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 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document}, H2O2, or ONOO- caused apoptosis, whereas ER stress was induced only by \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document} 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. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document} 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 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document} or cadmium was suppressed by dominant-negative inhibition of XBP1. These data elucidated 1) cadmium caused ER stress via generation of ROS, 2) \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document} 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 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{\overline{.}}\) \end{document}-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 O 2 . Endoplasmic reticulum (ER) 3 stress plays a crucial role in cadmium-induced apoptosis of renal tubular cells. It is based on our following findings: 1) Cadmium chloride (CdCl 2 ) 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 CdCl 2 -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 (1). 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 (2), which was associated with a decrease in glutathione levels and consequent cellular death (3). Another report showed that cadmium-triggered apoptosis of tubular cells was inhibited by an antioxidant (4). 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.
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 (
Detection of ROS-Cells were washed twice with phosphatebuffered saline and loaded with ROS-responsive fluorescent probe 2Ј,7Ј-dichlorofluorescein (100 M) for 3 h in the absence of fetal bovine serum. After washing with phosphate-buffered saline, cells were stimulated with CdCl 2 or H 2 O 2 for 2 h and subjected to fluorescent microscopy.
Assessment of Apoptosis-After induction of apoptosis, morphologic examination was performed by phase-contrast microscopy and fluorescent microscopy for Hoechst 33258 staining, as described before (1). Because, in some situations, round cells were easily detached from the bottoms of the plates, percentages of round cells were first evaluated by phase-contrast microscopy and then subjected to Hoechst staining. Apoptosis was identified using morphological criteria, including shrinkage of the cytoplasm (round shape), nuclear condensation, and membrane blebbing. Assays were performed in quadruplicate.
Luciferase Assay-Activity of luciferase was evaluated by Luciferase Assay System (Promega) following the protocol provided by the manufacturer. Assays were performed in quadruplicate.
Statistical Analysis-Data were expressed as means Ϯ S.E. Statistical analysis was performed using the non-parametric Mann-Whitney U test to compare data in different groups. p value Ͻ0.05 was considered to indicate a statistically significant difference.

ER Stress as an Event Downstream of Oxidative Stress in
Cadmium-exposed Cells-Previous reports indicated involvement of ROS in cadmium-induced renal tubular injury (2)(3)(4). We first confirmed generation of ROS in LLC-PK1 cells exposed to cadmium. Cells were loaded with an ROS-responsive fluorescence probe 2Ј,7Ј-dichlorofluorescein and stimulated with CdCl 2 or H 2 O 2 (positive control). Fluorescence microscopy showed that, under a basal culture condition, LLC-PK1 cells exhibited little fluorescence. Exposure of the cells to cadmium caused substantial and dose-dependent increases in the number of fluorescence-positive cells (Fig. 1A, bottom row). To examine whether the generation of ROS is causative of cadmium-induced apoptosis, cells were pretreated with antioxidant N-acetylcysteine (NAC) or SOD-like antioxidant MnTM-2-PyP, stimulated by CdCl 2 , and subjected to phase-contrast microscopy and Hoechst staining. As shown in Fig. 1B, CdCl 2 induced rounding and detachment of the cells with nuclear condensation typical of apoptosis. Treatment with NAC or MnTM-2-PyP attenuated these morphological changes. Quantitative analysis revealed that percentages of round cells and apoptotic cells with condensed nuclei were significantly reduced by NAC from 43.5 Ϯ 1.9% to 5.1 Ϯ 0.5% (Fig. 1C, left) and from 39.2 Ϯ 3.9% to 4.5 Ϯ 1.0% (Fig. 1C, right), respectively (means Ϯ S.E., p Ͻ 0.05). Similarly, percentages of round cells and apoptotic cells were reduced by MnTM-2-PyP from 43.5 Ϯ 1.9% to 13.9 Ϯ 0.9% and from 39.2 Ϯ 3.9% to 12.1 Ϯ 2.5%.
Cadmium-induced apoptosis of LLC-PK1 cells is mediated, at least in part, by ER stress (1). To investigate relationship between oxidative stress and ER stress, LLC-PK1 cells were treated with CdCl 2 in the absence or presence of NAC, and expression of endogenous ER stress markers GRP78 and GRP94 was examined. Northern blot analysis revealed that induction of GRPs by CdCl 2 was partially attenuated by NAC ( Fig. 2A,  top). Similarly, induction of CHOP by CdCl 2 was also inhibited by NAC ( Fig. 2A, bottom), suggesting that ER stress is an event downstream of oxidative stress. Of note, induction of c-fos, a marker of oxidative stress (22,23), by CdCl 2 was completely abolished by NAC (Fig. 2B), indicating involvement of ROS-dependent and -independent induction of ER stress by CdCl 2 .
To exclude a possibility that ER stress is located upstream of oxidative stress, LLC-PK1 cells were stably transfected with the ER chaperone GRP78, which attenuates ER stress. The established ER stress-resistant LL/GRP78 cells exhibited high levels of GRP78 mRNAs (supplemental Fig. S1) and resistance to tunicamycin-induced apoptosis (supplemental Fig. S2). Using LL/GRP78 cells, induction of c-fos by CdCl 2 was compared with that in mock transfected cells. As shown in Fig. 2C, expression of c-fos in LL/GRP78 cells was not attenuated when compared with that in LL/Mock cells. Similarly, attenuation of ER stress by overexpression of another ER chaperone ORP150 (1,9) did not affect induction of c-fos by CdCl 2 (Fig. 2D). Taken together, these results evidenced Asterisks indicate statistically significant differences versus CdCl 2 -exposed, antioxidant-untreated control (p Ͻ 0.05). FIGURE 2. Endoplasmic reticulum (ER) stress as an event downstream of oxidative stress in cadmium-exposed cells. A and B, LLC-PK1 cells were pretreated with (ϩ) or without (Ϫ) 1 mM NAC and exposed to 20 M CdCl 2 for 4 h. Expression of endogenous ER stress markers 78-kDa glucose-regulated protein (GRP78), GRP94, and CCAAT/enhancer-binding protein-homologous protein (CHOP) (A) and an oxidative stress marker c-fos (B) was examined by Northern blot analysis. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. C, LL/Mock cells and LL/GRP78 cells overexpressing GRP78 were treated with CdCl 2 , and expression of c-fos was evaluated. D, LL/Mock cells and LL/ORP150 cells expressing 150-kDa oxygenregulated protein were treated with CdCl 2 and subjected to Northern blot analysis of c-fos.
that ER stress is an event downstream of oxidative stress in cadmium-exposed cells.
Involvement  In E, the level of GRP78 was normalized by the level of glyceraldehyde-3-phosphate dehydrogenase, and its relative induction (versus untreated control) is shown on the right (n ϭ 4). An asterisk indicates a statistically significant difference (p Ͻ 0.05). H and I, LL/Mock cells and LL/MnSOD cells were exposed to CdCl 2 for 4 h and subjected to phase-contrast microscopy (H and I, left) and Hoechst staining to evaluate percentages of apoptotic cells (I, right). FEBRUARY 15, 2008 • VOLUME 283 • NUMBER 7 analysis confirmed abundant expression of MnSOD mRNA in LL/MnSOD-6 cells and LL/MnSOD-7 cells (Fig. 3B). Microscopic analysis revealed that these transfectants were resistant to menadione-induced apoptosis (Fig. 3C). Quantitative analysis showed that percentages of round cells and apoptotic cells with condensed nuclei (evaluated by Hoechst staining) were reduced by MnSOD from 52.6 Ϯ 2.3% to 15.9 Ϯ 0.3% (Fig. 3D,  left), and from 44.4 Ϯ 2.7 to 25.3 Ϯ 2.0% (Fig. 3D, right), respectively. Using these transfectants, induction of ER stress by cadmium was evaluated. As shown in Fig. 3E, expression of GRP78 mRNA in response to CdCl 2 was significantly reduced in LL/MnSOD cells (p Ͻ 0.05). The induction was approximately one-third of that in LL/Mock cells. Similar results were obtained in both LL/MnSOD-6 cells and LL/MnSOD-7 cells. The attenuated induction of GRP78 was associated with blunted accumulation of GRP78 protein (Fig. 3F). Furthermore, attenuation of cadmium-induced ER stress in LL/MnSOD cells was also evidenced by blunted induction of CHOP (Fig. 3G). Subsequent experiments revealed that apoptosis induced by CdCl 2 was attenuated in LL/MnSOD cells, when compared with that in LL/Mock cells (Fig. 3H). Quantitative analysis showed that percentages of round cells and apoptotic cells were significantly reduced by MnSOD from 55.0 Ϯ 2.9% to 16.4 Ϯ 0.7% (Fig. 3I, left), and from 57.6 Ϯ 1.9% to 19.8 Ϯ 1.3% (Fig. 3I,  right), respectively. These results suggested involvement of O 2 .

Selective ROS Upstream of Proapoptotic UPR
in cadmium-triggered ER stress and consequent apoptotic cell death.

Involvement of H 2 O 2 in Cadmium-triggered, ER Stress-independent
Apoptosis-We next examined involvement of H 2 O 2 in the induction of ER stress and apoptosis by cadmium. LLC-PK1 cells were treated with serial concentrations of H 2 O 2 , and expression of GRP78 as well as c-fos was examined by Northern blot analysis. As shown in Fig. 4A, H 2 O 2 induced expression of c-fos in a dose-dependent manner. In contrast, expression of GRP78 was not increased by H 2 O 2 at any concentration tested. This result indicated lack of involvement of H 2 O 2 in the induction of ER stress and ER stress-dependent apoptosis by cadmium. To further examine this possibility, we created LLC-PK1 cells stably overexpressing catalase. Northern blot analysis confirmed abundant expression of catalase mRNA in LL/Catalase-9 cells and LL/Catalase-11 cells (Fig. 4B). Microscopic analysis revealed that these transfectants were resistant to H 2 O 2 -induced apoptosis (Fig. 4C, left). Quantitative analysis by Hoechst staining showed that the percentage of apoptotic cells with condensed nuclei (supplemental Fig. S3) was markedly reduced by catalase from 50.4 Ϯ 4.2% to 7.3 Ϯ 0.5% (Fig. 4C,  right). Using these transfectants, induction of ER stress by cadmium was re-tested. As shown in Fig. 4D, induction of GRP78 by CdCl 2 was not different between LL/Mock cells and LL/Catalase cells. The similar results were obtained in both LL/Catalase-9 cells and LL/Catalase-11 cells. This result, together with the finding shown in Fig. 4A, indicated that H 2 O 2 did not contribute to the induction of ER stress and ER stress-dependent apoptosis in response to cadmium. Interestingly, however, apoptosis induced by CdCl 2 was significantly less in LL/Catalase cells, when compared with that in LL/Mock cells (Fig. 4E). Percentages of round cells and apoptotic cells were reduced by catalase from 55.0 Ϯ 2.6% to 21.5 Ϯ 2.9% (Fig. 4F, left), and from 57.6 Ϯ 1.9% to 37.6 Ϯ 5.1% (Fig. 4F, right), respectively. These results indicated that H 2 O 2 was generated in response to CdCl 2 and contributed to cadmium-induced apoptosis independent of ER stress.
Lack of Involvement of ONOO Ϫ in Cadmium-triggered ER Stress and Apoptosis-We further examined involvement of ONOO Ϫ in the induction of ER stress and apoptosis by cadmium. LLC-PK1 cells were treated with serial concentrations of 3-morpholinosydnonimine (SIN-1), a generator of ONOO Ϫ , and expression of GRP78 and CHOP, as well as c-fos, was evaluated by Northern blot analysis. Treatment with SIN-1 induced expression of c-fos, indicating induction of oxidative stress (supplemental Fig. S4). As shown in Fig. 5A, SIN-1 also induced expression of GRP78 and CHOP in a dose-dependent manner. Microscopic analyses revealed that treatment with SIN-1 induced membrane blebbing and nuclear condensation typical of apoptosis (Fig. 5B), suggesting the potential of ONOO Ϫ to induce ER stress and apoptosis in LLC-PK1 cells. To examine involvement of ONOO Ϫ in the effects of cadmium, we used uric acid, an inhibitor of ONOO Ϫ , as well as N -nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO that is required for the generation of ONOO Ϫ . As shown in Fig. 5C, both inhibitors did not affect induction of the ER stress markers by CdCl 2 . Furthermore, apoptosis induced by CdCl 2 was not attenuated by uric acid or L-NAME (Fig. 5D). Quantitative analysis showed that, even at the high concentration (5 mM), uric acid and L-NAME did not reduce percentages of round cells (Fig. 5E, left) and apoptotic cells (Fig. 5E, right). These results indicated that ONOO Ϫ was not involved in the induction of ER stress and apoptosis by cadmium in LLC-PK1 cells.
Activation of the ATF6-CHOP Pathway and the XBP1-JNK Pathway via Cadmium-triggered Generation of O 2 . -Cadmium activates the ATF6-CHOP pathway and the IRE1-XBP1 path-way, both of which contribute to the induction of apoptosis (1). We therefore examined roles of O 2 . in the activation of these proapoptotic cascades. First, LLC-PK1 cells were transiently transfected with FLAG-tagged p90 ATF6, exposed to menadione for up to 6 h, and subjected to Western blot analysis. As shown in Fig. 6A, treatment of the cells with menadione generated active p50 ATF6, suggesting the potential of O 2 . to activate the ATF6 pathway. To examine whether O 2 . produced by cadmium is causative of ATF6 activation, LL/Mock cells and LL/MnSOD cells were transiently transfected with FLAGtagged p90 ATF6, exposed to CdCl 2 , and subjected to Western blot analysis. Compared with LL/Mock cells, the increase in the level of p50 ATF6 was attenuated in LL/MnSOD cells (Fig. 6B). Furthermore, induction of CHOP in LL/MnSOD cells, but not in LL/Catalase cells, was lower than that in LL/Mock cells (Fig.  6C). These results suggested that the ATF6-CHOP proapoptotic pathway was activated, at least in part, by O 2 . generated in cadmium-exposed cells. We next examined a role of O 2 . in the activation of the IRE1-XBP1 pathway. LLC-PK1 cells were treated with menadione for up to 4 h, and splicing of XBP1 mRNA was examined by reverse transcription-PCR. As shown in Fig. 6D . is causative of activation of this proapoptotic pathway, LL/Mock cells and LL/MnSOD cells were transiently transfected with a reporter plasmid pCAX-F-XBP1⌬DBD-Luc. As described previously, the transfected cells produce luciferase exclusively under the conditions that trigger splicing of XBP1 mRNA (20). In CdCl 2 -exposed LL/Mock cells, . Arrows indicate membrane blebbing. C, cells were pretreated with (ϩ) or without (Ϫ) uric acid (UA; 1-5 mM) or L-NAME (1-5 mM) for 30 min, exposed to CdCl 2 for 4 h, and subjected to Northern blot analysis. D and E, cells were pretreated with (ϩ) or without (Ϫ) UA or L-NAME, exposed to CdCl 2 , and subjected to phase-contrast microscopy (D and E, left) and Hoechst staining (E, right) to evaluate apoptosis. N.S., not statistically significant.
luciferase activity was increased by 73.2% compared with untreated cells. However, this induction was significantly less in LL/MnSOD cells (39.3%) (Fig. 6E). These results suggested that O 2 . was also involved in the activation of the IRE1-XBP1 pathway in cadmium-exposed cells.
In general, the IRE1 pathway is proapoptotic via activation of apoptosis signal-regulating kinase 1 (ASK1) and JNK. Indeed, in our previous report, we demonstrated that JNK was rapidly activated following exposure to cadmium, and pharmacological suppression of JNK substantially attenuated apoptosis (1). However, we also found that dominant-negative inhibition of XBP1, which is generally not involved in the IRE1-JNK pathway, markedly attenuated cadmium-induced apoptosis independently of GRP78 or CHOP (1). We speculated that activation of JNK could be an event downstream of XBP1 in cadmium-exposed cells. To examine this possibility, LLC-PK1 cells were stably transfected with a gene coding for XBP1-DN, and LL/XBP1-DN cells were established. Northern blot analysis confirmed substantial expression of the transgene in the established transfectants (supplemental Fig. S5). LL/Mock cells and LL/XBP1-DN cells were then treated with menadione or CdCl 2 and subjected to Western blot analysis of phosphorylated JNK. As shown in Fig. 6, F and G (left), both menadione and CdCl 2 induced rapid phosphorylation of JNK in LL/Mock cells. In contrast, the activation of JNK was markedly suppressed in LL/XBP1-DN cells (Fig. 6, F and G, right). Furthermore, transfection of LLC-PK1 cells with XBP1(S) enhanced cadmiumtriggered phosphorylation of JNK (Fig. 6H), confirming involvement of XBP1 in the activation of JNK. Interestingly, the role of XBP1 was specific to the O 2 . -triggered JNK activation, because 1) phosphorylation of JNK by H 2 O 2 was observed to the same extent in LL/Mock cells and LL/XBP1-DN cells (Fig.  6I) and 2) CdCl 2 -induced phosphorylation of JNK was similarly observed in LL/Catalase cells (Fig. 6J). Taken together, these results evidenced that cadmium caused activation of the ATF6-CHOP and IRE1-XBP1 proapoptotic pathways via generation of O 2 . and that activation of JNK was located downstream of XBP1.

DISCUSSION
ROS is implicated in heavy metal-induced renal injury. However, cadmium-induced apoptosis of renal tubular cells is also dependent on ER stress (1). The present study was performed to elucidate roles of individual ROS and downstream events in cadmium-triggered, ER stress-mediated apoptosis. Our results elucidated that 1) cadmium caused ER stress via generation of ROS, 2) O 2 . , but not H 2 O 2 and ONOO Ϫ , mediated cadmiumtriggered, ER stress-induced apoptosis via activation of the  Our current data indicated that cadmium induces apoptosis of tubular cells through ER stress-dependent and -independent mechanisms. The fact that cadmium-induced apoptosis was markedly attenuated by antioxidants (Fig. 1, B and C) suggested that oxidative stress is the major mechanism for cadmium-induced apoptosis, as was reported previously (4,24). However, the mechanisms involved were found to be not so simple, i.e. oxidative stress-induced apoptosis is mediated by, at least in part, ER stress, and only O 2 . participates in the ER stress-dependent apoptotic process. H 2 O 2 mediates apoptosis independently of ER stress. Furthermore, ONOO Ϫ is not involved in both ER stress-dependent and -independent apoptotic processes. NAC completely inhibited oxidative stress but only partially attenuated the level of ER stress (Fig. 2, A and B), indicating a possibility that cadmium could also induce ER stress independently of oxidative stress. The relationship between oxidative stress and ER stress is not well understood. Haynes et al. recently reported that prolonged activation of UPR resulted in oxidative stress and consequent cellular death in Saccharomyces cerevisiae. Accumulation of ROS by UPR may be caused through two mechanisms; the oxidative folding machinery in the ER and the mitochondria-dependent ROS generation (29). However, our current results evidenced that oxidative stress was upstream, but not downstream of ER stress in cadmium-exposed cells. Consistent with our results, some recent reports also indicated that ER stress may be involved downstream of ROS. For example, in rat brain tumor cells, the anticancer agent geldanamycin caused expression of GRP78 via a ROS-dependent mechanism (30). In human vascular endothelial cells, ONOO Ϫ caused modest increases in GRP78 and GRP94 proteins (31). The source of ROS in cadmium-exposed cells was not investigated in the present study, but the mitochondrial pathway is possibly responsible for the generation of ROS in cadmium-exposed tubular cells (28).
Currently, it is still unclear how ROS induces ER stress. Previous reports showed that oxidative stress caused inhibition of Ca 2ϩ -ATPase (32, 33), a known trigger of ER stress. One possibility is, therefore, that ROS may cause depletion of the calcium store in the ER via inhibition of Ca 2ϩ -ATPase (34). Another possibility is that ROS might cause ER stress through generation and accumulation of oxidatively modified, abnormal proteins. Unfolded proteins may also be accumulated in the ER through ROS-induced functional perturbation of ER foldases and/or chaperones (35).
Among three major UPR branches, the ATF6 pathway and the IRE1 pathway mediate cadmium-triggered apoptosis, and induction of CHOP by ATF6 and activation of both XBP1 and JNK by IRE1 are involved in the apoptotic process (1). In the present investigation, we disclosed that O 2 . produced in FIGURE 7. Current hypothesis on the mechanism involved in cadmiumtriggered apoptosis. Cadmium causes generation of O 2 . and consequently induces ER stress that triggers proapoptotic branches of UPR, including the ATF6-CHOP pathway and the IRE1-XBP1-JNK pathway. Cadmium also causes generation of H 2 O 2 that contributes to apoptosis, whereas this proapoptotic signaling is independent of ER stress and subsequent proapoptotic UPR. Cadmium could cause generation of NO and ONOO Ϫ , but these RNS/ROS are not involved in the induction of ER stress and apoptosis by cadmium. Dashed lines indicate possible but not experimentally confirmed pathways. cadmium-exposed cells can trigger the ATF6-CHOP and the IRE1-XBP1 proapoptotic pathways. An interesting finding is that activation of JNK, an important mediator of cadmiuminduced apoptosis (1), was located downstream of XBP1. In general, it is believed that, in response to ER stress, activation of JNK occurs following recruitment of tumor necrosis factor receptor-associated factor 2 (TRAF2) by IRE1. ASK1 is also required for the TRAF2-dependent JNK activation, and the IRE1-TRAF2-ASK1-mediated phosphorylation of JNK plays a crucial role in ER stress-induced apoptosis independently of the IRE1-XBP1 pathway (36). To our knowledge, this is the first report demonstrating the existence of the XBP1-JNK proapoptotic pathway involved in ER stressinduced apoptosis.
ROS are involved in a wide range of cellular events under various pathophysiological circumstances. Our current finding that ER stress can locate downstream of oxidative stress raises a possibility that ER stress and UPR may also be involved not only in cadmium-induced apoptosis but also in a variety of other biological responses. Further investigation will be required to elucidate the spectrum of pathophysiological significance of the oxidative stress-ER stress axis in living cells and animals.