Taeko Nakagawa

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‐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.

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

The effect of regucalcin (RC), a regulatory protein in intracellular signaling pathway, on the gene expression of various mineral ion transport‐related proteins was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing RC. NRK52E cells (wild‐type) and stable RC/pCXN2 transfectant were cultured for 72 h in medium containing 5% bovine serum (BS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured 24–72 h in a medium containing either vehicle, aldosterone (10−8 or 10−7 M), or parathyroid hormone (PTH) (1–34) (10−8 or 10−7 M) without BS. RC was markedly localized in the nucleus of transfectants. Overexpression of RC caused a significant increase in rat outer medullary K+ channel (ROMK) mRNA expression, while it caused a remarkable decrease in L‐type Ca2+ channel and calcium‐sensing receptor (CaR) mRNA expressions. Overexpression of RC did not have an effect on epithelial sodium channel (ENaC), Na, K‐ATPase (alpha‐subunit), Type II Na‐Pi cotransporter (NaPi‐IIa), angiotensinogen, Na+‐Ca2+ exchanger, and glyceroaldehyde‐3‐phosphate dehydrogenase (G3PDH) mRNA expressions. Hormonal effect on gene expression, moreover, was examined. Culture with aldosterone (10−8 or 10−7 M) caused a significant increase in ENaC, Na, K‐ATPase, and ROMK mRNA expressions in the wild‐type cells. Those increases were weakened in the transfectants. Culture with PTH (10−8 or 10−7 M) significantly decreased NaPi‐IIa mRNA expression in the wild‐type cells. This effect was not altered in the transfectants. PTH significantly decreased angiotensinogen mRNA expression in the wild‐type cells and the transfectants, while aldosterone had no effect. Culture with PTH (10−8 or 10−7 M) caused a significant decrease in L‐type Ca2+ channel and CaR mRNA expressions in the wild‐type cells, while the hormone significantly increased Na+‐Ca2+ exchanger mRNA expression. The effects of PTH on L‐type Ca2+ channel, CaR, and Na+‐Ca2+ exchanger mRNA expressions were also seen in the transfectants. This study demonstrates that overexpression of RC caused a remarkable increase in its nuclear localization, and that it has suppressive effects on the gene expression of L‐type Ca2+ channel or CaR, which regulates intracellular Ca2+ signaling, among various regulator proteins for mineral ions in NRK52E cells. J. Cell. Biochem. 99: 1064–1077, 2006.

Hormonal regulation on regucalcin mRNA expression in cloned normal rat kidney proximal tubular epithelial NRK52E cells

Regucalcin is a regulatory protein in cell signaling. This study was undertaken to determine whether regucalcin mRNA expresses in the cloned normal rat kidney proximal tubular epithelial NRK52E cells and its expression regulates due to hormones and cell signaling‐related factors. Cells with subconfluency were cultured for 24, 48, or 72 h in a Dulbeccos modified Eagle medium supplemented with non‐essential amino acid without bovine serum (BS). The result of Western blot analysis showed that regucalcin protein was present in the NRK52E cells. The expression of regucalcin mRNA in the cells was determined using reverse transcription‐polymerase chain reaction (RT‐PCR). Regucalcin mRNA expression in the NRK52E cells was significantly increased by culture with parathyroid hormone (PTH, 10−8 or 10−7 M), aldosterone (10−8 or 10−7 M), or dexamethasone (10−8 M). The presence of 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3, 10−8 or 10−7 M) or calcitonin (10−9 or 10−8 M) did not have a significant effect on regucalcin mRNA levels in the cells. Culture with dibutyryl cyclic AMP (DcAMP, 10−5 or 10−4 M) or phorbol 12‐myristate 13‐acetate (PMA, 10−6 M), an activator of protein kinase C, caused a significant increase in regucalcin mRNA expression. The presence of staurosporine (10−8 M) caused a significant decrease in regucalcin mRNA expression. Dibucaine (10−7 M), PD98059 (10−7 M), or vanadate (10−6 or 10−5 M) did not have an effect on regucalcin mRNA levels. The present study demonstrates that regucalcin mRNA and its protein are expressed in the cloned normal rat kidney proximal tubular epithelial NRK52E cells, and that the expression is enhanced by hormones which regulate ion transport in the proximal tubule.

Regucalcin increases Ca2+-ATPase activity in the mitochondria of brain tissues of normal and transgenic rats.

The role of regucalcin, which is a regulatory protein in intracellular signaling, in the regulation of Ca2+‐ATPase activity in the mitochondria of brain tissues was investigated. The addition of regucalcin (10−10 to 10−8 M), which is a physiologic concentration in rat brain tissues, into the enzyme reaction mixture containing 25 µM calcium chloride caused a significant increase in Ca2+‐ATPase activity, while it did not significantly change in Mg2+‐ATPase activity. The effect of regucalcin (10−9 M) in increasing mitochondrial Ca2+‐ATPase activity was completely inhibited in the presence of ruthenium red (10−7 M) or lanthanum chloride (10−7 M), both of which are inhibitors of mitochondrial uniporter activity. Whether the effect of regucalcin is modulated in the presence of calmodulin or dibutyryl cyclic AMP (DcAMP) was examined. The effect of regucalcin (10−9 M) in increasing Ca2+‐ATPase activity was not significantly enhanced in the presence of calmodulin (2.5 µg/ml) which significantly increased the enzyme activity. DcAMP (10−6 to 10−4 M) did not have a significant effect on Ca2+‐ATPase activity. The effect of regucalcin (10−9 M) in increasing Ca2+‐ATPase activity was not seen in the presence of DcAMP (10−4 M). Regucalcin levels were significantly increased in the brain tissues or the mitochondria obtained from regucalcin transgenic (RC TG) rats. The mitochondrial Ca2+‐ATPase activity was significantly increased in RC TG rats as compared with that of wild‐type rats. This study demonstrates that regucalcin has a role in the regulation of Ca2+‐ATPase activity in the brain mitochondria of rats. J. Cell. Biochem. 104: 795–804, 2008.

Overexpression of regucalcin suppresses cell response for tumor necrosis factor-α or transforming growth factor-β1 in cloned normal rat kidney proximal tubular epithelial NRK52E cells

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.