Roland Blume

Characterization of a Novel Calcium Response Element in the Glucagon Gene

To maintain blood glucose levels within narrow limits, the synthesis and secretion of pancreatic islet hormones is controlled by a variety of extracellular signals. Depolarization-induced calcium influx into islet cells has been shown to stimulate glucagon gene transcription through the transcription factor cAMP response element-binding protein that binds to the glucagon cAMP response element. By transient transfection of glucagon-reporter fusion genes into islet cell lines, this study identified a second calcium response element in the glucagon gene (G2 element, from −165 to −200). Membrane depolarization was found to induce the binding of a nuclear complex with NFATp-like immunoreactivity to the G2 element. Consistent with nuclear translocation, a comigrating complex was found in cytosolic extracts of unstimulated cells, and the induction of nuclear protein binding was blocked by inhibition of calcineurin phosphatase activity by FK506. A mutational analysis of G2 function and nuclear protein binding as well as the effect of FK506 indicate that calcium responsiveness is conferred to the G2 element by NFATp functionally interacting with HNF-3β binding to a closely associated site. Transcription factors of the NFAT family are known to cooperate with AP-1 proteins in T cells for calcium-dependent activation of cytokine genes. This study shows a novel pairing of NFATp with the cell lineage-specific transcription factor HNF-3β in islet cells to form a novel calcium response element in the glucagon gene.

Gene-specific Transcriptional Activity of the Insulin cAMP-responsive Element Is Conferred by NF-Y in Combination with cAMP Response Element-binding Protein

Cyclic AMP stimulates insulin gene transcription through a cAMP response element (CRE). In the present study the insulin CRE-binding proteins and their functions were investigated. A mutational analysis of nuclear protein binding in electrophoretic mobility shift assays in combination with specific antisera showed that in the CRE of the rat insulin I gene the imperfect CRE octamer-like sequence TGACGTCC interacts weakly with CREB and overlaps with two sequence motifs (TTGTTGAC and CCAAT) that bind winged helix-like proteins and the transcription factor NF-Y, respectively. Transient transfection of wild-type and mutant insulin CRE-reporter fusion genes and the inactivation of cellular CREB or NF-Y by overexpression of the dominant negative mutants KCREB or NF-YA29, respectively, indicate that cAMP inducibility of the insulin CRE is mediated by CREB or closely related proteins; however, NF-Y binding to the insulin CRE confers constitutive, basal activity and decreases the ability of CREB to mediate cAMP-stimulated transcription and calcium responsiveness. Results from these studies demonstrate that NF-Y binds to the insulin CRE and modulates the function of CREB. Together with the nonpalindromic sequence of the CRE octamer motif, the interaction of NF-Y with CREB may be responsible for the gene-specific transcriptional activity of the insulin CRE and explain why it has considerable basal activity but is less responsive to cAMP stimulation than others.

Inhibition of CREB- and cAMP response element-mediated gene transcription by the immunosuppressive drugs cyclosporin A and FK506 in T cells

Abstract The clinically important immunosuppressant drugs cyclosporin A and FK506 (tacrolimus) inhibit in T-cells calcineurin phosphatase activity and nuclear translocation of the cytosolic component of the transcription factor nuclear factor of activated T-cells (NF-ATc) that is involved in the induction of early genes during T-cell activation. This effect has been proposed to explain at least part of the immunosuppressive effect of these drugs. Previous studies in pancreatic islet cell lines have shown that cyclosporin A and FK506 through inhibition of calcineurin interfere also with the function of the transcription factor cAMP response element binding protein (CREB) that is activated by cAMP and calcium signals and binds to cAMP/calcium response elements (CRE). By transient expression of CRE-reporter genes or GAL4-CREB fusion proteins, the present study shows that inhibition of CREB/ CRE-directed transcription by cyclosporin A and FK506 occurs in a great variety of cell types including in cell lines derived from tissues in which adverse effects of the immunosuppressants develop. CREB activity and CRE-mediated transcription was blocked by these drugs also in Jurkat T-cells. When taken together with recent evidence for an essential role of CREB in T-cell activation and proliferation, the present results suggest that inhibition of CREB/CRE-directed transcription may be a molecular mechanism of the immunosuppressive effect of cyclosporin A and FK506.

Activation of the Dual-Leucine-Zipper-Bearing Kinase and Induction of β-Cell Apoptosis by the Immunosuppressive Drug Cyclosporin A

Post-transplant diabetes is an untoward effect often observed under immunosuppressive therapy with cyclosporin A. Besides the development of peripheral insulin resistance and a decrease in insulin gene transcription, a β-cell toxic effect has been described. However, its molecular mechanism remains unknown. In the present study, the effect of cyclosporin A and the dual leucine-zipper-bearing kinase (DLK) on β-cell survival was investigated. Cyclosporin A decreased the viability of the insulin-producing pancreatic islet cell line HIT in a time- and concentration-dependent manner. Upon exposure to the immunosuppressant fragmentation of DNA, the activation of the effector caspase-3 and a decrease of full-length caspase-3 and BclXL were observed in HIT cells and in primary mature murine islets, respectively. Cyclosporin A and tacrolimus, both potent inhibitors of the calcium/calmodulin-dependent phosphatase calcineurin, stimulated the enzymatic activity of cellular DLK in an in vitro kinase assay. Immunocytochemistry revealed that the overexpression of DLK but not its kinase-dead mutant induced apoptosis and enhanced cyclosporin A-induced apoptosis to a higher extent than the drug alone. Moreover, in the presence of DLK, the effective concentration for cyclosporin A-caused apoptosis was similar to its known IC50 value for the inhibition of calcineurin activity in β cells. These data suggest that cyclosporin A through inhibition of calcineurin activates DLK, thereby leading to β-cell apoptosis. This action may thus be a novel mechanism through which cyclosporin A precipitates post-transplant diabetes.

In vitro Inhibition of Rat Intestinal Surface Hydrolysis of Disaccharides and Dipeptides by Guaran

The effect of guaran on intestinal surface hydrolases was studied in everted jejunal segments by measuring the hydrolysis of maltose and phenylalanylglycine. On the basis of kinetic experiments, guaran was found to inhibit competitively the hydrolysis of these substrates only if the cleavage was performed with intact tissue. Hydrolysis in mucosal homogenates was not affected by guaran, indicating that this polysaccharide may increase the diffusion barrier overlaying the mucosa and thus may retard final digestion of carbohydrates and peptides.

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.

The immunosuppressive drugs cyclosporin A and tacrolimus inhibit membrane depolarization-induced CREB transcriptional activity at the coactivator level

1 Cyclosporin A and tacrolimus are clinically important immunosuppressive drugs directly targeting the transcription factor nuclear factor of activated T cells (NFAT). Through inhibition of calcineurin phosphatase activity they block the dephosphorylation and thus activation of NFAT. Cyclosporin A and tacrolimus also inhibit other calcineurin‐dependent transcription factors including the ubiquitously expressed cAMP response element‐binding protein (CREB). Membrane depolarization by phosphorylating CREB on Ser119 leads to the recruitment of its coactivator CREB‐binding protein (CBP) that stimulates initiation of transcription. 2 It was unknown at what step in CREB‐mediated transcription cyclosporin A and tacrolimus interfere. 3 In transient transfection experiments, using GAL4‐CREB fusion proteins and a pancreatic islet β‐cell line, cyclosporin A inhibited depolarization‐induced activation of CREB proteins which carried various deletions or mutations throughout their sequence providing no evidence for the existence of a distinct CREB domain conferring cyclosporin A sensitivity. In a mammalian two‐hybrid assay, cyclosporin A did not inhibit Ser119‐dependent interaction of CREB with its coactivator CBP. 4 Using GAL4‐CBP fusion proteins, cyclosporin A inhibited depolarization‐induced CBP activity, with cyclosporin A‐sensitive domains mapped to both the N‐ (aa 1–451) and C‐terminal (aa 2040–2305) ends of CBP. The depolarization‐induced transcriptional activity of the CBP C‐terminus was enhanced by overexpression of calcineurin and was inhibited by cyclosporin A and tacrolimus in a concentration‐dependent manner with IC50 values (10 and 1 nM, respectively) consistent with their known IC50 values for inhibition of calcineurin. 5 These data suggest that, in contrast to NFAT, cyclosporin A and tacrolimus inhibit CREB transcriptional activity at the coactivator level.

The immunosuppressive drugs cyclosporin A and FK506 inhibit calcineurin phosphatase activity and gene transcription mediated through the cAMP-responsive element in a nonimmune cell line

SummaryCyclosporin A and the macrolide tacrolimus (FK506) are powerful immunosuppressive drugs that in T cells inhibit the calcium/calmodulin-dependent phosphatase calcineurin thereby preventing the activation of T-cell-specific transcription factors, such as NF-AT, involved in lymphokine gene expression. While this may explain, at least in part, the mechanism of cyclosporin A/FK506 immunosuppression, additional mechanisms have to be invoked in order to explain the pharmacological properties and toxic effects of these drugs, such as nephrotoxicity and neurotoxicity. We have studied the effects of cyclosporin A and FK506 on calcineurin phosphatase activity and gene transcription mediated by the cAMP-responsive element (CRE), a binding site of the ubiquitous transcription factor CREB. A reporter gene was placed under the transcriptional control of the CRE of the rat glucagon gene and transiently transfected into the glucagon-expressing cell line αTC2. Cyclosporin A and FK506 inhibited depolarization-induced gene transcription in a concentration-dependent manner (IC50 of about 1 nM and 30 nM for FK506 and cyclosporin A, respectively). Both cyclosporin A and FK506 inhibited calcineurin phosphatase activity at drug concentrations that inhibited gene transcription. The FK506 analogue rapamycin had no effect on calcineurin activity and gene transcription, but excess concentrations of rapamycin prevented the effects of FK506 on both calcineurin activity and gene transcription. These results support the notion that the interaction of drug-immunophilin complexes with calcineurin may be the molecular basis of cyclosporin A/FK506-induced inhibition of CREB/CRE-mediated gene transcription. The ability to interfere with CREB/CRE-mediated gene transcription represents a novel mechanism of cyclosporin A/FK506 action which may underlie pharmacological effects and toxic manifestations of these potent immunuosuppressive drugs.

Inhibition of MafA transcriptional activity and human insulin gene transcription by interleukin-1β and mitogen-activated protein kinase kinase kinase in pancreatic islet beta cells

Aims/hypothesisInappropriate insulin secretion and biosynthesis are hallmarks of beta cell dysfunction and contribute to the progression from a prediabetic state to overt diabetes mellitus. During the prediabetic state, beta cells are exposed to elevated levels of proinflammatory cytokines. In the present study the effect of these cytokines and mitogen-activated protein kinase kinase kinase 1 (MEKK1), which is known to be activated by these cytokines, on human insulin gene (INS) transcription was investigated.MethodsBiochemical methods and reporter gene assays were used in a beta cell line and in primary pancreatic islets from transgenic mice.ResultsIL-1β and MEKK1 specifically inhibited basal and membrane depolarisation and cAMP-induced INS transcription in the beta cell line. Also, in primary islets of reporter gene mice, IL-1β reduced glucose-stimulated INS transcription. A 5′- and 3′-deletion and internal mutation analysis revealed the rat insulin promoter element 3b (RIPE3b) to be a decisive MEKK1-responsive element of the INS. RIPE3b conferred strong transcriptional activity to a heterologous promoter, and this activity was markedly inhibited by MEKK1 and IL-1β. RIPE3b is also known to recruit the transcription factor MafA. We found here that MafA transcription activity is markedly inhibited by MEKK1 and IL-1β.Conclusions/interpretationThese data suggest that IL-1β through MEKK1 inhibits INS transcription and does so, at least in part, by decreasing MafA transcriptional activity at the RIPE3b control element. Since inappropriate insulin biosynthesis contributes to beta cell dysfunction, inhibition of MEKK1 might decelerate or prevent progression from a prediabetic state to diabetes mellitus.

Polycations. A new class of inhibitors for in vitro small intestinal transport of sugars and amino acids in the rat

Polycationic compounds like polylysine, protamine or polyethylenimine may interfere with a cation-related membrane transport system depending on superficially accessible binding sites for particular cations. In vitro experiments were performed using either everted segments of rat small intestine to measure tissue accumulation or everted sacs to determine mucosal-to-serosal transport. The effect of polycations was also tested using brush-border membrane vesicles of rat jejunum. Polycations inhibited the tissue accumulation of methyl alpha-D-glucoside as well as binding of phlorizin. Inhibition of accumulation was increased by raising the polycation concentration and by preincubation of the tissue with the polycations. Kinetic experiments revealed a competitive type of inhibition for the uptake of neutral amino acids and actively transported sugars. Using everted sacs to compare the monomeric cations with their corresponding polymeric forms for their inhibitory effect, it was found that only polymers applied to the mucosal compartment impaired active transport. The passive diffusion of solutes, e.g. 2-deoxy-D-glucose or mannitol, was slightly increased by polycations. With some intermediate oligomers of lysine it could be shown that more than 20 cationic groups are required for approximate complete inhibition. That membrane-related events are responsible for the observed inhibition is suggested by the reduced uptake of D-glucose by brush-border membrane vesicles in the presence of polycations. Therefore an interaction with transport-related cation binding sites, i.e. anionic residues, at the mucosal surface may be assumed.