Resveratrol and 4-hydroxynonenal act in concert to increase glutamate cysteine ligase expression and glutathione in human bronchial epithelial cells

Resveratrol has been shown to protect against oxidative stress through modulating antioxidant capacity. In this study, we investigated resveratrol-mediated induction of glutathione (GSH) and glutamate cysteine ligase (GCL), and the combined effect of resveratrol and 4-hydroxynonenal (HNE) on GSH synthesis in cultured HBE1 human bronchial epithelial cells. Resveratrol increased GSH and the mRNA contents of both the catalytic (GCLC) and modulatory subunit (GCLM) of GCL. Combined HNE and resveratrol treatment increased GSH content and GCL mRNAs to a greater extent than either compound did alone. Compared to individual agent, combining exposure to HNE and resveratrol also showed more protection against cell death caused by oxidative stress. These effects of combined exposure were additive rather than synergistic. In addition, Nrf2 silencing significantly decreased the combined effect of HNE and resveratrol on GCL induction. Our data suggest that resveratrol increases GSH and GCL gene expression and that there is an additive effect on GSH synthesis between resveratrol and HNE. The results also reveal that Nrf2-EpRE signaling was involved in the combined effects.     

4-hydroxynonenal induces glutamate cysteine ligase through JNK in HBE1 cells

Glutathione is the most abundant non-protein thiol in the cell, with roles in cell cycle regulation, detoxification of xenobiotics, and maintaining the redox tone of the cell. The glutathione content is controlled at several levels, the most important being the rate of de novo synthesis, which is mediated by two enzymes, glutamate cysteine ligase (GCL), and glutathione synthetase (GS), with GCL being rate-limiting generally. The GCL holoenzyme consists of a catalytic (GCLC) and a modulatory (GCLM) subunit, which are encoded by separate genes. In the present study, the signaling mechanisms leading to de novo synthesis of GSH in response to physiologically relevant concentrations of 4-hydroxy-2-nonenal (4HNE), an endproduct of lipid peroxidation, were investigated. We demonstrated that exposure to 4HNE resulted in increased content of both Gcl mRNAs, both GCL subunits, phosphorylated JNK1 and c-Jun proteins, as well as Gcl TRE sequence-specific AP-1 binding activity. These increases were attenuated by pretreating the cells with a novel membrane-permeable JNK pathway inhibitor, while chemical inhibitors of the p38 or ERK pathways were ineffective. These data reveal that de novo GSH biosynthesis in response to 4HNE signals through the JNK pathway and suggests a major role for AP-1 driven expression of both Gcl genes in HBE1 cells.     

Induction of glutathione synthesis and heme oxygenase 1 by the flavonoids butein and phloretin is mediated through the ERK/Nrf2 pathway and protects against oxidative stress

Butein and phloretin are chalcones that are members of the flavonoid family of polyphenols. Flavonoids have well-known antioxidant and anti-inflammatory activities. In rat primary hepatocytes, we examined whether butein and phloretin affect tert-butylhydroperoxide (tBHP)-induced oxidative damage and the possible mechanism(s) involved. Treatment with butein and phloretin markedly attenuated tBHP-induced peroxide formation, and this amelioration was reversed by l-buthionine-S-sulfoximine [a glutamate cysteine ligase (GCL) inhibitor] and zinc protoporphyrin [a heme oxygenase 1 (HO-1) inhibitor]. Butein and phloretin induced both HO-1 and GCL protein and mRNA expression and increased intracellular glutathione (GSH) and total GSH content. Butein treatment activated the ERK1/2 signaling pathway and increased Nrf2 nuclear translocation, Nrf2 nuclear protein-DNA binding activity, and ARE-luciferase reporter activity. The roles of the ERK signaling pathway and Nrf2 in butein-induced HO-1 and GCL catalytic subunit (GCLC) expression were determined by using RNA interference directed against ERK2 and Nrf2. Both siERK2 and siNrf2 abolished butein-induced HO-1 and GCLC protein expression. These results suggest the involvement of ERK2 and Nrf2 in the induction of HO-1 and GCLC by butein. In an animal study, phloretin was shown to increase GSH content and HO-1 expression in rat liver and decrease carbon tetrachloride-induced hepatotoxicity. In conclusion, we demonstrate that butein and phloretin up-regulate HO-1 and GCL expression through the ERK2/Nrf2 pathway and protect hepatocytes against oxidative stress.     

Variable regulation of glutamate cysteine ligase subunit proteins affects glutathione biosynthesis in response to oxidative stress

Glutamate cysteine ligase (GCL), composed of a catalytic (GCLC) and modulatory (GCLM) subunit, catalyzes the first step of glutathione (GSH) biosynthesis. Using 4-hydroxy-2-nonenal (4HNE), 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and tertiary-butylhydroquinone (tBHQ) as models of oxidative stress which are known to work through different mechanisms, we measured changes in cellular GSH, GCL mRNA, and GCL protein. 4HNE and tBHQ treatments increased cellular GSH levels, while DMNQ exposure depleted GSH. Furthermore, changes in the two GCL mRNAs largely paralleled changes in the GCL proteins; however, the magnitudes differed, suggesting some form of translational control. The molar ratio of GCLC:GCLM ranged from 3:1 to 17:1 in control human bronchial epithelial (HBE1) cells and all treatments further increased this ratio. Data from several mouse tissues show molar ratios of GCLC:GCLM that range from 1:1 to 10:1 in support of these findings. These data demonstrate that alterations in cellular GSH are clearly correlated with GCLC to a greater extent than GCLM. Surprisingly, both control HBE1 cells and some mouse tissues have more GCLC than GCLM and GCLM increases to a much lesser extent than GCLC, suggesting that the regulatory role of GCLM is minimal under physiologically relevant conditions of oxidative stress.     

Thyroid hormone promotes glutathione synthesis in astrocytes by up regulation of glutamate cysteine ligase through differential stimulation of its catalytic and modulator subunit mRNAs

To elucidate how thyroid hormone (TH) modulates glutathione (GSH) biogenesis in developing brain, the effect of the hormone on the activity of glutamate cysteine ligase (GCL), previously known as gamma-glutamyl synthetase (gamma-GCS), has been investigated. Hypothyroidism in developing rat brain declined the activity of GCL. Conversely, administration of TH to hypothyroid rats elicited an increase in the activity of the enzyme. TH treatment of astrocytes resulted in a rapid increase in the level of GSH and this up regulation was completely inhibited by L-buthionine S,R-sulfoximine. Kinetics of induction of GCL by TH in astrocytes were closely parallel to that of GSH and the induction was sensitive to both cycloheximide and actinomycin D. Quantitative RT-PCR analysis revealed that astrocytes contained a basal excess of GCLC (catalytic subunit of GCL) mRNA, relative to GCLM (modulator subunit of GCL) mRNA, the ratio being 4:1. TH treatment led to a differential increase in the expression of these two mRNAs, which resulted in a decline in the stoichiometric ratio of GCLC:GCLM mRNA that may favor holoenzyme formation with enhanced catalytic efficiency. TH treatment improved the antioxidative defense in astrocytes by enhancing their hydrogen peroxide scavenging ability with a decrease in peroxide half-life from 7.4 to 4.2 min. The overall results suggest that TH plays a positive role in maintaining GSH homeostasis in astrocytes and in protecting the brain from oxidative stress.     

Differential roles of 3H-1,2-dithiole-3-thione-induced glutathione, glutathione S-transferase and aldose reductase in protecting against 4-hydroxy-2-nonenal toxicity in cultured cardiomyocytes

4-hydroxy-2-nonenal (HNE) plays an important role in the pathogenesis of cardiac disorders. While conjugation with glutathione (GSH) catalyzed by GSH S-transferase (GST) has been suggested to be a major detoxification mechanism for HNE in target cells, whether chemically upregulated cellular GSH and GST afford protection against HNE toxicity in cardiac cells has not been investigated. In addition, the differential roles of chemically induced GSH and GST as well as other cellular factors in detoxifying HNE in cardiomyocytes are unclear. In this study, we have characterized the induction of GSH and GST by 3H-1,2-dithiole-3-thione (D3T) and the protective effects of the D3T-elevated cellular defenses on HNE-mediated toxicity in rat H9C2 cardiomyocytes. Treatment of cardiomyocytes with D3T resulted in a significant induction of both GSH and GST as well as the mRNA expression of gamma-glutamylcysteine ligase catalytic subunit and GSTA. Both GSH and GST remained elevated for at least 72 h after removal of D3T from the culture media. Treatment of cells with HNE led to a significant decrease in cell viability and an increased formation of HNE-protein adducts. Pretreatment of cells with D3T dramatically protected against HNE-mediated cytotoxicity and protein-adduct formation. HNE treatment caused a significant decrease in cellular GSH level, which preceded the loss of cell viability. Either depletion of cellular GSH by buthionine sulfoximine (BSO) or inhibition of GST by sulfasalazine markedly sensitized the cells to HNE toxicity. Co-treatment of cardiomyocytes with BSO was found to completely block the D3T-mediated GSH elevation, which however failed to reverse the cytoprotective effects of D3T, suggesting that other cellular factor(s) might be involved in D3T cytotprotection. In this regard, D3T was shown to induce cellular aldose reductase (AR). Surprisingly, inhibition of AR by sorbinil failed to potentiate HNE toxicity in cardiomyocytes. In contrast, sorbinil dramatically augmented HNE cytotoxicity in cells with GSH depletion induced by BSO. Similarly, in BSO-treated cells, D3T cytoprotection was also largely reversed by sorbinil, indicating that AR played a significant role in detoxifying HNE only under the condition of GSH depletion in cardiomyocytes. Taken together, this study demonstrates that D3T can induce GSH, GST, and AR in cardiomyocytes, and that the above cellular factors appear to play differential roles in detoxification of HNE in cardiomyocytes.     

Differential sensitivity to 4-hydroxynonenal for normal and malignant mesenchymal cells

Normal and malignant cells of various origin differ in their sensitivity to oxidative stress. Therefore, we used normal and malignant mesenchymal cells--human osteosarcoma cells (HOS and 143B), human fibroblasts (WI38) and two primary cultures of normal human osteoblasts to test sensitivity to reactive aldehyde 4-hydroxynonenal (HNE), known as a second messenger of free radicals and a signaling molecule. Upon HNE-treatment, decrease in cell viability (by Trypan-blue), apoptosis induction (by TiterTACS TUNEL assay), HNE-protein binding (by HNE-His ELISA) were higher in malignant than in normal cells, while glutathione content was higher in normal cells. These results indicate that HNE affects the growth of malignant mesenchymal cells more than normal and that this effect was mainly related to lower glutathione concentration and higher binding of HNE to the cellular proteins. We thus assume that HNE and GSH homeostasis play an important role in the growth regulation of normal and malignant mesenchymal cells.     

Induction of lung glutathione and glutamylcysteine ligase by 1,4-phenylenebis(methylene)selenocyanate and its glutathione conjugate: role of nuclear factor-erythroid 2-related factor 2

The synthetic organoselenium agent 1,4-phenylenebis(methylene)selenocyanate (p-XSC) and its glutathione (GSH) conjugate (p-XSeSG) are potent chemopreventive agents in several preclinical models. p-XSC is also an effective inducer of GSH in mouse lung. Our objectives were to test the hypothesis that GSH induction by p-XSC occurs through upregulation of the rate-limiting GSH biosynthetic enzyme glutamylcysteine ligase (GCL), through activation of antioxidant response elements (AREs) in GCL genes via activation of nuclear factor-erythroid 2-related factor 2 (Nrf2). p-XSC feeding (10 ppm Se) increased GSH (230%) and upregulated the catalytic subunit of GCL (GCLc) (55%), extracellular-related kinase (220%), and nuclear Nrf2 (610%) in lung but not liver after 14 days in the rat (P<0.05). Similarly, p-XSeSG feeding (10 ppm) induced lung GCLc (88%) and GSH (200%) (P<0.05), whereas the naturally occurring selenomethionine had no effect. Both p-XSC and p-XSeSG activated a luciferase reporter in HepG2 ARE-reporter cells up to threefold for p-XSC and greater than or equal to fivefold for p-XSeSG. Luciferase activation by p-XSeSG was associated with enhanced levels of GSH, GCLc, and nuclear Nrf2, which were significantly reduced by co-incubation with short interfering RNA targeting Nrf2. The dependence of GCL induction on Nrf2 was confirmed in Nrf2-deficient mouse embryonic fibroblasts, in which p-XSeSG induced GCL subunits in wild-type but not in Nrf2-deficient cells (P<0.05). These results indicate that p-XSC may act through the Nrf2 pathway in vivo and that p-XSeSG is the putative metabolite responsible for such activation, thus offering p-XSeSG as a less toxic, yet highly efficacious, inducer of GSH.     

Involvement of PI3K/PKG/ERK1/2 signaling pathways in cortical neurons to trigger protection by cotreatment of acetyl-L-carnitine and alpha-lipoic acid against HNE-mediated oxidative stress and neurotoxicity: implications for Alzheimer's disease

Oxidative stress has been shown to underlie neuropathological aspects of Alzheimer's disease (AD). 4-Hydroxy-2-nonenal (HNE) is a highly reactive product of lipid peroxidation of unsaturated lipids. HNE-induced oxidative toxicity is a well-described model of oxidative stress-induced neurodegeneration. GSH plays a key role in antioxidant defense, and HNE exposure causes an initial depletion of GSH that leads to gradual toxic accumulation of reactive oxygen species. In the current study, we investigated whether pretreatment of cortical neurons with acetyl-L-carnitine (ALCAR) and alpha-lipoic acid (LA) plays a protective role in cortical neuronal cells against HNE-mediated oxidative stress and neurotoxicity. Decreased cell survival of neurons treated with HNE correlated with increased protein oxidation (protein carbonyl, 3-nitrotyrosine) and lipid peroxidation (HNE) accumulation. Pretreatment of primary cortical neuronal cultures with ALCAR and LA significantly attenuated HNE-induced cytotoxicity, protein oxidation, lipid peroxidation, and apoptosis in a dose-dependent manner. Additionally, pretreatment of ALCAR and LA also led to elevated cellular GSH and heat shock protein (HSP) levels compared to untreated control cells. We have also determined that pretreatment of neurons with ALCAR and LA leads to the activation of phosphoinositol-3 kinase (PI3K), PKG, and ERK1/2 pathways, which play essential roles in neuronal cell survival. Thus, this study demonstrates a cross talk among the PI3K, PKG, and ERK1/2 pathways in cortical neuronal cultures that contributes to ALCAR and LA-mediated prosurvival signaling mechanisms. This evidence supports the pharmacological potential of cotreatment of ALCAR and LA in the management of neurodegenerative disorders associated with HNE-induced oxidative stress and neurotoxicity, including AD.     

Inhibition of Autophagy Potentiated the Antitumor Effect of Nedaplatin in Cisplatin-Resistant Nasopharyngeal Carcinoma Cells

Nedaplatin, a cisplatin analog, was developed to reduce the toxicity of cisplatin, whereas it can be cross-resistant with cisplatin in some circumstances. This study aimed to investigate the role of autophagy in nedaplatin induced cell death in cisplatin-resistant nasopharyngeal carcinoma cells. Here, we showed that HNE1/DDP and CNE2/DDP cells were resistant to nedaplatin-induced cell death with reduced apoptotic activity. Nedaplatin treatment resulted in autophagosome accumulation and increased expression of LC3-II, indicating the induction of autophagy by nedaplatin in HNE1/DDP and CNE2/DDP cells. Inhibition of autophagy by Bafilomycin A1 (Baf A1) and 3-Methyladenine (3-MA) remarkably enhanced the antitumor efficacy of nedaplatin in HNE1/DDP and CNE2/DDP cells, suggesting that the resistance to nedaplatin-induced cell death was caused by enhanced autophagy in nedaplatin-resistant NPC cells. Additionally, Baf A1 enhanced reactive oxygen species (ROS) generation and apoptosis induced by nedaplatin in HNE1/DDP cells. Mechanistically, nedaplatin treatment caused activation of ERK1/2 and suppression of Akt/mTOR signaling pathways. While inhibition of ERK1/2 by MEK1/2 inhibitor, U0126, could reduce the expression of LC3-II in nedaplatin-resistant NPC cells. Furthermore, suppression of ROS could inhibit nedaplatin-induced ERK activation in HNE1/DDP cells, indicating that ROS and ERK were involved in nedaplatin-induced autophagy. Together, these findings suggested that autophagy played a cytoprotective role in nedaplatin-induced cytotoxicity of HNE1/DDP and CNE2/DDP cells. Furthermore, our results highlighted a potential approach to restore the sensitivity of cisplatin-resistant nasopharyngeal cancer cells to nedaplatin in combination with autophagy inhibitors.     

Multiple signaling conduits regulate global differentiation-specific gene expression in PC12 cells

PC12 cells serve as a model for exploring nerve growth factor (NGF)-stimulated signal pathways that mediate neural differentiation. We previously demonstrated that neurofilament light chain (NFLC) gene induction by NGF requires collaborative extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling. Herein, we investigate the broader requirement for integrated ERK and JNK signaling in NGF-stimulated gene expression. NGF stimulates differentiation as well as maintenance of cell viability while insulin-like growth factor-1 (IGF-1) stimulates only trophic actions in PC12 cells. Affymetrix Genechips were used to identify genes whose expression specifically increased in response to NGF, but not IGF-1. From the set of NGF-specific genes, the induction by NGF of ten genes with diverse predicted cellular functions was tested for ERK and JNK pathway requirements using the protein kinase inhibitors, PD98059 and SP600125, respectively. Like NFLC, induction of urokinase plasminogen activator (uPAR), transin/matrix metalloproteinase 3 (MMP3), Fra-1 and transforming growth factor beta 1 (TGF beta 1) required collaborative ERK and JNK signaling while the increased expression of cortexin, rat collapsin response mediator protein 4 (rCRMP4), rat growth and transformation-dependent protein (RGT), and synapsin II required neither mitogen-activated protein kinase (MAPK) pathway. NGF-induction of the bradykinin B2 receptor and c-Ret mRNAs was partially inhibited by SP600125, but not PD98059. Reporter constructs containing the promoters for ERK/JNK-dependent genes (NFLC, transin, uPAR) as well as an ERK/JNK-independent gene (synapsin II) revealed that both sets of genes required functional Ras signaling for activation by NGF. Integrated signaling through the ERK and JNK MAPKs, therefore, represents a general conduit for NGF-dependent gene expression, but additional Ras-dependent signaling pathways distinct from the ERKs and JNKs must contribute as well. Thus, multiple signaling conduits control global differentiation-specific gene expression in PC12 cells.     

Cumulus cell‐enclosed oocytes acquire a capacity to synthesize GSH by FSH stimulation during in vitro maturation in pigs

We investigated (i) follicle stimulating hormone (FSH)‐modulated changes in the expression of glutathione (GSH) and its rate‐limiting enzyme, glutamate cysteine ligase (GCL), in porcine oocytes and cumulus cells, and (ii) the contribution of gap‐junctional communications (GJCs) in cumulus‐oocyte complexes (COCs) to intraoocyte GSH accumulation. In experiment (i), COCs were cultured for 48 h with (+FSH group) or without FSH (−FSH group). The GSH content of oocytes increased with cultivation time in the +FSH group, but decreased in the −FSH group. The GSH content of cumulus cells at 48 h was also higher in the +FSH group than that in the −FSH group. Expression of GCL subunit mRNAs in oocytes and cumulus cells was increased by FSH stimulation until 12 h, and then fell to the baseline level. On the other hand, the amount of GCL subunit proteins in oocytes and cumulus cells increased gradually throughout the period of culture with FSH. In experiment (ii), blocking of GJCs in COCs during 0–24 h of culture led to a decrease in the GSH content of oocytes at 24 h of culture, whereas the GSH content at 48 h of culture did not differ even after blocking of the GJCs during 24–48 h of culture. These findings indicate that FSH initiates GSH synthesis in cumulus cells and oocytes by modulating the expression of GCL, and that porcine oocytes are able to synthesize GSH without GJC‐mediated support from cumulus cells, at least in the later half of maturation culture. J. Cell. Physiol. 222: 294–301, 2010. © 2009 Wiley‐Liss, Inc.