Keiichi Tanaka

Publications of Keiichi Tanaka

• Oxidation and turnover of renal metallothioneins after an injection of ferric nitrilotriacetate.

  Authors: Kyong-Son Min, Naomi Kishi, Nozomu Yamashita, Keiichi Tanaka

  Chemico-biological interactions. 01/2012; 195(1):61-7.

  Metallothioneins (MTs) have demonstrated strong antioxidant properties, however the biological significance of their effect against hydroxyl radical toxicity remains unclear. We investigated theMetallothioneins (MTs) have demonstrated strong antioxidant properties, however the biological significance of their effect against hydroxyl radical toxicity remains unclear. We investigated the oxidation and turnover of renal MTs in MT-preinduced mice after an injection of ferric nitrilotriacetate (Fe-NTA). Incubation of MTs with Fe-NTA and H(2)O(2) resulted in a loss of their metal-binding properties and a decrease in their thiol concentration independent of binding potential and isoforms. Moreover, in vitro reduction of renal oxidized MT with dithiothreitol (DTT) reversed these oxidative changes. An injection of Fe-NTA oxidized renal preinduced MT in Zn- and Cd-pretreated mice. The metal-binding properties of renal MTs were lost when the Fe-NTA dose was increased. However, analysis of renal MTs using an immunoassay showed that its protein concentration did not decrease 4h after the injection with various Fe-NTA doses. Furthermore, in vitro reduction of renal oxidized MTs with DTT resulted in an increase in the concentration of metals in the MT fraction. These data indicate that radicals produced by Fe-NTA may oxidize MTs in vitro and in vivo. When we investigated the turnover of oxidized MTs in Fe-NTA-treated mice, effects on the concentration of renal (35)S-labeled MTs were opposite to those observed in Cd-pretreated mice. The concentration of preinduced (35)S-labeled MTs in the kidneys of Cd-pretreated mice showed a significant decrease (p<0.05), whereas that of newly synthesized (35)S-labeled MTs showed a considerable increase. These data suggest that degradation of oxidized MTs may be faster than intact MTs. Therefore, the radical scavenging system of MTs may include their induction and degradation during oxidative stress conditions.

• Enhanced in vivo gene transfer into the placenta using RGD fiber-mutant adenovirus vector.

  Authors: Kazufumi Katayama, Rie Furuki, Hideaki Yokoyama, Makoto Kaneko, Masashi Tachibana, Ichiro Yoshida, Hisamitsu Nagase, Keiichi Tanaka, Fuminori Sakurai, Hiroyuki Mizuguchi, Shinsaku Nakagawa, Tsuyoshi Nakanishi

  Biomaterials. 03/2011; 32(17):4185-93.

  Among viral vectors, the fiber-mutant adenovirus vector carrying the Arg-Gly-Asp (RGD) peptide sequence (Ad-RGD) seems to have potential for both clinical gene therapy and basic research. As a partAmong viral vectors, the fiber-mutant adenovirus vector carrying the Arg-Gly-Asp (RGD) peptide sequence (Ad-RGD) seems to have potential for both clinical gene therapy and basic research. As a part of a thorough evaluation of Ad-RGD in preclinical studies, we designed an experiment to investigate in detail the distribution of Ad-RGD compared with conventional adenovirus vector (WT-Ad) in pregnant mice. Surprisingly, Ad-RGD had substantial placental tropism, at 10-100 times that of WT-Ad. Transgene expression was sustained for at least 7 days, and Ad-RGD expressing firefly luciferase or red fluorescent protein has so far caused no placental dysfunction leading to fetal death. Ad-RGD showed high levels of transduction efficiency in in vitro-differentiated trophoblast stem cells, in which higher expression of αvβ3 integrin than in undifferentiated cells was observed. Our results suggest that the use of Ad-RGD or another RGD-mediated targeting strategy holds promise for drug delivery to the placenta.

• β-Citryl-L-glutamate acts as an iron carrier to activate aconitase activity.

  Authors: Michiko Hamada-Kanazawa, Masanori Narahara, Masaoki Takano, Kyong Son Min, Keiichi Tanaka, Masaharu Miyake

  Biological & pharmaceutical bulletin. 01/2011; 34(9):1455-64.

  The compound β-citryl-L-glutamate (β-CG) was initially isolated from developing brains, though its functional roles remain unclear. In in vitro experiments, the [Fe(II)(β-CG)] complex activatedThe compound β-citryl-L-glutamate (β-CG) was initially isolated from developing brains, though its functional roles remain unclear. In in vitro experiments, the [Fe(II)(β-CG)] complex activated aconitase in the presence of reducing reagents, whereas no Fe complex with citrate, glutamate, or deferoxamine displayed such an effect. β-CG and [Fe(II)(β-CG)] both bound to the fourth labile Fe atom (Fe(a)) in the [4Fe-4S] cluster of aconitase. Furthermore, [Fe(II)(β-CG)] reactivated aconitase damaged by ammonium peroxodisulfate (APS), while β-CG and citrate had no effect. These findings suggest that [Fe(II)(β-CG)] can transfer Fe to aconitase disassembled by APS. In intact mitochondria, both β-CG and [Fe(II)(β-CG)] bound to Fe(a) of aconitase, whereas only [Fe(II)(β-CG)] reactivated the enzyme disassembled by APS. In cultured neuronal cells, β-CG significantly enhanced cell viability by accelerating mitochondrial activity in primary cultures of neurons from newborn mouse cerebrum tissues. Thus, the β-CG plays a role as an Fe-carrier for mitochondrial aconitase, and then activates it. Taken together, these findings suggest that β-CG is an endogenous low molecular weight Fe chaperone for aconitase.

• Increased hepatic accumulation of ingested Cd is associated with upregulation of several intestinal transporters in mice fed diets deficient in essential metals.

  Authors: Kyong-Son Min, Hidenori Ueda, Tetsuya Kihara, Keiichi Tanaka

  Toxicological sciences : an official journal of the Society of Toxicology. 11/2008; 106(1):284-9.

  Essential metals (EMs) can affect the metabolism of nonessential metals. It has been suggested that Fe deficiency increases intestinal absorption of Cd via divalent metal transporter 1 (DMT1). ToEssential metals (EMs) can affect the metabolism of nonessential metals. It has been suggested that Fe deficiency increases intestinal absorption of Cd via divalent metal transporter 1 (DMT1). To investigate whether EM nutritional status is a host risk factor for Cd accumulation, we studied the effect of nutritional status of Ca, Cu, Mg, Zn, and Fe that most often ingested by humans at levels below recommended dietary allowances on tissue accumulation of orally administered Cd. Mice were divided into groups and given different EM-deficient (EMDF) diets (CaDF, CuDF, MgDF, ZnDF, or FeDF) for 4 weeks. EMDF mice had significantly (p < 0.05) lower plasma or hepatic concentrations of the deficient EM than did mice receiving control diets. Hepatic Cd accumulation was significantly (p < 0.05) increased after oral Cd administration in all EMDF mice, but not in any EM-supplemented mice. Intestinal expression of mRNAs for the Fe-transporters DMT1 and ferroportin was increased in FeDF mice, but not in other EMDF mice, causing an increase in hepatic Fe concentration. Similarly, intestinal expression of mRNA for calcium transporter 1 was significantly increased in CaDF mice, but not in other EMDF mice. These results suggest that DMT1 is not the sole transporter of Cd, and that Cd is absorbed and accumulated through multiple pathways that maintain EM homeostasis in EMDF condition. Therefore, EM nutritional status is a risk factor for increasing hepatic accumulation of ingested Cd.

• Effect of hemolytic and iron-deficiency anemia on intestinal absorption and tissue accumulation of cadmium.

  Authors: Kyong-Son Min, Naoyuki Iwata, Noriko Tetsutikawahara, Satomi Onosaka, Keiichi Tanaka

  Toxicology letters. 07/2008; 179(1):48-52.

  Abnormal iron (Fe) metabolism induces iron-deficiency anemia (FeDA) and also affects body cadmium (Cd) accumulation. However, whether hemolytic anemia also affects Cd metabolism is not known. WeAbnormal iron (Fe) metabolism induces iron-deficiency anemia (FeDA) and also affects body cadmium (Cd) accumulation. However, whether hemolytic anemia also affects Cd metabolism is not known. We compared the intestinal absorption and tissue accumulation of Cd after oral administration of Cd to mice with hemolytic anemia induced by treatment with phenylhydrazine (PHA mice) to that in mice with FeDA. Although the hematocrit decreased significantly in mice with either type of anemia, the Fe concentration decreased in the livers and kidneys of FeDA mice, but increased in those of PHA mice. After an oral administration with various amounts of Cd, hepatic and renal Cd concentrations significantly increased in both FeDA and PHA mice. An intraduodenal injection of Fe raised the hepatic Fe content in FeDA mice to the control level and raised the hepatic Fe content in PHA mice to 2.4 times that in control mice. Intestinal divalent metal transporter 1 (DMT1) expression increased significantly in mice with both types of anemia. These data indicate that, despite the accumulation of hepatic Fe associated with PHA, PHA also significantly increases hepatic and renal Cd accumulation according to an stimulation of intestinal DMT1 expression, as occurs in FeDA mice. This suggests that anemia may be a risk factor for Cd accumulation.

• Involvement of intestinal calcium transporter 1 and metallothionein in cadmium accumulation in the liver and kidney of mice fed a low-calcium diet.

  Authors: Kyong-Son Min, Hidenori Ueda, Keiichi Tanaka

  Toxicology letters. 02/2008; 176(1):85-92.

  Essential metals can affect the metabolism of nonessential metals. Calcium (Ca) is an essential mineral that is commonly lacking in the diet. When we fed 5-week-old male mice for 4 weeks on aEssential metals can affect the metabolism of nonessential metals. Calcium (Ca) is an essential mineral that is commonly lacking in the diet. When we fed 5-week-old male mice for 4 weeks on a purified diet containing 0.005% Ca (CaDF mice), the Ca concentration in the plasma, liver and kidneys did not decreased. Cd accumulation increased in the liver and kidneys of CaDF mice given 1mg/kg Cd orally each day for 5 days, but not in those given intraperitoneal injections of Cd or Cd-metallothionein (Cd-MT). The zinc (Zn) concentration increased significantly in the intestinal cytosol and plasma during the time the mice were fed the low-Ca diet, and expression of both MT-1 and ZnT-1 sharply increased with a similar time course. Intestinal mRNA expression of CaT1, a Ca transporter, was more than 10 times higher in CaDF mice than in controls, although expression of other transporters, including DMT1, decreased in CaDF mice. These results suggest that CaT1 may stimulate the intestinal absorption of Cd and Zn, and some Cd may be distributed to the kidneys along with MT induced by Zn.

• Ascorbic acid transported by sodium-dependent vitamin C transporter 2 stimulates steroidogenesis in human choriocarcinoma cells.

  Authors: Ximei Wu, Takuma Iguchi, Norio Itoh, Kousuke Okamoto, Tatsuya Takagi, Keiichi Tanaka, Tsuyoshi Nakanishi

  Endocrinology. 02/2008; 149(1):73-83.

  Reduced vitamin C [ascorbic acid (AA)], which is taken up into cells by sodium-dependent vitamin C transporter (SVCT) 1 and 2, is believed to be important for hormone synthesis, but its role inReduced vitamin C [ascorbic acid (AA)], which is taken up into cells by sodium-dependent vitamin C transporter (SVCT) 1 and 2, is believed to be important for hormone synthesis, but its role in generating placental steroids needed to maintain pregnancy and fetal development is not clear. To determine the steroidogenic effect of AA and the role of SVCT2 in AA-induced steroidogenesis, we tested the effects of AA treatment and SVCT2 knockdown on steroidogenesis in human choriocarcinoma cell lines. AA treatment of JEG-3, BeWo, and JAR cells for 48-h dose dependently increased progesterone and estradiol levels. In JEG-3 cells, AA increased the mRNA expression of P450 cholesterol side-chain cleavage enzyme, 3beta-hydroxysteroid dehydrogenase type 1, and aromatase, key enzymes for steroidogenesis. Stable knockdown of SVCT2 in JEG-3 cells by retrovirally mediated RNA interference decreased the maximal velocity of AA uptake by approximately 50%, but apparent affinity values were not affected. SVCT2 knockdown in JEG-3 cells significantly suppressed the AA-induced mRNA expression of placental P450 cholesterol side-chain cleavage enzyme, 3beta-hydroxysteroid dehydrogenase type 1, and aromatase. This suppression of the AA-induced mRNA expression of steroidogenic enzymes subsequently decreased progesterone and estradiol production. In addition, inhibition of MAPK kinase-ERK signaling, which is a major pathway for AA-regulated gene expression, failed to affect AA-induced steroidogenesis. Our observations indicate that SVCT2-mediated AA uptake into cells is necessary for AA-induced steroidogenesis in human choriocarcinoma cell, but MAPK kinase-ERK signaling is not involved in AA-induced steroidogenesis.

• Upregulation of sodium-dependent vitamin C transporter 2 expression in adrenals increases norepinephrine production and aggravates hyperlipidemia in mice with streptozotocin-induced diabetes.

  Authors: Ximei Wu, Takuma Iguchi, Junko Hirano, Isami Fujita, Hidenori Ueda, Norio Itoh, Keiichi Tanaka, Tsuyoshi Nakanishi

  Biochemical pharmacology. 11/2007; 74(7):1020-8.

  The hyperglycemia and hyperoxidation that characterize diabetes lead to reduced vitamin C (L-ascorbic acid, AA) levels in diabetic humans and animals. We examined the possibility thatThe hyperglycemia and hyperoxidation that characterize diabetes lead to reduced vitamin C (L-ascorbic acid, AA) levels in diabetic humans and animals. We examined the possibility that diabetes-induced low plasma AA levels impair AA distribution to various tissues and that these changes are closely related to the development of diabetic complications. AA levels were markedly decreased in the plasma and increased in the adrenals of mice with streptozotocin (STZ)-induced diabetes. Consistently with these results, in [1-(14)C]AA accumulation assays, the efficiency of [1-(14)C]AA accumulation was significantly higher in the adrenals (which had the greatest ability to accumulate [1-(14)C]AA) of diabetic mice than in those of controls. Expression of sodium-dependent vitamin C transporter (SVCT)-2, a transporter of AA, was upregulated in diabetic adrenals. Furthermore, increased AA incorporation into the diabetic adrenals by SVCT-2 led to increased plasma norepinephrine, triglyceride and free fatty acid levels in mice with STZ-induced diabetes. Therefore, oversupplementation with AA could be deleterious in diabetic patients, because overexpression of adrenal SVCT-2 in diabetes could lead to excessive AA uptake, thus enhancing norepinephrine production and exacerbating some diabetic complications. Interestingly, however, treatment with AA dose-dependently abolished the increased expression of adrenal SVCT-2 and normalized the abovementioned plasma parameters in diabetic mice. These results suggest SVCT-2-mediated increases in AA uptake by the adrenals followed by excessive production of plasma norepinephrine may play a pivotal role in the development of diabetic complications.

• Role of metal-responsive transcription factor-1 (MTF-1) in EGF-dependent DNA synthesis in primary hepatocytes.

  Authors: Tomoki Kimura, Norio Itoh, Tomomichi Sone, Masuo Kondoh, Keiichi Tanaka, Masakazu Isobe

  Journal of cellular biochemistry. 11/2006; 99(2):485-94.

  Metal-responsive transcription factor-1 (MTF-1), which is involved in sensing heavy metal load, induces the transcription of several protective genes. The mouse Mtf-1 gene is essential, andMetal-responsive transcription factor-1 (MTF-1), which is involved in sensing heavy metal load, induces the transcription of several protective genes. The mouse Mtf-1 gene is essential, and Mtf-1(-/-) embryos die from liver degeneration. We showed that DNA synthesis induced in hepatocytes by epidermal growth factor (EGF) was delayed by inhibition of MTF-1. To inhibit MTF-1 activity, MTFDeltaC, a C-terminal deletion mutant of MTF-1, was expressed by infection with the virus Ad5MTFDeltaC. Lactate dehydrogenase (LDH) release and/or caspase-3/7 activation was not observed under our experimental conditions. The inhibitory effect of MTFDeltaC on EGF-dependent DNA synthesis in hepatocytes was not eliminated by zinc addition. EGF-dependent extracellular signal-related kinase (ERK) phosphorylation, an essential reaction for EGF-dependent DNA synthesis, was decreased in MTF-1-inhibited hepatocytes. Moreover, decrease of ERK phosphorylation was observed by using siRNA in MTF-1-downregulated hepatocytes. These results indicate that MTF-1 is particularly important for proper hepatocyte proliferation. This is the first report to suggest the function of MTF-1 in the ERK pathway.

• Induction of zinc transporters by forskolin in human trophoblast BeWo cells.

  Authors: Nagayoshi Asano, Masuo Kondoh, Chiaki Ebihara, Makiko Fujii, Tsuyoshi Nakanishi, Naoki Utoguchi, Shuichi Enomoto, Keiichi Tanaka, Yoshiteru Watanabe

  Reproductive toxicology (Elmsford, N.Y.). 05/2006; 21(3):285-91.

  During pregnancy, the zinc level in fetal serum is up to two-fold higher than that in the maternal serum at the end of pregnancy, but the mechanism of zinc release from the placenta into fetalDuring pregnancy, the zinc level in fetal serum is up to two-fold higher than that in the maternal serum at the end of pregnancy, but the mechanism of zinc release from the placenta into fetal circulation is not well understood. In this study, we determined the expression profiles of zinc transporters in human trophoblast BeWo cells, a representative human trophoblast cell line. Zn transporter 1-8 (ZnT1-8), Zrt/IRT-like protein 1 and Zn transporter-like transporter 1 were detected in BeWo cells by reverse transcription-polymerase chain reaction. Forskolin (FK) is a representative inducer of differentiation of BeWo cells cytotrophoblast into syncytiotrophoblast. Treatment of BeWo cells with FK resulted in morphological changes of BeWo cells into syncytiotrophoblast cells and secretion of human chorionic gonadotropin, which is a characteristic of syncytiotrophoblast cells. Treatment of BeWo cells with FK elevated ZnT1, 2 and 4 mRNA levels. These data about expression profiles of ZnTs may be useful for further investigation of placental biology.

• Organotin compounds enhance 17beta-hydroxysteroid dehydrogenase type I activity in human choriocarcinoma JAr cells: potential promotion of 17beta-estradiol biosynthesis in human placenta.

  Authors: Tsuyoshi Nakanishi, Youhei Hiromori, Hideaki Yokoyama, Mihoko Koyanagi, Norio Itoh, Jun-ichi Nishikawa, Keiichi Tanaka

  Biochemical pharmacology. 04/2006; 71(9):1349-57.

  Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), are typical environmental contaminants and suspected endocrine-disrupting chemicals because they cause masculinization in femaleOrganotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), are typical environmental contaminants and suspected endocrine-disrupting chemicals because they cause masculinization in female mollusks. However, it remains unclear whether organotin compounds also cause crucial toxicities in human sexual development and reproductive functions. We investigated the effects of 17 tin compounds on the catalytic activity and mRNA expression of 17beta-hydroxysteroid dehydrogenase type I (17beta-HSD I) in human choriocarcinoma JAr cells. At nontoxic concentrations, both trialkyltins with propyl, butyl or cyclohexyl substituents on the tin atom and triphenyltin (TPT) enhanced 17beta-HSD I mRNA transcription and enzyme activity in a dose-dependent fashion. Although tetraalkyltin compounds such as tetrabutyltin and tributylvinyltin also increased the mRNA expression and enzyme activity of 17beta-HSD I, the concentrations necessary for activation were >30-100 times greater than those for trialkyltins. Inorganic tin had no effect on the catalytic activity and mRNA expression of 17beta-HSD I. Interestingly, diphenyltin and monophenyltin, which are metabolites of TPT, enhanced 17beta-HSD I activity with a concomitant increase in mRNA expression, whereas dibutyltin and monobutyltin, which are metabolites of tributyltin, enhanced 17beta-HSD I activity without a concomitant increase in mRNA expression. These results suggest that organotin compounds are potent stimulators of 17beta-estradiol biosynthesis to enhance 17beta-HSD I activity in the human placenta in vitro; the placenta represents a potential target organ for these compounds, whose endocrine-disrupting effects might be the result of local changes in 17beta-estradiol concentrations in pregnant women.

• Molecular targets of organotin compounds in endocrine disruption: do organotin compounds function as aromatase inhibitors in mammals?

  Authors: Tsuyoshi Nakanishi, Jun-ichi Nishikawa, Keiichi Tanaka

  Environmental sciences : an international journal of environmental physiology and toxicology. 02/2006; 13(2):89-100.

  Tributyltin (TBT) and triphenyltin (TPT) cause masculinization in female mollusks. These compounds may act as potential competitive inhibitors of aromatase, which converts androgens to estrogens,Tributyltin (TBT) and triphenyltin (TPT) cause masculinization in female mollusks. These compounds may act as potential competitive inhibitors of aromatase, which converts androgens to estrogens, although effective concentrations are high. TBT and TPT may, therefore, increase the levels of unconverted androgens in invertebrates and vertebrates. However, at concentrations effective for aromatase inhibition, they are generally toxic to mammalian cells. These compounds markedly enhance aromatase activity and human chorionic gonadotropin (hCG) production, along with their mRNA expression, at very low concentrations in human choriocarcinoma cells. In ovarian granulosa cells, these compounds suppress aromatase gene expression at the same low concentrations. Therefore, it is suspected that, in mammals, these organotin compounds affect target molecules that regulate the gene expressions of aromatase and hCG, rather than functioning as aromatase inhibitors. Recently, it has been demonstrated that TBT and TPT directly bind to the retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR) gamma with high affinity and function as transcriptional activators. These compounds promoted adipocyte differentiation, which is triggered by the PPAR gamma/RXR signaling pathway. They may, therefore, exert their toxic effects through the activation of these pathways in mammals. Here, we review the potential endocrine disruption of organotin compounds via these nuclear receptors in mammals.

• Reduced bactericidal activity and nitric oxide production in metallothionein-deficient macrophages in response to lipopolysaccharide stimulation.

  Authors: Norio Itoh, Hiroshi Shibayama, Masako Kanekiyo, Dunkokkuruad Namphung, Tsuyoshi Nakanishi, Akiko Matsuyama, Tomoyuki Odani, Keiichi Tanaka

  Toxicology. 01/2006; 216(2-3):188-96.

  This study was designed to investigate bactericidal activity of and nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated peritoneal exudate macrophages (Mvarphi) from metallothioneinThis study was designed to investigate bactericidal activity of and nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated peritoneal exudate macrophages (Mvarphi) from metallothionein (MT)-null mice. Control Mvarphi had a bactericidal effect on Staphylococcus aureus, but MT-null Mvarphi had significantly lower activity. NO is an important factor in the bactericidal function of Mvarphi. LPS-stimulated MT-null Mvarphi produced less NO than those of control mice. LPS-stimulated Mvarphi produce cytokines such as tumor necrosis factor (TNF)-alpha. TNF-alpha activate Mvarphi and stimulates NO production. We evaluated NO production by TNF-alpha-stimulated Mvarphi. MT-null Mvarphi produced less NO in response to TNF-alpha stimulation. Levels of expression of inducible NO synthase (iNOS) mRNA and production of iNOS protein in response to LPS stimulation were similar in MT-null and control cells, as were levels of expression of arginase, which competes in arginine metabolism with iNOS. No notable changes were found in arginine uptake or in expression of cationic amino acid transporter 2 (a major arginine transporter in Mvarphi) between control and MT-null Mvarphi. The rate of conversion of [(14)C]-l-arginine to citrulline, which is formed with NO by the action of iNOS, was much lower in MT-null Mvarphi than in control cells. These results indicate that the reduced production of NO in MT-deficient Mvarphi is due mainly to reduced activity of iNOS. Thus, MT plays important roles in bactericidal activity, NO production, and arginine metabolism in activated Mvarphi.

• Trialkyltin compounds bind retinoid X receptor to alter human placental endocrine functions.

  Authors: Tsuyoshi Nakanishi, Jun-ichi Nishikawa, Youhei Hiromori, Hideaki Yokoyama, Mihoko Koyanagi, Shinri Takasuga, Jun-ichi Ishizaki, Mai Watanabe, Shun-ichi Isa, Naoki Utoguchi, Norio Itoh, Yutaka Kohno, Tsutomu Nishihara, Keiichi Tanaka

  Molecular endocrinology (Baltimore, Md.). 11/2005; 19(10):2502-16.

  Retinoid X receptor (RXR) is a nuclear receptor that plays important and multiple roles in mammalian development and homeostasis. We previously reported that, in human choriocarcinoma cells,Retinoid X receptor (RXR) is a nuclear receptor that plays important and multiple roles in mammalian development and homeostasis. We previously reported that, in human choriocarcinoma cells, tributyltin chloride and triphenyltin hydroxide, which are typical environmental contaminants and cause masculinization in female mollusks, are potent stimulators of human chorionic gonadotropin production and aromatase activity, which play key endocrine functions in maintaining pregnancy and fetal development. However, the molecular mechanism through which these compounds stimulate these endocrine functions remains unclear. Our current study shows that trialkyltin compounds, including tributyltin chloride and triphenyltin hydroxide, function as RXR agonists. Trialkyltins directly bind to the ligand-binding domain of RXR with high affinity and function as transcriptional activators. Unlike the natural RXR ligand, 9-cis-retinoic acid, the activity of trialkyltins is RXR specific and does not activate the retinoic acid receptor pathway. In addition, trialkyltins activate RXR to stimulate the expression of a luciferase reporter gene containing the human placental promoter I.1 sequence of aromatase, suggesting that trialkyltins stimulate human placental endocrine functions through RXR-dependent signaling pathways. Therefore, our results suggest that activation of RXR may be a novel mechanism by which trialkyltins alter human endocrine functions.

• STAT3 mediates cardioprotection against ischemia/reperfusion injury through metallothionein induction in the heart.

  Authors: Yuichi Oshima, Yasushi Fujio, Tsuyoshi Nakanishi, Norio Itoh, Yasuhiro Yamamoto, Shinji Negoro, Keiichi Tanaka, Tadamitsu Kishimoto, Ichiro Kawase, Junichi Azuma

  Cardiovascular research. 03/2005; 65(2):428-35.

  OBJECTIVE: Activation of signal transducer and activator of transcription 3 (STAT3) was reported to be correlated with myocardial protection against ischemia/reperfusion (I/R) injury in ischemicOBJECTIVE: Activation of signal transducer and activator of transcription 3 (STAT3) was reported to be correlated with myocardial protection against ischemia/reperfusion (I/R) injury in ischemic preconditioning. Here, we tested the causality between STAT3 activity and cardioprotection. We also addressed the molecular mechanism for its cardioprotection. METHODS AND RESULTS: Cardiac-specific transgenic mice expressing constitutively active STAT3 (TG) were generated and exposed to I/R injury. TG hearts exhibited infarcts that reduced by 60.3% in size, compared with nontransgenic littermates (NTG). By measuring dichlorofluorescein (DCF) and 8-isoprostane, reactive-oxygen-species (ROS)-induced metabolites, it was revealed that ROS were generated to lesser extent in TG hearts than in NTG in response to I/R stress. In parallel, ROS scavengers, metallothionein1 (MT1), and metallothionein2 (MT2) were markedly up-regulated in TG hearts. Finally, homozygous deletion of the MT1 and MT2 genes abrogated cardioprotective effect of STAT3 against I/R injury with the cancellation of its ROS-scavenging effects. CONCLUSIONS: Activation of STAT3 protects myocardium from I/R injury in vivo. STAT3 mediates cardioprotection at least partially through MT1 and 2. STAT3 is a potential therapeutic target for I/R injury.

• Some organotin compounds enhance histone acetyltransferase activity.

  Authors: Shigehiro Osada, Jun-ichi Nishikawa, Tsuyoshi Nakanishi, Keiichi Tanaka, Tsutomu Nishihara

  Toxicology letters. 03/2005; 155(2):329-35.

  Eukaryotic DNA is packaged into chromatin, whose basic subunit is the nucleosome, which consists of DNA and a core histone octamer. Histone acetylation is important for the regulation of geneEukaryotic DNA is packaged into chromatin, whose basic subunit is the nucleosome, which consists of DNA and a core histone octamer. Histone acetylation is important for the regulation of gene expression and is catalyzed by histone acetyltransferase (HAT). We observed the effects of suspected endocrine-disrupting chemicals (EDCs) on HAT activity. We showed that some organotin compounds--tributyltin (TBT) and triphenyltin (TPT)--enhanced HAT activity of core histones in a dose-dependent way and other EDCs did not affect HAT activity. Organotin compounds have various influences on physical function including the hormone and immune systems, embryogenesis, and development. Dibutyltin and diphenyltin, metabolites of TBT and TPT, respectively, also promoted HAT activity, but monobutyltin, monophenyltin, and inorganic tin had no effect. Further, TBT and TPT enhanced HAT activity when nucleosomal histones were used as substrates. These data indicate that the organotin compounds have unique effects on HATs independent of their EDC activities and suggest that the varied toxicities of the organotin compounds may be caused by aberrant gene expression following altered histone acetylation.

• Expression profiles of zinc transporters in rodent placental models.

  Authors: Nagayoshi Asano, Masuo Kondoh, Chiaki Ebihara, Makiko Fujii, Tsuyoshi Nakanishi, Michael J Soares, Emi Nakashima, Keiichi Tanaka, Masao Sato, Yoshiteru Watanabe

  Toxicology letters. 01/2005; 154(1-2):45-53.

  Zinc is a vital metal that is a structural and functional component of many proteins. The precise mechanism of zinc transport in the placenta remains unclear. In this study, we investigated theZinc is a vital metal that is a structural and functional component of many proteins. The precise mechanism of zinc transport in the placenta remains unclear. In this study, we investigated the expression of zinc transporters (ZnTs) in the mouse placenta and in two rat trophoblast cell lines, TR-TBT cells, which are syncytiotrophoblast cells of the labyrinth zone, and Rcho-1 cells, which retain trophoblast cell features and differentiate into trophoblast giant cells of the junctional zone. All of the ZnTs that have been identified in mice (ZnT1-7) were detected in the mouse placenta by RT-PCR. The expression profiles of ZnTs in the placenta during pregnancy were different. The mRNA levels of ZnTs, with the exception of ZnT7, did not change during pregnancy. The ZnT7 mRNA level in placenta was elevated during pregnancy. In TR-TBT cells, ZnT1, ZnT3 and ZnT4 were detected by RT-PCR analysis. In Rcho-1 cells, all of the ZnTs that have been identified in rats (ZnT1-4) were detected by RT-PCR analysis. There were no differences between the mRNA expression levels of ZnT family members in undifferentiated Rcho-1 cells and differentiated Rcho-1 cells. This is the first report of expression profiles of ZnTs during differentiation of the placenta in the mouse placenta and rat placental cell models.

• C-terminal deletion mutant of MRE-binding transcription factor-1 inhibits MRE-driven gene expression.

  Authors: Tomoki Kimura, Norio Itoh, Tomomichi Sone, Keiichi Tanaka, Masakazu Isobe

  Journal of cellular biochemistry. 11/2004; 93(3):609-18.

  Heavy metal-induced transcriptional activation of the genes coding for metallothionein (MT) is mediated by a cis-acting DNA element, the metal-responsive element (MRE). MRE-binding transcriptionHeavy metal-induced transcriptional activation of the genes coding for metallothionein (MT) is mediated by a cis-acting DNA element, the metal-responsive element (MRE). MRE-binding transcription factor-1 (MTF-1) is a highly conserved heavy metal-induced transcriptional activator. MTF-1 also activates transcription in response to oxidative stress and regulates the expression of several cytoprotective factor genes, including MT, gamma-glutamylcysteine synthetase, and Cu/Zn-superoxide dismutase. It is thus thought that MTF-1 plays a role in cellular stress response. The physiological role of MTF-1 remains unclear because of the lack of MTF-1-specific activators and/or inhibitors. To obtain an MTF-1-specific inhibitor, we constructed an MTFDeltaC (amino acids 1-317), a C-terminal deletion mutant of MTF-1. MTFDeltaC could bind MRE and competed with MTF-1 for MTF-MRE complex formation. Transient expression of MTFDeltaC in HepG2 cells reduced MRE-driven gene expression, demonstrating that MTFDeltaC is dominant to MTF-1. HepG2 cells stably expressing MTFDeltaC showed increased susceptibility to the cytotoxic effects of tert-butyl hydroperoxide (tBH). Furthermore, we constructed Ad5MTFDeltaC, a recombinant adenovirus that expresses MTFDeltaC. Infection with the virus induced MTFDeltaC expression and increased susceptibility to the cytotoxic effects of tBH. These results indicate that MTF-1 participates in controlling the cellular redox state.

• Stimulation of differentiation in sodium-dependent vitamin C transporter 2 overexpressing MC3T3-E1 osteoblasts.

  Authors: Ximei Wu, Norio Itoh, Takashi Taniguchi, Junko Hirano, Tsuyoshi Nakanishi, Keiichi Tanaka

  Biochemical and biophysical research communications. 06/2004; 317(4):1159-64.

  Sodium-dependent vitamin C transporter (SVCT) 2 facilitates reduced ascorbic acid (AA) transport in MC3T3-E1 osteoblasts. Our previous studies suggested that Zn-induced osteoblast differentiation andSodium-dependent vitamin C transporter (SVCT) 2 facilitates reduced ascorbic acid (AA) transport in MC3T3-E1 osteoblasts. Our previous studies suggested that Zn-induced osteoblast differentiation and Ca2+-, PO4(3-)-stimulated osteopontin (OPN) expression might result from their up-regulation effect on SVCT2 expression and AA uptake. Here, we investigated the role of SVCT2 on osteoblast differentiation by using SVCT2-overexpressing cells. Two clones of SVCT2-introduced cells overexpressed SVCT2 mRNA by 2.8- and 3.1-fold those of control cells, which resulted in obvious increase of AA uptake by 2.1- and 2.4-fold in Vmax with no change in Km. Alkaline phosphatase activity, hydroxyproline content significantly increased in SVCT2-overexpressing cells, and the induction of OPN mRNA was through up-regulation of OPN promoter activity by SVCT2 overexpression. Moreover, SVCT2-overexpressing cells exhibited more ability to promote mineralization and increase calcium deposition under the stimulation of 5 mM beta-glycerophosphate. These findings indicate that SVCT2 stimulates osteoblast differentiation and mineralization.

• Zinc-induced sodium-dependent vitamin C transporter 2 expression: potent roles in osteoblast differentiation.

  Authors: Ximei Wu, Norio Itoh, Takashi Taniguchi, Tsuyoshi Nakanishi, Yoshiro Tatsu, Noboru Yumoto, Keiichi Tanaka

  Archives of biochemistry and biophysics. 01/2004; 420(1):114-20.

  Zinc is an essential trace element that increases osteoblast numbers and bone formation. However, the mechanisms involved in the Zn-induced differentiation of osteoblasts are poorly understood. WeZinc is an essential trace element that increases osteoblast numbers and bone formation. However, the mechanisms involved in the Zn-induced differentiation of osteoblasts are poorly understood. We examined the roles of L-ascorbic acid (AA) and its transporter, sodium-dependent vitamin C transporter (SVCT) 2, in the Zn-induced expression of osteoblastic differentiation markers. Zinc time- and dose-dependently induced SVCT2 mRNA expression in the absence or presence of AA. Western blotting and kinetic assays showed that Zn increased functional SVCT2 protein levels and AA transport. In the presence of AA, 50 microM Zn enhanced mRNA expression of the osteoblastic differentiation markers alkaline phosphatase, alpha(1)(I) procollagen, osteopontin (OPN), and osteocalcin (OCN) by 3.9-, 3.8-, 3.3-, and 3.5-fold, respectively; in the absence of AA, the Zn-induced increase was 2.8-, 2.5-, 1.3-, and 1.1-fold, respectively. These findings suggest that AA and SVCT2 mediate Zn-induced OPN and OCN expression and partly regulate Zn-induced osteoblastic differentiation.