Abstract
The role of regucalcin, which is a regulatory protein in intracellular signaling pathway, in the regulation of cell death was investigated by using the cloned rat hepatoma H4‐II‐E cells overexpressing regucalcin. The hepatoma cells (wild‐type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. The proliferation of the cells was significantly suppressed in transfectants cultured for 72 h, as shown previously (Tsurusaki and Yamaguchi [2003]: J Cell Biochem 90:619–626). After culture for 72 h, cells were further cultured for 24–72 h in medium without FBS containing either vehicle, tumor necrosis factor‐α (TNF‐α; 0.1, 1, or 10 ng/ml) or thapsigargin (10^−7^–10^−5^ M). The number of wild‐type cells was significantly decreased by culture for 42 or 72 h in the presence of TNF‐α (0.1, 1, or 10 ng/ml) or thapsigargin (10^−7^–10^−5^ M). The effect of TNF‐α (0.1 or 1 ng/ml) or thapsigargin (10^−7^ or 10^−6^ M) in decreasing the number of hepatoma cells was significantly prevented in transfectants overexpressing regucalcin. The presence of TNF‐α (10 ng/ml) or thapsigargin (10^−5^ M) caused a significant decrease in cell number of transfectants. Ca^2+^/calmodulin‐dependent nitric oxide (NO) synthase activity in wild‐type cells was significantly increased by culture with TNF‐α (10 ng/ml) for 48 or 72 h. This increase was significantly prevented in transfectants. Culture with thapsigargin (10^−5^ M) caused a significant increase in Ca^2+^/calmodulin‐dependent NO synthase activity in wild‐type cells or transfectants. TNF‐α‐induced decrease in the number of wild‐type cells was significantly prevented by culture with __N__ω‐nitro‐l‐arginine (10^−4^ M), an inhibitor of caspase. Agarose gel electrophoresis showed the presence of low‐molecular‐weight deoxyribonucleic acid (DNA) fragments of adherent wild‐type cells cultured with thapsigargin (10^−6^ M), and this DNA fragmentation was not suppressed by culture with caspase inhibitor. Thapsigargin‐induced DNA fragmentation was significantly suppressed in transfectants cultured with or without caspase inhibitor. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death induced by TNF‐α or thapsigargin. © 2004 Wiley‐Liss, Inc.