Cleavage of Protein Kinase C δ by Caspase-3 Mediates Pro-inflammatory Cytokine-Induced Apoptosis in the Pancreatic Islet

In type 1 diabetes (T1D), autoreactive immune cells infiltrate the pancreas and secrete proinflammatory cytokines that initiate cell death in insulin producing islet β-cells. Protein kinase C δ (PKCδ) plays a role in mediating cytokine-induced β-cell death; however, the exact mechanisms are not well understood. Here we explored the role of PKCδ in mediating pro-inflammatory cytokine-induced apoptosis in both mouse and human islets. Our results support a role for PKCδ activity in mediating cytokine-induced apoptosis and inhibiting PKCδ with a cell permeable inhibitor, δV1-1, also protected against cytokine-induced apoptosis in mouse and human islets. Pro-inflammatory cytokines increased PKCδ activity and nuclear translocation, and caspase-3 cleavage of PKCδ was required for cytokine-induced apoptosis. Our results support a role for PKCδ in regulation of pro-apoptotic signaling in pancreatic islets and suggest PKCδ may play a role in mediating cytokine-induced apoptosis in pancreatic β-cells in T1D. death. As caspase-3 activation plays a role in cytokine-mediated β-cell death, we hypothesized that cytokine-mediated activation of PKCδ leads to cleavage by caspase-3 and nuclear translocation, resulting in an increase in pro-apoptotic signaling. We utilized isolated islets from mice with a β-cell-specific knockout of PKCδ or human islets treated with a cell permeable PKCδ specific inhibitor, δV1-1. Improved understanding of the cytokine-mediated apoptotic pathway in human β-cells will further elucidate key mechanisms in the pathogenesis of T1D and will provide new avenues towards developing targeted therapies to preserve β-cell mass in T1D. roles of PKCδ in mediating β-cell death has provided further insight into apoptotic signaling mechanisms that may occur in T1D. Combined with the protective effects of PKCδ inhibition with δV1-1, the results of this study have the potential to aid in the development of novel β-cell targeted therapies to prevent or delay β-cell death and preserve β-cell function in T1D. support a role for PKCδ in human diabetes pathology. While no direct evidence for activation of PKCδ in islet β-cells has been shown in T1D or T2D, our results suggest that cytokine-induced activity of PKCδ may promote β-cell death under inflammatory conditions associated with diabetes. Future studies are warranted to investigate the role of PKCδ in β-cell death during diabetes pathogenesis and to determine therapeutic efficacy of δV1-1 in protecting against β-cell death. of the proapoptotic JNK. Overall, the results from this study provide novel insight into the role of PKCδ in mediating cytokine-induced apoptosis in pancreatic islets and supports a role for PKCδ in mediating cytokine-induced apoptosis in T1D in humans. Furthermore, the results of this study have clinical implications as targeted inhibition of this novel PKCδ mediated apoptotic pathway may preserve β-cell mass in T1D. activate PKCδ, in Activated PKCδ also promotes activation of JNK by phosphorylation, which translocates to the nucleus and promotes apoptosis through regulation of nuclear transcription factors. Activated PKCδ is translocated to the nucleus and cleaved by caspase-3. We propose that nuclear activity of cleaved fragment of PKCδ may also regulate nuclear transcription factors that promote apoptosis in the β-cell. Human and C57Bl/6 mouse islets were treated with a cytokine cocktail containing human or mouse recombinant cytokines respectively and 1µM of a PKCδ inhibitor δV1-1 for 24hr. Islets were stained with either propidium iodide or an apoptotic specific stain YO-PRO1 and NucBlue or fluorescein diacetate (FDA). Imaging was performed either on a Leica STELLARIS 5 LIAchroic laser supply unit, with a 40X water immersion objective. excitation laser for each respective dye. Nuclear translocation was determined in ImageJ by normalizing the GFP pixel intensity within each nucleus as determined by colocalization with NucRed or NucBlue, against its respective cytosolic GFP pixel intensity. The ratio of nuclear to cytosolic fluorescence intensity for all GFP-expressing cells was averaged for each islet, in 3-5 islets per experiment.