Willhart Knepel

The activation of the transcription factor cyclic AMP response element binding protein (CREB) by protein kinase A is inhibited by the human orthologue of the mitogen-activated protein kinase, dual-leucine-zipper-bearing kinase (DLK) in teratocarcinoma cells. However, pancreatic beta cells are electrically excitable and a major pathway regulating CREB in these cells is membrane depolarisation, leading to calcium influx and activation of the calcium/calmodulin-dependent protein phosphatase calcineurin. Therefore, the effect of DLK on CREB activity induced by membrane depolarisation was investigated in the beta cell line HIT.

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 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. 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. British Journal of Pharmacology (2005) 144, 982–993. doi:10.1038/sj.bjp.0706127

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 β-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.