Involvement of Selective Reactive Oxygen Species Upstream of Proapoptotic Branches of Unfolded Protein Response

Cadmium triggers apoptosis of LLC-PK1 cells through induction of endoplasmic reticulum (ER) stress. We found that cadmium caused generation of reactive oxygen species (ROS) and that cadmium-induced ER stress was inhibited by antioxidants. In contrast, suppression of ER stress did not attenuate cadmium-triggered oxidative stress, suggesting that ER stress occurs downstream of oxidative stress. Exposure of the cells to either O2.¯, H2O2, or ONOO- caused apoptosis, whereas ER stress was induced only by O2.¯ or ONOO-. Transfection with manganese superoxide dismutase significantly attenuated cadmium-induced ER stress and apoptosis, whereas pharmacological inhibition of ONOO- was ineffective. Interestingly, transfection with catalase attenuated cadmium-induced apoptosis without affecting the level of ER stress. O2.¯ caused activation of the activating transcription factor 6-CCAAT/enhancer-binding protein-homologous protein (CHOP) and the inositol-requiring ER-to-nucleus signal kinase 1-X-box-binding protein 1 (XBP1) proapoptotic cascades, and overexpression of manganese superoxide dismutase attenuated cadmium-triggered induction of both pathways. Furthermore, phosphorylation of proapoptotic c-Jun N-terminal kinase by O2.¯ or cadmium was suppressed by dominant-negative inhibition of XBP1. These data elucidated 1) cadmium caused ER stress via generation of ROS, 2) O2.¯ was selectively involved in cadmium-triggered, ER stress-mediated apoptosis through activation of the activating transcription factor 6-CHOP and inositol-requiring ER-to-nucleus signal kinase 1-XBP1 pathways, and 3) phosphorylation of JNK was caused by O2.¯-triggered activation of XBP1. Cadmium triggers apoptosis of LLC-PK1 cells through induction of endoplasmic reticulum (ER) stress. We found that cadmium caused generation of reactive oxygen species (ROS) and that cadmium-induced ER stress was inhibited by antioxidants. In contrast, suppression of ER stress did not attenuate cadmium-triggered oxidative stress, suggesting that ER stress occurs downstream of oxidative stress. Exposure of the cells to either O2.¯, H2O2, or ONOO- caused apoptosis, whereas ER stress was induced only by O2.¯ or ONOO-. Transfection with manganese superoxide dismutase significantly attenuated cadmium-induced ER stress and apoptosis, whereas pharmacological inhibition of ONOO- was ineffective. Interestingly, transfection with catalase attenuated cadmium-induced apoptosis without affecting the level of ER stress. O2.¯ caused activation of the activating transcription factor 6-CCAAT/enhancer-binding protein-homologous protein (CHOP) and the inositol-requiring ER-to-nucleus signal kinase 1-X-box-binding protein 1 (XBP1) proapoptotic cascades, and overexpression of manganese superoxide dismutase attenuated cadmium-triggered induction of both pathways. Furthermore, phosphorylation of proapoptotic c-Jun N-terminal kinase by O2.¯ or cadmium was suppressed by dominant-negative inhibition of XBP1. These data elucidated 1) cadmium caused ER stress via generation of ROS, 2) O2.¯ was selectively involved in cadmium-triggered, ER stress-mediated apoptosis through activation of the activating transcription factor 6-CHOP and inositol-requiring ER-to-nucleus signal kinase 1-XBP1 pathways, and 3) phosphorylation of JNK was caused by O2.¯-triggered activation of XBP1. Endoplasmic reticulum (ER) 3The abbreviations used are: ERendoplasmic reticulumGRPglucose-regulated proteinCHOPCCAAT/enhancer-binding protein-homologous proteinORP150150-kDa oxygen-regulated proteinUPRunfolded protein responseATF6activating transcription factor 6IRE1inositol-requiring ER-to-nucleus signal kinase 1XBP1X-box binding protein 1JNKc-Jun N-terminal kinaseROSreactive oxygen speciesSODsuperoxide dismutaseMnTM-2-PyPmanganese(III)-5,10,15,20-tetrakis(N-methylpyridinium-2-yl)porphyrin pentachlorideDNdominant-negative mutantNACN-acetylcysteineSIN-13-morpholinosydnoniminel-NAMENω-nitro-l-arginine methyl esterASK1apoptosis signal-regulating kinase 1TRAF2tumor necrosis factor receptor-associated factor 2MnSODmanganese SOD. stress plays a crucial role in cadmium-induced apoptosis of renal tubular cells. It is based on our following findings: 1) Cadmium chloride (CdCl2) induced expression of endogenous ER stress markers, 78-kDa glucose-regulated protein (GRP78), GRP94, and CCAAT/enhancer-binding protein-homologous protein (CHOP) in a dose-dependent manner. 2) Attenuation of ER stress by overexpression of GRP78 or 150-kDa oxygen-regulated protein (ORP150) significantly suppressed CdCl2-induced apoptosis. 3) Among three major branches of unfolded protein response (UPR), the activating transcription factor 6 (ATF6) pathway and the inositol-requiring ER-to-nucleus signal kinase 1 (IRE1)-X-box binding protein 1 (XBP1) pathway mediated apoptosis. 4) Induction of CHOP by ATF6, IRE1-initiated activation of XBP1, and phosphorylation of c-Jun N-terminal kinase (JNK) were responsible for the induction of apoptosis (1Yokouchi M. Hiramatsu N. Hayakawa K. Kasai A. Takano Y. Yao J. Kitamura M. Cell Death Differ. 2007; 14: 1467-1474Crossref PubMed Scopus (130) Google Scholar). However, several previous studies also indicated involvement of reactive oxygen species (ROS) in cadmium-induced renal tubular injury. For example, exposure of LLC-PK1 cells to cadmium caused generation of ROS (2Gennari A. Cortese E. Boveri M. Casado J. Prieto P. Toxicology. 2003; 183: 211-220Crossref PubMed Scopus (81) Google Scholar), which was associated with a decrease in glutathione levels and consequent cellular death (3Prozialeck W.C. Lamar P.C. Toxicol. Appl. Pharmacol. 1995; 134: 285-295Crossref PubMed Scopus (58) Google Scholar). Another report showed that cadmium-triggered apoptosis of tubular cells was inhibited by an antioxidant (4Thevenod F. Friedmann J.M. Katsen A.D. Hauser I.A. J. Biol. Chem. 2000; 275: 1887-1896Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). However, currently, it is unknown whether and how oxidative stress is linked to ER stress and, if so, what kind of ROS are involved in the induction of apoptosis in cadmium-exposed cells. endoplasmic reticulum glucose-regulated protein CCAAT/enhancer-binding protein-homologous protein 150-kDa oxygen-regulated protein unfolded protein response activating transcription factor 6 inositol-requiring ER-to-nucleus signal kinase 1 X-box binding protein 1 c-Jun N-terminal kinase reactive oxygen species superoxide dismutase manganese(III)-5,10,15,20-tetrakis(N-methylpyridinium-2-yl)porphyrin pentachloride dominant-negative mutant N-acetylcysteine 3-morpholinosydnonimine Nω-nitro-l-arginine methyl ester apoptosis signal-regulating kinase 1 tumor necrosis factor receptor-associated factor 2 manganese SOD. Oxygen normally accepts four electrons and is converted to water. In biological systems, partial reduction of oxygen occurs, resulting in the generation of cytotoxic ROS. That is, sequential reduction of oxygen leads to generation of superoxide anion (O2.¯) and hydrogen peroxide (H2O2) (5Jacobson M.D. Trends Biochem. Sci. 1996; 21: 83-86Abstract Full Text PDF PubMed Scopus (730) Google Scholar). Superoxide dismutase (SOD) scavenges O2.¯ by catalyzing conversion of O2.¯ to H2O2. O2.¯ also rapidly reacts with nitric oxide (NO), yielding another reactive species, peroxynitrite (ONOO-) (6Szabo C. Ohshima H. Nitric Oxide. 1997; 1: 373-385Crossref PubMed Scopus (393) Google Scholar). All of these ROS may be potential triggers of apoptosis (5Jacobson M.D. Trends Biochem. Sci. 1996; 21: 83-86Abstract Full Text PDF PubMed Scopus (730) Google Scholar, 6Szabo C. Ohshima H. Nitric Oxide. 1997; 1: 373-385Crossref PubMed Scopus (393) Google Scholar, 7Buttkea T.M. Sandstromb P.A. Immunol. Today. 1994; 15: 7-10Abstract Full Text PDF PubMed Scopus (2104) Google Scholar). In the present report, we first describe that cadmium induces generation of ROS and consequent ER stress and apoptosis in renal tubular cells. We show that, among ROS generated, O2.¯ but not downstream substances H2O2 and ONOO-, plays a crucial role in cadmium-triggered, ER stress-mediated apoptosis. We further demonstrate that cadmium induces both ER stress-dependent and -independent proapoptotic pathways, the latter of which involves H2O2, but not NO or ONOO-. We also provide evidence that O2.¯ has the potential to induce a novel, atypical proapoptotic pathway, i.e. the XBP1-JNK pathway, as well as the ATF6-CHOP pathway, and thereby critically contributes to cadmium-triggered apoptotic cell death. Reagents–CdCl2 was purchased from Wako Pure Chemical Industries (Osaka, Japan), manganese(III)-5,10,15,20-tetrakis(N-methylpyridinium-2yl)porphyrin pentachloride (MnTM-2-PyP) was from Calbiochem, and other reagents were from Sigma-Aldrich. Cells and Stable Transfectants–The porcine renal proximal tubular cell line LLC-PK1 was obtained from American Type Culture Collection (Manassas, VA). Experiments were performed in the presence of 1% fetal bovine serum. LLC-PK1 cells constitutively expressing GRP78, ORP150, manganese SOD (MnSOD), catalase or a dominant-negative mutant of XBP1 (XBP1-DN) were established by stable transfection (electroporation) with pcDNA3.1-GPR78 (8Watson L.M. Chan A.K. Berry L.R. Li J. Sood S.K. Dickhout J.G. Xu L. Werstuck G.H. Bajzar L. Klamut H.J. Austin R.C. J. Biol. Chem. 2003; 278: 17438-17447Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), pCIneo-ORP150 (9Ozawa K. Kuwabara K. Tamatani M. Takatsuji K. Tsukamoto Y. Kaneda S. Yanagi H. Stern D.M. Eguchi Y. Tsujimoto Y. Ogawa S. Tohyama M. J. Biol. Chem. 1999; 274: 6397-6404Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), pcDNA3-MnSOD (10Manna S.K. Zhang H.J. Yan T. Oberley L.W. Aggarwal B.B. J. Biol. Chem. 1998; 273: 13245-13254Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar), pCIneo-catalase (11Ushio-Fukai M. Alexander R.W. Akers M. Griendling K.K. J. Biol. Chem. 1998; 273: 15022-15029Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar), or pdn-XBP1 (12Lee A.H. Iwakoshi N.N. Anderson K.C. Glimcher L.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9946-9951Crossref PubMed Scopus (508) Google Scholar) and designated as LL/GRP78, LL/ORP150, LL/MnSOD, LL/Catalase, and LL/XBP1-DN, respectively. LL/Mock cells were established by stable transfection with neo alone (pcDNA3.1, Invitrogen). Northern Blot Analysis–Total RNA was extracted by the single-step method, and Northern blot analysis was performed as described before (13Kitamura M. Suto T. Yokoo T. Shimizu F. Fine L.G. J. Immunol. 1997; 156: 2964-2971Google Scholar). cDNAs for GRP78 (14Katayama T. Imaizumi K. Honda A. Yoneda T. Kudo T. Takeda M. Mori K. Rozmahel R. Fraser P. George-Hyslop P.S. Tohyama M. J. Biol. Chem. 2001; 276: 43446-43454Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), GRP94 (15Gazit G. Lu J. Lee A.S. Breast Cancer Res. Treat. 1999; 54: 135-146Crossref PubMed Scopus (136) Google Scholar), CHOP (16Wang X.Z. Harding H.P. Zhang Y. Jolicoeur E.M. M. J. 1998; PubMed Scopus Google Scholar), U. R. J. PubMed Scopus Google Scholar), catalase (11Ushio-Fukai M. Alexander R.W. Akers M. Griendling K.K. J. Biol. Chem. 1998; 273: 15022-15029Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar), and (10Manna S.K. Zhang H.J. Yan T. Oberley L.W. Aggarwal B.B. J. Biol. Chem. 1998; 273: 13245-13254Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar) were used for of of was used as a analysis was performed of were with and with for in the of fetal bovine serum. with cells were with or H2O2 for 2 and to of induction of apoptosis, was performed by and for as described before (1Yokouchi M. Hiramatsu N. Hayakawa K. Kasai A. Takano Y. Yao J. Kitamura M. Cell Death Differ. 2007; 14: 1467-1474Crossref PubMed Scopus (130) Google Scholar). in cells were from the of the of cells were first by and to was of the and were performed in Transfection cells were with A. S. Y. J. T. Hayakawa Y. T. K. J. Natl. Cancer PubMed Scopus Google Scholar), J. R. PubMed Scopus Google Scholar), or T. R. Biochem. Res. PubMed Scopus Google Scholar), with reagents and to blot analysis or Blot blot analysis of and GRP78 was performed and a the level of was of JNK was protein as described before K. Y. Hiramatsu N. Kasai A. K. Yao J. Kitamura M. J. Immunol. PubMed Scopus Google Scholar). were of XBP1 was by following and an expression of was the as and of was by following the by the were performed in were as analysis was performed the to data in was to a ER as an of in indicated involvement of ROS in cadmium-induced renal tubular (2Gennari A. Cortese E. Boveri M. Casado J. Prieto P. Toxicology. 2003; 183: 211-220Crossref PubMed Scopus (81) Google Scholar, W.C. Lamar P.C. Toxicol. Appl. Pharmacol. 1995; 134: 285-295Crossref PubMed Scopus (58) Google Scholar, F. Friedmann J.M. Katsen A.D. Hauser I.A. J. Biol. Chem. 2000; 275: 1887-1896Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). We first generation of ROS in LLC-PK1 cells to Cells were with an and with or H2O2 showed that, a LLC-PK1 cells Exposure of the cells to cadmium caused and dose-dependent in the of cells whether the generation of ROS is of cadmium-induced apoptosis, cells were with antioxidant N-acetylcysteine or antioxidant by and to and in induced and of the cells with of apoptosis. with or attenuated these analysis that of cells and apoptotic cells with were significantly by from to and from to of cells and apoptotic cells were by from to and from to apoptosis of LLC-PK1 cells is in by ER stress (1Yokouchi M. Hiramatsu N. Hayakawa K. Kasai A. Takano Y. Yao J. Kitamura M. Cell Death Differ. 2007; 14: 1467-1474Crossref PubMed Scopus (130) Google Scholar). oxidative stress and ER stress, LLC-PK1 cells were with in the or presence of and expression of endogenous ER stress GRP78 and GRP94 was Northern blot analysis that induction of by was attenuated by induction of CHOP by was also inhibited by suggesting that ER stress is an downstream of oxidative stress. induction of a of oxidative stress P. R. P. Google Scholar, M. P.A. Chem. Biol. 1994; PubMed Scopus Google Scholar), by was by involvement of and -independent induction of ER stress by a that ER stress is of oxidative stress, LLC-PK1 cells were with the ER GRP78, which ER stress. established ER cells levels of GRP78 and to apoptosis induction of by was with that in cells. in expression of in cells was not attenuated with that in LL/Mock cells. of ER stress by overexpression of another ER (1Yokouchi M. Hiramatsu N. Hayakawa K. Kasai A. Takano Y. Yao J. Kitamura M. Cell Death Differ. 2007; 14: 1467-1474Crossref PubMed Scopus (130) Google Scholar, K. Kuwabara K. Tamatani M. Takatsuji K. Tsukamoto Y. Kaneda S. Yanagi H. Stern D.M. Eguchi Y. Tsujimoto Y. Ogawa S. Tohyama M. J. Biol. Chem. 1999; 274: 6397-6404Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) did not induction of by these that ER stress is an downstream of oxidative stress in cadmium-exposed cells. of O2.¯ in ER sequential reduction of oxygen leads to generation of O2.¯ and H2O2 (5Jacobson M.D. Trends Biochem. Sci. 1996; 21: 83-86Abstract Full Text PDF PubMed Scopus (730) Google Scholar). O2.¯ also rapidly reacts with yielding another reactive species ONOO- (6Szabo C. Ohshima H. Nitric Oxide. 1997; 1: 373-385Crossref PubMed Scopus (393) Google Scholar). All of these ROS the potential for apoptosis (5Jacobson M.D. Trends Biochem. Sci. 1996; 21: 83-86Abstract Full Text PDF PubMed Scopus (730) Google Scholar, 6Szabo C. Ohshima H. Nitric Oxide. 1997; 1: 373-385Crossref PubMed Scopus (393) Google Scholar, 7Buttkea T.M. Sandstromb P.A. Immunol. Today. 1994; 15: 7-10Abstract Full Text PDF PubMed Scopus (2104) Google Scholar). previous report indicated that, in cadmium of ROS O2.¯ and H2O2 Toxicol. Appl. Pharmacol. PubMed Scopus Google Scholar). However, the major ROS that to CdCl2-induced apoptosis not It is also unknown which ROS to the induction of ER stress. these we first involvement of O2.¯ in the induction of ER stress and apoptosis. LLC-PK1 cells were with of a of O2.¯, and expression of was by Northern blot in induced expression of GRP78 and GRP94, suggesting that O2.¯ has the potential to induce ER stress. We LLC-PK1 cells to involvement of O2.¯ in the of Northern blot analysis expression of in cells and cells analysis that these were to apoptosis analysis showed that of cells and apoptotic cells with by were by from to and from to respectively. these induction of ER stress by cadmium was in expression of GRP78 in response to was significantly in cells induction was of that in LL/Mock cells. were obtained in both cells and cells. attenuated induction of GRP78 was associated with of GRP78 protein Furthermore, of cadmium-induced ER stress in cells was also by induction of CHOP that apoptosis induced by was attenuated in with that in LL/Mock cells analysis showed that of cells and apoptotic cells were significantly by from to and from to respectively. These involvement of O2.¯ in cadmium-triggered ER stress and consequent apoptotic cell death. of H2O2 in ER involvement of H2O2 in the induction of ER stress and apoptosis by LLC-PK1 cells were with of H2O2, and expression of GRP78 as well as was by Northern blot in H2O2 induced expression of in a dose-dependent manner. In contrast, expression of GRP78 was not by H2O2 indicated of involvement of H2O2 in the induction of ER stress and ER stress-dependent apoptosis by further we LLC-PK1 cells Northern blot analysis expression of catalase in cells and cells analysis that these were to apoptosis analysis by showed that the of apoptotic cells with was by catalase from to these induction of ER stress by cadmium was in induction of GRP78 by was not LL/Mock cells and cells. were obtained in both cells and cells. with the in indicated that H2O2 did not to the induction of ER stress and ER stress-dependent apoptosis in response to Interestingly, apoptosis induced by was significantly in with that in LL/Mock cells of cells and apoptotic cells were by catalase from to and from to respectively. These indicated that H2O2 was in response to and to cadmium-induced apoptosis of ER stress. of of ONOO- in ER and further involvement of ONOO- in the induction of ER stress and apoptosis by LLC-PK1 cells were with of 3-morpholinosydnonimine a of ONOO-, and expression of GRP78 and as well as was by Northern blot with induced expression of induction of oxidative stress in also induced expression of GRP78 and CHOP in a dose-dependent manner. that with induced and of apoptosis suggesting the potential of ONOO- to induce ER stress and apoptosis in LLC-PK1 cells. involvement of ONOO- in the of we used an of ONOO-, as well as Nω-nitro-l-arginine methyl ester an of NO that is for the generation of ONOO-. in both did not induction of the ER stress by Furthermore, apoptosis induced by was not attenuated by or analysis showed that, the and did not of cells and apoptotic cells These indicated that ONOO- was not involved in the induction of ER stress and apoptosis by cadmium in LLC-PK1 cells. of the ATF6-CHOP and the XBP1-JNK via of the ATF6-CHOP pathway and the pathway, both of which to the induction of apoptosis (1Yokouchi M. Hiramatsu N. Hayakawa K. Kasai A. Takano Y. Yao J. Kitamura M. Cell Death Differ. 2007; 14: 1467-1474Crossref PubMed Scopus (130) Google Scholar). We of O2.¯ in the activation of these proapoptotic LLC-PK1 cells were with ATF6, to for to 6 and to blot in of the cells with ATF6, suggesting the potential of O2.¯ to the whether O2.¯ by cadmium is of LL/Mock cells and cells were with ATF6, to and to blot with LL/Mock the in the level of was attenuated in cells Furthermore, induction of CHOP in but not in was that in LL/Mock cells These that the ATF6-CHOP proapoptotic pathway was in by O2.¯ in cadmium-exposed cells. We a role of O2.¯ in the activation of the LLC-PK1 cells were with for to and of XBP1 was by in following the exposure to O2.¯, the of XBP1 1 which was for the potential of O2.¯ to the whether cadmium-triggered O2.¯ is of activation of proapoptotic pathway, LL/Mock cells and cells were with a described the cells the that of XBP1 T. R. Biochem. Res. PubMed Scopus Google Scholar). In LL/Mock was by with cells. However, induction was significantly in cells These that O2.¯ was also involved in the activation of the pathway in cadmium-exposed cells. In the pathway is proapoptotic via activation of apoptosis signal-regulating kinase 1 and in our previous report, we that JNK was rapidly following exposure to and pharmacological suppression of JNK attenuated apoptosis (1Yokouchi M. Hiramatsu N. Hayakawa K. Kasai A. Takano Y. Yao J. Kitamura M. Cell Death Differ. 2007; 14: 1467-1474Crossref PubMed Scopus (130) Google Scholar). However, we also found that dominant-negative inhibition of XBP1, which is not involved in the pathway, attenuated cadmium-induced apoptosis of GRP78 or CHOP (1Yokouchi M. Hiramatsu N. Hayakawa K. Kasai A. Takano Y. Yao J. Kitamura M. Cell Death Differ. 2007; 14: 1467-1474Crossref PubMed Scopus (130) Google Scholar). We that activation of JNK be an downstream of XBP1 in cadmium-exposed cells. LLC-PK1 cells were with a for and cells were Northern blot analysis expression of the in the established LL/Mock cells and cells were with or and to blot analysis of in and both and induced phosphorylation of JNK in LL/Mock cells. In contrast, the activation of JNK was suppressed in cells and Furthermore, transfection of LLC-PK1 cells with cadmium-triggered phosphorylation of JNK involvement of XBP1 in the activation of Interestingly, the role of XBP1 was to the O2.¯-triggered JNK 1) phosphorylation of JNK by H2O2 was to the in LL/Mock cells and cells and 2) CdCl2-induced phosphorylation of JNK was in cells these that cadmium caused activation of the ATF6-CHOP and proapoptotic via generation of O2.¯ and that activation of JNK was downstream of XBP1. ROS is in renal injury. However, cadmium-induced apoptosis of renal tubular cells is also on ER stress (1Yokouchi M. Hiramatsu N. Hayakawa K. Kasai A. Takano Y. Yao J. Kitamura M. Cell Death Differ. 2007; 14: 1467-1474Crossref PubMed Scopus (130) Google Scholar). present was performed to of ROS and downstream in cadmium-triggered, ER stress-mediated apoptosis. elucidated that 1) cadmium caused ER stress via generation of ROS, 2) O2.¯, but not H2O2 and ONOO-, mediated cadmium-triggered, ER apoptosis via activation of the ATF6-CHOP and pathways, 3) phosphorylation of a of cadmium-induced apoptosis, was caused via O2.¯-triggered activation of XBP1, and 4) H2O2 was involved in cadmium-induced apoptosis of ER stress. of our is and in with our indicated that O2.¯ may be selectively involved in ER is a well in which both oxidative stress and ER stress are involved R. C. S. A. E. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). SOD induction of and of ER stress, in the T. A. S. M. Chan J. PubMed Scopus Google Scholar). In induced expression of and CHOP in the to apoptosis. In contrast, SOD showed only a of these and that O2.¯ is involved in both induction of ER stress and consequent death. In biological systems, O2.¯ is rapidly by SOD to H2O2. O2.¯ also rapidly reacts with yielding ONOO-. O2.¯ is generated, H2O2 and ONOO- may be and to the apoptotic In the present we found that these ROS the potential to induce apoptosis in LLC-PK1 cells. However, H2O2 did not ER stress in cell ONOO- ER stress, of ONOO- did not attenuate cadmium-induced ER stress and apoptosis. These and of ROS in the cadmium-triggered, ER stress-mediated apoptotic Interestingly, overexpression of catalase significantly attenuated cadmium-induced apoptosis without affecting the level of ER stress. indicated that, H2O2 is not involved in ER stress-dependent apoptosis, it to cadmium-induced apoptosis of ER stress, via the pathway F. 2003; Scopus Google Scholar). data indicated that cadmium induces apoptosis of tubular cells through ER stress-dependent and -independent that cadmium-induced apoptosis was attenuated by and that oxidative stress is the major for cadmium-induced apoptosis, as was (4Thevenod F. Friedmann J.M. Katsen A.D. Hauser I.A. J. Biol. Chem. 2000; 275: 1887-1896Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, Toxicol. Appl. Pharmacol. PubMed Scopus Google Scholar). However, the involved were found to be not i.e. oxidative apoptosis is mediated in ER stress, and only O2.¯ in the ER stress-dependent apoptotic H2O2 apoptosis of ER stress. Furthermore, ONOO- is not involved in both ER stress-dependent and -independent apoptotic inhibited oxidative stress but only attenuated the level of ER stress and a that cadmium also induce ER stress of oxidative stress. oxidative stress and ER stress is not well that activation of in oxidative stress and consequent cellular death in of ROS by may be caused through the oxidative in the ER and the ROS generation 15: Full Text Full Text PDF PubMed Scopus Google Scholar). However, our that oxidative stress was but not downstream of ER stress in cadmium-exposed cells. with our also indicated that ER stress may be involved downstream of ROS. For example, in tumor the caused expression of GRP78 via a Cell 2003; 15: PubMed Scopus Google Scholar). In ONOO- caused in GRP78 and GRP94 J.G. L.M. J. S. Austin R.C. Biol. PubMed Scopus Google Scholar). of ROS in cadmium-exposed cells was not in the present but the pathway is responsible for the generation of ROS in cadmium-exposed tubular cells F. 2003; Scopus Google Scholar). it is how ROS induces ER stress. showed that oxidative stress caused inhibition of P. E. J. Biochem. 2003; PubMed Scopus Google Scholar, P.C. Biol. 1998; PubMed Scopus Google Scholar), a of ER stress. is, that ROS may of the in the ER via inhibition of C. Res. 1996; PubMed Scopus Google Scholar). Another is that ROS ER stress through generation and of may also be in the ER through of ER S. L. G. N. E. Biol. 2000; PubMed Scopus Google Scholar). Among three major the pathway and the pathway cadmium-triggered apoptosis, and induction of CHOP by and activation of both XBP1 and JNK by are involved in the apoptotic (1Yokouchi M. Hiramatsu N. Hayakawa K. Kasai A. Takano Y. Yao J. Kitamura M. Cell Death Differ. 2007; 14: 1467-1474Crossref PubMed Scopus (130) Google Scholar). In the present we that O2.¯ in cadmium-exposed cells the ATF6-CHOP and the proapoptotic pathways. is that activation of an of cadmium-induced apoptosis (1Yokouchi M. Hiramatsu N. Hayakawa K. Kasai A. Takano Y. Yao J. Kitamura M. Cell Death Differ. 2007; 14: 1467-1474Crossref PubMed Scopus (130) Google Scholar), was downstream of XBP1. In it is that, in response to ER stress, activation of JNK occurs following of tumor necrosis factor receptor-associated factor 2 by is also for the JNK and the phosphorylation of JNK plays a crucial role in ER apoptosis of the pathway R. M. K. S. PubMed Scopus Google Scholar). our is the first report the of the XBP1-JNK proapoptotic pathway involved in ER apoptosis. ROS are involved in a of cellular that ER stress downstream of oxidative stress a that ER stress and may also be involved not only in cadmium-induced apoptosis but also in a of other biological be to the of of the oxidative stress in cells and We Imaizumi of and Ogawa of Cancer S. Lee of C. Austin H. Glimcher of and for with with

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