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Glucose-induced insulin secretion, a hallmark of mature β-cells, is achieved after birth and is preceded by a phase of intense proliferation. These events occurring in the neonatal period are decisive for establishing an appropriate functional β-cell mass that provides the required insulin throughout life. However, key regulators of gene expression involved in functional maturation of β-cells remain to be elucidated. Here, we addressed this issue by mapping open chromatin regions in newborn versus adult rat islets using the ATAC-seq assay. We obtained a genome-wide picture of chromatin accessible sites (~ 100,000) among which 20% were differentially accessible during maturation. An enrichment analysis of transcription factor binding sites identified a group of transcription factors that could explain these changes. Among them, Scrt1 was found to act as a transcriptional repressor and to control β-cell proliferation. Interestingly, Scrt1 expression was controlled by the transcriptional repressor RE-1 silencing transcription factor (REST) and was increased in an in vitro reprogramming system of pancreatic exocrine cells to β-like cells. Overall, this study led to the identification of several known and unforeseen key transcriptional events occurring during β-cell maturation. These findings will help defining new strategies to induce the functional maturation of surrogate insulin-producing cells.

Glucose-induced insulin secretion, a peculiar property of fully matureβ-cells, is only achieved after birth and is preceded by a phase of intense proliferation. These events occurring in the neonatal period are decisive for the establishment of an appropriate functionalβ-cell mass that provides the required insulin throughout life. However, key regulators of gene expression involved in cellular reprogramming along pancreatic islet maturation remain to be elucidated. The present study addressed this issue by mapping open chromatin regions in newborn versus adult rat islets using the ATAC-seq assay. Accessible regions were then correlated with the expression profiles of mRNAs to unveil the regulatory networks governing functional islet maturation. This led to the identification of Scrt1, a novel transcriptional repressor controllingβ-cell proliferation.

International audience; Type 1 diabetes is an autoimmune disease initiated by the invasion of pancreatic islets by immune cells that selectively kill the beta cells. We found that rodent and human T lymphocytes release exosomes containing the microRNAs (miRNAs) miR-142-3p, miR-142-5p, and miR-155, which can be transferred in active form to beta cells favoring apoptosis. Inactivation of these miRNAs in recipient beta cells prevents exosome-mediated apoptosis and protects non-obese diabetic (NOD) mice from diabetes development. Islets from protected NOD mice display higher insulin levels, lower insulitis scores, and reduced inflammation. Looking at the mechanisms underlying exosome action, we found that T lymphocyte exosomes trigger apoptosis and the expression of genes involved in chemokine signaling, including Ccl2, Ccl7, and Cxcl10, exclusively in beta cells. The induction of these genes may promote the recruitment of immune cells and exacerbate beta cell death during the autoimmune attack. Our data point to exosomal-miRNA transfer as a communication mode between immune and insulin-secreting cells.

Background Diabetes mellitus is a common metabolic disorder characterized by dysfunction of insulin-secreting pancreatic beta-cells. MicroRNAs are important regulators of beta-cell activities. These non-coding RNAs have recently been discovered to exert their effects not only inside the cell producing them but, upon exosome-mediated transfer, also in other recipient cells. This novel communication mode remains unexplored in pancreatic beta-cells. In the present study, the microRNA content of exosomes released by beta-cells in physiological and physiopathological conditions was analyzed and the biological impact of their transfer to recipient cells investigated. Results Exosomes were isolated from the culture media of MIN6B1 and INS-1 derived 832/13 beta-cell lines and from mice, rat or human islets. Global profiling revealed that the microRNAs released in MIN6B1 exosomes do not simply reflect the content of the cells of origin. Indeed, while a subset of microRNAs was preferentially released in exosomes others were selectively retained in the cells. Moreover, exposure of MIN6B1 cells to inflammatory cytokines changed the release of several microRNAs. The dynamics of microRNA secretion and their potential transfer to recipient cells were next investigated. As a proof-of-concept, we demonstrate that if cel-miR-238, a C. Elegans microRNA not present in mammalian cells, is expressed in MIN6B1 cells a fraction of it is released in exosomes and is transferred to recipient beta-cells. Furthermore, incubation of untreated MIN6B1 or mice islet cells in the presence of microRNA-containing exosomes isolated from the culture media of cytokine-treated MIN6B1 cells triggers apoptosis of recipient cells. In contrast, exosomes originating from cells not exposed to cytokines have no impact on cell survival. Apoptosis induced by exosomes produced by cytokine-treated cells was prevented by down-regulation of the microRNA-mediating silencing protein Ago2 in recipient cells, suggesting that the effect is mediated by the non-coding RNAs. Conclusions Taken together, our results suggest that beta-cells secrete microRNAs that can be transferred to neighboring beta-cells. Exposure of donor cells to pathophysiological conditions commonly associated with diabetes modifies the release of microRNAs and affects survival of recipient beta-cells. Our results support the concept that exosomal microRNAs transfer constitutes a novel cell-to-cell communication mechanism regulating the activity of pancreatic beta-cells. Electronic supplementary material The online version of this article (doi:10.1186/s12964-015-0097-7) contains supplementary material, which is available to authorized users.

The common leukocyte antigen CD45 plays a central role in T cell activation in coupling the T cell receptor (TCR) to the phosphatidylinositol pathway via interactions with TCR-associated protein tyrosine kinases lck and fyn. We here demonstrate that engagement of CD45 by monoclonal antibodies (mAb) on activated T cells induces tumor necrosis factor (TNF)-alpha as well as TNF-beta, interleukin (IL)-2 and IL-3 gene expression. When human alloreactive T cells are stimulated with mAb 4B2, which recognizes a determinant common to all CD45 isoforms, a vigorous production of TNF-alpha mRNA was detected, which peaked 2 h later. Anti-CD45 mAb cross-linking was required. In contrast, neither mAb 10G10, which recognizes an epitope distinct from the one recognized by mAb 4B2, nor mAb UCHL-1, a CD45RO-specific antibody, induced any significant increase in TNF-alpha transcription. Nuclear run-on transcription assays demonstrated that CD45 cross-linking caused transcriptional activation of the TNF-alpha gene. De novo protein synthesis was not required, since incubation with cycloheximide (CHX) did not block transcriptional activation. CHX in contrast up-regulated TNF-alpha gene expression and increased transcript half-life, an effect that was under control of post-transcriptional mechanisms. Engagement of CD45 by itself did not affect transcript stability. CD45 ligation resulted in TNF-alpha secretion. These results indicate that in addition to its role in TCR/CD3-mediated T cell activation, CD45, in an epitope-specific manner, may act as a primary signaling molecule, leading to the transcriptional regulation and secretion of a major pro-inflammatory cytokine.

Introduction Les microRNAs regulent l’expression genique a l’interieur des cellules les produisant, mais aussi dans des cellules receveuses suite a leur transfert par des microvesicules. Ce nouveau mode de communication intercellulaire est bien decrit dans certains systemes biologiques mais demeure peu caracterise dans les cellules beta pancreatiques. Dans la presente etude, nous avons analyse l’effet des conditions physiopathologiques associees au diabete sur le relâchement des microARNs par les cellules beta et determine l’impact biologique de leur transfert. Materiels et methodes Les microvesicules relâchees par des lignees insulinosecretrices et par des ilots pancreatiques ont ete isolees par ultra-centrifugation. Apres analyse de leur contenu, les microvesicules ont ete utilisees pour etudier l’impact du transfert des microARNs sur les fonctions des cellules beta receveuses. Resultats Des microARNs ont ete detectes dans les tous milieux de culture des cellules beta testees. L’analyse par micropuces a revele que le profil des microARNs relâches differe de celui des cellules. En effet, certains microARNs faiblement exprimes dans les cellules sont abondants dans les microvesicules et vice-versa. Ces resultats suggerent que certains microARNs sont preferentiellement relâches. De plus, l’incubation des cellules MIN6 en presence de cytokines ou de palmitate induit un changement dans les niveaux de plusieurs microARNs relâches. Fait interessant, l’incubation de MIN6 non-traitees en presence de microvesicules provenant du milieu de culture de MIN6 traitees aux cytokines conduit a une augmentation de l’apoptose et a une diminution de la proliferation des cellules receveuses. A l’oppose, les microvesicules purifiees du milieu de culture de MIN6 non-traitees n’affecte pas les fonctions des cellules receveuses. Conclusion L’ensemble des resultats obtenus suggerent que les cellules beta relâchent un groupe specifique de microARNs pouvant etre transferes a d’autres cellules beta et ainsi affecter leur fonction. Nos travaux supportent le concept que le transfert des microARNs constitue un mode de communication intercellulaire.

Introduction L’infiltration des ilots de Langerhans par les lymphocytes durant le developpement du diabete de type 1 conduit a la destruction des cellules beta. Les lymphocytes sont connus pour relâcher des cytokines pro-inflammatoires, mais aussi des microvesicules dont le contenu (ARNm, microRNAs et proteines) est transfere sous forme active a d’autres types cellulaires. Puisque les lymphocytes et les cellules beta sont en etroite proximite durant le developpement de l’insulite, nous avons emis l’hypothese que le transfert de certains micro-RNAs des lymphocytes T aux cellules beta contribuerait au dysfonctionnement des cellules insulinosecretrices durant le developpement du diabete de type 1. Materiels et methodes Les microvesicules ont ete isolees par ultra-centrifugation a partir du milieu de culture de cellules T (Jurkat ou cellules T de souris NOD). Apres analyse de leur contenu, ces microvesicules ont ete utilisees pour l’etude du transfert des microRNAs des cellules T aux cellules beta (MIN6 ou cellules beta primaires). Resultats Nous avons observe que l’expression de 4 microRNAs associes aux cellules T, miR-142.3p, miR-142.5p, miR-150 et miR-155, etait augmentee dans les ilots pancreatiques de souris NOD durant la progression de l’insulite. Ces microRNAs sont relâches dans les microvesicules des cellules T et peuvent etre transferes aux cellules beta. L’incubation de cellules beta en presence des microvesicules des cellules T a conduit a une augmentation de la mort cellulaire et a une diminution des fonctions secretoires des cellules insulinosecretrices. Afin de correler ces effets fonctionnels au transfert des microRNAs, nous avons determine l’effet de la surexpression de miR-142.3p et miR-142.5p dans les cellules beta. La surexpression de miR-142.3p et miR-142.5p a induit, respectivement, une diminution des fonctions secretoires et une elevation de l’apoptose. Conclusion Les resultats obtenus soutiennent l’hypothese que le transfert de microRNAs des cellules T aux cellules beta conduit au dysfonctionnement des cellules insulinosecretrices dans le contexte du diabete de type 1

While non-stimulated primary human monocytes exhibit very low levels of tumor necrosis factor (TNF)-alpha mRNA, direct binding of the staphylococcal exotoxin toxic shock syndrome toxin-1 (TSST-1) to major histocompatibility complex (MHC) class II molecules results in a fast (peak 1 h after stimulation), transient induction (sevenfold) of TNF-alpha mRNA. This induction correlates with a fourfold increase in transcription rates of the TNF-alpha gene, as detected by run-on assays, and does not require de novo protein synthesis. Mapping of DNase-I hypersensitive sites (DHS) discloses two constitutive DHS, one located far upstream (within the TNF-beta promoter) and the other centered at -39 +/- 40 bp relative to the major TNF-alpha transcription start site, suggesting that the TNF-alpha gene was transcriptionally competent even prior to MHC class II engagement. Furthermore, stimulation of human monocytes with either TSST-1 or lipopolysaccharide increases the translational efficiency of TNF-alpha mRNA, as shown by a shift in the distribution of this mRNA species in polysome gradients and the translation rates of TNF-alpha measured by immunoprecipitation from cells pulsed with [35S] methionine. The increase in translation efficiency of TNF-alpha mRNA is independent of the half-life of TNF-alpha transcripts, which under the conditions used is unchanged. Taken together, our data indicate that TNF-alpha expression is tightly regulated by MHC class II ligands, both at the transcriptional and translational levels.

Fine-tuning of insulin release from pancreatic β-cells is essential to maintain blood glucose homeostasis. Here, we report that insulin secretion is regulated by a circular RNA containing the lariat sequence of the second intron of the insulin gene. Silencing of this intronic circular RNA in pancreatic islets leads to a decrease in the expression of key components of the secretory machinery of β-cells, resulting in impaired glucose- or KCl-induced insulin release and calcium signaling. The effect of the circular RNA is exerted at the transcriptional level and involves an interaction with the RNA-binding protein TAR DNA-binding protein 43 kDa (TDP-43). The level of this circularized intron is reduced in the islets of rodent diabetes models and of type 2 diabetic patients, possibly explaining their impaired secretory capacity. The study of this and other circular RNAs helps understanding β-cell dysfunction under diabetes conditions, and the etiology of this common metabolic disorder., Circular RNAs contribute to the regulation of β-cell specific functions. Here the authors show that a circular RNA derived from one of the introns of the insulin gene is necessary for optimal insulin secretion from pancreatic islets and that its level is reduced in the islets of diabetic subjects.