Elke Oetjen

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.

Insulin Responsiveness of the Glucagon Gene Conferred by Interactions between Proximal Promoter and More Distal Enhancer-like Elements Involving the Paired-domain Transcription Factor Pax6

Regulation of gene transcription is an important aspect of insulins action. However, the mechanisms involved are poorly understood. Insulin inhibits glucagon gene transcription, and insulin deficiency is associated with hyperglucagonemia that contributes to hyperglycemia in diabetes mellitus. Transfecting glucagon-reporter fusion genes into a glucagon-producing pancreatic islet cell line, a 5′-, 3′-, and internal deletion analysis, and oligonucleotide cassette insertions failed in the present study to identify a single insulin-responsive element in the glucagon gene. They rather indicate that insulin responsiveness depends on the presence of both proximal promoter elements and more distal enhancer-like elements. When the paired domain transcription factor Pax6 binding sites within the proximal promoter element G1 and the enhancer-like element G3 were mutated into GAL4 binding sites, the expression of GAL4-Pax6 and GAL4-VP16 restored basal activity, whereas only GAL4-Pax6 restored also insulin responsiveness. Likewise, GAL4-CBP activity was inhibited by insulin within the glucagon promoter context. The results suggest that insulin responsiveness is conferred to the glucagon gene by the synergistic interaction of proximal promoter and more distal enhancer-like elements, with Pax6 and its potential coactivator the CREB-binding protein being critical components. These data thereby support concepts of insulin-responsive element-independent mechanisms of insulin action to inhibit gene transcription.

Cyclosporin A, but not everolimus, inhibits DNA repair mediated by calcineurin: implications for tumorigenesis under immunosuppression

Abstract:  Unlike other immunosuppressive drugs including everolimus, cyclosporin A causes a dramatic increase of UV‐induced skin cancer, a feature that is reminiscent of xeroderma pigmentosum (XP), where defective nucleotide excision repair (NER) of UV‐induced DNA damage results in cutaneous carcinogenesis. The molecular basis of the clinically important differential activities of cyclosporin A and everolimus is still unclear. We measured post‐UV cell survival of cyclosporin A‐ and everolimus‐treated human fibroblasts and lymphoblasts using a cell proliferation assay (MTT). The cellular NER capacity was assessed by host cell reactivation. Using an ELISA and specific antibodies, cyclobutane pyrimidine and pyrimidine‐6,4‐pyrimidone photoproduct removal from the cellular genome was measured. The effect of calcineurin on NER was investigated using a calcineurin A expression vector and specific RNAi. Cyclosporin A led to a dose dependent decrease in post‐UV cell survival, inhibited NER and blocked photoproduct removal. In contrast, none of these effects where seen in everolimus‐treated cells. Overexpression of calcineurin A resulted in increased NER and complemented the Cyclosporin A‐induced reduction of NER. Downregulation of calcineurin using RNAi inhibited NER comparable to cyclosporin A‐treatment. We conclude that cyclosporin A, but not everolimus, leads to an increased skin cancer risk via a calcineurin signalling‐dependent impairment of NER.

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.

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.

Functional characterization of a -100_-102delAAG deletion-insertion polymorphism in the promoter region of the HTR3B gene.

Objective The HTR3B gene encodes the B-subunit of the type 3 serotonin receptor (5-HT3). A -100_-102delAAG deletion in the promoter region has been associated with poor response to antiemetic medication and susceptibility to bipolar affective disorders. The molecular mechanisms underlying these associations, however, remained unclear. Methods We performed electrophoretic mobility shift and luciferase reporter gene assays to elucidate the effect of this polymorphism on the HTR3B promoter activity in PC-12 and HEK293 cells. The reporter constructs carried a 2171 bp fragment of the native HTR3B promoter or 30 bp of the polymorphic locus in tandem triplication upstream of the thymidine kinase minimal promoter. Results Deletion mapping indicated that the sequence around the -100_-102delAAG polymorphism had significant promoter activity. Electrophoretic mobility shift assays indicated differential binding of nuclear proteins to the polymorphic DNA region with stronger binding to the insertion than to the deletion allele. The activity of the native promoter carrying the deletion allele was 25% higher in PC-12 (P=0.016) and 40% higher in HEK cells (P=0.016) compared with the respective insertion construct. Constructs carrying the deletion allele in tandem triplicates showed 43% (PC-12 cells, P=0.002) and 28% (HEK293 cells, P=0.015) higher activity than those carrying the insertion allele. The polymorphism was not linked with known amino acid substitutions in HTR3A and HTR3B. Conclusions The -100_-102delAAG 3 bp deletion increases the HTR3B promoter activity in vitro. The consequences of this for the structure and the function of the resulting 5-HT3 receptors remain to be elucidated.

A Peroxisome Proliferator-Activated Receptor γ-Retinoid X Receptor Heterodimer Physically Interacts with the Transcriptional Activator PAX6 to Inhibit Glucagon Gene Transcription

The peptide hormone glucagon stimulates hepatic glucose output, and its levels in the blood are elevated in type 2 diabetes mellitus. The nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) has essential roles in glucose homeostasis, and thiazolidinedione PPARγ agonists are clinically important antidiabetic drugs. As part of their antidiabetic effect, thiazolidinediones such as rosiglitazone have been shown to inhibit glucagon gene transcription through binding to PPARγ and inhibition of the transcriptional activity of PAX6 that is required for cell-specific activation of the glucagon gene. However, how thiazolidinediones and PPARγ inhibit PAX6 activity at the glucagon promoter remained unknown. After transient transfection of a glucagon promoter-reporter fusion gene into a glucagon-producing pancreatic islet α-cell line, ligand-bound PPARγ was found in the present study to inhibit glucagon gene transcription also after deletion of its DNA-binding domain. Like PPARγ ligands, also retinoid X receptor (RXR) agonists inhibited glucagon gene transcription in a PPARγ-dependent manner. In glutathione transferase pull-down assays, the ligand-bound PPARγ-RXR heterodimer bound to the transactivation domain of PAX6. This interaction depended on the presence of the ligand and RXR, but it was independent of the PPARγ DNA-binding domain. Chromatin immunoprecipitation experiments showed that PPARγ is recruited to the PAX6-binding proximal glucagon promoter. Taken together, the results of the present study support a model in which a ligand-bound PPARγ-RXR heterodimer physically interacts with promoter-bound PAX6 to inhibit glucagon gene transcription. These data define PAX6 as a novel physical target of PPARγ-RXR.