Overexpression of regucalcin suppresses cell response for tumor necrosis factor-alpha or transforming growth factor-beta1 in cloned normal rat kidney proximal tubular epithelial NRK52E cells.

The regulatory role of regucalcin on cell responses for tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1) 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-alpha (0.1 or 1.0 ng/ml of medium), or TGF-beta1 (1.0 or 5.0 ng/ml). Culture with TNF-alpha or TGF-beta1 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-alpha (1.0 ng/ml) or TGF-beta1 (5.0 ng/ml). This DNA fragmentation was significantly suppressed in transfectants. TNF-alpha- or TGF-beta1-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 microM) and calmodulin (5 microg/ml) into the enzyme reaction mixture. This increase was significantly suppressed in transfectants. Culture with TNF-alpha caused a significant increase in NO synthase activity in wild-type cells. The effect of TNF-alpha was not seen in transfectants. Culture with TGF-beta1 did not cause a significant increase in NO synthase activity in wild-type cells and transfectants. Culture with TNF-alpha or TGF-beta1 caused a remarkable increase in alpha-smooth muscle actin in wild-type cells. This increase was significantly prevented in transfectants. The expression of Smad 2 or NF-kappaB 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-kappaB mRNA expression in wild-type cells was significantly increased with culture of TNF-alpha. Smad 2 mRNA expression was significantly enhanced in wild-type cells cultured with TGF-beta1. These effects of TNF-alpha or TGF-beta1 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-alpha or TGF-beta1 in kidney NRK52E cells.

Nakagawa T ，Yamaguchi M 《JOURNAL OF CELLULAR BIOCHEMISTRY》

Overexpression of regucalcin suppresses apoptotic cell death in cloned normal rat kidney proximal tubular epithelial NRK52E cells: change in apoptosis-related gene expression.

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-alpha (TNF-alpha; 0.1 or 1.0 ng/ml of medium), lipopolysaccharide (LPS; 0.1 or 1.0 microg/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-alpha (0.1 or 1.0 ng/ml), LPS (0.1 or 1.0 microg/ml), Bay K 8644 (10(-7)-10(-5) M), or thapsigargin (10(-8) or 10(-7) M). The effect of TNF-alpha (0.1 or 1.0 ng/ml), LPS (0.1 or 1.0 microg/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 microg/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-alpha (1.0 ng/ml). TNF-alpha-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 omega-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-alpha (1.0 ng/ml), LPS (1.0 microg/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 microg/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.

Nakagawa T ，Yamaguchi M 《JOURNAL OF CELLULAR BIOCHEMISTRY》

Overexpression of regucalcin suppresses cell death in cloned rat hepatoma H4-II-E cells induced by tumor necrosis factor-alpha or thapsigargin.

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-alpha (TNF-alpha; 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-alpha (0.1, 1, or 10 ng/ml) or thapsigargin (10(-7)-10(-5) M). The effect of TNF-alpha (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-alpha (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-alpha (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-alpha-induced decrease in the number of wild-type cells was significantly prevented by culture with N omega-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-alpha or thapsigargin.

Izumi T ，Yamaguchi M 《JOURNAL OF CELLULAR BIOCHEMISTRY》

Nuclear localization of regucalcin is enhanced in culture with protein kinase C activation in cloned normal rat kidney proximal tubular epithelial NRK52E cells.

Nakagawa T ，Yamaguchi M 《INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE》

Overexpression of regucalcin enhances its nuclear localization and suppresses L-type Ca2+ channel and calcium-sensing receptor mRNA expressions in cloned normal rat kidney proximal tubular epithelial NRK52E cells.

Nakagawa T ，Yamaguchi M 《JOURNAL OF CELLULAR BIOCHEMISTRY》