Exposure of pancreatic β-cells to cytokines, such as interleukin-1β (IL-1β), is thought to contribute to the β-cell apoptosis that underlies the onset of type 1 diabetes. One important event triggered by IL-1β is induction of nitric oxide synthase (iNOS), an enzyme that catalyzes intracellular generation of the cytotoxic free radical NO. We recently described a novel requirement for the protein kinase C (PKC) isozyme PKCδ in this process. Our current aim, therefore, was to assess whether PKCδ also plays a role in β-cell apoptosis. As assessed by either annexin V staining or DNA fragmentation, IL-1β caused INS-1 cells to undergo apoptosis. This was completely blocked by adenoviral overexpression of a dominant-negative, kinase-dead (KD) PKCδ mutant. The corresponding PKCα virus was without effect. However, apoptosis caused by the cytotoxic agent streptozotocin (STZ), which acts independent of iNOS, was also inhibited by overexpression of PKCδKD. STZ was additionally shown to activate the proteolytic enzyme caspase-3, a key biochemical effector of end-stage apoptosis. Moreover, STZ caused a caspase-dependent cleavage of PKCδ, thereby releasing a COOH-terminal fragment corresponding to the kinase catalytic domain. Thus, proteolytic activation of PKCδ seems to be important in the distal apoptotic pathway induced by STZ. That IL-1β also activated caspase-3 and promoted PKCδ cleavage suggests that this distal pathway also contributes in the apoptotic response to the cytokine. These data therefore support a dual role for PKCδ in IL-1β-mediated cell death: it is required for efficient NO generation through regulation of iNOS levels but also contributes to apoptotic pathways downstream of caspase activation.
Address correspondence and reprint requests to Dr. Trevor Biden, Cell Signalling Group, Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst, Sydney 2010, Australia. E-mail.
Received for publication 18 December 2000 and accepted in revised form 24 October 2001.
CAD, caspase-activated DNase; DNA-PK, DNA-dependent protein kinase; FACS, fluorescence-activated cell sorting; IL-1β, interleukin-1β; iNOS, inducible nitric oxide synthase; KD, kinase dead; NO, nitric oxide; PBS, phosphate-buffered saline; pfu, plaque-forming units; PI, propidium iodide; PKC, protein kinase C; PKCδCF, PKCδ constitutively active fragment; PS, phosphatidylserine; STZ, streptozotocin; TNF-α, tumor necrosis factor-α; WT, wild-type.