Expression of calbindin-D28k 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 beta -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 beta -cell destruction. Using the expression vector BSR alpha, rat calbindin-D-28k was stably expressed in the pancreatic islet beta -cell line, beta TC-3. Calbindin-D-28k expression resulted in increased cell survival in the presence of the cytotoxic combination of the cytokines IL-1 beta (30 U/ml), TNF alpha (10(3)U/ml), and interferon gamma (10(3)U/ml). The greatest protection was observed in the beta TC-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 beta TC-3 cells incubated with cytokines (14-15% apoptotic cells). The number of apoptotic cells was significantly decreased in calbindin-D-28k, overexpressing beta TC-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 beta TC-3 cells. Our findings indicate that calbindin-D-28k, by inhibiting free radical formation, can protect against cytokine-mediated apoptosis and destruction of beta -cells. These findings suggest that calbindin-D-28k may be an important regulator of cell death that can protect pancreatic islet beta -cells from autoimmune destruction in type 1 diabetes.