The NADPH oxidase NOX4 inhibits hepatocyte proliferation and liver cancer progression

The NADPH oxidase NOX4 has emerged as an important source of reactive oxygen species in signal transduction, playing roles in physiological and pathological processes. NOX4 mediates transforming growth factor-β-induced intracellular signals that provoke liver fibrosis, and preclinical assays have suggested NOX4 inhibitors as useful tools to ameliorate this process. However, the potential consequences of sustained treatment of liver cells with NOX4 inhibitors are yet unknown. The aim of this work was to analyze whether NOX4 plays a role in regulating liver cell growth either under physiological conditions or during tumorigenesis. In vitro assays proved that stable knockdown of NOX4 expression in human liver tumor cells increased cell proliferation, which correlated with a higher percentage of cells in S/G2/M phases of the cell cycle, downregulation of p21(CIP1/WAF1), increase in cyclin D1 protein levels, and nuclear localization of β-catenin. Silencing of NOX4 in untransformed human and mouse hepatocytes also increased their in vitro proliferative capacity. In vivo analysis in mice revealed that NOX4 expression was downregulated under physiological proliferative situations of the liver, such as regeneration after partial hepatectomy, as well as during pathological proliferative conditions, such as diethylnitrosamine-induced hepatocarcinogenesis. Xenograft experiments in athymic mice indicated that NOX4 silencing conferred an advantage to human hepatocarcinoma cells, resulting in earlier onset of tumor formation and increase in tumor size. Interestingly, immunochemical analyses of NOX4 expression in human liver tumor cell lines and tissues revealed decreased NOX4 protein levels in liver tumorigenesis. Overall, results described here strongly suggest that NOX4 would play a growth-inhibitory role in liver cells.     

Ghrelin stimulates angiogenesis via GHSR1a-dependent MEK/ERK and PI3K/Akt signal pathways in rat cardiac microvascular endothelial cells

Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHSR), is thought to exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function such as cell proliferation, migration, survival and angiogenesis. However, the effect of ghrelin on angiogenesis and the corresponding mechanisms have not yet been extensively studied in cardiac microvascular endothelial cells (CMECs) isolated from left ventricular myocardium of adult Sprague-Dawley (SD) rats. In our study, we found that ghrelin and GHSR are constitutively expressed in CMECs. Ghrelin significantly increases CMECs proliferation, migration, and in vitro angiogenesis. The ghrelin-induced angiogenic process was accompanied by phosphorylation of ERK and Akt. MEK inhibitor PD98059 abolished ghrelin-induced phosphorylation of ERK, but had no effect on Akt phosphorylation. PI3K inhibitor LY294002 abolished ghrelin-induced phosphorylation of Akt, but had no effect on ERK phosphorylation. Ghrelin-induced angiogenesis was partially blocked by treatment with PD98059 or LY294002. In addition, this angiogenic effect was almost completely inhibited by PD98059+LY294002. Pretreatment with GHSR1a blocker [D-Lys3]-GHRP-6 abolished ghrelin-induced phosphorylation of ERK, Akt and in vitro angiogenesis. In conclusion, this is the first demonstration that ghrelin stimulates CMECs angiogenesis through GHSR1a-mediated MEK/ERK and PI3K/Akt signal pathways, indicating that two pathways are required for full angiogenic activity of ghrelin. This study suggests that ghrelin may play an important role in myocardial angiogenesis.     

Endothelial NADPH oxidase 4 protects ApoE-/- mice from atherosclerotic lesions

Vascular reactive oxygen species (ROS) are known to be involved in atherosclerosis development and progression. NADPH oxidase 4 (Nox4) is a constitutively active ROS-producing enzyme that is highly expressed in the vascular endothelium. Nox4 is unique in its biology and has been implicated in vascular repair, however, the role of Nox4 in atherosclerosis is unknown. Therefore, to determine the effect of endothelial Nox4 on development of atherosclerosis, Apoe E-/- mice +/- endothelial Nox4 (ApoE-/- + EC Nox4) were fed a high cholesterol/high fat (Western) diet for 24 weeks. Significantly fewer atherosclerotic lesions were observed in the ApoE-/- + EC Nox4 mice as compared to the ApoE-/- littermates, which was most striking in the abdominal region of the aorta. In addition, markers of T cell populations were markedly different between the groups; T regulatory cell marker (FoxP3) was increased whereas T effector cell marker (T-bet) was decreased in aorta from ApoE-/- + EC Nox4 mice compared to ApoE-/- alone. We also observed decreased monokine induced by gamma interferon (MIG; CXCL9), a cytokine known to recruit and activate T cells, in plasma and tissue from ApoE-/- + EC Nox4 mice. To further investigate the link between endothelial Nox4 and MIG expression, we utilized cultured endothelial cells from our EC Nox4 transgenic mice and human cells with adenoviral overexpression of Nox4. In these cultured cells, upregulation of Nox4 attenuated endothelial cell MIG expression in response to interferon-gamma. Together these data suggest that endothelial Nox4 expression reduces MIG production and promotes a T cell distribution that favors repair over inflammation, leading to protection from atherosclerosis.     

Caveolin-1 is a negative regulator of NADPH oxidase-derived reactive oxygen species

Changes in the expression and function of caveolin-1 (Cav-1) have been proposed as a pathogenic mechanism underlying many cardiovascular diseases. Cav-1 binds to and regulates the activity of numerous signaling proteins via interactions with its scaffolding domain. In endothelial cells, Cav-1 has been shown to reduce reactive oxygen species (ROS) production, but whether Cav-1 regulates the activity of NADPH oxidases (Noxes), a major source of cellular ROS, has not yet been shown. Herein, we show that Cav-1 is primarily expressed in the endothelium and adventitia of pulmonary arteries (PAs) and that Cav-1 expression is reduced in isolated PAs from multiple models of pulmonary artery hypertension (PH). Reduced Cav-1 expression correlates with increased ROS production in the adventitia of hypertensive PA. In vitro experiments revealed a significant ability of Cav-1 and its scaffolding domain to inhibit Nox1–5 activity and it was also found that Cav-1 binds to Nox5 and Nox2 but not Nox4. In addition to posttranslational actions, in primary cells, Cav-1 represses the mRNA and protein expression of Nox2 and Nox4 through inhibition of the NF-κB pathway. Last, in a mouse hypoxia model, the genetic ablation of Cav-1 increased the expression of Nox2 and Nox4 and exacerbated PH. Together, these results suggest that Cav-1 is a negative regulator of Nox function via two distinct mechanisms, acutely through direct binding and chronically through alteration of expression levels. Accordingly, the loss of Cav-1 expression in cardiovascular diseases such as PH may account for the increased Nox activity and greater production of ROS.     

NADPH oxidase 4 mediates ROS production in radiation-induced senescent cells and promotes migration of inflammatory cells

Excessive DNA damage induced by ionising radiation (IR) to normal tissue cells is known to trigger cellular senescence, a process termed stress-induced premature senescence (SIPS). SIPS is often accompanied by the production of reactive oxygen species (ROS), and this is reported to be important for the initiation and maintenance of SIPS. However, the source of ROS during SIPS after IR and their significance in radiation-induced normal tissue damage remain elusive. In the present study, we tested the hypothesis that the NADPH oxidase (NOX) family of proteins mediates ROS production in SIPS-induced cells after IR and plays a role in SIPS-associated biological events. X-irradiation of primary mouse embryonic fibroblasts (MEFs) resulted in cellular senescence and the concomitant increase of intracellular ROS. Among all six murine NOX isoforms (NOX1–4 and DUOX1/2), only NOX4 was detectable under basal conditions and was upregulated following IR. In addition, radiation-induced ROS production was diminished by genetic or pharmacological inhibition of NOX4. Meanwhile, NOX4 deficiency did not affect the induction of cellular senescence after IR. Furthermore, the migration of human monocytic U937 cells to the culture medium collected from irradiated MEFs was significantly reduced by NOX4 inhibition, suggesting that NOX4 promotes the recruitment of inflammatory cells. Collectively, our findings imply that NOX4 mediates ROS production in radiation-induced senescent cells and contributes to normal tissue damage after IR via the recruitment of inflammatory cells and the exacerbation of tissue inflammation.     

Characterization of N-glycosylation sites on the extracellular domain of NOX1/NADPH oxidase

Extensive evidence demonstrates the pathophysiological importance of NOX1, the catalytic subunit of superoxide-generating enzyme NADPH oxidase, as a source of reactive oxygen species in nonphagocytic cells. However, the biochemical properties of NOX1 have not been extensively characterized due to a lack of specific immunological tools. We used a newly raised NOX1 polyclonal antibody to investigate posttranslational modifications of NOX1 overexpressed in cultured cells and in the colon, where endogenous NOX1 is highly expressed. Immunoblots of lysates from cells expressing NOX1 revealed a doublet of 56 and 60 kDa accompanied by a broad band of 60–90 kDa. Based on differential sensitivity to glycosidases, the doublet was identified as two high-mannose-type glycoforms of NOX1, whereas the broad band represented NOX1 with complex-type N-linked oligosaccharides. Deglycosylated NOX1 migrated at ~53 kDa and N-glycosylation was demonstrated in NOX1 derived from both rat and human. Site-directed mutagenesis identified N-glycosylation sites at Asn¹⁶¹ and Asn²⁴¹ on the extracellular loop of mouse NOX1. Elimination of N-glycosylation on NOX1 did not affect its electron transferase activity, protein stability, targeting to the cell surface, or localization in F-actin-positive membrane protrusions. Taken together, these data identify the two specific sites of N-linked glycosylation of murine NOX1 and demonstrate that they are not required for normal enzyme activity, protein stability, and membrane trafficking. As is true for NOX2, the contribution of glycosylation in NOX1 to its biologic function(s) merits further study.     

MsrA protects cardiac myocytes against hypoxia/reoxygenation induced cell death

Reactive oxygen species (ROS) are critical in tissue responses to ischemia-reperfusion. The enzyme methionine sulfoxide reductase-A (MsrA) is capable of protecting cells against oxidative damage by reversing damage to proteins caused by methionine oxidation or by decreasing ROS through a scavenger mechanism. The current study employed adenovirus mediated over-expression of MsrA in primary neonatal rat cardiac myocytes to determine the effect of this enzyme in protecting against hypoxia/reoxygenation in this tissue. Cells were transduced with MsrA encoding adenovirus and subjected to hypoxia/reoxygenation. Apoptotic cell death was decreased by greater than 45% in cells over-expressing MsrA relative to cells transduced with a control virus. Likewise total cell death as determined by levels of LDH release was dramatically decreased by MsrA over-expression. These observations indicate that MsrA is protective against hypoxia/reoxygenation stress in cardiac myocytes and point to MsrA as an important therapeutic target for ischemic heart disease.     

Characterization of NADPH oxidase 5 expression in human tumors and tumor cell lines with a novel mouse monoclonal antibody

Reactive oxygen species generated by NADPH oxidase 5 (Nox5) have been implicated in physiological and pathophysiological signaling pathways, including cancer development and progression. However, because immunological tools are lacking, knowledge of the role of Nox5 in tumor biology has been limited; the expression of Nox5 protein across tumors and normal tissues is essentially unknown. Here, we report the characterization and use of a mouse monoclonal antibody against a recombinant Nox5 protein (bp 600–746) for expression profiling of Nox5 in human tumors by tissue microarray analysis. Using our novel antibody, we also report the detection of endogenous Nox5 protein in human UACC-257 melanoma cells. Immunofluorescence, confocal microscopy, and immunohistochemical techniques were employed to demonstrate Nox5 localization throughout UACC-257 cells, with perinuclear enhancement. Tissue microarray analysis revealed, for the first time, substantial Nox5 overexpression in several human cancers, including those of prostate, breast, colon, lung, brain, and ovary, as well as in malignant melanoma and non-Hodgkin lymphoma; expression in most nonmalignant tissues was negative to weak. This validated mouse monoclonal antibody will promote further exploration of the functional significance of Nox5 in human pathophysiology, including tumor cell growth and proliferation.     

Ionizing irradiation induces apoptotic damage of salivary gland acinar cells via NADPH oxidase 1-dependent superoxide generation

Reactive oxygen species (ROS) have important roles in various physiological processes. Recently, several novel homologues of the phagocytic NADPH oxidase have been discovered and this protein family is now designated as the Nox family. We investigated the involvement of Nox family proteins in ionizing irradiation-induced ROS generation and impairment in immortalized salivary gland acinar cells (NS-SV-AC), which are radiosensitive, and immortalized ductal cells (NS-SV-DC), which are radioresistant. Nox1-mRNA was upregulated by γ-ray irradiation in NS-SV-AC, and the ROS level in NS-SV-AC was increased to approximately threefold of the control level after 10 Gy irradiation. The increase of ROS level in NS-SV-AC was suppressed by Nox1-siRNA-transfection. In parallel with the suppression of ROS generation and Nox1-mRNA expression by Nox1-siRNA, ionizing irradiation-induced apoptosis was strongly decreased in Nox1-siRNA-transfected NS-SV-AC. There were no large differences in total SOD or catalase activities between NS-SV-AC and NS-SV-DC although the post-irradiation ROS level in NS-SV-AC was higher than that in NS-SV-DC. In conclusion, these results indicate that Nox1 plays a crucial role in irradiation-induced ROS generation and ROS-associated impairment of salivary gland cells and that Nox1 gene may be targeted for preservation of the salivary gland function from radiation-induced impairment.     

Proopiomelanocortin gene delivery induces apoptosis in melanoma through NADPH oxidase 4-mediated ROS generation

Hypoxia in the tumor microenvironment triggers differential signaling pathways for tumor survival. In this study, we characterize the involvement of hypoxia and reactive oxygen species (ROS) generation in the antineoplastic mechanism of proopiomelanocortin (POMC) gene delivery in a mouse B16-F10 melanoma model in vivo and in vitro. Histological analysis revealed increased TUNEL-positive cells and enhanced hypoxic activities in melanoma treated with adenovirus encoding POMC (Ad-POMC) but not control vector. Because the apoptotic cells were detected mainly in regions distant from blood vessels, it was hypothesized that POMC therapy might render melanoma cells vulnerable to hypoxic insult. Using a hypoxic chamber or cobalt chloride (CoCl₂), we showed that POMC gene delivery elicited apoptosis and caspase-3 activation in cultured B16-F10 cells only under hypoxic conditions. The apoptosis induced by POMC gene delivery was associated with elevated ROS generation in vitro and in vivo. Blocking ROS generation using the antioxidant N-acetyl-l-cysteine abolished the apoptosis and caspase-3 activities induced by POMC gene delivery and hypoxia. We further showed that POMC-derived melanocortins, including α-MSH, β-MSH, and ACTH, but not γ-MSH, contributed to POMC-induced apoptosis and ROS generation during hypoxia. To elucidate the source of ROS generation, application of the NADPH oxidase inhibitor diphenyleneiodonium attenuated α-MSH-induced apoptosis and ROS generation, implicating the proapoptotic role of NADPH oxidase in POMC action. Of the NADPH oxidase isoforms, only Nox4 was expressed in B16-F10 cells, and Nox4 was also elevated in Ad-POMC-treated melanoma tissues. Silencing Nox4 gene expression with Nox4 siRNA suppressed the stimulatory effect of α-MSH-induced ROS generation and cell apoptosis during hypoxia. In summary, we demonstrate that POMC gene delivery suppressed melanoma growth by inducing apoptosis, which was at least partly dependent on Nox4 upregulation.     

5-Lipoxygenase plays an essential role in 4-HNE-enhanced ROS production in murine macrophages via activation of NADPH oxidase

Abstract

4-Hydroxynonenal (HNE) mediates oxidative stress-linked pathological processes; however, its role in the generation of reactive oxygen species (ROS) in macrophages is still unclear. Thus, this study investigated the sources and mechanisms of ROS generation in macrophages stimulated with HNE. Exposure of J774A.1 cells to HNE showed an increased production of ROS, which was attenuated by NADPH oxidase as well as 5-lipoxygenase (5-LO) inhibitors. Linked to these results, HNE increased membrane translocation of p47phox promoting NADPH oxidase activity, which was attenuated in peritoneal macrophages from 5-LO-deficient mice as well as in J774A.1 cells treated with a 5-LO inhibitor, MK886 or 5-LO siRNA. In contrast, HNE-enhanced 5-LO activity was not affected by inhibition of NADPH oxidase. Furthermore, leukotriene B₄, 5-LO metabolite, was found to enhance NADPH oxidase activity in macrophages. Altogether, these results suggest that 5-LO plays a critical role in HNE-induced ROS generation in murine macrophages through activation of NADPH oxidase.     

Involvement of NADPH oxidase 1 in UVB-induced cell signaling and cytotoxicity in human keratinocytes

Members of NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species (ROS) and are known to be involved in several physiological functions in response to various stimuli including UV irradiation. UVB-induced ROS have been associated with inflammation, cytotoxicity, cell death, or DNA damage in human keratinocytes. However, the source and the role of UVB-induced ROS remain undefined.Here, we show that Nox1 is involved in UVB-induced p38/MAPK activation and cytotoxicity via ROS generation in keratinocytes. Nox1 knockdown or inhibitor decreased UVB-induced ROS production in human keratinocytes. Nox1 knockdown impaired UVB-induced p38 activation, accompanied by reduced IL-6 levels and attenuated cell toxicity. Treatment of cells with N-acetyl-L-cysteine (NAC), a potent ROS scavenger, suppressed p38 activation as well as consequent IL-6 production and cytotoxicity in response to UVB exposure. p38 inhibitor also suppressed UVB-induced IL-6 production and cytotoxicity. Furthermore, the blockade of IL-6 production by IL-6 neutralizing antibody reduced UVB-induced cell toxicity.In vivo assay using wild-type mice, the intradermal injection of lysates from UVB-irradiated control cells, but not from UVB-irradiated Nox1 knockdown cells, induced inflammatory swelling and IL-6 production in the skin of ears. Moreover, administration of Nox1 inhibitor suppressed UVB-induced increase in IL-6 mRNA expression in mice skin.Collectively, these data suggest that Nox1-mediated ROS production is required for UVB-induced cytotoxicity and inflammation through p38 activation and inflammatory cytokine production, such as IL-6. Thus, our findings suggest Nox1 as a therapeutic target for cytotoxicity and inflammation in response to UVB exposure.     

高糖通过Nox4型NADPH氧化酶影响施旺细胞凋亡的机制研究

目的:探讨高糖通过Nox4型NADPH氧化酶影响施旺细胞凋亡的机制。方法:提取Wistar大鼠新生鼠的施旺细胞体外培养。分为对照组、高糖组、NOX4 siRNA组及对照siRNA组(n=10)。采用WST-1法检测细胞活力,DCFH-DA法检测细胞内活性氧自由基(ROS)含量,荧光实时定量RT-PCR检测Nox4和Caspase3 mRNA表达,蛋白印迹法检测Nox4和Caspase3蛋白表达。结果:高糖培养上调施旺细胞Nox4 mRNA及蛋白表达,降低施旺细胞活性,增加细胞内ROS含量,通过增加Caspase3 mRNA及蛋白表达促进细胞凋亡。NOX4 siRNA通过抑制Nox4基因表达,阻止高糖培养的施旺细胞内ROS蓄积,降低高糖对施旺细胞的活性损害,通过下调Caspase3 mRNA及蛋白表达减少细胞凋亡。结论:Nox4参与高糖引起的施旺细胞凋亡,针对Nox4表达或功能的调控方式可能成为治疗糖尿病周围神经病变的新途径。     

Oxygen free radical induced damage during intestinal ischemia/reperfusion in normal and xanthine oxidase deficient rats

This study looks at the role of xanthine oxidase (XO) in ischemia/reperfusion (I/R) induced intestinal mucosal damage using normal and xanthine oxidase deficient rats. Tungstate feeding for 3 days depleted the intestinal mucosal XO by 80%. A ligated loop of the rat small intestine (both normal and XO-deficient) was subjected to 1 h of total ischemia followed by 5 min revascularisation. The ensuing mucosal damage was assessed by biochemical and histological studies. Ischemia or I/R increased the XO levels in normal rats without any change in XO-deficient rats. Myeloperoxidase (a neutrophil marker) level was increased in both group of rats but it was comparatively higher in the XO-deficient rats. Accumulation of peroxidation products such as malondialdehyde, conjugated diene and increased production of hydroxyl radicals by microsomes were seen after ischemia and I/R and were similar in normal and XO-deficient rats. Studies on other parameters of peroxidation showed a decrease in polyunsaturated fatty acids and alpha-tocopherol, an increase in cysteine and cystine levels after I/R and were similar in both normal and XO-deficient rats. Histological results indicated gross morphological changes in the intestinal mucosa due to ischemia and I/R, and the damage was more severe in XO-deficient rats. These observations suggest that oxygen-derived free radicals are involved in the intestinal mucosal damage during I/R and infiltrated neutrophils rather than XO may be the primary source of free radicals under these conditions.     

Kaempferol suppresses collagen-induced platelet activation by inhibiting NADPH oxidase and protecting SHP-2 from oxidative inactivation

Reactive oxygen species (ROS) generated upon collagen stimulation act as second messengers to propagate various platelet-activating events. Among the ROS-generating enzymes, NADPH oxidase (NOX) plays a prominent role in platelet activation. Thus, NOX has been suggested as a novel target for anti-platelet drug development. Although kaempferol has been identified as a NOX inhibitor, the influence of kaempferol on the activation of platelets and the underlying mechanism have never been investigated. Here, we studied the effects of kaempferol on NOX activation, ROS-dependent signaling pathways, and functional responses in collagen-stimulated platelets. Superoxide anion generation stimulated by collagen was significantly inhibited by kaempferol in a concentration-dependent manner. More importantly, kaempferol directly bound p47^phox, a major regulatory subunit of NOX, and significantly inhibited collagen-induced phosphorylation of p47^phox and NOX activation. In accordance with the inhibition of NOX, ROS-dependent inactivation of SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) was potently protected by kaempferol. Subsequently, the specific tyrosine phosphorylation of key components (Syk, Vav1, Btk, and PLCγ2) of collagen receptor signaling pathways was suppressed by kaempferol. Kaempferol also attenuated downstream responses, including cytosolic calcium elevation, P-selectin surface exposure, and integrin-α_IIbβ₃ activation. Ultimately, kaempferol inhibited platelet aggregation and adhesion in response to collagen in vitro and prolonged in vivo thrombotic response in carotid arteries of mice. This study shows that kaempferol impairs collagen-induced platelet activation through inhibition of NOX-derived ROS production and subsequent oxidative inactivation of SHP-2. This effect suggests that kaempferol has therapeutic potential for the prevention and treatment of thrombovascular diseases.     

Protection of NAD(P)H:quinone oxidoreductase 1 against renal ischemia/reperfusion injury in mice

Ischemia/reperfusion (I/R) is the most common cause of acute renal injury. I/R-induced reactive oxygen species (ROS) are thought to be a major factor in the development of acute renal injury by promoting the initial tubular damage. NAD(P)H:quinone oxidoreductase 1 (NQO1) is a well-known antioxidant protein that regulates ROS generation. The purpose of this study was to investigate whether NQO1 modulates the renal I/R injury (IRI) associated with NADPH oxidase (NOX)-derived ROS production in an animal model. We analyzed renal function, oxidative stress, and tubular apoptosis after IRI. NQO1^−/− mice showed increased blood urea nitrogen and creatinine levels, tubular damage, oxidative stress, and apoptosis. In the kidneys of NQO1^−/− mice, the cellular NADPH/NADP⁺ ratio was significantly higher and NOX activity was markedly higher than in those of NQO1^+/+ mice. The activation of NQO1 by β-lapachone (βL) significantly improved renal dysfunction and reduced tubular cell damage, oxidative stress, and apoptosis by renal I/R. Moreover, the βL treatment significantly lowered the cellular NADPH/NADP⁺ ratio and dramatically reduced NOX activity in the kidneys after IRI. From these results, it was concluded that NQO1 has a protective role against renal injury induced by I/R and that this effect appears to be mediated by decreased NOX activity via cellular NADPH/NADP⁺ modulation. These results provide convincing evidence that NQO1 activation might be beneficial for ameliorating renal injury induced by I/R.     

Role of the plasma membrane ROS-generating NADPH oxidase in CD34+ progenitor cells preservation by hypoxia

Hypoxia favored the preservation of progenitor characteristics of hematopoietic stem and progenitor cells (HSPCs) in bone marrow. This work aimed at studying the role of reactive oxygen species (ROS)-generating NADPH oxidase system regulated by hypoxia in ex vivo cultures of cord blood CD34⁺ cells. The results showed that NADPH oxidase activity and ROS generation were reduced in hypoxia with respect to normal oxygen tension. Meanwhile the ROS generation was found to be inhibited by diphenyleneiodonium (the NADPH oxidase inhibitor), or N-acetylcysteine (the ROS scavenger). Accordingly NADPH oxidase mRNA and p67 protein levels decreased in hypoxia. The analysis of progenitor characteristics, including the proportion of cultured cells expressing the HSPCs marker CD34⁺CD38⁻, colony production ability of the colony-forming cells (CFCs), and the re-expansion capability of the cultured CD34⁺ cells, showed that either 5% pO₂ or reduced ROS favored preserving the characteristics of CD34⁺ progenitors, and promoted the expansion of CD34⁺CD38⁻ cells as well. The above results demonstrated that hypoxia effectively maintained biological characteristics of CD34⁺ cells through keeping lower intracellular ROS levels by regulating NADPH oxidase.     

NADPH oxidase involvement in cellular integrity

NADPH oxidase activity is involved in plant adaptation and development. The reactive oxygen species sourced by NADPH oxidase activity may contribute to wall strength and protoplast volume adjustment. Root hair bulge apices of the NADPH oxidase mutant rhd2/Atrbohc were more robust than the kjk cellulose synthase mutant, but burst more readily than the wild type (WT). Root epidermal wall appeared impaired in rhd2/Atrbohc, as revealed by the number of protoplasts released by wall-degrading enzymes. Root hair bulges of rhd2/Atrbohc burst more than the WT when challenged in situ with hypo-osmotic low ionic strength medium. Inhibition of NADPH oxidase activity with diphenylene iodonium caused WT to phenocopy the rhd2/Atrbohc bursting in response to hypo-osmotic shock. This implicates RHD2/AtRBOHC in softening the cell wall to permit protoplast expansion. Overall, the results point to a role for RHD2/AtRBOHC in contributing to wall strength.     

Oxidative stress and hepatic Nox proteins in chronic hepatitis C and hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common liver cancer and a leading cause of cancer-related mortality in the world. Hepatitis C virus (HCV) is a major etiologic agent of HCC. A majority of HCV infections lead to chronic infection that can progress to cirrhosis and, eventually, HCC and liver failure. A common pathogenic feature present in HCV infection, and other conditions leading to HCC, is oxidative stress. HCV directly increases superoxide and H₂O₂ formation in hepatocytes by elevating Nox protein expression and sensitizing mitochondria to reactive oxygen species generation while decreasing glutathione. Nitric oxide synthesis and hepatic iron are also elevated. Furthermore, activation of phagocytic NADPH oxidase (Nox) 2 of host immune cells is likely to exacerbate oxidative stress in HCV-infected patients. Key mechanisms of HCC include genome instability, epigenetic regulation, inflammation with chronic tissue injury and sustained cell proliferation, and modulation of cell growth and death. Oxidative stress, or Nox proteins, plays various roles in these mechanisms. Nox proteins also function in hepatic fibrosis, which commonly precedes HCC, and Nox4 elevation by HCV is mediated by transforming growth factor β. This review summarizes mechanisms of oncogenesis by HCV, highlighting the roles of oxidative stress and hepatic Nox enzymes in HCC.     

Phosphorylation of serine282 in NADPH oxidase activator 1 by Erk desensitizes EGF-induced ROS generation

Accumulating evidence indicates that protein phosphorylation regulates Nox activity. In this report, we show that serine282 residue of Nox activator 1 (NoxA1) is phosphorylated by Erk in response to EGF resulting in desensitization of Nox1 activity. Specifically, murine NoxA1 is detected as two independent protein bands in SDS PAGE, and the form of protein with higher mobility shifted to and merged with the one with lower mobility in response to EGF treatment. Pretreatment with PD98059 resulted in inhibition of NoxA1 migration in response to EGF indicating that Erk was involved in the process. Site-directed mutagenesis showed that S282A mutant but not S239A mutant failed to respond to EGF, demonstrating that serine282 is the target amino acid of Erk. Expression of S282A mutant of NoxA1 in these cells led to increased superoxide anion production in response to EGF compared to expression of the wild type, whereas the expression of S282E, a phosphomimetic mutant, resulted in significantly decreased superoxide anion generation. We also tested whether the phosphorylation of serine282 of NoxA1 affects Rac activation. Expression of S282A mutant NoxA1 up-regulated the Rac activity, whereas expression of S282E mutant led to the abrogation of Rac activation. Taken together, these results demonstrate that phosphorylation of NoxA1 is a part of the feedback mechanism that functions through activation of Rac with a net outcome of negative modulation of Nox1 activity.