Differential regulation of adaptive and apoptotic unfolded protein response signalling by cytokine-induced nitric oxide production in mouse pancreatic beta cells

Abstract

Aims/hypothesis Pro-inflammatory cytokines such as IL-1β, IFN-γ and TNF-α may contribute to pancreatic beta cell destruction in type 1 diabetes. A mechanism requiring nitric oxide, which is generated by inducible nitric oxide synthase (iNOS), in cytokine-induced endoplasmic reticulum (ER) stress and apoptosis has been proposed. Here, we tested the role of nitric oxide in cytokine-induced ER stress and the subsequent unfolded protein response (UPR) in beta cells. Methods Isolated islets from wild-type and iNos (also known as Nos2) knockout (iNos −/− ) mice, and MIN6 beta cells were incubated with IL-1β, IFN-γ and TNF-α for 24-48 h. N G -methyl-L-arginine was used to inhibit nitric oxide production in MIN6 cells. Protein levels and gene expression were assessed by western blot and realtime RT-PCR. Results In islets and MIN6 cells, inhibition of nitric oxide production had no effect on the generation of ER stress by cytokines, as evidenced by downregulation of Serca2b (also known as Atp2a2) mRNA and increased phosphorylation of PKR-like ER kinase, Jun N-terminal kinase (JNK) and eukaryotic translation initiation factor 2 α subunit. However, nitric oxide regulated the pattern of UPR signalling, which delineates the cellular decision to adapt to ER stress or to undergo apoptosis. Inhibition of nitric oxide production led to reduced expression of pro-apoptotic UPR markers, Chop (also known as Ddit3), Atf3 and Trib3. In contrast, adaptive UPR markers (chaperones, foldases and degradation enhancers) were increased. Further analysis of mouse islets showed that cytokine-induced Chop and Atf3 expression was also dependent on JNK activity. Conclusions/interpretation The mechanism by which cytokines induce ER stress in mouse beta cells is independent of nitric oxide production. However, nitric oxide may regulate the switch between adaptive and apoptotic UPR signalling.

Cross-talk between the unfolded protein response and nuclear factor-κB signalling pathways regulates cytokine-mediated beta cell death in MIN6 cells and isolated mouse islets

“…Cell/islet culture MIN6 cells and islets isolated from adult C57BL/6J mice (Australian BioResources, Moss Vale, NSW, Australia) were cultured as previously described [24]. Procedures were approved by the Garvan Institute/St Vincent's Hospital Animal Experimentation Ethics Committee.…”

“…BMS-345541 (IKKi, 50 μmol/l) and MG132 (10 μmol/l) (Sigma) were used to inhibit the activities of IKK and the proteasome. Cell death was determined with the use of a Cell Death Detection ELISA (Roche Diagnostics, Castle Hill, NSW, Australia) [24].…”

“…Since ER stress is capable of inducing apoptosis in beta cells [10,11], its potential involvement in cytokinemediated beta cell death in type 1 diabetes has been widely investigated [8,10,[12][13][14][15][16][17][18][19][20][21][22][23][24][25]. Cytokine-stimulated beta cells are characterised by suppression of the adaptive UPR, which may contribute to a lowering of defence mechanisms and activation of the proapoptotic UPR [24,26].…”

“…Cytokine-stimulated beta cells are characterised by suppression of the adaptive UPR, which may contribute to a lowering of defence mechanisms and activation of the proapoptotic UPR [24,26]. However, the ultimate fate of beta cells exposed to cytokines is likely to be influenced by the many signalling networks that are activated and the complex interactions between these systems.…”

Is ARE/poly(U)-binding factor 1 (AUF1) a new player in cytokine-mediated beta cell apoptosis?

“…iNOS production leads to the formation of NO, which has been shown to decrease endoplasmic reticulum (ER) calcium and induce ER stress and the unfolded protein response (UPR) in beta cells [23,24]. Of note, although induction of UPR is related to NO production in rat beta cells [24], NO does not seem to be required in mouse islets [25]. The mechanisms by which ER stress contributes to beta cell apoptosis are not completely clear, but induction of the pro-apoptotic proteins C/EBP homologous protein (CHOP), and death protein 5 (also called hara-kiri) (DP5) [26,27], and downregulation of the anti-apoptotic protein myeloid cell leukaemia sequence 1 (MCL-1), probably play a role in this process [28].…”