Abstract
The regulatory role of regucalcin on cell responses for tumor necrosis factor‐α (TNF‐α) or transforming growth factor‐β1 (TGF‐β1) was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin (RC)/pCXN2‐transfected cells (transfectant) were cultured for 72 h in a medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture, cells were further cultured for 24–72 h in medium without BS containing either vehicle, TNF‐α (0.1 or 1.0 ng/ml of medium), or TGF‐β1 (1.0 or 5.0 ng/ml). Culture with TNF‐α or TGF‐β1 caused a significant decrease in the number of wild‐type cells. This decrease 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 TNF‐α (1.0 ng/ml) or TGF‐β1 (5.0 ng/ml). This DNA fragmentation was significantly suppressed in transfectants. TNF‐α‐ or TGF‐β1‐induced cell death was significantly prevented in culture with caspase‐3 inhibitor (10^−8^ M). Nitric oxide (NO) synthase activity in wild‐type cells was significantly increased by addition of calcium chloride (10 µM) and calmodulin (5 µg/ml) into the enzyme reaction mixture. This increase was significantly suppressed in transfectants. Culture with TNF‐α caused a significant increase in NO synthase activity in wild‐type cells. The effect of TNF‐α was not seen in transfectants. Culture with TGF‐β1 did not cause a significant increase in NO synthase activity in wild‐type cells and transfectants. Culture with TNF‐α or TGF‐β1 caused a remarkable increase in α‐smooth muscle actin in wild‐type cells. This increase was significantly prevented in transfectants. The expression of Smad 2 or NF‐κB mRNAs was significantly increased in transfectants as compared with that of wild‐type cells. Smad 3 or glyceroaldehyde‐3‐phosphate dehydrogenase (G3PDH) mRNA expression was not significantly changed in transfectants. NF‐κB mRNA expression in wild‐type cells was significantly increased with culture of TNF‐α. Smad 2 mRNA expression was significantly enhanced in wild‐type cells cultured with TGF‐β1. These effects of TNF‐α or TGF‐β1 were not significantly enhanced in transfectants. This study demonstrates that overexpression of regucalcin has suppressive effects on cell responses which are mediated through intracellular signaling pathways of TNF‐α or TGF‐β1 in kidney NRK52E cells. J. Cell. Biochem. 100: 1178–1190, 2007. © 2006 Wiley‐Liss, Inc.