Nitric Oxide-Induced Apoptosis in Pancreatic ß Cells Is Mediated By

Nitric oxide-induced apoptosis in pancreatic ␤ cells is mediated by the endoplasmic reticulum stress pathway Seiichi Oyadomari*†, Kiyoshi Takeda‡, Masaki Takiguchi§, Tomomi Gotoh*, Makoto Matsumoto‡, Ikuo Wada¶, Shizuo Akira‡, Eiichi Araki†, and Masataka Mori*ʈ Departments of *Molecular Genetics and †Metabolic Medicine, Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto 860-0811, Japan; ‡Department of Host Defense, Research Institute for Microbial Diseases, and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan; §Department of Biochemistry, Chiba University School of Medicine, Inohana 1-8-1, Chiba 260-8670, Japan; and ¶Department of Biochemistry, Sapporo Medical University School of Medicine, South 1, West 17, Sapporo 060-8556, Japan Edited by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA, and approved July 13, 2001 (received for review April 28, 2001) Excessive nitric oxide (NO) production in cytokine-activated ␤ cells occurs when functions of the ER are severely impaired. This has been implicated in ␤ cell disruption in type 1 diabetes. ␤ cells apoptotic event is mediated by transcriptional activation of the are very vulnerable to NO-induced apoptosis. However, the mech- gene for CHOP͞GADD153, a member of the C͞EBP family of anism underlying this phenomenon is unclear. Low concentrations transcription factors (7, 8), and by activation of ER-associated of NO that lead to apoptosis apparently do not cause severe DNA caspase-12 (9). damage in mouse MIN6 ␤ cells. CHOP, a C͞EBP homologous protein Several lines of evidence indicate that CHOP is induced by ER that is induced by endoplasmic reticulum (ER) stress and plays a stress and plays a role in growth arrest and cell death (10). First, role in growth arrest and cell death, was induced by a NO donor, CHOP is induced under conditions that are known to increase S-nitroso-N-acetyl-D,L-penicillamine (SNAP). SNAP increased cyto- the level of unfolded proteins in the ER (10–13). Second, the 2؉ 2؉ solic Ca , and only agents depleting ER Ca induced CHOP unfolded protein response (14) and the induction of CHOP (10) expression and led to apoptosis, suggesting that NO depletes ER can be attenuated by the overexpression of an ER chaperone, 2؉ 2؉ Ca . Overexpression of calreticulin increased the Ca content of BiP͞GRP78 (14). Third, CHOP is induced by activation of Ire1␣ ER and afforded protection to cells against NO-mediated apoptosis. or Ire1␤ (15), ATF6 (16), and PERK (17). These results suggest Furthermore, pancreatic islets from CHOP knockout mice showed that the induction of CHOP is a direct consequence of the ER resistance to NO. We conclude that NO depletes ER Ca2؉, causes ER .؉ stress response stress, and leads to apoptosis. Thus, ER Ca2 stores are a new target ϩ We report here that ER Ca2 stores are a previously unrec- of NO, and the ER stress pathway is a major mechanism of ognized target of NO. NO depletes ER Ca2ϩ, causes ER stress, NO-mediated ␤ cell apoptosis. and leads to apoptosis through the ER stress pathway. Overex- pression of calreticulin (CRT) increased the Ca2ϩ content of the MEDICAL SCIENCES ype 1 diabetes is mediated by an autoimmune and inflam- ER and protected cells against NO-mediated apoptosis. NO Tmatory process characterized by selective disruption of pan- ␤ ␤ induced CHOP in mouse MIN6 cells, and the pancreatic islets creatic cells. Nitric oxide (NO) induced by inflammatory of CHOP-deficient mice were resistant to NO-mediated apo- cytokines, such as IL-1␤, tumor necrosis factor ␣ (TNF-␣), and ␥ ␤ ptosis. These findings highlight the importance of the ER stress IFN- , is a possible mediator of cell failure (1). NO is a pathway in NO-mediated apoptosis in ␤ cells. messenger molecule that mediates diverse biological functions, such as vasodilation, neurotransmission, and immunity; how- Materials and Methods ever, excessive production of NO leads to cytotoxic effects (2). Materials. S-nitroso-N-acetyl-D,L-penicillamine (SNAP), NG- It is generally thought that NO induces DNA damage leading to monomethyl-L-arginine, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin- cell death through induction of p53 and activation of poly(ADP- 1-one (ODQ), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazo- ribose)polymerase (1). However, several experiments suggested line-1-oxyl 3-oxide (carboxy-PT1O), and fura-2͞AM were that p53-independent signaling pathways operate during NO- purchased from Dojindo Laboratories (Kumamoto, Japan). mediated apoptosis (3). A consequence of severe DNA damage ␤ ␣ ␥ is the activation of poly(ADP-ribose)polymerase, which results Mouse IL-1 , TNF- , and IFN- were from Genzyme. Iono- in depletion of NADϩ and ATP and, subsequently, necrosis. Islet mycin, A23187, 2,5-di-tert-butylhydroquinone, cyclopiazonic acid, thapsigargin (TG), 1,2-bis-(o-aminophenoxy)-ethane- cells lacking poly(ADP-ribose)polymerase are more resistant to Ј Ј ͞ NO but do nevertheless undergo NO-induced cell death (4). N,N,N ,N -tetraacetic acid tetraacetoxy-methyl ester (BAPTA These results suggest that there is a cell death pathway distinct AM), 8-bromo-cGMP were from Biomol (Plymouth Meeting, from the DNA damage pathway. PA). KT5827 was from Calbiochem. The endoplasmic reticulum (ER) plays several important roles in the folding, export, and processing of newly synthesized This paper was submitted directly (Track II) to the PNAS ofﬁce. proteins. Various conditions can interfere with ER function, and Abbreviations: ER, endoplasmic reticulum; SNAP, S-nitroso-N-acetyl-D,L-penicillamine; these are collectively called ER stress. The unfolded protein SERCA, sarcoplasmic͞endoplasmic reticulum Ca2ϩ-ATPase; CRT, calreticulin; TNF-␣, tumor response is a mechanism by which cells self-protect against ER necrosis factor ␣; BAPTA͞AM, 1,2-bis-(o-aminophenoxy)-ethane-N,N,NЈ,NЈ-tetraacetic acid stress (5, 6). At least three functionally distinct responses have tetraacetoxy-methyl ester; TG, thapsigargin; HA, hemagglutinin peptide; GFP, green ﬂu- been identified. One response involves up-regulation of genes orescent protein; PKG, phosphoglycerate kinase; iNOS, inducible NO synthase. ʈ encoding ER chaperone proteins such as Bip͞GRP78 and To whom reprint requests should be addressed at: Department of Molecular Genetics, Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto 860-0811, Japan. GRP94 to increase protein folding activity and to prevent E-mail: [email protected]. protein aggregation. Another consists of translational attenua- The publication costs of this article were defrayed in part by page charge payment. This tion to reduce the load of protein synthesis and to prevent further article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. accumulation of unfolded proteins. Another is apoptosis, which §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.191207498 PNAS ͉ September 11, 2001 ͉ vol. 98 ͉ no. 19 ͉ 10845–10850 Downloaded by guest on September 26, 2021 Cell Culture and Transfection. Mouse insulinoma MIN6 cells and Generation of CHOP Knockout Mice. CHOP genomic DNA was mouse macrophage-derived RAW 264.7 cells were cultured in isolated by screening an 129͞Sv mouse genomic library (Strat- 60-mm collagen-coated dishes in DMEM supplemented with 25 agene). A targeting vector was designed to replace a 1.2-kb mM glucose and 10% FCS. Plasmid DNAs (5 ␮g in total) were genomic fragment containing parts of exons 3 and 4 with transfected into MIN6 cells, with the use of Lipofectamine 2000 pMC1-neo (Stratagene). The targeting vector was flanked by the (GIBCO). Plasmids pREP8 (control vector) (Invitrogen), 5.3-kb genomic fragment at the 3Ј end and the 1.2-kb fragment Ј Ј pCR(HA) (CRT expression vector) (18), pOPRSVI-Cat (con- at the 5 end and contained a HSV-tk cassette at the 3 end of trol vector) (Stratagene), and pOPRSVI-CHOP (CHOP expres- the vector. It was linearized with SalI and electroporated into sion vector) (8) were reported. pEGFP (enhanced green fluo- embryonic day 14.1 embryonic stem cells. Correctly targeted rescent protein expression vector; CLONTECH) and embryonic stem cell clones were identified within the clones pEYFP-ER (ER localization vector that expresses enhanced resistant to G418 and ganciclovir by PCR and Southern blot yellow fluorescent protein; CLONTECH) were purchased. analysis. Two independent targeted embryonic stem cell clones were microinjected into blastocysts of C57BL͞6 mice to generate ␮ chimeric mice. The chimeric male mice were mated with Northern and Western Blot Analyses. Northern blot analysis (2 g ͞ RNA per lane) was done as described (19). Digoxigenin-labeled C57BL 6 female mice. Two lines of mice contributed to the germ line. Heterozygous mice were intercrossed to obtain homozy- antisense RNA probes for mouse inducible NO synthase (iNOS) gous mice. (nucleotides 2344–3026) and mouse CHOP (nucleotides 68– 585) were synthesized with a transcription kit (Roche Molecular Isolation and Culture of Pancreatic Islets. Pancreatic islets were Biochemicals) (20). MIN6 cells were lysed in RIPA buffer (0.15 isolated from mice by digestion with collagenase (type V) and mM NaCl͞0.05 mM Tris⅐HCl, pH 7.2͞1% Triton X-100͞1% ͞ Ficoll gradient centrifugation, as described (21). Ten medium- sodium deoxycholate 0.1% SDS) containing complete mini sized islets per well were transferred into collagen-coated 96-well protease inhibitor mixture tablets (Roche Molecular Biochemi- plates and cultured for 3 days in DMEM containing penicillin G cals). Electrophoresis, immunoblotting, and detection were done (100 units͞ml), streptomycin (100 ␮g͞ml), and 10% FCS. as described (19). Rabbit anti-CRT polyclonal antibody (SPA- ͞ 600 StressGen, 1 1000) and mouse anti-human influenza hem- Reverse Transcription–PCR Analysis. The following primers were ͞ agglutinin peptide (HA) mAb (12CA5, 1 1000; Roche Molec- used with a reverse transcription–PCR kit (AMV version 2.1; ular Biochemicals) were used. Chemiluminescence signals were Takara Shuzo): mouse insulin II (GenBank accession no.

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