Ubiquitin editing enzymes and beta cell fate in type 1 diabetes

Type 1 Diabetes (T1D) is an autoimmune disease affecting around 0.1-0.8% of the population worldwide and is characterized by a progressive destruction of insulin-producing beta cells. Pro-inflammatory cytokines released by immune cells around the islets contribute for the “first wave” of beta cell apoptosis. Cytokine-mediated activation of the transcription factor nuclear factor kappa (NF-κB) contributes to beta cell demise in T1D. This is unusual, since NF-κB has anti-apoptotic effects in other cells. NF-κB is activated in most cells via the canonical pathway, while its activation via the non-canonical NF-κB pathway is restricted to few cell types, such as maturing/differentiating immune cell and osteoclasts. We have now observed that IL-1β+IFN-γ induces an atypical activation of the non-canonical NF-κB pathway in beta cells. This activation depends on different crosstalk mechanisms between the canonical and non-canonical NF-κB pathways, including the down-regulation of the E3 ligase Fbw7, which targets the p100 for proteasomal degradation, and up-regulation of another E3 ligase, βTrCP, which in turn induces cleavage of p100 to p52, a hallmark step in the non-canonical NF-κB activation. Importantly, cytokine-mediated activation of the non-canonical pathway regulates the expression of late NF-κB dependent genes, such as Ccl5, Ccl19, Ccl12, Fas that regulate both pro-inflammatory and pro-apoptotic responses and are implicated in beta cell loss in T1D. This atypical activation of the non-canonical NF-κB pathway by pro-inflammatory cytokines in beta cells constitutes a novel “feed-forward” mechanism that may explain the particular pro-apoptotic effect of this transcription factor in beta cells. Besides regulation of pro-death responses, NF-κB activation in beta cells triggers the expression of the ubiquitin-editing protein A20, encoded by TNFAIP3. A20 restricts NF-κB signalling and possess anti-apoptotic activities in beta cells. Importantly, genome-wide association studies have identified TNFAIP3 as a candidate gene for T1D. We presently demonstrated that A20 effects in beta cells are not restricted to inhibition of NF-κB. Thus, A20 also suppresses the pro-apoptotic mitogen-activated protein kinase c-Jun N-terminal kinase (JNK), and activates the survival signaling mediated via the v-akt murine thymoma viral oncogene homolog (Akt), thus inhibiting the intrinsic pathway of apoptosis. Finally, a cohort study of T1D children indicated that the risk allele of the rs2327832 single nucleotide polymorphism of TNFAIP3 predict lower C-peptide and higher hemoglobin A1c (HbA1c) levels 12 months after disease onset, indicating that this candidate gene contributes for reduced residual beta-cell function and impaired glycemic control in early T1D. In conclusion, our results indicate a critical role for A20 in the regulation of beta cell survival and unveil novel mechanisms by which A20 controls beta-cell fate. Moreover, we identified the single nucleotide polymorphism rs2327832 of TNFAIP3 as a prognostic marker for diabetes outcome in children with T1D.We have also observed that A20 protects beta cells against the pro-apoptotic effects of cytokines by preventing the degradation of the anti-apoptotic protein Mcl-1. Mcl-1 belongs to the Bcl-2 family of proteins that regulate the intrinsic apoptotic pathway. It was previously shown that Mcl-1 depletion contributes to apoptosis in rat beta cells and that its expression is downregulated in islets from T1D individuals infected by enteroviruses. We have now confirmed in human beta cells that decreased Mcl-1 expression contributes to cytokine-mediated beta cell death. We generated a beta cell specific Mcl-1 knockout mouse line (βMcl-1 KO) and observed that islets derived from these mice were more susceptible to pro-apoptotic stimuli exposure ex vivo. Of note, βMcl-1 KO mice were more vulnerable to multiple low dose streptozotocin-induced diabetes than their wild type littermates. One of the mechanisms by which cytokines mediate Mcl-1 degradation is via its phosphorylation by GSK3β. Overexpression of A20 increased AKT phosphorylation, providing a negative feedback on GSK3β activity and preventing Mcl-1 degradation. Cytokines also increase Mcl-1 ubiquitination, a process regulated by the E3 ligases Mule and βTrCP and the deubiquitinase USP9X. The present findings indicate that pro-inflammatory cytokines trigger post-translational modifications of Mcl-1 leading to its degradation. This contributes to exacerbation of pro-death responses and beta cell demise in T1D, but it can be prevented, at least in part, by A20. As a whole, our data unveil novel post-translational mechanisms and different ubiquitin editing proteins that regulate beta cell fate in T1D and are modulated by pro-inflammatory cytokines.Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)info:eu-repo/semantics/nonPublishe

The non-canonical NF-κB pathway and its contribution to β-cell failure in diabetes

The prevalence of diabetes has reached 8.8% in worldwide population and is predicted to increase up to 10.4% by 2040. Thus, there is an urgent need for the development of means to treat or prevent this major disease. Due to its role in inflammatory responses, several studies demonstrated the importance of the transcription factor nuclear factor-κB (NF-κB) in both type 1 diabetes (T1D) and type 2 diabetes (T2D). The two major NF-κB pathways are the canonical and the non-canonical. The later pathway is activated by the NF-κB-inducing kinase (NIK) that triggers p100 processing into p52, which forms with RelB its main dimer. Cytokines mediating the activation of this pathway are present in the serum of T1D and T2D patients. Conversely, limited information is available regarding the role of the alternative pathway on diabetes development and β-cell fate. In the present review, we will briefly describe the involvement of NF-κB on diabetes pathology and discuss new studies indicating an important role for the non-canonical NF-κB activation in β-cell function and survival. The non-canonical NF-κB pathway is emerging as a novel potential target for the development of therapeutic strategies to treat or prevent diabetes.SCOPUS: re.jinfo:eu-repo/semantics/publishe

Endoplasmic reticulum stress and the unfolded protein response in pancreatic islet inflammation.

Insulin-secreting pancreatic β-cells are extremely dependent on their endoplasmic reticulum (ER) to cope with the oscillatory requirement of secreted insulin to maintain normoglycemia. Insulin translation and folding rely greatly on the unfolded protein response (UPR), an array of three main signaling pathways designed to maintain ER homeostasis and limit ER stress. However, prolonged or excessive UPR activation triggers alternative molecular pathways that can lead to β-cell dysfunction and apoptosis. An increasing number of studies suggest a role of these pro-apoptotic UPR pathways in the downfall of β-cells observed in diabetic patients. Particularly, the past few years highlighted a cross talk between the UPR and inflammation in the context of both type 1 (T1D) and type 2 diabetes (T2D). In this article, we describe the recent advances in research regarding the interplay between ER stress, the UPR, and inflammation in the context of β-cell apoptosis leading to diabetes.SCOPUS: re.jinfo:eu-repo/semantics/publishe

The non-canonical NF-kB pathway is induced by cytokines in pancreatic beta cells and contributes to cell death and proinflammatory responses in vitro

Aims/hypothesis: Activation of the transcription factor nuclear factor (NF)-κB by proinflammatory cytokines plays an important role in beta cell demise in type 1 diabetes. Two main signalling pathways are known to activate NF-κB, namely the canonical and the non-canonical pathways. Up to now, studies on the role of NF-κB activation in beta cells have focused on the canonical pathway. The aim of this study was to investigate whether cytokines activate the non-canonical pathway in beta cells, how this pathway is regulated and the consequences of its activation on beta cell fate. Methods: NF-κB signalling was analysed by immunoblotting, promoter reporter assays and real-time RT-PCR, after knockdown or overexpression of key genes/proteins. INS-1E cells, FACS-purified rat beta cells and the human beta cell line EndoC-βH1 exposed to cytokines were used as models. Results: IL-1β plus IFN-γ induced stabilisation of NF-κB-inducing kinase and increased the expression and cleavage of p100 protein, culminating in the nuclear translocation of p52, the hallmark of the non-canonical signalling. This activation relied on different crosstalks between the canonical and non-canonical pathways, some of which were beta cell specific. Importantly, cytokine-mediated activation of the non-canonical pathway controlled the expression of ‘late’ NF-κB-dependent genes, regulating both pro-apoptotic and inflammatory responses, which are implicated in beta cell loss in early type 1 diabetes. Conclusions/interpretation: The atypical activation of the non-canonical NF-κB pathway by proinflammatory cytokines constitutes a novel ‘feed-forward’ mechanism that contributes to the particularly pro-apoptotic effect of NF-κB in beta cells.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

The type 1 diabetes candidate gene Src kinase associated phospho- protein 2 (SKAP2) controls beta cell sensitivity to pro-inflammatory cytokines

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Heterozygous inactivation of plasma membrane Ca(2+)-ATPase in mice increases glucose-induced insulin release and beta cell proliferation, mass and viability

AIMS/HYPOTHESIS: Calcium plays an important role in the process of glucose-induced insulin release in pancreatic beta cells. These cells are equipped with a double system responsible for Ca(2+) extrusion--the Na/Ca exchanger (NCX) and the plasma membrane Ca(2+)-ATPase (PMCA). We have shown that heterozygous inactivation of NCX1 in mice increased glucose-induced insulin release and stimulated beta cell proliferation and mass. In the present study, we examined the effects of heterozygous inactivation of the PMCA on beta cell function. METHODS: Biological and morphological methods (Ca(2+) imaging, Ca(2+) uptake, glucose metabolism, insulin release and immunohistochemistry) were used to assess beta cell function and proliferation in Pmca2 (also known as Atp2b2) heterozygous mice and control littermates ex vivo. Blood glucose and insulin levels were also measured to assess glucose metabolism in vivo. RESULTS: Pmca (isoform 2) heterozygous inactivation increased intracellular Ca(2+) stores and glucose-induced insulin release. Moreover, increased beta cell proliferation, mass, viability and islet size were observed in Pmca2 heterozygous mice. However, no differences in beta cell glucose metabolism, proinsulin immunostaining and insulin content were observed. CONCLUSIONS/INTERPRETATION: The present data indicates that inhibition of Ca(2+) extrusion from the beta cell and its subsequent intracellular accumulation stimulates beta cell function, proliferation and mass. This is in agreement with our previous results observed in mice displaying heterozygous inactivation of NCX, and indicates that inhibition of Ca(2+) extrusion mechanisms by small molecules in beta cells may represent a new approach in the treatment of type 1 and type 2 diabetes

Mcl-1 is a key anti-apoptotic protein in human and rodent pancreatic beta cells

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MCL-1 is a Key Anti-Apoptotic Protein in Human and Rodent Pancreatic Beta cells.

Induction of endoplasmic reticulum stress and activation of the intrinsic apoptotic pathway is widely believed to contribute to β-cell death in type 1 diabetes (T1D). MCL-1 is an anti-apoptotic member of the BCL-2 protein family, whose depletion causes apoptosis in rodent β-cells in vitro. Importantly, decreased MCL-1 expression was observed in islets from T1D patients. We report here that MCL-1 downregulation is associated with cytokine-mediated killing of human β-cells, a process partially prevented by MCL-1 overexpression. By generating a β-cell specific Mcl-1 knockout mouse strain (βMcl-1KO), we observed that, surprisingly, MCL-1 ablation does not affect islet development and function. β-cells from βMcl-1KO mice were, however, more susceptible to cytokine-induced apoptosis. Moreover, βMcl-1KO mice displayed higher hyperglycaemia and lower pancreatic insulin content after multiple low dose streptozotocin treatment. We found that the kinase GSK3β, the E3 ligases MULE and βTrCP and the deubiquitinase USP9x, regulate cytokine-mediated MCL-1 protein turnover in rodent β-cells. Our results identify MCL-1 as a critical pro-survival protein for preventing β-cell death and clarify the mechanisms behind its downregulation by pro-inflammatory cytokines. Development of strategies to prevent MCL-1 loss in the early stages of T1D may enhance β-cell survival and thereby delay or prevent disease progression.SCOPUS: ar.jinfo:eu-repo/semantics/publishe