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Aims: RGPR-p117 was originally discovered as a novel transcription factor, which specifically binds to a nuclear factor I (NFI) consensus motif TTGGC(N) 6 CC in the promoter region of the regucalcin gene. RGPR-p117 is also called as Lztr2 and SEC16B. The role of RGPR-p117 in cell regulation is poorly understood. This study was undertaken to determine whether the overexpression of RGPR-p117 impacts the proliferation of normal rat kidney proximal tubular epithelial NRK-52E cells in vitro., Main Methods: The NRK-52E wild-type cells and RGPR-p117-overexpressing NRK-52E cells were cultured in DMEM containing fetal bovine serum., Key Findings: The overexpression of RGPR-p117 repressed colony formation and proliferation of NRK-52E cells. Interestingly, RGPR-p117 overexpression blocked cell proliferation promoted by culturing with Bay K 8644, a calcium-entry agonist, and phorbol 12-myristate 13-acetate, an activator of protein kinase C. The depressive effects of RGPR-p117 overexpression on cell proliferation were not occurred by culturing with various inhibitors of cell cycle and intracellular signaling processes. RGPR-p117 overexpression increased the translocation of RGPR-p117 into the nucleus of NRK-52E cells. Mechanistically, RGPR-p117 overexpression diminished the levels of Ras, PI3 kinase, Akt, mitogen-activated protein kinase, and mTOR, while it raised the levels of p53, Rb, p21, and regucalcin. Furthermore, RGPR-p117 overexpression protected cell death caused by apoptosis-inducing factors, suggesting that the suppressive effects of RGPR-p117 on cell growth are independent of cell death., Significance: The present study demonstrates that the overexpressed transcription factor RGPR-p117 suppresses cell proliferation via targeting diverse signaling processes, suggesting a role of RGPR-p117 in cell regulation., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Prostate cancer is a bone metastatic cancer and is the second leading cause of cancer-related death in men. Prolonged progression-free survival of prostate cancer patients is associated with high regucalcin expression in the tumor tissues. This study investigates the underlying mechanism by which regucalcin prevents bone metastatic activity of prostate cancer cells., Methods: Human prostate cancer PC-3 or DU-145 wild-type cells or regucalcin-overexpressing PC-3 or DU-145 cells (transfectants) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum., Results: Overexpressed regucalcin suppressed the migration and invasion of bone metastatic human prostate cancer cells in vitro, and it reduced the levels of key proteins in metastasis including Ras, Akt, MAPK, RSK-2, mTOR, caveolin-1, and integrin β1. Invasion of prostate cancer cells was promoted by coculturing with preosteoblastic MC3T3-E1 or preosteoclastic RAW264.7 cells. Coculturing with cancer cells and bone cells repressed the growth of preosteoblastic cells and enhanced osteoclastogenesis of preosteoclastic cells, and these alterations were caused by a conditioned medium from cancer cell culture. Disordered differentiation of bone cells was prevented by regucalcin overexpression. Production of tumor necrosis factor-α (TNF-α) in cancer cells was blocked by overexpressed regucalcin. Of note, the effects of conditioned medium on bone cells were prevented by NF-κB inhibitor. TNF-α may be important as a mediator in the crosstalk between cancer cells and bone cells., Conclusion: Overexpression of regucalcin suppressed the migration, invasion, and bone metastatic activity of human prostate cancer cells. This study may provide a new strategy for therapy with the regucalcin gene transfer., (© 2022 Wiley Periodicals LLC.)

Regucalcin plays a multifunctional role in cell regulation as a suppressor in the processes of intracellular signaling and transcription, leading to inhibition of cell growth. The downregulated expression or activity of regucalcin has been shown to contribute to the development of carcinogenesis in various types of human cancer. The wild-type tumor suppressor TP53 gene encodes for a transcriptional factor p53. This protein may play a role in cell proliferation. Loss of p53 function may induce cell transformation during carcinogenesis and tumor progression of human cancer. We investigate whether or not extracellular regucalcin suppresses the proliferation of non-tumorigenic human mammary epithelial MCF 10A cells with loss of p53 in vitro. Loss of p53 did not impact colony formation and proliferation of the cells. Interestingly, p53 loss caused decrease in the cell cycle suppressor p21, but not retinoblastoma and regucalcin, as compared with those of wild-type MCF 10A cells. Notably, extracellular regucalcin suppressed colony formation and proliferation of wild-type MCF 10A cells and p53 (-/-) cells, while it did not have an effect on cell death. Mechanistically, extracellular regucalcin decreased levels of various signaling factors including Ras, phosphatidylinositol-3 kinase, mitogen-activated protein kinase (MAPK), phospho-MAPK, and signal transducer and activator of transcription 3 in wild-type MCF 10A cells and p53 (-/-) cells. Thus, extracellular regucalcin was found to suppress the growth of MCF 10A cells with loss of p53. Extracellular regucalcin may play a role as a suppressor in the growth of human mammary epithelial cells with p53 loss, providing a novel strategy for cancer., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Dysregulation of adipocyte differentiation and dysfunction play key roles in the pathogenesis of obesity and associated disorders such as diabetes and metabolic syndrome, and as such, a better understanding of the molecular mechanism of adipogenesis may help to elucidate the pathological condition of obesity and its associated disorders. Regucalcin (RGN) plays multiple regulatory roles in intracellular Ca 2+ signaling pathways in mammalian cells. Here, we report that overexpression of RGN enhances lipid accumulation in 3T3-L1 adipocyte cells after adipogenic stimulation, accompanied by upregulation of adipocyte differentiation marker proteins. In contrast, genetic disruption of RGN inhibited adipogenic stimulation-induced differentiation of 3T3-L1 cells. Furthermore, RGN overexpression in differentiated 3T3-L1 adipocytes blocked inflammatory crosstalk between 3T3-L1 adipocytes and RAW264.7 macrophages in a transwell coculture system. Knockdown of RGN expression in cocultured 3T3-L1 adipocytes enhanced their susceptibility to RAW264.7 macrophage-mediated inflammation. These results suggest that RGN is required for 3T3-L1 adipocyte differentiation and that it exerts anti-inflammatory activity against 3T3-L1 adipocyte inflammation after coculture with RAW264.7 macrophages. Thus, RGN may be a novel regulator of adipocyte differentiation and act as a suppressor of inflammation in macrophage-infiltrated adipocyte tissue., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Hepatocellular carcinoma is one of the most prevalent malignant diseases and causes a third of cancer-related death. The consequences of altered calcium homeostasis in cancer cells may contribute to tumor progression. Regucalcin plays an inhibitory role in calcium signaling linked to transcription regulation. Regucalcin gene expression is downregulated in the tumor tissues of liver cancer patients, suggesting an involvement as a suppressor in hepatocarcinogenesis. We investigated whether Bay K 8644, an agonist of the L-type Ca 2+ channel, promotes the growth of human liver cancer and if the effect of Bay K 8644 is suppressed by overexpressed regucalcin using the HepG2 cell model. The colony formation and growth of HepG2 cells were promoted by culturing with Bay K 8644 (0.1-10 nM). This effect was suppressed by inhibitors of signaling processes linked to cell proliferation, including PD98059 and wortmannin. Death of HepG2 cells was stimulated by Bay K 8644 with higher concentrations (25 and 100 nM). The effects of Bay K 8644 on cell growth and death were abolished by verapamil, an antagonist of calcium channel. Mechanistically, culturing with Bay K 8644 increased levels of mitogen-activated protein kinase (MAPK) and phospho-MAPK. Notably, overexpressed regucalcin suppressed Bay K 8644-promoted growth and death of HepG2 cells. Furthermore, overexpressed regucalcin prevented growth and increased death induced by thapsigargin, which induces the release of intracellular stored calcium. Thus, higher regucalcin expression suppresses calcium signaling linked to the growth of liver cancer cells, providing a novel strategy in treatment of hepatocellular carcinoma with delivery of the regucalcin gene.

Regucalcin plays a pivotal role as a suppressor of human carcinogenesis, and downregulation of regucalcin expression may contribute to the promotion of human osteosarcoma. Overexpression of regucalcin suppressed the proliferation of Saos-2 human osteosarcoma cells in vitro and decreased the protein levels of multiple signaling components, transcription factors, and tumor suppressors. Interestingly, extracellular regucalcin repressed colony formation and proliferation of Saos-2 cells, and reduced the protein levels of multiple signaling components, cell cycle inhibitor, and various transcription factors. Thus, regucalcin suppressed the growth of human osteosarcoma cells, providing a novel strategy with the gene therapy for treatment of osteosarcoma.

Renal cell carcinoma (RCC), which is a type of cancer found in the kidney tubule, is among the 10 most frequently occurring human cancers. Regucalcin plays a potential role as a regulator of transcriptional activity, and its downregulated expression or activity may contribute to the promotion of human cancers. In this study, we investigated the involvement of regucalcin in human RCC. Regucalcin expression was compared in 23 normal and 29 tumor samples of kidney cortex tissues of patients with clear cell RCC obtained through the Gene Expression Omnibus (GEO) database (GSE36895). Regucalcin expression was downregulated in the tumor tissues. The prolonged survival of patients with clear cell RCC was demonstrated to be associated with a higher regucalcin gene expression in the TCGA dataset. The overexpression of regucalcin suppressed the colony formation, proliferation and the death of human clear cell RCC A498 cells in vitro. Mechanistically, the overexpression of regucalcin induced the G1 and G2/M phase cell cycle arrest of A498 cells through the suppression of multiple signaling components, including Ras, PI3 kinase, Akt and mitogen‑activated protein (MAP) kinase. Importantly, the overexpression of regucalcin led to an elevation in the levels of the tumor suppressors, p53, Rb and the cell cycle inhibitor, p21. The levels of the transcription factors, c‑fos, c‑jun, nuclear factor‑κB p65, β‑catenin and signal transducer and activator of transcription 3, were suppressed by regucalcin overexpression. On the whole, the findings of this study suggest that regucalcin plays a suppressive role in the promotion of human RCC. The overexpression of regucalcin by gene delivery systems may thus prove to be a novel therapeutic strategy for RCC.

Regucalcin plays a crucial role as a regulator of transcriptional signaling activity, and its decreased expression or activity may contribute to the promotion of human carcinogenesis. A higher regucalcin expression in the tumor tissues has been demonstrated to prolong the survival of patients with various types of cancer, including pancreatic cancer, breast cancer, liver cancer and lung adenocarcinoma. The involvement of regucalcin in human colorectal cancer was investigated in the current study. Regucalcin gene expression and the survival data of 62 patients with colorectal cancer were obtained though the Gene Expression Omnibus (GEO) database (GSE12945) for outcome analysis. The data of gene expression revealed that the prolonged survival of patients with colorectal cancer was associated with a higher regucalcin gene expression in tumor tissues. The overexpression of regucalcin suppressed colony formation and proliferation, and induced the death of human colorectal carcinoma RKO cells cultured in a medium containing fetal bovine serum in vitro. Mechanistically, the overexpression of regucalcin induced the G1 and G2/M phase cell cycle arrest of the RKO cells through the suppression of multiple signaling pathways, including Ras, Akt, mitogen-activated protein (MAP) kinase and SAPK/JNK. Of note, the overexpression of regucalcin induced an increase in the levels of the tumor suppressors, p53 and Rb, and the cell cycle inhibitor, p21. Moreover, the levels of the transcription factors, c‑fos, c‑jun, nuclear factor (NF)‑κB p65, β-catenin and signal transducer and activator of transcription 3 (Stat3), were suppressed by the overexpression of regucalcin. On the whole, the findings of this study suggest that regucalcin plays a crucial role as a suppressor in human colorectal cancer, and that the suppressed expression of the regucalcin gene may predispose patients to the promotion of colorectal cancer. The overexpression of regucalcin by gene delivery may thus prove to be a novel therapeutic strategy for colorectal cancer.

Regucalcin, which its gene is localized on the X chromosome, plays a pivotal role as a suppressor protein in signal transduction in various types of cells and tissues. Regucalcin gene expression has been demonstrated to be suppressed in various tumor tissues of animal and human subjects, suggesting a potential role of regucalcin in carcinogenesis. Regucalcin, which is produced from the tissues including liver, is found to be present in the serum of human subjects and animals. This study was undertaken to determine the effects of exogenous regucalcin on the proliferation in cloned human hepatoma HepG2 cells in vitro. Proliferation of HepG2 cells was suppressed after culture with addition of regucalcin (0.01 – 10 nM) into culture medium. Exogenous regucalcin did not reveal apoptotic cell death in HepG2 cells in vitro. Suppressive effects of regucalcin on cell proliferation were not enhanced in the presence of various signaling inhibitors including tumor necrosis factor-α (TNF-α), Bay K 8644, PD98059, staurosporine, worthomannin, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) or gemcitabine, which were found to suppress the proliferation. In addition, exogenous regucalcin suppressed the formation of colonies of cultured hepatoma cells in vitro. These findings demonstrated that exogenous regucalcin exhibits a suppressive effect on the growth of human hepatoma HepG2 cells, proposing a strategy with the gene therapy for cancer treatment.

Regucalcin plays a crucial role as a suppressor of transcription signaling, and its diminished expression or activity may play a key role in human carcinogenesis. Higher regucalcin expression has been demonstrated to prolong survival of the patients of pancreatic cancer, breast cancer, and hepatocellular carcinoma. Moreover, we investigated an involvement of regucalcin in human lung cancer. Human non-small cell lung cancer (NSCLC) accounts for over 80% in human lung cancer and is one of the leading causes of malignancy-related mortality with fewer than 16% patients surviving beyond 5 years. In this study, gene expression and survival data of 204 lung adenocarcinoma patients were obtained through the gene expression omnibus database (GSE31210) for outcome analysis. Gene expression data demonstrated that prolonged survival in lung cancer patients is associated with higher regucalcin gene expression. Overexpression of regucalcin suppressed the proliferation, cell death, and migration of human lung adenocarcinoma NSCLC A549 cells in vitro. Mechanistically, regucalcin induced G1 and G2/M phase cell cycle arrest of A549 cells through suppression of multiple signaling pathways including Ras, Akt, MAP kinase, and SAPK/JNK. Moreover, overexpression of regucalcin caused decreases in the oncogenes c-fos and c-myc and elevation of the tumor suppressers p53 and Rb. These findings suggest that regucalcin may play a potential role as a suppressor of human lung cancer, and that downregulation of regucalcin expression may predispose patients to development of lung cancer. Overexpression of regucalcin using gene delivery may constitute a novel therapeutic approach to treating lung cancer.

Hepatocellular carcinoma (HCC) is one of the most common malignant cancers worldwide and ranks third in overall global cancer-related mortality rates. Importantly, in this study gene expression data demonstrate that prolonged survival in HCC patients is associated with increased regucalcin gene expression. Regucalcin has been shown to play a pivotal role as a transcription repressor and diminished expression or activity of regucalcin may play a key role in the development of human carcinogenesis. Indeed, overexpression of regucalcin suppressed the proliferation, cell death, and migration of human HCC HepG2 cells in vitro. Mechanistically, regucalcin induced G1 and G2/M phase cell cycle arrest of HepG2 cells through suppression of multiple signaling pathways including Ras, Akt, MAP kinase and SAPK/JNK and by increasing the tumor suppressors p53 and Rb. Furthermore, the oncogenes c-fos and c-myc were suppressed by overexpression of regucalcin, and overexpression of regucalcin caused an increase in p21 and a decrease in NF-κB p65 and β-catenin. These findings suggest that regucalcin may play a potential role as a suppressor of human HCC, and that diminished expression of regucalcin may predispose patients to development of HCC. Overexpression of regucalcin may constitute a novel therapeutic approach to treating HCC.

Human breast cancer is highly metastatic to bone and drives bone turnover. Breast cancer metastases cause osteolytic lesions and skeletal damage that leads to bone fractures. Regucalcin, which plays a pivotal role as an inhibitor of signal transduction and transcription activity, has been suggested to act as a suppressor of human cancer. In the present study, we compared the clinical outcome between 44 breast cancer patients with higher regucalcin expression and 43 patients with lower regucalcin expression. Prolonged relapse-free survival was identified in the patients with increased regucalcin gene expression. We further demonstrated that overexpression of full length, but not alternatively spliced variants of regucalcin, induces G1 and G2/M phase cell cycle arrest, suppressing the proliferation of MDA-MB-231 cells, a commonly used in vitro model of human breast cancer that metastasize to bone causing osteolytic lesions. Overexpression of regucalcin was found to suppress multiple signaling pathways including Akt, MAP kinase and SAPK/JNK, and NF-κB p65 and β-catenin along with increased p53, a tumor suppressor, and decreased K-ras, c-fos and c-jun. Moreover, we found that co-culture of regucalcin-overexpressing MDA-MB-231 cells with mouse bone marrow cells prevented enhanced osteoclastogenesis and suppressed mineralization in mouse bone marrow cells in vitro. Taken together, the present study suggests that regucalcin may have important anticancer properties in human breast cancer patients. Mechanistically, these effects are likely mediated through suppression of multiple signaling pathways, upregulation of p53 and downregulation of oncogenes leading to anti-proliferative effects and reduced metastases to bone, a phenotype associated with poor clinical outcome.

Approximately 90% of all pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC). PDAC is a highly aggressive malignancy and is one of the deadliest. This poor clinical outcome is due to the prominent resistance of pancreatic cancer to drug and radiation therapies. Regucalcin plays a pivotal role as a suppressor protein in signal transduction in various types of cells including tumor tissues. We demonstrated that the prolonged survival is induced in PDAC patients with increased regucalcin gene expression using a dataset of PDAC obtained from GEO database (GSE17891) together with the clinical annotation data file. Moreover, overexpression of regucalcin with full length was demonstrated to suppress the proliferation, cell death and migration in human pancreatic cancer MIA PaCa-2 (K-ras mutated) cells that possess resistance to drug and radiation therapies. Suppressive effects of regucalcin on cell proliferation and death were not seen in the cells overexpressed with regucalcin cDNA alternatively spliced variants (deleted exon 4 or deleted exon 4 and 5). Regucalcin was suggested to induce G1 and G2/M phase cell cycle arrest in MIA PaCa-2 cells. Suppressive effects of regucalcin on cell proliferation were independent of cell death. Overexpression of regucalcin was found to suppress signaling pathways including Akt, MAP kinase and SAPK/JNK, to increase the protein levels of p53, a tumor suppresser, and to decrease K-ras, c-fos and c-jun, a oncogene, by suppressing signaling pathways that are related to signaling of K-ras. Regucalcin may play a potential role as a suppressor protein in human pancreatic cancer.

Regucalcin serves a pivotal role as a suppressor protein in signal transduction in various types of cells and tissues. The regucalcin gene, which is localized on the X chromosome, consists of seven exons and six introns. Reductions in the gene expression of regucalcin have been suggested to serve a role in hepatocarcinogenesis in animal models and human patients, indicating a potential role as a suppressor protein in cancer. The aim of the current study was to investigate the effect of exogenous regucalcin on cell proliferation in the cloned human breast cancer MDA‑MB‑231 bone metastatic cell line in vitro. The proliferation of MDA‑MB‑231 cells was suppressed following the addition of regucalcin (0.1‑10 nM) in vitro. The suppression of proliferation was not enhanced in the presence of tumor necrosis factor‑α, PD98059, staurosporine, Bay K8644, wortmannin, 5,6‑dichloro‑1‑β‑D‑ribofuranosylbenzimidazole or gemcitabine. Exogenous regucalcin did not induce cell death in MDA‑MB‑231 cells in vitro. These data suggest that exogenous regucalcin possesses suppressive effects on the proliferation of human breast cancer MDA‑MB‑231 bone metastatic cells, and that this effect may be mediated through various intracellular signaling pathways in vitro. Exogenous regucalcin is suggested to function as a suppressor in cancer cell proliferation.

Regucalcin, which its gene is located on the X chromosome, plays a multifunctional role as a suppressor protein in cell signal transduction in various types of cells and tissues. The suppression of regucalcin gene expression has been shown to involve in carcinogenesis. Regucalcin gene expression was uniquely downregulated in carcinogenesis of rat liver in vivo, although the expression of other many genes was upregulated, indicating that endogenous regucalcin plays a suppressive role in the development of hepatocarcinogenesis. Overexpression of endogenous regucalcin was found to suppress proliferation of rat cloned hepatoma cells in vitro. Moreover, the regucalcin gene and its protein levels were demonstrated specifically to downregulate in human hepatocellular carcinoma by analysis with multiple gene expression profiles and proteomics. Regucalcin gene expression was also found to suppress in human tumor tissues including kidney, lung, brain, breast and prostate, suggesting that repressed regucalcin gene expression leads to the development of carcinogenesis in various tissues. Regucalcin may play a role as a suppressor protein in carcinogenesis. Overexpression of endogenous regucalcin is suggested to reveal preventive and therapeutic effects on carcinogenesis. Delivery of the regucalcin gene may be a novel useful tool in the gene therapy of carcinogenesis. This review will discuss regarding to an involvement of regucalcin as a suppressor protein in human carcinogenesis in insight into the gene therapy.

Regucalcin plays a pivotal role as a suppressor protein in signal transduction in various types of cells and tissues. The regucalcin gene is localized on the X chromosome. and its expression has been shown to be suppressed in various types of tumor tissue in animal and human subjects, suggesting a potential role of regucalcin in carcinogenesis. This study was undertaken to determine the effects of exogenous regucalcin on the proliferation of cloned human pancreatic cancer MIA PaCa-2 cells in vitro. The proliferation of the MIA PaCa-2 cells was suppressed following culture with regucalcin (0.01-10 nM). Such an effect was also observed in pancreatic cancer Pt45P1 cells, that highly expressed tissue factor (high TF), or Pt45P1 cells, that highly expressed alternativly spliced variants of tissue factor (asTF). In the MIA PaCa-2 cells, the suppressive effects of regucalcin on cell proliferation were not enhanced either in the presence of tumor necrosis factor-α (TNF-α), or in the presence of Bay K 8644, PD98059, staurosporine, wortmannin or 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB). However, this was not the case for gemcitabine, which was shown to suppress cell proliferation. Exogenous regucalcin did not cause apoptotic cell death in the MIA PaCa-2 cells in vitro. These findings demonstrate that exogenous regucalcin exerts suppressive effects on the proliferation of human pancreatic MIA PaCa-2 cells and that these effects are mediated through the inhibition of various signaling pathways related to nuclear factor-κB (NF-κB), extracellular signal-regulated kinase (ERK), protein kinase C, calcium signaling, phosphatidylinositol 3-kinase (PI3K) or nuclear transcription activity in vitro. Our data suggest that exogenous regucalcin exerts suppressive effects on the proliferation of human pancreatic cancer cells.

In this study, we first investigated the expressions of Jagged1, Notch2, the receptor activator of nuclear factor-kappa B ligand (RANKL), and interleukin (IL)-6 in areas of root resorption during experimental tooth movement in rats in vivo. We then assessed the effects of compression force (CF) with or without GSI (an inhibitor of Notch signaling) on Jagged1, RANKL, and IL-6 release from human periodontal ligament (hPDL) cells. Twelve male 6-wk-old Wistar rats were subjected to an orthodontic force of 50 g to induce mesially tipping movement of the upper first molars for 7 d. The expression levels of tartrate-resistant acid phosphatase, Jagged1, Notch2, IL-6, and RANKL proteins in the dental root were determined using an immunohistochemical analysis. Furthermore, the effects of the CF on Jagged1, IL-6, and RANKL production were investigated using hPDL cells in vitro. The effects of the cell-conditioned medium obtained from the hPDL cells subjected to CF (CFM) and Jagged 1 on osteoclastogenesis of human osteoclast precursor cells (hOCPs) were also investigated. Under the conditions of experimental tooth movement in vivo, resorption lacunae with multinucleated cells were observed in the 50 g group. In addition, immunoreactivity for Jagged1, Notch2, IL-6, and RANKL was detected on day 7 in the PDL tissue subjected to the orthodontic force. In the in vitro study, the compression force increased the production of Jagged1, IL-6, and RANKL from the hPDL cells, whereas treatment with GSI inhibited the production of these factors in vitro. The osteoclastogenesis increased with the CFM and rhJagged1, and the increase in the osteoclastogenesis was almost inhibited by GSI. These results suggest that the Notch signaling response to excessive orthodontic forces stimulates the process of root resorption via RANKL and IL-6 production from hPDL cells., (© International & American Associations for Dental Research 2014.)

Regucalcin plays a pivotal role as a suppressor protein in signal transduction in various cell types. The regucalcin gene, which is localized on the X chromosome, consists of 7 exons and 6 introns. Decreased liver regucalcin gene expression has been suggested to play a suppressive role in the development of hepatocellular carcinogenesis in animal models. This study was undertaken to determine the changes in regucalcin gene expression in various human normal and tumor tissues, including liver, kidney, brain and lung tissues. The full-length and alternatively spliced variants of regucalcin mRNA were found to be expressed in various human tissues. This expression was suppressed in tumor tissues of hepatocellular carcinoma, kidney transitional cell carcinoma, brain malignant meningioma and lung non-small cell carcinoma. The full-length regucalcin protein was found to be highly expressed in normal human liver and kidney tissues; its expression was suppressed, however, in the liver and kidney tumor tissues. The spliced variant proteins were found to be expressed in the normal liver and kidney tissues, and decreased in the tumor tissues. Such alternative variants were not observed in the liver and kidneys of rats and mice. The alternatively spliced variants of the regucalcin gene were found to be expressed in various human normal and tumor tissues.

Regucalcin was initially discovered in 1978 as a regulatory protein in calcium signaling. The regucalcin gene, which is localized on the X chromosome, is found in vertebrate and invertebrate species. Regucalcin has been shown to play a pivotal role in cell regulation: maintaining of intracellular calcium homeostasis, suppressions of signal transduction, inhibition of translational protein synthesis, nuclear deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis, regulation of gene expression, and anti-effects on proliferation and apoptosis in many cell types. The expression of the regucalcin gene and its protein has been shown to alter with various metabolic diseases, and regucalcin plays an important role in the development of many pathophysiologic states. Serum regucalcin has been found to increase with liver injury, and also urinary regucalcin is elevated with kidney damage, suggesting a useful tool as biomarker for diagnosis. Moreover, regucalcin has been shown to be good tool in early diagnosis for Alzheimer's disease and other brain diseases. This review will discuss a significance of regucalcin as a clinical biomarker in various diseases.

Regucalcin, which was discovered as a calcium-binding protein in 1978, has been demonstrated to play a multifunctional role in the regulation of various tissues and cell types. Regucalcin plays a pivotal role in the regulation of intracellular calcium homeostasis, various enzyme activities, cell signal transduction, nuclear function and gene expression, and cell proliferation and apoptosis. Moreover, regucalcin has been found to play a role in the regulation of bone homeostasis. Overexpression of regucalcin induces bone loss in regucalcin transgenic rats in vivo and deficiency causes osteomalacia in vivo. Regucalcin mRNA and its protein are expressed in rat femoral tissues, bone marrow cells, and osteoblastic cells. Exogenous regucalcin has suppressive effects on the differentiation and mineralization of osteoblastic MC3T3-E1 cells and stimulates osteoclastogenesis in mouse bone marrow culture in vitro. Moreover, regucalcin has been found to suppress osteoblastogenesis and stimulate adipogenesis in the bone marrow culture system in vitro. Regucalcin shows enhancing effects on activation of NF-κB, which is mediated through tumor necrosis factor-α (TNF-α) or the receptor activator of the NF-κB ligand (RANKL) in preosteoblastic cells and preosteoclastic cells. Exogenous regucalcin may play a pivotal role in the regulation of bone homeostasis as a suppressor in osteoblastogenesis and an enhancer in osteoclastogenesis, suggesting its role as a cytokine.

Regucalcin (RGN/SMP30) was discovered in 1978 as a calcium (Ca(2+))-binding protein that contains no EF-hand motif of the Ca(2+)-binding domain. The name of regucalcin was proposed for this Ca(2+)-binding protein, which can regulate various Ca(2+)-dependent enzyme activations in liver cells. The regucalcin gene is localized on the X chromosome. Regucalcin plays a multifunctional role in cell regulation through maintaining intracellular Ca(2+) homeostasis and suppressing signal transduction in various cell types. The cytoplasmic regucalcin is translocated into the nucleus and inhibits nuclear Ca(2+)-dependent and -independent protein kinases and protein phosphatases, Ca(2+)-activated deoxyribonucleic acid (DNA) fragmentation and DNA and ribonucleic acid (RNA) synthesis. Moreover, overexpression of endogenous regucalcin regulates the gene expression of various proteins that are related to cell proliferation and apoptosis. This review will discuss the role of regucalcin in the regulation of cell nuclear function and an involvement in gene expression as a novel transcription factor.

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 Ca(2+)-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 Ca(2+)-activated DNA fragmentation, Ca(2+)/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.

Regucalcin (RGN/SMP30) was originally discovered in 1978 as a unique calcium-binding protein that does not contain the EF-hand motif of calcium-binding domain. The regucalcin gene (rgn) is localized on the X chromosome and is identified in over 15 species consisting the regucalcin family. Regucalcin has been shown to play a multifunctional role in cell regulation; maintaining of intracellular calcium homeostasis and suppressing of signal transduction, translational protein synthesis, nuclear deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis, proliferation, and apoptosis in many cell types. Moreover, regucalcin may play a pathophysiological role in metabolic disorder. The expression of regucalcin is stimulated through the action of insulin in liver cells in vitro and in vivo and it is decreased in the liver of rats with type I diabetes induced by streptozotocin administration in vivo. Overexpression of endogenous regucalcin stimulates glucose utilization and lipid production in liver cells with glucose supplementation in vitro. Regucalcin reveals insulin resistance in liver cells. Deficiency of regucalcin induces an impairment of glucose tolerance and lipid accumulation in the liver of mice in vivo. Overexpression of endogenous regucalcin has been shown to decrease triglyceride, total cholesterol and glycogen contents in the liver of rats, inducing hyperlipidemia. Leptin and adiponectin mRNA expressions in the liver tissues are decreased in regucalcin transgenic rats. Decrease in hepatic regucalcin is associated with the development and progression of nonalcoholic fatty liver disease and fibrosis in human patients. Regucalcin may be a key molecule in lipid metabolic disorder and diabetes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Regucalcin (RGN/SMP30) was discovered in 1978 and is a unique calcium-binding protein contains no EF-hand motif calcium-binding domain. Its name, regucalcin, was proposed as it suppresses activation of enzymes related to calcium signalling. The regucalcin gene (rgn) is localized on the X chromosome. Regucalcin plays its role of suppressor protein in intracellular signalling pathways, including of protein kinases and protein phosphatase activities, protein synthesis, and DNA and RNA synthesis in liver cells. Overexpression of endogenous regucalcin has a suppressive effect on cell proliferation in modelled rat hepatoma H4-II-E cells, which are induced by various signalling stimulations in vitro. This suppressive effect is independent of apoptosis. Endogenous regucalcin plays a suppressive role on overproduction of proliferating cells in regenerating rat liver in vivo. Regucalcin mRNA expression is uniquely down-regulated in development of carcinogenesis in liver of rats in vivo. Regucalcin mRNA and protein expressions are also depressed in human hepatoma HepG2 cells, MCF-7 breast cancer cells, and prostate cancer LNCaP cells. Depression of regucalcin expression may be associated with activity progression of carcinogens. Regucalcin may be a key molecule suppressor protein in cell proliferation and carcinogenesis., (© 2013 Blackwell Publishing Ltd.)

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 hyperlipidemia. Consistent with these effects exogenous regucalcin has been shown to promote osteoclastogenesis in mouse bone marrow cultures and to suppress the differentiation and mineralization of MC3T3 osteoblast precursors. Regucalcin may induce hyperlipidemia in vivo by suppressing osteoblast differentiation and stimulating adipogenesis in bone marrow mesenchymal stem cells. The present study demonstrates that exogenous regucalcin suppresses differentiation to osteoblasts and stimulates adipogenesis in mouse bone marrow cell culture ex vivo. Moreover, exogenous regucalcin was found to enhance adipogenesis stimulated by insulin which is involved in the extracellular signal-related kinase pathway in 3T3-L1 adipocytes in vitro.

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.

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.

Neural circuitry formation depends on the molecular control of axonal projection during development. By screening with fluorophore-assisted light inactivation in the developing mouse brain, we identified cartilage acidic protein-1B as a key molecule for lateral olfactory tract (LOT) formation and named it LOT usher substance (LOTUS). We further identified Nogo receptor-1 (NgR1) as a LOTUS-binding protein. NgR1 is a receptor of myelin-derived axon growth inhibitors, such as Nogo, which prevent neural regeneration in the adult. LOTUS suppressed Nogo-NgR1 binding and Nogo-induced growth cone collapse. A defasciculated LOT was present in lotus-deficient mice but not in mice lacking both lotus- and ngr1. These findings suggest that endogenous antagonism of NgR1 by LOTUS is crucial for normal LOT formation.

Regucalcin was discovered in 1978 as a calcium-binding protein that does not contain EF-hand motif of calcium-binding domain (Yamaguchi and Yamamoto Chem Pharm Bull 26:1915-1918, 1978). The name regucalcin was proposed for this calcium-binding protein, which can regulate various Ca(2+)-dependent enzyme activations in liver cells. The regucalcin gene is localized on the chromosome X, and the organization of the regucalcin gene consists of seven exons and six introns. AP-1, NF1-A1, and RGPR-p117 bind to the promoter region of the rat regucalcin gene and enhance transcription activity of regucalcin gene expression that is mediated through calcium signaling. Regucalcin plays a pivotal role in the keep of intracellular calcium ion (Ca(2+)) homeostasis due to activating Ca(2+) pump enzymes in the plasma membrane (basolateral membrane), microsomes (endoplasmic reticulum), mitochondria, and nuclei of many cell types. Regucalcin has a suppressive effect on calcium signaling from the cytoplasm to the nucleus in the proliferative cells. Regucalcin has also been demonstrated to transport to the nucleus, and it can inhibit Ca(2+)-dependent protein kinase and protein phosphatase activities, Ca(2+)-activated deoxyribonucleic acid (DNA) fragmentation, and DNA and ribonucleic acid (RNA) synthesis in the nucleus. Overexpression of regucalcin suppresses cell death and apoptosis in the cloned rat hepatoma cells induced by various signaling factors. Regucalcin can inhibit the enhancement of cell proliferation due to hormonal stimulation. Regucalcin plays an important role as a regulatory protein in cell signaling system, and it is proposed to play a pivotal role in keep of cell homeostasis and function.

Regucalcin, which is discovered as a calcium-binding protein in 1978, has been shown to play a multifunctional role in many tissues and cell types; regucalcin has been proposed to play a pivotal role in keeping cell homeostasis and function for cell response. Regucalcin and its gene are identified in over 15 species consisting of regucalcin family. Comparison of the nucleotide sequences of regucalcin from vertebrate species is highly conserved in their coding region with throughout evolution. The regucalcin gene is localized on the chromosome X in rat and human. The organization of rat regucalcin gene consists of seven exons and six introns and several consensus regulatory elements exist upstream of the 5'-flanking region. AP-1, NF1-A1, RGPR-p117, β-catenin, and other factors have been found to be a transcription factor in the enhancement of regucalcin gene promoter activity. The transcription activity of regucalcin gene is enhanced through intracellular signaling factors that are mediated through the phosphorylation and dephosphorylation of nuclear protein in vitro. Regucalcin mRNA and its protein are markedly expressed in the liver and kidney cortex of rats. The expression of regucalcin mRNA in the liver and kidney cortex has been shown to stimulate by hormonal factors (including calcium, calcitonin, parathyroid hormone, insulin, estrogen, and dexamethasone) in vivo. Regucalcin mRNA expression is enhanced in the regenerating liver after partial hepatectomy of rats in vivo. The expression of regucalcin mRNA in the liver and kidney with pathophysiological state has been shown to suppress, suggesting an involvement of regucalcin in disease. Liver regucalcin expression is down-regulated in tumor cells, suggesting a suppressive role in the development of carcinogenesis. Liver regucalcin is markedly released into the serum of rats with chemically induced liver injury in vivo. Serum regucalcin has a potential sensitivity as a specific biochemical marker of chronic liver injury with hepatitis. Regucalcin has been proposed to be a key molecule in cellular regulation and metabolic disease.

Syntrophin components of the dystrophin glycoprotein complex (DGC) feature multiple protein interaction domains that may act in molecular scaffolding, recruiting signaling proteins to membranes and the DGC. Drosophila Syntrophin-1 (Syn1) and Syntrophin-2 (Syn2) are counterparts of human alpha1/beta1/beta2-syntrophins and gamma1/gamma2-syntrophins, respectively. alpha1/beta1/beta2-syntrophins are well documented, while little is known about gamma1/gamma2-syntrophins. Here, we performed immunohistochemical analyses with a specific antibody to Syn2 and demonstrated predominant expression in the larval and adult central nervous system. To investigate the in vivo functions of Syn2, we have generated Drosophila Syn2 deficiency mutants. Although the Syn2 mutants exhibit no overt phenotype, the combination of Syn1 knockdown and Syn2(37) mutation dramatically shortened life span, synergistically reduced locomotion ability and synergistically enhanced overgrowth of neuromuscular junctions in N-ethylmaleimide sensitive factor 2 mutants. From these data we conclude that Syn1 and Syn2 are required for locomotion and are involved in regulation of synaptic morphology. In addition, the two syntrophins can at least partially compensate for each other's functions.

Regucalcin transgenic (TG) rat has been generated to determine the role in metabolic disorders. Regucalcin homozygote male and female rats induce a prominent increase in regucalcin protein in the various tissues. Bone loss has been found to induce in regucalcin TG rats with growing (5 weeks old) and aging (50 weeks old). Osteoclastogenesis has been shown to stimulate in culture with the bone marrow cells obtained from regucalcin TG rats. Exogenous regucalcin stimulates osteoclastogenesis in mouse marrow culture in vitro. Regucalcin has a suppressive effect on the differentiation and mineralization in osteoblastic MC3T3-E1 cells in vitro. The mechanism by which regucalcin TG rat induces bone loss may result from the enhancement of osteoclastic bone resorption and the suppression of osteoblastic bone formation. Moreover, regucalcin TG rat has been found to induce hyperlipidemia with increasing age (14-50 weeks); serum triglyceride, high-density lipoprotein (HDL)-cholesterol, free fatty acid, albumin and calcium concentrations are markedly increased in regucalcin TG male and female rats with increasing age. The decrease in lipid and glycogen contents in liver tissues is induced in regucalcin TG rats. The gene expression of leptin and adiponectin is suppressed in the TG rats. 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 insulin resistance is seen in the cells. The expression of glucose transporter 2 mRNA is increased in the transfectants, while it has been shown to suppress insulin receptor and phosphatidylinositol 3-kinase mRNA expressions that are involved in insulin signaling. This review proposes that regucalcin relates in osteoporosis and hyperlipidemia, and that the regucalcin TG rat model may be useful in determining the pathophysiologic state and the development of therapeutic tool for osteoporosis and hyperlipidemia.

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.

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.

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.

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.

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.

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.

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.

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., (c 2006 Wiley-Liss, Inc.)

The role of regucalcin, which is a regulatory protein in intracellular signaling pathway, in the regulation of glucose utilization and lipid production was investigated using the cloned rat hepatoma H4-II-E cells overexpressing regucalcin. The hepatoma cells (wild-type) and stable regucalcin/pCXN2-transfected cells (transfectant) 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 or 72 h in medium containing either vehicle or insulin (10(-8) or 10(-7) M) with or without supplementation of glucose (10, 25, or 50 mg/ml of medium) in the absence of insulin. The production of triglyceride and free fatty acid was significantly increased in transfectants cultured without insulin and glucose supplementation as compared with that of wild-type cells. The supplementation of glucose (10, 25, or 50 mg/ml) caused a remarkable increase in medium glucose consumption, triglyceride, and free fatty acid productions in transfectants cultured without insulin. The presence of insulin (10(-7) M) caused a significant increase in medium glucose consumption, triglyceride, and free fatty acid productions in wild-type cells cultured with glucose supplementation. These increases were significantly prevented in transfectants cultured for 72 h. The expression of acetyl-CoA carboxylase, HMG-CoA reductase, glucokinase, pyruvate kinase, and glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNAs in wild-type cells was not significantly changed by culture with or without glucose supplementation in the presence of insulin. These gene expressions were not significantly changed in transfectants. The expression of glucose transporter 2 mRNA was significantly increased in transfectants as compared with that of wild-type cells. Such an increase was not seen in transfectants cultured in the presence of insulin with or without glucose supplementation. This study demonstrates that overexpression of regucalcin enhances glucose utilization and lipid production in the cloned rat hepatoma H4-II-E cells, and that it regulates the effect of insulin., ((c) 2006 Wiley-Liss, Inc.)

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.

A novel protein RGPR-p117 was discovered as regucalcin gene promoter region-related protein that binds to the TTGGC motif using a yeast one-hybrid system. RGPR-p117 is localized in the nucleus of kidney cells, and overexpression of RGPR-p117 can modulate regucalcin protein and its mRNA expression in the cloned normal rat kidney proximal tubular epithelial NRK52E cells. This study was undertaken to determine whether overexpression of RGPR-p117 enhances the regucalcin promoter activity using the -710/+18 LUC construct (wild-type) or -710/+18 LUC construct (mutant) with deletion of -523/-435 including TTGGC motif. NRK52E cells (wild-type) or stable HA-RGPR-p117/phCMV2-transfected cells (transfectant) were cultured in Dulbecco's minimum essential medium (DMEM) containing 5% bovine serum (BS). Wild-type cells or transfectants were transfected with the -710/+18 LUC construct vector or the -710/+18 LUC construct with deletion of -523/-435. Wild-type cells or transfectants with subconfluency were cultured for 48 h in a DMEM medium containing either vehicle, BS (5%), or parathyroid hormone (1-34) (PTH; 10(-7) M). Luciferase activity in wild-type cells was significantly increased with culture of BS or PTH. This increase was significantly blocked in the presence of various protein kinase inhibitors (staurosporine and PD 98059). Luciferase activity in transfectants was significantly increased as compared with that of wild-type cells in the absence of BS or PTH. The increase in luciferase activity in transfectants was completely decreased in mutant with deletion of -523/-435 sequence of regucalcin promoter. This was also seen using the -710/+18 LUC construct with deletion of -523/-503 sequence containing TTGGC motif. The increase in luciferase activity in transfectants was not significantly enhanced with culture of BS (5%), PTH (10(-7) M), Bay K 8644 (10(-6) M), phorbol 12-myristate 13-acetate (PMA; 10(-6) M), or N(6), 2'-dibutyryl cyclic adenosine 3', 5'-monophosphate (DcAMP; 10(-4) M). The increase in luciferase activity in transfectants was completely inhibited with culture of dibucaine (10(-6) M), staurosporine (10(-9) M), PD 98059 (10(-8) M), wortmannin (10(-8) M), genistein (10(-6) M), vanadate (10(-6) M), or okadaic acid (10(-6) M) which are inhibitors of various kinases and protein phosphatases. This study demonstrates that RGPR-p117 can enhance the regucalcin promoter activity which is related to the NF-1 consensus sequences including TTGGC motif, and that its enhancing effect is partly mediated through phosphorylation and dephosphorylation in NRK52E cells., (Copyright 2006 Wiley-Liss, Inc.)

Nuclear factor I-A1 (NF1-A1) can bind to the TTGGC motif in the rat regucalcin gene promoter region. This study was undertaken to determine whether NF1-A1 is involved in the enhancement of the rat regucalcin gene promoter activity using the -710/+18 LUC construct (wild-type) or -710/+18 LUC construct with the deletion of -523/-435 including the TTGGC motif (mutant) in cloned normal rat kidney proximal tubular epithelial NRK52E cells. Cells were transfected with the -710/+18 LUC construct vector or the -710/+18 LUC construct with the deletion of -523/-435. NRK52E cells (wild-type) or NRK52E cells transiently transfected with HA-NF1-A1/phCMV2 were cultured for 48 h in a medium containing either vehicle or BS (5%) in the presence or absence of various factors. HA-NF1-A1 was localized in the nucleus of wild-type cells. Luciferase activity was significantly increased as compared to that of wild-type cells. This increase was significantly enhanced in the presence of phorbol 12-myristate 13-acetate (PMA; 10(-6) M). Such an enhancement was not seen by culture with Bay K 8644 (10(-6) M) or dibutyryl cyclic AMP (10(-4) M). The increase in luciferase activity in NRK52E cells transfected with HA-NF1-A1 was not observed in the presence of dibucaine (10(-6) M), staurosporine (10(-9) M), or PD 98059 (10(-8) M), which is an inhibitor of various protein kinases. Such an inhibition was also seen in the presence of vanadate (10(-6) M) or okadaic acid (10(-6) M), an inhibitor of protein phosphatase. The increase in luciferase activity in NRK52E cells transfected with HA-NF1-A1/ phCMV2 was not seen in the mutant with deletion of -523/-435. The increase in luciferase activity in HA-NF1-A1/ phCMV2-transfected NRK52E cells was not significantly enhanced in the cells transiently co-transfected with HA-RGPR-p117/phCMV2, which could increase the regucalcin gene promoter activity using the -710/+18 LUC construct (wild-type). This study demonstrates that NF1-A1 enhances the regucalcin promoter activity which is related to the TTGGC motif, and that its enhancing effect is partly mediated through phosphorylation in NRK52E cells.

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.

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.

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., (Copyright 2005 Wiley-Liss, Inc.)

A 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. Whether overexpression of RGPR-p117 can modulate gene expression in the cloned normal rat kidney proximal tubular epithelial NRK52E cells was investigated. NRK52E cells (wild-type) or HA-RGPR-p117/phCMV2-transfected NRK52E cells were cultured in Dulbecco's minimum essential medium (DMEM) containing 5% bovine serum (BS). Proliferation of NRK52E cells (wild-type) was not significantly altered by overexpression of HA-RGPR-p117. The expression of rat regucalcin, alpha-fetoprotein, albumin, glucokinase, 11beta-hydroxy-steroid dehydrogenase, phosphoenolpyruvate carboxykinase, which contains TTGGC motif in the promoter region of their genes, was seen in NRK52E cells (wild-type) by using reverse transcription-polymerase chain reaction (RT-PCR). Of these genes, regucalcin mRNA levels were significantly enhanced in transfectants. The expression of p21 or glycero-aldehyde-3-phosphate dehydrogenase mRNA was not significantly changed in transfectants. The results of Western blot analysis showed that regucalcin protein was significantly increased in transfectants. The enhancement of regucalcin mRNA expression in transfectants was significantly suppressed in the presence of staurosporine (10(-10) M), an inhibitor of protein kinase C. This enhancement was not significantly changed in the presence of dibucaine (10(-8) M), PD98059 (10(-8) M) or vanadate (10(-6) M). This study demonstrates that overexpression of RGPR-p117 enhances the expression of regucalcin mRNA and its protein level in NRK52E cells. RGPR-p117 may play a role as a transcriptional factor.

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., ((c) 2005 Wiley-Liss, Inc.)

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.