Regulation of dual leucine zipper kinase activity through its interaction with calcineurin.

Hyperglycemia enhancing the intracellular levels of reactive oxygen species (ROS) contributes to dysfunction and progressive loss of beta cells and thereby to diabetes mellitus. The oxidation sensitive calcium/calmodulin dependent phosphatase calcineurin promotes pancreatic beta cell function and survival whereas the dual leucine zipper kinase (DLK) induces apoptosis. Therefore, it was studied whether calcineurin interferes with DLK action. In a beta cell line similar concentrations of H2O2 decreased calcineurin activity and activated DLK. DLK interacted via its φLxVP motif (aa 362-365) with the interface of the calcineurin subunits A and B. Mutation of the Val prevented this protein protein interaction, hinting at a distinct φLxVP motif. Indeed, mutational analysis revealed an ordered structure of DLK's φLxVP motif whereby Val mediates the interaction with calcineurin and Leu maintains an enzymatically active conformation. Overexpression of DLK wild-type but not the DLK mutant unable to bind calcineurin diminished calcineurin-induced nuclear localisation of the nuclear factor of activated T-cells (NFAT), suggesting that both, DLK and NFAT compete for the substrate binding site of calcineurin. The calcineurin binding-deficient DLK mutant exhibited increased DLK activity measured as phosphorylation of the downstream c-Jun N-terminal kinase, inhibition of CRE-dependent gene transcription and induction of apoptosis. These findings show that calcineurin interacts with DLK; and inhibition of calcineurin increases DLK activity. Hence, this study demonstrates a novel mechanism regulating DLK action. These findings suggest that ROS through inhibition of calcineurin enhance DLK activity and thereby lead to beta cell dysfunction and loss and ultimately diabetes mellitus.

TNFα-induced DLK activation contributes to apoptosis in the beta-cell line HIT.

Reduction in beta-cell mass and function contributes to the pathogenesis of diabetes mellitus type 2. The proinflammatory cytokines tumor necrosis factor (TNF)α and interleukin (IL)-1ß have been implicated in the pathogenesis of this disease. Overexpression of the dual leucine zipper kinase (DLK) inhibits beta-cell function and induces apoptosis in the beta-cell line HIT. In the present study, it was investigated whether TNFα or IL-1ß stimulates DLK enzymatic activity. Immunoblot analysis, transient transfection with luciferase reporter gene assays, and immunofluorescence were used. In contrast to IL-1ß, TNFα stimulated DLK kinase activity, which was dependent on the c-Jun N-terminal kinase (JNK). Furthermore, DLK contributed to TNFα-induced JNK phosphorylation. The phosphorylation of DLK on Ser-302 within the activation loop was required for DLK to stimulate JNK and to inhibit CREB-dependent gene transcription. TNFα induced apoptosis in a time- and concentration-dependent manner and inhibited CREB-directed gene transcription in HIT cells. The reduction of endogenous DLK by small interfering or small hairpin RNA attenuated TNFα's effects on apoptosis and CREB-dependent transcription. These data suggest that TNFα induces beta-cell apoptosis through activation of DLK thereby inhibiting the beta-cell protective transcription factor CREB. Furthermore, activation of DLK by a well-known diabetic risk factor supports the role of DLK in the pathogenesis of diabetes mellitus. Thus, the inhibition of DLK might prevent or retard the pathogenesis of diabetes mellitus type 2.

Distinct functions of the dual leucine zipper kinase depending on its subcellular localization.

The dual leucine zipper kinase DLK induces ß-cell apoptosis by inhibiting the transcriptional activity conferred by the ß-cell protective transcription factor cAMP response element binding protein CREB. This action might contribute to ß-cell loss and ultimately diabetes. Within its kinase domain DLK shares high homology with the mixed lineage kinase (MLK) 3, which is activated by tumor necrosis factor (TNF) α and interleukin (IL)-1ß, known prediabetic signals. In the present study, the regulation of DLK in ß-cells by these cytokines was investigated. Both, TNFα and IL-1ß induced the nuclear translocation of DLK. Mutations within a putative nuclear localization signal (NLS) prevented basal and cytokine-induced nuclear localization of DLK and binding to the importin receptor importin α, thereby demonstrating a functional NLS within DLK. DLK NLS mutants were catalytically active as they phosphorylated their down-stream kinase c-Jun N-terminal kinase to the same extent as DLK wild-type but did neither inhibit CREB-dependent gene transcription nor transcription conferred by the promoter of the anti-apoptotic protein BCL-xL. In addition, the ß-cell apoptosis-inducing effect of DLK was severely diminished by mutation of its NLS. In a murine model of prediabetes, enhanced nuclear DLK was found. These data demonstrate that DLK exerts distinct functions, depending on its subcellular localization and thus provide a novel level of regulating DLK action. Furthermore, the prevention of the nuclear localization of DLK as induced by prediabetic signals with consecutive suppression of ß-cell apoptosis might constitute a novel target in the therapy of diabetes mellitus.

Inhibition of human insulin gene transcription and MafA transcriptional activity by the dual leucine zipper kinase.

Insulin biosynthesis is an essential ß-cell function and inappropriate insulin secretion and biosynthesis contribute to the pathogenesis of diabetes mellitus type 2. Previous studies showed that the dual leucine zipper kinase (DLK) induces ß-cell apoptosis. Since ß-cell dysfunction precedes ß-cell loss, in the present study the effect of DLK on insulin gene transcription was investigated in the HIT-T15 ß-cell line. Downregulation of endogenous DLK increased whereas overexpression of DLK decreased human insulin gene transcription. 5'- and 3'-deletion human insulin promoter analyses resulted in the identification of a DLK responsive element that mapped to the DNA binding-site for the ß-cell specific transcription factor MafA. Overexpression of DLK wild-type but not its kinase-dead mutant inhibited MafA transcriptional activity conferred by its transactivation domain. Furthermore, in the non-ß-cell line JEG DLK inhibited MafA overexpression-induced human insulin promoter activity. Overexpression of MafA and DLK or its kinase-dead mutant into JEG cells revealed that DLK but not its mutant reduced MafA protein content. Inhibition of the down-stream DLK kinase c-Jun N-terminal kinase (JNK) by SP600125 attenuated DLK-induced MafA loss. Furthermore, mutation of the serine 65 to alanine, shown to confer MafA protein stability, increased MafA-dependent insulin gene transcription and prevented DLK-induced MafA loss in JEG cells. These data suggest that DLK by activating JNK triggers the phosphorylation and degradation of MafA thereby attenuating insulin gene transcription. Given the importance of MafA for ß-cell function, the inhibition of DLK might preserve ß-cell function and ultimately retard the development of diabetes mellitus type 2.

Activation of the dual-leucine-zipper-bearing kinase and induction of beta-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 beta-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 beta-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 Bcl(XL) 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 IC(50) value for the inhibition of calcineurin activity in beta cells. These data suggest that cyclosporin A through inhibition of calcineurin activates DLK, thereby leading to beta-cell apoptosis. This action may thus be a novel mechanism through which cyclosporin A precipitates post-transplant diabetes.

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 alpha-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 alpha-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 alpha-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.

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

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. It was unknown at what step in CREB-mediated transcription cyclosporin A and tacrolimus interfere. In transient transfection experiments, using GAL4-CREB fusion proteins and a pancreatic islet beta-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. 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. These data suggest that, in contrast to NFAT, cyclosporin A and tacrolimus inhibit CREB transcriptional activity at the coactivator level.

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.

Inhibition of human insulin gene transcription by the immunosuppressive drugs cyclosporin A and tacrolimus in primary, mature islets of transgenic mice.

Cyclosporin A and tacrolimus are clinically important immunosuppressive drugs. They share a diabetogenic action as one of their most serious adverse effects. The underlying mechanism is unknown. Previous studies have shown that tacrolimus can inhibit insulin gene transcription at high concentrations in tumor cell lines. To study insulin gene transcription in normal, mature pancreatic islet cells, we used a novel approach in the present study. Transgenic mice that carry a human insulin promoter-reporter gene were generated. The human insulin promoter directed transcription in pancreatic islets and conferred a normal, physiological glucose response to reporter gene expression in isolated islets. After stimulation with glucose, human insulin promoter-mediated gene expression was inhibited in normal, mature islet cells by both tacrolimus and cyclosporin A to a large extent (approximately 70%) and with high potency at concentrations that are known to inhibit calcineurin phosphatase activity (IC50 values of 1 and 35 nM, respectively). Furthermore, glucose stimulated calcineurin phosphatase activity in mouse pancreatic islets, further supporting the view that calcineurin phosphatase activity is an essential part of glucose signaling to the human insulin gene. The high potency of cyclosporin A and tacrolimus in normal islets suggests that inhibition of insulin gene transcription by cyclosporin A and tacrolimus is clinically important and is one mechanism of the diabetogenic effect of these immunosuppressive drugs.

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 IC(50) 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.