Activating transcription factor 3 (ATF3) is a stress-inducible gene and encodes a member of the ATF/CREB family of transcription factors. However, the physiological significance of ATF3 induction by stress signals is not clear. In this report, we describe several lines of evidence supporting a role of ATF3 in stress-induced ␤-cell apoptosis. First, ATF3 is induced in ␤ cells by signals relevant to ␤-cell destruction: proinflammatory cytokines, nitric oxide, and high concentrations of glucose and palmitate. Second, induction of ATF3 is mediated in part by the NF-B and Jun N-terminal kinase/stress-activated protein kinase signaling pathways, two stress-induced pathways implicated in both type 1 and type 2 diabetes. Third, transgenic mice expressing ATF3 in ␤ cells develop abnormal islets and defects secondary to ␤-cell deficiency. Fourth, ATF3 knockout islets are partially protected from cytokine-or nitric oxide-induced apoptosis. Fifth, ATF3 is expressed in the islets of patients with type 1 or type 2 diabetes, and in the islets of nonobese diabetic mice that have developed insulitis or diabetes. Taken together, our results suggest ATF3 to be a novel regulator of stress-induced ␤-cell apoptosis.It is widely accepted that autoimmunity is the main cause of type 1 but not type 2 diabetes. Despite this difference, ␤-cell death plays an important role in the pathophysiological progression of both diseases (15,43,45). On one hand, proinflammatory cytokines (interleukin-1␤ , tumor necrosis factor alpha [TNF-␣], and gamma interferon [IFN-␥]) destroy ␤ cells in the islets of Langerhans, leading to the pathogenesis of type 1 diabetes (11,14,15,42); on the other hand, elevated glucose and free fatty acids (FFAs)-common metabolic abnormalities in type 2 diabetes-induce ␤-cell death, contributing to the progression of the disease (13,32,35,53,62). Emerging evidence indicates that activation of the NF-B and Jun N-terminal kinase/stress-activated protein kinase (JNK/ SAPK) signaling pathways is a key event leading to cell death, when ␤ cells are exposed to these signals: proinflammatory cytokines, elevated glucose, and elevated FFAs (12,15,16,43,49). Furthermore, activation of these pathways has been demonstrated to impair insulin signaling (1,17,18,36) and play a role in type 2 diabetes (63,71). Therefore, these stress-activated signaling pathways constitute a common molecular mechanism in the pathophysiological progression of type 1 and type 2 diabetes.Thus far, inducible nitric oxide (NO) synthase (iNOS), whose expression leads to NO production, is one of the best known target genes for these pathways (14-16, 42, 54). Several lines of evidence indicate that iNOS plays an important role in the pathogenesis of diabetes. (i) iNOS is induced in the islets by cytokines (16) and is expressed in the islets of diabetes prone BB rats (33) and nonobese diabetic (NOD) mice (55,58). (ii) Transgenic mice expressing iNOS in ␤ cells develop ␤-cell destruction and diabetes (60). (iii) ␤ cells lacking functional iNOS are partially protected ...

A20 is a stress response gene in endothelial cells (ECs). A20 serves a dual cytoprotective function, protecting from tumor necrosis factor (TNF)-mediated apoptosis and inhibiting inflammation via blockade of the transcription factor nuclear factor-B (NF-B). In this study, we evaluated the molecular basis of the cytoprotective function of A20 in EC cultures and questioned whether its protective effect extends beyond TNF to other apoptotic and necrotic stimuli. Our data demonstrate that A20 targets the TNF apoptotic pathway by inhibiting proteolytic cleavage of apical caspases 8 and 2, executioner caspases 3 and 6, Bid cleavage, and release of cytochrome c, thus preserving mitochondrion integrity. A20 also protects from Fas/CD95 and significantly blunts natural killer cell-mediated EC apoptosis by inhibiting caspase 8 activation. In addition to protecting ECs from apoptotic stimuli, A20 safeguards ECs from complement-mediated necrosis. These data demonstrate, for the first time, that the cytoprotective effect of A20 in ECs is not limited to TNF-triggered apoptosis. Rather, A20 affords broad EC protective functions by effectively shutting down cell death pathways initiated by inflammatory and immune offenders. IntroductionA20 is a zinc finger protein originally identified as a tumor necrosis factor (TNF)-responsive gene in endothelial cells (ECs). 1 A20 is expressed in multiple cell types in response to a variety of stimuli that activate the transcription factor nuclear factor-B (NF-B), including interleukin 1 (IL-1), lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), H 2 O 2 , and CD40 ligand. [2][3][4][5][6][7] We and others have demonstrated that A20, initially described as an antiapoptotic gene, is also a potent inhibitor of the transcription factor NF-B. 7-9 A20-null mice fail to terminate TNF-induced NF-B activation, develop severe inflammation and cachexia, and die prematurely, indicating the importance of A20 in the hierarchy of anti-inflammatory defense processes. 6,10 Elucidating the molecular basis and binding partner(s) that determine the inhibitory effect of A20 upon NF-B activation is the focus of ongoing research. 8 A20 has been shown to interact with components of the NF-B signaling cascade upstream of inhibitor of NF-B (IB), including TNF receptor-associated factors (TRAFs) 11,12 TRAF-1, TRAF-2, and TRAF-6; A20 binding inhibitors of NF-B; and the signalosome IB kinase-␥ (IKK␥)/NF-B essential modulator (NEMO) unit. [13][14][15] Further work is required to test the relevance of these interactions in blocking NF-B activation in response to stimuli other than TNF. 16 In contrast to its so far universal inhibitory effect on NF-B activation, the antiapoptotic activity of A20 remains controversial and appears to be specific to cell type and stimulus. Overexpression of A20 protects human breast carcinoma MCF-7 cells, murine fibrosarcoma WEHI 164, and murine embryonic NIH3T3, but not Hela and lung epithelial A459 cells from TNF-mediated apoptosis. 9,[17][18][19] In cultures derived from prima...

Transplantation of an excessive number of islets of Langerhans (two to four pancreata per recipient) into patients with type I diabetes is required to restore euglycemia. Hypoxia, nutrient deprivation, local inflammation, and the β cell inflammatory response (up-regulation of NF-κB-dependent genes such as inos) result in β cell destruction in the early post-transplantation period. Genetic engineering of islets with anti-inflammatory and antiapoptotic genes may prevent β cell loss and primary nonfunction. We have shown in vitro that A20 inhibits NF-κB activation in islets and protects from cytokine- and death receptor-mediated apoptosis. In vivo, protection of newly transplanted islets would reduce the number of islets required for successful transplantation. Transplantation of 500 B6/AF1 mouse islets into syngeneic, diabetic recipients resulted in a cure rate of 100% within 5 days. Transplantation of 250 islets resulted in a cure rate of only 20%. Transplantation of 250 islets overexpressing A20 resulted in a cure rate of 75% with a mean time to cure of 5.2 days, comparable to that achieved with 500 islets. A20-expressing islets preserve functional β cell mass and are protected from cell death. These data demonstrate that A20 is an ideal cytoprotective gene therapy candidate for islet transplantation.

A20 is a NF-kappaB-dependent gene that has dual anti-inflammatory and antiapoptotic functions in endothelial cells (EC). The function of A20 in smooth muscle cells (SMC) is unknown. We demonstrate that A20 is induced in SMC in response to inflammatory stimuli and serves an anti-inflammatory function via blockade of NF-kappaB and NF-kappaB-dependent proteins ICAM-1 and MCP-1. A20 inhibits SMC proliferation via increased expression of cyclin-dependent kinase inhibitors p21waf1 and p27kip1. Surprisingly, A20 sensitizes SMC to cytokine- and Fas-mediated apoptosis through a novel NO-dependent mechanism. In vivo, adenoviral delivery of A20 to medial rat carotid artery SMC after balloon angioplasty prevents neointimal hyperplasia by blocking SMC proliferation and accelerating re-endothelialization, without causing apoptosis. However, expression of A20 in established neointimal lesions leads to their regression through increased apoptosis. This is the first demonstration that A20 exerts two levels of control of vascular remodeling and healing. A20 prevents neointimal hyperplasia through combined anti-inflammatory and antiproliferative functions in medial SMC. If SMC evade this first barrier and neointima is formed, A20 has a therapeutic potential by uniquely sensitizing neointimal SMC to apoptosis. A20-based therapies hold promise for the prevention and treatment of neointimal disease.

A1 and A20 exert differential cytoprotective effects in RPTECs. A1 is antiapoptotic. A20 is anti-inflammatory via blockade of NF-kappaB. We propose that A1 and A20 are both required for optimal protection of RPTECs from apoptosis (A1) and inflammation (A20) in conditions leading to renal damage.