing agents such as cytochalasin D and latrunculin B and actin-polymerizing compounds such as jasplakinolide induced NF-kappaB activation in myelomonocytic cells. The transduction pathway involved the IkappaB (inhibitory kappaB) kinase complex and a degradation of IkappaBalpha. We have shown that NF-kappaB activation in response to the perturbation of actin dynamics required reactive oxygen species, as demonstrated by the effect of antioxidants. Actin cytoskeleton disruption by cytochalasin D induced O2- release from human monocytes, through the activation of the NADPH oxidase, as confirmed by the phosphorylation and by the membrane translocation of p47phox. NF-kappaB activation after actin cytoskeleton disruption could be physiologically relevant during monocyte activation and/or recruitment into injured tissues, where cellular attachment, migration and phagocytosis result in cyclic shifts in cytoskeletal organization and disorganization.

Crucial Role of Two Potential Cytosolic Regions of Nox2, 191TSSTKTIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH Oxidase Activation

Assembly of cytosolic factors p67(phox) and p47(phox) with cytochrome b(558) is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop (191)TSSTKTIRRS(200) and the C-terminal (484)DESQANHFAVHHDEEKD(500) of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47(phox) translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)-activated R199Q-Nox2 and D-loop(Nox4)-Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/R/G mutants of the alpha-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47(phox) membrane translocation. This suggests that the alpha-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD.

Mechanical stretch enhances mRNA expression and proenzyme release of matrix metalloproteinase-2 (MMP-2) via NAD(P)H oxidase-derived reactive oxygen species.

Mechanical stretch is a hallmark of arterial hypertension and leads to vessel wall remodeling, which involves matrix metalloproteinases (MMPs). Because mechanical stretch is further capable of inducing reactive oxygen species (ROS) formation via the NAD(P)H oxidase, we assessed whether mechanical stretch enhances MMP expression and activity in a NAD(P)H oxidase-dependent manner. Therefore, vascular smooth muscle cells (VSMCs) isolated from C57BL/6 mice were exposed to cyclic mechanical stretch. The impact of ROS was assessed using VSMCs isolated from p47phox-/- mice, deficient for a NAD(P)H oxidase subunit responsible for ROS formation. Transcript levels were investigated by cDNA array and confirmed by RT-PCR. ROS formation was determined by DCF fluoroscopy and MMP-2 activity by zymography. Mechanical stretch of wild-type VSMCs resulted in a rapid ROS formation and p47phox membrane translocation that is followed by an increase in Nox-1 transcripts. ROS formation was completely abrogated in p47phox-/- VSMCs. cDNA array further revealed an increase of MMP-2 mRNA in response to mechanical stretch, which was validated by RT-PCR. Using p47phox-/- VSMCs, this increase in MMP-2 mRNA was completely blunted. mRNA expression of tissue inhibitor of MMP-2 TIMP-1 and TIMP-2 and membrane-type 1 MMP was unaffected by mechanical stretch. Gelatinolytic activity of pro-MMP-2 has been increased rapidly in wild-type VSMCs and was completely abolished in p47phox-/- VSMCs. These results indicate that mechanical stretch induces ROS formation via the NAD(P)H oxidase and thereby enhances MMP-2 mRNA expression and pro-MMP-2 release. These results are consistent with the notion that in arterial hypertension, reactive oxygen species are involved in vascular remodeling via MMP activation. The full text of this article is available online at http://www.circresaha.org.

Vascular NAD(P)H oxidase triggers delayed cerebral vasospasm after subarachnoid hemorrhage in rats.

To clarify the role of vascular NAD(P)H oxidase in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH), both the activity and/or activation mechanisms of NAD(P)H oxidase in the cerebral vasculature and the effect of oxidase inhibition on SAH-induced cerebral vasospasm were assessed. The changes in the luminal perimeter of the middle cerebral artery were measured histologically after SAH was induced according to a 2-hemorrhage model in rats. The NAD(P)H oxidase activity in the cerebral vasculature was measured with a lucigenin assay at different time intervals from 12 hours to 14 days after injection of autologous blood into cisterna magna. The membrane translocation of p47phox and the protein expression of membrane subunits (gp91phox and p22phox) of NAD(P)H oxidase were analyzed using Western blot analysis. The luminal perimeter of the middle cerebral artery started to decrease on day 1 and peaked on day 5 after a second injection of blood, and these changes were significantly ameliorated by treatment with an NAD(P)H oxidase inhibitor, diphenyleneiodonium. At 24 hours after the second injection of blood, both vascular production of superoxide anion and NAD(P)H oxidase activity were markedly increased with enhanced membrane translocation of p47phox, but by 48 hours the enzyme activity had regained normal values. However, no significant changes in the expression of gp91phox and p22phox were observed throughout the experiments. These findings suggest that the activation of NAD(P)H oxidase through enhanced assembly of the oxidase components in the early stages of SAH might contribute to the delayed cerebral vasospasm in SAH rats.

The Fatty Acid Bimodal Action on Superoxide Anion Production by Human Adherent Monocytes under Phorbol 12-Myristate 13-Acetate or Diacylglycerol Activation Can Be Explained by the Modulation of Protein Kinase C and p47phox Translocation

We studied the translocation of protein kinase C (PKC), the endogenous phosphorylation and presence in the membrane fraction of p47phox (the 47-kDa cytosolic component of the phagocyte NADPH oxidase), and the O-.2 production in human adherent monocytes (HAMs). This was performed under phorbol myristate acetate (PMA) or diacylglycerol stimulation after cell preincubation in the presence of either 13-methyltetradecanoate or arachidonate. At 3 nM and 30 microM, both fatty acids had enhancing and depressing effects, respectively, on PKC translocation and O-.2 production strictly depending on the PMA- or diacylglycerol-stimulated state of the cell. Endogenous phosphorylation and membrane presence of p47phox were markedly reinforced in PMA-stimulated HAMs in the presence as compared to the absence of 13-methyltetradecanoate. These results emphasize the fact that in intact cells the capacity of both FAs to potentiate or depress the HAM O-.2 production is mediated by a direct action on the PKC membrane translocation leading to a simultaneous endogenous phosphorylation and membrane translocation of p47phox. They confirm the recent findings (Kadri-Hassani, N., Léger, C. L., and Descomps, B. (1995) J. Lipid Med. Cell Signal. 11, 159-173) on the PKC-mediated, adherent monocyte-specific capacity of these fatty acids and others (with the exception of linear saturated fatty acids) to enhance the PMA-stimulated O-.2 production at nanomolar concentrations and to depress it at micromolar concentrations.

Translocation of p21rac2 from Cytosol to Plasma Membrane Is Neither Necessary nor Sufficient for Neutrophil NADPH Oxidase Activity

Activation of the membrane-associated NADPH oxidase of neutrophils requires several cytosolic factors including p47phox, p67phox and p21rac2. We compared NADPH oxidase activity with the membrane translocation of p47phox, p67phox, and p21rac2. In a cell-free system, GTP gamma S stimulated translocation of p47phox and p67phox to the plasma membrane only in the presence of arachidonate, and this translocation correlated with NADPH oxidase activity of the reisolated plasma membranes (R = 0.94 and 0.97, respectively). In contrast, GTP gamma S-stimulated p21rac2 translocation with or without arachidonate, and the extent of translocation did not correlate with oxidase activity (R = 0.17). Neutrophil cytoplasts were used to relate membrane translocation of p47phox, p67phox and p21rac2 to membrane oxidase activity in response to the inflammatory agonists. Whereas N-formyl-methionyl-leucyl-phenylalanine stimulated equimolar, transient membrane translocation of p47phox and p67phox which kinetically paralleled NADPH oxidase activity, relatively little p21rac2 translocated (moles of p47phox/p21rac2 = 16.6). Moreover, although phorbol 12-myristate 13-acetate stimulated both the stable translocation of p47phox and p67phox and sustained NADPH oxidase activity, it did not stimulate p21rac2 translocation. From these data we conclude that membrane translocation of p21rac2 does not regulate NADPH oxidase activity stoichiometrically.