Structural basis of human NOX5 activation.

NADPH oxidase 5 (NOX5) catalyzes the production of superoxide free radicals and regulates physiological processes from sperm motility to cardiac rhythm. Overexpression of NOX5 leads to cancers, diabetes, and cardiovascular diseases. NOX5 is activated by intracellular calcium signaling, but the underlying molecular mechanism of which - in particular, how calcium triggers electron transfer from NADPH to FAD - is still unclear. Here we capture motions of full-length human NOX5 upon calcium binding using single-particle cryogenic electron microscopy (cryo-EM). By combining biochemistry, mutagenesis analyses, and molecular dynamics (MD) simulations, we decode the molecular basis of NOX5 activation and electron transfer. We find that calcium binding to the EF-hand domain increases NADPH dynamics, permitting electron transfer between NADPH and FAD and superoxide production. Our structural findings also uncover a zinc-binding motif that is important for NOX5 stability and enzymatic activity, revealing modulation mechanisms of reactive oxygen species (ROS) production.

Proteasome inhibitors induce apoptosis by superoxide anion generation via NADPH oxidase 5 in human neuroblastoma SH-SY5Y cells.

The ubiquitin-proteasome system (UPS) is a major proteolytic system that plays an important role in the regulation of various cell processes, such as cell cycle, stress response, and transcriptional regulation, especially in neurons, and dysfunction of UPS is considered to be a cause of neuronal cell death in neurodegenerative diseases. However, the mechanism of neuronal cell death caused by UPS dysfunction has not yet been fully elucidated. In this study, we investigated the mechanism of neuronal cell death induced by proteasome inhibitors using human neuroblastoma SH-SY5Y cells. Z-Leu-D-Leu-Leu-al (MG132), a proteasome inhibitor, induced apoptosis in SH-SY5Y cells in a concentration- and time-dependent manner. Antioxidants N-acetylcysteine and EUK-8 attenuated MG132-induced apoptosis. Apocynin and diphenyleneiodonium, inhibitors of NADPH oxidase (NOX), an enzyme that produces superoxide anions, also attenuated MG132-induced apoptosis. It was also found that MG132 treatment increased the expression of NOX5, a NOX family member, and that siRNA-mediated silencing of NOX5 and BAPTA-AM, which inhibits NOX5 by chelating calcium, suppressed MG132-induced apoptosis and production of reactive oxygen species in SH-SY5Y cells. These results suggest that MG132 induces apoptosis in SH-SY5Y cells through the production of superoxide anion by NOX5.

Endothelial NOX5 overexpression induces changes in the cardiac gene profile: potential impact in myocardial infarction?

Cardiovascular diseases and the ischemic heart disease specifically constitute the main cause of death worldwide. The ischemic heart disease may lead to myocardial infarction, which in turn triggers numerous mechanisms and pathways involved in cardiac repair and remodeling. Our goal in the present study was to characterize the effect of the NADPH oxidase 5 (NOX5) endothelial expression in healthy and infarcted knock-in mice on diverse signaling pathways. The mechanisms studied in the heart of mice were the redox pathway, metalloproteinases and collagen pathway, signaling factors such as NFκB, AKT or Bcl-2, and adhesion molecules among others. Recent studies support that NOX5 expression in animal models can modify the environment and predisposes organ response to harmful stimuli prior to pathological processes. We found many alterations in the mRNA expression of components involved in cardiac fibrosis as collagen type I or TGF-ß and in key players of cardiac apoptosis such as AKT, Bcl-2, or p53. In the heart of NOX5-expressing mice after chronic myocardial infarction, gene alterations were predominant in the redox pathway (NOX2, NOX4, p22phox, or SOD1), but we also found alterations in VCAM-1 and ß-MHC expression. Our results suggest that NOX5 endothelial expression in mice preconditions the heart, and we propose that NOX5 has a cardioprotective role. The correlation studies performed between echocardiographic parameters and cardiac mRNA expression supported NOX5 protective action.

Structure, regulation, and physiological functions of NADPH oxidase 5 (NOX5).

NOX5 is the last member of the NADPH oxidase (NOXs) family to be identified and presents some specific characteristics differing from the rest of the NOXs. It contains four Ca2+ binding domains at the N-terminus and its activity is regulated by the intracellular concentration of Ca2+. NOX5 generates superoxide (O2•-) using NADPH as a substrate, and it modulates functions related to processes in which reactive oxygen species (ROS) are involved. Those functions appear to be detrimental or beneficial depending on the level of ROS produced. For example, the increase in NOX5 activity is related to the development of various oxidative stress-related pathologies such as cancer, cardiovascular, and renal diseases. In this context, pancreatic expression of NOX5 can negatively alter insulin action in high-fat diet-fed transgenic mice. This is consistent with the idea that the expression of NOX5 tends to increase in response to a stimulus or a stressful situation, generally causing a worsening of the pathology. On the other hand, it has also been suggested that it might have a positive role in preparing the body for metabolic stress, for example, by inducing a protective adipose tissue adaptation to the excess of nutrients supplied by a high-fat diet. In this line, its endothelial overexpression can delay lipid accumulation and insulin resistance development in obese transgenic mice by inducing the secretion of IL-6 followed by the expression of thermogenic and lipolytic genes. However, as NOX5 gene is not present in rodents and human NOX5 protein has not been crystallized, its function is still poorly characterized and further extensive research is required.

NOX5 mediates the crosstalk between tumor cells and cancer-associated fibroblasts via regulating cytokine network.

Activation of cancer-associated fibroblasts (CAFs) is a crucial feature for tumor malignancy. The reciprocal interplay between tumor cells and CAFs not only facilitates tumor progression and metastasis but also sustains the tumor-promoting function of CAFs. Nevertheless, how tumor cells readily adapt to these functional CAFs is still unclear. NADPH oxidase 5 (NOX5) is a strong reactive oxygen species producer overexpressed in esophageal squamous cell carcinoma (ESCC) cells. In this study, we showed that NOX5-positive ESCC cells induced normal fibroblasts (NFs) or adipose-derived mesenchymal stem cells (MSCs) to express the marker of CAFs-α smooth muscle actin. Moreover, these tumor cells reprogrammed the cytokine profile of the activated CAFs, which further stimulated NFs or MSCs to CAFs and induced lymphangiogenesis to facilitate ESCC malignancy. NOX5 activated intratumoral Src/nuclear factor-κB signaling to stimulate secretion of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and lactate from tumor cells. Subsequently, TNF-α, IL-1ß, and lactate activated CAFs, and facilitated the secretion of IL-6, IL-7, IL-8, CCL5, and transforming growth factor-ß1 from CAFs. These CAFs-derived cytokines reciprocally induced the progression of NOX5-positive ESCC cells. Our findings together indicate that NOX5 serves as the driving oncoprotein to provide a niche that is beneficial for tumor malignant progression.

NADPH oxidase 5 has a crucial role in cellular motility of colon cancer cells.

NADPH oxidases (NOXs) are a family of transmembrane proteins that generate reactive oxygen species. It was previously reported that patients with colon cancer who had high NOX5 expression had poor prognosis. However, no studies have investigated the cellular functions of NOX5 in colon cancer. The present study aimed to clarify the relationship between NOX5 and cancer development using an in vitro model. Reverse transcription­quantitative PCR was performed to determine the NOX5 expression levels of colon cancer cell lines. NOX5­knockdown experiments were conducted, and the effect on cell proliferation, migration, and invasion were analyzed. In addition, mRNA microarray was conducted to assess changes in gene profile. NOX5 mRNA expression was high in HCT116 cells and moderate in SW48 cells. NOX5 knockdown significantly inhibited cell migration and invasion in both HCT116 and SW48 cells; however, NOX5 knockdown reduced cell proliferation in only HCT116 cells. mRNA microarrays revealed a strong relationship between NOX5 expression levels and integrin­linked kinase signaling pathways. The NOX5 expression in colon cancer cells affected cancer progression, especially cell motility. NOX5 may be a novel therapeutic target for the future development of treatments for colon cancer.

An Integrative Gene Expression and Mathematical Flux Balance Analysis Identifies Targetable Redox Vulnerabilities in Melanoma Cells.

Melanomas harboring BRAF mutations can be treated with BRAF inhibitors (BRAFi), but responses are varied and tumor recurrence is inevitable. Here we used an integrative approach of experimentation and mathematical flux balance analyses in BRAF-mutated melanoma cells to discover that elevated antioxidant capacity is linked to BRAFi sensitivity in melanoma cells. High levels of antioxidant metabolites in cells with reduced BRAFi sensitivity confirmed this conclusion. By extending our analyses to other melanoma subtypes in The Cancer Genome Atlas, we predict that elevated redox capacity is a general feature of melanomas, not previously observed. We propose that redox vulnerabilities could be exploited for therapeutic benefits and identify unsuspected combination targets to enhance the effects of BRAFi in any melanoma, regardless of mutational status. SIGNIFICANCE: An integrative bioinformatics, flux balance analysis, and experimental approach identify targetable redox vulnerabilities and show the potential for modulation of cancer antioxidant defense to augment the benefits of existing therapies in melanoma.

Epoxyazadiradione exhibit activities in head and neck squamous cell carcinoma by targeting multiple pathways.

The head and neck squamous cell carcinoma (HNSCC) constitute about 90% of all head and neck cancers. HNSCC falls in the top 10 cancers in men globally. Epoxyazadiradione (EPA) and Azadiradione (AZA) are the limonoids derived from the medicinal plant Azadirachta indica (popularly known as Neem). Whether or not the limonoids exhibit activities against HNSCC and the associated mechanism remains elusive. Herein, we demonstrate that EPA exhibits stronger activity in HNSCC in comparison to AZA. The limonoids obeyed the Lipinski's rule of 5. EPA exhibited activities in a variety of HNSCC lines like suppression of the proliferation and the induction of apoptosis. The limonoid suppressed the level of proteins associated with anti-apoptosis (survivin, Bcl-2, Bcl-xL), proliferation (cyclin D1), and invasion (MMP-9). Further, the expression of proapoptotic Bax and caspase-9 cleavage was induced by the limonoid. Exposure of EPA induced reactive oxygen species (ROS) generation in the FaDu cells. N-acetyl-L-cysteine (ROS scavenger) abrogated the down-regulation of tumorigenic proteins caused by EPA exposure. EPA induced NOX-5 while suppressing the expression of programmed death-ligand 1 (PD-L1). Further, hydrogen peroxide induced NF-κB-p65 nuclear translocation and EPA inhibited the translocation. Finally, EPA modulated the expression of lncRNAs in HNSCC lines. Overall, these results have shown that EPA exhibit activities against HNSCC by targeting multiple cancer related signalling molecules. Currently, we are evaluating the efficacy of this molecule in mice models.

Membranous NOX5-derived ROS oxidizes and activates local Src to promote malignancy of tumor cells.

Reactive oxygen species (ROS) localized at the precise subcellular compartments are essential for regulating the activity of signaling proteins. Furthermore, ROS are master regulators of tumor malignant progression that respond to a diverse set of environmental stress, especially hypoxia. NADPH oxidases (NOXs) appear to be activated within discrete subcellular compartments to facilitate local ROS production. However, the subcellular function of NOXs in hypoxic tumor is still unclear. In this study, we demonstrated that NOX5 was greatly upregulated in clinical esophageal squamous cell carcinoma (ESCC) tumors, ESCC cell lines or primary ESCC cells, and elevated NOX5 was correlated to malignancy of ESCC tumors and poor prognosis. NOX5 induced the malignant progression of ESCC by activating Src, especially under hypoxic condition. Mechanistically, we showed that hypoxia promoted the interaction between NOX5 and Pyk2 on cell membrane via facilitating Ca2+-mediated Pyk2 Tyr402 site phosphorylation. Subsequently, Pyk2 acted as a scaffold for c-Abl phosphorylating the catalytic domain of NOX5 Tyr476/478 sites, which in turn upregulated hydrogen peroxide (H2O2) inside the Pyk2/NOX5 complex to oxidize and activate local Src. These findings provide insights into the biological significance of NOX5 in the development of ESCC.

Induction of Cyclooxygenase-2 by Overexpression of the Human NADPH Oxidase 5 (NOX5) Gene in Aortic Endothelial Cells.

Oxidative stress is a main molecular mechanism that underlies cardiovascular diseases. A close relationship between reactive oxygen species (ROS) derived from NADPH oxidase (NOX) activity and the prostaglandin (PG) biosynthesis pathway has been described. However, little information is available about the interaction between NOX5 homolog-derived ROS and the PG pathway in the cardiovascular context. Our main goal was to characterize NOX5-derived ROS effects in PG homeostasis and their potential relevance in cardiovascular pathologies. For that purpose, two experimental systems were employed: an adenoviral NOX5-ß overexpression model in immortalized human aortic endothelial cells (TeloHAEC) and a chronic infarction in vivo model developed from a conditional endothelial NOX5 knock-in mouse. NOX5 increased cyclooxygenase-2 isoform (COX-2) expression and prostaglandin E2 (PGE2) production through nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in TeloHAEC. Protein kinase C (PKC) activation and intracellular calcium level (Ca++) mobilization increased ROS production and NOX5 overexpression, which promoted a COX-2/PGE2 response in vitro. In the chronic infarction model, mice encoding endothelial NOX5 enhanced the cardiac mRNA expression of COX-2 and PGES, suggesting a COX-2/PGE2 response to NOX5 presence in an ischemic situation. Our data support that NOX5-derived ROS may modulate the COX-2/PGE2 axis in endothelial cells, which might play a relevant role in the pathophysiology of heart infarction.

Inhibition of NADPH Oxidase 5 (NOX5) Suppresses High Glucose-Induced Oxidative Stress, Inflammation and Extracellular Matrix Accumulation in Human Glomerular Mesangial Cells.

BACKGROUND The aim of this study was to explore the effects of NADPH oxidase 5 (NOX5) in high glucose-stimulated human glomerular mesangial cells (HMCs). MATERIAL AND METHODS Cells were cultured under normal glucose (NG) or high glucose (HG) conditions. Then, NOX5 siRNA was transfected into HG-treated HMCs. A Cell Counting Kit-8 assay, colony formation assay and 5-ethynyl-20-deoxyuridine (EDU) incorporation assay were applied to measure cell proliferative ability. In addition, the levels of oxidative stress factors including reactive oxygen species (ROS), malonaldehyde (MDA), NADPH, superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX), inflammatory cytokines including tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, IL-1ß, and monocyte chemoattractant protein-1 (MCP-1) in HMCs were detected by kits. Moreover, the expression of TLR4/NF-kappaB signaling and extracellular matrix (ECM) associated genes were evaluated by western blotting. RESULTS The results revealed that the NOX5 was overexpressed in HG-treated HMCs. Silencing of NOX5 decreased proliferation of HMCs induced by HG. And NOX5 silencing alleviated the production of MDA and NADPH accompanied by an increase of SOD and GSH-PX levels. Additionally, the contents of TNF-alpha, IL-6, IL-1ß, and MCP-1 were reduced after transfection with NOX5 siRNA. Furthermore, silencing of NOX5 deceased the expression of collagen I, collagen IV, TGF-ß1, and fibronectin induced by HG stimulation. TLR4, MyD88, and phospho-NF-kappaB p65 expression were downregulated notably in NOX5 silencing group. CONCLUSIONS Taken together, these findings demonstrated that inhibition of NOX5 attenuated HG-induced HMCs oxidative stress, inflammation, and ECM accumulation, suggesting that NOX5 may serve as a potential therapeutic target for diabetic nephropathy (DN) treatment.

Dopamine 1 receptor activation protects mouse diabetic podocytes injury via regulating the PKA/NOX-5/p38 MAPK axis.

Diabetic nephropathy (DN) is a major microvascular complication of diabetes that can lead to end-stage renal disease. Podocytes constitute the last barrier of glomerular filtration, whose damage are the direct cause of proteinuria. Dopamine receptors are involved in the regulation of diabetes-induced glomerular hyperfiltration, and only dopamine 1 receptor (D1R) can be amplified in cultured mouse podocytes. However, the exact effect of D1R on diabetic podocytes remains unclear. This study aims to investigate the protective role of D1R activation on diabetic podocytes injury in vivo and vitro as well as its potential mechanism. We observed D1R protective effect respectively in streptozotocin (STZ)-induced type 1 diabetes (T1D) mice as well as mouse podocytes (MPC5) cultured in high glucose (HG, 40 mM) medium. It showed that D1R and podocyte-associated proteins (Podocin, CD2AP and Nephrin) expression were significantly decreased both in the T1D mice (fed for 8 and 12 weeks) and HG-cultured MPC5 cells, while the NOX-5 expression increased. In T1D mice, the levels of 24-h urine protein, serum creatinine and urinary 8-OHdG were increased in a time-dependent manner, at the same time, hematoxylin-eosin (HE) staining and electron microscope observed the kidney lesion and podocytes injury. In vitro, HG induced podocytes oxidative stress and apoptosis, which could be inhibited by SKF38393 (a D1R agonist) and N-acetyl-l-cysteine (NAC, a reactive oxygen species scavenger). Furthermore, there was a decreasing Podocin expression and a significant increasing NOX-5 expression in podocytes transfected with D1R-small interfering RNA (siRNA). More importantly, the expression of phospho-CREB (the PKA downstream transcription factor) was decreased and phospho-p38 MAPK was increased in HG-induced podocytes, which can respectively be activated or blocked by SKF38393, 8-Bromo-CAMP (a PKA activator), NAC, and SB20380 (a p38 MAPK inhibitor). In conclusion, D1R activation can protect diabetic podocytes from apoptosis and oxidative damage, in part through the PKA/NOX-5/p38 MAPK pathway.

Beta-Cell-Specific Expression of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 5 Aggravates High-Fat Diet-Induced Impairment of Islet Insulin Secretion in Mice.

Aims: Nicotinamide adenine dinucleotide phosphate oxidases (NOX-es) produce reactive oxygen species and modulate ß-cell insulin secretion. Islets of type 2 diabetic subjects present elevated expression of NOX5. Here, we sought to characterize regulation of NOX5 expression in human islets in vitro and to uncover the relevance of NOX5 in islet function in vivo using a novel mouse model expressing NOX5 in doxycycline-inducible, ß-cell-specific manner (RIP/rtTA/NOX5 mice). Results:In situ hybridization and immunohistochemistry employed on pancreatic sections demonstrated NOX5 messenger ribonucleic acid (mRNA) and protein expressions in human islets. In cultures of dispersed islets, NOX5 protein was observed in somatostatin-positive (δ) cells in basal (2.8 mM glucose) conditions. Small interfering ribonucleic acid (siRNA)-mediated knockdown of NOX5 in human islets cultured in basal glucose concentrations resulted in diminished glucose-induced insulin secretion (GIIS) in vitro. However, when islets were preincubated in high (16.7 mM) glucose media for 12 h, NOX5 appeared also in insulin-positive (ß) cells. In vivo, mice with ß-cell NOX5 expression developed aggravated impairment of GIIS compared with control mice when challenged with 14 weeks of high-fat diet. Similarly, in vitro palmitate preincubation resulted in more severe reduction of insulin release in islets of RIP/rtTA/NOX5 mice compared with their control littermates. Decreased insulin secretion was most distinct in response to theophylline stimulation, suggesting impaired cyclic adenosine monophosphate (cAMP)-mediated signaling due to increased phosphodiesterase activation. Innovation and Conclusions: Our data provide the first insight into the complex regulation and function of NOX5 in islets implying an important role for NOX5 in δ-cell-mediated intraislet crosstalk in physiological circumstances but also identifying it as an aggravating factor in ß-cell failure in diabetic conditions.

Effect of Proton Pump Inhibitor Therapy on NOX5, mPGES1 and iNOS expression in Barrett's Esophagus.

Acid reflux may contribute to the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). However, it is not clear whether the molecular changes present in BE patients are reversible after proton pump inhibitor (PPI) treatment. In this study we examined whether PPI treatment affects NOX5, microsomal prostaglandin E synthase (mPGES)-1 and inducible nitric oxide synthase (iNOS) expression. We found that NADPH oxidase 5 (NOX5), mPGES-1 and iNOS were significantly increased in BE mucosa. One-month PPI treatment significantly decreased NOX5, mPGES1 and iNOS. In BAR-T cells, NOX5 mRNA and p16 promoter methylation increased after pulsed acid treatment in a time-dependent manner. Four or eight-week-acid induced increase in NOX5 mRNA, NOX5 protein and p16 methylation may be reversible. Twelve-week acid treatment also significantly increased NOX5, mPGES1 and iNOS mRNA expression. However, twelve-week-acid-induced changes only partially restored or did not recover at all after the cells were cultured at pH 7.2 for 8 weeks. We conclude that NOX5, mPGES1 and iNOS may be reversible after PPI treatment. Short-term acid-induced increase in NOX5 expression and p16 methylation might be reversible, whereas long-term acid-induced changes only partially recovered 8 weeks after removal of acid treatment.

Histone Acetyltransferase-Dependent Pathways Mediate Upregulation of NADPH Oxidase 5 in Human Macrophages under Inflammatory Conditions: A Potential Mechanism of Reactive Oxygen Species Overproduction in Atherosclerosis.

Histone acetylation plays a major role in epigenetic regulation of gene expression. Monocyte-derived macrophages express functional NADPH oxidase 5 (Nox5) that contributes to oxidative stress in atherogenesis. The mechanisms of Nox5 regulation are not entirely elucidated. The aim of this study was to investigate the expression pattern of key histone acetyltransferase subtypes (p300, HAT1) in human atherosclerosis and to determine their role in mediating the upregulation of Nox5 in macrophages under inflammatory conditions. Human nonatherosclerotic and atherosclerotic tissue samples were collected in order to determine the expression of p300 and HAT1 isoforms, H3K27ac, and Nox5. In vitro determinations were done on human macrophages exposed to lipopolysaccharide in the absence or presence of histone acetyltransferase inhibitors. Western blot, immunohistochemistry, immunofluorescence, real-time PCR, transfection, and chromatin immunoprecipitation assay were employed. The protein levels of p300 and HAT1 isoforms, H3K27ac, and Nox5 were found significantly elevated in human atherosclerotic specimens. Immunohistochemistry/immunofluorescence staining revealed that p300, HAT1, H3K27ac, H3K9ac, and Nox5 proteins were colocalized in the area of CD45+/CD68+ immune cells and lipid-rich deposits within human atherosclerotic plaques. Lipopolysaccharide induced the levels of HAT1, H3K27ac, H3K9ac, and Nox5 and the recruitment of p300 and HAT1 at the sites of active transcription within Nox5 gene promoter in cultured human macrophages. Pharmacological inhibition of histone acetyltransferase significantly reduced the Nox5 gene and protein expression in lipopolysaccharide-challenged macrophages. The overexpression of p300 or HAT1 enhanced the Nox5 gene promoter activity. The histone acetyltransferase system is altered in human atherosclerosis. Under inflammatory conditions, HAT subtypes control Nox5 overexpression in cultured human macrophages. The data suggest the existence of a new epigenetic mechanism underlying oxidative stress in atherosclerosis.

Clinical Significance of NADPH Oxidase 5 in Human Colon Cancer.

BACKGROUND/AIM: Recent studies have reported the involvement of NADPH oxidases (NOXs) in tumor progression. However, the role of NOX5 in colon cancer is unclear. We examined the clinical significance of NOX5 expression in colon cancer. PATIENTS AND METHODS: NOX5 expression was evaluated by immunohistochemistry in 119 patients with stage II or III colon cancer, and the relationship between NOX5 expression and clinicopathological data was analyzed. RESULTS: Of all tissues, 39.5% were negative and 60.5% were positive for NOX5 expression. Positive expression was significantly associated with undifferentiated histology (p=0.037) and lymph node metastasis (p=0.023). The 5-year progression-free survival rate of NOX5-positive patients was significantly worse than that of NOX5-negative patients (p=0.046). The rates of local recurrence observed in NOX5-positive patients were higher than that in NOX5-negative patients. CONCLUSION: NOX5 expression may be related to poor prognostic factors and could be useful as a prognostic biomarker.

Dihydrotanshinone-Induced NOX5 Activation Inhibits Breast Cancer Stem Cell through the ROS/Stat3 Signaling Pathway.

Cancer stem cells (CSCs) are known to mediate metastasis and recurrence and are therefore a promising therapeutic target. In this study, we found that dihydrotanshinone (DHTS) inhibits CSC formation. DHTS inhibited mammosphere formation in a dose-dependent manner and showed significant tumor growth inhibition in a xenograft model. This compound reduced the CD44high/CD24low- and aldehyde dehydrogenase- (ALDH-) expressing cell population and the self-renewal-related genes Nanog, SOX2, OCT4, C-Myc, and CD44. DHTS induced NOX5 activation by increasing calcium, and NOX5 activation induced reactive oxygen species (ROS) production. ROS production reduced the nuclear phosphorylation levels of Stat3 and secreted IL-6 levels in the mammospheres. DHTS deregulated the dynamic equilibrium from non-stem cancer cells to CSCs by dephosphorylating Stat3 and decreasing IL-6 secretion and inhibiting CSC formation. These novel findings showed that DHTS-induced ROS deregulated the Stat3/IL-6 pathway and induced CSC death. NOX5 activation by DHTS inhibits CSC formation through ROS/Stat3/IL-6 signaling, and DHTS may be a promising potential therapeutic agent against breast CSCs.

NOX5: Molecular biology and pathophysiology.

NEW FINDINGS: What is the topic of this review? This review provides a comprehensive overview of Nox5 from basic biology to human disease and highlights unique features of this Nox isoform What advances does it highlight? Major advances in Nox5 biology relate to crystallization of the molecule and new insights into the pathophysiological role of Nox5. Recent discoveries have unravelled the crystal structure of Nox5, the first Nox isoform to be crystalized. This provides new opportunities to develop drugs or small molecules targeted to Nox5 in an isoform-specific manner, possibly for therapeutic use. Moreover genome wide association studies (GWAS) identified Nox5 as a new blood pressure-associated gene and studies in mice expressing human Nox5 in a cell-specific manner have provided new information about the (patho) physiological role of Nox5 in the cardiovascular system and kidneys. Nox5 seems to be important in the regulation of vascular contraction and kidney function. In cardiovascular disease and diabetic nephropathy, Nox5 activity is increased and this is associated with increased production of reactive oxygen species and oxidative stress implicated in tissue damage. ABSTRACT: Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox), comprise seven family members (Nox1-Nox5 and dual oxidase 1 and 2) and are major producers of reactive oxygen species in mammalian cells. Reactive oxygen species are crucially involved in cell signalling and function. All Noxs share structural homology comprising six transmembrane domains with two haem-binding regions and an NADPH-binding region on the intracellular C-terminus, whereas their regulatory systems, mechanisms of activation and tissue distribution differ. This explains the diverse function of Noxs. Of the Noxs, NOX5 is unique in that rodents lack the gene, it is regulated by Ca2+ , it does not require NADPH oxidase subunits for its activation, and it is not glycosylated. NOX5 localizes in the perinuclear and endoplasmic reticulum regions of cells and traffics to the cell membrane upon activation. It is tightly regulated through numerous post-translational modifications and is activated by vasoactive agents, growth factors and pro-inflammatory cytokines. The exact pathophysiological significance of NOX5 remains unclear, but it seems to be important in the physiological regulation of sperm motility, vascular contraction and lymphocyte differentiation, and NOX5 hyperactivation has been implicated in cardiovascular disease, kidney injury and cancer. The field of NOX5 biology is still in its infancy, but with new insights into its biochemistry and cellular regulation, discovery of the NOX5 crystal structure and genome-wide association studies implicating NOX5 in disease, the time is now ripe to advance NOX5 research. This review provides a comprehensive overview of our current understanding of NOX5, from basic biology to human disease, and highlights the unique characteristics of this enigmatic Nox isoform.

BIAM switch assay coupled to mass spectrometry identifies novel redox targets of NADPH oxidase 4.

AIM: NADPH oxidase (Nox) -derived reactive oxygen species have been implicated in redox signaling via cysteine oxidation in target proteins. Although the importance of oxidation of target proteins is well known, the specificity of such events is often debated. Only a limited number of Nox-oxidized proteins have been identified thus far; especially little is known concerning redox-targets of the constitutively active NADPH oxidase Nox4. In this study, HEK293 cells with tetracycline-inducible Nox4 overexpression (HEK-tet-Nox4), as well as podocytes of WT and Nox4-/- mice, were utilized to identify Nox4-dependent redox-modified proteins. RESULTS: TGFß1 induced an elevation in Nox4 expression in podocytes from WT but not Nox4-/- mice. Using BIAM based redox switch assay in combination with mass spectrometry and western blot analysis, 142 proteins were identified as differentially oxidized in podocytes from wild type vs. Nox4-/- mice and 131 proteins were differentially oxidized in HEK-tet-Nox4 cells upon Nox4 overexpression. A predominant overlap was found for peroxiredoxins and thioredoxins, as expected. More interestingly, the GRB2-associated-binding protein 1 (Gab1) was identified as being differentially oxidized in both approaches. Further analysis using mass spectrometry-coupled BIAM switch assay and site directed mutagenesis, revealed Cys374 and Cys405 as the major Nox4 targeted oxidation sites in Gab1. INNOVATION & CONCLUSION: BIAM switch assay coupled to mass spectrometry is a powerful and versatile tool to identify differentially oxidized proteins in a global untargeted way. Nox4, as a source of hydrogen peroxide, changes the redox-state of numerous proteins. Of those, we identified Gab1 as a novel redox target of Nox4.

Association analysis of NOX5 polymorphisms with Hirschsprung disease.

BACKGROUND/PURPOSE: Hirschsprung disease (HSCR) is a developmental disease characterized by the absence of ganglion cells in the intestinal region. NADPH oxidase5 (NOX5) has been identified as one of the possible candidate genes for risk of Hirschsprung disease in our recent genome wide association study (GWAS). In this study, we performed a replication study to analyze the association of NOX5 polymorphisms with HSCR risk and conducted an extended analysis to investigate further associations for sub-groups and haplotypes. METHODS: A total of 23 NOX5 single nucleotide polymorphisms (SNPs) were genotyped in 187 HSCR patients and 283 unaffected controls. Statistical analysis was performed to examine the effects of genotype on risk of HSCR and HSCR subtype. RESULTS: Logistic regression analyses revealed that six SNPs (rs59355559, rs62010828, rs34990910, rs11856030, rs311905, and rs8024894) were associated with risk of HSCR (minimum p = 0.007 at rs62010828). Moreover, three SNPs (rs59355559, rs62010828, and rs8024894) were significantly associated with risk of long-segment HSCR (L-HSCR) subtype and 5 SNPs (rs59355559, rs62010828, rs34990910, rs11856030, and rs8024894) were found to be associated with risk of TCA subtype. CONCLUSION: Our results demonstrate that genetic variants in NOX5 have genetic effects on risk of HSCR, which may serve as useful preliminary information for further study. LEVELS OF EVIDENCE: Level III of prognosis study.