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186 NATURE CELL BIOLOGY VOLUME 7 | NUMBER 2 | FEBRUARY 2005

OASIS, a CREB/ATF-family member, modulates UPR signalling in astrocytesShinichi Kondo1,2, Tomohiko Murakami1,2, Kouko Tatsumi3, Maiko Ogata1,2, Soshi Kanemoto1,2, Kumi Otori2, Ken Iseki4, Akio Wanaka3 and Kazunori Imaizumi1,5

Endoplasmic reticulum (ER) stress transducers IRE1, PERK and ATF6 are well known to transduce signals from the ER to the cytoplasm and nucleus when unfolded proteins are accumulated in the ER. Here, we identified OASIS as a novel ER stress transducer. OASIS is a basic leucine zipper (bZIP) transcription factor of the CREB/ATF family with a transmembrane domain that allows it to associate with the ER. The molecule is cleaved at the membrane in response to ER stress, and its cleaved amino-terminal cytoplasmic domain, which contains the bZIP domain, translocates into the nucleus where it activates the transcription of target genes that are mediated by ER stress-responsive and cyclic AMP-responsive elements. Intriguingly, OASIS was induced at the transcriptional level during ER stress in astrocytes of the central nervous system, but not in other cell types examined. Furthermore, overexpression of OASIS resulted in induction of BiP and suppression of ER-stress-induced cell death, whereas knockdown partially reduced BiP levels and led to ER stress in susceptible astrocytes. Our results reveal pivotal roles for OASIS in modulating the unfolded protein response in astrocytes, and the possibility that cell type-specific UPR signalling also exists in other cells.

Eukaryotic cells have adapted to deal with unfolded protein accumulated in the ER by producing diverse signals in the ER lumen that signal to the cytoplasm and nucleus. This system is termed the unfolded protein response (UPR)1–3. The three major transducers of the UPR are IRE1, PERK and ATF6, which all sense the presence of unfolded proteins in the ER lumen and transduce signals to the nucleus to activate the transcrip-tion of UPR target genes. The mammalian ER stress-response element (ERSE) is present in the promoter regions of most UPR target genes, including BiP4. IRE1, a transmembrane kinase, processes the XBP1 mRNA to generate mature XBP1 mRNA5–9. Spliced XBP1 binds directly to ERSE and activates the transcription of BiP. ATF6, a transmembrane

1Department of Anatomy, Faculty of Medicine, University of Miyazaki, Kihara 5200, Kiyotake, Miyazaki 889-1692, Japan. 2Division of Structural Cellular Biology, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. 3Department of Anatomy, Nara Medical University, 840 Shijyo-cho, Kashihara, Nara 634-8522, Japan. 4Department of Emergency Medicine, Yamagata University, School of Medicine, 2-2-2 Iidanishi Yamagata city, Yamagata 990-9585, Japan. 5Correspondence should be addressed to K.I. (e-mail: imaizumi@med.miyazaki-u.ac.jp)

Published online: 23 January 2005, DOI: 10.1038/ncb1213

transcription factor, is cleaved by site-1 and site-2 proteases (S1P and S2P) in response to ER stress10,11. The cleaved ATF6 N-terminal frag-ment migrates to the nucleus to activate the transcription of BiP through direct binding to ERSE. ATF4, a transcription factor whose translation is up-regulated by the PERK-eIF2α pathway3, can activate the BiP pro-moter independent of ERSE. The ATF4-activating site is localized to the cAMP-responsive element (CRE)-like sequence12.

Excessive or long-term ER stresses result in apoptotic cell death13,14, and cell fate after ER stress is regulated by a balance between apoptosis and UPR signalling. The nervous system is composed of heterogeneous cells, including neuronal and glial cells, and among these cell populations astrocytes have the unique ability to tolerate, and even proliferate, under ischemic and hypoxic stress conditions that lead to ER stress15. These observations raise the possibility that astrocytes may adapt to ER stress using different signalling pathways from those of neurons or other cells.

We searched for gene homologues to ER-stress transducers using nucleotide databases to identify novel ER-stress transducers involved in UPR signalling in astrocytes. As a result, we found OASIS (old astrocyte specifically induced substance), which is a transmembrane transcription factor with a bZIP domain in its cytoplasmic portion16,17. The overall structure of OASIS is similar to that of ATF6 (Fig. 1a). In the luminal segment, OASIS contains the sequence Arg-Ser-Leu-Leu (RSLL) begin-ning at amino acid 423 (Fig. 1b), which fits the RXXL consensus for S1P, a membrane-anchored serine protease in the Golgi lumen18,19, suggesting that OASIS is cleaved by regulated intramembrane proteolysis (RIP), similarly to ATF6 and SREBP-2. To detect OASIS expression, western blot analysis with an anti-OASIS antibody was performed on primary astrocytes after exposure to ER stressors: 1 µM thapsigargin, an inhibitor of ER Ca2+-ATPase, or 3 µg ml–1 tunicamycin, an inhibitor of N-glyco-sylation. In the absence of these agents, OASIS was expressed as a protein with a relative molecular mass (Mr) of 80,000 (Fig. 1c). Treatment with ER stressors led to the appearance of a new 50K band (p50OASIS) in addition to the 80K band, suggesting that OASIS is cleaved in response to ER stress. The same findings were observed in astrocyte-derived C6

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Figure 1 OASIS is upregulated and cleaved at the transmembrane region in response to ER stress in astrocytes. (a) Predicted peptide features of human OASIS and ATF6. The basic region, leucine-zipper, putative transmembrane domain and luminal domain are indicated. (b) Comparison of amino-acid sequences of transmembrane regions of human OASIS, ATF6 and SREBP-2. The consensus sequence of the S1P site (RXXL; red) is conserved within these proteins. The proline residue, boxed in red, in the transmembrane region of OASIS is a putative S2P recognition site10. The box denotes the position of the transmembrane domains. (c) Expression of endogenous OASIS in primary astrocytes. Cells were incubated with 1 µM thapsigargin (TG) or 3 µg ml–1 tunicamycin (Tm) for 15 h, and lysates subjected to western blot analysis with an anti-OASIS antibody. A 50K band (p50OASIS) appears in cells treated with ER stress. Asterisk denotes partially N-glycosylated or non-glycosylated forms of OASIS. The anti-KDEL antibody recognizes BiP. Bottom, quantitative analysis of OASIS protein levels (OASIS-FL and p50OASIS) determined

by band intensity. (d) Subcellular localization of OASIS. C6 glioma cells stably transfected with OASIS were incubated in the presence or absence of 1 µM thapsigargin for 12 h and then co-stained with anti-OASIS and anti-KDEL antibodies. Note, OASIS immunoreactivity completely overlaps that of KDEL under normal conditions, but after ER stress the OASIS signal accumulates in the nucleus. Bottom, OASIS-374 localized in the nucleus. (e) OASIS mRNA is specifically induced by ER stress. C6 glioma cells were exposed to tunicamycin (Tm; 3 µg ml–1), thapsigargin (TG; 1 µM) or staurosporine (STS; 200 nM) for the indicated times. Total RNA was isolated from each culture and subjected to northern blot analysis with probes for OASIS, BiP or β-actin mRNAs. (f) Expression of OASIS mRNA in various cell types; each type treated with 1 µM thapsigargin for the indicated times. OASIS mRNA is notably induced in primary astrocytes after ER stress, but not in other cells. Ethidium bromide-stained 28S ribosomal RNA indicates that the amounts of total RNA were nearly equivalent in each lane.

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glioma (see Supplementary Information, Fig. S1a). Interestingly, the total amounts of OASIS proteins (80K + 50K proteins) increased in primary astrocytes during ER stress. However, the level of OASIS was much lower in mouse embryonic fibroblasts (MEFs) and was non-detectable in neu-roblastoma Neuro 2A cells. In C6 glioma cell lines stably transfected with full-length OASIS, p50OASIS was also generated after treatment with thapsigargin (see Supplementary Information, Fig. S1b). After transfec-tion of a cDNA encoding OASIS with mutations at both the S1P and S2P sites, no p50OASIS could be detected (see Supplementary Information, Fig. S1c), suggesting that OASIS is cleaved by S1P and S2P in response to ER stress, similarly to ATF6.

Immunocytochemistry with an anti-OASIS antibody showed fine reticular staining surrounding the nucleus in unstressed C6 glioma cells (Fig. 1d). The staining pattern was very similar to that obtained with the anti-KDEL antibody, which recognizes the ER molecular chaper-ones BiP and GRP94. When cells were treated with thapsigargin, OASIS immunoreactivity accumulated in the nucleus and did not overlap with

those of BiP and GRP94. We thus concluded that OASIS is cleaved in response to ER stress, and that the released p50OASIS is translocated into the nucleus. In addition to immunocytochemistry, fractionation examination was used to confirm that full-length OASIS and p50OASIS were recovered exclusively in the ER and nuclear fractions, respectively (see Supplementary Information, Fig. S1d).

We next examined the temporal changes in OASIS mRNA levels in C6 glioma cells after treatment with various ER stressors. A relatively low level of OASIS mRNA expression was detected before treatment with tunicamycin (Fig. 1e). The signal increased 6 h after treatment and a higher level of expression was observed at 12 h. Treatment with thapsi-gargin also induced expression of OASIS mRNA. However, the stresses induced by 200 nM staurosporine, which do not activate the UPR, did not induce expression of OASIS mRNA, indicating that OASIS induction may be regulated by UPR signalling. We further examined OASIS mRNA expression after ER stress in various other cells. Among the cells exam-ined, only primary astrocytes induced OASIS mRNA to a similar degree

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Figure 2 Activation of the BiP promoter by OASIS. (a) Schematic structures of full-length OASIS (OASIS-FL), its carboxy-terminal deletion mutant (OASIS-374), a dominant-negative form of OASIS (OASIS-Δ56)17, full-length ATF6 (ATF6-FL) and a C-terminal deletion mutant of ATF6 (ATF6-373). The locations of the basic region (basic), leucine zipper (ZIP) and transmembrane domain (TM) are indicated. Bottom, expression level of each construct. Lysates of transfected C6 glioma cells were subjected to western blot analysis with anti-OASIS (left) or anti-ATF6 (right) antibodies. (b) C6 glioma cells were transfected with the indicated constructs and the reporter plasmid pGL2-long BiP promoter (–366)–luc. The relative luciferase activities in the cells incubated in the presence (solid boxes) or absence (open boxes) of 1 µM thapsigargin (TG) for 16 h were determined; the means ± s.d. of the results from four independent experiments are shown. Top, schematic structure of the reporter construct. (c,d) Northern blot analysis of BiP mRNA. In c, HEK293T cells were

transiently transfected with the indicated expression plasmids. In d. C6 glioma cells stably transfected with mock or OASIS-FL expression plasmids were stimulated with 1 µM thapsigargin for the indicated times and total RNA extracted to examine the levels of BiP mRNA. Bottom, quantitative analyses of the BiP mRNA expression levels. (e) OASIS binds to the BiP promoter in response to ER stress in vivo. Top, schematic representation of the rat BiP promoter and the annealing sites of the primer set used in the ChIP assays. Bottom, C6 glioma cells were treated with 1 µM thapsigargin for the indicated times and then formaldehyde crosslinked, before immunoprecipitation of the chromatin with the antibodies indicated at the top. Purified, immunoprecipitated and input DNAs were analysed by PCR using the primers shown in the top panel. The PCR products of the input DNA and DNAs immunoprecipitated with the anti-OASIS and anti-histone H3 antibodies were electrophoresed in a 5% polyacrylamide gel and stained with ethidium bromide.

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as C6 glioma cells (Fig. 1f). OASIS has been reported to be expressed in the pancreas, prostate17 and osteoblasts during osteogenesis20; therefore, whether or not the induction of OASIS mRNA is specific to astrocytes remains to be clarified. ATF6 mRNA was expressed at high levels in all cells examined (see Supplementary Information, Fig. S1e), suggesting that ATF6 functions ubiquitously, whereas OASIS may function in a cell type-specific manner.

We next examined the effects of OASIS on the expression of a reporter gene carrying the human BiP promoter (–366 to +52), which contains a CRE-like site and three tandem ERSE sites, immediately upstream of the firefly luciferase gene (pGL2-long BiP promoter (–366)–luc). Thapsigargin treatment of C6 glioma cells transfected with the vector alone enhanced the reporter expression about twofold (Fig. 2b). Under normal conditions, transcription of the reporter gene was activated by overexpression of full-length OASIS (OASIS-FL), as it was after overex-pression of full-length ATF6 (ATF6-FL). This elevation of the reporter transcription is most probably the result of constitutively activate pro-teolysis of OASIS and ATF6 by transient overexpression (Fig. 2a). The reporter activities in cells transfected with OASIS-FL or ATF6-FL were

enhanced by thapsigargin treatment, suggesting that OASIS is cleaved in response to ER stress and that p50OASIS activates the BiP promoter. Indeed, transfection of OASIS carboxy-terminal deletion mutant (OASIS-374) into HEK293T cells elevated the level of endogenous BiP mRNA, but it was rather lower compared with that by ATF6-373 (Fig. 2c). We also observed that stable transfectants containing full-length OASIS achieved higher induction of BiP mRNA after ER stress than mock-transfected control cells (Fig. 2d).

We attempted to detect direct binding of OASIS to the BiP promoter region (–25 to –249) in C6 glioma cells treated with thapsigargin, using chromatin immunoprecipitation (ChIP) assays. When the anti-OASIS antibody was used for immunoprecipitation with equal amounts of input DNA, no binding of OASIS to the BiP promoter was detected in non-stressed cells. However, binding was detected 2 h after thap-sigargin treatment and a high level of binding was observed at 4 h (Fig. 2e). In contrast, binding of core histone H3 to the same promoter region was similar before and after thapsigargin treatment. These find-ings indicate that endogenous OASIS binds to the BiP promoter in response to ER stress.

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Figure 3 Effects of OASIS on CRE and ERSE sites. (a) CRE and ERSE1-3 of the BiP promoter were disrupted by mutating their sequences. Intact or mutant promoters were inserted into the pGL3-Basic vector (pGL3-short BiP promoter (–304)–luc). Left, schematic representations of intact and mutant constructs. The columns show the relative activities of reporter assays after transfection of the indicated expression plasmids. (b) Primary astrocytes (top) and Neuro 2A (bottom). Cells were transfected with each construct, and then 5 h after transfection were either left untreated or were

treated with thapsigargin (TG) for 16 h. The luciferase activity in each sample was normalized and plotted with each non-treated reporter activity set as one. (c) Direct binding of OASIS to the CRE site. The 32P-labelled CRE sequence of the BiP promoter was incubated with in vitro-translated Flag-OASIS-374. Note that competition by a 100-fold excess of the unlabelled CRE sequence (lane 3) and a supershift after incubation with an anti-Flag antibody (lane 5) are detected. Bottom, western blot analysis of in vitro-translated OASIS-374 and ATF6-373.

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Figure 4 Requirement for OASIS in the UPR signal and protective effects on ER stress-induced cell death. (a) Decreased induction of BiP mRNA after ER stress in C6 glioma cells treated with OASIS siRNA (OASIS-K13). Cells were treated with the indicated siRNAs, incubated for 36 h, and then stimulated with 1 µM thapsigargin (TG) for the indicated times. RT–PCR was performed to examine the levels of the indicated genes. OASIS siRNA effectively reduces OASIS mRNA expression. Bottom, quantitative analysis of the BiP mRNA expression levels. Twelve and 24 h after ER stress, the levels of BiP mRNA are decreased in OASIS siRNA-treated cells. By contrast, XBP1 splicing is not affected. XBP1-u, unspliced form of XBP1; XBP1-s, spliced form of XBP1. (b) Expression of various genes after treatment with OASIS siRNA. C6 glioma cells treated with the indicated siRNA were stimulated with 1 µM thapsigargin for 12 h. Note that BiP and PDI mRNA expression are significantly reduced, whereas the other genes remain almost unchanged. (c) Parent C6 glioma cells (C6) or

cells stably transfected with full-length OASIS (two cell lines, OA1 and OA2) or mock vectors (Mock) were exposed to 1 µM thapsigargin for the indicated periods. Representative phase-contrast micrographs of mock and OASIS-expressing cells (OA2) are shown. Right, TUNEL staining 36 h after treatment. In cells expressing OASIS, cell death is delayed and suppressed compared with mock-vector-transfected cells. (d) Quantitative analysis of cell death 30 h after treatment with thapsigargin or staurosporine. Cell death was determined by the morphology (round and shrunken cells were counted as dead cells), TUNEL-positive cell number and released LDH activity. Data are the means ± s.d. from four independent experiments. Asterisk indicates P < 0.05; double asterisk indicates P < 0.01 relative to the control (Student’s t-test). (e,f) Cells were treated with thapsigargin or staurosporine for 24 h. Cell death assay of C6 glioma cells treated with OASIS siRNA (e). Cell death assays for neuroblastoma Neuro 2A and MEF cells treated with OASIS siRNA (f).

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Figure 5 OASIS expression after cryo-injury to the cortex. (a) Expression of OASIS, BiP, XBP1, ATF6 and β-actin mRNAs in injured brains. Seven days after injury, control and injured regions were collected for RNA extraction and RT–PCR performed. (b) Protein levels of OASIS, BiP and β-actin in the injured brains. Bottom, quantitative analysis of OASIS protein levels (OASIS-FL and p50OASIS) in control and injured sides. (c) Photomicrograph of cryo-injured mouse brain (Haematoxylin and eosin (HE) stain, 7 days after the injury). Cryo-injury was inflicted to the right cerebral cortex (see Methods section). (d) Higher magnification of the boxed area in c. The lesion reproducibly consists of a necrotic core region (star) and a surrounding reactive region (arrows). (e) In situ hybridization histochemistry of OASIS mRNA. OASIS mRNA (arrows) is detected in the region surrounding the necrotic lesion (star). (f, g) Immunohistochemistry of OASIS in control (f)

and injured (g) brain. Note that OASIS is expressed in reactive astrocytes in the injured region (arrows). (h–j) Combined in situ hybridization and immunohistochemistry analyses of OASIS mRNA and GFAP protein. OASIS mRNA expression is primarily observed in cells close to the necrotic core. These cells have a moderate level of GFAP-like immunoreactivity (proximal reactive astrocytes24). Arrows show cells that are double-labelled for OASIS mRNA and GFAP protein. By contrast, cells distant from the necrotic core show intense immunoreactivity for GFAP and prominent cellular processes (distal reactive astrocytes), and are mostly negative for OASIS mRNA. (k–m) Double-labelling in situ hybridization for OASIS and BiP mRNAs in injured brain. (n–p) Double-labelling immunohistochemistry for OASIS and BiP proteins. OASIS immunoreactivity is primarily detected in the cytoplasm and overlaps markedly with BiP immunoreactivity.

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To examine whether OASIS activates ERSE or CRE sites, we mutated the ERSE and/or CRE sites in reporter constructs (pGL3-short BiP pro-moter (–304)–luc); Fig. 3a). Mutations of ERSEs alone reduced the reporter activity induced by OASIS-374 by about 20% compared with the wild-type promoter; a rather small reduction. Mutation of the CRE site decreased the activity to less than 50%, and this more severe effect suggests that OASIS targets both the ERSE and CRE sites, but preferentially acts on the CRE site as a transcription activator. The CRE site was found to be necessary for induction of the BiP promoter in astrocytes; that is, the activities of a reporter carrying mutant CRE with intact ERSE was decreased by about 35% compared with those of the wild-type promoter when primary astro-cytes were treated with thapsigargin (Fig. 3b). Electrophoretic mobility shift assays (EMSAs) were used to confirm that OASIS directly bound to the CRE sequence in the BiP promoter (Fig. 3c). By contrast, binding of OASIS to the ERSE probe was not detected. The reason for this is not known, although it is possible that the binding of OASIS is either very weak or requires other nuclear factors in astrocytes to bind directly to the ERSE.

To more directly assess the requirement for OASIS in UPR signal-ling, we used small interfering RNAs (siRNAs). We confirmed that OASIS siRNA (OASIS-K13, OASIS-K26) specifically targeted OASIS mRNA and efficiently degraded it (see Supplementary Information, Fig. S2a). Treatment of C6 glioma cells with OASIS siRNA (OASIS-K13) led to a marked reduction in levels of OASIS protein (Fig. 4a; also see Supplementary Information, Fig. S2). In cells treated with OASIS siRNA, BiP mRNA was induced to similar levels as in control cells 6 h after expo-sure to thapsigargin; however, at 12 h and thereafter, the level decreased by 50% compared with the control cells at each time point (Fig. 4a). This suggests that OASIS is important for BiP induction during ER stress, particularly at the late phase of UPR signalling, which may be related to the transcriptional induction of OASIS mRNA after ER stress. ATF6 (see Supplementary Information, Fig. S1e), IRE1 and PERK (data not shown) were all expressed in astrocytes, and therefore the roughly 50% reduction in BiP mRNA induction by OASIS knockdown may be due to other intact UPR signalling pathways. No significant reductions in the expression of other genes induced during ER stress was detected, except for protein disulphide isomerase (PDI), in OASIS siRNA-treated cells (Fig. 4b).

BiP functions as a cytoprotective protein in stressed cells21–23. As OASIS activates BiP induction during ER stress, OASIS might protect cells from ER stress. To investigate whether OASIS suppresses ER stress-induced cell death, C6 stable transfectants of OASIS-FL were exposed to thapsigargin. Apoptotic cell death was induced in parent C6 glioma or mock-transfected cells (Fig. 4c, d; also see Supplementary Information, Fig. S3a). By contrast, OASIS-transfected cells were significantly more resistant to ER stress-induced cell death. OASIS transfectants were also resistant to other ER stressors, including tunicamycin and calcium iono-phore A23187 (data not shown). Alternatively, cells treated with OASIS siRNA markedly underwent cell death (Fig. 4e; also see Supplementary Information, Fig. S3b). Treatment of neuroblastoma Neuro 2A and MEF cells with OASIS siRNA did not affect their vulnerability to ER stress (Fig. 4f). Thus, we conclude that OASIS is required to protect astrocytes from ER stress, but does not protect other cells, such as neuroblastoma cells and fibroblasts. Although detailed mechanisms remain unclear, one possibility is that activation of the UPR, including induction of molecu-lar chaperones by OASIS, may contribute to protection from cell death induced by ER stress. Another possibility is that OASIS may transcrip-tionally upregulate anti-apoptotic genes.

To analyse the in vivo function of OASIS, we assessed the expression of OASIS and its targets in cryo-injured brains. Both mRNA and protein lev-els of OASIS were elevated, and the cleaved fragments of OASIS appeared in the injured cortex (Fig. 5a, b). Cells expressing OASIS appeared in the region surrounding necrotic tissue 7 days after injury (Fig. 5e, g), and showed a similar distribution to glial fibrillary acidic protein (GFAP)-like immunoreactivity (Fig. 5h–j). These expression patterns suggest that the cells expressing OASIS mRNA are proximal reactive astrocytes with weak GFAP immunoreactivity24. By contrast, distal reactive astrocytes with intense GFAP immunoreactivity and prominent cellular processes were negative for OASIS mRNA. Most reactive astrocytes expressing OASIS mRNA showed induction of BiP mRNA (Fig. 5k–m). Double-labelling immunocytochemistry revealed expression of OASIS protein in BiP-positive reactive astrocytes and these signals notably overlapped each other, suggesting that OASIS mainly resides in the ER (Fig. 5n–p). However, accumulation of the OASIS N-terminal fragment was not detected in nuclei of reactive astrocytes, probably because it is difficult to detect small amounts of cleaved OASIS translocated into nuclei using immunohistochemical techniques in vivo. Expression of OASIS in reac-tive astrocytes may be involved in UPR activation in injured brains, but further analyses, including investigations using OASIS-deficient mice, are required to elucidate the in vivo functions of OASIS.

This study has characterized a gene product, OASIS, that seems to function as an ER-stress transducer for the mammalian UPR. Our hypothesis that OASIS functions as an ER-stress transducer is supported by the following points: first, OASIS is an ER-resident transmembrane protein that is cleaved at the membrane in response to ER stress; second, the cleaved OASIS N-terminal fragment containing the bZIP domain is translocated into the nucleus; third, endogenous OASIS binds to the BiP promoter in response to ER stress, and the OASIS N-terminal fragment acts on the ERSE and CRE elements in the BiP promoter; fourth, OASIS activates the transcription of endogenous BiP; and finally, knockdown of OASIS suppresses the induction level of BiP mRNA at the late stage of UPR. Putative mechanisms for the activation of the UPR by OASIS in astrocytes are shown in Supplementary Information, Fig. S4.

CREB-H and BBF2H7 — transcription factors that possess both bZIP and transmembrane domains and are structurally very similar to OASIS, and for which authentic biological roles have not yet been defined — localize to the ER25. These factors may be processed in a similar manner to OASIS and ATF6, which are sequestered in cellular membranes in response to ER stress, and are activated by RIP — a process that allows cells to respond rapidly to physiological crises by activating pre-made transcription factors. Understanding RIP regulation of these transcription factors and determin-ing the target genes should enable the discovery of novel signal pathways involved in the UPR or cell type-specific ER stress responses.

METHODS

Plasmids, transfection, antibodies and western blot analysis. Flag-tagged mouse OASIS cDNA26 was inserted into pCDNA3.1+. Site-directed mutagen-esis was performed and a deletion mutation of OASIS made by PCR to create Flag–OASIS-374, OASIS-Δ56 and OASISP392L/R423A/L426V. Expression plasmids for full-length ATF6, ATF6-373, XBP-1 and pGL3-short BiP promoter (–304)–luc were gifts from K. Mori (Kyoto University), whereas pGL2-long BiP promoter (–366)–luc was provided by Y. Omori (Tokyo University). C6 glioma or HEK293T cells were transfected with each expression plasmid using Lipofectamine or Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA), and each stable trans-formant was established by selection with G418 (Invitrogen).

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Rabbit antisera were raised against recombinant OASIS (amino acids 17–253) fused to maltose-binding protein and affinity purified using a ProtOn kit1 (Multiple Peptide Systems, San Diego, CA). Anti-Flag (Eastman Kodak Company, New Haven, CT), anti-KDEL, anti-calnexin and anti-HSP60 (StressGen Biotechnologies Corporation, Victoria, BC, Canada), anti-PARP (Cell Signaling Technology, Beverly, MA), anti-CHOP (GADD153; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-actin (Chemicon, Temecula, CA) antibodies were pur-chased commercially. K. Mori also provided an anti-ATF6 antibody. For western blot analysis of OASIS, cells were lysed in hot SDS as described27, before electro-phoresis in 10% SDS–PAGE gels. The protein concentration of each sample was quantified by the Lowry assay (DC protein assay; Bio-Rad, Hercules, CA), and protein-equivalent samples were subjected to western blot analysis.

Subcellular fractionation. Stable transfectants of OASIS cultured to 70% con-fluence in 150-mm dishes were incubated in the absence or presence of 1 µM thapsigargin for 12 h. Cells were harvested, disrupted using a Dounce-type homogenizer and centrifuged at 600g for 10 min to obtain nuclear pellets. The resultant supernatants were then centrifuged at 3,000g for 10 min to produce mitochondrial pellets. Supernatants were further centrifuged at 100,000g for 1 h and pellets collected. Extracts were subjected to SDS–PAGE and then analysed by western blotting with anti-OASIS and various other antibodies.

Northern blotting and RT–PCR assays. Northern blotting and RT–PCR assays were performed as described28. The oligonucleotides used for amplification of each cDNA can be found in the Supplementary Information.

Luciferase assay. C6 glioma cells or primary astrocytes plated onto 24-well plates were transfected with 0.2 µg of a reporter plasmid carrying the firefly luciferase gene and 0.02 µg of the reference plasmid pRL-SV40 carrying the renilla luciferase gene under the control of the SV40 enhancer and promoter (Promega, Madison, WI) with or without 0.1 µg of an effector protein expression plasmid. After 46 h, cells were lysed in 200 µl of Passive Lysis Buffer (Promega). For induction of ER stress, cells were treated with 1 µM thapsigargin for 16 h before harvesting. Firefly luciferase and renilla luciferase activities were measured in 10 µl of cell lysate using the Dual-Luciferase Reporter Assay System (Promega) and a luminometer (Berthold Technologies, Bad Wildbad, Germany). Relative activity was defined as the ratio of firefly luciferase activity to renilla luciferase activity.

Chromatin immunoprecipitation assays. C6 glioma cells were grown to 80% confluence in 10-cm plates under normal cell culture conditions and then treated with 1 µM thapsigargin for 2 or 4 h. Protein–DNA crosslinking was initiated by adding formaldehyde directly to the culture medium to a final concentration of 1% and was stopped after 15 min by adding 1.5 M glycine to a final concentration

of 0.15 M. To harvest the C6 glioma cells, plates were rinsed with cold PBS and scraped. Chromatin was prepared using a kit (Upstate, Lake Placid, NY) accord-ing to the manufacturer’s recommendation of 24× 5-s sonication pulses at 10-s intervals, which yielded chromatin fragments with apparent sizes of 100–400-base-pairs (bp). An aliquot from each sample representing 5% of the total vol-ume was removed for use as the input fraction and processed with the eluted immunoprecipitates beginning at the crosslink reversal step. Equal amounts of chromatin from each sample were incubated overnight at 4 °C with 5 µl of anti-OASIS or 5 µl of anti-H3 (Santa Cruz Biotechnology) antibodies. Formaldehyde-induced cross-linking was reversed (6 h at 65 °C) and the DNA was purified by phenol–chloroform extraction and ethanol precipitation. Purified DNA from the input and immunoprecipitation samples was subjected to 35 cycles of PCR and the products were electrophoresed in 5% polyacrylamide gels and visual-ized by ethidium bromide staining. The primers used for the endogenous rat BiP promoter were: 5′-CATTGGCGGCCGTTAAGAATGACCAG-3′ (forward) and 5′-AGTATCGAGCGCGCCGGTC-3′ (reverse), yielding a 225-bp product.

Electrophoretic mobility shift assay. In vitro translation of genes of interest was performed using each cDNA and TNT Quick Coupled Transcription/Translation Systems (Promega). Double-stranded synthetic oligonucleotides were radiola-belled using γ-32P-dATP (3,000 Ci mmol–1; Amersham, Piscataway, NJ) and T4 polynucleotide kinase (Promega). Two microlitres of translation products were incubated with each of the oligonucleotide probes (0.1 pmol; ~9,000 cpm) at 4 °C for 1 h in binding buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol (DTT), 50 mM NaCl, 10 mM Tris-HCl (at pH 7.5) and 0.05 mg ml–1

of poly(dI-dC)•poly(dI-dC)). Samples were loaded onto non-denaturing poly-acrylamide gels and electrophoresed at 200 V at 4 °C for 100 min in running buffer (0.5× TBE). The sequences of the oligonucleotides used in the binding were: 5′-CAGCTGGGGGGGCGGAGCAGTGACGTTTATTGCGGAGGGG-3′ (CRE-WT) and 5′-CAGCTGGGGGGGCGGAGCctgtcgactcATTGCGGAGGGG-3′ (CRE-MT) (lower-case letters indicate mutations). For supershift experiments, samples were treated with various antibodies at 4 °C for 1 h before incubation with a radiolabelled probe.

Cell death assay. The numbers of dead cells were counted based on morpho-logical changes, and the activity of LDH released in the culture medium was quantified as described29. TUNEL assays were performed using the DeadEndTM Fluorometric TUNEL System (Promega), and the cells were analysed directly under a fluorescence microscope.

Knockdown of OASIS. Annealed double-stranded siRNAs were obtained from Dharmacon (Lafayette, CO). The sequences of the OASIS siRNA can be found in the Supplementary Information. BLAST searches (www.ncbi.nlm.nih.gov/BLAST/) confirmed that these sequences were not homologous to any genes other than OASIS. For control siRNA, lamin A/C siRNA and non-silencing siRNA (Qiagen, Valencia, CA) were used. Cells at 60% confluence in 24-well plates were transfected with 0.8 µg of each of the above siRNAs using the RNAiFect transfec-tion reagent (Qiagen) according to the manufacturer’s protocol. The transfected cells were incubated at 37 °C for 24–36 h and then stimulated by ER stressors.

Animal surgery, immunohistochemistry and in situ hybridization. Adult male ICR mice were divided randomly into sham-operated and 1-min cryo-injury groups. All operation procedures were performed under pentobarbital anaes-thesia. For the 1-min cryo-injury group, a lead probe pre-chilled with liquid nitrogen was placed in unilateral contact with the cranium for 1 min. The mice were sacrificed 7 days after surgery. Brain tissues were dissected out and cut into 12-µm sections using a cryostat. The sections were then subjected to in situ hybridization and immunohistochemistry as described16.

Note: Supplementary Information is available on the Nature Cell Biology website.

ACKNOWLEDGEMENTSWe thank K. Mori and Y. Omori for providing plasmids and antibodies, and M. Tohyama and S. Shiosaka for helpful discussions and critical reading of this manuscript. This work was partly supported by the UEHARA Foundation, and grants from the Japan Society for the Promotion of Science KAKENHI (14208093).

COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests.

Received 8 November 2004; accepted 24 December 2004Published online at http://www.nature.com/naturecellbiology.

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Figure S1 Biochemical characteristics and expression of OASIS. a, Protein expression levels of OASIS in C6 glioma, mouse embryonic fibroblasts (MEF) and neuroblastoma Neuro 2A. Note that OASIS is expressed at a higher level and its cleaved fragments appear in C6 glioma. The total amounts of OASIS proteins (Full-length OASIS + p50OASIS) are increased after ER stress (lower panel). In contrast, the expression levels are much lower in MEF and Neuro 2A. F, Full-length OASIS; p50, p50OASIS. The asterisk denotes the partially glycosylated or non-glycosylated forms of OASIS. b, Cleavage of OASIS in C6 glioma cells stably transfected with full-length OASIS. Full-length OASIS and the cleaved fragment (p50OASIS) are indicated by arrows. c, Mutations of S1P and S2P recognition sites in OASIS alter its proteolytic processing in transfected C6 glioma cells. C6 glioma cells were transfected with plasmids encoding FLAG-tagged wild-type OASIS or S1P and S2P double mutations (OASIS (R423A/L426V, P392L)). After 24 h, cells were

treated with 1 µM thapsigargin, then harvested after 4 h and subjected to immunoblotting analysis with an anti-FLAG antibody. d, Distributions of full-length OASIS and its N-terminal portion. Subcellular fractionation was performed using cell lysates from stable transfectants of OASIS cultured in the presence or absence of 1 µM thapsigargin for 12 h, and the fractions were subjected to western blotting for the indicated proteins. The positions of full-length OASIS and its N-terminal fragment (p50OASIS) are marked. PARP, calnexin and Hsp60 were used as nuclear, ER and mitochondrial protein markers, respectively. e, Expression of ATF6 and OASIS mRNAs in various cells. Cells were treated with 1 µM thapsigargin for 12 h and then RT-PCR performed using specific primer sets for the indicated genes. ATF6 mRNA is expressed at similar levels in all cells. In contrast, OASIS mRNA is induced in C6 glioma cells after ER stress, whereas MEF and Neuro 2A cells show no induction of OASIS mRNA expression, or an extremely low level.

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Figure S2 Control experiments for siRNA. a, Effects of various siRNAs on OASIS mRNA knockdown. C6 glioma cells were treated with the indicated siRNAs, incubated for 36 h, and then stimulated with 1 µM thapsigargin (TG) for 12 h. Total RNA was extracted and RT-PCR was performed to examine the levels of OASIS mRNA. The lower panel shows the target sites of each siRNA against OASIS mRNA. Note that two types of OASIS siRNAs (OASIS-K13 and OASIS-K26) effectively reduce the expression of OASIS mRNA. b, HEK293T cells were cotransfected with GFP-tagged OASIS expression vectors and OASIS siRNA or non-silencing siRNA (control siRNA). Non-siRNA means that the cells were transfected with GFP-OASIS, but not treated with a siRNA. At 30 h after transfection, the cells were observed by fluorescence microscopy. Note that treatment with OASIS RNAi resulted in no GFP-OASIS expression, whereas control siRNA showed no effects on

GFP-OASIS expression. c, OASIS siRNA does not knockdown other genes that are homologous to the OASIS nucleotide sequence. Database searches showed that three genes contain sequences homologous to the OASIS-K13 siRNA sequence. The lower panel shows the extent of the match of the partial sequence of each gene to the OASIS-K13 siRNA sequence. The RT-PCR analysis shows that OASIS-K13 siRNA does not affect the expressions of these genes. d, Effects of OASIS siRNA at low concentrations on OASIS mRNA knockdown. C6 glioma cells were treated with the indicated doses of OASIS siRNA. OASIS siRNA is effective at very low concentrations. e, Each protein level in cells treated with OASIS or control siRNA (Cont). In OASIS siRNA-treated cells, the protein levels of OASIS are reduced and the induction of BiP protein after ER stress is attenuated, but CHOP is not.

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Figure S3 The expression levels of OASIS and cell death assay. a, The levels of OASIS protein in OA1 and OA2 cell lines are considerably higher than the physiological levels (C6). b, Representative phase-contrast images. Cells

treated with OASIS siRNA show an increase in ER stress-induced cell death compared with non- or control (cont) siRNA-treated cells.

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Figure S4 Putative mechanisms responsible for activation of the UPR by OASIS in astrocytes. OASIS is induced at a transcriptional level during ER stress in astrocytes. Translated OASIS is cleaved at the membrane by S1P and S2P in response to ER stress, and its cleaved N-terminal cytoplasmic

domain translocates into the nucleus; and then activates transcription of target genes via acting to the CRE and ERSE sites. This signal pathway mediated by OASIS may contribute to cell survival after ER stress in astrocytes.

Northern blotting and RT–PCR assays. The oligonucle-otides used for amplification of each cDNA were as fol-lows: OASIS-5’, 5’-ATGGACGCCGTCTTGGAACCTT-3’ and OASIS-3’, 5’-CTCAGCCTTGGTGAGGGGGAG-3’; BiP-5’, 5’-GAAAGGATGGTCAATGACGCCGAG-3’ and BiP-3’, 5’-GTCTTCAATGTCTGCATCCTGGTGG-3’; XBP1-5’, 5’-ACACGCTTGGGGATGAATGC-3’ and XBP1-3’, 5’-CCATGGGAAGATGTTCTGGG-3’; β-actin-5’, 5’-TCCTCCCTGGAGAAGAGCTAC-3’ and β-actin-3’, 5’-TCCTGCTTGCTGATCCACAT-3’; GRP94-5’,5’-TACTATGCCAGTCAGAAGAAAACG-3’ and GRP94-3’, 5’-CATCCTTTCTATCCTGTCTCCATA-3’; ATF6-5’, 5’-GGATTTGATGCCTTGGGAGTCAGAC-3’ and ATF6-3’, 5’-ATTTTTTTCTTTGGAGTCAGTCCAT-3’; CHOP-5’, 5’-ACAGTCATGGCAGCTGAGTCTCTGCCTTTC-3’ and CHOP-3’, 5’-CAGACAGGAGGTGATGCCAACAGTTCATGC-3’; PDI-5’, 5’-TCCAGCTGTGCGGCTTATTA-3’ and PDI-3’, 5’-TTCTCAGCTGTCAGCTCGTCTG-3’; LYRIC-5’, 5’-TTGAGTGCAGGTGA

GGAGAAGTGGAAC-3’ and LYRIC-3’, 5’-TGCATTCCAGTCTGAGCTAGGATCAG-3’; D9UIA10-5’, 5’-CCTTTTATTACTGATATTGACATGGGGAAG-3’ and D9UIA10-3’, 5’- GGCTTTNGGATTAANTTTCCAANGGAC-3’; protease16-5’, 5’- AGTAAACCTTGAACCTCCAGGTCCTCTTAC-3’ and pro-tease16-3’, 5’- ATGCGGTCCACAAAACTTGGGATTG-3’.

Knockdown of OASIS. The sequence of each OASIS siRNA was follows: OASIS-K2 siRNA, 5’-GCUUCCUGGACUUGGGAGAdTdT-3’ (sense) and 5’-UCUCCCAAGUCCAGGAAGCdTdT-3’ (antisense); OASIS-K9 siRNA, 5’-GAGCACAGCUACUCCCUGAdTdT-3’ (sense) and 5’-UCAGGGAGUAGCUGUGCUCdTdT-3’ (antisense); OASIS-K13 siRNA, 5’-GAAAUGAGCCAGUUUCUCAdTdT-3’ (sense) and 5’-UGAGAAACUGGCUCAUUUCdTdT-3’ (antisense); OASIS-K26 siRNA, 5’-GAGACAUAUACAUCAGAGAdTdT-3’ (sense) and 5’-UCUCUGAUGUAUAUGUCUCdT dT-3’ (antisense).

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