Regulation of human insulin gene transcription by the immunosuppressive drugs cyclosporin A and tacrolimus at concentrations that inhibit calcineurin activity and involving the transcription factor CREB

Cyclosporin A and tacrolimus are important immunosuppressive drugs. They share a diabetogenic action as one of their most serious adverse effects. In a single study, tacrolimus (100 nM) inhibited human insulin gene transcription in the beta-cell line HIT. Using transfections of a human insulin-reporter gene into HIT cells, the present study shows that this inhibition is seen only at high concentrations of tacrolimus and is not caused by cyclosporin A. However, after stimulation by the major second messengers in the regulation of the insulin gene, cAMP and depolarization-induced calcium influx, both tacrolimus and cyclosporin A inhibited human insulin gene transcription in a concentration-dependent manner with IC50 values of 1 nM and 30 nM, respectively. A further analysis offers a mechanism for this effect by revealing that the activation by cAMP and calcium of human insulin gene transcription is mediated by the transcription factor cAMP-responsive element binding protein (CREB) whose activity is inhibited by the immunosuppressants. These data demonstrate for the first time that cAMP- and calcium-induced activity of the human insulin gene is mediated by CREB and blocked by both tacrolimus and cyclosporin A at concentrations that inhibit calcineurin phosphatase activity. Since also the immunosuppressive effects of cyclosporin A and tacrolimus are thought to be secondary to inhibition of calcineurin, the present study suggests that inhibition of human insulin gene transcription by the immunosuppressants is clinically important and may contribute to their diabetogenic effect

Protein kinase B activity is sufficient to mimic the effect of insulin on glucagon gene transcription

Insulin inhibits glucagon gene transcription, and insulin deficiency is associated with hyperglucagonemia that contributes to hyperglycemia in diabetes mellitus. However, the insulin signaling pathway to the glucagon gene is unknown. Protein kinase B (PKB) is a key regulator of insulin signaling and glucose homeostasis. Impaired PKB function leads to insulin resistance and diabetes mellitus. Therefore, the role of PKB in the regulation of glucagon gene transcription was investigated. After transient transfections of glucagon promoter-reporter genes into a glucagon-producing islet cell line, the use of kinase inhibitors indicated that the inhibition of glucagon gene transcription by insulin depends on phosphatidylinositol (PI) 3-kinase. Furthermore, insulin caused a PI 3-kinase-dependent phosphorylation and activation of PKB in this cell line as revealed by phospho-immunoblotting and kinase assays. Overexpression of constitutively active PKB mimicked the effect of insulin on glucagon gene transcription. Both insulin and PKB responsiveness of the glucagon promoter were abolished when the binding sites for the transcription factor Pax6 within the G1 and G3 promoter elements were mutated. Recruitment of Pax6 or its potential coactivator, the CREB-binding protein (CBP), to G1 and G3 by using the GAL4 system restored both insulin and PKB responsiveness. These data suggest that insulin inhibits glucagon gene transcription by signaling via PI 3-kinase and PKB, with the transcription factor Pax6 and its potential coactivator CBP being critical components of the targeted promoter-specific nucleoprotein complex. The present data emphasize the importance of PKB in insulin signaling and glucose homeostasis by defining the glucagon gene as a novel target gene for PKB

Characterization of a novel Foxa (hepatocyte nuclear factor-3) site in the glucagon promoter that is conserved between rodents and humans

The pancreatic islet hormone glucagon stimulates hepatic glucose production and thus maintains blood glucose levels in the fasting state. Transcription factors of the Foxa [Fox (forkhead box) subclass A; also known as HNF-3 (hepatocyte nuclear factor-3)] family are required for cell-specific activation of the glucagon gene in pancreatic islet α-cells. However, their action on the glucagon gene is poorly understood. In the present study, comparative sequence analysis and molecular characterization using protein–DNA binding and transient transfection assays revealed that the well-characterized Foxa-binding site in the G2 enhancer element of the rat glucagon gene is not conserved in humans and that the human G2 sequence lacks basal enhancer activity. A novel Foxa site was identified that is conserved in rats, mice and humans. It mediates activation of the glucagon gene by Foxa proteins and confers cell-specific promoter activity in glucagon-producing pancreatic islet α-cell lines. In contrast with previously identified Foxa-binding sites in the glucagon promoter, which bind nuclear Foxa2, the novel Foxa site was found to bind preferentially Foxa1 in nuclear extracts of a glucagon-producing pancreatic islet α-cell line, offering a mechanism that explains the decrease in glucagon gene expression in Foxa1-deficient mice. This site is located just upstream of the TATA box (between −30 and −50), suggesting a role for Foxa proteins in addition to direct transcriptional activation, such as a role in opening the chromatin at the start site of transcription of the glucagon gene

Studies in Greek epigraphy and history in honor of Stefen V. Tracy

Ce recueil d'articles en épigraphie grecque est destiné à honorer l'œuvre de Stephen V. Tracy dont la recherche méticuleuse sur les "mains" - ou styles - des graveurs a transformé notre perception des inscriptions d'Attique et d'au-delà. Les vingt-neuf érudits qui ont contribué à ce volume présentent des articles qui dévoilent de nouvelles inscriptions ou de nouvelles études sur des textes connus depuis longtemps, mais aussi traitent de questions historiques, religieuses et sociales. Ainsi sont étudiées la Grèce, la Mer Egée et l'Asie Mineure occidentale sur une période qui s'étend de l'époque archaïque à l'Empire romain. Le point commun de ces Mélanges réside dans le caractère central de l'épigraphie, tant dans les problèmes soulevés que dans la conviction qu'un soin minutieux porté au moindre détail d'un témoignage épigraphique peut élargir et enrichir notre compréhension de l'histoire grecque.This collection of studies in Greek epigraphy honors the work of Stephen V. Tracy. His meticulous research on the hands of Attic letter-cutters has transformed the way we think about Greek inscriptions in Attica and beyond. The twenty-nine scholars who have contributed to this volume offer papers ranging from publication of new inscriptions and studies of others long-known to wide-ranging discussions of historical, religious, and social matters. Chronologically and geographically they cover Greece, the Aegean, and western Asia Minor from the Archaic period to the Roman Empire. What unites the work here offered to Tracy is the centrality of epigraphy to the questions addressed and conviction that careful attention to even the smallest details of the epigraphic evidence can advance our understanding of the Greek past in rich and unexpected ways