ROCK2 interacts with p22phox to phosphorylate p47phox and to control NADPH oxidase activation in human monocytes.

Monocytes play a key role in innate immunity by eliminating pathogens, releasing high levels of cytokines, and differentiating into several cell types, including macrophages and dendritic cells. Similar to other phagocytes, monocytes produce superoxide anions through the NADPH oxidase complex, which is composed of two membrane proteins (p22phox and gp91phox/NOX2) and four cytosolic proteins (p47phox, p67phox, p40phox and Rac1). The pathways involved in NADPH oxidase activation in monocytes are less known than those in neutrophils. Here, we show that p22phox is associated with Rho-associated coiled-coil kinase 2 (ROCK2) in human monocytes but not neutrophils. This interaction occurs between the cytosolic region of p22phox (amino acids 132 to 195) and the coiled-coil region of ROCK2 (amino acids 400 to 967). Interestingly, ROCK2 does not phosphorylate p22phox, p40phox, p67phox, or gp91phox in vitro but phosphorylates p47phox on Ser304, Ser315, Ser320 and Ser328. Furthermore, KD025, a selective inhibitor of ROCK2, inhibited reactive oxygen species (ROS) production and p47phox phosphorylation in monocytes. Specific inhibition of ROCK2 expression in THP1-monocytic cell line by siRNA inhibited ROS production. These data show that ROCK2 interacts with p22phox and phosphorylates p47phox, and suggest that p22phox could be a shuttle for ROCK2 to allow p47phox phosphorylation and NADPH oxidase activation in human monocytes.

Hypoxia stabilizes the H2 O2 -producing oxidase Nox4 in cardiomyocytes via suppressing autophagy-related lysosomal degradation.

The hydrogen peroxide (H2 O2 )-producing NADPH oxidase Nox4, forming a heterodimer with p22phox , is expressed in a variety of cells including those in the heart to mediate adaptive responses to cellular stresses such as hypoxia. Since Nox4 is constitutively active, H2 O2 production is controlled by its protein abundance. Hypoxia-induced Nox4 expression is observed in various types of cells and generally thought to be regulated at the transcriptional level. Here we show that hypoxia upregulates the Nox4 protein level and Nox4-catalyzed H2 O2 production without increasing the Nox4 mRNA in rat H9c2 cardiomyocytes. In these cells, the Nox4 protein is stabilized under hypoxic conditions in a manner dependent on the presence of p22phox . Cell treatment with the proteasome inhibitor MG132 results in a marked decrease of the Nox4 protein under both normoxic and hypoxic conditions, indicating that the proteasome pathway does not play a major role in Nox4 degradation. The decrease is partially restored by the autophagy inhibitor 3-methyladenine. Furthermore, the Nox4 protein level is upregulated by the lysosome inhibitors bafilomycin A1 and chloroquine. Thus, in cardiomyocytes, Nox4 appears to be degraded via an autophagy-related pathway, and its suppression by hypoxia likely stabilizes Nox4, leading to upregulation of Nox4-catalyzed H2 O2 production.

Characterization of missense mutations in the NADPH oxidase partner p22phox in the A22° subtype of chronic granulomatous disease.

Defective superoxide production by NADPH oxidase 2 (Nox2) in phagocyte cells results in the development of chronic granulomatous disease (CGD), a hereditary disease characterized by recurrent and life-threatening infections. The partner protein p22phox is a membrane-spanning protein which forms a stable heterodimer with Nox2 in the endoplasmic reticulum. This interaction ensures the stability of each protein and their accurate trafficking to the cell membrane. The present paper describes the characterization of p22phox missense mutations that were identified in a patient with CGD who presented with undetectable levels of p22phox . Using a reconstitution system, it was found that p22phox expression decreased when R90Q, A117E, S118R, A124S, A124V, A125T, or E129K mutations were introduced, suggesting that these mutations destabilize the protein. In contrast, introducing an L105R mutation did not affect protein expression, but did inhibit p22phox binding to Nox2. Thus, the missense mutations discussed here contribute to the development of CGD by either disrupting protein stability or by impairing the interaction between p22phox and Nox2.

Interpretation of Dihydrorhodamine-1,2,3 Flow Cytometry in Chronic Granulomatous Disease: an Atypical Exemplar.

PURPOSE: This is a functional characterization of a novel CYBA variant associated with normal DHR flow cytometry. Chronic granulomatous disease (CGD) is an inborn error of immunity characterized by recurrent bacterial and fungal infections and dysregulated inflammatory responses due to defective phagocytic cell function leading to the formation of granulomas. CGD patients have pathogenic variants in any of the five components of the phagocytic NADPH oxidase, which transfers electrons through the phagosomal membrane and produces superoxide upon bacterial uptake. Here, we report a pediatric female patient with a novel homozygous missense variant (c.293C > T, p.(Ser98Leu)) in CYBA, encoding the p22phox protein, associated with autosomal recessive CGD. METHODS AND RESULTS: The patient presented with severe recurrent pneumonia. Specific pathogens identified included Burkholderia and Serratia species suggesting neutrophil functional abnormalities; however, the dihydrorhodamine-1,2,3 (DHR) flow cytometric and cytochrome c reduction assays for neutrophil respiratory burst fell within the low side of the normal range. Western blot and flow cytometric analysis of individual NADPH oxidase components revealed reduced levels of p22phox and gp91phoxphox proteins. The pathological consequence of the p.Ser98Leu variant was further evaluated in heterologous expression systems, which confirmed reduced p22phox protein stability and oxidase activity. CONCLUSIONS: Although this patient did not exhibit all the classic features of CGD, such as granulomas and skin infections, she had recurrent pneumonias with oxidant-sensitive pathognomonic organisms, resulting in appropriate targeted CGD testing. This case emphasizes the need to contextually interpret laboratory data, especially using clinical findings to direct additional assessments including genetic analysis.

Connexin32 ameliorates epithelial-to-mesenchymal-transition in diabetic renal tubular via inhibiting NOX4.

Renal tubulointerstitial fibrosis (RIF), characterized by epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells (TECs), is the main cause of diabetic renal fibrosis. Oxidative stress plays a pivotal role in the development of diabetic RIF. Connexin32 (Cx32), prominently expressed in renal TECs, has emerged as an important player in the regulation of oxidative stress. However, the role of Cx32 in diabetic RIF has not been explored yet. Here, we showed that adenovirus-mediated Cx32 overexpression suppressed EMT to ameliorate RIF and renal function in STZ-induced diabetic mice, while knockout (KO) of Cx32 exacerbated RIF in diabetic mice. Moreover, overexpression of Cx32 inhibited EMT and the production of extra cellular matrix (ECM) in high glucose (HG) induced NRK-52E cells, whereas knockdown of Cx32 showed the opposite effects. Furthermore, we showed that NOX4, the main source of ROS in renal tubular, was down-regulated by Cx32. Mechanistically, Cx32 down-regulated the expression of PKC alpha in a carboxyl-terminal-dependent manner, thereby inhibiting the phosphorylation at Thr147 of p22phox triggered by PKC alpha, which ultimately repressed the formation of the p22phox-NOX4 complex to reduce the protein level of NOX4. Thus, we establish Cx32 as a novel target and confirm the protection mechanism in RIF.

RNF34 ablation promotes cerebrovascular remodeling and hypertension by increasing NADPH-derived ROS generation.

Cerebrovascular remodeling is the most common cause of hypertension and stroke. Ubiquitin E3 ligase RING finger protein 34 (RNF34) is suggested to be associated with the development of multiple neurological diseases. However, the importance of RNF34 in cerebrovascular remodeling and hypertension is poorly understood. Herein, we used mice with a global RNF34 knockout as well as RNF34 floxed mice to delete RNF34 in endothelial cells and smooth muscle cells (SMCs). Our results showed that global RNF34 knockout mice substantially promoted angiotensin II (AngII)-induced middle cerebral artery (MCA) remodeling, hypertension, and neurological dysfunction. Endothelial cell RNF34 did not regulate the development of hypertension. Rather, SMC RNF34 expression is a critical regulator of hypertension and MCA remodeling. Loss of RNF34 enhanced AngII-induced mouse brain vascular SMCs (MBVSMCs) proliferation, migration and invasion. Furthermore, MCA and MBVSMCs from SMC RNF34-deficient mice showed increased superoxide anion and reactive oxygen species (ROS) generation as well as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, but exhibited no marked effect on mitochondria-derived ROS. Knockout of RNF34 promoted p22phox expression, leading to increased binding of p22phox/p47phox and p22phox/NOX2, and eventually NADPH oxidase complex formation. Immunoprecipitation assay identified that RNF34 interacted with p22phox. RNF34 deletion increased p22phox protein stability by inhibiting ubiquitin-mediated degradation. Blockade of NADPH oxidase activity or knockdown of p22phox significantly abolished the effects of RNF34 deletion on cerebrovascular remodeling and hypertension. Collectively, our study demonstrates that SMC RNF34 deficiency promotes cerebrovascular SMC hyperplasia and remodeling by increased NADPH-derived ROS generation via reducing p22phox ubiquitin-dependent degradation.

HGF/c-Met regulates p22phox subunit of the NADPH oxidase complex in primary mouse hepatocytes by transcriptional and post-translational mechanisms.

INTRODUCTION AND OBJECTIVES: It is well-known that signaling mediated by the hepatocyte growth factor (HGF) and its receptor c-Met in the liver is involved in the control of cellular redox status and oxidative stress, particularly through its ability to induce hepatoprotective gene expression by activating survival pathways in hepatocytes. It has been reported that HGF can regulate the expression of some members of the NADPH oxidase family in liver cells, particularly the catalytic subunits and p22phox. In the present work we were focused to characterize the mechanism of regulation of p22phox by HGF and its receptor c-Met in primary mouse hepatocytes as a key determinant for cellular redox regulation. MATERIALS AND METHODS: Primary mouse hepatocytes were treated with HGF (50 ng/mL) at different times. cyba expression (gene encoding p22phox) or protein content were addressed by real time RT-PCR, Western blot or immunofluorescence. Protein interactions were explored by immunoprecipitation and FRET analysis. RESULTS: Our results provided mechanistic information supporting the transcriptional repression of cyba induced by HGF in a mechanism dependent of NF-κB activity. We identified a post-translational regulation mechanism directed by p22phox degradation by proteasome 26S, and a second mechanism mediated by p22phox sequestration by c-Met in plasma membrane. CONCLUSION: Our data clearly show that HGF/c-Met exerts regulation of the NADPH oxidase by a wide-range of molecular mechanisms. NADPH oxidase-derived reactive oxygen species regulated by HGF/c-Met represents one of the main mechanisms of signal transduction elicited by this growth factor.

NAPDH Oxidase-Specific Flow Cytometry Allows for Rapid Genetic Triage and Classification of Novel Variants in Chronic Granulomatous Disease.

PURPOSE: Chronic granulomatous disease (CGD) is an innate immune deficiency, primarily affecting the phagocytic compartment, and presenting with a diverse phenotypic spectrum ranging from severe childhood infections to monogenic inflammatory bowel disease. Dihydrorhodamine (DHR) flow cytometry is the standard diagnostic test for CGD, and correlates with NADPH oxidase activity. While there may be genotype correlation with the DHR flow pattern in some patients, in several others, there is no correlation. In such patients, assessment by flow cytometric evaluation of NADPH oxidase-specific (NOX) proteins provides a convenient and rapid means of genetic triage, though immunoblotting has long been used for this purpose. METHODS AND RESULTS: We describe the clinical utility of the NOX flow cytometry assay through assessment of X-linked and autosomal recessive CGD patients and their first-degree relatives. The assessment of specific NOX proteins was correlated with overall NADPH oxidase function (DHR flow), clinical phenotype and genotype. NOX-specific protein assessment is a valuable adjunct to DHR assessment and genotyping to classify and characterize CGD patients. CONCLUSIONS: The atypical clinical presentation of some CGD patients can make genotype-phenotype correlation with DHR flow data challenging. Genetic testing, while useful for confirmation of diagnosis, can take several weeks, and in some patients does not provide a conclusive answer. However, NADPH-oxidase-specific protein flow assessment offers a rapid alternative to identification of the underlying genetic defect in cellular subsets, and can be utilized as a reflex test to an abnormal DHR flow. Further, it can provide insight into correlation between oxidative burst relative to protein expression in granulocytes and monocytes.

Direct Binding of Cisplatin to p22phox, an Endoplasmic Reticulum (ER) Membrane Protein, Contributes to Cisplatin Resistance in Oral Squamous Cell Carcinoma (OSCC) Cells.

Prolonged treatment with cisplatin (CDDP) frequently develops chemoresistance. We have previously shown that p22phox, an endoplasmic reticulum (ER) membrane protein, confers CDDP resistance by blocking CDDP nuclear entry in oral squamous cell carcinoma (OSCC) cells; however, the underlying mechanism remains unresolved. Using a fluorescent dye-labeled CDDP, here we show that CDDP can bind to p22phox in both cell-based and cell-free contexts. Subsequent detection of CDDP-peptide interaction by the Tris-Tricine-based electrophoresis revealed that GA-30, a synthetic peptide matching a region of the cytosolic domain of p22phox, could interact with CDDP. These results were further confirmed by liquid chromatography-mass spectrometry (LC-MS) analysis, from which MA-11, an 11-amino acid subdomain of the GA-30 domain, could largely account for the interaction. Amino acid substitutions at Cys50, Met65 and Met73, but not His72, significantly impaired the binding between CDDP and the GA-30 domain, thereby suggesting the potential CDDP-binding residues in p22phox protein. Consistently, the p22phox point mutations at Cys50, Met65 and Met73, but not His72, resensitized OSCC cells to CDDP-induced cytotoxicity and apoptosis. Finally, p22phox might have binding specificity for the platinum drugs, including CDDP, carboplatin and oxaliplatin. Together, we have not only identified p22phox as a novel CDDP-binding protein, but further highlighted the importance of such a drug-protein interaction in drug resistance.

Quantitative interaction analysis permits molecular insights into functional NOX4 NADPH oxidase heterodimer assembly.

Protein-protein interactions critically regulate many biological systems, but quantifying functional assembly of multipass membrane complexes in their native context is still challenging. Here, we combined modeling-assisted protein modification and information from human disease variants with a minimal-size fusion tag, split-luciferase-based approach to probe assembly of the NADPH oxidase 4 (NOX4)-p22phox enzyme, an integral membrane complex with unresolved structure, which is required for electron transfer and generation of reactive oxygen species (ROS). Integrated analyses of heterodimerization, trafficking, and catalytic activity identified determinants for the NOX4-p22phox interaction, such as heme incorporation into NOX4 and hot spot residues in transmembrane domains 1 and 4 in p22phox Moreover, their effect on NOX4 maturation and ROS generation was analyzed. We propose that this reversible and quantitative protein-protein interaction technique with its small split-fragment approach will provide a protein engineering and discovery tool not only for NOX research, but also for other intricate membrane protein complexes, and may thereby facilitate new drug discovery strategies for managing NOX-associated diseases.

p22phox promotes Ang-II-induced vascular smooth muscle cell phenotypic switch by regulating KLF4 expression.

NADPH oxidase (Nox) is the main source of reactive oxygen species in vascular diseases, which have been implicated in promoting VSMCs phenotypic switch. P22phox, the indispensable component of the complex Nox, is required for their activity and stability. Krüppel-like factor 4 (KLF4) is an important transcriptional regulator of VSMCs phenotypic switch. Both KLF4 and p22phox are involved in the proliferation, migration and differentiation of VSMC. This study aims to determine whether and how p22phox regulates KLF4 expression in phenotypic switching of VSMCs. In cultured primary rat VSMCs, we noticed that the expression of P22phox was significantly increased in combination with VSMCs phenotypic switch and up-regulated KLF4 expression in Ang-II-treated cells. Ang-II-induced VSMC dedifferentiation, proliferation, migration, KLF4 expression, H2O2 production and the phosphorylation of AKT, ERK1/2 were all inhibited by knockdown of P22phox. Furthermore, H2O2 treatment effectively enhanced the phosphorylation of AKT, ERK1/2 and the expression of KLF4, whereas LY294002 (a specific inhibitor of PI3K), U0126 (a specific inhibitor of ERK1/2) significantly attenuated the H2O2-induced up-regulation of KLF4. In conclusion, these results demonstrated that p22phox promotes Ang-II-induced VSMC phenotypic switch via the H2O2-ERK1/2/AKT-KLF4 signaling pathway.

Pulmonary metastatic colonisation and granulomas in NOX2-deficient mice.

Metastasis is the leading cause of death in cancer patients, and successful colonisation of a secondary organ by circulating tumour cells (CTCs) is the rate-limiting step of this process. We used tail-vein injection of B16-F10 melanoma cells into mice to mimic the presence of CTCs and to allow for the assessment of host (microenvironmental) factors that regulate pulmonary metastatic colonisation. We found that mice deficient for the individual subunits of the NADPH oxidase of myeloid cells, NOX2 (encoded by Cyba, Cybb, Ncf1, Ncf2, and Ncf4), all showed decreased pulmonary metastatic colonisation. To understand the role of NOX2 in controlling tumour cell survival in the pulmonary microenvironment, we focused on Cyba-deficient (Cybatm1a ) mice, which showed the most significant decrease in metastatic colonisation. Interestingly, histological assessment of pulmonary metastatic colonisation was not possible in Cybatm1a mice, owing to the presence of large granulomas composed of galectin-3 (Mac-2)-positive macrophages and eosinophilic deposits; granulomas of variable penetrance and severity were also found in Cybatm1a mice that were not injected with melanoma cells, and these contributed to their decreased survival. The decreased pulmonary metastatic colonisation of Cybatm1a mice was not due to any overt defects in vascular permeability, and bone marrow chimaeras confirmed a role for the haematological system in the reduced metastatic colonisation phenotype. Examination of the lymphocyte populations, which are known key regulators of metastatic colonisation, revealed an enhanced proportion of activated T and natural killer cells in the lungs of Cybatm1a mice, relative to controls. The reduced metastatic colonisation, presence of granulomas and altered immune cell populations observed in Cybatm1a lungs were mirrored in Ncf2-deficient (Ncf2tm1a ) mice. Thus, we show that NOX2 deficiency results in both granulomas and the accumulation of antitumoural immune cells in the lungs that probably mediate the decreased pulmonary metastatic colonisation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

NADPH oxidase activation in neutrophils: Role of the phosphorylation of its subunits.

Neutrophils are key cells of innate immunity and during inflammation. Upon activation, they produce large amounts of superoxide anion (O2 -. ) and ensuing reactive oxygen species (ROS) to kill phagocytized microbes. The enzyme responsible for O2 -. production is called the phagocyte NADPH oxidase. This is a multicomponent enzyme system that becomes active after assembly of four cytosolic proteins (p47phox , p67phox , p40phox and Rac2) with the transmembrane proteins (p22phox and gp91phox , which form the cytochrome b558 ). gp91phox represents the catalytic subunit of the NADPH oxidase and is also called NOX2. NADPH oxidase-derived ROS are essential for microbial killing and innate immunity; however, excessive ROS production induces tissue injury and prolonged inflammatory reactions that contribute to inflammatory diseases. Thus, NADPH oxidase activation must be tightly regulated in time and space to limit ROS production. NADPH oxidase activation is regulated by several processes such as phosphorylation of its components, exchange of GDP/GTP on Rac2 and binding of p47phox and p40phox to phospholipids. This review aims to provide new insights into the role of the phosphorylation of the NADPH oxidase components, that is gp91phox , p22phox , p47phox , p67phox and p40phox , in the activation of this enzyme.

Three novel mutations in CYBA among 22 Iranians with Chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by defect in one of the components of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase enzyme. The enzyme is at least composed of membrane-bound subunits gp91-phox and p22-phox (also named cytochrome b558 ), and cytosolic ones p40-phox, p47-phox and p67-phox. A defect in the enzyme activity leads to impaired intracellular killing of phagocytic cells. The CYBA gene encoding p22-phox is located on chromosome 16q24. In this study, new genetic changes of CYBA gene in 22 Iranian patients with autosomal recessive-CGD (AR-CGD) were identified. Twenty-two patients with CGD were referred to Immunology, Asthma and Allergy Research Institute (IAARI) and enrolled in this study based on defect in NADPH oxidase activity, demographic data and clinical histories. All patients had p22-phox deficiency based on Western blotting. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs), and PCR followed by direct sequencing was performed to find p22-phox mutations. Mutation analysis of CYBA revealed 12 different mutations, including three novel mutations: one was deletion of exon 1, and two were point mutations in exon 3 (c.136G>A (p.Gly46Ser)), and exon 6 (c.388C>T (p.Gln130X)). Three new mutations of CYBA gene in four of 22 Iranian patients with AR-CGD were found. These three novel mutations can partly complete the database of Human Gene Mutation Database (HGMD) and other related ones. It can also be helpful for further prenatal diagnosis in the affected families. Given that currently bone marrow transplantation is considered to be the curative treatment for patients with CGD, finding mutations will also be useful for timely decision-making in bone marrow transplantation.

Association between NAD(P)H oxidase p22phox gene variants and acute myocardial infarction in a Han Chinese population.

BACKGROUND: We investigated the association between five selected single-nucleotide polymorphisms (rs12933505, rs3180279, rs3794264, rs4673, rs1049255) in the NAD(P)H p22phox gene and acute myocardial infarction (AMI) as well as severity of coronary artery stenosis in a Han Chinese population. PATIENTS AND METHODS: A total of 168 patients with AMI and 138 healthy controls were recruited. The TaqMan allelic discrimination assay was used to genotype five single-nucleotide polymorphisms. RESULTS: The frequency of the rs1049255 G allele was significantly lower in patients with AMI than in controls (p = 0.022). Compared with subjects with an AA genotype, subjects with a GG or AG genotype had a lower risk of AMI [multivariate-adjusted odds ratio (OR), 0.53; 95 % CI, 0.29-0.95; p = 0.031). Subjects with the GG and AG genotypes of rs1049255 showed a decreased susceptibility for triple-vessel disease (TVD) as compared with controls (multivariate-adjusted OR, 0.43; 95 % CI, 0.19-0.98; p = 0.042). Multiple logistic regression analysis revealed that the rs1049255G variant was an independent protective factor for AMI/TVD. CONCLUSION: The results suggest there is an association between the p22phox rs1049255 polymorphism with the prevalence of AMI and the severity of coronary artery stenosis in the Han Chinese population.

Association of the p22phox polymorphism C242T with the risk of late-onset Alzheimer's disease in a northern Han Chinese population.

The C242T polymorphism of the CYBA gene that encodes p22phox, a component of nicotinamide adenine dinucleotide phosphate oxidase, has been found to modulate reactive oxygen species (ROS) production. Oxidative stress is thought to play a pivotal role in the pathophysiology of Alzheimer's disease (AD), which is manifested as increased availability of ROS because of an imbalanced redox state. Therefore, the aim of this study was to investigate potential associations of the p22phox C242T polymorphism with the risk of late-onset AD (LOAD) in a northern Han Chinese population. Patients with LOAD (n = 276) and 320 control subjects were recruited for the study. Polymerase chain reaction-restriction fragment length polymorphism was used to detect the genotypes. No significant differences were found between LOAD and p22phox C242T polymorphism, but a significant association was obtained in the genotype and allele distributions of p22phox C242T between LOAD patients and controls in apolipoprotein E (ApoE) ϵ4 carriers. These results suggested that p22phox C242T polymorphism has a possible role in changing the genetic susceptibility to LOAD in ApoE ϵ4 carriers of this northern Han Chinese population.

Activation of Rap1 inhibits NADPH oxidase-dependent ROS generation in retinal pigment epithelium and reduces choroidal neovascularization.

Activation of Rap1 GTPase can improve the integrity of the barrier of the retina pigment epithelium (RPE) and reduce choroidal neovascularization (CNV). Inhibition of NADPH oxidase activation also reduces CNV. We hypothesize that Rap1 inhibits NADPH oxidase-generated ROS and thereby reduces CNV formation. Using a murine model of laser-induced CNV, we determined that reduced Rap1 activity in RPE/choroid occurred with CNV formation and that activation of Rap1 by 2'-O-Me-cAMP (8CPT)-reduced laser-induced CNV via inhibiting NADPH oxidase-generated ROS. In RPE, inhibition of Rap1 by Rap1 GTPase-activating protein (Rap1GAP) increased ROS generation, whereas activation of Rap1 by 8CPT reduced ROS by interfering with the assembly of NADPH oxidase membrane subunit p22phox with NOX4 or cytoplasmic subunit p47phox. Activation of NADPH oxidase with Rap1GAP reduced RPE barrier integrity via cadherin phosphorylation and facilitated choroidal EC migration across the RPE monolayer. Rap1GAP-induced ROS generation was inhibited by active Rap1a, but not Rap1b, and activation of Rap1a by 8CPT in Rap1b(-/-) mice reduced laser-induced CNV, in correlation with decreased ROS generation in RPE/choroid. These findings provide evidence that active Rap1 reduces CNV by interfering with the assembly of NADPH oxidase subunits and increasing the integrity of the RPE barrier.

Possible roles of tumor necrosis factor-α and angiotensin II type 1 receptor on high glucose-induced damage in renal proximal tubular cells.

Recent studies have identified that high glucose-induced renal tubular cell damage. We previously demonstrated that high glucose treatment induced oxidative stress in human renal proximal tubular epithelial cells (RPTECs), and angiotensin II type 1 (AT1) receptor blockers reduce high glucose-induced oxidative stress in RPTEC possibly via blockade of intracellular as well as extracellular AT1 receptor. However, exact roles of tumor necrosis factor (TNF)-α and AT1 receptor on high glucose-induced renal tubular function remain unclear. N-acetyl-beta-glucosaminidase (NAG), concentrations of TNF-α/angiotensin II and p22(phox) protein levels after high glucose treatment with or without AT1 receptor blocker or thalidomide, an inhibitor of TNF-α protein synthesis, were measured in immortalized human renal proximal tubular epithelial cells (HK2 cells). AT1 receptor knockdown was performed with AT1 receptor small interfering RNA (siRNA). High glucose treatment (30 mM) significantly increased NAG release, TNF-α/angiotensin II concentrations in cell media and p22(phox) protein levels compared with those in regular glucose medium (5.6 mM). Candesartan, an AT1R blocker, showed a significant reduction on high glucose-induced NAG release, TNF-α concentrations and p22(phox) protein levels in HK2 cells. In addition, significant decreases of NAG release, TNF-α concentrations and p22(phox) protein levels in HK2 cells were observed in high glucose-treated group with thalidomide. AT1R knockdown with siRNA markedly reversed high glucose, angiotensin II or TNF-α-induced p22(phox) protein levels in HK2 cells. TNF-α may be involved in high glucose-induced renal tubular damage in HK2 cells possibly via AT1 receptor signaling.

NADPH oxidase subunit p22(phox)-mediated reactive oxygen species contribute to angiogenesis and tumor growth through AKT and ERK1/2 signaling pathways in prostate cancer.

Excessive generation of reactive oxygen species (ROS) in cancer cells is associated with cancer development, but the underlying mechanisms and therapeutic significance remain elusive. In this study, we reported that levels of ROS and p22(phox) expression are greatly increased in human prostate cancer tissues, and knockdown of p22(phox) by specific small interfering RNA (siRNA) decreased ROS levels in prostate cancer cells. We also showed that stable downregulation of p22(phox) in prostate cancer cells inhibited cell proliferation and colony formation, which was mediated by AKT and extracellular signal-regulated kinase (ERK)1/2 signaling pathways and their downstream molecules hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF). The NADPH oxidase subunit NOX1 was also elevated in prostate cancer cells, and was involved in activation of AKT/ERK/HIF-1/VEGF pathway and regulation of cell proliferation. Knockdown of p22(phox) resulted in inhibition of tumor angiogenesis and tumor growth in nude mice. These findings reveal a new function of p22(phox) in tumor angiogenesis and tumor growth, and suggest that p22(phox) is a potential novel target for prostate cancer treatment.

Attenuation of urokinase activity during experimental ischaemia protects the cerebral barrier from damage through regulation of matrix metalloproteinase-2 and NAD(P)H oxidase.

Ischaemic injury impairs the integrity of the blood-brain barrier (BBB). In this study, we investigated the molecular causes of this defect with regard to the putative correlations among NAD(P)H oxidase, plasminogen-plasmin system components, and matrix metalloproteinases. Hence, the activities of NAD(P)H oxidase, matrix metalloproteinase-2, urokinase-type plasminogen activator (uPA), and tissue-type plasminogen activator (tPA), and superoxide anion levels, were assessed in human brain microvascular endothelial cells (HBMECs) exposed to oxygen-glucose deprivation (OGD) alone or OGD followed by reperfusion (OGD + R). The integrity of an in vitro model of BBB comprising HBMECs and astrocytes was studied by measuring transendothelial electrical resistance and the paracellular flux of albumin. OGD with or without reperfusion (OGD ± R) radically perturbed barrier function while concurrently enhancing uPA, tPA and NAD(P)H oxidase activities and superoxide anion release in HBMECs. Pharmacological inactivation of NAD(P)H oxidase attenuated OGD ± R-mediated BBB damage through modulation of matrix metalloproteinase-2 and tPA, but not uPA activity. Overactivation of NAD(P)H oxidase in HBMECs via cDNA electroporation of its p22-phox subunit confirmed the involvement of tPA in oxidase-mediated BBB disruption. Interestingly, blockade of uPA or uPA receptor preserved normal BBB function by neutralizing both NAD(P)H oxidase and matrix metalloproteinase-2 activities. Hence, selective targeting of uPA after ischaemic strokes may protect cerebral barrier integrity and function by concomitantly attenuating basement membrane degradation and oxidative stress.