Effect Of Regucalcin Research Articles

Molecular Reproduction & Development Volume 84, Issue 3, March 2017

The proteins secreted from the accessory sex glands are important for interacting with and modulating sperm function. For example, Regucalcin, a secreted calcium‐regulating protein (green), is highly expressed by the glandular epithelium of buffalo prostate gland shown in this image. Harikrishna et al. (this issue) identified the suppressive effect of Regucalcin during in vitro capacitation of buffalo spermatozoa.

The protective effect of regucalcin against radiation-induced damage in testicular cells

AimsRegucalcin (RGN), a protein broadly expressed in the male reproductive tract, has shown to have beneficial effects on spermatogenesis suppressing chemical-induced apoptosis. This study aimed to evaluate whether RGN overexpression ameliorates the spermatogenic phenotype after radiation treatment. Main methodsTransgenic rats overexpressing RGN (Tg-RGN) and their wild-type (Wt) counterparts were exposed to a single dose of X-rays (6Gy), and at ten weeks after irradiation, the testicular status and the epididymal sperm parameters were evaluated. The expression of RGN and several cell cycle and apoptosis regulators, the enzymatic activity of caspase-3, and RGN immunostaining were also assessed. Key findingsTg-RGN animals displayed higher gonadosomatic index, and augmented sperm viability and motility relatively to their Wt counterparts after irradiation, as well as higher frequency of normal sperm morphology and a diminished incidence of head-defects. The differences in reproductive parameters were underpinned by a lower rate of apoptosis, as evidenced by the reduced activity of caspase-3, lower levels of caspase-8, and increased Bcl-2/Bax ratio in the testis of Tg-RGN animals. Supporting the involvement of RGN in the anti-apoptotic response, an enhanced expression of RGN was observed in irradiated rats. SignificanceTransgenic-overexpression of RGN protected against radiation-induced testicular damage, which strengthens the role of this protein protecting cells from the damage of external agents. These findings also indicated that the modulation of RGN testicular levels would be a mechanism for fertility preservation in men undergoing oncological treatment.

The anti-apoptotic effect of regucalcin is mediated through multisignaling pathways

Regucalcin (RGN/SMP30) was originally discovered in 1978 as a calcium-binding protein that does not contain the EF-hand motif of as a calcium-binding domain. The name, regucalcin, was proposed for this calcium-binding protein, which can regulate various Ca2+-dependent enzymes activation in liver cells. The regucalcin gene is localized on the X chromosome, and its expression is mediated through many signaling factors. Regucalcin plays a pivotal role in regulation of intracellular calcium homeostasis in various cell types. Regucalcin also has a suppressive effect on various signaling pathways from the cytoplasm to nucleus in proliferating cells and regulates nuclear function in including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis. Overexpression of endogenous regucalcin was found to suppress apoptosis in modeled rat hepatoma cells and normal rat kidney proximal epithelial NRK52 cells induced by various signaling factors. Suppressive effect of regucalcin on apoptosis is related to inhibition of nuclear Ca2+-activated DNA fragmentation, Ca2+/calmodulin-dependent nitric oxide synthase, caspase-3, Bax, cytochrome C, protein tyrosine kinase, protein tyrosine phosphatase in the cytoplasm and nucleus. Moreover, regucalcin stimulates Bcl-2 mRNA expression and depresses enhancement of caspase-3, Apaf-1 and Akt-1 mRNAs expression. This review discusses that regucalcin plays a pivotal role in rescue of apoptotic cell death, which is mediated through various signaling factors.

Role of regucalcin in brain calcium signaling: involvement in aging

Regucalcin was discovered in 1978 to be a calcium-binding protein that does not contain the EF-hand motif of the calcium-binding domain [M. Yamaguchi and T. Yamamoto, Chem. Pharm. Bull., 26, 1915-1918, 1978]. The regucalcin gene is localized on the X chromosome and its expression is enhanced through various transcription factors. Regucalcin is known to play a multifunctional role as a suppressor protein of cell signaling in many cell types. Regucalcin is expressed in rat brain neurons and it is decreased in the cerebral cortex and hippocampus of the brain with aging. Neuronal Ca(2+) signaling has been implicated in mechanisms of neuronal plasticity like long-term potentiation, which is likely to play an important role in learning and memory. The disturbance of brain Ca(2+) homeostasis may play a pivotal role in the revelation of brain disease. The intracellular Ca(2+) in brain tissues is increased with aging. Aging enhances the entry of Ca(2+) into brain neuronal cells across the plasma membranes. An increase in the brain microsomal Ca(2+)-ATPase activity of rats with aging resulted in calcium accumulation in the microsomes of the Ca(2+)-sequestrating system that is partly related to the brain toxicity by calcium. Regucalcin had an inhibitory effect on rat brain microsomal Ca(2+)-ATPase activity. The suppressive effect of regucalcin on brain microsomal Ca(2+)-ATPase activity was weakened in aged rats. Regucalcin was found to inhibit brain cytosolic protein kinase C. Brain microsomal Ca(2+)-ATPase activity was enhanced by protein kinase C in aged rats. Regucalcin could also inhibit activity of Ca(2+)/calmodulin-dependent protein kinase, protein phosphatase, and Ca(2+)/calmodulin-dependent nitric oxide synthase, which is linked to Ca(2+) signaling, in the cytosol of rat brain neurons. These inhibitory effects of regucalcin were weakened with aging. Regucalcin may play a pivotal role in the regulation of Ca(2+) signaling which is stimulated through a neurotransmitter in the brain neurons with aging.

Exogenous regucalcin stimulates osteoclastogenesis and suppresses osteoblastogenesis through NF-κB activation

Regucalcin plays a pivotal role in regulating intracellular calcium homeostasis and consequently has a profound effect on multiple intracellular signal transduction pathways. The regucalcin transgenic rat displays pronounced bone loss, and bone marrow from these animals exhibits significantly elevated osteoclast formation. Consistent with these effects exogenous regucalcin promotes osteoclastogenesis in mouse bone marrow cultures, but interestingly regucalcin suppresses the differentiation and mineralization of MC3T3 osteoblast precursors. However, the molecular mechanisms involved are presently unclear. As the nuclear factor-kappa B (NF-κB) signal transduction pathway is critical to osteoclastogenesis but inhibitory of osteoblastogenesis, we hypothesized that regucalcin may promote osteoclastogenesis and suppress osteoblastogenesis upregulating NF-κB signal transduction. In this study, we examined the effect of regucalcin on receptor activator of NF-κB (RANK) ligand (RANKL) -induced osteoclast formation using the RAW264.7 monocytic cell line and osteoblast formation using the pre-osteoblastic cell line MC3T3. As expected, culture with exogenous regucalcin was found to enhance RANKL-induced osteoclastogenesis. Consistent with this effect regucalcin increased basal and RANKL-induced NF-κB activation as assessed by NF-κB luciferase assay. The capacity of regucalcin to augment RANKL-induced NF-κB activity was inhibited by menaquinone-7, a potent NF-κB antagonist, while the Erk inhibitor PD98059 and staurosporine had no effect, demonstrating a specific effect on NF-κB signaling. By contrast, regucalcin inhibited mineralization of MC3T3 cells and enhanced tumor necrosis factor-α (TNFα)-induced NF-κB activation. As with NF-κB induction in osteoclasts, NF-κB activation was abolished by addition of the NF-κB antagonist menaquinone-7, but not by PD98059 and staurosporine. Transforming growth factor-β (TGFβ) and bone morphogenic protein-2 (BMP2) are potent early commitment and late osteoblast differentiation factors, respectively, and both mediate their actions through the Smad-signal transduction pathway, a system that is extremely sensitive to and inhibited by TNFα-induced NF-κB. We consequently examined the effect of regucalcin on TGFβ and BMP2-induced Smad activation in the presence and absence of TNFα. While regucalcin had no effect on basal Smad activation by TGFβ and BMP2, it enhanced the suppressive effect of TNFα on both TGFβ- and BMP2-induced Smad activations. Taken together, present data suggest that regucalcin may induce bone loss in vivo by promoting osteoclasts and simultaneously suppressing osteoblasts through amplification of basal and/or cytokine-induced NF-κB activation. Regucalcin may have a role as a modulator in NF-κB activation.

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.

Overexpression of regucalcin suppresses gene expression of insulin signaling-related proteins in cloned rat hepatoma H4-II-E cells: Involvement of insulin resistance

Overexpression of regucalcin has been shown to enhance glucose utilization and lipid production in the cloned rat hepatoma H4-II-E cells in vitro, and it induces insulin resistance. The effect of regucalcin on the gene expression of insulin signaling-related proteins was investigated in the cloned rat hepatoma H4-II-E cells overexpressing regucalcin in vitro. The hepatoma cells (wild-type) and stable regucalcin/ pCXN2-transfected cells (transfectants) were cultured for 72 h in a medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. Cells with subconfluency were cultured for 24, 48, or 72 h in a medium containing either vehicle or insulin (10(-9)-10(-7) M) with or without supplementation of glucose (10, 25, or 50 mg/ml of medium). The expression of rat insulin receptor (Insr), phosphatidylinositol 3-kinase (PI3K), glucose transporter 2 (GLUT 2), or glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNAs was examined using reverse transcription-polymerase chain reaction analysis with specific primers. GLUT 2 mRNA expression was significantly increased in the transfectants, while Insr, PI3K, and G3PDH mRNA levels were not significantly changed in the transfectants. Culture with insulin (10(-8) or 10(-7) M) caused a significant increase in PI3K mRNA levels in wild-type cells cultured for 24 or 48 h, while it had no effect on Insr mRNA levels. The supplementation of glucose (10, 25, or 50 mg/ml) caused a significant increase in Insr and PI3K mRNA levels in wild-type cells. The effect of insulin or glucose supplementation on these gene expression levels was not seen in the transfectants. The combination of insulin (10(-7) M) and glucose (50 mg/ml) caused a significant increase in Ins and PI3K mRNA levels in wild-type cells. Such an effect was not seen in the transfectants. Culture with insulin or glucose supplementation failed to have a significant effect on GLUT2 and G3PDH mRNA levels in the wild-type cells or transfectants. This study demonstrates that overexpression of regucalcin suppresses the enhancing effect of insulin or glucose on the gene expression of insulin signaling-related proteins in the cloned rat hepatoma H4-II-E cells.

Regulatory effect of exogenous regucalcin on cell function in osteoblastic MC3T3-E1 cells: Involvement of intracellular signaling factor

Bone loss is induced in regucalcin transgenic rats. The role of exogenous regucalcin in the regulation of osteoblastic cell function was investigated. Osteoblastic MC3T3-E1 cells with subconfluent monolayers were cultured for 24-72 h in medium containing regucalcin (10(-10) or 10(-9) M) without fetal bovine serum. The presence of regucalcin did not have a significant effect on cell number. Culture with regucalcin (10(-9) M) for 24 h caused a significant decrease in protein and DNA contents in osteoblastic cells. The effect of regucalcin in decreasing cellular protein content was significantly inhibited in the presence of various kinase inhibitors including staurosporine (10(-7) M), dibucaine (10(-6) M), PD98059 (10(-8) M), or wortmannin (10(-8) M). Meanwhile, culture with regucalcin caused a significant decrease in cellular DNA content in the presence of various kinase inhibitors. The presence of regucalcin did not have a significant effect on protein and DNA contents in the cells cultured with cycloheximide (10(-7) M), an inhibitor of protein synthesis, or 5,6-dichloro -1-beta-D-ribofuranosylbenzimidazole (10(-6) M), an inhibitor of transcription activity; which each inhibitor caused a significant decrease in those contents. The effect of regucalcin in decreasing cellular protein content was seen in the presence of insulin-like growth factor-I (IGF-I; 10(-9) or 10(-8) M). Such an effect was not observed in cellular DNA content. The results of reverse transcription-polymerase chain reaction analysis with specific primers showed that the expression of Runx 2 (Cbfa 1) and alkaline phosphatase mRNAs in osteoblastic cells was significantly suppressed in the presence of regucalcin (10(-10) or 10(-9) M). Glyceraldhyde-3-phosphate dehydrogenase mRNA level was not significantly changed with culture of regucalcin (10(-10) or 10(-9) M). This study supports the view that exogenous regucalcin regulates the function of osteoblastic cells, and that the effect of protein is mediated through signaling factors.

Regucalcin increases Ca2+-ATPase activity in the heart mitochondria of normal and regucalcin transgenic rats

The role of regucalcin, a regulatory protein in intracellular signaling system, in the regulation of Ca2+-ATPase activity in rat heart mitochondria was investigated. Mitochondrial Ca2+-ATPase activity was significantly increased by increasing concentrations of CaCl2 (2.5-50 microM). An increase in the enzyme activity was saturated at 50 microM CaCl2. The addition of regucalcin (10(-11)-10(-8) M) in the enzyme reaction mixture caused a significant increase in Ca2+-ATPase activity in heart mitochondria in the presence of 50 microM CaCl2. Regucalcin did not have a significant effect on mitochondrial Mg2+-ATPase activity. Regucalcin (10(-9) M) did not have a significant effect on Ca2+-ATPase activity in the presence of digitonin (10(-3) or 10(-2) %), which is a solubilization effect on membranous lipids. The effect of regucalcin in increasing mitochondrial Ca2+-ATPase activity was not observed in the presence of ruthenium red (10(-7) M) or lanthanum chloride (10(-7) M), which is an inhibitor of Ca2+ uniporter. The effect of regucalcin (10(-9) M) in increasing mitochondrial Ca2+-ATPase activity was not significantly enhanced in the presence of calmodulin (5 microg/ml) or dibutyryl cyclic AMP (10(-4) M), which is an intracellular signaling factor that can cause a significant increase in the enzyme activity. Mitochondrial regucalcin localization was significantly increased in the heart of regucalcin transgenic rats as compared with that of normal rats using Western blot analysis. Ca2+-ATPase activity was significantly increased in the heart mitochondria of regucalcin transgenic rats. This study demonstrates that regucalcin has an activating effect on Ca2+-ATPase in rat heart mitochondria, suggesting its role in the regulation of heart mitochondrial function.

Overexpression of regucalcin suppresses Ca2+‐sensing receptor and L‐type Ca2+ channel mRNA expression in cloned rat kidney proximal tubular NRK52E cells

The effect of regucalcin, a regulatory protein in intracellular signaling pathway [Yamaguchi M, Int J Mol Med 15:372–389, 2005 in Review], on various gene expressions was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin /pCXN2 transfectants were cultured for 72 hours in medium containing 5% bovine serum to obtain subconfluent monolayers. After culture, cells were further cultured for 24–72 hours in serum free medium. Regucalcin was localized in the nucleus of wild type cells. This localization was markedly increased in transfectants. Overexpression of regucalcin caused a significant increase in rat outer medullary K+ channel mRNA expression, while it did not cause epithelial sodium channel, Na+-K+-ATPase, type II Na-Pi cotransporter, angiotensinogen and Na+/Ca+ exchanger mRNA expressions in transfectants. The expression of Ca2+-sensing receptor and L-type Ca2+ channel mRNA was markedly suppressed in transfectants. Thses decreases were changed in the presence of parathyroid hormone that can suppress the gene expression. This study demonstrates that regucalcin has a role in the regulation of gene expression for Ca2+-sensing receptor and L-type Ca2+ channel that is involved in intracellular Ca2+ transport in kidney cells.

Overexpression of regucalcin enhances its nuclear localization and suppressesL-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.

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.

Suppressive effect of regucalcin on cell differentiation and mineralization in osteoblastic MC3T3‐E1 cells

The role of regucalcin in the regulation of osteoblastic cell function was investigated. Osteoblastic MC3T3-E1 cells with subconfluent monolayers were cultured in a medium containing regucalcin (10(-10)-10(-8) M) without fetal bovine serum (FBS). The proliferation of osteoblastic cells was not significantly altered in the presence of regucalcin. The results of reverse transcription-polymerase chain reaction (RT-PCR) analysis with specific primers showed that the expression of Runx2 (Cbfa1) and insulin-like growth factor-I (IGF-I) mRNAs in osteoblastic cells was significantly suppressed in the presence of regucalcin (10(-10) or 10(-9) M). Transforming growth factor-beta1 mRNA levels were significantly enhanced in the 24 h-culture with regucalcin (10(-10) or 10(-9) M). Alpha1(I) collagen and glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA levels were not significantly changed by culture with regucalcin (10(-10) or 10(-9) M). Alkaline phosphatase activity was significantly decreased in the lysate of cells cultured for 24 or 48 h with regucalcin (10(-10)-10(-8) M). Moreover, the expression of regucalcin in osteoblastic cells was demonstrated by RT-PCR and Western blot analysis. When regucalcin (10(-7) M) was added into the enzyme reaction mixture containing the lysate of osteoblastic cells cultured in the absence of regucalcin, alkaline phosphatase activity was significantly decreased. Also, Ca2+/calmodulin-dependent nitric oxide (NO) synthase activity in the cell lysate was significantly decreased by addition of regucalcin (10(-10)-10(-8) M) into the reaction mixture. The presence of anti-regucalcin monoclonal antibody (5 or 10 ng/ml) in the enzyme reaction mixture caused a significant increase in NO synthase activity in the cell lysate in the presence or absence of Ca2+/calmodulin, suggesting a role of endogenous regucalcin. When osteoblastic cells with subconfluency were cultured in the presence of regucalcin (10(-10) or 10(-9) M) for 3, 9, or 18 days, the results with Alizarin red staining showed that the mineralization was markedly suppressed by culture with regucalcin for 3, 9, or 18 days. This study demonstrates that regucalcin regulates the function of osteoblastic cells, and that the protein suppresses cell differentiation and mineralization.

Overexpression of regucalcin suppresses apoptotic cell death in the cloned rat hepatoma H4-II-E cells induced by a naturally occurring isothiocyanate sulforaphane

The effect of regucalcin, a regulatory protein in intracellular signaling system, on cell death and apoptosis was investigated. Sulforaphane, a naturally occurring isothiocyanate, is known to induce cell cycle arrest and apoptosis in cancer cells, although its effect has not been clarified in the cloned rat hepatoma H4-II-E cells. Hepatoma H4-II-E cells (wild-type) and stable regucalcin/pCXN2-transfected cells were cultured for 72 h in a medium containing 10% fetal bovine serum (FBS). Cells with subconfluency were changed to a medium containing either vehicle or sulforaphane (10(-7) or 10(-6) M) in the absence of FBS. After medium change, cells were cultured for 24, 48, or 72 h. The number of wild-type cells was significantly decreased in the presence of sulforaphane (10(-7) or 10(-6) M). Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with sulforaphane (10(-7) or 10(-6) M) for 24 h. Sulforaphane (10(-7) or 10(-6) M)-induced cell number and DNA fragmentation was significantly suppressed in transfectants. The effect of sulforaphane (10(-6) M) in decreasing the number of wild-type cells was significantly prevented in the presence of caspase-3 inhibitor (10(-9) M), while the presence of Nomega-nitro-L-arginine methylester (NAME), an inhibitor of nitric oxide synthase, did not prevent sulforaphane-induced death of wild-type cells. Sulforaphane (10(-6) M) did not have a significant effect on cell number of transfectants in the presence of caspase-3 inhibitor or NAME. This study demonstrates that sulforaphane induces cell death and apoptosis in the cloned rat hepatoma H4-II-E cells, and that overexpression of regucalcin suppresses sulforaphane-induced apoptotic cell death which is partly mediated through caspase-3..

Regucalcin stimulates osteoclast‐like cell formation in mouse marrow cultures

The effect of regucalcin, a regulatory protein in intracellular signaling, on osteoclastic cell formation in mouse bone marrow culture is investigated. The bone marrow cells were cultured for 7 days in an alpha-minimal essential medium containing either vehicle or regucalcin (10(-10)-10(-8)M). Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of regucalcin (10(-10)-10(-8)M) caused a remarkable increase in osteoclast-like multinucleated cells (MNCs). The effect of regucalcin in stimulating osteoclast-like cell formation was significantly inhibited in the presence of calcitonin (CT; 10(-9)M), 17beta-estradiol (10(-9)M), beta-cryptoxanthin (CX; 10(-6)M), or zinc sulfate (10(-4)M), which is an anti-bone resorbing factor. The effect of regucalcin on osteoclast-like cell formation was not significantly blocked in the presence of cycloheximide, an inhibitor of protein synthesis, or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an inhibitor of transcriptional activity. The effect of parathyroid hormone (10(-7)M), 1,25-dihydroxyvitamin D(3) (10(-7)M), prostaglandin E(2) (10(-5)M), or tumor necrosis factor-alpha (10 ng/ml) in increasing osteoclast-like cell formation was significantly enhanced in the presence of regucalcin (10(-8)M). Moreover, when rat femoral-diaphyseal or -metaphyseal tissues were cultured for 48 h in the presence of regucalcin (10(-10)-10(-8)M), the diaphyseal or metaphyseal calcium content was significantly decreased in the presence of regucalcin (10(-10)-10(-8)M) in vitro. The consumption of glucose and the production of lactic acid in culture medium by the diaphyseal or metaphyseal tissues was significantly raised in the presence of regucalcin (10(-10)-10(-8)M). This study demonstrates that regucalcin directly stimulates osteoclast-like cell formation in mouse marrow culture in vitro, and that the protein stimulates bone resorption in rat femoral tissues in vitro.

Regucalcin increases superoxide dismutase activity in the heart cytosol of normal and regucalcin transgenic rats.

The effect of regucalcin, a regulatory protein in the intracellular signaling system, on superoxide dismutase (SOD) activity in the heart cytosol of normal rats and regucalcin transgenic (TG) rats was investigated. The addition of regucalcin (10(-10) to 10(-8) M) with a physiologic concentration in the enzyme reaction mixture containing the heart cytosol obtained from normal rats caused a significant increase in SOD activity, indicating that regucalcin directly activates the enzyme. The effect of regucalcin (10(-8) M) in increasing SOD activity was not seen in the presence of dithiothreitol (DTT; 0.1 or 1.0 mM), a protecting reagent for sulfhydryl group, or N-ethylmaleimide (NEM; 0.1 or 1.0 mM), a modifying reagent for sulfhydryl group, in the reaction mixture, indicating that regucalcin does not affect the sulfhydryl group. The addition of zinc sulfate (10(-6) to 10(-4) M) in the reaction mixture did not cause a significant change in SOD activity, while the enzyme activity was markedly decreased in the presence of cupric sulfate (10(-6) to 10(-4) M). The activatory effect of regucalcin (10(-8) M) on SOD was seen in the presence of zinc (10(-4) M), while not observed in the presence of copper (10(-4) M). Moreover, SOD activity was significantly enhanced in the heart cytosol of regucalcin TG rats as compared with that of normal rats. This study demonstrates that regucalcin increases SOD activity in the heart cytosol of rats, and that its effect is not related to the sulfhydryl group of enzymes.

Overexpression of regucalcin suppresses cell death and apoptosis in cloned rat hepatoma H4-II-E cells induced by lipopolysaccharide, PD 98059, dibucaine, or Bay K 8644.

The effect of regucalcin, a regulatory protein in intracellular signaling pathway, on 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. After culture for 72 h, cells were further cultured for 12-72 h in medium without FBS containing either vehicle or lipopolysaccharide (LPS; 0.1 or 1.0 microg/ml). The number of wild-type cells was significantly decreased by culture for 24 or 48 h in the presence of LPS (0.1 or 1.0 microg/ml). The effect of LPS (0.1 or 1.0 microg/ml) in decreasing the number of hepatoma cells was significantly prevented in transfectants overexpressing regucalcin. However, the culture with LPS (0.1 or 1.0 microg/ml) for 72 h caused a significant decrease in cell number of transfectants. Ca(2+)/calmodulin-dependent nitric oxide (NO) synthase activity was significantly decreased by culture with LPS (1.0 microg/ml) for 24-72 h of wild-type cells. This decrease was significantly prevented in transfectants. LPS (0.1 or 1.0 microg/ml)-induced decrease in the number of wild-type cells was significantly prevented by culture with caspase-3 inhibitor (10(-8) M). Moreover, the number of wild-type cells was significantly decreased by culture with PD 98059 (10(-6) M), dibucaine (10(-6) M), or staurosporine (10(-6) M), which is an inhibitor of various protein kinases. The effect of PD 98059 or dibucaine on the number of wild-type cells was not observed in transfectants, although the effect of staurosporine was seen in transfectants. Culture with Bay K 8644 (2.5 x 10(-6) M), an agonist of Ca(2+) entry in cells, caused a significant decrease in the number of wild-type cells. Such an effect was not seen in transfectants. The presence of LPS did not significantly decrease the number of wild-type cells in the presence of Bay K 8644. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with Bay K 8644, and this DNA fragmentation was significantly prevented in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death induced by LPS or various intracellular signaling-related factors.

Suppressive effect of regucalcin on protein phosphatase activity in the heart cytosol of normal and regucalcin transgenic rats

The role of regucalcin, a regulatory protein in intracellular signaling pathway, in the regulation of protein phosphatase activity in the heart muscle cytosol was investigated by using normal (wild-type) and regucalcin transgenic (TG) rats. Protein phosphatase activity was assayed in a reaction mixture containing the cytosolic protein in the presence of phosphotyrosine, phosphoserine, and phosphothreonine. The addition of calcium chloride (10 and 20 microM) in the enzyme reaction mixture caused a significant increase in protein phosphatase activity toward three phosphoaminoacids. Trifluoperazine (10 and 20 microM), an antagonist of calmodulin, completely inhibited calcium (10 microM) addition-increased protein phosphatase activity toward three phosphoaminoacids. Moreover, the calcium (10 microM)-increased enzyme activity toward phosphoserine and phosphothreonine was significantly enhanced by the addition of calmodulin (2.5 or 5 microg/ml). Such an enhancement was not seen in the presence of phosphotyrosine. Regucalcin (10(-9) and 10(-8) M) significantly inhibited protein phosphatase activity toward three phosphoaminoacids in the presence of ethylene glycol bis (2-aminoethlether) N,N,N',N'-tetraacetic acid (EGTA; 1 mM), without Ca2+ addition. The inhibitory effect of regucalcin (10(-10)-10(-8) M) was also seen in the presence of calcium chloride (10 microM). Western blot analysis showed a remarkable expression of regucalcin protein in the cytosol of heart of regucalcin TG female rats as compared with that of wild-type female rats. Protein phosphatase activity toward three phosphoaminoacids was significantly decreased in the heart cytosol of TG rats. The enhancing effect of calcium (10 microM) addition on protein phosphatase activity toward three phosphoaminoacids was not seen in the heart cytosol of TG rats. This study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in rat heart cytoplasm.

Regucalcin increases superoxide dismutase activity in rat liver cytosol.

The effect of regucalcin, a regulatory protein in the intracellular signaling process, on superoxide dismutase (SOD) activity in the cytosol of rat liver was investigated. The presence of zinc sulfate (10(-6) or 10(-5) m) or cupric sulfate (10(-6) m) in the enzyme reaction mixture caused a significant increase in SOD activity, indicating that Cu/Zn-SOD may be present in the liver cytosol. SOD activity was significantly increased by the addition of regucalcin (0.1, 0.25, or 0.5 microM) to the reaction mixture. The presence of dithiothreitol (DTT; 0.1, 0.5, or 1.0 mM), a protective reagent for the sulfhydryl group, caused a significant decrease in SOD activity. The effect of regucalcin (0.25 microM) in increasing SOD activity was not seen in the presence of DTT (1.0 mM). Meanwhile, SOD activity was significantly raised by the addition of N-ethylmaleimide (NEM; 0.5 or 1.0 mM), a modifying reagent for the sulfhydryl reagent. Regucalcin (0.25 microM) caused a significant increase in SOD activity in the presence of NEM (1.0 mM). The effect of regucalcin in increasing SOD activity may not involve the sulfhydryl group of SOD. This study demonstrates that regucalcin has an activatory effect on SOD in the liver cytosol of rats.

Overexpression of regucalcin modulates tumor‐related gene expression in cloned rat hepatoma H4‐II‐E cells

Regucalcin is a regulatory protein in intracellular signaling pathway which is related to various protein kinases and protein phosphatases in many cells. The effect of regucalcin on the expression of tumor-related genes was investigated in the cloned rat hepatoma H4-II-E cells and the hepatoma cells (transfectants) overexpressing regucalcin. Hepatoma cells were cultured for 24-72 h in the presence of fetal bovine serum (FBS; 10%). The proliferation of hepatoma cells was significantly suppressed at 24-72 h of culture in regucalcin transfectants as compared with that of wild-type or mock-type cells. Western blot analysis showed that regucalcin was markedly expressed in transfectants. The expression of c-myc, c-fos, c-jun, Ha-ras, and p53 mRNAs was determined using reverse transcription-polymerase chain reaction (RT-PCR). Of these genes, the expression of c-myc or Ha-ras mRNAs was significantly suppressed in regucalcin transfectants. The suppression of c-myc mRNA expression in transfectants was confirmed by using Northern blot analysis; significant suppression was seen at 24, 48, or 72 h of culture in the presence of 10% FBS. Culture with 10% FBS significantly enhanced c-myc mRNA expression in the hepatoma cells (wild-type) as compared with that of 1% FBS. The enhancement was significantly abolished in the transfectants. Meanwhile, the expression of p53 mRNA in the hepatoma cells was significantly enhanced in regucalcin-overexpressing hepatoma cells. This study demonstrates that the expression of oncogene c-myc and Ha-ras mRNA in hepatoma cells overexpressing regucalcin is suppressed, and that the tumor suppression gene p53 is enhanced in the transfectants.