In type 1 diabetes (T1D) autoreactive T-cells secrete cytokines such as tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), and interferon-γ (IFNγ) that can initiate β-cell apoptosis. Protein kinase C delta (PKCδ) plays a role in mediating cytokine-induced β-cell apoptosis. The exact mechanisms of PKCδ regulation in β-cell death and role in T1D are unknown. In cancer cells PKCδ mediates apoptosis by translocating to the nucleus and being cleaved by caspase 3. We hypothesize that PKCδ mediates cytokine-induced β-cell apoptosis by translocating to the nucleus and cleavage by caspase 3. Islets from WT or mice with a β-cell specific PKCδ knockout were cultured for 24hr with cytokines (10ng/ml TNFα, 5ng/ml IL-1β, 100ng/ml IFNγ), caspase 3 inhibitor (Z-DEVD-FMK, 33µg/ml), or adenovirus to produce GFP-PKCδ, GFP with a constitutively active nuclear localization sequence, and a PKCδ mutant that cannot be cleaved by caspase 3 (CM-GFP-PKCδ). Human islets were treated with 10μM PKCδ inhibitor (δV1-1) with cytokines. Apoptosis was determined by fluorescent live/dead staining. PKCδ translocation was determined by GFP colocalization with a nuclear stain and activity was determined with a FRET-based sensor. Decreasing PKCδ activity protected mouse (p<0.01) and human (p=0.02) islets against cytokine-induced apoptosis. Cytokines induced PKCδ translocation to the nucleus at 3 (p=0.05) and 24hr (p<0.01) and increased nuclear PKCδ activity at 3hr (p=0.04). WT islets with Z-DEVD-FMK and CM-GFP-PKCδ islets were protected from cytokine-induced death. Production of PKCδ with an active nuclear localization sequence did not impact apoptosis. Inhibiting PKCδ protected islets from cytokine-induced β-cell death. Pro-inflammatory cytokines induce nuclear translocation and activity of PKCδ. Nuclear localization of PKCδ alone is insufficient for apoptosis but caspase-3 cleavage is required. Our data suggests that targeted inhibition of PKCδ has the potential to prevent or delay β-cell death in T1D onset.
J. Collins: None. R. K. Benninger: None. N. L. Farnsworth: None.