Expression of calbindin-D_28k in a pancreatic islet β-cell line protects against cytokine-induced apoptosis and necrosis

Cytokines produced by immune system cells that infiltrate pancreatic islets are candidate mediators of islet β-cell destruction in autoimmune (type 1) diabetes mellitus. Because the calcium binding protein, calbindin-D_28k, can prevent apoptotic cell death in different cell types, we investigated the possibility that calbindin-D_28k may prevent cytokine-mediated islet β-cell destruction. Using the expression vector BSRα, rat calbindin-D_28k was stably expressed in the pancreatic islet β-cell line, βTC-3. Calbindin-D_28k expression resulted in increased cell survival in the presence of the cytotoxic combination of the cytokines IL-1β (30 U/ml), TNFα, (10³ U/ml), and interferon y (10³ U/ml). The greatest protection was observed in the βTC-3 cell clone expressing the highest concentration of calbindin-D_28k. Apoptotic cell death was detected by annexin V staining and by the TdT-mediated dUTP-X nick end labeling assay in vector-transfected βTC-3 cells incubated with cytokines (14-15% apoptotic cells). The number of apoptotic cells was significantly decreased in calbindin-D^28k-overexpressing βTC-3 cells incubated with cytokines (5-6% apoptotic cells). To address the mechanism of the antiapoptotic effects of calbindin, studies were done to examine whether calbindin inhibits free radical formation. The stimulatory effects of the cytokines on lipid hydroperoxide, nitric oxide, and peroxynitrite production were significantly decreased in the calbindin -D_28k-expressing βTC-3 cells. Our findings indicate that calbindin-D_28k, by inhibiting free radical formation, can protect against cytokine-mediated apoptosis and destruction of β-cells. These findings suggest that calbindin-D_28k may be an important regulator of cell death that can protect pancreatic islet β-cells from autoimmune destruction in type 1 diabetes.