Role of MAPK phosphatase-1 in sustained activation of JNK during ethanol-induced apoptosis in hepatocyte-like VL-17A cells

Ethanol metabolism plays a central role in activating the mitogen-activated protein kinase (MAPK) cascade leading to inflammation and apoptosis. Sustained activation of c-Jun N-terminal kinase (JNK), one of the MAPKs, has been shown to induce apoptosis in hepatocytes. MAPK phosphatase-1 (MKP-1) has been shown to dephosphorylate MAPKs in several cells. The aim of the study is to evaluate the role of MKP-1 in sustained JNK activation as a mechanism to explain ethanol-induced hepatocyte apoptosis. VL-17A cells (HepG2 cells overexpressing alcohol dehydrogenase and cytochrome P450-2E1) were exposed to ethanol for different time periods. Western blots were performed for MKP-1, phospho-JNK, phosphotyrosine, and protein kinase Cdelta (PKCdelta). Electrophoretic mobility shift assays for AP-1 were performed. Apoptosis was measured by caspase-3 activity assay, TUNEL, and 4',6-diamidino-2-phenylindole staining. Reactive oxygen species were neutralized by overexpressing both superoxide dismutase-3 and catalase genes using lentiviral vectors in VL-17A cells. Ethanol incubation markedly decreased the MKP-1 protein levels to 15% of control levels and was associated with sustained phosphorylation of p46 JNK and p54 JNK, as well as increased apoptosis. VL-17A cells overexpressing superoxide dismutase-3 and catalase, treatment with a tyrosine kinase inhibitor, or incubation of the cells with PKCdelta small interference RNAs significantly inhibited the ethanol-induced MKP-1 degradation and apoptosis. Ethanol-induced oxidative stress enhanced the tyrosine phosphorylation of PKCdelta, which in turn caused the proteasomal degradation of MKP-1, leading to sustained JNK activation and increased apoptosis in VL-17A cells.     

Recombinant Hep G2 cells that express alcohol dehydrogenase and cytochrome P450 2E1 as a model of ethanol-elicited cytotoxicity

HepG2 cells were transfected with recombinant plasmids, one carrying the murine alcohol dehydrogenase (ADH) gene and the other containing the gene encoding human cytochrome P450 2E1 (CYP2E1). One of recombinant clones called VL-17A exhibited ADH and CYP2E1 specific activities comparable to those in isolated rat hepatocytes. VL-17A cells oxidized ethanol and generated acetaldehyde, the levels of which depended upon the initial ethanol concentration. Compared with unexposed VL-17A cells, ethanol exposure increased the cellular redox (lactate:pyruvate ratio) and caused cell toxicity, indicated by increased leakage of lactate dehydrogenase into the medium,. Exposure of VL-17A cells to 100mM ethanol significantly elevated caspase 3 activity, an indicator of apoptosis, but this ethanol concentration did not affect caspase 3 activity in parental HepG2 cells. Because ethanol consumption causes a decline in hepatic protein catabolism, we examined the influence of ethanol exposure on proteasome activity in HepG2, VL-17A, E-47 (CYP2E1(+)) and VA-13 (ADH(+)) cells. Exposure to 100mM ethanol caused a 25% decline in the chymotrypsin-like activity of the proteasome in VL-17A cells, but the enzyme was unaffected in the other cell types. This inhibitory effect on the proteasome was blocked when ethanol metabolism was blocked by 4-methyl pyrazole. We conclude that recombinant VL-17A cells, which express both ADH and CYP2E1 exhibit hepatocyte-like characteristics in response to ethanol. Furthermore, the metabolism of ethanol by these cells via ADH and CYP2E1 is sufficient to bring about an inhibition of proteasome activity that may lead to apoptotic cell death.     

Inhibition of CYP2E1 leads to decreased malondialdehyde–acetaldehyde adduct formation in VL-17A cells under chronic alcohol exposure

AimEthanol metabolism leads to the formation of acetaldehyde and malondialdehyde. Acetaldehyde and malondialdehyde can together form malondialdehyde–acetaldehyde (MAA) adducts. The role of alcohol dehydrogenase (ADH) and cytochrome P4502E1 (CYP2E1) in the formation of MAA-adducts in liver cells has been investigated.Main methodsChronic ethanol treated VL-17A cells over-expressing ADH and CYP2E1 were pretreated with the specific CYP2E1 inhibitor — diallyl sulfide or ADH inhibitor — pyrazole or ADH and CYP2E1 inhibitor — 4-methyl pyrazole. Malondialdehyde, acetaldehyde or MAA-adduct formation was measured along with assays for viability, oxidative stress and apoptosis.Key findingsInhibition of CYP2E1 with 10 μM diallyl sulfide or ADH with 2 mM pyrazole or ADH and CYP2E1 with 5 mM 4-methyl pyrazole led to decreased oxidative stress and toxicity in chronic ethanol (100 mM) treated VL-17A cells. In vitro incubation of VL-17A cell lysates with acetaldehyde and malondialdehyde generated through ethanol led to increased acetaldehyde (AA)-, malondialdehyde (MDA)-, and MAA-adduct formation. Specific inhibition of CYP2E1 or ADH and the combined inhibition of ADH and CYP2E1 greatly decreased the formation of the protein aldehyde adducts. Specific inhibition of CYP2E1 led to the greatest decrease in oxidative stress, toxicity and protein aldehyde adduct formation, implicating that CYP2E1 accelerates the formation of protein aldehyde adducts which can be an important mechanism for alcohol mediated liver injury.SignificanceCYP2E1-mediated metabolism of ethanol leads to increased AA-, MDA-, and MAA-adduct formation in liver cells which may aggravate liver injury.     

Inhibition of CYP2E1 leads to decreased advanced glycated end product formation in high glucose treated ADH and CYP2E1 over-expressing VL-17A cells

Background

In recent years, there has been a growing interest to explore the association between liver injury and diabetes. Advanced glycated end product (AGE) formation which characterizes diabetic complications is formed through hyperglycemia mediated oxidative stress and is itself a source for ROS. Further, in VL-17A cells over-expressing ADH and CYP2E1, greatly increased oxidative stress and decreased viability have been observed with high glucose exposure.

Methods

In VL-17A cells treated with high glucose and pretreated with the different inhibitors of ADH and CYP2E1, the changes in cell viability, oxidative stress parameters and formation of AGE, were studied.

Results

Inhibition of CYP2E1 with 10 μM diallyl sulfide most effectively led to decreases in the oxidative stress and toxicity as compared with ADH inhibition with 2 mM pyrazole or the combined inhibition of ADH and CYP2E1 with 5 mM 4-methyl pyrazole. AGE formation was decreased in VL-17A cells when compared with HepG2 cells devoid of the enzymes. Further, AGE formation was decreased to the greatest extent with the inhibitor for CYP2E1 suggesting that high glucose inducible CYP2E1 and the consequent ROS aid AGE formation.

Conclusions

Thus, CYP2E1 plays a pivotal role in the high glucose induced oxidative stress and toxicity in liver cells as observed through direct evidences obtained utilizing the different inhibitors for ADH and CYP2E1.

General significance

The study demonstrates the role of CYP2E1 mediated oxidative stress in aggravating hyperglycemic insult and suggests that CYP2E1 may be a vital component of hyperglycemia mediated oxidative injury in liver.     

Detection of Mercury in Aquatic Environments Using EPRE Reporter Zebrafish

It has been proposed that transgenic zebrafish could be designed to detect low levels of chemical contaminants that cause oxidative stress in aquatic environments, such as heavy metals or pesticides. In this paper, we describe such a transgenic zebrafish that produces a luciferase–green fluorescent protein (LUC–GFP) fusion protein under conditions of oxidative stress. The reporter gene expression is under the regulation of the electrophile responsive element (EPRE), which activates gene expression in response to oxidative stressors. The GFP component of this fusion protein allows us to visually detect reporter gene activity in live animals to determine if activity is localized to a particular tissue. The luciferase component is capable of returning a quantitative assessment of reporter gene activity that allows us to determine if reporter gene activity is directly correlated to the concentration of the chemical inducer. We have tested this reporter construct in both transient and stable transgenic fish after exposure to a range of HgCl₂ concentrations. GFP expression from the EPRE–LUC–GFP construct was inducible in transient assays but was below the limit of detection in stable lines. In contrast, we observed inducible luciferase activity in both transient assays and stable lines treated with HgCl₂. We conclude that the EPRE is capable of driving reporter gene expression in a whole animal assay under conditions of oxidative stress. Furthermore, expression was induced at HgCl₂ concentrations that do not result in obvious morphological defects, making this approach useful for the detection of low levels of oxidative contaminants in aquatic environments.     

Suppression of Grb2 expression improved hepatic steatosis, oxidative stress, and apoptosis induced by palmitic acid in vitro partly through insulin signaling alteration

In this study, we aimed to study the role of growth factor receptor-bound protein 2 (Grb2) in palmitic acid-induced steatosis and other “fatty liver” symptoms in vitro. HepG2 cells, with or without stably suppressed Grb2 expression, were incubated with palmitic acid for 24 h to induce typical clinical “fatty liver” features, including steatosis, impaired glucose metabolism, oxidative stress, and apoptosis. MTT and Oil Red O assays were applied to test cell viability and fat deposition, respectively. Glucose uptake assay was used to evaluate the glucose utilization of cells. Quantitative polymerase chain reaction and Western blot were used to measure expressional changes of key markers of insulin signaling, lipid/glucose metabolism, oxidative stress, and apoptosis. After 24-h palmitic acid induction, increased fat accumulation, reduced glucose uptake, impaired insulin signaling, enhanced oxidative stress, and increased apoptosis were observed in HepG2 cells. Suppression of Grb2 in HepG2 significantly reduced fat accumulation, improved glucose metabolism, ameliorated oxidative stress, and restored the activity of insulin receptor substrate-1/Akt and MEK/ERK pathways. In addition, Grb2 deficiency attenuated hepatic apoptosis shown by reduced activation of caspase-3 and fluorescent staining. Modulation of Bcl-2 and Bak1 also contributed to reduced apoptosis. In conclusion, suppression of Grb2 expression in HepG2 cells improved hepatic steatosis, glucose metabolism, oxidative stress, and apoptosis induced by palmitic acid incubation partly though modulating the insulin signaling pathway.     

miR-23b-3p regulates apoptosis and autophagy via suppressing SIRT1 in lens epithelial cells

Age-related cataract is one of the prior causes of blindness and the incidence rates of cataract are even rising. Oxidative stress plays an important role in the pathogenesis of cataracts. Under oxidative stress, lens epithelial cell (LEC cell) apoptosis is activated, which might lead to the opacity of the lens and accelerate the progression of cataract development. Meanwhile, autophagy is also active to face oxidative stress. miRNAs have been reported to involve cataract. However, the underlying mechanism is not clear. The present study aimed to investigate the regulatory effect of miR23b-3p on apoptosis and autophagy in LEC cells under oxidative stress. The expression levels of miR-23b-3p were examined in age-related cataract tissues and LEC cells treated with hydrogen peroxide, showing that miR23b-3p expression levels were upregulated. Knockdown of miR23b-3p expression in LEC cells brought about apoptosis significantly decreased while autophagy significantly increased during hydrogen peroxide. We predicted microRNA miRNA-23b-3p might participate in regulating silent information regulator 1 (SIRT1) by bioinformatics database of TargetScan. Luciferase reporter assays confirmed that miRNA-23b-p could suppress SIRT1 expression by binding its 3′UTR. In addition, overexpression or knockdown of miR-23b-3p could decrease or increase SIRT1 expression, which indicated that Mir-23b-3p could suppress SIRT1 expression. In addition, enhanced SIRT1 could attenuate the regulation of cell apoptosis and autophagy induced by overexpression of miR-23b-3p. Taken together, our findings revealed that miR-23b-3p regulated apoptosis and autophagy via suppressing SIRT1 in LEC cell under oxidative stress, which could provide new ideas for clinical treatment of cataract.     

GM-CSF provides autocrine protection for murine alveolar epithelial cells from oxidant-induced mitochondrial injury

Exposure of mice to hyperoxia induces alveolar epithelial cell (AEC) injury, acute lung injury and death. Overexpression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the lung protects against these effects, although the mechanisms are not yet clear. Hyperoxia induces cellular injury via effects on mitochondrial integrity, associated with induction of proapoptotic members of the Bcl-2 family. We hypothesized that GM-CSF protects AEC through effects on mitochondrial integrity. MLE-12 cells (a murine type II cell line) and primary murine type II AEC were subjected to oxidative stress by exposure to 80% oxygen and by exposure to H(2)O(2). Exposure to H(2)O(2) induced cytochrome c release and decreased mitochondrial reductase activity in MLE-12 cells. Incubation with GM-CSF significantly attenuated these effects. Protection induced by GM-CSF was associated with Akt activation. GM-CSF treatment also resulted in increased expression of the antiapoptotic Bcl-2 family member, Mcl-1. Primary murine AEC were significantly more tolerant of oxidative stress than MLE-12 cells. In contrast to MLE-12 cells, primary AEC expressed significant GM-CSF at baseline and demonstrated constitutive activation of Akt and increased baseline expression of Mcl-1. Treatment with exogenous GM-CSF further increased Akt activation and Mcl-1 expression in primary AEC. Conversely, suppression of AEC GM-CSF expression by use of GM-CSF-specific small interfering RNA resulted in decreased tolerance of oxidative stress, Furthermore, silencing of Mcl-1 prevented GM-CSF-induced protection. We conclude that GM-CSF protects alveolar epithelial cells against oxidative stress-induced mitochondrial injury via the Akt pathway and its downstream components, including Mcl-1. Epithelial cell-derived GM-CSF may contribute to intrinsic defense mechanisms limiting lung injury.     

H2S, endoplasmic reticulum stress, and apoptosis of insulin-secreting beta cells

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway, which uses L-cysteine to produce hydrogen sulfide (H2S). Functional changes of pancreatic beta cells induced by endogenous H2S have been reported, but the effect of the CSE/H2S system on pancreatic beta cell survival has not been known. In this study, we demonstrate that H2Sat physiologically relevant concentrations induced apoptosis of INS-1E cells, an insulin-secreting beta cell line. Transfection of INS-1E cells with a recombinant defective adenovirus containing the CSE gene (Ad-CSE) resulted in a significant increase in CSE expression and H2S production. Ad-CSE transfection also stimulated apoptosis. The other two end products of CSE-catalyzed enzymatic reaction, ammonium and pyruvate, had no effects on INS-1E cell apoptosis, indicating that overexpression of CSE may stimulate INS-1E cell apoptosis via increased endogenous production of H2S. Both exogenous H2S (100 microM) and Ad-CSE transfection inhibited ERK1/2 but activated p38 MAPK. Interestingly, BiP and CHOP, two indicators of endoplasmic reticulum (ER) stress, were up-regulated in H2S-and CSE-mediated apoptosis in INS-1E cells. After suppressing CHOP mRNA expression, H2S-induced apoptosis of INS-1E cells was significantly decreased. Inhibition of p38 MAPK, but not of ERK1/2, inhibited the expression of BiP and CHOP and decreased H2S-stimulated apoptosis, suggesting that p38 MAPK activation functions upstream of ER stress to initiate H2S-induced apoptosis. It is concluded that H2S induces apoptosis of insulin-secreting beta cells by enhancing ER stress via p38 MAPK activation. Our findings may help unmask a novel role of CSE/H2S system in regulating pancreatic functions under physiological condition and in diabetes.     

A rapid in vivo zebrafish model to elucidate oxidative stress-mediated PCB126-induced apoptosis and developmental toxicity

Dioxin-like 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) is one of the most potent and widespread environmental pollutants. Although PCB126-induced toxicity is related to the aryl hydrocarbon receptor pathway, there is still no study that has constructed an in vivo visual model to clarify the role of the Nrf2/ARE signaling pathway in the oxidative stress mechanism of PCB126-induced toxicity. In the present study, an in vivo zebrafish model of nrf2a fused to enhanced green fluorescent protein (nrf2a-eGFP) was constructed. The zebrafish embryos microinjected with nrf2a-eGFP (72 h postfertilization) were exposed to various concentrations of PCB126 (0, 25, 50, 100, 200 μg/L) or 30 mM N-acetylcysteine (NAC)+200 μg/L PCB126. After 72 h exposure, PCB126 significantly increased the malformation rates and induced eGFP expression in a dose-dependent manner in several zebrafish tissue types. The distribution of eGFP fluorescence coincided with developmental deformity sites. NAC pretreatment effectively counteracted PCB126-induced developmental toxicity including heart rate, pericardial edema, and body length. The highest PCB126 dose, 200 μg/L, produced marked apoptosis in the eye, gill, and trunk detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. At 48 and 72 h exposure, 200 μg/L PCB126 affected glutathione metabolism as evidenced by decreased glutathione and increased glutathione disulfide concentrations, indicative of oxidative stress. These effects were also counteracted by NAC pretreatment. Furthermore, the Nrf2-regulated genes gclc, gpx, gstp1, and hmox1 were significantly induced at 24, 48, and 72 h at the highest PCB126 exposures but not in the NAC-pretreated group. In addition, a significant increase in ROS generation was detected in zebrafish larvae at 72 h PCB126 exposure, which might offer a link for future mechanistic studies. Collectively, these data suggest that PCB126-induced developmental toxicity and apoptosis in the nrf2a-eGFP-injected zebrafish model are due to oxidative stress mediated by disruption to glutathione metabolism and changes in Nrf2-regulated gene expression.     

Cholesterol induces pancreatic β cell apoptosis through oxidative stress pathway

Type 2 diabetes is often associated with high blood cholesterol. Here, we investigated the effect of cholesterol loading on MIN6 cells derived from pancreatic β cells. Exposure of MIN6 cells to cholesterol-induced apoptosis in time- and dose-dependent manner. Treatment with methyl-β-cyclodextrin that removes cholesterol from plasma membrane prevented the cells from cholesterol-induced apoptosis. Western blot analysis revealed that the levels of phosphorylated-p38 mitogen-activated protein kinase (P-p38 MAPK) and c-Jun N-terminal kinases (P-JNK) were significantly increased after the cholesterol loading, suggesting that the stress-activated protein kinase signaling was stimulated. A specific p38 inhibitor rescued MIN6 cells from cholesterol-induced apoptosis, while JNK inhibitor failed, suggesting the importance of activation of p38 MAPK signaling in response to cholesterol. The expression of Bip and CHOP, the endoplasmic reticulum (ER) stress markers, remained unaffected, indicating that the ER stress may not be involved in the cytotoxicity of cholesterol on the ΜΙΝ6 cells. The intracellular concentration of reactive oxygen species measured by use of 2′,7′-dichlorofluorescin diacetate was significantly increased after cholesterol loading, demonstrating the induced apoptosis was mediated through oxidative stress. Addition of reduced form of glutathione in the medium rescued MIN6 cells from apoptosis induced by cholesterol loading. Taken together, these results demonstrate that the free cholesterol loading can induce apoptosis of MIN6 cells mediated by oxidative stress and the activation of p38 MAPK signaling.     

Oxidative stress induces the expression of Fas and Fas ligand and apoptosis in murine intestinal epithelial cells

Intestinal epithelial cell function is compromised by local immune and inflammatory responses. In this study, we examined the possibility that intestinal epithelial cell injury occurs in the presence of activated inflammatory cells, such as neutrophils and macrophages, via production of reactive oxygen species (ROS). Following exposure to 50–150 μM H₂O₂, levels of mRNA and protein for Fas and, to a lesser degree, Fas-L were increased and intestinal epithelial cells underwent apoptosis. Treatment of H₂O₂-exposed cells with agonistic anti-Fas antibody, but not isotype control antibody, significantly enhanced apoptosis. Apoptosis was associated with the activation of caspase 8, while Z-IETD, an inhibitor of caspase 8, blocked apoptosis of H₂O₂-exposed intestinal epithelial cells. Thus, ROS induced Fas and Fas-L expression in association with intestinal epithelial cell apoptosis. These data support the hypothesis that, following exposure to oxidative stress, enterocytes are primed for cell death via Fas-mediated pathways.     

FBXL10 regulates cardiac dysfunction in diabetic cardiomyopathy via the PKC β2 pathway

Diabetic cardiomyopathy (DCM) is a condition associated with significant structural changes including cardiac tissue necrosis, localized fibrosis, and cardiomyocyte hypertrophy. This study sought to assess whether and how FBXL10 can attenuate DCM using a rat streptozotocin (STZ)‐induced DCM model system. In the current study, we found that FBXL10 expression was significantly decreased in diabetic rat hearts. FBXL10 protected cells from high glucose (HG)‐induced inflammation, oxidative stress, and apoptosis in vitro. In addition, FBXL10 significantly activated PKC β2 signaling pathway in H9c2 cells and rat model. The cardiomyocyte‐specific overexpression of FBXL10 at 12 weeks after the initial STZ administration attenuated oxidative stress and inflammation, thereby reducing cardiomyocyte death and preserving cardiac function in these animals. Moreover, FBXL10 protected against DCM via activation of the PKC β2 pathway. In conclusion, FBXL has the therapeutic potential for the treatment of DCM.