Peroxynitrite induces HO-1 expression via PI3K/Akt-dependent activation of NF-E2-related factor 2 in PC12 cells

Peroxynitrite is a strong oxidant produced by rapid interaction between superoxide anion and nitric oxide radicals and induces oxidative stress and cell death. Treatment of PC12 cells with 3-morpholinosydnonimine (SIN-1), a generator of peroxynitrite, induced the expression of heme oxygenase-1 (HO-1), an antioxidant cytoprotective enzyme. Inhibition of the HO activity by zinc protoporphyrin IX or knockdown of HO-1 gene expression with siRNA exacerbated the SIN-1-induced apoptosis. After SIN-1 treatment, there was a time-related increase in nuclear localization and subsequent binding of NF-E2-related factor 2 (Nrf2) to the antioxidant-responsive element (ARE). Transfection of PC12 cells with dominant-negative Nrf2 abolished the SIN-1-induced increase in Nrf2-ARE binding and subsequent upregulation of HO-1 expression, leading to enhanced cell death. Upon exposure of PC12 cells to SIN-1, the phosphatidylinositol 3-kinase (PI3K) activity was increased in a time-dependent manner. Pretreatment of cells with LY294002, a pharmacologic inhibitor of PI3K or transfection with the kinase-dead mutant Akt abrogated the SIN-1-induced Nrf2 activation and HO-1 expression. Taken together, these results suggest that peroxynitrite activates Nrf2 via PI3K/Akt signaling and enhances Nrf2-ARE binding, which leads to upregulation of HO-1 expression. The SIN-1-induced HO-1 upregulation may confer the adaptive survival response against nitrosative stress.     

Salvianolic acid A protects RPE cells against oxidative stress through activation of Nrf2/HO-1 signaling

Reactive oxygen species (ROS) impair the physiological functions of retinal pigment epithelial (RPE) cells, which is known as one major cause of age-related macular degeneration. Salvianolic acid A (Sal A) is the main effective aqueous extract of Salvia miltiorrhiza. The aim of this study was to test the potential role of Sal A against oxidative stress in cultured RPE cells and to investigate the underlying mechanistic signaling pathways. We observed that Sal A significantly inhibited hydrogen peroxide (H₂O₂)-induced primary and transformed RPE cell death and apoptosis. H₂O₂-stimulated mitogen-activated protein kinase activation, ROS production, and subsequent proapoptotic AMP-activated protein kinase activation were largely inhibited by Sal A. Further, Sal A stimulation resulted in a fast and dramatic activation of Akt/mammalian target of rapamycin complex 1 (mTORC1) signaling, followed by phosphorylation, accumulation, and nuclear translocation of the NF-E2-related factor 2 (Nrf2), along with increased expression of the antioxidant-response element-dependent gene heme oxygenase-1 (HO-1). Both Nrf2 and HO-1 were required for Sal A-mediated cytoprotective effect, as Nrf2/HO-1 inhibition abolished Sal A-induced beneficial effects against H₂O₂. Meanwhile, the PI3K/Akt/mTORC1 chemical inhibitors not only suppressed Sal A-induced Nrf2/HO-1 activation, but also eliminated its cytoprotective effect in RPE cells. These observations suggest that Sal A activates the Nrf2/HO-1 axis in RPE cells and protects against oxidative stress via activation of Akt/mTORC1 signaling.     

Involvement of heme oxygenase-1 induction via Nrf2/ARE activation in protection against H2O2-induced PC12 cell death by a metabolite of sesamin contained in sesame seeds

Induction of phase II antioxidant enzymes by activation of Nrf2/ARE (antioxidant response element) signaling has been considered as a promising strategy to combat with oxidative stress-related diseases. In the present study, we tested for potential effects of sesamin, a major lignan contained in sesame seeds, its stereoisomer episesamin, and their metabolites on Nrf2/ARE activation in rat pheochromocytoma PC12 cells. Luciferase reporter assays showed that primary metabolites of sesamin and episesamin, SC-1 and EC-1 were the most potent ARE activators among all tested compounds. SC-1 {(1R,2S,5R,6S)-6-(3,4-dihydroxyphenyl)-2-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo-[3,3,0]octane} enhanced nuclear translocation of Nrf2 and up-regulated expression of phase II antioxidant enzymes including heme oxygenase-1 (HO-1). Treatment with SC-1 resulted in increased phosphorylation of p38 MAP kinase and transient increase in intracellular ROS levels. N-acetylcysteine (NAC) treatment abolished p38 phosphorylation as well as HO-1 induction caused by SC-1, indicating that ROS are upstream signals of p38 in Nrf2/ARE activation by SC-1. Furthermore, preconditioning with SC-1 attenuated H(2)O(2)-induced cell death in a dose-dependent manner. Finally, treatment with a HO-1 inhibitor, Zn-protoporphyrin (ZnPP), and overexpression of a dominant-negative mutant of Nrf2 diminished SC-1-mediated neuroprotection. Our results demonstrate that SC-1 is capable of protecting against oxidative stress-induced neuronal cell death in part through induction of HO-1 via Nrf2/ARE activation, suggesting its potential to reduce oxidative stress and ameliorate oxidative stress-related neurodegenerative diseases.     

3',4'-didemethylnobiletin induces phase II detoxification gene expression and modulates PI3K/Akt signaling in PC12 cells

Oxidative stress is considered a major cause of neurodegenerative disorders. In this work, we investigated the cytoprotective effects and mechanisms of the citrus flavonoid nobiletin (NOB) and its metabolite, 3',4'-didemethylnobiletin (3',4'-dihydroxy-5,6,7,8-tetramethoxyflavone; DTF), in PC12 cells. Both NOB and DTF exhibited strong potency in attenuating serum withdrawal- and H(2)O(2)-caused cell death and increased intracellular GSH level via upregulation of both catalytic and modifier subunits of glutamate-cysteine ligase (GCL). However, only DTF suppressed intracellular ROS accumulation in H(2)O(2)-treated cells, induced heme oxygenase-1 (HO-1) expression, and enhanced nuclear factor E2-related factor 2 (Nrf2) binding to the ARE. Nevertheless, DTF-mediated HO-1 upregulation was independent of Nrf2 activation because knockdown of Nrf2 expression by siRNA did not affect its expression. DTF suppressed NF-κB activation, and addition of NF-κB inhibitor, pyrrolidine dithiocarbamate or Bay 11-7082, synergistically enhanced DTF-mediated HO-1 expression, indicating that HO-1 induction is associated with NF-κB suppression. NOB and DTF also activated the ERK, JNK, and Akt pathways in PC12 cells that had undergone serum starvation. Addition of pharmacological kinase inhibitors, U0126, SP600125, and LY294002, caused cytotoxicity and the last significantly attenuated NOB- and DTF-mediated antiapoptotic actions, indicating the involvement of PI3K/Akt signaling in their cytoprotective effects. In conclusion, HO-1 and GCL upregulation and intrinsic ROS-scavenging activity may contribute to DTF-mediated cytoprotection. Furthermore, modulation of PI3K/Akt signaling is involved in channeling the DTF stimulus for cell survival against oxidative insults.     

Significance of HLA class I antibody-induced antioxidant gene expression for endothelial cell protection against complement attack

It has been observed that a graft organ continues to survive and function normally even in the presence of anti-graft antibodies. However, the mechanisms behind acquirement of this condition remain unknown. Here we report that the anti-HLA ligation on endothelial cells induces PI3K/AKT activation followed by antioxidant gene induction through Nrf2-mediated antioxidant-responsive element (ARE) activation. Activation of PI3K/AKT in endothelial cells by a low concentration of anti-HLA ligation enhances protection from complement attack. A real-time quantitative PCR and flow-cytometry experiment showed that ferritin H and HO-1 mRNAs were induced in a PI3K/AKT-dependent manner, while CD55 and CD59 expression were not enhanced by anti-HLA ligation. Anti-HLA ligation on endothelial cells activates ferritin H ARE and induces Nrf2 binding on its enhancer element. Finally, overexpression of Nrf2 in endothelial cells attenuates complement-mediated cytotoxicity. These experiments suggest that induction of PI3K/AKT-dependent cytoprotective genes by Nrf2 is an important mechanism to prevent complement attack. Thus, a protocol to activate this pathway would be a potential strategy for avoidance of graft rejection in transplantation.     

A novel kavalactone derivative protects against H2O2-induced PC12 cell death via Nrf2/ARE activation

Oxidative stress is involved in the pathogenesis of neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. Natural kavalactones isolated from Piper methysticum (Piperaceae) are capable of activating the Nrf2/ARE (antioxidant response element) pathway and thus enhancing the expression of phase II antioxidant enzymes such as heme oxygenase-1 (HO-1). In an attempt to identify kavalactone derivatives that are more potent in Nrf2/ARE activation than natural compounds, we synthesized a series of chemically-modified kavalactones and studied their effects on the ARE enhancer activity in rat pheochromocytoma PC12 cells. Among 81 compounds tested, a kavalactone derivative, 2′,6′-dichloro-5-methoxymethyl-5,6-dehydrokawain [(E)-6-(2′,6′-dichlorostyryl)-4-methoxy-5-(methoxymethyl)-2H-pyran-2-one] (1), exhibited the strongest ARE enhancer activity. The ARE activation and HO-1 protein induction by the compound 1 were higher than those by natural kavalactones. The compound did not affect cell viability and induced expression of various phase II enzymes. Nuclear translocation of Nrf2 after treatment with 1 was preceded by phosphorylation of ERK1/2 and p38. The compound transiently increased intracellular ROS levels. Finally, pretreatment with the compound ameliorated H₂O₂-induced cell death, which was associated with increased expression of HO-1. These results suggest that the compound 1 protects against oxidative stress-induced neuronal cell death via a preconditioning effect on the Nrf2/ARE activation.     

Preconditioning by sesquiterpene lactone enhances H2O2-induced Nrf2/ARE activation

The Nrf2/ARE pathway plays a pivotal role in chemoprevention and neuroprotection. Here, we report that sesquiterpene lactones extracted from Calea urticifolia and feverfew increased enhancer activity of the ARE. ARE activation was dependent on the number of α,β-unsaturated carbonyl groups each compound bears and calealactone A (CL-A) harboring 3 of those was the most potent ARE inducer. At subtoxic doses, CL-A induced expression of heme oxygenase-1 (HO-1) gene, one of ARE target genes, through activation of the Nrf2/ARE pathway involving transient ROS generation and activation of PI3-K/Akt and MAPK pathways. Interestingly, H₂O₂-induced ARE activation and HO-1 induction were potentiated by pretreatment with CL-A at lower concentrations, at which Nrf2/ARE activation by the compound was minimal. These results suggest a possibility that preconditioning by sesquiterpene lactone may enhance activation of the Nrf2/ARE pathway and induction of phase II detoxification/antioxidant enzymes upon oxidative stress, thereby resulting in increased resistance to oxidative damage.     

Dermato-protective properties of ergothioneine through induction of Nrf2/ARE-mediated antioxidant genes in UVA-irradiated Human keratinocytes

UVA irradiation-induced skin damage and redox imbalance have been shown to be ameliorated by ergothioneine (EGT), a naturally occurring sulfur-containing amino acid. However, the responsible molecular mechanism with nanomolar concentrations of EGT remains unclear. We investigated the dermato protective efficacies of EGT (125–500 nM) against UVA irradiation (15 J/cm²), and elucidated the underlying molecular mechanism in human keratinocyte-derived HaCaT cells. We found that EGT treatment prior to UVA exposure significantly increased the cell viability and prevented lactate dehydrogenase release into the medium. UVA-induced ROS and comet-like DNA formation were remarkably suppressed by EGT with a parallel inhibition of apoptosis, as evidenced by reduced DNA fragmentation (TUNEL), caspase-9/-3 activation, and Bcl-2/Bax dysregulation. Furthermore, EGT alleviated UVA-induced mitochondrial dysfunction. Dose-dependent increases of antioxidant genes, HO-1, NQO-1, and γ-GCLC and glutathione by EGT were associated with upregulated Nrf2 and downregulated Keap-1 expressions. This was confirmed by increased nuclear accumulation of Nrf2 and inhibition of Nrf2 degradation. Notably, augmented luciferase activity of ARE may explain Nrf2/ARE-mediated signaling pathways behind EGT dermato-protective properties. We further demonstrated that Nrf2 translocation was mediated by PI3K/AKT, PKC, or ROS signaling cascades. This phenomenon was confirmed with suppressed nuclear Nrf2 activation, and consequently diminished antioxidant genes in cells treated with respective pharmacological inhibitors (LY294002, GF109203X, and N-acetylcysteine). Besides, increased basal ROS by EGT appears to be crucial for triggering the Nrf2/ARE signaling pathways. Silencing of Nrf2 or OCTN1 (EGT carrier protein) signaling with siRNA showed no such protective effects of EGT against UVA-induced cell death, ROS, and apoptosis, which is evidence of the vitality of Nrf2 translocation and protective efficacy of EGT in keratinocytes. Our findings conclude that EGT at nanomolar concentrations effectively ameliorated UVA-induced skin damage, and may be considered as a desirable food supplement for skin protection and/or preparation of skin care products.     

Genipin up-regulates heme oxygenase-1 via PI3-kinase-JNK1/2-Nrf2 signaling pathway to enhance the anti-inflammatory capacity in RAW264.7 macrophages

Genipin, an aglycon of geniposide, has been reported to exhibit diverse pharmacological functions such as antitumor and anti-inflammatory effects. This study aimed to elucidate the anti-inflammatory mechanism of genipin, focusing particularly on the role of heme oxygenase-1 (HO-1), a potent anti-inflammatory enzyme. In RAW264.7 cells, genipin increased HO-1 expression and its enzyme activity via a NF-E2-related factor 2 (Nrf2)–antioxidant response element (ARE) pathway. These effects were significantly inhibited by exposure to the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, or by expression of a dominant negative mutant of PI 3-kinase. Additional experiments showed that the activation of c-Jun NH₂-terminal kinase 1/2 (JNK1/2) is required for genipin-induced phosphorylation and nuclear translocation of Nrf2 and antioxidant response element (ARE)-driven induction of HO-1, and acts as a downstream effector of PI 3-kinase. Furthermore, functional significance of HO-1 induction was revealed by genipin-mediated inhibition of lipopolysaccharide-stimulated inducible nitric oxide synthase expression or cyclooxygenase-2 promoter activity, the response was reversed by the blocking of HO-1 protein synthesis or HO-1 enzyme activity. Therefore, identification of PI 3-kinase-JNK1/2-Nrf2-linked signaling cascade in genipin-mediated HO-1 expression defines the signaling event that could participate in genipin-mediated anti-inflammatory response.