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 -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 -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  cells. These data suggest that cyclosporin A through inhibition of calcineurin activates DLK, thereby leading to -cell apoptosis. This action may thus be a novel mechanism through which cyclosporin A precipitates post-transplant diabetes.
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)-1β 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-1β stimulates DLK enzymatic activity. Immunoblot analysis, transient transfection with luciferase reporter gene assays, and immunofluorescence were used. In contrast to IL-1β, 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.
Die Wirkung vonCyclosporin A (CsA), TNF- und IL-1 auf die Apoptoseinduktion der pankreatischen Betazelllinie HIT-T15 in An-und Abwesenheit von überexprimierter DLK, untersucht im Immunfluoreszenz-Assay.
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