294-OR: RIPK3-Mediated Necroptosis Is an Alternative Form of TNFa-Induced ß-Cell Death

Abstract

Necroptosis is an inflammatory and immunogenic form of lytic cell death that can occur downstream of TNFα signaling. Receptor interacting protein kinase 3 (RIPK3) mediates TNFα-induced necroptosis when caspase activity is repressed, but this pathway has not been examined in insulin-producing β-cells. As TNFα contributes to β-cell death in the setting of diabetes and since we found RIPK3 is expressed in INS-1 β-cells and mouse islets, we hypothesized that β-cells are susceptible to necroptosis. To test this hypothesis, INS-1 cells were treated with TNFα (40 ng/ml) in the absence or presence of a synthetic pan-caspase inhibitor (zVAD, 50 μM) for 24 h, cell death was monitored in real-time, and caspase activity was determined. TNFα increased INS-1 cell death (1.63 ± 0.13 vs. 2.44 ± 0.13 fold, n=7, p<0.001) and caspase activity, consistent with apoptosis. When caspases were inhibited with zVAD, TNFα no longer increased caspase activity, but induced a similar degree of cell death (1.63 ± .013 vs. 2.82 ± 0.23 fold, n=7, p<0.001), suggesting an alternate mechanism under these conditions. Moreover, when TNFα-induced cell death was amplified by the SMAC/DIABLO mimetic BV6 (5 μM), we again observed similar levels of cell death with or without zVAD. To further investigate the mechanism of cell death under caspase inhibition, we used the RIPK3 inhibitor GSK ‘872 (5 μM). GSK ‘872 had no effect on TNFα+BV6-induced cell death when caspases were activated (3.35 ± 0.61 vs. 3.48 ± 1.21 fold, n=3, p=0.93), but reduced cell death when caspases were inhibited (4.69 ± 0.57 vs. 1.81 ± 0.57 fold, n=3, p=0.04). These data indicate that TNFα induces β-cell death independent of caspase activity level. However, the mechanism by which TNFα elicits β-cell death does depend on caspase activity, with apoptosis occurring when caspases are activated, and RIPK3-mediated necroptosis occurring when caspases are inhibited. Additional studies are required to understand the factors that may promote β-cell necroptosis in the setting of human diabetes. Disclosure C. J. Contreras: None. L. Lin: None. M. F. Hogan: None. A. Oberst: None. S. E. Kahn: Advisory Panel; Self; Bayer AG, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Merck & Co., Inc., Novo Nordisk, Pfizer Inc. A. T. Templin: None. Funding U.S. Department of Veterans Affairs (IK2BX004659, I01BX001060)