Orthologs of NOX5 and EC-SOD/SOD3: dNox and dSod3 Impact Egg Hardening Process and Egg Laying in Reproductive Function of Drosophila melanogaster.

The occurrence of ovarian dysfunction is often due to the imbalance between the formation of reactive oxygen species (ROS) and the ineffectiveness of the antioxidative defense mechanisms. Primary sources of ROS are respiratory electron transfer and the activity of NADPH oxidases (NOX) while superoxide dismutases (SOD) are the main key regulators that control the levels of ROS and reactive nitrogen species intra- and extracellularly. Because of their central role SODs are the subject of research on human ovarian dysfunction but sample acquisition is low. The high degree of cellular and molecular similarity between Drosophila melanogaster ovaries and human ovaries provides this model organism with the best conditions for analyzing the role of ROS during ovarian function. In this study we clarify the localization of the ROS-producing enzyme dNox within the ovaries of Drosophila melanogaster and by a tissue-specific knockdown we show that dNox-derived ROS are involved in the chorion hardening process. Furthermore, we analyze the dSod3 localization and show that reduced activity of dSod3 impacts egg-laying behavior but not the chorion hardening process.

Endothelial NOX5 Obliterates the Reno-Protective Effect of Nox4 Deletion by Promoting Renal Fibrosis via Activation of EMT and ROS-Sensitive Pathways in Diabetes.

Chronic hyperglycemia induces intrarenal oxidative stress due to the excessive production of reactive oxygen species (ROS), leading to a cascade of events that contribute to the development and progression of diabetic kidney disease (DKD). NOX5, a pro-oxidant NADPH oxidase isoform, has been identified as a significant contributor to renal ROS in humans. Elevated levels of renal ROS contribute to endothelial cell dysfunction and associated inflammation, causing increased endothelial permeability, which can disrupt the renal ecosystem, leading to progressive albuminuria and renal fibrosis in DKD. This study specifically examines the contribution of endothelial cell-specific human NOX5 expression in renal pathology in a transgenic mouse model of DKD. This study additionally compares NOX5 with the previously characterized NADPH oxidase, NOX4, in terms of their relative roles in DKD. Regardless of NOX4 pathway, this study found that endothelial cell-specific expression of NOX5 exacerbates renal injury, albuminuria and fibrosis. This is attributed to the activation of the endothelial mesenchymal transition (EMT) pathway via enhanced ROS formation and the modulation of redox-sensitive factors. These findings underscore the potential therapeutic significance of NOX5 inhibition in human DKD. The study proposes that inhibiting NOX5 could be a promising approach for mitigating the progression of DKD and strengthens the case for the development of NOX5-specific inhibitors as a potential therapeutic intervention.

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-β 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 β-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. (AU)

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.

Regorafenib induces NOX5-mediated endoplasmic reticulum stress and potentiates the anti-tumor activity of cisplatin in non-small cell lung cancer cells.

Lung cancer is one of the most commonly diagnosed cancers worldwide. Although cisplatin-based chemotherapy regimens serve a pivotal role in non-small cell lung cancer (NSCLC) treatment, drug resistance and serious side effects limited its further clinical application. Regorafenib, a small-molecule multi-kinase inhibitor, was demonstrated to have promising anti-tumor activity in various solid tumors. In the present study, we found that regorafenib markedly enhanced cisplatin-induced cytotoxicity in lung cancer cells by activating reactive oxygen species (ROS)-mediated endoplasmic reticulum stress (ER Stress), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways. Regorafenib increased ROS generation by promoting NADPH oxidase 5 (NOX5) expression, and knocking down NOX5 attenuated ROS-mediated cytotoxicity of regorafenib in lung cancer cells. Additionally, mice xenograft model validated that synergistic anti-tumor effects of combined treatment with regorafenib and cisplatin. Our results suggested that combination therapy with regorafenib and cisplatin may serve as a potential therapeutic strategy for some NSCLC patients.

Functional interactions between NADPH oxidase 5 and actin.

NADPH oxidase 5 (NOX5) is a transmembrane oxidative signaling enzyme which produces superoxide in response to intracellular calcium flux. Increasing evidence indicates that NOX5 is involved in a variety of physiological processes as well as human disease, however, details of NOX5 signaling pathways and targets of NOX5 mediated oxidative modifications remain poorly resolved. Actin dynamics have previously been shown to be modulated by oxidative modification, however, a direct connection to NOX5 expression and activity has not been fully explored. Here we show that NOX5 and actin interact in the cell, and each modulate the activity of the other. Using actin effector molecules jasplakinolide, cytochalasin D and latrunculin A, we show that changes in actin dynamics affect NOX5 superoxide production. In tandem, NOX5 oxidatively modifies actin, and shifts the ratio of filamentous to monomeric actin. Finally, we show that knockdown of NOX5 in the pancreatic cancer cell line PSN-1 impairs cell migration. Together our findings indicate an important link between actin dynamics and oxidative signaling through NOX5.

Ca2+ -binding-region-dependent cell surface localization of NADPH oxidase Nox5.

Six gene splice variants of superoxide-generating NADPH oxidase 5 (Nox5) have been identified in humans, and they differ in the sequence of their N-terminal cytoplasmic domains, which comprise four EF-hand motifs. Here, we demonstrated that the Ca2+ -dependent association and dissociation between the N- and C-terminal cytoplasmic domains of the Nox5ß variant are affected by the alanine substitution of the conserved Ile-113 or Leu-115 at the connecting loop between the third and fourth EF-hand motifs. These substitutions impair the cell surface localization of Nox5ß. In addition, the Nox5ε/S variant, lacking all EF-hand motifs, does not localize to the plasma membrane. Thus, the Ca2+ -sensitive intramolecular interaction determines the Nox5 subcellular localization, that is, whether Nox5 variants generate superoxide in the extracellular or intracellular space.

NOX2 and NOX5 are increased in cardiac microvascular endothelium of deceased COVID-19 patients.

BACKGROUND: Cardiac injury and inflammation are common findings in COVID-19 patients. Autopsy studies have revealed cardiac microvascular endothelial damage and thrombosis in COVID-19 patients, indicative of microvascular dysfunction in which reactive oxygen species (ROS) may play a role. We explored whether the ROS producing proteins NOX2, NOX4 and NOX5 are involved in COVID-19-induced cardio-microvascular endothelial dysfunction. METHODS: Heart tissue were taken from the left (LV) and right (RV) ventricle of COVID-19 patients (n = 15) and the LV of controls (n = 14) at autopsy. The NOX2-, NOX4-, NOX5- and Nitrotyrosine (NT)-positive intramyocardial blood vessels fractions were quantitatively analyzed using immunohistochemistry. RESULTS: The LV NOX2+, NOX5+ and NT+ blood vessels fractions in COVID-19 patients were significantly higher than in controls. The fraction of NOX4+ blood vessels in COVID-19 patients was comparable with controls. In COVID-19 patients, the fractions of NOX2+, NOX5+ and NT+ vessels did not differ significantly between the LV and RV, and correlated positively between LV and RV in case of NOX5 (r = 0.710; p = 0.006). A negative correlation between NOX5 and NOX2 (r = -0.591; p = 0.029) and between NOX5 and disease time (r = -0.576; p = 0.034) was noted in the LV of COVID-19 patients. CONCLUSION: We show the induction of NOX2 and NOX5 in the cardiac microvascular endothelium in COVID-19 patients, which may contribute to the previously observed cardio-microvascular dysfunction in COVID-19 patients. The exact roles of these NOXes in pathogenesis of COVID-19 however remain to be elucidated.

NADPH Oxidase Isoforms in COPD Patients and Acute Cigarette Smoke-Exposed Mice: Induction of Oxidative Stress and Lung Inflammation.

Cigarette smoke (CS) is a major risk factor for chronic obstructive pulmonary disease (COPD), which represents the third leading cause of death worldwide. CS induces reactive oxygen species (ROS) production, leading to pulmonary inflammation and remodeling. NADPH oxidases (NOXs) represent essential sources of ROS production in the cardiovascular system. Whether and how NOX isoforms are activated in COPD patients and in response to acute cigarette smoke (ACS) remains incompletely understood. In the present study, the expression of NOX isoforms was examined in the lungs of end-stage COPD patients. In addition, mice silenced of NOX1 or NOX4 expression using in vivo RNA interference (RNAi), and NOX2-deficient (NOX2-/y) mice, were exposed to ACS for 1 h using a standard TE-10B smoking machine. In lung sections isolated from COPD patients undergoing lung transplantation, protein expression of NOX1, NOX2, NOX4, or NOX5 was markedly upregulated compared to non-smoking donor controls. Likewise, ACS upregulated protein expression of NOX1, NOX2, and NOX4, production of ROS, inflammatory cell infiltration, and mRNA expression of proinflammatory cytokines TNF-α and KC in the mouse lung. In vivo RNAi knockdown of NOX1 or NOX4 decreased ACS induced ROS production, inflammatory cell influx, and the expression of TNF-α and KC, which were accompanied by inhibition of the NF-κB-COX-2 axis. Although ACS induced ROS production was reduced in the lungs of NOX2-/y mice, inflammatory cell influx and expression of NF-κB/COX-2 were increased. Taken together, our results demonstrate for the first time that NOX isoforms 1, 2, 4 and 5 all remain activated in end-stage COPD patients, while NOX1 and NOX4 mediate oxidative stress and inflammatory responses in response to acute cigarette smoke. Therefore, targeting different isoforms of NOX might be necessary to treat COPD at different stages of the disease, which represents novel mechanistic insights enabling improved management of the devastating disease.

Impact of calcium and reactive oxygen species on human sperm function: Role of NOX5.

NOX5 is introduced as a new therapeutic target for infertility treatment. This study aimed to compare the basal and stimulated reactive oxygen species (ROS) production and sperm function in human teratozoospermic (n = 15) and normozoospermic (n = 17) semen samples following calcium overload and NOX5 activation. Washed spermatozoa incubated for 1 h under five various conditions: control group, adding a calcium ionophore A23187, phorbol myristate acetate (PMA), A23187 + PMA, and diphenylene iodonium (DPI) + A23187 + PMA. ROS generation was measured immediately after treatment for 30 min. Motility, viability, acrosome reaction, and apoptosis were evaluated after 1-h incubation. ROS production significantly increased when A23187 or PMA was added to the sperm medium. DPI had suppressive effects on ROS generation. Progressive and total motility significantly decreased following calcium elevation and NOX5 activation, which was somewhat returned by DPI. Necrotic and live cells in teratozoospermia was, respectively, higher and lower than normozoospermia samples. Incubation with A23187 significantly increased the percentage of early and late apoptosis. Teratozoosperm are more vulnerable than normal spermatozoa, and produce more basal and stimulated ROS. It seems that calcium overload induces apoptosis in spermatozoa and loss of viability through MPT pore opening and increased intracellular ROS.

Central role of c-Src in NOX5- mediated redox signalling in vascular smooth muscle cells in human hypertension.

AIMS: NOX-derived reactive oxygen species (ROS) are mediators of signalling pathways implicated in vascular smooth muscle cell (VSMC) dysfunction in hypertension. Among the numerous redox-sensitive kinases important in VSMC regulation is c-Src. However, mechanisms linking NOX/ROS to c-Src are unclear, especially in the context of oxidative stress in hypertension. Here, we investigated the role of NOX-induced oxidative stress in VSMCs in human hypertension focusing on NOX5, and explored c-Src, as a putative intermediate connecting NOX5-ROS to downstream effector targets underlying VSMC dysfunction. METHODS AND RESULTS: VSMC from arteries from normotensive (NT) and hypertensive (HT) subjects were studied. NOX1,2,4,5 expression, ROS generation, oxidation/phosphorylation of signalling molecules, and actin polymerization and migration were assessed in the absence and presence of NOX5 (melittin) and Src (PP2) inhibitors. NOX5 and p22phox-dependent NOXs (NOX1-4) were down-regulated using NOX5 siRNA and p22phox-siRNA approaches. As proof of concept in intact vessels, vascular function was assessed by myography in transgenic mice expressing human NOX5 in a VSMC-specific manner. In HT VSMCs, NOX5 was up-regulated, with associated oxidative stress, hyperoxidation (c-Src, peroxiredoxin, DJ-1), and hyperphosphorylation (c-Src, PKC, ERK1/2, MLC20) of signalling molecules. NOX5 siRNA reduced ROS generation in NT and HT subjects. NOX5 siRNA, but not p22phox-siRNA, blunted c-Src phosphorylation in HT VSMCs. NOX5 siRNA reduced phosphorylation of MLC20 and FAK in NT and HT. In p22phox- silenced HT VSMCs, Ang II-induced phosphorylation of MLC20 was increased, effects blocked by melittin and PP2. NOX5 and c-Src inhibition attenuated actin polymerization and migration in HT VSMCs. In NOX5 transgenic mice, vascular hypercontractilty was decreased by melittin and PP2. CONCLUSION: We define NOX5/ROS/c-Src as a novel feedforward signalling network in human VSMCs. Amplification of this system in hypertension contributes to VSMC dysfunction. Dampening the NOX5/ROS/c-Src pathway may ameliorate hypertension-associated vascular injury.

Nicotine promotes vascular calcification via intracellular Ca2+-mediated, Nox5-induced oxidative stress, and extracellular vesicle release in vascular smooth muscle cells.

AIMS: Smokers are at increased risk of cardiovascular events. However, the exact mechanisms through which smoking influences cardiovascular disease resulting in accelerated atherosclerosis and vascular calcification are unknown. The aim of this study was to investigate effects of nicotine on initiation of vascular smooth muscle cell (VSMC) calcification and to elucidate underlying mechanisms. METHODS AND RESULTS: We assessed vascular calcification of 62 carotid lesions of both smoking and non-smoking patients using ex vivo micro-computed tomography (µCT) scanning. Calcification was present more often in carotid plaques of smokers (n = 22 of 30, 73.3%) compared to non-smokers (n = 11 of 32, 34.3%; P < 0.001), confirming higher atherosclerotic burden. The difference was particularly profound for microcalcifications, which was 17-fold higher in smokers compared to non-smokers. In vitro, nicotine-induced human primary VSMC calcification, and increased osteogenic gene expression (Runx2, Osx, BSP, and OPN) and extracellular vesicle (EV) secretion. The pro-calcifying effects of nicotine were mediated by Ca2+-dependent Nox5. SiRNA knock-down of Nox5 inhibited nicotine-induced EV release and calcification. Moreover, pre-treatment of hVSMCs with vitamin K2 ameliorated nicotine-induced intracellular oxidative stress, EV secretion, and calcification. Using nicotinic acetylcholine receptor (nAChR) blockers α-bungarotoxin and hexamethonium bromide, we found that the effects of nicotine on intracellular Ca2+ and oxidative stress were mediated by α7 and α3 nAChR. Finally, we showed that Nox5 expression was higher in carotid arteries of smokers and correlated with calcification levels in these vessels. CONCLUSION: In this study, we provide evidence that nicotine induces Nox5-mediated pro-calcific processes as novel mechanism of increased atherosclerotic calcification. We identified that activation of α7 and α3 nAChR by nicotine increases intracellular Ca2+ and initiates calcification of hVSMCs through increased Nox5 activity, leading to oxidative stress-mediated EV release. Identifying the role of Nox5-induced oxidative stress opens novel avenues for diagnosis and treatment of smoking-induced cardiovascular disease.

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.

Expression of Endothelial NOX5 Alters the Integrity of the Blood-Brain Barrier and Causes Loss of Memory in Aging Mice.

Blood-Brain barrier (BBB) disruption is a hallmark of central nervous system (CNS) dysfunction, and oxidative stress is one of the molecular mechanisms that may underlie this process. NADPH oxidases (NOX) are involved in oxidative stress-mediated vascular dysfunction and participate in the pathophysiology of its target organs. The NADPH oxidase 5 (NOX5) isoform is absent in rodents, and although little is known about the role it may play in disrupting the BBB, it has recently been implicated in experimental stroke. Our aim was to investigate the role of NADPH oxidase 5 (NOX5) in promoting vascular alterations and to identify its impact on the cognitive status of aged mice. No differences were detected in the arterial blood pressure or body weight between knock-in mice expressing endothelial NOX5 and the control mice. The Morris water maze test showed memory impairments in the aged knock-in mice expressing NOX5 compared with their control littermates. For assessing the BBB integrity, we studied the protein expression of two tight junction (TJ) proteins: Zonula occludens-1 (ZO-1) and occludin. Compared to the control animals, Aged NOX5 mice exhibited reduced levels of both proteins, demonstrating an alteration of the BBB integrity. Our data indicate that vascular NOX5 may favor behavioral changes with aging through oxidative stress-mediated BBB breakdown.

Suppressed mitochondrial respiration via NOX5-mediated redox imbalance contributes to the antitumor activity of anlotinib in oral squamous cell carcinoma.

Anlotinib, a novel multitarget tyrosine kinase inhibitor, has shown promising results in the management of various carcinomas. This study aimed to investigate the antitumor activity of anlotinib in oral squamous cell carcinoma (OSCC) and the underlying molecular mechanism. A retrospective clinical study revealed that anlotinib improved the median progression-free survival (mPFS) and median overall survival (mOS) of patients with recurrent and metastatic (R/M) OSCC, respectively. Functional studies revealed that anlotinib markedly inhibited in vitro proliferation of OSCC cells and impeded in vivo tumor growth of OSCC patient-derived xenograft models. Mechanistically, RNA-sequencing identified that oxidative stress, oxidative phosphorylation and AKT/mTOR signaling were involved in anlotinib-treated OSCC cells. Anlotinib upregulated NADPH oxidase 5 (NOX5) expression, elevated reactive oxygen species (ROS) production, impaired mitochondrial respiration, and promoted apoptosis. Moreover, anlotinb also inhibited phospho-Akt (p-AKT) expression and elevated p-eIF2α expression in OSCC cells. NOX5 knockdown attenuated these inhibitory effects and cytotoxicity in anlotinib-treated OSCC cells. Collectively, we demonstrated that anlotinib monotherapy demonstrated favorable anticancer activity and manageable toxicities in patients with R/M OSCC. The antitumor activity of anlotinib in OSCC may be mainly involved in the suppression of mitochondrial respiration via NOX5-mediated redox imbalance and the AKT/eIF2α pathway.

Lysophosphatidylcholine induces oxidative stress in human endothelial cells via NOX5 activation - implications in atherosclerosis.

OBJECTIVE: The mechanisms involved in NOX5 activation in atherosclerotic processes are not completely understood. The present study tested the hypothesis that lysophosphatidylcholine (LPC), a proatherogenic component of oxLDL, induces endothelial calcium influx, which drives NOX5-dependent reactive oxygen species (ROS) production, oxidative stress, and endothelial cell dysfunction. APPROACH: Human aortic endothelial cells (HAEC) were stimulated with LPC (10-5 M, for different time points). Pharmacological inhibition of NOX5 (Melittin, 10-7 M) and NOX5 gene silencing (siRNA) was used to determine the role of NOX5-dependent ROS production in endothelial oxidative stress induced by LPC. ROS production was determined by lucigenin assay and electron paramagnetic spectroscopy (EPR), calcium transients by Fluo4 fluorimetry, and NOX5 activity and protein expression by pharmacological assays and immunoblotting, respectively. RESULTS: LPC increased ROS generation in endothelial cells at short (15 min) and long (4 h) stimulation times. LPC-induced ROS was abolished by a selective NOX5 inhibitor and by NOX5 siRNA. NOX1/4 dual inhibition and selective NOX1 inhibition only decreased ROS generation at 4 h. LPC increased HAEC intracellular calcium, important for NOX5 activation, and this was blocked by nifedipine and thapsigargin. Bapta-AM, selective Ca2+ chelator, prevented LPC-induced ROS production. NOX5 knockdown decreased LPC-induced ICAM-1 mRNA expression and monocyte adhesion to endothelial cells. CONCLUSION: These results suggest that NOX5, by mechanisms linked to increased intracellular calcium, is key to early LPC-induced endothelial oxidative stress and pro-inflammatory processes. Since these are essential events in the formation and progression of atherosclerotic lesions, the present study highlights an important role for NOX5 in atherosclerosis.

[Role of p22phox and NOX5 in autophagy and apoptosis of osteoblasts induced by hypoxia].

OBJECTIVE: To investigate the role of p22phox and NOX5 in autophagy and apoptosis of osteoblasts induced by hypoxia. METHODS: The skull tissue of newborn rats was cut into small pieces, and the osteoblasts were separated and purified by the tissue block adherent method and the differential adherent method. The first generation cells were harvested and identified by HE staining, Alizarin red staining, alkaline phosphatase (ALP) staining, and flow cytometry. A three-gas incubator was used to prepare a hypoxia model of osteoblasts. At 0, 3, 6, 12, and 24 hours of hypoxia, the expressions of p22phox, NOX5, and LC3Ⅱ/Ⅰ were detected by Western blot, and the level of reactive oxygen species (ROS) and cell apoptosis rate were detected by flow cytometry. And the time point of the highest level of ROS was selected as the hypoxia time point for subsequent experiments. The first generation osteoblasts were divided into normal group, si-p22phox hypoxia group, and si-NOX5 hypoxia group and subjected to corresponding transfection and hypoxia treatment. The inhibition efficiency of si-p22phox and si-NOX5 were detected by RT-PCR. Then the osteoblasts were divided into normal group, si-NC hypoxia group, si-p22phox hypoxia group, and si-NOX5 hypoxia group. After transfection and hypoxia treatment, Western blot was used to detect the expressions of p22phox, NOX5, autophagy-related proteins (LC3Ⅱ/Ⅰ, Beclin), and apoptosis-related proteins (Bcl-2, Bax), and flow cytometry was used to detect the cell apoptosis rate and level of ROS. The first generation osteoblasts were divided into a hypoxia group for 12 hours (hypoxia group) and a group that simultaneously inhibited si-p22phox and si-NOX5 and hypoxia for 12 hours (inhibition+hypoxia group). The expressions of Beclin and Bax were observed by immunofluorescence staining after the corresponding treatment. RESULTS: After identification, the isolated cells were osteoblasts. After hypoxia treatment, the relative expressions of p22phox, NOX5, and LC3Ⅱ/Ⅰ proteins and the apoptosis rate of osteoblasts gradually increased ( P<0.05), and the level of ROS also significantly increased ( P<0.05) and reached the peak value at 12 hours. The 12-hour hypoxia model was selected for subsequent experiments. Silencing the p22phox gene did not affect the expression of NOX5, and silencing the NOX5 gene did not affect the expression of p22phox. Compared with hypoxia treatment, the relative expressions of LC3Ⅱ/Ⅰ, Beclin, and Bax proteins after inhibiting the expression of p22phox or NOX5 gene significantly decreased ( P<0.05), the relative expression of Bcl-2 protein significantly increased ( P<0.05), the cell apoptosis rate and level of ROS also significantly decreased ( P<0.05). After silencing the expressions of p22phox and NOX5 genes at the same time, the immunofluorescence staining showed that the fluorescence of Beclin and Bax were weak. CONCLUSION: Inhibiting the expressions of p22phox and NOX5 genes can reduce the level of ROS in osteoblasts under hypoxia-induced conditions, and at the same time reduce autophagy and apoptosis, especially attenuate the excessive apoptosis of cells in the early to late stages, and strengthen the hypoxic osteoblasts proliferation.

Nitric oxide and heme-NO stimulate superoxide production by NADPH oxidase 5.

Nitric oxide (NO) is a ubiquitous cell signaling molecule which mediates widespread and diverse processes in the cell. These NO dependent effects often involve activation (e.g. NO binding to the heme group of soluble guanylyl cyclase for cGMP production) or inactivation (e.g. S-nitrosation) of protein targets. We studied the effect of NO and heme-NO on the transmembrane signaling enzyme NADPH oxidase 5 (NOX5), a heme protein which produces superoxide in response to increases in intracellular calcium. We found that treatment with NO donors increases NOX5 activity through heme-dependent effects, and that this effect could be recapitulated by the addition of heme-NO. This work adds to our understanding of NOX5 regulation in the cell but also provides a framework for understanding how NO could cause widespread changes in hemeprotein activity based on different affinities for heme v. heme-NO, and helps explain the opposing roles NO plays in activation and inactivation of hemeprotein targets.

NADPH Oxidase 5 and Melatonin: Involvement in Ram Sperm Capacitation.

Reactive oxygen species (ROS) play an essential role in mammalian sperm capacitation. NADPH oxidase 5 (NOX5) has been described as the main source of ROS production in some mammalian spermatozoa, such as human and equine. On the other hand, melatonin can decrease cellular ROS levels and regulates NOX activity in somatic cells. Therefore, the objectives of this work were (1) to identify NOX5 in ram spermatozoa and analyze its possible changes during in vitro capacitation and (2) to investigate the effect of melatonin on NOX5 expression and localization and on superoxide levels in capacitated ram spermatozoa. Protein bands associated with NOX5 were detected by Western blot analysis. Likewise, indirect immunofluorescence (IIF) revealed six different immunotypes for NOX5, which varied throughout in vitro capacitation. Superoxide (O2 ⋅-), evaluated by DHE/Yo-Pro-1, rose after in vitro capacitation and in the presence of the calcium ionophore A23187 but decreased in the presence of the NOX inhibitor GKT136901. GKT also reduced the percentage of capacitated and acrosome-reacted spermatozoa that had increased during incubation in capacitating conditions. The presence of melatonin at micromolar concentrations avoided the increment in O2 ⋅- and the changes in NOX5 immunotypes provoked by capacitation. In conclusion, NOX5 is present in ram spermatozoa and the changes in its distribution, associated with sperm capacitation, can be prevented by melatonin. To this extent, it could imply that melatonin exerts its antioxidant role, at least in part, by modulating NOX5 activity during ram sperm capacitation.

NOX5 is expressed aberrantly but not a critical pathogenetic gene in Hirschsprung disease.

BACKGROUND: Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of intramural ganglion cells in the distal gastrointestinal tract (GI), which results in tonic contraction of the aganglionic gut segment and functional intestinal obstruction. Recent studies have suggested NADPH oxidase 5 (NOX5) as a candidate risk gene for HSCR. In this study, we examined the function of NOX5 to verify its role in the development of the enteric nervous system (ENS). METHODS: HSCR tissue specimens (n = 10) were collected at the time of pull-through surgery and control specimens (n = 10) were obtained at the time of colostomy closure in patients. The NOX5 expression in aganglionic and ganglionic segments of HSCR colon and normal colon were analyzed by immunohistochemistry (IHC), western blot and real-time quantitative PCR (qPCR). The gene expression levels and spatiotemporal expression spectrum of NOX5 in different development stages of zebrafish embryo were determined using qPCR and in-situ hybridization (ISH). The enteric nervous system in NOX5 Morpholino (MO) knockdown and wild type (WT) zebrafish embryo was analyzed by whole-mount immunofluorescence (IF). Intestinal transit assay was performed to analyze the gastrointestinal motility in NOX5 knockdown and control larvae. RESULTS: NOX5 is strongly expressed in the ganglion cells in the proximal segment of HSCR colons and all segments of normal colons. Moreover, the expression of NOX5 is markedly decreased in the aganglionic segment of HSCR colon compared to the ganglionic segment. In zebrafish, NOX5 mRNA level is the highest in the one cell stage embryos and it is decreased overtime with the development of the embryos. Interestingly, the expression of NOX5 appears to be enriched in the nervous system. However, the number of neurons in the GI tract and the GI motility were not affected upon NOX5 knockdown. CONCLUSIONS: Our study shows that NOX5 markedly decreased in the aganglionic segment of HSCR but didn't involve in the ENS development of zebrafish. It implies that absence of intestinal ganglion cells may lead to down-regulation of NOX5.