Expression of calbindin-D-28k in a pancreatic islet beta-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 (typ e 1) diabetes mellitus. Because the calcium binding protein, calbindin-D-28 k, can prevent apoptotic cell death in different cell types, we investigate d the possibility that calbindin-D-28k may prevent cytokine-mediated islet beta -cell destruction. Using the expression vector BSR alpha, rat calbindi n-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 pro tection was observed in the beta TC-3 cell clone expressing the highest con centration 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 vecto r-transfected beta TC-3 cells incubated with cytokines (14-15% apoptotic ce lls). The number of apoptotic cells was significantly decreased in calbindi n-D-28k, overexpressing beta TC-3 cells incubated with cytokines (5-6% apop totic cells). To address the mechanism of the antiapoptotic effects of calb indin, 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 i n the calbindin-D-28k-expressing beta TC-3 cells. Our findings indicate tha t calbindin-D-28k, by inhibiting free radical formation, can protect agains t cytokine-mediated apoptosis and destruction of beta -cells. These finding s suggest that calbindin-D-28k may be an important regulator of cell death that can protect pancreatic islet beta -cells from autoimmune destruction i n type 1 diabetes.