In type 1 diabetes (T1D) , autoreactive immune cells secrete cytokines, such as tumor necrosis factor-α (TNF-α) , interleukin-1β (IL-1β) , and interferon-γ (IFN-γ) that can initiate β-cell apoptosis. Protein kinase C delta (PKCδ) plays a role in mediating cytokine-induced β-cell apoptosis; however, the exact mechanisms and role in T1D are unknown. We hypothesize that PKCδ mediates cytokine-induced β-cell apoptosis via nuclear translocation and activation of pro-apoptotic signaling. Islets from WT or mice with a β-cell specific knockout of PKCδ (PKCδβ-KO) were cultured for 24hr with cytokines (10ng/ml TNFα, 5ng/ml IL-1β, 100ng/ml IFN-γ) , a caspase-3 inhibitor (Z-DEVD-FMK, 10µg/ml) , or an adenoviral vector for GFP-tagged PKCδ, GFP only, and a PKCδ mutant that cannot be cleaved by caspase-3 (CM-GFP-PKCδ) . Human islets were treated with 10μM PKCδ inhibitor (δV1-1) for 24hr with or without cytokines. Translocation was determined by GFP colocalization with a nuclear stain. Apoptosis was determined by fluorescent microscopy. PKCδ activity was determined with a FRET-based sensor in MIN6 cells. PKCδ’s role in activating pro-apoptotic signaling was determined via western blot. Decreasing PKCδ activity protected against cytokine-induced apoptosis in mouse (p=0.003) and human (p=0.020) islets. PKCδ translocation and activity increased in nuclei of cytokine treated islets (p<0.001, p=0.039) compared to untreated controls. Islets treated with caspase inhibitor (p<0.001) and CM-GFP-PKCδ islets were protected from cytokine-induced death. Western blot analysis demonstrates that PKCδ regulates pro-apoptotic signaling. Inhibiting PKCδ protected islets from cytokine-induced death. The role we identified for PKCδ in mediating pro-apoptotic proteins will provide novel insight to protect against cytokine-induced β-cell death. Our data suggests targeted inhibition of PKCδ has the potential to prevent cytokine-mediated β-cell death in T1D.
J.Collins: None. N.L.Farnsworth: None. R.K.Benninger: n/a.
American Diabetes Association (7-21-JDF-020) ; Juvenile Diabetes Research Foundation (1-FAC-2020-891-A-N)