Abstract
The effect of regucalcin, a regulatory protein in intracellular signaling pathway, on cell death and apoptosis was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in a medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured for 24–72 h in a medium without BS containing either vehicle, tumor necrosis factor‐α (TNF‐α; 0.1 or 1.0 ng/ml of medium), lipopolysaccharide (LPS; 0.1 or 1.0 µg/ml), Bay K 8644 (10^−9^–10^−7^ M), or thapsigargin (10^−9^–10^−7^ M). The number of wild‐type cells was significantly decreased by culture for 42–72 h in the presence of TNF‐α (0.1 or 1.0 ng/ml), LPS (0.1 or 1.0 µg/ml), Bay K 8644 (10^−7^–10^−5^ M), or thapsigargin (10^−8^ or 10^−7^ M). The effect of TNF‐α (0.1 or 1.0 ng/ml), LPS (0.1 or 1.0 µg/ml), Bay K 8644 (10^−7^–10^−6^ M), or thapsigargin (10^−7^ M) in decreasing the number of wild‐type cells cultured for 24–72 h was significantly prevented in transfectants overexpressing regucalcin. Agarose gel electrophoresis showed the presence of low‐molecular‐weight deoxyribonucleic acid (DNA) fragments of adherent wild‐type cells cultured with LPS (1.0 µg/ml), Bay K 8644 (10^−7^ M), or thapsigargin (10^−8^ M) for 24 h, and this DNA fragmentation was significantly suppressed in transfectants. DNA fragmentation in adherent cells was not seen by culture with TNF‐α (1.0 ng/ml). TNF‐α‐induced decrease in the number of wild‐type cells was significantly prevented by culture with caspase‐3 inhibitor (10^−8^ M), while LPS‐ or Bay K 8644‐induced decrease in cell number was significantly prevented by caspase‐3 inhibitor or N ω‐nitro‐L‐arginine methylester (NAME) (10^−5^ M), an inhibitor of nitric oxide (NO) synthase. Thapsigargin‐induced decrease in cell number was not prevented in the presence of two inhibitors. Bcl‐2 and Akt‐1 mRNA levels were significantly increased in transfectants cultured for 24 h as compared with those of wild‐type cells, while Apaf‐1, caspase‐3, or glyceroaldehyde‐3‐phosphate dehydrogenase (G3PDH) mRNA expressions were not significantly changed in transfectants. Culture with TNF‐α (1.0 ng/ml), LPS (1.0 µg/ml), Bay K 8644 (l0^−7^ M), or thapsigargin (10^−8^ M) caused a significant increase in caspase‐3 mRNA levels in wild‐type cells. LPS (1.0 µg/ml) significantly decreased Bcl‐2 mRNA expression in the cells. Their effects on the gene expression of apoptosis‐related proteins were not significantly changed in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death and apoptosis induced by various factors which their action are mediated through many intracellular signaling pathways, and that it modulates the gene expression of apoptosis‐related proteins. J. Cell. Biochem. © 2005 Wiley‐Liss, Inc.