Protective Effect of Selenoprotein X Against Oxidative Stress-Induced Cell Apoptosis in Human Hepatocyte (LO2) Cells via the p38 Pathway

Oxidative stress, as mediated by ROS (reactive oxygen species), is a significant factor in initiating the cells damaged by affecting cellular macromolecules and impairing their biological functions; SelX, a selenoprotein also known as MsrB1 belonging to the methionine sulfoxide reductase (Msr) family, is the redox repairing enzyme and involved in redox-related functions. In order to more precisely analyze the relationship between oxidative stress, cell oxidative damage, and SelX, we stably overexpressed porcine Selx full-length cDNA in human normal hepatocyte (LO2) cells. Cell viability, cell apoptosis rate, intracellular ROS, and the expression levels of mRNA or protein of apoptosis-related genes under H₂O₂-induced oxidative stress were detected. We found that overexpression of SelX can prevent the oxidative damage caused by H₂O₂ and propose that the main mechanism underlying the protective effects of SelX is the inhibition of LO2 cell apoptosis. The results revealed that overexpressed SelX reduced the H₂O₂-induced intracellular ROS generation, inhibited the H₂O₂-induced upregulation of Bax and downregulation of Bcl-2, and increased the mRNA and protein ratio of Bcl-2/Bax. Furthermore, it inhibited H₂O₂-induced p38 MAPK phosphorylation. Taken together, our findings suggested that SelX played important roles in protecting LO2 cells against oxidative damage and that its protective effect is partly via the p38 pathway by acting as a ROS scavenger.     

Modulation of Notch Signaling Pathway to Prevent H2O2/Menadione-Induced SK-N-MC Cells Death by EUK134

The brain in Alzheimer’s disease is under increased oxidative stress, and this may have a role in the pathogenesis and neural death in this disorder. It has been verified that numerous signaling pathways involved in neurodegenerative disorders are activated in response to reactive oxygen species (ROS). EUK134, a synthetic salen–manganese antioxidant complex, has been found to possess many interesting pharmacological activities awaiting exploration. The present study is to characterize the role of Notch signaling in apoptotic cell death of SK-N-MC cells. The cells were treated with hydrogen peroxide (H₂O₂) or menadione to induce oxidative stress. The free-radical scavenging capabilities of EUK134 were studied through the MTT assay, glutathione peroxidase (GPx) enzyme activity assay, and glutathione (GSH) Levels. The extents of lipid peroxidation, protein carbonyl formation, and intracellular ROS levels, as markers of oxidative stress, were also studied. Our results showed that H₂O₂/menadione reduced GSH levels and GPx activity. However, EUK134 protected cells against ROS-induced cell death by down-regulation of lipid peroxidation and protein carbonyl formation as well as restoration of antioxidant enzymes activity. ROS induced apoptosis and increased NICD and HES1 expression. Inhibition of NICD production proved that Notch signaling is involved in apoptosis through p53 activation. Moreover, H₂O₂/menadione led to Numb protein down-regulation which upon EUK134 pretreatment, its level increased and subsequently prevented Notch pathway activation. We indicated that EUK134 can be a promising candidate in designing natural-based drugs for ROS-induced neurodegenerative diseases. Collectively, ROS activated Notch signaling in SK-N-MC cells leading to cell apoptosis.     

Mitofusin 2 deficiency leads to oxidative stress that contributes to insulin resistance in rat skeletal muscle cells

Mitofusin 2 (Mfn2) is a dynamin-like protein anchored in the outer mitochondrial membrane that plays a crucial role in ensuring optimal mitochondrial morphological homeostasis. It has been shown that reduced expression of Mfn2 is associated with insulin resistance, but the mechanism is still unclear. We investigated whether Mfn2 deficiency leads to impaired insulin sensitivity via elevated oxidative stress. L6 skeletal muscle cells were treated with palmitate and Mfn2 expression was repressed by transfection with antisense Mfn2. Levels of antioxidant enzymes, reactive oxygen species (ROS), the phosphorylation of c-Jun N-terminal Kinase (JNK) and nuclear factor-κB (NF-κB) and the mitochondrial membrane potential (Δψ_m) were measured. The results showed palmitate-induced insulin resistance of skeletal muscle cells was accompanied by Mfn2 repression. Meanwhile, the cells had decreased Δψ_m and activity of antioxidant enzymes which could increase production of ROS, phosphorylation of JNK and NF-κB. When Mfn2 was up-regulated in palmitate-treated cells, oxidative stress and insulin resistance was alleviated. Furthermore, knock-down of Mfn2 in control cells enhanced oxidative stress. Mfn2 deficiency led to increased superoxide concentration and activation of JNK as well as NF-κB associated with insulin signaling. In conclusion, Mfn2 is a potent repressor for oxidative stress and regulation of Mfn2 expression may prove to be a potential method to circumvent insulin resistance.     

HOX-1 and COX-2: Two differentially regulated key mediators of skeletal myoblast tolerance under oxidative stress

Abstract

The exact physiological role of oxidative stress as a primary cause for skeletal muscle pathological conditions involving muscle degeneration remains elusive. Therefore, the present study was performed so as to decipher the signalling pathways orchestrating the potential cytoprotective role of heme oxygenase 1 (HOX-1) as well as cyclooxygenase 2 (COX-2) in skeletal myoblasts exposed to H₂O₂. Cell treatment with H₂O₂ (0.5 mM) resulted in a time- and dose-dependent response of HOX-1 and COX-2 mRNA and protein levels, with ERK1/2, p38-MAPK and MSK1 found to mediate these effects. Furthermore, Src and JNKs blockade attenuated COX-2 response. Collectively, these novel findings highlight for the first time HOX-1 and COX-2 fundamental contribution to skeletal myoblast tolerance under oxidative stress, since their inhibition significantly attenuated viability of skeletal myoblasts. The data also delineate the various effectors regulating HOX-1 and COX-2 expression, probably alleviating muscle degeneration in related disorders.     

Neuroprotective Effects of Bikaverin on H2O2-Induced Oxidative Stress Mediated Neuronal Damage in SH-SY5Y Cell Line

The generation of free radicals and oxidative stress has been linked to several neurodegenerative diseases including Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and Amyotrophic lateral sclerosis. The use of free radical scavenging molecules for the reduction of intracellular reactive oxygen species is one of the strategies used in the clinical management of neurodegeneration. Fungal secondary metabolism is a rich source of novel molecules with potential bioactivity. In the current study, bikaverin was extracted from Fusarium oxysporum f. sp. lycopersici and its structural characterization was carried out. Further, we explored the protective effects of bikaverin on oxidative stress and its anti-apoptotic mechanism to attenuate H₂O₂-induced neurotoxicity using human neuroblastoma SH-SY5Y cells. Our results elucidate that pretreatment of neurons with bikaverin attenuates the mitochondrial and plasma membrane damage induced by 100 µM H₂O₂ to 82 and 26 % as evidenced by MTT and LDH assays. H₂O₂ induced depletion of antioxidant enzyme status was also replenished by bikaverin which was confirmed by Realtime Quantitative PCR analysis of SOD and CAT genes. Bikaverin pretreatment efficiently potentiated the H₂O₂-induced neuronal markers, such as BDNF, TH, and AADC expression, which orchestrate the neuronal damage of the cell. The H₂O₂-induced damage to cells, nuclear, and mitochondrial integrity was also restored by bikaverin. Bikaverin could be developed as a preventive agent against neurodegeneration and as an alternative to some of the toxic synthetic antioxidants.     

Parthenolide regulates oxidative stress-induced mitophagy and suppresses apoptosis through p53 signaling pathway in C2C12 myoblasts

Muscle redox disturbances and oxidative stress have emerged as a common pathogenetic mechanism and potential therapeutic intervention in some muscle diseases. Parthenolide (PTL), a sesquiterpene lactone found in large amounts in the leaves of feverfew, possesses anti-inflammatory, anti-migraine, and anticancer properties. Although PTL was reported to alleviate cancer cachexia and improve skeletal muscle characteristics in a cancer cachexia model, its actions on oxidative stress-induced damage in C2C12 myoblasts have not been reported and the regulatory mechanisms have not yet been defined. In our study, PTL attenuated H₂O₂-induced growth inhibition and morphological changes. Furthermore, PTL exhibited scavenging activity against reactive oxygen species and protected C2C12 cells from apoptosis in response to H₂O₂. Meanwhile, PTL suppressed collapse of the mitochondrial membrane potential, thereby contributing to normalizing H₂O₂-induced autophagy flux and mitophagy, correlating with inhibiting degradation of mitochondrial marker protein TIM23, the increase in LC3-II expression and the reduction of mitochondria DNA. Besides its protective effect on mitochondria, PTL also prevented H₂O₂-induced lysosomes damage in C2C12 cells. In addition, the phosphorylation of p53, cathepsin B, and Bax/Bcl-2 protein levels, and the translocation of Bax from the cytosol to mitochondria induced by H₂O₂ in C2C12 cells was significantly reduced by PTL. In conclusion, PTL modulates oxidative stress-induced mitophagy and protects C2C12 myoblasts against apoptosis, suggesting a potential protective effect against oxidative stress-associated skeletal muscle diseases.     

Upstream reactive oxidative species (ROS) signals in exogenous oxidative stress-induced mitochondrial dysfunction

Exogenous oxidative stress induces cell death, but the upstream molecular mechanisms involved of the process remain relatively unknown. We determined the instant dynamic reactions of intracellular reactive oxygen species (ROS, including hydrogen peroxide (H₂O₂), superoxide radical (O₂⁻), and nitric oxide (NO)) in cells exposed to exogenous oxidative stress by using a confocal laser scanning microscope. Stimulation with extracellular H₂O₂ significantly increased the production of intracellular H₂O₂, O₂⁻, and NO (P < 0.01) through certain mechanisms. Increased levels of intracellular ROS resulted in mitochondrial dysfunction, involving the impairment of mitochondrial activity and the depolarization of mitochondrial membrane potential. Mitochondrial dysfunction significantly inhibited the proliferation of human hepatoblastoma G2 (HepG2) cells and resulted in mitochondrial cytochrome c (cyt c) release. The results indicate that upstream ROS signals play a potential role in exogenous oxidative stress-induced cell death through mitochondrial dysfunction and cyt c release.     

Impact of oxidative stress on cellular biomechanics and rho signaling in C2C12 myoblasts

Although cells often can tolerate oxidative environments, abnormal oxidative stress has been identified in inflammation, cardiovascular and neurodegenerative diseases, and aging. The impact of oxidative stress on the cellular biomechanics is poorly understood, however. In this study, we used C2C12 myoblasts to investigate the effect of oxidative stress, mimicked by hydrogen peroxide (H₂O₂), on the cell elasticity (i.e., Young?s modulus), viability, and production of intracellular reactive oxygen species (ROS). To better understand the mechanisms underlying the impact of H₂O₂, we examined various effectors of the Rho signaling pathway, which has been shown to play a key role in the control of cell mechanics. H₂O₂ decreased the cell stiffness in a dose-dependent manner, caused cell death, and reduced the RhoA expression that was accompanied by down-regulation of α-actin, cytoskeleton-membrane linker proteins (ezrin–radixin–moesion proteins), and focal adhesion. Modulating the Rho signaling by using a Rho activator partially restored the cell stiffness, enhanced the cell viability, and decreased the intracellular ROS level, suggesting a potential intervention strategy to maintain the cellular biomechanical homeostasis and rescue cell damage in the threat of oxidative stresses.     

Cytoprotective propensity of green tea polyphenols against citrinin-induced skeletal-myotube damage in C2C12 cells

The mycotoxin citrinin, is produced by several species of Penicillium, Aspergillus and Monascus, and is capable of inducing cytotoxicity, oxidative stress and apoptosis. The aim of the present study was to investigate the effect of citrinin in mouse skeletal muscle cells (C2C12) and to overcome the cellular adverse effects by supplementing green tea extract (GTE) rich in polyphenols. C2C12 myoblasts were differentiated to myotubes and were exposed to citrinin in a dose dependent manner (0–100 µM) for 24 h and IC₅₀ value was found to be 100 µM that resulted in decreased cell viability, increased LDH leakage and compromised membrane integrity. Mitochondrial membrane potential loss, increased accumulation of intracellular ROS and sub G1 phase of cell cycle was observed. To ameliorate the cytotoxic effects of CTN, C2C12 cells were pretreated with GTE (20, 40, 80 µg/ml) for 2 h followed by citrinin (100 µM) treatment for 24 h. GTE pretreatment combated citrinin-induced cytotoxicity and oxidative stress. GTE at 40 and 80 µg/ml significantly promoted cell survival and upregulated antioxidant enzyme activities (CAT, SOD, GPx) and endogenous antioxidant GSH, while the gene and protein expression levels were significantly restored through its effective antioxidant mechanism. Present study results suggested the antioxidant properties of GTE as a herbal source in ameliorating the citrinin-induced oxidative stress.     

Correlation between oxidative stress and G6PD activity in neonatal jaundice

Fetal distress represents a pathophysiological condition in which oxygen is not available to the fetus in sufficient quantities. In cases of glucose 6-phosphate dehydrogenase (G6PD) deficiency, under conditions of oxidative stress, the residual G6PD and complimentary antioxidant mechanisms may become insufficient to neutralize the large amounts of ROS and to prevent severe hemolysis. Alteration in the oxidant–antioxidant profile is also known to occur in neonatal jaundice. The study group included 22 neonates presented with fetal distress during labor and 24 neonates with no evidence of fetal distress (control group). Umbilical cord blood samples were taken immediately after delivery, and the following blood tests were carried out after birth and at discharge from the hospital: erythrocyte count, total bilirubin, G6PD activity, and parameters presenting oxidative status [thiobarbituric acid reactive substances (TBARS), NO, O₂ ^?, H₂O₂, SOD, CAT, O₂ ^?/SOD, and H₂O₂/CAT]. There were no significant differences in TBARS and NO values among neonates with or without fetal distress. However, the values of O₂ ^?, H₂O₂, SOD, O₂ ^?/SOD, and H₂O₂/CAT among neonates born after fetal distress were significantly higher than in neonates without fetal distress (p < 0.01). In neonates with fetal distress, the total number of RBCs at delivery was significantly lower, accompanied with higher bilirubin content. Also neonates with fetal distress had lower activity of G6PD and lower CAT activity. Higher values of oxidative stress parameters in newborns delivered after fetal distress do not indicate strictly what occurred first—oxidative stress or basic lower G6PD activity.     

Nox4-Derived H2O2 Mediates Endoplasmic Reticulum Signaling through Local Ras Activation

The unfolded-protein response (UPR) of the endoplasmic reticulum (ER) has been linked to oxidant production, although the molecular details and functional significance of this linkage are poorly understood. Using a ratiometric H₂O₂ sensor targeted to different subcellular compartments, we demonstrate specific production of H₂O₂ by the ER in response to the stressors tunicamycin and HIV-1 Tat, but not to thapsigargin or dithiothreitol. Knockdown of the oxidase Nox4, expressed on ER endomembranes, or expression of ER-targeted catalase blocked ER H₂O₂ production by tunicamycin and Tat and prevented the UPR following exposure to these two agonists, but not to thapsigargin or dithiothreitol. Tat also triggered Nox4-dependent, sustained activation of Ras leading to ERK, but not phosphatidylinositol 3-kinase (PI3K)/mTOR, pathway activation. Cell fractionation studies and green fluorescent protein (GFP) fusions of GTPase effector binding domains confirmed selective activation of endogenous RhoA and Ras on the ER surface, with ER-associated K-Ras acting upstream of the UPR and downstream of Nox4. Notably, the Nox4/Ras/ERK pathway induced autophagy, and suppression of autophagy unmasked cell death and prevented differentiation of endothelial cells in 3-dimensional matrix. We conclude that the ER surface provides a platform to spatially organize agonist-specific Nox4-dependent oxidative signaling events, leading to homeostatic protective mechanisms rather than oxidative stress.Coupled in part to its function as a major site of protein synthesis, the endoplasmic reticulum (ER) has emerged as an important signaling organelle, responding to various cell stresses and controlling cell fate. Much of this signaling is initiated on the ER membrane surface. In response to an overload of misfolded client proteins in the ER lumen, for example, transmembrane ER stress sensors such as IRE1, PERK, and ATF6 initiate signals on the cytosolic face of the ER to reduce global protein synthesis, promote protein folding, and increase the degradation of misfolded proteins (36). Failure of this response to alleviate protein misfolding stress leads to late expression of proteins such as CHOP, culminating in cell death. In addition to factors controlling this unfolded-protein response (UPR), cyclins, pro- and anti-apoptotic BH3 domain proteins, caspases, and signaling adapters associate with the ER surface and control cell cycle entry, cell death, Ca²⁺ flux, amino acid metabolism, oxidative-stress response, and autophagy (15, 17-19, 41). Thus, the ER integrates a variety of stresses (metabolic, protein misfolding, and oxidative) to coordinate cellular stress responses.Another ER transmembrane protein is the NADPH oxidase Nox4. Like other Nox family members, Nox4 produces H₂O₂ and is thought to function primarily in cell signaling. Consistent with its ER localization, Nox4 mediates oxidative inactivation of the ER-resident phosphatase PTP1B and responds to ER stress induced by the LDL oxysterol 7-ketocholesterol (10, 34). The presence of Nox4 on the ER surface is notable, since ER stress is associated with the production of reactive oxygen species (ROS), though the nature of this association is poorly understood. Agents that cause ER stress initiate ATF4-dependent glutathione (GSH) synthesis, which is necessary to diminish ROS production and subsequent cell death (15). The mechanism of ROS production is also unclear, but it is thought to be a consequence of the UPR, possibly downstream of CHOP (25, 26). For the most part, such studies suggest a role for oxidants as late effectors of cell death, downstream from the UPR. The ER itself may be a source of oxidants, as knockdown of the ER oxidase Ero-1 in pek-1-null worms diminishes oxidant production (15). To some extent, the excessive production of ROS by the stressed ER would seem to run counter to recent observations that chemical and physiologic ER stressors converge on the production of a hyperreduced ER interior (28). In addition, [rho⁰] Saccharomyces cerevisiae lacking mitochondria also fails to produce ROS in response to ER stress, suggesting a mitochondrial source (16). In part, such studies linking the ER and oxidative stress have been hampered by reliance on probes, such as dichlorofluorescein, that are untargeted and nonspecific, reacting with a broad variety of oxidants as well as non-ROS compounds, such as cytosolic cytochrome c.In this study, using an H₂O₂-specific probe targeted to the ER, we found that Nox4 participates early in ER stress signaling in an agonist-specific fashion through a novel process involving focal activation of Ras on the ER. This Nox4-dependent pathway leads to activation of autophagy, which prevents the progression of the UPR to cell death, thus distinguishing Nox4 oxidant signaling from oxidative stress.     

Pterostilbene protects against H2O2-induced oxidative stress by regulating GAS6/Axl signaling in HL-1 cells

Pterostilbene (PTE, trans-3,5-dimethoxy-4′-hydroxystilbene), a natural plant polyphenol, possesses numerous pharmacological effects, including antioxidant, antidiabetic, antiatherosclerotic, and neuroprotective aspects. This study aims to investigate whether PTE plays a protective role against oxidative stress injury by GAS6/Axl signaling pathway in cardiomyocytes. Hydrogen peroxide (H₂O₂)-induced oxidative stress HL-1 cells were used as models. The mechanism by which PTE protected oxidative stress is investigated by combining cell viability, cell ROS levels, apoptosis assay, molecular docking, quantitative real-time PCR, and western blot analysis. GAS6 shRNA was performed to investigate the involvement of GAS6/Axl pathways in PTE's protective role. The results showed that PTE treatment improved the cell morphology and viability, and inhibited the apoptosis rate and ROS levels in H₂O₂-injured HL-1 cells. Particularly, PTE treatment upregulated the levels of GAS6, Axl, and markers related to oxidative stress, apoptosis, and mitochondrial function related. Molecular docking showed that PTE and GAS6 have good binding ability. Taken together, PTE plays a protective role against oxidative stress injury through inhibiting oxidative stress and apoptosis and improving mitochondrial function. Particularly, GAS6/Axl axis is the surprisingly prominent in the PTE-mediated pleiotropic effects.     

Antioxidant effect of human placenta hydrolysate against oxidative stress on muscle atrophy

Sarcopenia, which refers to the muscle loss that accompanies aging, is a complex neuromuscular disorder with a clinically high prevalence and mortality. Despite many efforts to protect against muscle weakness and muscle atrophy, the incidence of sarcopenia and its related permanent disabilities continue to increase. In this study, we found that treatment with human placental hydrolysate (hPH) significantly increased the viability (approximately 15%) of H₂O₂-stimulated C2C12 cells. Additionally, while H₂O₂-stimulated cells showed irregular morphology, hPH treatment restored their morphology to that of cells cultured under normal conditions. We further showed that hPH treatment effectively inhibited H₂O₂-induced cell death. Reactive oxygen species (ROS) generation and Mstn expression induced by oxidative stress are closely associated with muscular dysfunction followed by atrophy. Exposure of C2C12 cells to H₂O₂ induced abundant production of intracellular ROS, mitochondrial superoxide, and mitochondrial dysfunction as well as myostatin expression via nuclear factor-κB (NF-κB) signaling; these effects were attenuated by hPH. Additionally, hPH decreased mitochondria fission–related gene expression (Drp1 and BNIP3) and increased mitochondria biogenesis via the Sirt1/AMPK/PGC-1α pathway and autophagy regulation. In vivo studies revealed that hPH-mediated prevention of atrophy was achieved predominantly through regulation of myostatin and PGC-1α expression and autophagy. Taken together, our findings indicate that hPH is potentially protective against muscle atrophy and oxidative cell death.     

Nox2 Mediates Skeletal Muscle Insulin Resistance Induced by a High Fat Diet

Inflammation and oxidative stress through the production of reactive oxygen species (ROS) are consistently associated with metabolic syndrome/type 2 diabetes. Although the role of Nox2, a major ROS-generating enzyme, is well described in host defense and inflammation, little is known about its potential role in insulin resistance in skeletal muscle. Insulin resistance induced by a high fat diet was mitigated in Nox2-null mice compared with wild-type mice after 3 or 9 months on the diet. High fat feeding increased Nox2 expression, superoxide production, and impaired insulin signaling in skeletal muscle tissue of wild-type mice but not in Nox2-null mice. Exposure of C2C12 cultured myotubes to either high glucose concentration, palmitate, or H₂O₂ decreases insulin-induced Akt phosphorylation and glucose uptake. Pretreatment with catalase abrogated these effects, indicating a key role for H₂O₂ in mediating insulin resistance. Down-regulation of Nox2 in C2C12 cells by shRNA prevented insulin resistance induced by high glucose or palmitate but not H₂O₂. These data indicate that increased production of ROS in insulin resistance induced by high glucose in skeletal muscle cells is a consequence of Nox2 activation. This is the first report to show that Nox2 is a key mediator of insulin resistance in skeletal muscle.     

Metabolic derangement and cardiac injury early after reperfusion following intermittent cross-clamp fibrillation in patients undergoing coronary artery bypass graft surgery using conventional or miniaturized cardiopulmonary bypass

Reactive oxygen species (ROS)-induced oxidative stress increases in skeletal muscle with aging and decreases the viability of implanted cells. Type 1 insulin-like growth factor (IGF-1) promotes the survival of skeletal muscle cells under oxidative stress. It is unknown whether IGF-1 protects muscle-derived stem cells (MDSCs) from oxidative stress. In this study, we genetically engineered rat MDSCs to overexpress IGF-1 and determined cell viability, apoptosis, and VEGF secretion under oxidative stress. Overexpression of IGF-1 prevented MDSCs from H₂O₂-induced caspase-dependent apoptotic cell death by upregulating the PI3K/AKT pathway, accompanied with an increase of NF-κB, p-NF-κB, Bcl-2, and VEGF, as well as a decrease of Bax. In contrast, pre-administration of picropodophyllinb, wortmannin, 1L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate), or pyrrolidine-dithiocarbamate, specific inhibitors of IGF-1R, PI3K, AKT, and NF-κB, respectively, followed by treatment with H₂O₂, resulted in cell death of MDSCs. Our data indicated that IGF-1 suppresses apoptosis and enhances the paracrine function of MDSCs under oxidative stress via enhancing IGF-1R/PI3K/AKT signaling. Thus, IGF-1 gene-modified MDSCs present a potential application in the treatment of muscle wasting, such as urethra intrinsic sphincter deficiency.     

A novel small-molecule MRCK inhibitor blocks cancer cell invasion

Background

Dual oxidase maturation factor 1 (DUOXA1) has been associated with the maturation of the reactive oxygen species (ROS) producing enzyme, dual oxidase 1 (DUOX1) in the adult thyroid. However, ROS have also been implicated in the development of several tissues. We found that activated muscle satellite cells and primary myoblasts isolated from mice express robust levels of DUOXA1 and that its levels are altered as cells differentiate.

Results

To determine whether DUOXA1 levels affect muscle differentiation, we used an adenoviral construct (pCMV5-DUOXA1-GFP) to drive constitutive overexpression of this protein in primary myoblasts. High levels of DUOXA1 throughout myogenesis resulted in enhanced H₂O₂ production, fusion defects, reduced expression of early (myogenin) and late (myosin heavy chain) markers of differentiation, and elevated levels of apoptosis compared to control cells infected with an empty adenoviral vector (pCMV5-GFP). DUOXA1 knockdown (using a DUOXA1 shRNA construct) resulted in enhanced differentiation compared to cells subjected to a control shRNA, and subjecting DUOXA1 overexpressing cells to siRNAs targeting DUOX1 or apoptosis signal-regulating kinase 1 (ASK1) rescued the phenotype.

Conclusions

This study represents the first to demonstrate the importance of DUOXA1 in skeletal muscle myoblasts and that DUOXA1 overexpression in muscle stem cells induces apoptosis and inhibits differentiation through DUOX1 and ASK1.     

Cytosolic ascorbate peroxidase 1 protects organelles against oxidative stress by wounding- and jasmonate-induced H2O2 in Arabidopsis plants

Background

Reactive oxygen species (ROS) are not only cytotoxic compounds leading to oxidative damage, but also signaling molecules for regulating plant responses to stress and hormones. Arabidopsis cytosolic ascorbate peroxidase 1 (APX1) is thought to be a central regulator for cellular ROS levels. However, it remains unclear whether APX1 is involved in plant tolerance to wounding and methyl jasmonate (MeJA) treatment, which are known to enhance ROS production.

Methods

We studied the effect of wounding and MeJA treatment on the levels of H₂O₂ and oxidative damage in the Arabidopsis wild-type plants and knockout mutants lacking APX1 (KO-APX1).

Results

The KO-APX1 plants showed high sensitivity to wounding and MeJA treatment. In the leaves of wild-type plants, H₂O₂ accumulated only in the vicinity of the wound, while in the leaves of the KO-APX1 plants it accumulated extensively from damaged to undamaged regions. During MeJA treatment, the levels of H₂O₂ were much higher in the leaves of KO-APX1 plants. Oxidative damage in the chloroplasts and nucleus was also enhanced in the leaves of KO-APX1 plants. These findings suggest that APX1 protects organelles against oxidative stress by wounding and MeJA treatment.

General significance

This is the first report demonstrating that H₂O₂-scavenging in the cytosol is essential for plant tolerance to wounding and MeJA treatment.