Nox4 Protein Research Articles

The role of Nox-mediated oxidation in the regulation of cytoskeletal dynamics.

Nox generated ROS, particularly those derived from Nox1, Nox2 and Nox4, have emerged as important regulators of the actin cytoskeleton and cytoskeleton-supported cell functions, such as migration and adhesion. The effects of Nox-derived ROS on cytoskeletal remodeling may be largely attributed to the ability of ROS to directly modify proteins that constitute or are associated with the cytoskeleton. Additionally, Nox-derived ROS may participate in signaling pathways governing cytoskeletal remodeling. In addition to these more extensively studied signaling pathways involving Nox-derived ROS, there also exist redox sensitive pathways for which the source of ROS is unclear. ROS from as of yet undetermined sources play a role in modifying, and thus regulating, the activity of several proteins critical for remodeling of the actin cytoskeleton. In this review we discuss ROS sensitive targets that are likely to affect cytoskeletal dynamics, as well as the potential involvement of Nox proteins.

Abstract B31: NOX2 generated reactive oxygen species promotes glioblastoma growth through inhibition of PTEN

Abstract The tumor suppressor protein Phosphatase and Tensin Homolog (PTEN) is a negative regulator of the Akt pathway which promotes proliferation, migration, and self-renewal. PTEN is mutated or deleted in ~40% of glioblastoma (GBM) resulting in increased Akt activity; however almost all GBM's have increased Akt activity, regardless of PTEN status. This raises the possibility that GBM may inactivate PTEN through means other than mutation or deletion. Treatment with exogenous reactive oxygen species (ROS), like hydrogen peroxide, inactivates PTEN through oxidation in the phosphatase domain. The low oxygen environment of tumors (1%O2) increases ROS levels as compared to normal body tissue (4% O2), and tissue culture conditions (20% O2). Recent data indicate that low to moderate levels of ROS are required for cell maintenance and stem cell renewal while moderate increases in ROS may promote migration, invasion, and proliferation. Indeed, our lab recently found that normal neural stem cells have higher ROS levels, which is required for stem cell renewal. Moreover, data from our lab and others, have found that growing cells under physiological oxygen levels increases ROS and promotes renewal and proliferation. This suggests that the low-oxygen environment of tumors may promote proliferation and tumorigenesis through post-translational modification of proteins, like PTEN. NADPH oxidase (NOX) is a membrane bound complex of proteins that produces a significant amount of endogenous intracellular ROS. While this protein was originally identified in macrophages as being important for immune surveillance, new data suggests that the family of NOX proteins (1-4) has many other biological functions in a variety of different cell types. New data has found that the constitutively active NOX4 acts as an oncogene in several different cancer types by promoting migration, invasion, and proliferation. Within the brain, NOX2 promotes ROS-induced stem cell renewal in normal neural stem cells. Additionally, increased NOX2 expression in GBM patients correlates with shorter survival, while patients with low NOX2 expression generally live longer. Together these data suggest that, through NOX2, the low-oxygen environment of GBM increases ROS levels that oxidize and inhibit PTEN. This novel post-translational modification may increase proliferation and promote tumorigenicity through activation of Akt in cells with intact PTEN. We examined endogenous ROS levels in patient-derived GBM samples and found that increased ROS correlated with faster proliferating cells. Furthermore, low levels of O2 (1%) increased proliferation and Akt activation in a PTEN dependent manner. Both PTEN null and knockdown cells showed no increase in either proliferation or Akt activation over cells grown at room air (20%). Moreover, pharmacological inhibition of Akt abrogated ROS-induced proliferation, suggesting this mechanism functions through the PTEN/Akt pathway. This response was due directly to ROS production, as treatment with antioxidants decreased both ROS production and proliferation at low oxygen and room air. Additionally, we found that growth at 1% O2 led to the oxidation and inhibition of PTEN as seen by Akt phosphorylation. Treatment with apocynin, a NOX2 inhibitor decreased ROS production, proliferation, and Akt phosphorylation, suggesting that ROS-induced proliferation may be mediated through the NOX2 protein. This data not only show PTEN oxidation and inhibition under physiological conditions for the first time, it suggests that the low-oxygen tumor microenvironment may alter PTEN positive GBM to up-regulate the Akt pathway and promote tumorigenesis. Citation Format: Kirsten Ludwig, Harley Kornblum. NOX2 generated reactive oxygen species promotes glioblastoma growth through inhibition of PTEN. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B31.

Abstract 19752: Nox4 Expression Regulates Inflammation in Tgfβ-treated Vascular Smooth Muscle Cells

NADPH oxidases (NOX) are a major source of reactive oxygen species in the cardiovascular system, contributing to the pathogenesis of disease. We recently reported that increased NOX4 expression in aging results in enhanced cellular and mitochondrial oxidative stress, vascular inflammation, dysfunction, and atherosclerosis. However, the role of NOX4 in the activation of inflammatory molecular pathways in vascular smooth muscle cells (VSMC) under pathophysiological conditions has not been elucidated. In this study, we investigated whether inhibition of NOX4 expression modulates proinflammatory gene expression in mouse VSMC by suppressing its expression with a novel peptide-mediated siRNA transfection approach. Using melittin derived peptide p5RHH, we achieved significantly higher transfection efficiency (92% vs 85% with Lipofectamine) and decreased toxicity (p<0.01 vs Lipofectamine in MTT assay). TGFβ, a proinflammatory cytokine, known to induce NOX4 expression significantly upregulated NOX4 mRNA (p<0.01) and protein expression (p<0.01) in VSMC. p5RHH mediated NOX4 siRNA transfection attenuated TGFβ induced upregulation of NOX4 mRNA (p<0.01) and protein (p<0.01) levels. Analysis of inflammatory marker genes, CCL2, CCL5, IL6, and VCAM-1, showed that NOX4 knockdown significantly attenuated the expression of these genes in TGFβ-treated VSMC, with the highest inhibition seen in the expression of IL6. Congruent with this, inflammatory marker gene expression, which increases with aging, was significantly lower in VSMC isolated from aged (16 months old) NOX4 knockout mice compared with the aged wild-type. TGFβ-activated kinase 1 (TAK1, also known as MAP3K7) is an upstream regulator of inflammatory signaling in cells treated with cytokines. p5RHH mediated TAK1 siRNA transfection downregulated AP1 and NFκB activation and inhibited NOX4 expression in wild-type VSMC, indicating that TAK1 regulates NOX4 expression via regulation of AP1 and NFκB. Because plasma TGFβ levels increase with aging, it may be of one of the factors contributing to increased NOX4 expression with aging. Our data indicate that NOX4 is a potential therapeutic target for modulation of inflammation in aging and aging-associated cardiovascular disease.

NADPH Oxidase Accounts for Changes in Cerebrovascular Redox Status in Hindlimb Unweighting Rats

ObjectiveThe roles of cerebrovascular oxidative stress in vascular functional remodeling have been described in hindlimb-unweighting (HU) rats. However, the underlying mechanism remains to be established. MethodsWe investigated the generation of vascular reactive oxygen species (ROS), Nox2/Nox4 protein and mRNA levels, NADPH oxidase activity, and manganese superoxide dismutase (MnSOD) and glutathione peroxidase-1 (GPx-1) mRNA levels in cerebral and mesenteric smooth muscle cells (VSMCs) of HU rats. ResultsROS production increased in cerebral but not in mesenteric VSMCs of HU rats compared with those in control rats. Nox2 and Nox4 protein and mRNA levels were increased significantly but MnSOD/GPx-1 mRNA levels decreased in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly more in cerebral but not in mesenteric arteries of HU rats. NADPH oxidase inhibition with apocynin attenuated cerebrovascular ROS production and partially restored Nox2/Nox4 protein and mRNA levels, NADPH oxidase activity, and MnSOD/GPx-1 mRNA levels in cerebral VSMCs of HU rats. ConclusionThese results suggest that vascular NADPH oxidases regulate cerebrovascular redox status and participate in vascular oxidative stress injury during simulated microgravity.

Mechanisms of bradykinin-induced expression of connective tissue growth factor and nephrin in podocytes.

Diabetic nephropathy (DN) is the main cause of morbidity and mortality in diabetes and is characterized by mesangial matrix deposition and podocytopathy, including podocyte loss. The risk factors and mechanisms involved in the pathogenesis of DN are still not completely defined. In the present study, we aimed to understand the cellular mechanisms through which activation of B2 kinin receptors contribute to the initiation and progression of DN. Stimulation of cultured rat podocytes with bradykinin (BK) resulted in a significant increase in ROS generation, and this was associated with a significant increase in NADPH oxidase (NOX)1 and NOX4 protein and mRNA levels. BK stimulation also resulted in a signicant increase in the phosphorylation of ERK1/2 and Akt, and this effect was inhibited in the presence of NOX1 and Nox4 small interfering (si)RNA. Furthermore, podocytes stimulated with BK resulted in a significant increase in protein and mRNA levels of connective tissue growth factor (CTGF) and, at the same time, a significant decrease in protein and mRNA levels of nephrin. siRNA targeted against NOX1 and NOX4 significantly inhibited the BK-induced increase in CTGF. Nephrin expression was increased in response to BK in the presence of NOX1 and NOX4 siRNA, thus implicating a role for NOXs in modulating the BK response in podocytes. Moreover, nephrin expression in response to BK was also significantly increased in the presence of siRNA targeted against CTGF. These findings provide novel aspects of BK signal transduction pathways in pathogenesis of DN and identify novel targets for interventional strategies.

NRROS Negatively Regulates Osteoclast Differentiation by Inhibiting RANKL-Mediated NF-N:B and Reactive Oxygen Species Pathways.

Negative regulator of reactive oxygen species (NRROS) is known to repress ROS generation in phagocytes. In this study, we examined the roles of NRROS in both osteoclasts and osteoblasts. Our results demonstrate that NRROS negatively regulates the differentiation of osteoclasts, but not osteoblasts. Further, overexpression of NRROS in osteoclast precursor cells attenuates RANKL-induced osteoclast differentiation. Conversely, osteoclast differentiation is enhanced upon siRNA-mediated knockdown of NRROS. Additionally, NRROS attenuates RANKL-induced NF-κB activation, as well as degradation of the NOX1 and NOX2 proteins, which are required for ROS generation. Based on our observations, we present NRROS as a novel negative regulator of RANKL-induced osteoclastogenesis.

NADPH OXIDASE 2, 4 AND MITOCHONDRIA-DERIVED REACTIVE OXYGEN SPECIES CONTRIBUTE TO ANGIOPOIETIN-1 INDUCED ANGIOGENESIS

The NOX proteins are reactive oxygen species (ROS) generating enzymes and together with Mitochondrial-derived ROS, they play an important role in pathological as well as physiological processes of the vasculature. Recent evidences indicate that ROS generated by NOX and the mitochondria may play important roles in growth factors downstream signaling. Our own study have pointed out that Angiopoietin-1 (Ang-1) induced ROS production is important for Ang-1/Tie-2 receptor signaling in endothelial cells, however, we did not identify the molecular sources of ROS generation in this respect. In this study, we have identified the origins of Ang-1 induced ROS production in endothelial cells and their involvement in Ang-1 induced angiogenesis. The most abundant isoforms of NOX in human umbilical endothelial cells were knocked down using adenoviruses expressing shRNA against NOX2 and NOX4. Mitochondrial-derived ROS were inhibited using SS31 peptide or Mito-Tempol. Cellular ROS levels were measured upon Ang-1 treatment using Amplex Red and Mitochondrial ROS level were measured with MitoSox. To evaluate the involvement of NOX or Mitochondrial-derived ROS in Ang-1 induced angiogenesis we evaluated cell survival by cell count and Caspase-3 cleavage, tubulogenesis by matrigel assay and migration by scratch wound assay. Activation level of the MAPK and PI3K/AKT pathways downstream of Ang-1/Tie-2 were measured by immunoblotting. Ang-1treatment leads to NOX-derived and Mitochondrial ROS production. NOX4 and the mitochondrial ROS are required for Ang-1 induced ROS production. NOX4 knockdown and Mitochondrial ROS blockage lead to impaired Ang-1 induced migration. NOX2 minimally contributes to Ang-1 induced ROS production and appears to be required for Ang-1 induced cell survival. In addition, NOX4 and mitochondrial-derived ROS are required for optimal activation of p38 and inactivation of ERK and AKT. On the other hand NOX2 appears to be required for optimal AKT activation and ERK and p38 inactivation. We conclude that NOX4 and mitochondrial ROS are required for Ang-1 induced migration through p38 activation. We also concluded that NOX2-derived ROS regulates Ang-1 induced cell survival by activation of AKT. This study provides new insight into cellular signaling mechanism downstream of Ang-1/Tie-2 signaling axis.

Increased expression of NAPDH oxidase 4 in systemic sclerosis dermal fibroblasts: regulation by transforming growth factor β.

Systemic sclerosis (SSc) is characterized by severe and often progressive fibrosis of the skin and multiple internal organs. The mechanisms responsible for these alterations remain obscure, although excessive reactive oxygen species (ROS)-mediated oxidative stress has been implicated. NOX-4 is 1 of 7 isoforms of NADPH oxidase responsible for the generation of ROS. The purpose of this study was to examine NOX-4 expression in skin and cultured dermal fibroblasts from SSc patients and to examine its regulation by transforming growth factor β1 (TGFβ1). NOX-4 was assessed in normal and SSc skin by immunohistologic analysis and in normal and SSc cultured dermal fibroblasts by quantitative polymerase chain reaction analysis, fluorescence microscopy, and Western blotting. ROS levels were assessed by fluorescence measurement of H2 O2 production. Specific kinase inhibitors were used to study the TGFβ1 signaling involved in NOX-4 stimulation. NOX-4 inhibition/down-regulation was induced with a selective NOX-4 small-molecule inhibitor and NOX-4 small interfering RNA (siRNA). In contrast with normal skin fibroblasts, those from SSc skin showed intense NOX-4 staining. Cultured SSc fibroblasts displayed increased NOX-4 expression. TGFβ1 caused potent NOX-4 protein and messenger RNA stimulation in normal and SSc fibroblasts, which was mediated by the protein kinase Cδ (PKCδ) and Smad2/3 pathways. NOX-4 knockdown in SSc fibroblasts reduced the production of ROS and lowered the expression of type I collagen. NOX-4 expression and production were found to be constitutively elevated in SSc skin and cultured SSc dermal fibroblasts. TGFβ1 stimulated NOX-4 expression in normal and SSc fibroblasts through PKCδ and Smad2/3 signaling pathways. A small-molecule NOX-4 inhibitor decreased collagen and fibronectin production by normal and SSc fibroblasts, and NOX-4 siRNA knockdown reduced ROS and collagen production by SSc fibroblasts. These results demonstrate the involvement of NOX-4 in SSc-associated fibrosis and indicate NOX-4 inhibitors as novel therapeutic approaches for SSc.

Abstract 096: Sequential Activation of Nox1 and Gremlin1 Leads to Endothelial Proliferation in Human Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a rapidly degenerating and devastating disease of increased pulmonary vessel resistance leading to eventual right heart failure. Until now, palliative modalities have targeted the reduction of vascular tone with little success. Recent studies have delved into the mechanisms regulating increased pulmonary vascular resistance: aberrant vascular remodeling and occlusion. However, little is known of the molecular mechanisms responsible for endothelial proliferation, a root cause of PAH-associated vascular remodeling. We provide the first evidence to our knowledge of an upregulation of NADPH oxidase 1 (Nox1) at the transcript and protein (2.1±0.62 -fold, P < 0.05) level in resistance vessels from PAH vs. non-PAH subjects. This coincided with an increase in bone morphogenetic protein (BMP) antagonist Gremlin1 protein expression (2.3±0.47 [[Unable to Display Character: –]]fold vs. non-PAH, P < 0.05) and reactive oxygen species (ROS) production (iodonium-inhibitable hydrogen peroxide production: 0.69±0.06 vs. 0.43±0.032 nmol/min/mg protein for PAH vs. non-PAH, respectively, P < 0.05). In vitro studies in human pulmonary artery endothelial cells (HPAEC) demonstrate that hypoxia (24 hr, 1 % O 2 ) drives Nox1 subunit expression (Nox1 protein: 1.4±0.075-fold vs. normoxia, P < 0.05), assembly and oxidase activity (superoxide production, nmol/min/mg protein: 14.0±1.9 vs. 6.00±0.94 for normoxia, P < 0.01) leading to elevation in sonic hedgehog (SHH; 1.5±0.011 fold, P < 0.05) and Gremlin1 (1.90±0.32-fold, P < 0.01) expression. Nox1 gene-silencing in hypoxia-exposed HPAEC abrogated this cascade. Moreover, hypoxia-induced endothelial cell proliferation (1.18±0.038-fold vs. normoxia, P < 0.05) was attenuated with loss of either Nox1 or Gremlin1. Finally, incubation of normoxic HPAEC with conditioned media from hypoxia-exposed HPAEC resulted in increased proliferation, which was abrogated by Nox1 suppression of donor cells. Together these data support a Nox1-Gremlin1 signaling axis in pulmonary vascular endothelium that is likely to contribute to pathophysiological endothelial proliferation and the progression of pulmonary hypertension. The findings also support targeting of Nox1 as a viable therapeutic option to combat PAH.

Abstract 098: MicroRNA Regulation of NADPH Oxidase Mediates the Antioxidant Effect of Renal Paraoxonase 2

Increased renal generation of reactive oxygen species (ROS) is important in the pathogenesis of hypertension caused by absent or dysfunctional dopamine receptor subtype. Germline deletion of the dopamine 2 receptor in mice increases renal NADPH oxidase (NOX) activity and decreases expression paraoxonase 2 (PON2) and results in ROS-dependent hypertension. We determined if microRNA (miR) is involved in PON2-mediated regulation of NOX. Silencing PON2 in human renal proximal tubules cells decreased PON2 (-60±4%, n=3,*P<0.05) and increased NOX2 (110±15%, n=3,*P<0.05) and NOX4 (80±10%, n=3,*P<0.05) proteins, NOX activity (50±6%, n=3,*P<0.05), and ROS production (57±3%, n=4,*P<0.05). Inhibition of NOX activity by diphenyleneiodonium normalized the increase in ROS caused by PON2 silencing. Renal-selective silencing of Pon2 in mice by the renal subcapsular infusion of Pon2 siRNA decreased PON2 (~50%), and increased NOX2 (191±11%,n=3, P<0.05), NOX4 (60±4%,n=3, P<0.05), NOX activity (94±23%, n=3, P<0.05), and blood pressure (BP) (+41±6 mmHg, n=3, P<0.05). Pon2-/- mice also had higher BP than wild-type littermates (+15±2 mmHg,n=3/4,*P<0.05) but less than observed with renal-selective silencing indicating extrarenal compensation. Renal NOX2 (220±64%, n=3/4,*P<0.05) and NOX activity (195±77%, n=3,*P<0.05) were also increased in Pon2-/- mice. However, the renal expression of NOX4 was similar in Pon2-/- and wild-type littermates. The renal expressions of miR-23b, miR-34a and miR-155 (reported to regulate NOX expression) were also similar in Pon2-/- mice and wild-type littermates. However, renal miR-146a expression was decreased (-25±4%, n=3/4,*P<0.05) while miR-204 (150±12%, n=3/4,*P<0.05) and NFAT expressions (21±7%, n=3/4,*P<0.05) were increased in Pon2-/- mice. The increase in miR-204 could be a compensatory response because miR-204 has been shown to decrease NFAT expression. It is known that NFAT and NOX2 can positively regulate each other’s expression while miR-146a negatively regulates NOX4 expression and inflammation. We conclude that PON2 by increasing miR-146a and decreasing NFAT expression negatively regulates NOX activity and reduce ROS production that would contribute to the maintenance of normal BP.

Effect of short-term acumagnetotherapy on diabetic kidney disease in patients with type II diabetes and study on the molecular mechanism

ObjectiveTo explore whether magnetotherapy is preventive in retarding diabetic kidney disease (DKD) progression and investigate underling molecular mechanisms related to its therapeutic efficacy. MethodsTwenty-eight patients with type II diabetes (T2D) were undergone pulsed electromagnetic fields (PEMF) stimulation at the acupoints of Píshū (▪ BL 20), Zúsānl▪ (▪ ST 36), Shènshū (▪ BL 23) and Yíshū (▪, EX-B3) for a period of 4 weeks. Urinary micro-albumin (U-mAlb) excretion, plasma methane dicarboxylic aldehyde (MDA and lipopolysaccharide (LPS) of the patients were used for evaluating therapeutic efficacies. ResultsAfter the acumagnetotherapy, U-mAlb excretion in the participated patients was markedly reduced (27.21±3.51 vs 8.51±0.95, P<0.001) accompanied with decreased MDA (16.46±1.17 vs 12.40±1.86, P<0.05) and LPS (37.41±3.84 vs 21.63±3.61, P<0.05) levels in plasma while the metabolic control of these patients was not significantly altered. Acumagnetotherapy increased IκBα content (0.69±1.17 vs 1.30±0.29, P<0.01), an inhibitory protein of inflammatory response, and correspondingly reduced the protein levels of inflammatory activating proteins, NF-κB p65 (0.98±0.42 vs 0.43±0.28, P<0.05) and NF-κB p50 (1.19±0.40 vs 0.76±0.30, P<0.05). The acumagnetotherapy also inhibited the oxidant-producing enzyme, Nox4 protein expression (1.32±0.40 vs 0.37±0.23, P<0.05) in patient's blood lymphocytes. ConclusionShort-term intervention of acumagnetotherapy in patients with T2D mitigates DKD progress potentially by its anti-oxidative and anti-inflammatory effects.

INF-γ Enhances Nox2 Activity by Upregulating phox Proteins When Applied to Differentiating PLB-985 Cells but Does Not Induce Nox2 Activity by Itself.

BackgroundThe cytokine and drug interferon-γ enhances superoxide anion production by the antimicrobicidal Nox2 enzyme of neutrophils. Because mature neutrophils have a short lifespan, we hypothesized that the effects of interferon-γ on these cells might be mediated by its prolonged exposure to differentiating neutrophil precursors in the bone marrow rather than its brief exposure to mature circulating neutrophils.Effects of INF-γ on Nox2 ActivityTo address this possibility we exposed the myeloid PLB-985 cell line to interferon-γ for 3 days in the presence of dimethyl sulfoxide which induces terminal differentiation of these cells. Interferon-γ was found to enhance superoxide production by Nox2 in a concentration dependent manner. In contrast, application of interferon-γ alone for 3 days failed to induce detectible Nox2 activity. Additionally, application of interferon-γ for 3 hours to pre-differentiated PLB-985 cells, which models studies using isolated neutrophils, was much less effective at enhancing superoxide anion production.Effects of INF-γ on phox Protein LevelsAddition of interferon-γ during differentiation was found to upregulate the Nox2 proteins gp91phox and p47phox in concert with elevated transcription of their genes. The p22phox protein was upregulated in the absence of increased transcription presumably reflecting stabilization resulting from binding to the elevated gp91phox. Thus, increased levels of gp91phox, p47phox and p22phox likely account for the interferon-γ mediated enhancement of dimethyl sulfoxide-induced Nox2 activity. In contrast, although interferon-γ alone also increased various phox proteins and their mRNAs, the pattern was very different to that seen with interferon-γ plus dimethyl sulfoxide. In particular, p47phox was not induced thus explaining the inability of interferon -γ alone to enhance Nox2 activity. Short application of interferon-γ to already differentiated cells failed to increase any phox proteins.ConclusionsOur findings indicate that interferon-γ has complex effects on phox protein expression and that these are different in cells undergoing terminal differentiation. Understanding these changes may indicate additional therapeutic uses for this cytokine in human disorders.

NOX4 in Mitochondria: Yeast Two-Hybrid-Based Interaction with Complex I Without Relevance for Basal Reactive Oxygen Species?

NADPH oxidases (NOXs) represent the only known dedicated source of reactive oxygen species (ROS) and thus a prime therapeutic target. Type 4 NOX is unique as it produces H2O2, is constitutively active, and has been suggested to localize to cardiac mitochondria, thus possibly linking mitochondrial and NOX-derived ROS formation. The aim of this study was to identify NOX4-binding proteins and examine the possible physiological localization of NOX4 to mitochondria and its impact on mitochondrial ROS formation. We here provide evidence that NOX4 can, in principle, enter protein–protein interactions with mitochondrial complex I NADH dehydrogenase subunits, 1 and 4L. However, under physiological conditions, NOX4 protein was neither detectable in the kidney nor in cardiomyocyte mitochondria. The NOX inhibitor, GKT136901, slightly reduced ROS formation in cardiomyocyte mitochondria, but this effect was observed in both wild-type and Nox4−/− mice. NOX4 may thus associate with mitochondrial complex I proteins, but in cardiac and renal mitochondria under basal conditions, expression is beyond our detection limits and does not contribute to ROS formation. Antioxid. Redox Signal. 23, 1106–1112.

Abstract 2215: Transcriptional co-regulation of Nox4 by p53 and SMAD3

Abstract Recent studies suggest p53 plays an important role in TGF-beta-mediated cell signaling and migration. Previously, we showed that wild-type (WT) and mutant forms of p53 differentially regulate reactive oxygen species (ROS) generation by NADPH oxidase-4 (Nox4). We found that WT-p53 is a potent suppressor of TGF-beta-induced Nox4, ROS production, and cell migration, whereas tumor-associated mutant p53 proteins enhanced Nox4 mRNA and protein expression and cell migration by both TGF-beta-dependent and independent mechanisms (Boudreau et al. Br J Cancer. 2014 May 13;110(10):2569-82). In this study, we examined the basis of human Nox4 promoter regulation by p53 and SMAD3. By constructing a deletion series of Nox4 promoter-reporter constructs, we discovered two regions (-4.76kb to -3.9kb and -1.8kb to - 0.2kb) upstream of the transcription start site that induce promoter activity upon activation of the TGF-beta/SMAD3 pathway. Interestingly, TGF-beta/SMAD3-mediated Nox4 promoter activity was abolished by heterologous expression of p53-WT in p53-null cell lines Hep3B (hepatocytes) and H1299 (lung epithelial). This activity was repressed by wild-type p53 in a dose-dependent manner. In contrast, expression of common tumor-associated mutant-p53 proteins (R175H, R248H, R249Q, R273H) did not repress but enhanced Nox4 promoter activity. We observed this enhancement of Nox4 promoter activity by mutant p53 (R273H) in H1299 cells expressing T204D, a constitutively active TGF-beta receptor. When individual p53 response elements were mutated, however, both untreated and R273H-expressing cells showed 50% reduction in promoter activity. These results indicate that the Nox4 promoter region between -3.97kb and -4.76kb, containing candidate p53 response elements, plays a role in modulating Nox4 expression. Preliminary chromatin immunoprecipitation results suggest that endogenous mutant p53 R249S in PLC/PRF/5 hepatoma cells complexes with the Nox4 promoter region between -3.97kb and -4.76kb. Furthermore, constructs lacking this region show significantly reduced Nox4 promoter activity in response to TGF-beta. Our results provide a basis for Nox4 co-regulation by SMAD3 and mutant p53 that may be involved in tumor progression and metastasis. Citation Format: Howard E. Boudreau, Jonathan J. Park, Thomas L. Leto. Transcriptional co-regulation of Nox4 by p53 and SMAD3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2215. doi:10.1158/1538-7445.AM2015-2215

Metformin decreases growth of pancreatic cancer cells by decreasing reactive oxygen species: Role of NOX4

BackgroundRetrospective epidemiologic studies show that metformin reduced the incidence of pancreatic cancer in diabetic patients. One potential mechanism may be by altering reactive oxygen species (ROS) and apoptosis. In this in vitro study, we explore the role of ROS and apoptosis in mediating the growth-inhibitory effects of metformin in pancreatic cancer cells. MethodsWe cultured MIA PaCa and Panc1 pancreatic cancer cells in medium containing physiological concentrations of glucose (5 mM) or supra-physiological concentrations of glucose and then treated cells with metformin. Cell viability, ROS production, apoptosis, and protein levels of manganese superoxide dismutase (MnSOD), NADPH oxidase (NOX) 2, and NOX4 were measured. ResultsMetformin decreased viability of MIA PaCa and Panc1 cells under physiological glucose conditions in comparison to untreated cells; metformin did not have any effect on human pancreatic normal epithelial (HPNE) cells. The decrease in cell survival was associated with decreased intracellular ROS, increased protein levels of MnSOD, and decreased levels of NOX2 and NOX4 proteins in MIA PaCa and Panc1 cells but not HPNE cells. Transfecting MIA PaCa and Panc1 cells with pcDNA3NOX4 protected against the anti-survival effects of metformin. ConclusionOur findings suggest that metformin decreases cell survival by reducing ROS production, in part through down regulation of NOX4 protein expression.

Deficiency of NOX1 or NOX4 Prevents Liver Inflammation and Fibrosis in Mice through Inhibition of Hepatic Stellate Cell Activation

Reactive oxygen species (ROS) produced by nicotinamide adenine dinucleotide phosphate oxidase (NOX) play a key role in liver injury and fibrosis. Previous studies demonstrated that GKT137831, a dual NOX1/4 inhibitor, attenuated liver fibrosis in mice as well as pro-fibrotic genes in hepatic stellate cells (HSCs) as well as hepatocyte apoptosis. The effect of NOX1 and NOX4 deficiency in liver fibrosis is unclear, and has never been directly compared. HSCs are the primary myofibroblasts in the pathogenesis of liver fibrosis. Therefore, we aimed to determine the role of NOX1 and NOX4 in liver fibrosis, and investigated whether NOX1 and NOX4 signaling mediates liver fibrosis by regulating HSC activation. Mice were treated with carbon tetrachloride (CCl4) to induce liver fibrosis. Deficiency of either NOX1 or NOX4 attenuates liver injury, inflammation, and fibrosis after CCl4 compared to wild-type mice. NOX1 or NOX4 deficiency reduced lipid peroxidation and ROS production in mice with liver fibrosis. NOX1 and NOX4 deficiency are approximately equally effective in preventing liver injury in the mice. The NOX1/4 dual inhibitor GKT137831 suppressed ROS production as well as inflammatory and proliferative genes induced by lipopolysaccharide (LPS), platelet-derived growth factor (PDGF), or sonic hedgehog (Shh) in primary mouse HSCs. Furthermore, the mRNAs of proliferative and pro-fibrotic genes were downregulated in NOX1 and NOX4 knock-out activated HSCs (cultured on plastic for 5 days). Finally, NOX1 and NOX4 protein levels were increased in human livers with cirrhosis compared with normal controls. Thus, NOX1 and NOX4 signaling mediates the pathogenesis of liver fibrosis, including the direct activation of HSC.

Role of glomerular mesangial cell in the pathogenetic progress of lupus nephritis fibrosis

To explore the roles of proliferative lesions of mesangial cell and reactive oxygen species (ROS) in the pathogenetic progress of lupus nephritis fibrosis. Control mice C57BL/6 and model mice NZBWF1 were selected. Kidney tissues were collected for hematoxylin and eosin (HE) and Masson staining at Week 8, 12 and 16. The proliferation of glomerular cells was assessed by BrdU incorporation light microscopy. And the expression of NOX4 protein was detected by Western blot. HE and Masson staining showed that model mice began to develop interstitial fibrosis and worsened at Weeks 12 and 16. The numbers of mesangial cell increased at Week 8 and became prominent at Weeks 12 and 16. The expression of NOX4 protein increased with the progression of lupus nephritis fibrosis. Glomerular mesangial cell and ROS play an role in the pathogenetic progress of lupus nephritis fibrosis.

Molecular characterization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins.

LC3-associated phagocytosis (LAP) is a process wherein elements of autophagy conjugate LC3 to phagosomal membranes. We characterize the molecular requirements for LAP, and identify Rubicon as being required for LAP but not autophagy. Rubicon is recruited to LAPosomes and is required for the activity of a Class III PI(3)K complex containing UVRAG but lacking ATG14 and Ambra1. This allows for the sustained localization of PtdIns(3)P, which is critical for recruitment of downstream autophagic proteins and stabilization of the NOX2 complex to produce reactive oxygen species. Both PtdIns(3)P and reactive oxygen species are required for conjugation of LC3 to LAPosomes and subsequent association with LAMP1(+) lysosomes. LAP is induced by engulfment of Aspergillus fumigatus, a fungal pathogen that commonly afflicts immunocompromised hosts, and is required for its optimal clearance in vivo. Therefore, we have identified molecules that distinguish LAP from canonical autophagy, thereby elucidating the importance of LAP in response to A. fumigatus infection.

Impairment of Endothelial Cell Function Induced by Hemoglobin A(1c) and the Potential Mechanisms.

Hemoglobin A(1c) (HbA(1c)) concentrations reflect glycemic control and diabetic complications. However, there is little evidence supporting the pathological role of HbA(1c) in the development and progression of diabetic complications. We investigated the impact of HbA(1c) on endothelial cell function and the potential mechanisms. The effects of HbA(1c) on the viability and migration of human umbilical vein endothelial cells (HUVECs) were measured by the Cell Counting Kit-8 and a wound healing scratch assay, respectively. Production of nitric oxide (NO) and reactive oxygen species was measured by the nitrate reductase colorimetric method and flow cytometry, respectively. The expression of endothelial nitric oxide synthase (eNOS) mRNA was quantitated by reverse-transcriptase PCR. The expression of eNOS, p-AMPK, and NOX4 proteins was detected by Western blot. High concentrations of HbA(1c) reduced the viability and migration of HUVECs in a dose- and time-dependent manner. High concentrations of HbA(1c) inhibited production of NO but increased production of ROS. Incubation with increasing concentrations of HbA(1c) downregulated the expression of eNOS mRNA, decreased expression of eNOS and p-AMPK, and upregulated expression of NOX4. These findings provide direct evidence that HbA(1c) is involved in the development and progression of the cardiovascular complications of diabetes.

184 Increasing Insulin Sensitivity in the Endothelium Leads to Reduced Nitric Oxide Bioavailability

<h3>Introduction</h3> Insulin resistance is known to precede Type 2 diabetes (T2DM). Insulin mediated release of the endothelial cell (EC) derived anti-atherosclerotic molecule, nitric oxide (NO) is blunted in patients suffering from insulin resistance T2DM. We examined the effects of enhancing EC insulin sensitivity <i>in vivo</i>, by generating a novel transgenic mouse, over-expressing Type A human Insulin Receptor (HIRECO) restricted to EC. <h3>Methods</h3> Western blotting and RT-PCR were carried out on tissues and isolated endothelial cells from lungs to measure protein levels and mRNA expression, respectively. NADPH-dependent lucigenin-enhanced chemiluminescence was used to measure superoxide anion levels. Isolated thoracic aortic rings suspended in an organ bath were used to determine vasomotor functions. eNOS activity was examined by citrulline assay with ¹⁴C-labelled L-arginine. HIRECO were compared to wild type littermates. <h3>Results</h3> Over-expressing human insulin receptors in EC had no significant effect on morphological features, metabolic phenotype or blood pressure of HIRECO. HIRECO demonstrated significant EC dysfunction measured by a blunted endothelium-dependent vasorelaxation to acetylcholine and reduced basal NO release. EC-independent response to sodium nitroprusside remained unchanged. EC dysfunction observed in the organ bath was normalised by a NADPH oxidase-specific inhibitor peptide, gp91ds-tat as well as the superoxide dismutase mimetic, MnTmPyp. HIRECO demonstrated significant increase in superoxide anion release compared to WT littermates. This data was supported by a concomitant increase in NADPH oxidase isoform, NOX2 protein expression. Basal eNOS and Akt phosphorylation levels in isolated EC of HIRECO mice were enhanced compared to WT mice. Interestingly, insulin-stimulated eNOS phosphorylation and activation was decreased, whereas Akt phosphorylation remained unchanged. eNOS tyrosine phosphorylation mediated by proline-rich tyrosine kinase (PYK2) was significantly enhanced in EC from HIRECO mice. In order to investigate, if the perturbations of insulin signalling in the EC have a pathological outcome, HIRECO mice were crossed with ApoE-knockout mice. These mice demonstrated a significant enhancement of plaque formation in aorta, as well as the aortic root. <h3>Conclusions/implications</h3> These data show that increasing EC insulin sensitivity leads to reduced bioavailability of NO. These data demonstrate for the first time that increased insulin signalling in EC increases the generation of superoxide anion via activation of NOX₂ NADPH oxidase and reduced NO production in response to insulin due at least in part to increased EC PYK2 activity leading to a pro-atherosclerotic state.