Superoxide Anion Production and Expression of gp91phox and p47phox Are Increased in Glomeruli and Proximal Tubules of Cisplatin‐Treated Rats

The chemotherapeutic drug cisplatin has some side effects including nephrotoxicity that has been associated with reactive oxygen species production, particularly superoxide anion. The major source of superoxide anion is nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase. However, the specific segment of the nephron in which superoxide anion is produced has not been identified. Rats were sacrificed 72 h after cisplatin injection (7.5 mg/kg), and kidneys were obtained to isolate glomeruli and proximal and distal tubules. Cisplatin induced superoxide anion production in glomeruli and proximal tubules but not in distal tubules. This enhanced superoxide anion production was prevented by diphenylene iodonium, an inhibitor of NADPH oxidase. Consistently, this effect was associated with the increased expression of gp91^phox and p47^phox, subunits of NADPH oxidase. The enhanced superoxide anion production in glomeruli and proximal tubules, associated with the increased expression of gp91^phox and p47^phox, is involved in the oxidative stress in cisplatin‐induced nephrotoxicity.     

Molecular Insights of p47phox Phosphorylation Dynamics in the Regulation of NADPH Oxidase Activation and Superoxide Production

Phagocyte superoxide production by a multicomponent NADPH oxidase is important in host defense against microbial invasion. However inappropriate NADPH oxidase activation causes inflammation. Endothelial cells express NADPH oxidase and endothelial oxidative stress due to prolonged NADPH oxidase activation predisposes many diseases. Discovering the mechanism of NADPH oxidase activation is essential for developing novel treatment of these diseases. The p47^phox is a key regulatory subunit of NADPH oxidase; however, due to the lack of full protein structural information, the mechanistic insight of p47^phox phosphorylation in NADPH oxidase activation remains incomplete. Based on crystal structures of three functional domains, we generated a computational structural model of the full p47^phox protein. Using a combination of in silico phosphorylation, molecular dynamics simulation and protein/protein docking, we discovered that the C-terminal tail of p47^phox is critical for stabilizing its autoinhibited structure. Ser-379 phosphorylation disrupts H-bonds that link the C-terminal tail to the autoinhibitory region (AIR) and the tandem Src homology 3 (SH3) domains, allowing the AIR to undergo phosphorylation to expose the SH3 pocket for p22^phox binding. These findings were confirmed by site-directed mutagenesis and gene transfection of p47^phox−/− coronary microvascular cells. Compared with wild-type p47^phox cDNA transfected cells, the single mutation of S379A completely blocked p47^phox membrane translocation, binding to p22^phox and endothelial O₂^⨪ production in response to acute stimulation of PKC. p47^phox C-terminal tail plays a key role in stabilizing intramolecular interactions at rest. Ser-379 phosphorylation is a molecular switch which initiates p47^phox conformational changes and NADPH oxidase-dependent superoxide production by cells.     

Aryl hydrocarbon receptor-dependent induction of the NADPH oxidase subunit NCF1/p47 expression leading to priming of human macrophage oxidative burst

Polycyclic aromatic hydrocarbons such as benzo(a)pyrene (BaP) are toxic environmental contaminants known to regulate gene expression through activation of the aryl hydrocarbon receptor (AhR). In the present study, we demonstrated that acute treatment by BaP markedly increased expression of the NADPH oxidase subunit gene neutrophil cytosolic factor 1 (NCF1)/p47^phox in primary human macrophages; NCF1 was similarly up-regulated in alveolar macrophages from BaP-instilled rats. NCF1 induction in BaP-treated human macrophages was prevented by targeting AhR, through its chemical inhibition or small interference RNA-mediated down-modulation of its expression. BaP moreover induced activity of the NCF1 promoter sequence, containing a consensus AhR-related xenobiotic-responsive element (XRE), and electrophoretic mobility shift assays and chromatin immunoprecipitation experiments indicated that BaP-triggered binding of AhR to this XRE. Finally, we showed that BaP exposure resulted in p47^phox protein translocation to the plasma membrane and in potentiation of phorbol myristate acetate (PMA)-induced superoxide anion production in macrophages. This BaP priming effect toward NADPH oxidase activity was inhibited by the NADPH oxidase specific inhibitor apocynin and the chemical AhR inhibitor α-naphtoflavone. These results indicated that BaP induced NCF1/p47^phox expression and subsequently enhanced superoxide anion production in PMA-treated human macrophages, in an AhR-dependent manner; such an NCF1/NADPH oxidase regulation by polycyclic aromatic hydrocarbons may participate in deleterious effects toward human health triggered by these environmental contaminants, including atherosclerosis and smoking-related diseases.     

FPRL1-mediated induction of superoxide in LL-37-stimulated IMR90 human fibroblast

Molecular mechanisms underlying the generation of reactive oxygen species in LL-37-stimulated cells are poorly understood. Previously, we demonstrated that in human fibroblasts the exposure to WKYMVm induced p47^phox phosphorylation and translocation and, in turn, NADPH oxidase activation. These effects were mediated by the activation of the Formyl-peptide receptor-like 1 (FPRL1) and the downstream signaling involved ERKs phosphorylation and PKCα- and PKCδ-activation. Since LL-37 uses FPRL1 as a receptor to mediate its action on several cell types, we investigated in LL-37-stimulated IMR90 cells molecular mechanisms involved in NADPH-dependent superoxide generation. The exposure to LL-37, which is expressed in fibroblasts, induced ERKs activation, p47^phox phosphorylation and translocation as well as NADPH oxidase activation. These effects were prevented by pertussis toxin, PD098059 and WRWWWW, a FPRL1-selective antagonist. Furthermore, the stimulation with LL-37 of HEK293 cells, transfected to stably express FPRL1, induced a rapid activation of ERKs and p47^phox phosphorylation.     

Nox4 overexpression activates reactive oxygen species and p38 MAPK in human endothelial cells

Nicotine adenine dinucleotide phosphate (NADPH) oxidase (Nox) complexes are the main sources of reactive oxygen species (ROS) formation in the vessel wall. We have used DNA microarray, real-time PCR and Western blot to demonstrate that the subunit Nox4 is the major Nox isoform in primary human endothelial cells; we also found high levels of NADPH oxidase subunit p22^phox expression. Nox4 was localized by laser scanning confocal microscopy within the cytoplasm of endothelial cells. Endothelial Nox4 overexpression enhanced superoxide anion formation and phosphorylation of p38 MAPK. Nox4 down-regulation by shRNA has in contrast to TGF-β no effect on p38 MAPK phosphorylation. We conclude that Nox4 is the major Nox isoform in human endothelial cells, and forms an active complex with p22^phox. The Nox4-containing complex mediates formation of reactive oxygen species and p38 MAPK activation. This is a novel mechanism of redox-sensitive signaling in human endothelial cells.     

NADPH oxidase is involved in protein kinase CKII down-regulation-mediated senescence through elevation of the level of reactive oxygen species in human colon cancer cells

We have shown that protein kinase CKII (CKII) inhibition induces senescence through the p53-dependent pathway in HCT116 cells. Here we examined the molecular mechanism through which CKII inhibition activates p53 in HCT116 cells. CKII inhibition by treatment with CKII inhibitor or CKIIα small-interfering RNA (siRNA) increased intracellular hydrogen peroxide and superoxide anion levels. These effects were significantly blocked by pretreatment of cells with the antioxidant N-acetylcysteine. Additionally, NADPH oxidase (NOX) inhibitor apocynin and p22^phox siRNA significantly reduced p53 expression and suppressed the appearance of senescence markers. CKII inhibition did not affect mitochondrial superoxide generation. These data demonstrate that CKII inhibition induces superoxide anion generation via NOX activation, and subsequent superoxide-dependent activation of p53 acts as a mediator of senescence in HCT116 cells after down-regulation of CKII.     

Phosphorylation of p22phox on Threonine 147 Enhances NADPH Oxidase Activity by Promoting p47phox Binding

NADPH oxidase comprises both cytosolic and membrane-bound subunits, which, when assembled and activated, initiate the transfer of electrons from NADPH to molecular oxygen to form superoxide. This activity, known as the respiratory burst, is extremely important in the innate immune response as indicated by the disorder chronic granulomatous disease. The regulation of this enzyme complex involves protein-protein and protein-lipid interactions as well as phosphorylation events. Previously, our laboratory demonstrated that the small membrane subunit of the oxidase complex, p22^phox, is phosphorylated in neutrophils and that its phosphorylation correlates with NADPH oxidase activity. In this study, we utilized site-directed mutagenesis in a Chinese hamster ovarian cell system to determine the phosphorylation sites within p22^phox. We also explored the mechanism by which p22^phox phosphorylation affects NADPH oxidase activity. We found that mutation of threonine 147 to alanine inhibited superoxide production in vivo by more than 70%. This mutation also blocked phosphorylation of p22^phox in vitro by both protein kinase C-α and -δ. Moreover, this mutation blocked the p22^phox-p47^phox interaction in intact cells. When phosphorylation was mimicked in vivo through mutation of Thr-147 to an aspartyl residue, NADPH oxidase activity was recovered, and the p22^phox-p47^phox interaction in the membrane was restored. Maturation of gp91^phox was not affected by the alanine mutation, and phosphorylation of the cytosolic component p47^phox still occurred. This study directly implicates threonine 147 of p22^phox as a critical residue for efficient NADPH oxidase complex formation and resultant enzyme activity.     

Constitutive activation of phosphatidylinositol 3-kinase signaling pathway down-regulates TLR4-mediated tumor necrosis factor-alpha release in alveolar macrophages from asymptomatic HIV-positive persons in vitro

Alveolar macrophages represent critical effector cells of innate immunity to infectious challenge in the lungs and recognize bacterial pathogens through pattern recognition receptors such as Toll-like receptors (TLRs). Phosphatidylinositol 3-kinase (PI3K) regulates TLR-mediated cytokine release, but whether HIV infection influences PI3K signaling pathway and alters TLR4-mediated macrophage response has not been investigated. In the current study, surface TLR4 expression were similar but TLR4 activation (lipid A, 10 microg/ml) resulted in lower TNF-alpha release by HIV+ human macrophages compared with healthy cells. Pharmacological inhibition of PI3K (LY294002) normalized TNF-alpha release in HIV+ macrophages and augments ERK1/2 mitogen-activated protein kinase phosphorylation in response to lipid A. Importantly, HIV+ macrophages demonstrated increased constitutive phosphatidylinositol 3,4,5-trisphosphate formation, increased phosphorylation of downstream signaling molecules Akt and glycogen synthase kinase-3beta (GSK-3beta) at Ser9, and reduced PTEN protein expression. As a functional assessment of GSK-3beta phosphorylation, TLR4-mediated interleukin-10 release was significantly higher in HIV+ human macrophages compared with healthy cells. Incubation of human macrophages with exogenous HIV Nef protein induced phosphorylation of Akt and GSK-3beta (whereas phosphorylation was reduced by PI3K inhibition) and promoted interleukin-10 release. Taken together, these data demonstrate increased constitutive activation of the PI3K signaling pathway in HIV+ macrophages and support the concept that PI3K activation (by HIV proteins such as Nef) may contribute to reduced TLR4-mediated TNF-alpha release in HIV+ human macrophages and impair host cell response to infectious challenge.     

胞浆Ca~(2+)和某些激酶对NADPH氧化酶激活和肌动蛋白聚合的作用

 为澄清中性粒细胞胞浆 Ca2 +和某些 O-·2 产生相关激酶对 NADPH氧化酶激活和肌动蛋白聚合的作用 ,利用分化为中性粒细胞样的 HL- 60细胞研究了胞浆 Ca2 +螯合剂 BAPTA- AM和激酶抑制剂对这些激酶激活、NADPH氧化酶激活和肌动蛋白聚合的影响 .使用 1 0 μmol/L的 Ca2 +螯合剂 BAPTA- AM去除胞浆 Ca2 +后 ,趋化肽 f MLP诱导的 O-·2 产生明显减少 ,但不影响 f MLP诱导的肌动蛋白聚合 ;8μmol/L的 PKC激酶抑制物 GF1 0 92 0 3x几乎完全抑制 O-·2 产生 ;50 μmol/L的p38激酶抑制物 SB2 0 3580、50 μmol/L的 ERK激酶抑制物 PD0 980 59和 0 .1 μmol/L的 PI3激酶抑制物渥曼青霉素 (Wortmannin)使 f MLP诱导的 O-·2 产生大约减少一半 ;其中 Wortmannin还抑制 f MLP诱导的肌动蛋白聚合 ;f MLP刺激细胞后 ,PI3- K、p38和 ERK激酶迅速激活 ,但这些激酶的激活对 Ca2 +是非必需的 .这些结果说明 Ca2 +依赖途径 (PKC)和 Ca2 +非依赖途径 (PI3- K、p38和ERK)对 NADPH氧化酶激活都起着重要作用 ,而 Ca2 +非依赖途径中的 PI3- K激酶还参与中性粒细胞样 HL- 60细胞的肌动蛋白聚合 .     

Oxidative stress in rats fed a high-fat high-sucrose diet and preventive effect of polyphenols: Involvement of mitochondrial and NAD(P)H oxidase systems

Mitochondrial and NADPH oxidase systems and oxidative stress were investigated in 12 week high-fat high-sucrose (HFHS) diet-fed rats. A protective effect of wine polyphenol (PP) extract was also examined. In liver, maximal activities of CII and CII+III mitochondrial complexes were decreased but NADPH oxidase expression (p22^phox and p47^phox) and NADPH oxidase-dependent superoxide anion production were not modified, whereas oxidative stress (lipid and protein oxidation products and antioxidant systems) was increased with HFHS diet. In muscle, anion superoxide production was slightly increased while mitochondrial complex activities and lipid and protein oxidation products were not modified with HFHS diet. In heart, NADPH oxidase expression and superoxide anion production were increased, and maximal activity of mitochondrial respiratory chain complexes or oxidative stress parameters were not modified. Wine polyphenol extract had an inhibiting effect on liver oxidative stress and on heart NADPH oxidase expression and superoxide anion production, and on induction of hepatic steatosis with HFHS diet. Induction of mitochondrial dysfunction could be a primary event in the development of oxidative stress in liver, while in skeletal muscle and in heart the NADPH oxidase system seems to be mainly involved in oxidative stress. Wine polyphenol extract was shown to partially prevent oxidative stress in liver and heart tissues and to nearly completely prevent steatosis development in liver.     

Homocysteine stimulates NADPH oxidase-mediated superoxide production leading to endothelial dysfunction in rats

Elevation of blood homocysteine (Hcy) levels (hyperhomocysteinemia) is a risk factor for cardiovascular disorders. We previously reported that oxidative stress contributed to Hcy-induced inflammatory response in vascular cells. In this study, we investigated whether NADPH oxidase was involved in Hcy-induced superoxide anion accumulation in the aorta, which leads to endothelial dysfunction during hyperhomocysteinemia. Hyperhomocysteinemia was induced in rats fed a high-methionine diet. NADPH oxidase activity and the levels of superoxide and peroxynitrite were markedly increased in aortas isolated from hyperhomocysteinemic rats. Expression of the NADPH oxidase subunit p22 phox increased significantly in these aortas. Administration of an NADPH oxidase inhibitor (apocynin) not only attenuated aortic superoxide and peroxynitrite to control levels but also restored endothelium-dependent relaxation in the aortas of hyperhomocysteinemic rats. Transfection of human endothelial cells or vascular smooth muscle cells with p22 phox siRNA to inhibit NADPH oxidase activation effectively abolished Hcy-induced superoxide anion production, thus indicating the direct involvement of NADPH oxidase in elevated superoxide generation in vascular cells. Taken together, these results suggest that Hcy-stimulated superoxide anion production in the vascular wall is mediated through the activation of NADPH oxidase, which leads to endothelial dysfunction during hyperhomocysteinemia.     

Characterization of P-Rex1 for its role in fMet-Leu-Phe-induced superoxide production in reconstituted COSphox cells

P-Rex1 (phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1) is a Rac-specific guanine nucleotide exchange factor activated by Gβγ subunits and by PtdIns_(3,4,5)P₃. Recent studies indicate that P-Rex1 plays an important role in signaling downstream of neutrophil chemoattractant receptors. Here we report that heterologous expression of P-Rex1, but not Vav1, reconstitutes formyl peptide receptor 1 (FPR1)-mediated NADPH oxidase activation in the transgenic COS^phox cells expressing gp91^phox, p22^phox, p67^phox and p47^phox. A successful reconstitution requires the expression of a full-length P-Rex1 with intact DH and PH domains, and is accompanied by P-Rex1 membrane localization as well as Rac1 activation. P-Rex1-dependent superoxide generation in the reconstituted COS^phox cells was further enhanced by expression of the novel PKC isoform PKCδ and by overexpression of Akt. Heterologous expression of P-Rex1 in COS^phox cells potentiated fMet-Leu-Phe-induced Akt phosphorylation, whereas expression of a constitutively active form of Akt enhanced Rac1 activation. In contrast, a dominant negative Akt mutant reduced the fMet-Leu-Phe stimulated superoxide generation as well as Rac1 activation. These results demonstrate that in COS^phox cells, P-Rex1 is a critical component for FPR1-mediated signaling leading to NADPH oxidase activation, and there is a crosstalk between the P-Rex1-Rac pathway and Akt in superoxide generation.     

Phospholipase D-mediated Activation of IQGAP1 through Rac1 Regulates Hyperoxia-induced p47phox Translocation and Reactive Oxygen Species Generation in Lung Endothelial Cells

Phosphatidic acid generated by the activation of phospholipase D (PLD) functions as a second messenger and plays a vital role in cell signaling. Here we demonstrate that PLD-dependent generation of phosphatidic acid is critical for Rac1/IQGAP1 signal transduction, translocation of p47^phox to cell periphery, and ROS production. Exposure of [³²P]orthophosphate-labeled human pulmonary artery endothelial cells (HPAECs) to hyperoxia (95% O₂ and 5% CO₂) in the presence of 0.05% 1-butanol, but not tertiary-butanol, stimulated PLD as evidenced by accumulation of [³²P]phosphatidylbutanol. Infection of HPAECs with adenoviral constructs of PLD1 and PLD2 wild-type potentiated hyperoxia-induced PLD activation and accumulation of /reactive oxygen species (ROS). Conversely, overexpression of catalytically inactive mutants of PLD (hPLD1-K898R or mPLD2-K758R) or down-regulation of expression of PLD with PLD1 or PLD2 siRNA did not augment hyperoxia-induced [³²P]phosphatidylbutanol accumulation and ROS generation. Hyperoxia caused rapid activation and redistribution of Rac1, and IQGAP1 to cell periphery, and down-regulation of Rac1, and IQGAP1 attenuated hyperoxia-induced tyrosine phosphorylation of Src and cortactin and ROS generation. Further, hyperoxia-mediated redistribution of Rac1, and IQGAP1 to membrane ruffles, was attenuated by PLD1 or PLD2 small interference RNA, suggesting that PLD is upstream of the Rac1/IQGAP1 signaling cascade. Finally, small interference RNA for PLD1 or PLD2 attenuated hyperoxia-induced cortactin tyrosine phosphorylation and abolished Src, cortactin, and p47^phox redistribution to cell periphery. These results demonstrate a role of PLD in hyperoxia-mediated IQGAP1 activation through Rac1 in tyrosine phosphorylation of Src and cortactin, as well as in p47^phox translocation and ROS formation in human lung endothelial cells.Phagocytic cells of the immune system (neutrophils, eosinophils, monocytes, and macrophages) generate superoxide ()² instrumental in the killing of invading pathogens solely by NADPH oxidase (1-3). Deficiency of results in the genetically inherited disorder chronic granulomatous disease, a condition in which the affected individuals are susceptible to infection (4). Phagocytic NADPH oxidase is activated when cytosolic p47^phox, p67^phox, and Rac2 translocate to the phagosomes and plasma membrane and form a complex with integral membrane cytochrome b₅₅₈, which, in turn, is a Nox2 (gp91^phox)/p22^phox heterodimer (5, 6). Assembly of phagocytic NADPH oxidase is initiated by two signals. The first is the phosphorylation of multiple serine and tyrosine residues in the p47^phox domain, which leads to unmasking of p47^phox SH3 domains that bind to a proline-rich target in the C terminus of p22^phox (7-10). The interaction between p47^phox and p22^phox seems to be an essential requirement for the translocation of other cytosolic components of the oxidase. The second signal is the binding of GTP to Rac2, which leads to the dissociation of Rac from Rho-GDI and binding to p67^phox, followed by translocation of p67^phox/GTP-Rac2 to the membrane (11). Nonphagocytic cells express predominantly Rac1, Tiam1 (a GEF involved in Rac1 activation), Nox1-5, and most of the other cytosolic phagocytic oxidase components (12); however, the oxidative output of non-phagocytes is much smaller compared with the phagocytes. A recent study indicates that IQGAP1, an effector of Rac1, may link Nox2 to actin, thereby enhancing ROS production and contributing to cell motility in ECs (13). The one or more mechanisms responsible for differences in the oxidative burst between the phagocytic and non-phagocytic cells are yet to be defined.We have demonstrated previously that hyperoxia activates lung endothelial NADPH oxidase, which in part is mediated by ERK, p38 MAPK (14, 15), and Src (16), and hyperoxia-induced p47^phox tyrosine phosphorylation and translocation to cell periphery is dependent on Src (16). Further, tyrosine phosphorylation of cortactin mediated by Src is essential for hyperoxia-induced p47^phox translocation and /ROS generation in HPAECs (17). In addition to Src, phosphatidic acid (PA) or diacylglycerol also stimulated phosphorylation of p47^phox and p22^phox in neutrophils both in vivo and in vitro (18-20). PA is generated in mammalian cells via de novo biosynthesis or hydrolysis of membrane phospholipids catalyzed by phospholipase D (PLD) (21-25). Activation of polymorphonuclear leukocytes with formyl-Met-Leu-Phe enhanced the oxidative burst that correlated with PA accumulation, and inclusion of short-chain primary alcohols attenuated the NADPH oxidase mediated /ROS generation, suggesting a potential role for PLD in the regulation of NADPH oxidase (12, 26, 27). However, the downstream targets of PLD that signal NADPH oxidase activation have not been fully characterized.Here, we identify for the first time that activation of IQGAP1 by Rac1 is downstream of PLD in hyperoxia-induced ROS generation. In addition, we show that activation of Rac1/IQGAP1 by PLD also regulates Src-dependent tyrosine phosphorylation of cortactin and p47^phox translocation to cell periphery. Thus, our results define a novel molecular mechanism for hyperoxia-induced NADPH oxidase activation by PLD/PA-mediated p47^phox membrane translocation via Rac1/IQGAP1/Src/cortactin signaling cascade.     

Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and p67phox subunits in monocytes via protein kinase Cbeta 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.     

Inorganic arsenic activates reduced NADPH oxidase in human primary macrophages through a Rho kinase/p38 kinase pathway

Inorganic arsenic is an immunotoxic environmental contaminant to which millions of humans are chronically exposed. We recently demonstrated that human primary macrophages constituted a critical target for arsenic trioxide (As(2)O(3)), an inorganic trivalent form. To specify the effects of arsenic on macrophage phenotype, we investigated in the present study whether As(2)O(3) could regulate the activity of NADPH oxidase, a major superoxide-generating enzymatic system in human phagocytes. Our results show that superoxide levels were significantly increased in a time-dependent manner in blood monocyte-derived macrophages treated with 1 muM As(2)O(3) for 72 h. Concomitantly, As(2)O(3) induced phosphorylation and membrane translocation of the NADPH oxidase subunit p47(phox) and it also increased translocation of Rac1 and p67(phox). Apocynin, a selective inhibitor of NADPH oxidases, prevented both p47(phox) translocation and superoxide production. NADPH oxidase activation was preceded by phosphorylation of p38-kinase in As(2)O(3)-treated macrophages. The p38-kinase inhibitor SB-203580 prevented phosphorylation and translocation of p47(phox) and subsequent superoxide production. Pretreatment of macrophages with the Rho-kinase inhibitor Y-27632 was found to mimic inhibitory effects of SB-203580 and to prevent As(2)O(3)-induced phosphorylation of p38 kinase. Treatment with As(2)O(3) also resulted in an increased secretion of the proinflammatory chemokine CCL18 that was fully inhibited by both apocynin and SB-203580. Taken together, our results demonstrate that As(2)O(3) induced a marked activation of NADPH oxidase in human macrophages, likely through stimulation of a Rho-kinase/p38-kinase pathway, and which may contribute to some of the deleterious effects of inorganic arsenic on macrophage phenotype.     

Role of VPO1, a newly identified heme-containing peroxidase, in ox-LDL induced endothelial cell apoptosis

Myeloperoxidase (MPO) is an important enzyme involved in the genesis and development of atherosclerosis. Vascular peroxidase 1 (VPO1) is a newly discovered member of the peroxidase family that is mainly expressed in vascular endothelial cells and smooth muscle cells and has structural characteristics and biological activity similar to those of MPO. Our specific aims were to explore the effects of VPO1 on endothelial cell apoptosis induced by oxidized low-density lipoprotein (ox-LDL) and the underlying mechanisms. The results showed that ox-LDL induced endothelial cell apoptosis and the expression of VPO1 in endothelial cells in a concentration- and time-dependent manner concomitant with increased intracellular reactive oxygen species (ROS) and hypochlorous acid (HOCl) generation, and up-regulated protein expression of the NADPH oxidase gp91^phox subunit and phosphorylation of p38 MAPK. All these effects of ox-LDL were inhibited by VPO1 gene silencing and NADPH oxidase gp91^phox subunit gene silencing or by pretreatment with the NADPH oxidase inhibitor apocynin or diphenyliodonium. The p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor DEVD-CHO significantly inhibited ox-LDL-induced endothelial cell apoptosis, but had no effect on intracellular ROS and HOCl generation or the expression of NADPH oxidase gp91^phox subunit or VPO1. Collectively, these findings suggest for the first time that VPO1 plays a critical role in ox-LDL-induced endothelial cell apoptosis and that there is a positive feedback loop between VPO1/HOCl and the now-accepted dogma that the NADPH oxidase/ROS/p38 MAPK/caspase-3 pathway is involved in ox-LDL-induced endothelial cell apoptosis.     

Arjunolic acid ameliorates reactive oxygen species via inhibition of p47phox-serine phosphorylation and mitochondrial dysfunction

Impaired cardiovascular function during acute myocardial infarction (MI) is partly associated with recruitment of activated polymorphonuclear neutrophils. The protective role of arjunolic acid (AA; 2,3,23-trihydroxy olean-12-en-28-oic acid) is studied in the modulation of neutrophil functions in vitro by measuring the reactive oxygen species (ROS) generation. Neutrophils were isolated from normal and acute MI mice to find out the efficacy of AA in reducing oxidative stress. Stimulation of neutrophils with phorbol-12-myristate-13-acetate (PMA) resulted in an oxidative burst of superoxide anion (O₂⁻) and enhanced release of lysosomal enzymes. The treatment of neutrophils with PMA induced phosphorylation of Ser345 on p47^phox, a cytosolic component of NADPH oxidase. Furthermore, we observed activated ERK induced phosphorylation of Ser345 in MI neutrophils. Treatment with AA significantly inhibited the phosphorylation of P47^phox and ERK in the stimulated controls and MI neutrophils. Oxidative phosphorylation activities in MI cells were lower than in control, while the glycolysis rates were elevated in MI cells compared to the control. In addition, we observed AA decreased intracellular oxidative stress and reduced the levels of O₂⁻ in neutrophils. This study therefore identifies targets for AA in activated neutrophils mediated by the MAPK pathway on p47^phox involved in ROS generation.     

Rac1 activation induces tumour necrosis factor‐α expression and cardiac dysfunction in endotoxemia

Induction of tumour necrosis factor‐α (TNF‐α) expression leads to myocardial depression during sepsis. However, the underlying molecular mechanisms are not fully understood. The aim of this study was to investigate the role of Rac1 in TNF‐α expression and cardiac dysfunction during endotoxemia and to determine the involvement of phosphoinositide‐3 kinase (PI3K) in lipopolysaccharide (LPS)‐induced Rac1 activation. Our results showed that LPS‐induced Rac1 activation and TNF‐α expression in cultured neonatal mouse cardiomyocytes. The response was inhibited in Rac1 deficient cardiomyocytes or by a dominant‐negative Rac1 (Rac1N17). To determine whether PI3K regulates Rac1 activation, cardiomyocytes were treated with LY294002, a PI3K selective inhibitor. Treatment with LY294002 decreased Rac1 activity as well as TNF‐α expression stimulated by LPS. Furthermore, inhibition of PI3K and Rac1 activity decreased LPS‐induced superoxide generation which was associated with a significant reduction in ERK1/2 phosphorylation. To investigate the role of Rac1 in myocardial depression during endotoxemia in vivo, wild‐type and cardiomyocyte‐specific Rac1 deficient mice were treated with LPS (2 mg/kg, i.p.). Deficiency in Rac1 significantly decreased myocardial TNF‐α expression and improved cardiac function during endotoxemia. We conclude that PI3K‐mediated Rac1 activation is required for induction of TNF‐α expression in cardiomyocytes and cardiac dysfunction during endotoxemia. The effect of Rac1 on TNF‐α expression seems to be mediated by increased NADPH oxidase activity and ERK1/2 phosphorylation.     

Cigarette smoke particle-phase extract induces HO-1 expression in human tracheal smooth muscle cells: role of the c-Src/NADPH oxidase/MAPK/Nrf2 signaling pathway

Heme oxygenase-1 (HO-1) is known as an oxidative stress protein that is up-regulated by various stimuli. HO-1 has been shown to protect cells against oxidative damage. Cigarette smoke is a potential inflammatory mediator that causes chronic obstructive pulmonary disease and asthma. In this study, we report that cigarette smoke particle-phase extract (CSPE) is an inducer of HO-1 expression mediated through various signaling pathways in human tracheal smooth muscle cells (HTSMCs). CSPE-induced HO-1 protein, mRNA expression, and promoter activity were attenuated by pretreatment with a ROS scavenger (N-acetyl-l-cysteine) and inhibitors of c-Src (PP1), NADPH oxidase [diphenylene iodonium chloride (DPI) and apocynin (APO)], MEK1/2 (U0126), p38 MAPK (SB202190), and JNK1/2 (SP600125) or transfection with siRNAs for Src, p47^phox, NOX2, p42, p38, JNK2, or NF-E2-related factor 2 (Nrf2). CSPE-stimulated translocation of p47^phox and Nrf2, ROS production, and NADPH oxidase activity was attenuated by transfection with siRNAs for Src, p47^phox, and NOX2 or pretreatment with PP1, DPI, or APO. Furthermore, CSPE-induced NOX2, c-Src, and p47^phox complex formation was revealed by immunoprecipitation using an anti-NOX2, anti-p47^phox, or anti-c-Src Ab followed by Western blot against anti-NOX2, anti-p47^phox, or anti-c-Src Abs. These results demonstrate that CSPE-induced ROS generation is mediated through a c-Src/NADPH oxidase/MAPK pathway and in turn initiates the activation of Nrf2 and ultimately induces HO-1 expression in HTSMCs.     

A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91^phox serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91^phox requires the cytosolic proteins p67^phox, p47^phox, and Rac (a small GTPase). p67^phox, comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47^phox and there interacts with Rac; these processes are prerequisite for gp91^phox activation. Here we show that a region of p67^phox (amino acids 190–200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67^phox to support superoxide production by gp91^phox-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91^phox-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67^phox interaction with the gp91^phox NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67^phox (amino acids 190–210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91^phox.