bims-noxint 2020-01-19 papers

bims-noxint

Biomed News

on NADPH oxidases in tumorigenesis

Issue of 2020–01–19
eight papers selected by
Laia Caja Puigsubira, Uppsala University

Strenuous Acute Exercise Induces Slow and Fast Twitch-Dependent NADPH Oxidase Expression in Rat Skeletal Muscle.
APX-115A, a pan-NADPH oxidase inhibitor, induces caspase-dependent cell death by suppressing NOX4-ROS signaling in EBV-infected retinal epithelial cells.
Nox4-mediated ROS production is involved, but not essential for TGFβ-induced lens EMT leading to cataract.
miR-124-5p/NOX2 Axis Modulates the ROS Production and the Inflammatory Microenvironment to Protect Against the Cerebral I/R Injury.
Beta-cell-specific expression of NOX5 aggravates high fat diet-induced impairment of islet insulin secretion in mice.
Non-phagocytic Activation of NOX2 is Implicated in Progressive Non-alcoholic Steatohepatitis During Aging.
Induction of HO-1 by Mevastatin Mediated via a Nox/ROS-Dependent c-Src/PDGFRα/PI3K/Akt/Nrf2/ARE Cascade Suppresses TNF-α-Induced Lung Inflammation.
NADPH oxidase controls pulmonary neutrophil infiltration in the response to fungal cell walls by limiting LTB4.

Antioxidants (Basel). 2020 Jan 08. pii: E57. [Epub ahead of print]9(1):

Strenuous Acute Exercise Induces Slow and Fast Twitch-Dependent NADPH Oxidase Expression in Rat Skeletal Muscle.

Juliana Osório Alves, Leonardo Matta Pereira, Igor Cabral Coutinho do Rêgo Monteiro, Luiz Henrique Pontes Dos Santos, Alex Soares Marreiros Ferraz, Adriano Cesar Carneiro Loureiro, Crystianne Calado Lima, José Henrique Leal-Cardoso, Denise Pires Carvalho, Rodrigo Soares Fortunato, Vânia Marilande Ceccatto.

  The enzymatic complex Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase (NOx) may be the principal source of reactive oxygen species (ROS). The NOX2 and NOX4 isoforms are tissue-dependent and are differentially expressed in slow-twitch fibers (type I fibers) and fast-twitch fibers (type II fibers) of skeletal muscle, making them different markers of ROS metabolism induced by physical exercise. The aim of this study was to investigate NOx signaling, as a non-adaptive and non-cumulative response, in the predominant fiber types of rat skeletal muscles 24 h after one strenuous treadmill exercise session. The levels of mRNA, reduced glycogen, thiol content, NOx, superoxide dismutase, catalase, glutathione peroxidase activity, and PPARGC1α and SLC2A4 gene expression were measured in the white gastrocnemius (WG) portion, the red gastrocnemius (RG) portion, and the soleus muscle (SOL). NOx activity showed higher values in the SOL muscle compared to the RG and WG portions. The same was true of the NOX2 and NOX4 mRNA levels, antioxidant enzymatic activities, glycogen content. Twenty-four hours after the strenuous exercise session, NOx expression increased in slow-twitch oxidative fibers. The acute strenuous exercise condition showed an attenuation of oxidative stress and an upregulation of antioxidant activity through PPARGC1α gene activity, antioxidant defense adaptations, and differential gene expression according to the predominant fiber type. The most prominent location of detoxification (indicated by NOX4 activation) in the slow-twitch oxidative SOL muscle was the mitochondria, while the fast-twitch oxidative RG portion showed a more cytosolic location. Glycolytic metabolism in the WG portion suggested possible NOX2/NOX4 non-regulation, indicating other possible ROS regulation pathways.

Keywords:  antioxidant defenses; glycolytic metabolism fibers; oxidative metabolism fibers; reactive oxygen species; skeletal muscle fibers

DOI:  https://doi.org/10.3390/antiox9010057
Curr Eye Res. 2020 Jan 17.

APX-115A, a pan-NADPH oxidase inhibitor, induces caspase-dependent cell death by suppressing NOX4-ROS signaling in EBV-infected retinal epithelial cells.

Seung-Woo Hong, Min Hye Noh, Yeong Seok Kim, Dong-Hoon Jin, Sung Hwan Moon, Jae Wook Yang, Dae Young Hur.

  Purpose: Epstein-Barr virus is a γ-herpes virus that infects primary B cells and can transform infected cells into immortalized lymphoblastoid cell lines (LCL). The role of EBV in malignancies such as Burkitt's lymphoma and nasopharyngeal carcinoma is well understood, however, its role in EBV-infected retinal cells remains poorly understood. Therefore, we investigated the effect of EBV on the growth of retinal cells.Methods: Previously, we established and reported a cell line model to address the relationship between EBV infection and retinal cell proliferation that used adult retinal pigment epithelium (ARPE-19) and EBV infection. To determine the effect of EBV on ARPE-19 cells, cell death was measured by propidium iodine/annexin V staining and reactive oxygen species (ROS) were measured by FACS, and protein expression was evaluated using western blot analysis. Also, downregulation of LMP1 and NADPH oxidase 4 (NOX4) expression was accomplished using siRNA technology.Results: We found that ROS were dramatically increased in EBV-infected ARPE19 cells (APRE19/EBV) relative to the parental cell line. Additionally, the expression level of NOX4, a main source of ROS, was upregulated by EBV infection. Interestingly, downregulation of LMP1, one of the EBV viral onco-proteins, completely decreased EBV-induced ROS accumulation and the upregulation of NOX4. Treatment with APX-115A, a pan-NOX inhibitor, induced apoptotic cell death of only the EBV-infected ARPE19 cells but not the parental cell line. Pretreatment with z-VAD, a pan-caspase inhibitor, inhibited NOX inhibitor-induced cell death in ARPE19/EBV cells. Furthermore, APX-115A-induced cell death mediated the activation of JNK and ERK. Finally, we confirmed the expression level of NOX4, and APX-115A induced cell death of EBV-infected human primary retina epithelial cells and the activation of JNK and ERK.Conclusion Taken together, these our results suggest that APX-115A could be a therapeutic agent for treating EBV-infected retinal cells or diseases by inhibiting LMP1-NOX4-ROS signaling.

Keywords:  EBV-infection; LMP1; NOX inhibitor; NOX4; Retinal pigment epithelial cell

DOI:  https://doi.org/10.1080/02713683.2020.1718164
Exp Eye Res. 2020 Jan 08. pii: S0014-4835(19)30591-3. [Epub ahead of print] 107918

Nox4-mediated ROS production is involved, but not essential for TGFβ-induced lens EMT leading to cataract.

S J Das, T Wishart, K Jandeleit-Dahm, F J Lovicu.

  The reactive oxygen species (ROS) producing enzyme, NADPH oxidase 4 (Nox4),is upregulated in response to TGFβ in lens epithelial cells in vitro, and its selective inhibition was shown to block aspects of TGFβ-induced epithelial-mesenchymal transition (EMT). In the present in situ study we validate the role(s) of Nox4 in TGFβ-induced lens EMT leading to anterior subcapsular cataract (ASC) formation. Mice overexpressing TGFβ in the lens, that develop ASC, were crossed to Nox4-deficient mice. When comparing mice overexpressing TGFβ in lens, to mice that were also deficient for Nox4, we see the delayed onset of cataract, along with a delay in EMT protein markers normally associated with TGFβ-induced fibrotic cataracts. In the absence of Nox4, we also see elevated levels of ERK1/2 activity that was shown to be required for TGFβ/Smad2/3-signaling. qRT-PCR revealed upregulation of Nox2 and its regulatory subunit in TGFβ-overexpressing lens epithelial cells devoid of Nox4. Taken together, these findings provide an improved platform to delineate putative Nox4 (and ROS) interactions with Smad2/3 and/or ERK1/2, in particular in the development of fibrotic diseases, such as specific forms of cataract.

Keywords:  Anterior subcapsular cataract; Cataract; ERK1/2; Epithelial-mesenchymal transition (EMT); Fibrosis; Lens; Myofibroblast; Nox2; Nox4; ROS; Smad; Transforming growth factor-beta (TGFβ)

DOI:  https://doi.org/10.1016/j.exer.2020.107918
Neurochem Res. 2020 Jan 16.

miR-124-5p/NOX2 Axis Modulates the ROS Production and the Inflammatory Microenvironment to Protect Against the Cerebral I/R Injury.

Yakun Wu, Jia Yao, Kai Feng.

  The reperfusion after an acute ischemic stroke can lead to a secondary injury, which is ischemia-reperfusion (I/R) injury. During ischemia, the reactive oxygen species (ROS) is over-produced, mostly from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). Besides, miRNAs are also associated with neuronal death in ischemic stroke. MiR-124-5p is selectively expressed within central nervous system (CNS) and is predicted to bind to NOX2 directly. Herein, we successfully set up cerebral I/R injury model in rats through middle cerebral artery occlusion (MCAO) surgery. After 12 h or 24 h of refusion, the superoxide dismutase (SOD) activity was significantly inhibited, accompanied by NOX2 protein increase within MCAO rat infarct area. In vitro, oxygen-glucose deprivation/refusion (OGD/R) stimulation on PC-12 cells significantly increased NOX2 protein levels, ROS production, and the cell apoptosis, while a significant suppression on SOD activity; OGD/R stimulation-induced changes in PC-12 cells described above could be significantly attenuated by NOX2 silence. In vivo, miR-124 overexpression improved, whereas miR-124 inhibition aggravated I/R injury in MCAO rats. miR-124-5p directly bound to the CYBB 3'-untranslated region (UTR) to negatively regulate CYBB expression and NOX2 protein level. In vitro, miR-124 overexpression improved, while NOX2 overexpression aggravated OGD/R-induced cellular injuries; NOX2 overexpression significantly attenuated the effects of miR-124 overexpression. Besides, miR-124 overexpression significantly repressed NF-κB signaling activation and TNFα and IL-6 production through regulating NOX2. In conclusion, miR-124-5p/NOX2 axis modulates NOX-mediated ROS production, the inflammatory microenvironment, subsequently the apoptosis of neurons, finally affecting the cerebral I/R injury.

Keywords:  Ischemia–reperfusion (I/R) injury; NF-κB signaling; Nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2); Reactive oxygen species (ROS); miR-124-5p

DOI:  https://doi.org/10.1007/s11064-019-02931-0
Antioxid Redox Signal. 2020 Jan 13.

Beta-cell-specific expression of NOX5 aggravates high fat diet-induced impairment of islet insulin secretion in mice.

Karim Bouzakri, Christelle Veyrat-Durebex, Chet E Holterman, Caroline Arous, Charlotte Barbieux, Domenico Bosco, Jordi Altirriba, Mohamed Alibashe, Benjamin Tournier, Jenny E Gunton, Sarah Mouche, William Bietiger, Alexis Forterre, Thierry Berney, Michel Pinget, Gerhard Christofori, Chris Rj Kennedy, Ildiko Szanto.

NADPH oxidases (NOX-es) produce reactive oxygen species and modulate β-cell insulin secretion. Islets of Type2 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, β-cell-specific manner (RIP/rtTA/NOX5 mice). In situ hybridization and immunohistochemistry employed on pancreatic sections demonstrated NOX5 mRNA and protein expressions in human islets. In cultures of dispersed islets NOX5 protein was observed in somatostatin-positive (δ) cells in basal (2.8mM glucose) conditions. siRNA-mediated knock-down of NOX5 in human islets cultured in basal glucose concentrations resulted in diminished glucose-induced insulin secretion in vitro. However, when islets were preincubated in high (16.7mM) glucose media for 12 hours, NOX5 appeared also in insulin-positive (β) cells. In vivo, mice with β-cell NOX5 expression developed aggravated impairment of glucose-induced insulin secretion compared to control mice when challenged with 14 weeks of high-fat diet. Similarly, in vitro palmitate pre-incubation resulted in more severe reduction of insulin release in islets of RIP/rtTA/NOX5 mice compared to their control littermates. Decreased insulin secretion was most distinct in response to theophylline stimulation suggesting impaired cAMP-mediated signaling due to increased phophodiesterase activation. 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 intra-islet crosstalk in physiological circumstances but also identifying it as an aggravating factor in β-cell failure in diabetic conditions.

DOI: https://doi.org/10.1089/ars.2018.7579
Hepatology. 2020 Jan 17.

Non-phagocytic Activation of NOX2 is Implicated in Progressive Non-alcoholic Steatohepatitis During Aging.

Joy X Jiang, Sarah R Fish, Alexey Tomilov, Yuan Li, Weiguo Fan, Ali Dehnad, David Gae, Suvarthi Das, Gergely Mozes, Gregory W Charville, Jon Ramsey, Gino Cortopassi, N J Török.

  Older patients with obesity/type II DM frequently present with advanced non-alcoholic steatohepatitis (NASH). Whether this is due to specific molecular pathways that accelerate fibrosis during aging, is unknown. Activation of the Src homology 2 domain containing collagen-related (Shc) proteins and redox stress have been recognized in aging, however their link to NASH has not been explored. Shc expression increased in livers of older patients with NASH, as assessed by RTqPCR or western blots. Fibrosis, Shc expression, markers of senescence and NADPH oxidases (NOXs) were studied in young/old mice on fast food diet (FFD). To inhibit Shc in old mice LV-shShc vs. control-LV were used during FFD. For hepatocyte-specific effects, fl/fl Shc mice on FFD were injected with AAV8-TBG-Cre vs. control. Fibrosis was accelerated in older mice on FFD, and Shc inhibition by LV in older mice, or hepatocyte-specific deletion resulted in significantly improved inflammation, reduction in senescence markers in older mice, lipid peroxidation and fibrosis. To study NOX2 activation, the interaction of p47phox (NOX2 regulatory subunit) and p52Shc was evaluated by proximity ligation, and co-IPs. Palmitate induced p52Shc binding to p47phox activating the NOX2 complex, more so at older age. Kinetics of binding were assessed in SH2 or PTB deletion mutants by biolayer interferometry, revealing the role of SH2 and the PTB domains. Lastly, an in silico model of p52Shc/p47phox interaction using RosettaDock was generated. Conclusion Accelerated fibrosis in the aged is modulated by p52Shc/NOX2. We show a novel pathway for direct activation of the phagocytic NOX2 in hepatocytes by p52Shc binding and activating the p47phox subunit that results in redox stress, and accelerated fibrosis in the aged.

Keywords:  NADPH oxidase; Src homology 2 domain containing collagen-related (Shc) proteins; liver fibrosis

DOI:  https://doi.org/10.1002/hep.31118
J Clin Med. 2020 Jan 15. pii: E226. [Epub ahead of print]9(1):

Induction of HO-1 by Mevastatin Mediated via a Nox/ROS-Dependent c-Src/PDGFRα/PI3K/Akt/Nrf2/ARE Cascade Suppresses TNF-α-Induced Lung Inflammation.

Chih-Chung Lin, Wei-Ning Lin, Rou-Ling Cho, Chien-Chung Yang, Yi-Cheng Yeh, Li-Der Hsiao, Hui-Ching Tseng, Chuen-Mao Yang.

   BACKGROUND: Mevastatin (MVS), a 3-hydroxy-3-methylglutaryl coenzyme, a reductase (HMG-CoA) inhibitor, has anti-inflammatory effects potentially via up-regulation of heme oxygenase-1 (HO-1). However, the mechanisms underlying MVS-induced HO-1 expression remain largely unknown in human pulmonary alveolar epithelial cells (HPAEpiCs).
METHODS: HO-1 and intercellular adhesion molecule (ICAM)-1 expression were determined using real-time PCR, Western blotting, and promoter reporter analyses. The signaling components were investigated using pharmacological inhibitors or specific small interfering RNA (siRNA)s. Interaction between Nrf2 and the antioxidant response element (ARE) binding site for the HO-1 promoter was determined by chromatin immunoprecipitation (ChIP) assay.
RESULTS: Upregulation of HO-1 by MVS attenuated the tumor necrosis factor (TNF)-α-stimulated ICAM-1 expression associated with THP-1 adhesion to HPAEpiCs. These inhibitory effects of HO-1 were reversed by tin protoporphyrin (SnPP)IX or by transfection with HO-1 siRNA. MVS-induced HO-1 expression was mediated via NADPH oxidase (Nox)-derived reactive oxygen species (ROS) generation. Activation of Nox2/ROS further stimulated the phosphorylation of p47phox, proto-oncogene tyrosine-protein kinase (c-Src), platelet-derived growth factor receptor (PDFGR)α, protein kinase B (Akt), and Nrf2, which were inhibited by siRNAs. Pretreatment with pharmacological inhibitors, including diphenyleneiodonium (DPI), apocynin (APO), N-acetyl-L-cysteine (NAC), PP1, AG1296, or LY294002, reduced the MVS-activated Nrf2 nuclear-translocation binding to the ARE on the HO-1 promoter.
CONCLUSIONS: MVS-induced HO-1 is, at least in part, mediated through a p47phox/Nox2/ROS-dependent activation of c-Src/PDGFRα/PI3K/Akt-regulated Nrf2/ARE axis and suppresses the TNF-α-mediated inflammatory responses in HPAEpiCs.

Keywords:  AREs; Nrf2; ROS; heme oxygenase-1; mevastatin

DOI:  https://doi.org/10.3390/jcm9010226
Blood. 2020 Jan 17. pii: blood.2019003525. [Epub ahead of print]

NADPH oxidase controls pulmonary neutrophil infiltration in the response to fungal cell walls by limiting LTB4.

Zhimin Song, Guangming Huang, Luana Chiquetto Paracatu, Derayvia J Grimes, Jiwei Gu, Cliff J Luke, Regina A Clemens, Mary C Dinauer.

Leukocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a key role in host defense and immune regulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD), characterized by recurrent bacterial and fungal infections and aberrant inflammation. Key drivers of hyper-inflammation induced by fungal cell walls in CGD are still incompletely defined. Here, we found that CGD (CYBB-null) neutrophils produced higher amounts of leukotriene B4 (LTB4) in vitro following activation with zymosan or Immune complexes, as compared to wild type (WT) neutrophils. This correlated with increased calcium influx in CGD neutrophils, which is restrained in WT neutrophils by the electrogenic activity of the NADPH oxidase. Increased LTB4 generation by CGD neutrophils was also augmented by paracrine cross-talk with the LTB4 receptor BLT1. CGD neutrophils formed more numerous and larger clusters in the presence of zymosan in vitro compared to WT, which was also LTB4- and BLT1-dependent. In zymosan-induced lung inflammation, focal neutrophil infiltrates were increased in CGD compared to WT mice and associated with higher LTB4 levels. Inhibiting LTB4 synthesis or antagonizing the BLT1 receptor following zymosan challenge reduced lung neutrophil recruitment in CGD to WT levels. Thus, LTB4 was the major driver of excessive neutrophilic lung inflammation in CGD mice in the early response to fungal cell walls, likely by a dysregulated feed-forward loop involving amplified neutrophil production of LTB4. This study identifies neutrophil LTB4 generation as a target of NADPH oxidase regulation, which could potentially be exploited therapeutically to reduce excessive inflammation in CGD.

DOI: https://doi.org/10.1182/blood.2019003525