Phytocomponent p-hydroxycinnamic acid stimulates mineralization in osteoblastic MC3T3-E1 cells.

Phytocomponent p-hydroxycinnamic acid (HCA) has been shown to have stimulatory effects on bone calcification and inhibitory effects on bone resorption in rat femoral tissues in vitro. Whether HCA has a stimulatory effect on mineralization in osteoblastic cells is unknown. This study was undertaken to determine the effect of HCA on mineralization in osteoblastic MC3T3-E1 cells in vitro. Cells were cultured for 72 h in a minimum essential medium (alpha-MEM) containing 10% fetal bovine serum (FBS), and the cells with subconfluency were changed to a medium containing either vehicle or HCA (10(-7)-10(-5) M) without FBS. Culture with HCA (10(-7)-10(-5) M) did not have a significant effect on cell proliferation and cell death. Deoxyribonucleic acid (DNA) content in osteoblastic cells was significantly increased after culture with HCA (10(-6) or 10(-5) M) for 48 or 72 h. Alkaline phosphatase activity in osteoblastic cells was significantly increased after culture with HCA (10(-7)-10(-5) M) for 24, 48, or 72 h. The results with Alizarin red staining for calcium showed that mineralization was significantly stimulated after culture with HCA (10(-8)-10(-5) M) for 7, 14, or 21 days. This study demonstrates that HCA has stimulatory effects on mineralization in osteoblastic MC3T3-E1 cells.

Hormonal regulation of regucalcin mRNA expression in osteoblastic MC3T3-E1 cells.

The effect of various hormones on regucalcin mRNA expression in osteoblastic MC3T3-E1 cells in vitro was investigated. Cells with subconfluency were cultured for 24 or 48 h in a medium containing either vehicle or various hormones without fetal bovine serum. Regucalcin mRNA expression was significantly increased after culture with parathyroid hormone (synthetic human PTH; 10(-7) M), insulin-like growth factor-I (IGF-I; 10(-8) M), or 17beta-estradiol (10(-10) or 10(-9) M) for 48 h. Culture with 1,25-dihydroxyvitamin D3 (10(-7) M) for 48 h caused a significant decrease in regucalcin mRNA expression. Regucalcin mRNA expression was significantly decreased after culture with tumor necrosis factor-alpha (1 or 10 ng/ml of medium) for 24 or 48 h. The effect of PTH or IGF-I in increasing regucalcin mRNA expression was not seen in the presence of staurosporine (10(-8) M), an inhibitor of protein kinase C, or PD98059 (10(-7) M), an inhibitor of mitosis-activated protein kinase (MAP kinase), respectively, suggesting that regucalcin mRNA expression is enhanced through intracellular signaling factors. This study demonstrated that regucalcin mRNA expression in osteoblastic MC3T3-E1 cells is regulated by various hormones.

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

In this study we investigated whether the nuclear localization of regucalcin in cloned normal rat kidney tubular epithelial NRK52E cells is regulated after culture with hormonal signaling factors. Stable regucalcin/pCXN2 transfectants with subconfluent monolayers were further cultured for 24 or 48 h in a serum-free medium containing either vehicle, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta1 (TGF-beta1), parathyroid hormone (PTH), phorbol 12-myristate 13-acetate (PMA), or other factors. Culture with TNF-alpha (1.0 ng/ml of medium) or TGF-beta1 (5.0 ng/ml) for 48 h caused a significant decrease in regucalcin mRNA levels in NRK52E cells (wild-type), while regucalcin mRNA levels were markedly increased in the presence of PMA (10(-6) M), an activator of protein kinase C, in wild-type cells. Immunocytochemical observation showed that HA-regucalcin was markedly localized in the nucleus of HA-regucalcin/ phCMV2-transfected cells. The nuclear localization was enhanced in culture with BS (5%), PTH (10(-7) M), Bay K 8644 (2.5x10(-6) M), or PMA (10(-6) M) for 24 or 48 h. Culture with staurosporine, an inhibitor of protein kinase C, caused a remarkable decrease in the localization of HA-regucalcin in the nucleus of HA-RGPR-p117/phCMV2-transfected cells with PMA. Culture with PMA (10(-6) M) for 24 or 48 h caused a remarkable increase in nuclear regucalcin protein levels. The effect of PMA in increasing nuclear regucalcin levels was completely absent in culture with staurosporine (10(-8) M). The nuclear localization of regucalcin in the stable regucalcin/pCXN2-transfected cells (transfectant) increased markedly as compared with that of wild-type cells, whereas the increase was less evident in the transfectants cultured with staurosporine. This study demonstrated that regucalcin localizes in the nucleus of cloned normal rat kidney proximal tubular epithelial NRK52E cells, and that its nuclear localization is enhanced through an intracellular signaling process which involves protein kinase C.

Effect of zinc on gene expression in osteoblastic MC3T3-E1 cells: enhancement of Runx2, OPG, and regucalcin mRNA expressions.

The effect of zinc sulfate on the mRNA expressions in Runx2, osteocalcin, alpha1(I) collagen, insulin-like growth factor-I (IGF-I), transforming growth factor-beta1 (TGF-beta1), osteoprotegerin (OPG), regucalcin, zinc transporter 1 (ZIP1), or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) in osteoblastic MC3T3-E1 cells in vitro was investigated. Cells with subconfluency were cultured for 48 h in a medium containing either vehicle or zinc sulfate (10(-6)-10(-4) M) without fetal bovine serum. Culture with zinc sulfate (10(-5) M) caused a significant increase in Runx2, OPG, or regucalcin mRNA expressions in the cells, while it did not have a significant effect on osteocalcin, alpha1(I) collagen, IGF-I, TGF-beta1, ZIP1, or G3PDH mRNA expressions. The effect of zinc sulfate (10(-4) M) in increasing Runx2 mRNA expression was seen at 24-72 h after culture. A significant increase in OPG mRNA expression was observed at 24 or 48 h after culture. Regucalcin mRNA expression was significantly increased at 48 or 72 h after culture with zinc sulfate (10(-4) M). The stimulatory effects of zinc sulfate on Runx2, OPG, or regucalcin mRNAs were significantly prevented in the presence of cycloheximide (10(-7) M), an inhibitor of protein synthesis, or 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (10(-6) M), an inhibitor of transcription activity. Culture with beta-alanyl-L-histidinato zinc (10(-5) M) caused a significant increase in Runx2 or regucalcin mRNA expressions, while zinc acexamate (10(-5) M) did not have a significant effect on Runx2, OPG, ZIP1, or regucalcin mRNA expressions. This study demonstrates that zinc sulfate has a role in the enhancement of Runx2, OPG, or regucalcin mRNA expression in osteoblastic cells in vitro, suggesting its role in the regulation of gene expression in the cells.

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 Ca(2+)-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 microM calcium chloride caused a significant increase in Ca(2+)-ATPase activity, while it did not significantly change in Mg(2+)-ATPase activity. The effect of regucalcin (10(-9) M) in increasing mitochondrial Ca(2+)-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 Ca(2+)-ATPase activity was not significantly enhanced in the presence of calmodulin (2.5 microg/ml) which significantly increased the enzyme activity. DcAMP (10(-6) to 10(-4) M) did not have a significant effect on Ca(2+)-ATPase activity. The effect of regucalcin (10(-9) M) in increasing Ca(2+)-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 Ca(2+)-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 Ca(2+)-ATPase activity in the brain mitochondria of rats.

Oral administration of phytocomponent p-hydroxycinnamic acid prevents bone loss in ovariectomized rats.

The preventive effect of phytocomponent p-hydroxycinnamic acid (HCA) on ovariectomy (OVX)-induced bone loss was investigated. HCA (250 or 500 microg/100 g body weight) was orally administered once daily for 30 days to OVX rats. The analysis using a peripheral quantitative computed tomography (pQCT) showed that OVX caused bone loss in the femoral-metaphyseal tissues. This change was significantly restored after the administration of HCA (250 or 500 microg/100 g body weight) to OVX rats. Mineral content, mineral density, and polar strength strain index in the femoral-metaphyseal tissues were significantly decreased in OVX rats. These decreases were significantly restored after the administration of HCA (500 microg/100 g) to OVX rats. Moreover, OVX caused a significant decrease in calcium content or alkaline phosphatase activity in the femoral-diaphyseal and -metaphyseal tissues. These decreases were significantly restored after the administration of HCA (250 or 500 microg/100 g) to OVX rats. Deoxyribonucleic acid (DNA) content in the diaphyseal or metaphyseal tissues was significantly increased in OVX rats. These increases were significantly restored after oral administration of HCA (500 microg/100 g). This study demonstrates that HCA has preventive effects on OVX-induced bone loss of rats in vivo.

Overexpression of RGPR-p117 suppresses apoptotic cell death and its related gene expression in cloned normal rat kidney proximal tubular epithelial NRK52E cells.

The novel protein RGPR-p117 was discovered as a regucalcin gene promoter region-related protein that binds to the TTGGC motif using a yeast one-hybrid system. The role of RGPR-p117 in cell function has not been fully clarified. This study was undertaken to determine whether overexpression of RGPR-p117 regulates various types of signaling factor-induced apoptotic cell death in the cloned normal rat kidney proximal tubular epithelial NRK52E cells. NRK52E cells (wild-type) or stable RGPR-p117/phCMV2-transfected cells (transfectant) were cultured in Dulbecco's modified Eagle's medium containing 5% bovine serum (BS). NRK52E cells with subconfluent monolayers were cultured for 24-72 h in a medium without BS. The presence of tumor necrosis factor-alpha (TNF-alpha; 1.0 or 10 ng/ml of medium), lipopolysaccharide (LPS; 0.1 or 1.0 microg/ml), Bay K 8644 (10(-6) or 10(-5) M), or thapsigargin (10(-8) or 10(-7) M) caused a significant decrease in the number of NRK52E wild-type cells or phCMV2-transfected (mock-type) cells. The effect of TNF-alpha, LPS, Bay K 8644, or thapsigargin in decreasing cell number was significantly suppressed in the presence of the caspase-3 inhibitor (10(-8) M) in wild-type cells cultured for 48 h. The effect of TNF-alpha, LPS, or Bay K 8644 in decreasing cell number was significantly inhibited in the transfectants, while the effect of thapsigargin on cell death was not inhibited in the transfectants. Culture with TNF-alpha or LPS caused DNA fragmentation in wild-type cells. These effects were significantly suppressed in the transfectants. The result of reverse transcription-polymerase chain reaction analysis using specific primers for the genes of apoptotic cell death-related proteins showed that IAP-1, FADD, caspase-8, caspase-9, and caspase-3 mRNA levels were significantly decreased in the transfectants, while Akt-1, Bid, Apaf-1, and glyceroaldehyde-3-phosphate dehydrogenase mRNA levels were not significantly altered in the transfectants. Culture with TNF-alpha, LPS, Bay K 8644, or thapsigargin caused a significant increase in Apaf-1 or caspase-3 mRNA levels. Such an effect was not seen in the transfectants. This study demonstrates that overexpression of RGPR-p117 has a suppressive effect on cell death and apoptosis induced by TNF-alpha, LPS, or Bay K 8644 whose actions are mediated through intracellular signaling pathways. This study also demonstrates that RGPR-p117 regulates the gene expression of apoptosis-related proteins.

Change in lipid components in the adipose and liver tissues of regucalcin transgenic rats with increasing age: suppression of leptin and adiponectin gene expression.

Regucalcin plays a multifunctional role as a regulatory protein in intracellular signaling pathway in many cell types. Regucalcin transgenic (TG) rats have been shown to experience hyperlipidemia with increasing age. This study was undertaken to determine whether lipid components in the adipose and liver tissues are changed in regucalcin TG rats in vivo. Female regucalcin TG rats were used at 7 or 50 weeks of age. Serum triglyceride or HDL-cholesterol concentrations were significantly increased in 7-week-old regucalcin TG rats as compared with those in 7-week-old normal rats. Serum triglyceride, total cholesterol, HDL-cholesterol, or free fatty acid concentrations were significantly increased in 50-week-old regucalcin TG rats. Meanwhile, triglyceride content in the adipose tissues was significantly increased in 50-week-old regucalcin TG rats,while the free fatty acid content was not significantly changed. Triglyceride, total cholesterol, or free fatty acid content in the liver tissues was significantly decreased in 50-week-old regucalcin TG rats. Liver glycogen content was significantly decreased in 7- or 50-week-old regucalcin TG rats. In addition, regucalcin mRNA and its protein levels were seen in the adipose tissues of normal rats. Those levels were not significantly changed in regucalcin TG rats at 50 weeks of age. Leptin mRNA expression in the adipose or liver tissues was significantly decreased in 50-week-old regucalcin TG rats. Adiponectin mRNA levels were not significantly changed in the adipose tissues of 50-week-old regucalcin TG rats, while the levels were significantly decreased in the liver tissues. This study demonstrates that the disorder of lipid metabolism in the adipose and liver tissues is induced in regucalcin TG rats with aging, and that the gene expression of leptin or adiponectin is suppressed in TG rats.

Suppression of regucalcin mRNA expression in the hearts of rats administered with free radical compound: the administration-induced death is accelerated in regucalcin transgenic rats.

The expression of regucalcin, a regulatory protein in the intracellular signaling system, in the hearts of rats was investigated. Regucalcin expression was examined using reverse transcription-polymerase chain reaction and Western blot analysis. Regucalcin mRNA and its protein levels in the hearts of male and female rats were significantly decreased with increasing age (50 weeks old) as compared with that of 5-week-old rats. The effect of 1,1-diphenyl-2-picryl-hydrazyl (DPPH), a compound that produces free radical, on regucalcin mRNA expression in the hearts of female rats (5 weeks old) was examined. Heart regucalcin mRNA levels were significantly decreased at 60 or 180 min after a single intraperitoneal administration of DPPH (0.5 mg /100 g body weight), suggesting that free radical stress has a suppressive effect on the gene expression. Normal (wild) female rats died at approximately 300 min after a single intraperitoneal administration of DPPH (0.5 mg/100 g), while regucalcin transgenic (TG) female rats died at approximately 150 min after the administration. Heart regucalcin protein in DPPH-administered rats was greater in regucalcin TG rats than in normal (wild) rats. This study demonstrates that the death of regucalcin TG rats is accelerated after the administration of free radical compound, indicating that overexpression of regucalcin does not have effects as the suppressor for free radical stress and the scavenger for free radical in rats.

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.

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 Ca(2+) 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(+)-Ca(2+) 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 Ca(2+) channel and CaR mRNA expressions in the wild-type cells, while the hormone significantly increased Na(+)-Ca(2+) exchanger mRNA expression. The effects of PTH on L-type Ca(2+) channel, CaR, and Na(+)-Ca(2+) 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 Ca(2+) channel or CaR, which regulates intracellular Ca(2+) signaling, among various regulator proteins for mineral ions in NRK52E cells.

Nuclear localization of a novel protein, RGPR-p117, in cloned normal rat kidney proximal tubular epithelial cells.

A novel protein RGPR-p117 was discovered as a regucalcin gene promoter region-related protein that binds to the TTGGC motif. The role of RGPR-p117 in cell function is unknown. The nuclear localization of RGPR-p117 was investigated using cloned normal rat kidney proximal tubular epithelial NRK52E cells in vitro. RGPR-p117 mRNA was expressed in NRK52E cells, and its expression was stimulated by culture with parathyroid hormone (10(-7) M) or phorbol 12-myristate (10(-6) M). RGPR-p117 was found to localize in the cytoplasm and nucleus with immunocytochemical and Western blot analysis using HA-RGPR-p117/phCMV2-transfected NRK52E cells. Overexpression of HA-RGPR-p117 was found to have a significant stimulatory effect on regucalcin mRNA expression in NRK52E cells. This study demonstrates that RGPR-p117 is localized in the nucleus of kidney cells, and may be involved in gene expression.

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.

Overexpression of regucalcin suppresses cell proliferation of cloned normal rat kidney proximal tubular epithelial NRK52E cells.

The role of regucalcin, which is a regulatory protein in intracellular signaling pathway, in the regulation of cell proliferation was investigated by using the cloned normal rat kidney proximal tubular epithelial NRK52E cells over-expressing regucalcin. NRK52E cells were transfected with regucalcin (RC) pCXN2 vector and the multiple neomycin-resistant clones that stably overexpress regucalcin were selected. The regucalcin content of RC/pCXN2-transfected cells used in this study was about 21-fold as compared with that of the parental wild-type NRK52E cells. Wild-type NRK52E cells, pCXN2 vector-transfected cells (mock-type), and RC/pCXN2-transfected cells (transfectants) were cultured for 24, 48, and 72 h in the presence of bovine serum (5%). The cell numbers of wild- and mock-type were significantly increased with the time course of the culture. Cell numbers of transfectants were significantly suppressed as compared with that of wild- and mock-type. The decrease in cell number of wild-type cultured for 72 h in the presence of butyrate (8.3 x 10(-6) or 8.3 x 10(-5) M), rescovitine (10(-8) or 10(-7) M), or sulforaphane (10(-9) M), which is an inhibitor of the cell cycle, was not observed in transfectants. The effect of PD98059 (10(-8) M), staurosporine (10(-10) M) or dibucaine (10(-8)-10(-6) M), which is an inhibitor of protein kinases, in decreasing cell number of wild-type was not seen in transfectants. Moreover, the culture with wortmannin (10(-8) or 10(-7) M), an inhibitor of phosphatidylinositol 3 (PI3)-kinase, or Bay K 8644 (10(-8) or 10(-7) M), an agonist of calcium entry in cells, caused a significant decrease in cell number of the wild-type. This decrease was not observed in transfectants. The result of reverse transcription-polymerase chain reaction (RT-PCR) analysis using specific primers showed that c-jun and chk2 mRNA levels were significantly decreased in transfectant. p53 mRNA level was significantly increased in transfectants. The expression of c-myc, c-fos, cdc2, p21, and G3PDH mRNAs in transfectants was not significantly changed. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell proliferation, which is mediated through various signaling pathways, in the cloned normal rat kidney proximal tubular epithelial NRK52E cells.

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 Dulbecco's 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 D(3) (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.