Suppressive effect of endogenous regucalcin on nitric oxide synthase activity in cloned rat hepatoma H4‐II‐E cells overexpressing regucalcin

Izumi, Takako; Tsurusaki, Yoshinori; Yamaguchi, Masayoshi

doi:10.1002/jcb.10544

Cited by 23 publications

(32 citation statements)

References 44 publications

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“…We have noted further that SMP30 is localized in both the nuclei and cytoplasm of cultured murine hepatocytes and that a domain consisting of 51 amino acid residues has a 60 -66% similarity to bacterial and yeast RNA polymerases (18). The diverse subcellular localization of SMP30 may support its many functions in intracellular signaling processes such as activation of Akt on apoptosis (30) or suppression of nuclear DNA and RNA synthesis (21,22). These divergent properties of SMP30 seem to explain why VC supplementation did not completely restore lung morphology or levels of collagen I mRNA and ROS in SMP30 VC(ϩ) mice to those of WT mice despite the comparable VC level attained in their lungs.…”

Section: Discussionmentioning

confidence: 78%

Complete lack of vitamin C intake generates pulmonary emphysema in senescence marker protein-30 knockout mice

Koike

Kondo

Sekiya

et al. 2010

American Journal of Physiology-Lung Cellular and Molecular Phys

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Vitamin C (VC) is a potent antioxidant and plays an essential role in collagen synthesis. As we previously reported, senescence marker protein-30 (SMP30) knockout (KO) mice cannot synthesize VC due to the genetic disruption of gluconolactonase (i.e., SMP30). Here, we utilized SMP30 KO mice deprived of VC and found that VC depletion caused pulmonary emphysema due to oxidative stress and a decrease of collagen synthesis by the third month of age. We grew SMP30 KO mice and wild-type (WT) mice on VC-free chow and either VC water [VC(ϩ)] or plain water [VC(Ϫ)] after weaning at 4 wk of age. Morphometric findings and reactive oxygen species (ROS) in the lungs were evaluated at 3 mo of age. No VC was detected in the lungs of SMP30 KO VC(Ϫ) mice, but their ROS increased 50.9% over that of the VC(ϩ) group. Moreover, their collagen content in the lungs markedly decreased, and their collagen I mRNA decreased 82.2% compared with that of the WT VC(Ϫ) group. In the SMP30 KO VC(Ϫ) mice, emphysema developed [21.6% increase of mean linear intercepts (MLI) and 42.7% increase of destructive index compared with VC(ϩ) groups], and the levels of sirtuin 1 (Sirt1) decreased 16.8%. However, VC intake increased the MLI 16.2% and thiobarbituric acid reactive substances 22.2% in WT mice, suggesting that an excess of VC can generate oxidative stress and may be harmful during this period of lung development. These results suggest that VC plays an important role in lung development through affecting oxidant-antioxidant balance and collagen synthesis. chronic obstructive pulmonary disease; collagen VITAMIN C (VC) is a water-soluble, hexonic sugar acid that has two dissociable protons (40) and is abundant in fluids of the lung epithelial lining (50). VC scavenges free radicals such as superoxide (34), singlet oxygen (4), and hydroxyl radicals (2). Several reports indicate that VC has a beneficial effect on lung function but, when depleted during aging, may promote chronic obstructive pulmonary disease (COPD; Refs. 41, 44). On the other hand, VC also plays an essential role in collagen synthesis because it acts as a cofactor for prolyl hydroxylase to catalyze hydroxyproline synthesis, which is involved in collagen helix stabilization (33, 38). Furthermore, VC stimulates collagen biosynthesis not only by promoting the activity of the prolyl hydroxylase, but also by increasing the mRNA of collagen (I and III; Refs. 13,35).Senescence marker protein-30 (SMP30) is characterized by its ever-decreasing content in the liver, kidney, and lung with aging, a decrease that is androgen-independent (11). To clarify the physiological role of SMP30 in age-associated organ disorders, we used gene targeting to establish the SMP30 knockout (KO) mouse from C57BL/6 mice (17). Recently, we (24) reported that SMP30 has gluconolactonase (GNL) activity. Since GNL is a key enzyme in the VC biosynthetic pathway of mammals, mice deprived of GNL (SMP30 KO mice) lack the ability to synthesize VC (19). We (42) further discovered that oxidative stress is greater in the lungs o...

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Section: Discussionmentioning

confidence: 78%

Complete lack of vitamin C intake generates pulmonary emphysema in senescence marker protein-30 knockout mice

Koike

Kondo

Sekiya

et al. 2010

American Journal of Physiology-Lung Cellular and Molecular Phys

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“…Regucalcin plays a role in maintaining intracellular Ca 2þ homeostasis, the inhibitory regulation of various Ca 2þ -dependent protein kinases and tryrosine kinases, protein phosphatases, nitric oxide (NO) synthase, and the control of the enhancement of nuclear deoxyribonucleic acid (DNA) and RNA synthesis in proliferative cell [Tsurusaki and Yamaguchi, 2002a,b;Izumi et al, 2003;Yamaguchi, 2005]. Recent study has demonstrated that regucalcin has a suppressive effect on cell proliferation and apoptosis, which is mediated through many signaling factors, in the cloned rat hepatoma H4-II-E cells overexpressing regucalcin Izumi and Yamaguchi, 2004a,b].…”

Section: Introductionmentioning

confidence: 99%

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

Yamaguchi

Kobayashi

Uchiyama

2005

J of Cellular Biochemistry

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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.

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“…Regucalcin plays a role in the maintenance of intracellular Ca 2þ homeostasis, the inhibitory regulation of various Ca 2þ -dependent protein kinases and thyrosine kinases, protein phosphatases, NO synthase, protein synthesis, and the control of the enhancement of DNA and RNA syntheses in proliferative cells [Yamaguchi, 2000a[Yamaguchi, ,b, 2005Tsurusaki and Yamaguchi, 2002a,b;Izumi et al, 2003]. Regucalcin may play a pivotal role in the regulation of cell function.…”

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confidence: 99%

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

Nakagawa

Yamaguchi

2005

J of Cellular Biochemistry

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101

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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.

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Suppressive effect of endogenous regucalcin on nitric oxide synthase activity in cloned rat hepatoma H4‐II‐E cells overexpressing regucalcin

Cited by 23 publications

References 44 publications

Complete lack of vitamin C intake generates pulmonary emphysema in senescence marker protein-30 knockout mice

Complete lack of vitamin C intake generates pulmonary emphysema in senescence marker protein-30 knockout mice

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

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

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