SFEBES2014 Oral Communications Endocrine regulation of cell behaviour (6 abstracts)
1Keele University, Stoke-on-Trent, UK; 2University of Ulster, Londonderry, UK.
Abstract
Background: β-cell apoptosis is a cardinal feature of type 1 diabetes. A20 is an important negative regulator of inflammation and apoptosis and protects against β-cell death in response to inflammatory cytokines, and β-cell rejection following islet transplantation1. Preliminary experiments showed rapid induction of A20 (1560 min) in β-cells in response to treatment with TNFα. Experimental data suggests that TNF-related apoptosis-inducing ligand (TRAIL), an immune system modulator protein, is important in the pathogenesis of type 1 diabetes2. Recent evidence from glioblastoma cells now suggests that A20 regulates TRAIL-mediated apoptosis through inhibition of caspase-83. However, this relationship has not been examined in the β-cell.
Methods: The BRINBD11 β-cell line was used for all experiments, which included untreated cells as a control group, cells treated with a commercially available A20 siRNA, and cells treated with recombinant A20 (100 ng/ml). A20 knockdown was achieved using siRNA against A20 (Qiagen). All cells were subsequently treated with recombinant TRAIL (100 ng/ml) for 0, 1, 2, 4 and 24 h. The induction of A20 and cleaved caspase-8 was assessed over time by qPCR, while the effect on cellular viability was measured by MTT assay.
Results: We confirmed the induction of A20 in response to TNFα in BRINBD11 cells initially, and subsequently showed that TRAIL enhanced A20 mRNA expression in a similar manner. Peak TRAIL-induced A20 expression was observed after 1 h (P<0.01). In A20-silenced cells, TRAIL treatment provoked significant cell death (P<0.001), which was reversed following addition of recombinant A20. A significant increase in caspase-8 mRNA expression was observed in A20 silenced cells exposed to TRAIL (P<0.001, 4 and 24 h).
Conclusion: The data suggest that A20 may offer protection against TRAIL induced apoptosis in pancreatic β-cells.
References:
1. Liuwantara et al. Diabetes 2006.
2. Bernardi et al. Curr Pharm Des 2012.
3. Bellail et al. Cancer Discov 2012.