Abstract
The effect of regucalcin, a regulatory protein in intracellular signaling pathway, on 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. After culture for 72 h, cells were further cultured for 12–72 h in medium without FBS containing either vehicle or lipopolysaccharide (LPS; 0.1 or 1.0 μg/ml). The number of wild‐type cells was significantly decreased by culture for 24 or 48 h in the presence of LPS (0.1 or 1.0 μg/ml). The effect of LPS (0.1 or 1.0 μg/ml) in decreasing the number of hepatoma cells was significantly prevented in transfectants overexpressing regucalcin. However, the culture with LPS (0.1 or 1.0 μg/ml) for 72 h caused a significant decrease in cell number of transfectants. Ca^2+^/calmodulin‐dependent nitric oxide (NO) synthase activity was significantly decreased by culture with LPS (1.0 μg/ml) for 24–72 h of wild‐type cells. This decrease was significantly prevented in transfectants. LPS (0.1 or 1.0 μg/ml)‐induced decrease in the number of wild‐type cells was significantly prevented by culture with caspase‐3 inhibitor (10^−8^ M). Moreover, the number of wild‐type cells was significantly decreased by culture with PD 98059 (10^−6^ M), dibucaine (10^−6^ M), or staurosporine (10^−6^ M), which is an inhibitor of various protein kinases. The effect of PD 98059 or dibucaine on the number of wild‐type cells was not observed in transfectants, although the effect of staurosporine was seen in transfectants. Culture with Bay K 8644 (2.5 × 10^−6^ M), an agonist of Ca^2+^ entry in cells, caused a significant decrease in the number of wild‐type cells. Such an effect was not seen in transfectants. The presence of LPS did not significantly decrease the number of wild‐type cells in the presence of Bay K 8644. Agarose gel electrophoresis showed the presence of low‐molecular‐weight deoxyribonucleic acid (DNA) fragments of adherent wild‐type cells cultured with Bay K 8644, and this DNA fragmentation was significantly prevented in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death induced by LPS or various intracellular signaling‐related factors. © 2004 Wiley‐Liss, Inc.