bims-noxint 2021-04-11 papers

Issue of 2021–04–11
six papers selected by
Laia Caja Puigsubira, Uppsala University

Invest Ophthalmol Vis Sci. 2021 Apr 01. 62(4): 4

TGF-β2 Promotes Oxidative Stress in Human Trabecular Meshwork Cells by Selectively Enhancing NADPH Oxidase 4 Expression.

Vidhya R Rao, Evan B Stubbs.

Purpose: The multifunctional profibrotic cytokine TGF-β2 is implicated in the pathophysiology of primary open angle glaucoma (POAG). While the underlying cause of POAG remains unclear, TGF-β2 dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment is considered an early pathologic consequence associated with impaired aqueous humor (AH) outflow and elevated IOP. Mitochondrial-targeted antioxidants have been recently shown by our group to markedly attenuate TGF-β2 profibrotic responses, strongly implicating oxidative stress as a key facilitator of TGF-β2 signaling in human TM cells. In this study, we determined the mechanism by which oxidative stress facilitates TGF-β2 profibrotic responses in cultured primary human TM cells.
Methods: Semiconfluent cultures of primary or transformed human TM cells were conditioned overnight in serum-free media and subsequently challenged without or with TGF-β2 (5 ng/mL). Relative changes in the mRNA content of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) isoforms, connective tissue growth factor (CTGF), collagen 1α1 and 4α1 isoforms or relative changes in the protein content of Nox4, phospho- and total-Smad2 and -Smad3, collagens I and IV were determined in the absence or presence of GKT137831, a Nox1-Nox4 dual enzyme inhibitor, and quantified by real-time qPCR or by immunoblot, respectively. Relative in situ changes in collagens I and IV and in alpha smooth muscle actin (αSMA) were semiquantified by immunocytochemistry, whereas relative changes in filamentous actin stress fiber formation was semiquantified by phalloidin staining.
Results: Quiescent primary human TM cells cultured in the presence of TGF-β2 exhibited a marked selective increase in endogenous Nox4 mRNA and Nox4 protein expression. Actinomycin D prevented TGF-β2 mediated increases in Nox4 mRNA expression. TM cells reverse transfected with siRNA against Smad3 prevented TGF-β2 mediated increases in Nox4 mRNA expression. Pre-incubating TM cells with GKT137831 attenuated TGF-β2 mediated increases in intracellular reactive oxygen species (ROS), in COL1A1, COL4A1, and CTGF mRNA expression, in Smad3 protein phosphorylation, in collagens I, collagens IV, and αSMA protein expression, and in filamentous actin stress fiber formation.
Conclusions: TGF-β2 promotes oxidative stress in primary human TM cells by selectively increasing expression of NADPH oxidase 4. Dysregulation of redox equilibrium by induction of NADPH oxidase 4 expression appears to be a key early event involved in the pathologic profibrotic responses elicited by TGF-β2 canonical signaling, including ECM remodeling, filamentous actin stress fiber formation, and αSMA expression. Selective inhibition of Nox4 expression/activation, in combination with mitochondrial-targeted antioxidants, represents a novel strategy by which to slow the progression of TGF-β2 elicited profibrotic responses within the TM.

DOI: https://doi.org/10.1167/iovs.62.4.4

Proc Natl Acad Sci U S A. 2021 Mar 16. pii: e2020152118. [Epub ahead of print]118(11):

Genetic deletion of Nox4 enhances cancerogen-induced formation of solid tumors.

Valeska Helfinger, Florian Freiherr von Gall, Nina Henke, Michael M Kunze, Tobias Schmid, Flavia Rezende, Juliana Heidler, Ilka Wittig, Heinfried H Radeke, Viola Marschall, Karen Anderson, Ajay M Shah, Simone Fulda, Bernhard Brüne, Ralf P Brandes, Katrin Schröder.

Reactive oxygen species (ROS) can cause cellular damage and promote cancer development. Besides such harmful consequences of overproduction of ROS, all cells utilize ROS for signaling purposes and stabilization of cell homeostasis. In particular, the latter is supported by the NADPH oxidase 4 (Nox4) that constitutively produces low amounts of H2O2 By that mechanism, Nox4 forces differentiation of cells and prevents inflammation. We hypothesize a constitutive low level of H2O2 maintains basal activity of cellular surveillance systems and is unlikely to be cancerogenic. Utilizing two different murine models of cancerogen-induced solid tumors, we found that deletion of Nox4 promotes tumor formation and lowers recognition of DNA damage. Nox4 supports phosphorylation of H2AX (γH2AX), a prerequisite of DNA damage recognition, by retaining a sufficiently low abundance of the phosphatase PP2A in the nucleus. The underlying mechanism is continuous oxidation of AKT by Nox4. Interaction of oxidized AKT and PP2A captures the phosphatase in the cytosol. Absence of Nox4 facilitates nuclear PP2A translocation and dephosphorylation of γH2AX. Simultaneously AKT is left phosphorylated. Thus, in the absence of Nox4, DNA damage is not recognized and the increased activity of AKT supports proliferation. The combination of both events results in genomic instability and promotes tumor formation. By identifying Nox4 as a protective source of ROS in cancerogen-induced cancer, we provide a piece of knowledge for understanding the role of moderate production of ROS in preventing the initiation of malignancies.

Keywords: AKT; Nox4; genomic instability; solid tumors

DOI: https://doi.org/10.1073/pnas.2020152118

Cell Rep. 2021 Apr 06. pii: S2211-1247(21)00263-1. [Epub ahead of print]35(1): 108949

NOX1-dependent redox signaling potentiates colonic stem cell proliferation to adapt to the intestinal microbiota by linking EGFR and TLR activation.

Sjoerd van der Post, George M H Birchenough, Jason M Held.

The colon epithelium is a primary point of interaction with the microbiome and is regenerated by a few rapidly cycling colonic stem cells (CSCs). CSC self-renewal and proliferation are regulated by growth factors and the presence of bacteria. However, the molecular link connecting the diverse inputs that maintain CSC homeostasis remains largely unknown. We report that CSC proliferation is mediated by redox-dependent activation of epidermal growth factor receptor (EGFR) signaling via NADPH oxidase 1 (NOX1). NOX1 expression is CSC specific and is restricted to proliferative CSCs. In the absence of NOX1, CSCs fail to generate ROS and have a reduced proliferation rate. NOX1 expression is regulated by Toll-like receptor activation in response to the microbiota and serves to link CSC proliferation with the presence of bacterial components in the crypt. The TLR-NOX1-EGFR axis is therefore a critical redox signaling node in CSCs facilitating the quiescent-proliferation transition and responds to the microbiome to maintain colon homeostasis.

Keywords: EGFR; NADPH oxidases; NOX1; ROS; TLR; colon; microbiome; microbiota; redox signaling; stem cells

DOI: https://doi.org/10.1016/j.celrep.2021.108949

Oxid Med Cell Longev. 2021 ;2021 6668971

NOX4-Derived ROS Promotes Collagen I Deposition in Bronchial Smooth Muscle Cells by Activating Noncanonical p38MAPK/Akt-Mediated TGF-β Signaling.

Binwei Hao, Ruiting Sun, Xiaotong Guo, Lili Zhang, Jieda Cui, Yumin Zhou, Wei Hong, Yanan Zhang, Jinxi He, Xiaoming Liu, Bing Li, Pixin Ran, Juan Chen.

Background: Airway smooth muscle (ASM) remodeling is a hallmark in chronic obstructive pulmonary disease (COPD). NADPH oxidase 4- (NOX4-) mediated reactive oxygen species (ROS) production plays a crucial role in cell differentiation and extracellular matrix (ECM) synthesis in ASM remodeling. However, the precise mechanisms underpinning its pathogenic roles remain elusive.
Methods: The expression of NOX4 and TGF-β 1 in the airway of the lung was measured in COPD patients and the control group. Cigarette smoke- (CS-) induced emphysema mice were generated, and the alteration of α-SMA, NOX4, TGF-β 1, and collagen I was accessed. The changes of the expression of ECM markers, NOX4, components of TGF-β/Smad, and MAPK/Akt signaling in human bronchial smooth muscle cells (HBSMCs) were ascertained for delineating mechanisms of NOX4-mediated ROS production on cell differentiation and remodeling in human ASM cells.
Results: An increased abundance of NOX4 and TGF-β 1 proteins in the epithelial cells and ASM of lung was observed in COPD patients compared with the control group. Additionally, an increased abundance expression of NOX4 and α-SMA was observed in the lungs of the CS-induced emphysema mouse model. TGF-β 1 displayed abilities to increase the oxidative burden and collagen I production, along with enhanced phosphorylation of ERK, p38MAPK, and p-Akt473 in HBSMCs. These effects of TGF-β 1 could be inhibited by the ROS scavenger N-acetylcysteine (NAC), siRNA-mediated knockdown of Smad3 and NOX4, and pharmacological inhibitors SB203580 (p38MAPK inhibitor) and LY294002 (Akt inhibitor).
Conclusions: NOX4-mediated ROS production alters TGF-β 1-induced cell differentiation and collagen I protein synthesis in HBSMCs in part through the p38MAPK/Akt signaling pathway in a Smad-dependent manner.

DOI: https://doi.org/10.1155/2021/6668971

Brain Res. 2021 Mar 31. pii: S0006-8993(21)00320-6. [Epub ahead of print] 147463

An Inhibitory and Beneficial Effect of Chlorpromazine and Promethazine (C+P) on Hyperglycolysis through HIF-1α Regulation in Ischemic Stroke.

Sichao Guo, Eric Cosky, Fengwu Li, Longfei Guan, Yu Ji, Wenjing Wei, Changya Peng, Xiaokun Geng, Yuchuan Ding.

BACKGROUND: After ischemic stroke, the increased catabolism of glucose (hyperglycolysis) results in the production of reactive oxygen species (ROS) via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). A depressive or hibernation-like effect of C+P on brain activity was reported to induce neuroprotection. The current study assesses the effect of C+P on hyperglycolysis and NOX activation.
METHODS: Adult male Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) followed by 6 or 24 h of reperfusion. At the onset of reperfusion, rats received C+P with or without temperature control, or phloretin [glucose transporter (GLUT)-1 inhibitor], or cytochalasin B (GLUT-3 inhibitor). We detected brain ROS, apoptotic cell death, and ATP levels along with HIF-1α expression. Cerebral hyperglycolysis was measured by glucose, protein expression of GLUT-1/3, and phosphofructokinase-1 (PFK-1), as well as lactate and lactate dehydrogenase (LDH) at 6 and 24 h of reperfusion. The enzymatic activity of NOX and protein expression of its subunits (gp91phox) were detected. Neural SHSY5Y cells were placed under 2 h of oxygen-glucose deprivation (OGD) followed by reoxygenation for 6 and 24 h with C+P treatment. Cell viability and protein levels of HIF-1α, GLUT-1/3, PFK-1, LDH, and gp91phox were measured. A HIF-1α overexpression vector was transfected into the cells, and then protein levels of HIF-1α, GLUT-1/3, PFK-1, and LDH were quantitated. In sham-operated rats and control cells, the protein levels of HIF-1α, GLUT-1/3, PFK-1, LDH, and gp91phox were measured at 6 and 24 h after C+P administration.
RESULTS: C+P reduced the protein elevations after stroke in HIF-1α, glycolytic enzymes, as well as in ROS, cell death, glucose and lactate, but raised ATP levels in the brain. In ischemic rats exposed to GLUT-1/3 inhibitors, ROS, cell death, glucose, and lactate were all decreased, as well as GLUT-1, GLUT-3, LDH, and PFK-1 protein levels. C+P decreased ischemia-induced NOX activation by reducing the enzymatic activity and protein expression of the NOX subunit gp91phox, as was observed in the presence of GLUT-1/3 inhibitors. These markers were significantly decreased following C+P administration with the induced hypothermia, while C+P administration with temperature control at 37°C induced lesser protection after ischemia stroke. In the OGD/reoxygenation model, C+P treatment increased cell viability and diminished protein levels of HIF-1α, GLUT-1, GLUT-3, PFK-1, LDH, and gp91phox. However, in OGD with HIF-1α overexpression, C+P was unable to effectively reduce the upregulated GLUT-1, GLUT-3, and LDH. In normal conditions, C+P reduced HIF-1α and the levels of key glycolytic enzymes depending on its pharmacological effect.
CONCLUSION: C+P, partially depending on hypothermia, attenuates hyperglycolysis and NOX activation through HIF-1α regulation.

Keywords: Chlorpromazine and Promethazine (C+P); Ischemia/reperfusion; hypoxia-induced factor-1α (HIF-1α); pharmacological hypothermia; phenothiazines; stroke

DOI: https://doi.org/10.1016/j.brainres.2021.147463

J Cell Physiol. 2021 Jul;236(7): 5052-5068

Hydrogen sulfide restores sevoflurane postconditioning mediated cardioprotection in diabetic rats: Role of SIRT1/Nrf2 signaling-modulated mitochondrial dysfunction and oxidative stress.

Jing Zhang, Xia Cai, Qin Zhang, Xiaozhong Li, Siyuan Li, Jianyong Ma, Wengen Zhu, Xiao Liu, Meilin Wei, Wei Tu, Yunfeng Shen, Jianping Liu, Xiaoyang Lai, Peng Yu.

Diabetic hearts are vulnerable to myocardial ischemia/reperfusion injury (IRI), but are insensitive to sevoflurane postconditioning (SPC), activating peroxiredoxins that confer cardioprotection. Previous studies have demonstrated that hydrogen sulfide (H2 S) can suppress oxidative stress of diabetic rats through increasing the expression of silent information regulator factor 2-related enzyme 1 (SIRT1), but whether cardioprotection by SPC can be restored afterward remains unclear. Diabetic rat was subjected to IRI (30 min of ischemia followed by 120 min reperfusion). Postconditioning treatment with sevoflurane was administered for 15 min upon the onset of reperfusion. The diabetic rats were treated with GYY4137 (H2 S donor) 5 days before the experiment. Myocardial infarct size, mitochondrial structure and function, ATP content, activities of complex I-IV, marker of oxidative stress, SIRT1, nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and NADPH Oxidase-2 (Nox-2) protein expression were detected after reperfusion, and cardiac function was evaluated by echocardiography at 24 h after reperfusion. After H2 S activated SIRT1 in the impaired myocardium of diabetic rats, SPC significantly upregulated the expression of Nrf2 and its downstream mediator HO-1, thus reduced the expression of Nox-2. In addition, H2 S remarkably increased cytoplasmic and nuclear SIRT1 which was further enhanced by SPC. Furthermore, H2 S combined with SPC reduced the production of reactive oxygen species, increased the content of ATP, and maintained mitochondrial enzyme activity. Finally, myocardial infarct size and myocardium damage were decreased, and cardiac function was improved. Taken together, our study proved that H2 S could restore SPC-induced cardioprotection in diabetic rats by enhancing and promoting SIRT1/Nrf2 signaling pathway mediated mitochondrial dysfunction and oxidative stress.

Keywords: SIRT1; cardiac ischemia reperfusion injury; diabetes; hydrogen sulfide; oxidative stress; sevoflurane

DOI: https://doi.org/10.1002/jcp.30214