A Critical Role for A20 in -Cell Protection David Liuwantara,1 Mark Elliot,2 Mariya W
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Nuclear Factor-B Regulates -Cell Death A Critical Role for A20 in -Cell Protection David Liuwantara,1 Mark Elliot,2 Mariya W. Smith,2 Andrew O. Yam,1 Stacy N. Walters,1 Eliana Marino,1 Andy McShea,2 and Shane T. Grey1 Apoptotic -cell death is central to the pathogenesis of and as the final effector mechanism (5,6). Evidence from type 1 diabetes and may be important in islet graft rejec- the NOD mouse (a widely studied model of autoimmune tion. Despite this, genetic control of -cell apoptosis is only diabetes) (7) indicates that autoreactive cytolytic T-cells poorly understood. We report that inhibition of gene tran- (8), as well as soluble mediators including proinflamma- scription sensitized -cells to tumor necrosis factor (TNF)- tory cytokines and free radicals, contribute to increased ␣–induced apoptosis, indicating the presence of a regulated -cell apoptotic destruction during the pathogenesis of antiapoptotic response. Using oligonucleotide microarrays type 1 diabetes (6,9). Transplantation of islets is consid- and real-time PCR, we identified TNFAIP3/A20 as the most ered to be one potential approach that could restore highly regulated antiapoptotic gene expressed in cytokine- stimulated human and mouse islets. Cytokine induction of normal metabolic control for the cure of type 1 diabetes. A20 mRNA in primary islets and insulinoma cells was rapid However, multiple obstacles are faced in islet transplants, and observed within 1 h, consistent with A20 being an including cellular rejection akin to the mechanisms in- immediate early response gene in -cells. Regulation of volved in autoimmune destruction of -cells, and also A20 was nuclear factor-B (NF-B)–dependent, two NF-B primary nonfunction, a phenomena related to lack of sites within the A20 promoter were found to be necessary nutrients, hypoxia, and nonspecific inflammatory media- and sufficient for A20 expression in -cells. Activation of tors (10–12). NF-B by TNF receptor–associated factor (TRAF) 2, Despite the importance of apoptosis in the pathophysi- TRAF6, NF- B–inducing kinase, or protein kinase D, which ology of -cell death, the genetic control of apoptosis in transduce signals downstream of Toll-like receptors, TNF islets is poorly understood. Determining the molecular receptors, and free radicals, respectively, were all potent  activators of the A20 promoter. Moreover, A20 expression basis of -cell susceptibility to apoptosis would increase was induced in transplanted islets in vivo. Finally, A20 our understanding of the mechanisms underscoring -cell expression was sufficient to protect -cells from TNF- loss, as well as reveal potential gene therapy candidates induced apoptosis. These data demonstrate that A20 is the for the creation of “death-defying” islets (13), capable of cardinal antiapoptotic gene in -cells. Further, A20 expres- resisting immune and nonimmune insults. With this goal in sion is NF-B dependent, thus linking islet proinflamma- mind, we generated a custom microarray, a so-called tory gene responses with protection from apoptosis. “death-CHIP,” to map the immediate early antiapoptotic Diabetes 55:2491–2501, 2006 gene expression profile of cytokine-activated islets. Though later changes in islet gene expression after cyto- kine activation have been mapped in a number of studies (14,15), we focused only on the immediate early response, poptosis or programmed cell death is a geneti- as it is this response that determines cell fate after cally controlled response of the cell to commit inflammatory insult (16–19). Surprisingly, we found that in suicide (1,2). Apoptosis is the physiological contrast to other cell types, primary islets have a highly A process for cell deletion in normal, reorganiz- restricted immediate early antiapoptotic gene response, ing, or involuting tissue and is required for shaping of the with TNFAIP3/A20 being the most highly regulated anti- endocrine pancreas (3,4). Aside from its role in normal cell apoptotic gene. Significantly, we demonstrate that A20 is biology, -cell apoptosis has been implicated in the patho- regulated at the level of gene transcription in pancreatic physiology of type 1 diabetes, both at its initiation phase -cells by the proinflammatory transcription factor nu- clear factor-B (NF-B), thus linking islet inflammatory From the 1Arthritis and Inflammation Program, Garvan Institute for Medical gene responses with protection from apoptosis. Together, Research, Darlinghurst, New South Wales, Australia; and the 2Biology and with our previous studies demonstrating an anti-inflamma- Chemistry Section, CombiMatrix Corporation, Mukilteo, Washington. tory and antiapoptotic function for A20 (13,20–22), these Address correspondence and reprint requests to Dr. Shane T Grey, Gene Therapy & Autoimmunity Group, Arthritis and Inflammation Program, Garvan present data indicate that A20 is a critical component of Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW 2010 the islet-regulated response to inflammatory stress and Australia. E-mail: [email protected]. injury. Thus, we demonstrate that loss of A20 expression Received for publication 31 January 2006 and accepted in revised form 1  June 2006. renders -cells susceptible to apoptotic death; conversely, FACS, fluorescence-activated cell sorter; FADD, Fas-associated death do- enhancing A20 expression in -cells may improve their main; FADD-DN, FADD dominant negative inhibitor; -gal, -galactosidase; survival in the face of inflammatory and autoimmune IL, interleukin; IFN, interferon; IB␣, inhibitor of NF-B; NF-B, nuclear insults (13,21). factor-B; NIK, NF-B-inducing kinase; PKD, protein kinase D; Th, T-helper; TLR, Toll-like receptor; TNF, tumor necrosis factor; TNF-R, TNF receptor; TRAF, TNF receptor–associated factor. RESEARCH DESIGN AND METHODS DOI: 10.2337/db06-0142 BALB/c and C57BL/6 mice were purchased from ARC (Perth, WA, Australia). © 2006 by the American Diabetes Association. NOD mice were purchased from The Walter and Eliza Hall Institute (Mel- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance bourne, VIC, Australia). Procedures complied with the Australian Code of with 18 U.S.C. Section 1734 solely to indicate this fact. Practice for the Care and Use of Animals for Scientific Purposes. DIABETES, VOL. 55, SEPTEMBER 2006 2491 CRITICAL ROLE FOR A20 IN -CELL PROTECTION Cell death assays. Assays were performed using a modified crystal violet   method (23,24). Briefly, -insulinoma ( -TC3) cells were pretreated (30 min) with 0.01 mol/l actinomycin D (Calbiochem, Kilsyth, Australia) before the addition of 100, 500, or 1000 units/ml of recombinant mouse tumor necrosis factor (TNF)-␣ (R&D Systems, Minneapolis, MN). Twenty-four hours later, cell death was determined as described (23). Cytokine stimulation. Human primary islets, provided by Prof. Phillip O’Connell (Westmead Hospital, Sydney, NSW, Australia), were isolated from cadaveric organ donors and cultured as described (25). Mouse islets were isolated and cultured as we have described (13,26). For cytokine stimulation, islets (150–200 islets/treatment) were stimulated with 200 units/ml of the appropriate cytokine for the indicated times. Human and mouse recombinant T-helper (Th) 1 cytokines interleukin (IL)-1, TNF-␣,or␥-interferon (IFN),  and the Th2 cytokines IL-4, IL-13, or IL-15 (R&D Systems). -TC3 and Min6 cells were cultured as described (27). Cells were stimulated with 200 units/ml FIG. 1. -Cells have an inducible antiapoptotic response. Survival of of mouse recombinant IL-1 or TNF-␣ for the indicated time points. In some -TC cells treated with increasing concentrations of TNF-␣ with (f) 3 cases islets were first pretreated with 100 mol/l of the pharmacological or without (Ⅺ) Actinomycin D. Data represent mean ؎ SD percentage NF-B inhibitor, pyrrolidine dithiocarbamate (Sigma, St. Louis, MO) as survival from four independent experiments, performed in duplicate. described (28). Data with 500 and 1,000 units are significant (**P < 0.005). Gene expression in -cells sorted with a fluorescence-activated cell sorter. For cell sorting, primary mouse islets were first stimulated with cytokines as above and then washed (4% EDTA-PBS), and dispersed into RESULTS single cells by a brief incubation with 0.05% trypsin-EDTA (Invitrogen). -Cells have an inducible antiapoptotic response. Dispersed islets were then washed free of trypsin and resuspended in 1% Despite the importance of cell death in pathological -cell BSA-PBS and sorted in a fluorescence-activated cell sorter (FACS) based upon autofluorescence (FL-1) as described (29). Sorted cells were then analyzed for loss, little is known about the genetic control of apoptosis A20 expression as described below. The percentage of insulin-positive -cells in these cells. Cell death is a highly regulated event, in the FL-1Hi and FL-1Lo sorted populations was determined by insulin (mouse controlled in a proinflammatory setting by an inducible set anti-insulin; Zymed Laboratories, South San Francisco, CA) and glucagon of antiapoptotic genes rapidly upregulated in response to (rabbit anti-glucagon; ICN Immunologicals, Lisle, IL) specific labeling on this same inflammatory stress and/or injury (16–19). We cytospun cells. were particularly interested in determining whether Real-time PCR. mRNA was extracted as described (26); we obtained ϳ3–5  g mRNA per 200 islets. cDNA was produced from 0.5 g of RNA using the -cells