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.     

Activation of the UPR Protects against Cigarette Smoke-induced RPE Apoptosis through Up-Regulation of Nrf2

Recent studies have revealed a role of endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) in the regulation of RPE cell activity and survival. Herein, we examined the mechanisms by which the UPR modulates apoptotic signaling in human RPE cells challenged with cigarette smoking extract (CSE). Our results show that CSE exposure induced a dose- and time-dependent increase in ER stress markers, enhanced reactive oxygen species (ROS), mitochondrial fragmentation, and apoptosis of RPE cells. These changes were prevented by the anti-oxidant NAC or chemical chaperone TMAO, suggesting a close interaction between oxidative and ER stress in CSE-induced apoptosis. To decipher the role of the UPR, overexpression or down-regulation of XBP1 and CHOP genes was manipulated by adenovirus or siRNA. Overexpressing XBP1 protected against CSE-induced apoptosis by reducing CHOP, p-p38, and caspase-3 activation. In contrast, XBP1 knockdown sensitized the cells to CSE-induced apoptosis, which is likely through a CHOP-independent pathway. Surprisingly, knockdown of CHOP reduced p-eIF2α and Nrf2 resulting in a marked increase in caspase-3 activation and apoptosis. Furthermore, Nrf2 inhibition increased ER stress and exacerbated cell apoptosis, while Nrf2 overexpression reduced CHOP and protected RPE cells. Our data suggest that although CHOP may function as a pro-apoptotic gene during ER stress, it is also required for Nrf2 up-regulation and RPE cell survival. In addition, enhancing Nrf2 and XBP1 activity may help reduce oxidative and ER stress and protect RPE cells from cigarette smoke-induced damage.     

Carbon monoxide protects PC12 cells from peroxynitrite-induced apoptotic death by preventing the depolarization of mitochondrial transmembrane potential

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in catalyzing heme degradation into biliverdin, free iron, and carbon monoxide (CO), serves as a protective enzyme against oxidative and nitrosative stresses. In the present study, we investigated the cytoprotective effects of HO-1 upregulation and its product CO against the peroxynitrite-induced PC12 cell death. PC12 cells treated with 3-morphoinosydonimine (SIN-1), a generator of peroxynitrite (ONOO-), underwent apoptotic cell death as evidenced by dissipation of mitochondrial transmembrane potential (DeltaPsim), release of mitochondrial cytochrome c into cytoplasm, cleavage of poly(ADP-ribose)polymerase and fragmentation of internucleosomal DNA. Pretreatment of PC12 cells with a low non-toxic concentration of SIN-1 (0.5 mM) induced HO-1 expression and abrogated the cell death caused by subsequent challenge with high dose SIN-1 (2.5 mM). Furthermore, pretreatment of PC12 cells with SnCl2, a potent inducer of HO-1 expression, increased endogenous production of CO (HO activity) and rescued the PC12 cells from peroxynitrite-induced apoptosis. The cytoprotective effect of SnCl2 was abolished when the HO activity was inhibited by zinc protoporphyrin IX (ZnPP IX). PC12 cells treated directly with the CO-releasing molecule, tricarbonyldichlororuthenium (II) dimer ([Ru(CO)3Cl2]2) became tolerant to the depolarization of DeltaPsim and apoptosis induced by high dose peroxynitrite. Taken together, these data demonstrate that the adaptive protection against peroxynitrite-induced apoptotic death in PC12 cells is mediated by CO formed as a consequence of HO-1 induction.     

Carbon monoxide produced by heme oxygenase-1 in response to nitrosative stress induces expression of glutamate-cysteine ligase in PC12 cells via activation of phosphatidylinositol 3-kinase and Nrf2 signaling

Induction of heme oxygenase-1 (HO-1) expression has been associated with adaptive cytoprotection against a wide array of toxic insults, but the underlying molecular mechanisms remain largely unresolved. In this study, we investigated the potential role of carbon monoxide (CO), one of the by-products of the HO-1 reaction, in the adaptive survival response to peroxynitrite-induced PC12 cell death. Upon treatment of rat pheochromocytoma (PC12) cells with the peroxynitrite generator 3-morpholinosydnonimine hydrochloride (SIN-1), the cellular GSH level decreased initially, but was gradually restored to the basal level. This was accompanied by increased expression of the catalytic subunit of glutamate-cysteine ligase (GCLC), the rate-limiting enzyme in GSH biosynthesis. The SIN-1-induced GCLC up-regulation was preceded by induction of HO-1 and subsequent CO production. Inhibition of HO activity by zinc protoporphyrin IX or knockdown of HO-1 gene expression by small interfering RNA abrogated the up-regulation of GCLC expression and the subsequent GSH restoration induced by SIN-1. In contrast, additional exposure to the CO-releasing molecule (CO-RM) restored the GSH level previously reduced by inhibition of CO production using zinc protoporphyrin IX. Furthermore, CO-RM treatment up-regulated GCLC expression through activation of Nrf2. The CO-RM-induced activation of Nrf2 was under the control of the phosphatidylinositol 3-kinase/Akt signaling pathway. In conclusion, CO produced by HO-1 rescues PC12 cells from nitrosative stress through induction of GCLC, which is mediated by activation of phosphatidylinositol 3-kinase/Akt and subsequently Nrf2 signaling.     

Peroxynitrite activates NF-E2-related factor 2/antioxidant response element through the pathway of phosphatidylinositol 3-kinase: the role of nitric oxide synthase in rat glutathione S-transferase A2 induction.

The protective adaptive response to electrophiles and reactive oxygen species is mediated by the induction of phase II detoxifying genes through antioxidant response elements (AREs). Our previous study showed that sulfur amino acid deprivation (SAAD) produces peroxides and induces rat glutathione S-transferase A2 (rGSTA2) through NF-E2-related factor 2 (Nrf2)/ARE activation via the pathway of phosphatidylinositol 3-kinase (PI3-kinase). The current study was designed to investigate the role of peroxynitrite in Nrf2/ARE activation and rGSTA2 induction. L-Arginine deficiency or N(G)-nitro-L-arginine methyl ester (L-NAME) reduced peroxide production induced by SAAD in H4IIE cells. Northern and Western blot analyses revealed that the levels of rGSTA2 mRNA and protein were significantly increased 24h after incubation of the cells in SAAD medium, which was inhibited by L-arginine deficiency or L-NAME. Subcellular fractionation and gel shift analyses revealed that SAAD increased the level of nuclear Nrf2 and activated ARE, which were also blocked by L-arginine deficiency or L-NAME. Whereas the exogenous NO donor S-nitroso-N-acetyl-penicillamine (SNAP) alone failed to significantly induce rGSTA2, SNAP enhanced SAAD-inducible rGSTA2 expression, verifying the notion that peroxynitrite derived from NO contributes to rGSTA2 induction. 3-Morpholinosydnonimine (SIN-1), which decomposes and yields peroxynitrite, increased the rGSTA2 mRNA and protein levels in a dose-dependent manner. SIN-1 increased the level of nuclear Nrf2 and activated Nrf2/ARE, which was supershifted by anti-Nrf2 and anti-Maf antibodies. SIN-1 increased the activity of PI3-kinase, as monitored by phosphorylation of Akt. SIN-1-inducible rGSTA2 expression was inhibited by PI3-kinase inhibitors. These results provide evidence that peroxynitrite plays an essential role in nuclear translocation of Nrf2 and ARE activation through the pathway of PI3-kinase and that nitric oxide synthase is involved in the induction of rGSTA2.     

DDIT3/CHOP promotes autophagy in chondrocytes via SIRT1-AKT pathway

Endoplasmic reticulum (ER) stress can initiate autophagy via unfolded protein response (UPR). As a key downstream gene of UPR, DDIT3/CHOP is expressed in chondrocytes. However, the regulation mechanism of DDIT3/CHOP on autophagy in chondrocytes remains unclear. In this study, the expression levels of autophagic markers Beclin1 and LC3B were found to decrease while p62 increase in the tibial growth plate and costal primary chondrocytes from DDIT3/CHOP KO mice. In vitro, overexpressing DDIT3/CHOP induced autophagy in ATDC5 chondrocytes, displaying an elevated immunofluorescence signal of LC3B and elevated numbers of autophagosomes and autolysosomes. Analysis of the gain- and loss-of-function indicated that the protein level of Beclin1 and the ratio of LC3BII/I increased in DDIT3/CHOP overexpression cells, whereas decreased in DDIT3/CHOP knockdown cells. The decreased level of p62 and additional accumulation of LC3BII caused by chloroquine (CQ) further indicated that DDIT3/CHOP enhanced autophagic flux. Mechanistically, we found that DDIT3/CHOP binds directly to the promoter of SIRT1 to promote its expression by CHIP, qRT-PCR, and Western blot analysis. In addition, SIRT1 enhanced autophagic activity in ATDC5 cells, and inhibition or activation of SIRT1 partially reversed the effect of overexpressing or downregulating DDIT3/CHOP on autophagy. Furthermore, AKT signaling was found to be responsible for DDIT3/CHOP-regulated autophagy in ATDC5 cells. SIRT1 knockdown reversed the effect of DDIT3/CHOP overexpression on AKT signaling. In conclusion, our data clarifies that DDIT3/CHOP promotes autophagy in ATDC5 chondrocytes through the SIRT1-AKT pathway. These results were also confirmed in the primary chondrocytes.     

Docosahexaenoic acid inhibition of inflammation is partially via cross-talk between Nrf2/heme oxygenase 1 and IKK/NF-κB pathways

We examined the underlying mechanisms involved in n-3 docosahexaenoic acid (DHA) inhibition of inflammation in EA.hy926 cells. The present results demonstrated that pretreatment with DHA (50 and 100 μM) inhibited tumor necrosis factor-alpha (TNF-α)-induced intercellular adhesion molecule 1 (ICAM-1) protein, mRNA expression and promoter activity. In addition, TNF-α-stimulated inhibitory kappa B (IκB) kinase (IKK) phosphorylation, IκB phosphorylation and degradation, p65 nuclear translocation, and nuclear factor-κB (NF-κB) and DNA binding activity were attenuated by pretreatment with DHA. DHA triggered early-stage and transient reactive oxygen species (ROS) generation and significantly increased the protein expression of heme oxygenase 1 (HO-1), induced nuclear factor erythroid 2-related factor 2 (Nrf2) translocation to the nucleus and up-regulated antioxidant response element (ARE)-luciferase reporter activity. Moreover, DHA inhibited Nrf2 ubiquitination and proteasome activity. DHA activated Akt, p38 and ERK1/2 phosphorylation, and specific inhibitors of respective pathways attenuated DHA-induced Nrf2 nuclear translocation and HO-1 expression. Transfection with HO-1 siRNA knocked down HO-1 expression and partially reversed the DHA-mediated inhibition of TNF-α-induced p65 nuclear translocation and ICAM-1 expression. Importantly, we show for the first time that HO-1 plays a down-regulatory role in NF-κB nuclear translocation, and inhibition of Nrf2 ubiquitination and proteasome activity are involved in increased cellular Nrf2 level by DHA. In this study, we show that HO-1 plays a down-regulatory role in NF-κB nuclear translocation and that the protective effect of DHA against inflammation is partially via up-regulation of Nrf2-mediated HO-1 expression and inhibition of IKK/NF-κB signaling pathway.     

Sofalcone, a gastric mucosa protective agent, increases vascular endothelial growth factor via the Nrf2-heme-oxygenase-1 dependent pathway in gastric epithelial cells

Sofalcone, 2′-carboxymethoxy-4,4-bis(3-methyl-2-butenyloxy)chalcone, is an anti-ulcer agent that is classified as a gastric mucosa protective agent. Recent studies indicate heat shock proteins such as HSP32, also known as heme-oxygenase-1(HO-1), play important roles in protecting gastrointestinal tissues from several stresses. We have previously reported that sofalcone increases the expression of HO-1 in adipocytes and pre-adipocytes, although the effect of sofalcone on HO-1 induction in gastrointestinal tissues is not clear. In the current study, we investigated the effects of sofalcone on the expression of HO-1 and its functional role in rat gastric epithelial (RGM-1) cells. We found that sofalcone increased HO-1 expression in RGM-1 cells in both time- and concentration-dependent manners. The HO-1 induction was associated with the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in RGM-1 cells. We also observed that sofalcone increased vascular endothelial growth factor (VEGF) production in the culture medium. Treatment of RGM-1 cells with an HO-1 inhibitor (tin-protoporphyrin), or HO-1 siRNA inhibited sofalcone-induced VEGF production, suggesting that the effect of sofalcone on VEGF expression is mediated by the HO-1 pathway. These results suggest that the gastroprotective effects of sofalcone are partly exerted via Nrf2-HO-1 activation followed by VEGF production.     

Inhibition of stearoyl-CoA desaturase 1 expression induces CHOP-dependent cell death in human cancer cells

Background

Cancer cells present a sustained de novo fatty acid synthesis with an increase of saturated and monounsaturated fatty acid (MUFA) production. This change in fatty acid metabolism is associated with overexpression of stearoyl-CoA desaturase 1 (Scd1), which catalyses the transformation of saturated fatty acids into monounsaturated fatty acids (e.g., oleic acid). Several reports demonstrated that inhibition of Scd1 led to the blocking of proliferation and induction of apoptosis in cancer cells. Nevertheless, mechanisms of cell death activation remain to be better understood.

Principal Findings

In this study, we demonstrated that Scd1 extinction by siRNA triggered abolition of de novo MUFA synthesis in cancer and non-cancer cells. Scd1 inhibition-activated cell death was only observed in cancer cells with induction of caspase 3 activity and PARP-cleavage. Exogenous supplementation with oleic acid did not reverse the Scd1 ablation-mediated cell death. In addition, Scd1 depletion induced unfolded protein response (UPR) hallmarks such as Xbp1 mRNA splicing, phosphorylation of eIF2α and increase of CHOP expression. However, the chaperone GRP78 expression, another UPR hallmark, was not affected by Scd1 knockdown in these cancer cells indicating a peculiar UPR activation. Finally, we showed that CHOP induction participated to cell death activation by Scd1 extinction. Indeed, overexpression of dominant negative CHOP construct and extinction of CHOP partially restored viability in Scd1-depleted cancer cells.

Conclusion

These results suggest that inhibition of de novo MUFA synthesis by Scd1 extinction could be a promising anti-cancer target by inducing cell death through UPR and CHOP activation.     

Epigallocatechin-gallate stimulates NF-E2-related factor and heme oxygenase-1 via caveolin-1 displacement

Flavonoids, such as the tea catechin epigallocatechin-gallate (EGCG), can protect against atherosclerosis by decreasing vascular endothelial cell inflammation. Heme oxygenase-1 (HO-1) is an enzyme that plays an important role in vascular physiology, and its induction may provide protection against atherosclerosis. Heme oxygenase-1 can be compartmentalized in caveolae in endothelial cells. Caveolae are plasma microdomains important in vesicular transport and the regulation of signaling pathways associated with the pathology of vascular diseases. We hypothesize that caveolae play a role in the uptake and transport of EGCG and mechanisms associated with the anti-inflammatory properties of this flavonoid. To test this hypothesis, we explored the effect of EGCG on the induction of NF-E2-related factor (Nrf2) and HO-1 in endothelial cells with or without functional caveolae. Treatment with EGCG activated Nrf2 and increased HO-1 expression and cellular production of bilirubin. In addition, EGCG rapidly accumulated in caveolae, which was associated with caveolin-1 displacement from the plasma membrane towards the cytosol. Similar to EGCG treatment, silencing of caveolin-1 by siRNA technique also resulted in up-regulation of Nrf2, HO-1 and bilirubin production. These data suggest that EGCG-induced caveolin-1 displacement may reduce endothelial inflammation.     

CO-releasing molecules and increased heme oxygenase-1 induce protein S-glutathionylation to modulate NF-κB activity in endothelial cells

Protein glutathionylation is a protective mechanism that functions in response to mild oxidative stress. Carbon monoxide (CO) can increase the reactive oxygen species concentration from a low level via the inhibition of cytochrome c oxidase. We therefore hypothesized that CO would induce NF-κB-p65 glutathionylation and then show anti-inflammatory effects. In this study, we found that CO-releasing molecules suppress TNFα-induced monocyte adhesion to endothelial cells (ECs) and reduce ICAM-1 expression. Moreover, CO donors were further found to exert their inhibitory effects by blocking NF-κB-p65 nuclear translocation, but do so independent of IκBα degradation, in TNFα-treated ECs. In addition, p65 protein glutathionylation represents the response signal to CO donors and is reversed by the reducing agent dithiothreitol. Thiol modification of the cysteine residue in the p65 RHD region was required for the CO-modulated NF-κB activation. The suppression of p65 glutathionylation by a GSH synthesis inhibitor, BSO, and by catalase could also attenuate TNFα-induced p65 nuclear translocation and ICAM-1 expression. CO donors induce Nrf2 activation and Nrf2 siRNA suppresses CO-induced p65 glutathionylation and inhibition. Furthermore, we found that the CO donors induce heme oxygenase-1 (HO-1) expression, which increases p65 glutathionylation. In contrast, HO-1 siRNA attenuates CO donor- and hemin-induced p65 glutathionylation. Our results thus indicate that the glutathionylation of p65 is likely to be responsible for CO-mediated NF-κB inactivation and that the HO-1-dependent pathway may prolong the inhibitory effects of CO donors upon TNFα treatment of ECs.     

Sulforaphane induces autophagy through ERK activation in neuronal cells

Sulforaphane (SFN), an activator of nuclear factor E2-related factor 2 (Nrf2), has been reported to induce autophagy in several cells. However, little is known about its signaling mechanism of autophagic induction. Here, we provide evidence that SFN induces autophagy with increased levels of LC3-II through extracellular signal-regulated kinase (ERK) activation in neuronal cells. Pretreatment with NAC (N-acetyl-l-cysteine), a well-known antioxidant, completely blocked the SFN-induced increase in LC3-II levels and activation of ERK. Knockdown or overexpression of Nrf2 did not affect autophagy. Together, the results suggest that SFN-mediated generation of reactive oxygen species (ROS) induces autophagy via ERK activation, independent of Nrf2 activity in neuronal cells.     

Antiarrhythmic effect of lithium in rats after myocardial infarction by activation of Nrf2/HO-1 signaling

Glycogen synthase kinase-3 (GSK-3) signaling has been shown to play a role in the regulation of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of antioxidant genes, including heme oxygenase-1 (HO-1). We assessed whether lithium, a GSK-3 inhibitor, attenuates cardiac sympathetic reinnervation after myocardial infarction, a status of high reactive oxygen species (ROS), by attenuating nerve growth factor (NGF) expression and whether Nrf2/HO-1 signaling is involved in the protection. Twenty-four hours after ligation of the left anterior descending artery, male Wistar rats were treated for 4 weeks. The postinfarction period was associated with increased oxidative–nitrosative stress, as measured by myocardial superoxide, nitrotyrosine, and dihydroethidium fluorescent staining. In concert, myocardial norepinephrine levels and immunohistochemical analysis of sympathetic nerve revealed a significant increase in innervation in vehicle-treated rats compared with sham-operated rats. Arrhythmic scores during programmed stimulation in the vehicle-treated rats were significantly higher than those in sham. This was paralleled by a significant upregulation of NGF protein and mRNA in the vehicle-treated rats, which was reduced after administration of LiCl. LiCl stimulated the nuclear translocation of Nrf2 and the transactivation of the Nrf2 target gene HO-1. Inhibition of phosphoinositide 3-kinase by wortmannin reduced the increase in Nrf2 nucleus translocation and HO-1 expression compared with lithium alone. In addition, the lithium-attenuated NGF levels were reversed in the presence of the Nrf2 inhibitor trigonelline, HO-1 inhibitor SnPP, and peroxynitrite generator SIN-1, indicating the role of Nrf2/HO-1/ROS. In conclusion, lithium protects against ventricular arrhythmias by attenuating NGF-induced sympathetic innervation via antioxidant activation of the Nrf2/HO-1 axis.