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 (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 BSRalpha, rat calbindin-D(28k) was stably expressed in the pancreatic islet beta-cell line, betaTC-3. Calbindin-D(28k) expression resulted in increased cell survival in the presence of the cytotoxic combination of the cytokines IL-1beta (30 U/ml), TNFalpha (10(3) U/ml), and interferon gamma (10(3) U/ml). The greatest protection was observed in the betaTC-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 betaTC-3 cells incubated with cytokines (14-15% apoptotic cells). The number of apoptotic cells was significantly decreased in calbindin-D(28k)-overexpressing betaTC-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 betaTC-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.
Rabinovitch
A
A
Department of Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2S2. alex.rabinovitch@ualberta.ca
Suarez-Pinzon
W L
WL
Sooy
K
K
Strynadka
K
K
Christakos
S
S
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D064026:Calbindins; D016207:Cytokines / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D004338:Drug Combinations; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D007515:Islets of Langerhans / Q000187:drug effects* / Q000473:pathology / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics; D009336:Necrosis; D064030:S100 Calcium Binding Protein G / Q000235:genetics / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D064026:Calbindins; D016207:Cytokines; D004338:Drug Combinations; D007375:Interleukin-1; D064030:S100 Calcium Binding Protein G; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma