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

Abstract

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. 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. Heme oxygenase (HO) 2The abbreviations used are: HOheme oxygenaseMAPKmitogen-activated protein kinaseROSreactive oxygen speciesGCLglutamate-cysteine ligaseGCLCglutamate-cysteine ligase catalytic subunitGCLMglutamate-cysteine ligase modifier subunitPI3Kphosphatidylinositol 3-kinaseAREantioxidant-responsive elementCO-RMCO-releasing moleculeBSObuthionine (SR)-sulfoximineMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideZnPP IXzinc protoporphyrin IXPBSphosphate-buffered salinesiRNAsmall interfering RNARTreverse transcriptionDNdominant-negative. is the rate-limiting enzyme involved in the oxidative degradation of free heme and produces biliverdin/bilirubin, free iron, and carbon monoxide (CO) (1Maines M.D. FASEB J. 1988; 2: 2557-2568Crossref PubMed Scopus (1583) Google Scholar). Of the two known isoforms of HO (i.e. HO-1 and HO-2), the expression of only HO-1 is induced in response to external stresses. Formerly known as the stress response protein HSP32 in rats, HO-1 confers cytoprotection against oxidative and inflammatory injuries caused by a wide array of noxious or pathogenic insults (2Vile G.F. Basu-Modak S. Waltner C. Tyrrell R.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2607-2610Crossref PubMed Scopus (464) Google Scholar, 3Srisook K. Cha Y.-N. Nitric Oxide. 2005; 12: 70-79Crossref PubMed Scopus (63) Google Scholar, 4Abraham N.G. Kushida T. McClung J. Weiss M. Quan S. Lafaro R. Darzynkiewicz Z. Wolin M. Circ. Res. 2003; 93: 507-514Crossref PubMed Scopus (143) Google Scholar). In a previous study, we observed that up-regulation of HO-1 prevents the rat pheochromocytoma (PC12) cells from apoptotic death caused by peroxynitrite (5Li M.-H. Cha Y.-N. Surh Y.-J. Free Radic. Biol. Med. 2006; 41: 1079-1091Crossref PubMed Scopus (127) Google Scholar). CO produced by elevated HO activity has been found to be responsible, at least in part, for the anti-apoptotic function of up-regulated HO-1 (6Li M.-H. Cha Y.-N. Surh Y.-J. Biochem. Biophys. Res. Commun. 2006; 342: 984-990Crossref PubMed Scopus (48) Google Scholar, 7Liu X.M. Chapman G.B. Peyton K.J. Schafer A.I. Durante W. Cardiovasc. Res. 2002; 55: 396-405Crossref PubMed Scopus (161) Google Scholar, 8Choi B.M. Pae H.O. Chung H.T. Free Radic. Biol. Med. 2003; 34: 1136-1145Crossref PubMed Scopus (69) Google Scholar). However, the underlying mechanisms by which CO exerts the cytoprotective effects against peroxynitrite-induced cell death remain largely unresolved. heme oxygenase mitogen-activated protein kinase reactive oxygen species glutamate-cysteine ligase glutamate-cysteine ligase catalytic subunit glutamate-cysteine ligase modifier subunit phosphatidylinositol 3-kinase antioxidant-responsive element CO-releasing molecule buthionine (SR)-sulfoximine 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide zinc protoporphyrin IX phosphate-buffered saline small interfering RNA reverse transcription dominant-negative. CO is reported to increase the production of cGMP and to exert vasodilatory and anti-apoptotic effects (9Foresti R. Hammad J. Clark J.E. Johnson T.R. Mann B.E. Friebe A. Green C.J. Motterlini R. Br. J. Pharmacol. 2004; 142: 453-460Crossref PubMed Scopus (269) Google Scholar). Liu et al. (7Liu X.M. Chapman G.B. Peyton K.J. Schafer A.I. Durante W. Cardiovasc. Res. 2002; 55: 396-405Crossref PubMed Scopus (161) Google Scholar) reported that the anti-apoptotic function of CO is partially dependent on the activation of soluble guanylate cyclase in vascular smooth muscle cells. Furthermore, a recent study has revealed that the anti-apoptotic effect of CO is associated with activation of p38 mitogen-activated protein kinase (MAPK) (10Brouard S. Otterbein L.E. Anrather J. Tobiasch E. Bach F.H. Choi A.M. Soares M.P. J. Exp. Med. 2000; 192: 1015-1026Crossref PubMed Scopus (880) Google Scholar). In the HO-1-deficient cells, exogenous CO is able to suppress the excessive generation of reactive oxygen species (ROS) and expression of plasminogen activator inhibitor-1, leading to preservation of viability (11Matsumoto H. Ishikawa K. Itabe H. Maruyama Y. Mol. Cell. Biochem. 2006; 291: 21-28Crossref PubMed Scopus (115) Google Scholar). It is interesting that, as one of the end products of HO-1-catalyzed heme degradation, CO by itself has been shown to induce HO-1 expression via Nrf2 (NF-E2-related factor-2) activation in hepatoma cells (12Lee B.S. Heo J. Kim Y.M. Shim S.M. Pae H.O. Kim Y.M. Chung H.T. Biochem. Biophys. Res. Commun. 2006; 343: 965-972Crossref PubMed Scopus (86) Google Scholar). A ubiquitous cellular non-protein thiol, reduced GSH is an important antioxidant responsible for the maintenance of intracellular redox balance. The rate-limiting enzyme in GSH biosynthesis is glutamate-cysteine ligase (GCL; formerly known as γ-glutamylcysteine synthetase), which consists of catalytic (GCLC) and modifier (GCLM) subunits (13Griffith O.W. Mulcahy R.T. Adv. Enzymol. Relat. Areas Mol. Biol. 1999; 73: 209-267PubMed Google Scholar). Overexpression of both GCL subunits has been shown to elevate intracellular GSH levels and to protect cells from apoptosis (14Botta D. Franklin C.C. White C.C. Krejsa C.M. Dabrowski M.J. Pierce R.H. Fausto N. Kavanagh T.J. Free Radic. Biol. Med. 2004; 37: 632-642Crossref PubMed Scopus (39) Google Scholar, 15Mulcahy R.T. Bailey H.H. Gipp J.J. Cancer Res. 1995; 55: 4771-4775PubMed Google Scholar, 16Manna S.K. Kuo M.T. Aggarwal B.B. Oncogene. 1999; 18: 4371-4382Crossref PubMed Scopus (142) Google Scholar). As GSH plays a pivotal role in cellular defense against a wide array of toxic insults (17Lim S.Y. Jang J.-H. Na H.-K. Lu S.C. Rahman I. Surh Y.-J. J. Biol. Chem. 2004; 279: 46263-46270Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 18Trocino R.A. Akazawa S. Ishibashi M. Matsumoto K. Matsuo H. Yamamoto H. Goto S. Urata Y. Kondo T. Nagataki S. Diabetes. 1995; 44: 992-998Crossref PubMed Scopus (127) Google Scholar), we attempted to examine whether CO could elevate the GSH level in PC12 cells challenged with peroxynitrite, thereby providing cytoprotection against nitrosative stress. Here we report that the CO produced as a consequence of peroxynitrite-induced up-regulation of HO-1 expression induces the expression of GCLC via activation of phosphatidylinositol 3-kinase (PI3K)/Akt and subsequently Nrf2/antioxidant-responsive element (ARE) signaling. Materials—Tricarbonyldichlororuthenium(II) dimer ((Ru(CO)3 Cl2)2, a CO-releasing molecule (CO-RM)), buthionine (S, R)-sulfoximine (BSO), poly-d-lysine, N-acetyl-l-cysteine, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma. The peroxynitrite generator 3-morpholinosydnonimine hydrochloride (SIN-1) was supplied by BIOMOL (Plymouth Meeting, PA). Dulbecco's modified Eagle's medium, fetal bovine serum, horse serum, nutrient mixture F-12, and N-2 supplement were from protoporphyrin IX was the of was purchased from against was a from J. Kavanagh of and cells were at in Dulbecco's modified Eagle's with horse and fetal bovine in a of and cells were in were at an to the of and the was at a cells were to a N-2 supplement for treatment with CO-RM viability was by the as previously M.-H. Jang J.-H. Surh Y.-J. Free Radic. Biol. Med. 2005; PubMed Scopus Google Scholar). GSH intracellular GSH levels in PC12 cells were using a exposure to PC12 cells were and in of of in was to of by the of of the mixture was at for the of the was at The protein was using a protein treatment with or cells in a were with phosphate-buffered saline and was at by in cell at for the was and at The protein was using the protein with protein were on a were to at for protein the was for at using the in saline of and and and and were in with and the were with for at in additional with and the were for in to the and by exposure to of CO of CO from degradation of to the cell was by as by et al. K. Choi M.-H. H. Kim C. Cha Y.-N. Biochem. Pharmacol. 2005; PubMed Scopus Google Scholar). the of cells, were in and by and were to the the was at for for The was at by of through the of was and a with a Areas under the CO and the CO production were using the by the of activity was by the of production to the previously K. Cha Y.-N. Nitric Oxide. 2005; 12: 70-79Crossref PubMed Scopus (63) Google Scholar, R. S.K. M. Green C.J. Motterlini R. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). PC12 cells in a were with in the of and a mixture of and and at for at The was and at for at The were in and The was to the mixture of and of rat as the of were at for in the and the were in an at least for to the was by the in at and of for HO activity is as of of of HO-1 RNA and for HO-1 was to HO-1 expression using The rat and were by and the gene for and were by A. Johnson of Johnson Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar, Johnson Biochem. Biophys. 1999; PubMed Scopus Google Scholar). and with the were from Chung of and D. Kim S. H. Chung Chung J. FASEB J. PubMed Scopus Google Scholar). The GCLC was C. Lu of H. J. Lu S.C. Biochem. J. PubMed Scopus (48) Google Scholar). and cells were at a of in cells were with of or using to the the was and the cells were with The cells were with and in activity was using a and the protein was using the protein RNA was from PC12 cells using the was to The used in this study were as GCLC, and and Biochem. Biophys. 342: PubMed Scopus Google and and products were by with and under were purchased from of treatment with the PC12 cells in a were with by and in cell and in an for the cells were the was and the was in and at for the was and the of the was at for the The protein of was by the using the protein for the of the (ARE) was with using kinase and from the free by using a The of in the used in this study were and The was by to the of the of the was on for in and The were on a at for and to of was used to intracellular of treatment with CO-RM for in the or of N-acetyl-l-cysteine, cells on a were with and with a at cells were under a with an were as the and was used to for a The for was GCLC and GSH by by the from cells were with a used peroxynitrite and the GSH levels were at the intracellular GSH level decreased for to the GSH level increased and by the intracellular GSH was restored to the basal level whether restoration of GSH in cells is mediated via induction of GSH we the expression of GCLC, the rate-limiting enzyme in GSH in GCLC expression were at treatment with SIN-1. However, GCLC expression was increased at of exposure to HO-1 is an important antioxidant enzyme known to be induced by oxidative or that of GSH R. Clark J.E. Green C.J. Motterlini R. Biochem. J. 1999; PubMed Scopus Google Scholar, K. Cha Y.-N. Biochem. Pharmacol. 2004; PubMed Scopus Google Scholar). In with the expression of HO-1 protein was increased in PC12 cells as as exposure to However, the expression of the In with HO-1 the CO production from increased HO activity was elevated in a with a increase observed at the treatment whether the increase in GCLC expression is associated with the up-regulation of we the HO IX. IX only HO-1 activity but GCLC expression induced by in PC12 cells the of HO-1 induction with the SIN-1-induced up-regulation of GCLC we HO-1 expression knockdown of the gene The SIN-1-induced up-regulation of GCLC expression was by with of the PC12 cells with the of was with the of GCLC expression by IX treatment the GSH level was reduced It is interesting that the of GSH as a of inhibition of GCLC up-regulation was by the of exogenous CO from CO-RM with the of CO-RM elevated GSH that CO from the elevated HO-1 activity the induction of GCLC expression and subsequent GSH in PC12 cells challenged with of HO-1 in SIN-1-induced up-regulation of GCLC expression and GSH biosynthesis. effect of IX on the increase in HO activity as by production. were for in the and of IX and for an additional with effect of an of HO on SIN-1-induced GCLC PC12 cells were to for with and IX for of activation of GCLC expression as a consequence of HO-1 inhibition was by of activation of HO-1 expression by by effect of HO-1 on the SIN-1-induced up-regulation of GCLC protein were and as the of HO-1 to PC12 cells were with or with the of to effects of IX on SIN-1-induced GSH were for with or IX and with for in the or of CO-RM The intracellular GSH level was as under the the with CO-RM the GSH and against by a previous study, we reported that CO protect PC12 cells from SIN-1-induced apoptotic death (6Li M.-H. Cha Y.-N. Surh Y.-J. Biochem. Biophys. Res. Commun. 2006; 342: 984-990Crossref PubMed Scopus (48) Google Scholar). the by which CO could protect against apoptosis caused by we the level of GSH in cells with of PC12 cells with of CO-RM for the of GSH caused by exposure to for in a It is interesting that the GSH level in cells with the of CO-RM was that in control cells to SIN-1. examine whether the increased GSH biosynthesis induced by to CO-RM is responsible for the cytoprotective effect against the caused by we the cytoprotective effects of CO-RM in the and of an and of As shown in the CO-RM-induced of PC12 cells against SIN-1-induced was GSH was with the intracellular GSH level in PC12 cells with CO-RM was but the cells with the to the GSH biosynthesis that the anti-apoptotic effect of CO in PC12 cells is at least in part, by induced GSH from up-regulation of GCLC CO-RM GCLC the increased GSH biosynthesis CO-RM the effect of CO-RM on GCL expression was PC12 cells were with CO-RM up-regulation of GCLC and was at The induction of GCLC and gene transcription by CO-RM was by Furthermore, the GCLC activity was elevated CO-RM treatment as by the gene CO-RM GCLC through of of both GCL subunit known to that in the cellular response to oxidative stress R.T. Bailey H.H. Gipp J.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. Free Radic. Biol. Med. 2004; 37: PubMed Scopus Google Scholar). Nrf2 is a activator protein that the oxidative stress and is involved in the expression of and antioxidant H. K. Yamamoto M. Y. 2005; PubMed Scopus Google Scholar, M.J. K. Johnson J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). As in CO-RM treatment to a increase in the levels of Nrf2 protein in the CO-RM induced a increase in the of Nrf2 and the activity a the was in the the increase in activity was that the up-regulation of GCLC expression in PC12 cells is mediated by we a of Nrf2 with a In PC12 cells with the was a in the induction of activity The activity of was in cells with revealed that PC12 cells with levels of GCLC expression CO-RM treatment with control cells with the only PC12 cells to increase the GSH level exposure to CO-RM of SIN-1-induced GCLC up-regulation in PC12 cells by effect of on the activity of Nrf2 at CO-RM on activity on the effect of on the CO-RM-induced activity of activity was at CO-RM effect of on the CO-RM-induced up-regulation of GCLC expression at as by effect of on the CO-RM-induced increase in the GSH level. the the was used as a control CO-RM-induced Nrf2 of GCLC and against in PC12 via of the has been reported that and an important role in the activation of Nrf2 and the up-regulation of antioxidant enzyme gene expression in of cells T. Mann Free Radic. Biol. Med. 2005; PubMed Scopus Google Scholar, D. A.I. M. R. J. C.M. A. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, Mulcahy R.T. Sci. 2003; 73: PubMed Scopus Google Scholar). A increase in the of was observed in the PC12 cells, the level of of PC12 cells with CO-RM increased the of p38 which to the basal level at the of and p38 to cytoprotection against SIN-1-induced we of As in the abrogated the cytoprotection by CO-RM against cell death caused by of p38 the In with this the up-regulation of GCLC expression was by but by the of signaling in Nrf2 activation and induction of GCLC expression in PC12 cells to cells were with or with the and with expression of the of and the up-regulation of GCLC expression that the up-regulation of GCLC expression induced by CO-RM is mediated through activation of the of activation in adaptive GCLC and Nrf2 activation in PC12 cells. PC12 cells were for with the or the p38 in the of CO-RM and to for viability was by the the PC12 cells were with CO-RM for in the or of or GCLC protein expression was by and the levels were to protein PC12 cells were with or and to CO-RM for activity was by the the effects of on CO-RM-induced GCLC expression CO-RM-induced GCLC by CO has been reported to and to of leading to of in the C. J. S. J. Motterlini R. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). examine whether cells to CO-RM oxidative stress and the for up-regulation of GCLC expression and of intracellular was using a that is to the cell and that produces with as with CO-RM a increase in of the intracellular of of the cells with the antioxidant reduced the increase in the up-regulation of GCLC expression induced by CO-RM was by that the up-regulation of GCLC expression is mediated by production of which signaling. is a reactive and by and that produced under of peroxynitrite from and has been in the of and D. M.P. Google Scholar, J. Res. 1994; PubMed Scopus Google Scholar). However, the peroxynitrite produced in has been reported to as an intracellular molecule that induces the adaptive survival response under cellular (17Lim S.Y. Jang J.-H. Na H.-K. Lu S.C. Rahman I. Surh Y.-J. J. Biol. Chem. 2004; 279: 46263-46270Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, Choi Kim Nitric Oxide. 2002; PubMed Scopus Google Scholar, R.M. Free Radic. Biol. Med. PubMed Scopus Google Scholar). In of the adaptive survival response in PC12 cells challenged with peroxynitrite, we that exposure to a of caused a but of which was by a to the control level. It is interesting that restoration of the intracellular GSH was to up-regulated GCLC which was preceded by induction of HO-1 expression with production of inhibition of HO activity with the peroxynitrite-induced up-regulation of GCLC expression was but was restored by of an end of HO-1 in cells to SIN-1. that CO from elevated HO activity is responsible for the up-regulation of GCLC expression and restoration of the of which was peroxynitrite increased expression of HO-1 with subsequent generation of CO is to be a of the by which PC12 cells protect from that CO from elevated HO activity as a consequence of exposure to PC12 cells from apoptotic death by the of the potential and the of caused by subsequent exposure to of (5Li M.-H. Cha Y.-N. Surh Y.-J. Free Radic. Biol. Med. 2006; 41: 1079-1091Crossref PubMed Scopus (127) Google Scholar, M.-H. Cha Y.-N. Surh Y.-J. Biochem. Biophys. Res. Commun. 2006; 342: 984-990Crossref PubMed Scopus (48) Google Scholar). This to examine whether CO that be from up-regulated HO-1 expression could the biosynthesis of a ubiquitous cellular thereby adaptive cytoprotection against peroxynitrite-induced apoptosis in PC12 cells. The in this study that CO increase GCL expression and subsequently the intracellular GSH level to the adaptive survival response in PC12 cells. The cytoprotective effect of CO was by the cells with a of to the that CO the cellular signaling responsible for GSH biosynthesis. The which the stress and been reported to important in CO L.E. Bach F.H. J. Soares M. Lu M. R.A. Choi A.M. Med. 2000; PubMed Scopus Google Scholar, J. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The a of transcription of to for up-regulation of or antioxidant GCLC Y.M. Gipp J.J. Mulcahy R.T. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus (86) Google Scholar, Liu R.M. Free Radic. Biol. Med. 2005; PubMed Scopus Google Scholar). In cells, the anti-apoptotic effect of CO was found to be dependent on the activation of p38 (10Brouard S. Otterbein L.E. Anrather J. Tobiasch E. Bach F.H. Choi A.M. Soares M.P. J. Exp. Med. 2000; 192: 1015-1026Crossref PubMed Scopus (880) Google Scholar). However, in cells, CO against apoptosis through activation of the which is of p38 signaling J. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). In this we found that CO induced activation of the p38 and via However, inhibition of p38 the CO-RM-induced increase in GCLC expression cytoprotection in PC12 cells with SIN-1. Furthermore, of cells with a of the up-regulation of GCLC expression and thereby the cytoprotective effect of In in PC12 cells with kinase the increase in and up-regulation of GCLC expression were the of is to be the enzyme responsible for the and of leading to up-regulation of GCLC expression and biosynthesis of GSH in the PC12 cells. The molecular by which the CO Nrf2 and induces GCLC expression is In this study, we observed that CO-RM intracellular production in PC12 cells and that with the antioxidant both the increase in production and the subsequent up-regulation of GCLC In this is interesting that CO redox signaling in smooth muscle cells by with the heme of and the C. J. S. J. Motterlini R. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). from an study of the molecular mechanisms of Nrf2 activation by oxidative stress. Nrf2 signaling is to be by a protein enzyme or or of in K. N. Y. Yamamoto M. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar, Y.-J. 2003; PubMed Scopus Google Scholar, Chem. Res. 2005; 18: PubMed Scopus Google Scholar). As a could N. A. Yamamoto M. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar) and Nrf2 W. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). In of Nrf2 at and by as S. 2006; PubMed Scopus Google Scholar), protein kinase T. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), and E. R. C. R. S. PubMed Scopus Google Scholar) is to the of Nrf2 from et al. Kim Mol. Pharmacol. 2002; PubMed Scopus Google Scholar) reported that Nrf2 through in response to oxidative stress. Nrf2 is through by CO-RM-induced oxidative stress or as to the mechanisms by which CO-RM induces production of and the Nrf2 signaling in PC12 cells. In we for the that CO by up-regulation of HO-1 expression the increased expression of GCL and of the intracellular GSH level in PC12 cells challenged with The peroxynitrite-induced nitrosative stress to and of the cellular GSH level. In this the up-regulation of HO-1 leading to degradation of free The CO as an oxidative molecule to the and up-regulation of GCLC This which been by exposure to peroxynitrite This a potential of previously as a noxious for the of a of caused by excessive oxidative or nitrosative stress.