Chronic free fatty acid (FFA) exposure induces pancreatic beta-cell death, which may contribute to the development of type 2 diabetes. The mechanisms involved in FFA-induced cell death are not completely understood. Here we have investigated the effect of FFA on endoplasmic reticulum (ER) stress pathways in INS-1 pancreatic beta-cells. INS-1 cells exposed to palmitate for 16-24 h under serum-free conditions showed marked apoptosis and increased protein levels of phosphorylated eukaryotic translation initiation factor 2alpha (eIF2alpha), activating transcription factor 4 (ATF4), X box-binding protein 1 (XBP-1), and C/EBP homologous transcription factor (CHOP) compared with control cells. The CHOP transcription factor has been implicated in mediating ER stress-induced apoptosis. Unexpectedly, the levels of the ER chaperone proteins Grp78/BiP and PDI were not affected by palmitate treatment, suggesting that the cell protective aspects of the unfolded protein response (UPR) are not up-regulated by palmitate. Palmitate-treated cells had markedly altered distribution of ER chaperones and altered ER morphology, suggesting that accumulation of misfolded proteins might trigger the ER stress response. In contrast, oleate treatment did not significantly induce the UPR pathways, nor was it as detrimental to INS-1 beta-cells. The results suggest that activation of the UPR may significantly contribute to palmitate- but not oleate-induced pancreatic beta-cell death.

IFN-␥ induction of the CIITA (class II transactivator) promoter (pIV) requires Brahma-related gene 1 (BRG1), a chromatin-remodeling enzyme. However, the events that lead to pIV activation are only partially understood, and the point at which BRG1 acts is unknown. The first IFN-␥-induced event triggers nuclear translocation of STAT1 (signal transducer and activator of transcription 1), which binds IFN-␥-responsive promoters. BRG1 is recruited after activator binding at several other inducible loci, and STAT family members are known to bind BRG1, suggesting that BRG1 might act downstream of STAT1. Here, we delineate a comprehensive view of factor assembly and detailed histone modifications at pIV and show that all events, even STAT1 binding, require BRG1 at CIITA pIV and other IFN-␥ target promoters. Recruitment of IFN-stimulated gene factor-3 (ISGF3) [STAT1͞STAT2͞IFN regulatory factor 9 (IRF9)] to several IFN-␣-responsive promoters is also BRG1-dependent. In contrast, constitutive BRG1 association at IFN targets is STAT1-independent. Furthermore, BRG1 is required for IFN-induced restriction enzyme and DNase I accessibility at promoters. Thus, BRG1 has an apical role in cytokine-induced promoter assembly, acting upstream of STAT complexes at multiple IFN target loci.BRG1-associated factor ͉ chromatin ͉ interferon ͉ SWI͞SNF

Macrophages (M ) are activated by IFN␥ and are important cellular targets for infection by human and murine cytomegalovirus (MCMV), making it advantageous for CMVs to block IFN␥-induced M differentiation. We found that MCMV infection inhibited IFN␥ regulation of many genes in M . MCMV infection blocked IFN␥ responses at the level of transcription without blocking Janus kinase͞signal transducer and activator of transcription pathway activation and targeted IFN response factor 1-and class II transactivator-dependent and independent promoters. MCMV did not alter basal transcription from IFN␥-responsive promoters and left the majority of cellular transcripts unchanged even after 48 h of infection. The effects of MCMV infection were specific to chromosomal rather than transiently transfected promoters. Characterization of the IFN␥-responsive chromosomal class II transactivator promoter revealed that MCMV infection blocked IFN␥-induced promoter assembly, allowing the virus to transcriptionally paralyze infected M responses while allowing basal transcription to proceed.immune evasion ͉ microarray F or the ␤-herpesviruses human cytomegalovirus (HCMV) and murine CMV (MCMV), macrophages (M ) and their progenitors play an important role in pathogenesis by providing vehicles for dissemination and a cellular site for viral latency (1-7). This reliance on M creates a problem for CMVs, because M are activated by antiviral cytokines such as IFN␥ in vivo (8,9) and are critical for control of MCMV infection (2). The M activating cytokine IFN␥ is crucial for controlling persistent MCMV replication in vivo (9) and reversibly inhibits reactivation of MCMV from latency, in part by blocking viral growth (9-11). M are activated to express increased MHC class II in vivo in response to IFN␥ during MCMV infection (8,9,12), and IFN␥ treatment of M decreases HCMV and MCMV growth up to 100-fold (10, 13).Given the importance of M and IFN␥ for control of CMV infection, it is not surprising that these viruses have strategies for altering differentiation of infected cells such as dendritic cells and M (14-18). For example, infection with HCMV or MCMV effectively inhibits IFN␥-induced antigen presentation by MHC class II by inhibiting IFN␥ induction of genes involved in antigen presentation (15,(19)(20)(21).Although HCMV and MCMV inhibit M and DC differentiation, the molecular mechanisms responsible for paralysis of cytokine-driven accessory immune cell differentiation are unknown. Because these viruses rely on cellular proteins during their prolonged replication cycle, it is likely that blockade of differentiation will involve mechanisms that preserve cellular functions critical for viral replication.We report here that MCMV infection inhibits expression of many IFN␥-responsive genes in M at the transcriptional level without affecting proximal signaling machinery or basal transcription. To accomplish this, MCMV inhibits IFN␥-induced chromosomal promoter assembly, providing an explanation for how global blockade of IFN␥-induced gene expression ca...

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