Specific Silencing of the REST Target Genes in Insulin-Secreting Cells Uncovers Their Participation in Beta Cell Survival

The absence of the transcriptional repressor RE-1 Silencing Transcription Factor (REST) in insulin-secreting beta cells is a major cue for the specific expression of a large number of genes. These REST target genes were largely ascribed to a function of neurotransmission in a neuronal context, whereas their role in pancreatic beta cells has been poorly explored. To identify their functional significance, we have generated transgenic mice expressing REST in beta cells (RIP-REST mice), and previously discovered that REST target genes are essential to insulin exocytosis. Herein we characterized a novel line of RIP-REST mice featuring diabetes. In diabetic RIP-REST mice, high levels of REST were associated with postnatal beta cell apoptosis, which resulted in gradual beta cell loss and sustained hyperglycemia in adults. Moreover, adenoviral REST transduction in INS-1E cells led to increased cell death under control conditions, and sensitized cells to death induced by cytokines. Screening for REST target genes identified several anti-apoptotic genes bearing the binding motif RE-1 that were downregulated upon REST expression in INS-1E cells, including Gjd2, Mapk8ip1, Irs2, Ptprn, and Cdk5r2. Decreased levels of Cdk5r2 in beta cells of RIP-REST mice further confirmed that it is controlled by REST, in vivo. Using siRNA-mediated knockdown in INS-1E cells, we showed that Cdk5r2 protects beta cells against cytokines and palmitate-induced apoptosis. Together, these data document that a set of REST target genes, including Cdk5r2, is important for beta cell survival

RNA Profiling and Chromatin Immunoprecipitation-Sequencing Reveal that PTF1a Stabilizes Pancreas Progenitor Identity via the Control of MNX1/HLXB9 and a Network of Other Transcription Factors

Pancreas development is initiated by the specification and expansion of a small group of endodermal cells. Several transcription factors are crucial for progenitor maintenance and expansion, but their interactions and the downstream targets mediating their activity are poorly understood. Among those factors, PTF1a, a basic helix-loop-helix (bHLH) transcription factor which controls pancreas exocrine cell differentiation, maintenance, and functionality, is also needed for the early specification of pancreas progenitors. We used RNA profiling and chromatin immunoprecipitation (ChIP) sequencing to identify a set of targets in pancreas progenitors. We demonstrate that Mnx1, a gene that is absolutely required in pancreas progenitors, is a major direct target of PTF1a and is regulated by a distant enhancer element. Pdx1, Nkx6.1, and Onecut1 are also direct PTF1a targets whose expression is promoted by PTF1a. These proteins, most of which were previously shown to be necessary for pancreas bud maintenance or formation, form a transcription factor network that allows the maintenance of pancreas progenitors. In addition, we identify Bmp7, Nr5a2, RhoV, and P2rx1 as new targets of PTF1a in pancreas progenitors

Dissecting the role of glucocorticoids on pancreas development

To determine whether glucocorticoids are involved in pancreas development, glucocorticoid treatment of rat pancreatic buds in vitro was combined with the analysis of transgenic mice lacking the glucocorticoid receptor (GR) in specific pancreatic cells. In vitro treatment of embryonic pancreata with dexamethasone, a glucocorticoid agonist, induced a decrease of insulin-expressing cell numbers and a doubling of acinar cell area, indicating that glucocorticoids favored acinar differentiation; in line with this, expression of Pdx-1, Pax-6, and Nkx6.1 was downregulated, whereas the mRNA levels of Ptf1-p48 and Hes-1 were increased. The selective inactivation of the GR gene in insulin-expressing beta-cells in mice (using a RIP-Cre transgene) had no measurable consequences on beta- or alpha-cell mass, whereas the absence of GR in the expression domain of Pdx-1 (Pdx-Cre transgene) led to a twofold increased beta-cell mass, with increased islet numbers and size but normal alpha-cell mass in adults. These results demonstrate that glucocorticoids play an important role in pancreatic beta-cell lineage, acting before hormone gene expression onset and possibly also modulating the balance between endocrine and exocrine cell differentiation

Glucocorticoid signalling affects pancreatic development through both direct and indirect effects.: Direct and indirect effects of glucocorticoids on pancreas

AIMS/HYPOTHESIS: Beta cell development is sensitive to glucocorticoid levels. Although direct effects of glucocorticoids on pancreatic precursors have been shown to control beta cell mass expansion, indirect effects of these hormones on pancreatic development remain unexplored. This issue was addressed in mice lacking the glucocorticoid receptor (GR) in the whole organism. MATERIALS AND METHODS: The pancreatic phenotype of GR(null/null) mice was studied at fetal ages (embryonic day [E]) E15.5 and E18 by immunohistochemistry and beta cell fraction measurements. To distinguish between direct and indirect effects, mutant E15.5 fetal pancreata were grafted under the kidney capsule of immunodeficient mice and analysed after 1 week. RESULTS: E18 GR(null/null) fetuses had smaller digestive tracts and tiny pancreata. Massive pancreatic disorganisation and apoptosis were observed despite the presence of all cell types. E15.5 GR(null/null) mutants were indistinguishable from wild-type regarding pancreatic size, tissue structure and organisation, beta cell fraction and production of exocrine transcription factor Ptf1a, neurogenin 3 and Pdx-1. Grafting E15.5 GR(null/null) pancreata into a GR-expressing environment rescued the increased apoptosis and mature islets were observed, suggesting that GR(null/null) pancreatic cell death can be attributed to indirect effects of glucocorticoids on this tissue. Heterozygous GR(+/null) mutants with reduced GR numbers showed no apoptosis but increased beta cell fraction at E18 and the adult age, strengthening the importance of an accurate GR dosage on beta cell mass expansion. CONCLUSIONS/INTERPRETATION: Our results provide evidence for GR involvement in pancreatic tissue organisation and survival through indirect effects. GR does not appear necessary for early phases, but its accurate dosage is critical to modulate beta cell mass expansion at later fetal stages, presumably through direct effects

<i>REST</i> expression is associated with a major loss of beta cell mass.

<p><i>A</i>. Immunostaining for insulin (Ins, red) and glucagon (Glu, green) reveals, when compared to wild type mice (WT, left panel), a major loss of insulin-positive cells, within a low number of disorganized islets in pancreas of adult diabetic RIP-REST mice (middle panel). REST nuclear staining (green, right panel) indicates that only a few surviving beta cells still express REST. Blue staining is the DAPI labeling of nuclei. Scale bar, 25 μm. <i>B</i>. Immunostaining for insulin (Ins, red) and glucagon (Glu, green) shows that, even if disorganized when compared to islets from wild type animals (WT, left panel), islets from diabetic RIP-REST mice at postnatal day 7 (P7) show a significant number of insulin-positive cells (middle panel). REST nuclear staining (green, right panel) indicates that a majority of beta cells express REST. Blue staining is the DAPI labeling of nuclei. Scale bar, 50 μm. <i>C</i>. Quantification of beta-(Ins+ cells) and alpha- (Gluc+ cells) cell mass in P2, P7 and adult diabetic RIP-REST (black bars, n = 3 each) and wild type mice (white bars, n = 3 each). A 30, 45 and 90% reduction in beta cell mass is observed in P2, P7, and adult diabetic RIP-REST mice, respectively, when compared with corresponding mass of controls. Results are mean ± SD. *<i>P</i><0.05, ***<i>P</i><0.001 versus values of wild type mice. <i>D</i>. Confocal analysis of insulin (Ins, red) and glucagon (Glu, green) immunofluorescence shows scarce double insulin-and glucagon-expressing cells (arrows) in pancreatic sections of adult diabetic RIP-REST mice. Blue staining is the DAPI labeling of nuclei. Scale bar, 25 μm.</p

CDK5R2 protects beta cells against cytokines and palmitate.

<p><i>A</i>. Left panel, immunoblotting of total proteins from INS-1E cells, 72 h after transfection with a negative siRNA (siNeg) or siRNAs against <i>Cdk5r2</i> (siCdk5r2#1 and siCdk5r2#2). Right panel, quantification of the corresponding Cdk5r2 protein levels. Results are mean ± SEM of three independent experiments. **<i>P</i><0.01 versus values of siNeg-transduced INS-1E cells. <i>B</i>. Quantification of apoptotic nuclei in non-transfected INS-1E cells (NT) and INS-1E cells transfected with a negative siRNA (siNeg) or with siRNAs against <i>Cdk5r2</i> (siCdk5r2#1 and siCdk5r2#2), treated 24 h with a mix of cytokines (black bars) or not (white bars). Results are mean ± SEM of six independent experiments. **<i>P</i><0.01 versus controls. <i>C</i>. Immunoblotting of total proteins from INS-1E cells transfected with a negative siRNA (siNeg) or siRNAs against <i>Cdk5r2</i> (siCdk5r2#1 and siCdk5r2#2), treated 24 h with a mix of cytokines (Cyt) or not (Ctrl). The cleavage of caspase-9 and −3, induced by cytokines, is higher in siCdk5r2- than in siNeg-treated cells. <i>D</i>. Quantification of apoptotic nuclei in non-transfected INS-1E cells (NT) and INS-1E cells transfected with a negative siRNA (siNeg) or siRNAs against <i>Cdk5r2</i> (siCdk5r2#1 and siCdk5r2#2), exposed 15 h to control medium (white bars) or palmitate (black bars). Results are mean ± SEM of six independent experiments. **<i>P</i><0.01 versus controls.</p

Diabetic RIP-REST transgenic mice are hyperglycemic and show poor insulin secretion.

<p><i>A</i>. Blood glucose levels were assessed at different ages in male and female diabetic RIP-REST transgenic (dark diamonds; n = 7) and wild type mice (open diamonds; n = 5). Diabetic RIP-REST mice feature hyperglycemia from weaning onward. Results are mean ± SD. ***<i>P</i><0.001 versus values of wild type mice. <i>B</i>. Diabetic RIP-REST mice (black circles, n = 3) and wild type littermates (open squares, n = 4) were subjected to <i>in situ</i> pancreatic perfusion at 1.5 ml/min rate. After a 30-min equilibration period at basal 1.4 mmol/l glucose, the pancreas was perfused sequentially at different glucose concentrations, first at 1.4 mmol/l for 20 min, next at 8.0 mmol/l for 20 min, then at 16.0 mmol/l for 20 min, followed by a 30-min stimulation at 8.0 mmol/l plus 1 nmol/l GLP-1, and finally at 1.4 mmol/l for 15 min. Results are mean ± SD.</p

Identification of RE-1-containing genes potentially involved in beta cell survival.

<p><i>A</i>. Alignment of identified RE-1 sequences in human (hum) or murine (mus) genes with the consensus RE-1. Left alignment identifies members of the insulin-like growth factor transduction pathway: <i>Igfbp7</i>: insulin-like growth factor binding protein 7; <i>IRS</i>: insulin receptor substrate; <i>Akt3</i>: PKB gamma; <i>CTNND2</i>: delta2 catenin; <i>Ctnnb1</i>: beta catenin; <i>MAPK10</i>: JNK3; <i>MAP4K3</i>: MEK kinase kinase 3. Upper right alignment identifies members of the cyclin-related family of mitogenic factors: <i>Cdk5r2</i>: cyclin-dependent kinase 5 regulatory subunit 2; <i>CKS2</i>: CDC28 protein kinase regulatory subunit 2; <i>CNNG1</i>: cyclin G1; <i>Cnnm2</i>: cyclin M2. Lower right alignment identifies <i>Api5</i>: apoptosis inhibitor 5 and <i>Nrg1</i>: neuregulin1. <i>B</i>. Quantitative RT-PCR analysis of mRNA levels from control INS-1E cells (white bars) and INS-1E cells infected with GFP-expressing adenovirus (gray bars) or <i>REST</i>-expressing adenovirus (black bars). <i>Ptprn</i>: protein tyrosine phosphatase, receptor type, N; <i>Irs2</i>: insulin receptor substrate2; <i>Cdk5r2</i>: cyclin-dependent kinase 5 regulatory subunit 2; <i>Nrg1</i>: neuregulin1; <i>Ctnnd2</i>: delta2 catenin; <i>Api5</i>: apoptosis inhibitor 5. Results are mean ± SD of six independent experiments. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 versus INS-1E cells transduced with GFP. <i>C</i>. Quantitative RT-PCR analysis of mRNA levels from islets of WT (white bars) (n = 5) and RIP-REST (black bars) (n = 5) mice. <i>Gjd2</i>: Connexin36; <i>Mapk8ip1</i>: Ib1; <i>Cdk5r2</i>: cyclin-dependent kinase 5 regulatory subunit 2; <i>Ptprn</i>: protein tyrosine phosphatase, receptor type, N; <i>Irs2</i>: insulin receptor substrate2; <i>Nrg1</i>: neuregulin1; <i>Ctnnd2</i>: delta2 catenin. Results are mean ± s.e.m. **<i>P</i><0.01 versus values of wild type animals. D. Left panel, immunoblotting of total proteins from non infected (NI) and infected (Ad-GFP, Ad-REST) INS-1E cells showing decreased protein levels of Cdk5r2 upon REST expression. The right panel shows the corresponding quantifications of REST protein levels in INS-1E cells after infection. Data are mean ± SEM of 4 independent experiments. **<i>P</i><0.01, ***<i>P</i><0.001 versus the respective Ad-GFP-infected and NI conditions.</p

Beta cell loss in diabetic RIP-REST mice occurs through apoptosis.

<p><i>A</i>. Nuclear PCNA (green) and insulin (red) staining show same level of proliferating beta cells (arrows) in both wild type (WT, left panel) and diabetic RIP-REST mice (right panel) at P2. Blue staining is the DAPI labeling of nuclei. Scale bar, 50 μm. <i>B</i>. Representative images of nuclear TUNEL (green) and insulin (red) staining showing apoptotic nuclei of beta cells in sections of P2 diabetic RIP-REST mice. No apoptotic beta cells were detected in wild type animals (not shown). Blue staining is the DAPI labeling of nuclei. Scale bar, 25 μm. C. Quantification of apoptotic nuclei in non-infected INS-1E cells (NI) and INS-1E cells transfected with a control (Ad-GFP) or REST-expressing adenovirus (Ad-REST) at different multiplicity of infection (MOI) as indicated, and treated 24 h with a mix of cytokines (black bars) or not (white bars). Results are mean ± SD of six independent experiments. *<i>P</i><0.05 versus respective controls in treated or untreated conditions. # <i>P</i><0.05 versus MOI 2 of Ad-REST infection.</p

Increasing REST levels in beta cells lead to worsened glucose homeostasis.

<p><i>A</i>. Blood glucose levels in 6 month-old wild type (open triangles; n = 8), RIP-REST 5 (gray triangles; n = 8) and RIP-REST males (dark triangles; n = 10) during an IPGTT. After a 14 h fasting, blood samples were taken before (t = 0) and after (t = 15, 30, 60 and 120 min) intraperitoneal injection of glucose (2 g/kg). Results are mean ± SD. *<i>P</i><0.05, **<i>P</i><0.01. <i>B</i>. Insulin content in pancreas of 5 month-old RIP-REST 5 (light gray bar; n = 8), RIP-REST (dark gray bar; n = 6) and diabetic RIP-REST (black bar; n = 6) transgenic mice reveal a 30, 50 and 85% reduction, respectively, when compared with wild type mice (white bars, n = 6). Results are mean ± SD. **<i>P</i><0.01, ***<i>P</i><0.001 versus values of wild type animals. <i>C</i>. Quantitative RT-PCR analysis of human <i>REST</i> mRNA levels in islets of 5 month-old WT (white bar; n = 5), RIP-REST 5 (light gray bar; n = 6) and RIP-REST (dark gray bar; n = 6) mice. <i>REST</i> mRNA levels are six-fold higher in RIP-REST than in RIP-REST 5 animals. Results are mean ± SD. **<i>P</i><0.01 versus values of wild type animals. <i>D</i>. REST transgene abundance in islets from P2 RIP-REST 5, RIP-REST and diabetic RIP-REST mice. Upper panel shows representative images of REST protein levels revealed using specific antibodies against REST and AEC staining of peroxydase activity (nuclear black dots). Parallel immunostaining of insulin (red) and the merge shows colocalization of the two signals. Scale bar, 25 μm. Lower panel shows the quantification of the corresponding average pixel intensity per nuclei for each group. *<i>P</i><0.05 versus the two other groups.</p