論文 - 田村　悦臣

New diterpenoids with estrogen sulfotransferase inhibitory activity from Leonurus sibiricus L.

Narukawa Y, Niimura A, Noguchi H, Tamura H, Kiuchi F

Journal of natural medicines （Journal of Natural Medicines）  68(1) （ 1 ） 125-131 - 31 2014年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1340-3443

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Four new diterpenoids (1-4), along with eight known diterpenoids (5-12) were isolated from the acetone extract of Leonurus herb (aerial parts of Leonurus sibiricus L.). The structures of the compounds were determined by spectroscopic methods. Among the isolated compounds, compounds 2, 3, 8, 9 and 10 showed inhibitory activity against human liver cytosol estrogen sulfotransferase (E-ST), which plays a key role in the maintenance of cellular estrogen levels. Compound 2 showed the strongest activity with an IC 50 value of 7.9 μM, which is comparable to the activity of the positive control, meclofenamic acid (IC50 5.4 μM). © The Japanese Society of Pharmacognosy and Springer Japan 2013.

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Dexamethasone suppresses neurosteroid biosynthesis via downregulation of steroidogenic enzyme gene expression in human glioma GI-1 cells.

Koibuchi F, Ritoh N, Aoyagi R, Funakoshi-Tago M, Tamura H

Biological & pharmaceutical bulletin （Biological and Pharmaceutical Bulletin）  37 （ 7 ） 1241 - 7 2014年

研究論文（学術雑誌）,  ISSN  0918-6158

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Emerging evidence indicates that stress hormone glucocorticoids (GC) are an important modulator of brain development and function. To investigate whether GCs modulate neurosteroid biosynthesis in neural cells, we studied the effects of GCs on steroidogenic gene expression in human glioma GI-1 cells. The GC dexamethasone (Dex) reduced steroidogenic acute regulatory protein (StAR), CYP11A1 and 3β-hydroxysteroid dehydrogenase gene expression in a dose- and GC receptor-dependent manner. In addition to its effects on steroidogenic gene expression, Dex also reduced de novo synthesis of progesterone (PROG). Furthermore, Dex inhibited all-trans retinoic acid (ATRA) and vitamin D 3-induced steroidogenic gene expression and PROG production. This suggests that GC regulates steroidogenic gene expression in neural cells via cross-talk with the two fat-soluble vitamins, A and D. The relationship between the effects of GCs on neurosteroid biosynthesis and on cognitive behaviors and hippocampal neural activity is also discussed herein. © 2014 The Pharmaceutical Society of Japan.

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Coffee inhibits adipocyte differentiation via inactivation of PPARγ.

Aoyagi R, Funakoshi-Tago M, Fujiwara Y, Tamura H

Biological & pharmaceutical bulletin （Biological and Pharmaceutical Bulletin）  37 （ 11 ） 1820 - 5 2014年

研究論文（学術雑誌）,  ISSN  0918-6158

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© 2014 The Pharmaceutical Society of Japan. Recent epidemiological studies showed that coffee consumption is associated with a lower risk of type 2 diabetes, presumably due to suppression of excess fat accumulation in adipocytes. However, the mechanism underlying the effect of coffee on adipocyte differentiation has not been well documented. To elucidate the mechanism, we investigated the effect of coffee on the differentiation of mouse preadipocyte 3T3-L1 cells. Coffee reduced the accumulation of lipids during adipocytic differentiation of 3T3-L1 cells. At 5% coffee, the accumulation of lipids decreased to half that of the control. Coffee also inhibited the expression of the peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor controlling the differentiation of adipocytes. Furthermore, coffee reduced the expression of other differentiation marker genes, aP2, adiponectin, CCAAT-enhancer-binding protein α (C/EBPα), glucose transporter 4 (GLUT4), and lipoprotein lipase (LPL), during adipocyte differentiation. Major bioactive constituents in coffee extracts, such as caffeine, trigonelline, chlorogenic acid, and caffeic acid, showed no effect on PPARγ gene expression. The inhibitory activity was produced by the roasting of the coffee beans.

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Human keratinocyte caspase-14 expression is altered in human epidermal 3D models by dexamethasone and by natural products used in cosmetics.

Kataoka S, Hattori K, Date A, Tamura H

Archives of dermatological research （Archives of Dermatological Research）  305 （ 8 ） 683-689 - 9 2013年08月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0340-3696

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Caspase-14 is a cysteinyl-aspartate-specific proteinase that is specifically expressed in epidermal keratinocytes. Dysregulation of caspase-14 expression is implicated in impaired skin barrier formation. To elucidate the regulation of caspase-14 in differentiated keratinocytes, we characterized the expression of caspase-14 in normal human epidermal keratinocytes (NHEKs) and two types of three-dimensional (3D) human epidermis culture models, EPI-200 and EPI-201, via RT-PCR and immunoblot analyses. Caspase-14 expression was absent in subconfluent NHEKs, but was present in confluent NHEKs as well as those induced to differentiate by calcium. Caspase-14 expression levels in the 3D epidermis models were almost equal to that in the Ca2+-treated differentiated NHEKs. Despite the presence of caspase-14 expression in these models, caspase-14 activity was found only in the mature 3D skin model, EPI-200. This was confirmed by detection of a 17 kDa cleaved fragment of caspase-14 present only in the EPI-200 model. Since glucocorticoid (GC) receptor is required for skin barrier competence, we investigated whether the GC dexamethasone (Dex) and various natural components of common skin moisturizers affect caspase-14 expression in keratinocytes. Dex decreased caspase-14 expression in undifferentiated, but not differentiated, NHEKs. Conversely, Dex increased caspase-14 expression in both 3D skin models, although it did not alter caspase protease activity. Similar to treatment with Dex, treatment of the premature 3D skin mode, EPI-201 with a Galactomyces ferment filtrate markedly increased expression of caspase-14. Further, these results suggest that the effect of Dex, or lack thereof, on caspase-14 expression is dependent on the stage of keratinocyte differentiation. © 2013 Springer-Verlag Berlin Heidelberg.

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Coffee reduces SULT1E1 expression in human colon carcinoma Caco-2 cells.

Isshiki M, Ohta H, Tamura H

Biological & pharmaceutical bulletin （Biological and Pharmaceutical Bulletin）  36(2) （ 2 ） 229-304 - 304 2013年02月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0918-6158

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Recent epidemiological studies have shown that moderate coffee consumption is associated with a lower risk of certain types of cancers, particularly colon cancer in postmenopausal women. To elucidate the molecular basis for the preventive action of coffee, we investigated the effect of coffee on estrogen sulfotransferase (SULT) because sulfation is the major pathway involved in the inactivation of estrogens. We found that coffee reduced SULT1E1 gene expression in human colon carcinoma Caco-2 cells. Treatment with 2.5% (v/v) coffee for 24 h resulted in a 60% reduction of the expression of the SULT1E1 gene in Caco-2 cells. Corresponding to reduced SULT1E1 gene expression, cytosolic estrogen SULT activity toward E2 (20 nm) decreased by 25%. In addition, an accumulation of E2 sulfates in the medium, which reflects cellular activity of estrogen SULT, decreased after the cells were treated with coffee. Major bioactive constituents in coffee such as caffeine, chlorogenic acid and caffeic acid did not show any effect. The inhibitory activity was extractable by using ethyl acetate. We also found that the inhibitory activity was produced by roasting the coffee beans. Mithramycin, an inhibitor of the transcription factor stimulating protein 1 (Sp1), was able to restore coffee-reduced SULT1E1 gene expression. Our data suggest that daily coffee consumption may reduce estrogen SULT activity, thereby enhancing estrogenic activity in the colon. © 2013 The Pharmaceutical Society of Japan.

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Vitamin D3 enhances all-trans retinoic acid (ATRA)-mediated neurosteroid biosynthesis in human glioma GI-1 cells.

Yagishita T, Kushida A, Tamura H*.

J Biochem 152(3)   285-292 2012年10月

研究論文（学術雑誌）, 共著, 査読有り

Interferon gamma induces steroid sulfatase expression in human keratinocytes.

Hattori K, Yamaguchi N, Umezawa K, Tamura H

Biological & pharmaceutical bulletin （Biological and Pharmaceutical Bulletin）  35(9) （ 9 ） 1588-1593 - 93 2012年09月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0918-6158

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Steroid sulfatase (STS) plays an important role in steroid metabolism in which estrogens and dehydroepiandrosterone (DHEA) are produced from their sulfates. However, little is known about the transcriptional regulation of the STS gene in keratinocytes. Since keratinocytes are thought to be a primary target of interferon gamma (IFNγ) in inflammatory and immune responses, we assessed the effects of this cytokine upon STS gene expression in the human keratinocyte cell line SVHK and in normal human keratinocytes (NHEK). Stimulation of SVHK cells with 50 ng/mL of IFNγ for 24 h induced an approximately three-fold increase in STS activity and in its mRNA levels compared to non-treated cells. IFNγ treatment also induced an approximately 1.5-fold increase in STS mRNA levels in NHEK cells. This induction was completely inhibited by treatment with phosphatidylinositol (PI) 3-kinase inhibitors such as LY294002 or wortmannin, and by the nuclear factor-kappa B (NF-κB) inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ). These data suggest that activation of the PI 3-kinase signal transduction pathway mediates induction of STS gene expression by IFNγ through activation of NF-κB. The anti-inflammatory agent dexamethasone inhibited IFNγ induction of STS gene expression, suggesting involvement of a glucocorticoid receptor in the regulation of STS gene expression in keratinocytes. Regulation of STS gene expression in skin as a novel target of drugs for therapy of psoriasis in the skin is discussed. © 2012 The Pharmaceutical Society of Japan.

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Vitamin D(3) enhances ATRA-mediated neurosteroid biosynthesis in human glioma GI-1 cells.

Yagishita T, Kushida A, Tamura H

Journal of biochemistry （Journal of Biochemistry）  152 （ 3 ） 285 - 92 2012年09月

研究論文（学術雑誌）,  ISSN  0021-924X

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Emerging evidence indicates that vitamin D (VD) is an important modulator of brain development and function. To investigate whether VD modulates neurosteroid biosynthesis in neural cells, we investigated the effect of VD 3 on steroidogenic gene expression in human glioma GI-1 cells. We found that VD3 enhanced CYP11A1 and 3β-hydroxysteroid dehydrogenase gene expression. The induction of CYP11A1 gene expression by VD3 was dose-and incubation time-dependent. Calcipotriol, a VD 3 receptor (VDR) agonist, also induced CYP11A1 gene expression in GI-1 cells, indicating that VDR is involved in this induction. The induction of progesterone (PROG) de novo synthesis was observed along with the induction of steroidogenic genes by VD3. Furthermore, VD3 enhanced all-trans retinoic acid (ATRA)-induced CYP11A1 gene expression and PROG production. This suggests cooperative regulation of steroidogenic gene expression by the two fat-soluble vitamins, A and D. In addition, a mixed culture of neuronal IMR-32 cells and GI-1 cells treated with ATRA and VD 3 resulted in the induction of PROG-responsive gene expression in the IMR-32 cells. This result shows a paracrine action of PROG that is induced in and released by the GI-1 cells. The relationship between neurological dysfunction associated with VD deficiency and neurosteroid induction by VD is discussed. © 2012 The Authors.

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Xanthuretic acid is the endogenous substrate of silkworm cytosolic sulfotransferase bmST1.

Kushida A, Horie R, Hattori K, Sekimizu K, Tamura H*.

J Insect Physiol 58(1)   83-88 2012年01月

研究論文（学術雑誌）, 共著, 査読有り

Xanthurenic acid is an endogenous substrate for the silkworm cytosolic sulfotransferase, bmST1.

Kushida A, Horie R, Hattori K, Hamamoto H, Sekimizu K, Tamura H

Journal of insect physiology （Journal of Insect Physiology）  58 （ 1 ） 83 - 8 2012年01月

研究論文（学術雑誌）,  ISSN  0022-1910

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Sulfotransferase enzymes are known to regulate physiologically active substances such as steroids and catecholamines in mammals. Although invertebrates also express sulfotransferases, their biological function is mostly unclear. In a previous study, we reported that 4-nitrocatechol and the gallete ester are substrates for the silkworm sulfotransferase bmST1. The Km of bmST1 for these substrates is high. However, endogenous substrates of bmST1 have not yet been determined. We therefore investigated endogenous bmST1 substrates and carried out a detailed expression profile analysis of bmST1. We found that xanthurenic acid, a tryptophan metabolite, is a possible endogenous substrate of bmST1. The Km of bmST1 for xanthurenic acid is low, in the μM range, which is lower than that for previously reported substrates. Additionally, xanthurenic acid is a tryptophan metabolite that characteristically shows toxicity in vivo. High dose administration of xanthurenic acid resulted in inhibition of cuticular biosynthesis. The expression of the bmST1 gene reached a maximal level in the Malpighian tubule at the 4th molting stage, when amino acid metabolism might be activated. Our results suggest that bmST1 plays a role in detoxification of xanthurenic acid in the silkworm. © 2011 Elsevier Ltd.

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Coffee induces 11β-hydroxysteroid dehydrogenase type 2 activity in human placental choriocarcinoma cells, JEG-3

Arao T., Tamura H.

Journal of Health Science （Journal of Health Science）  57 （ 5 ） 436 - 441 2011年10月

研究論文（学術雑誌）,  ISSN  13449702

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Coffee is the beverage consumed during pregnancy that is most commonly associated with adversely affecting the developing fetoplacental unit. Since placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD-2) is an important determinant for maintenance of fetal glucocorticoid levels during pregnancy, we investigated the effect of coffee on 11β-HSD-2 expression in the human placental choriocar-cinoma cell line, JEG-3. Addition of coffee to the culture medium of JEG-3 cells increased 11β-HSD-2 gene expression after 24 hr in a dose-dependent manner. Cellular 11β-HSD-2 activity, which was calculated by the amount of cortisol that was converted to cortisone in the culture medium, also increased in the coffee-treated JEG-3 cells. None of the major constituents of coffee, such as caffeine, caffeic acid or chlorogenic acid, showed any ability to induce 11β-HSD-2 gene expression. The 11β-HSD-2-inducing activity in coffee was extractable with ethyl acetate. Increasing the degree of roasting improved the ability of the coffee extract to induce 11β-HSD-2 gene expression indicating that the active constituent(s) is produced during the roasting process. © 2011 The Pharmaceutical Society of Japan.

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Coffee induces 11b-hydroxysteroid dehydrogenase type 2 activity in human placental choriocarcinoma cells, JEG-3.

Arao T, Tamura H*.

J Health Sci 57   436-441 2011年10月

研究論文（学術雑誌）, 共著, 査読有り

Coffee induces breast cancer resistance protein expression in Caco-2 cells.

Isshiki M, Umezawa K, Tamura H

Biological & pharmaceutical bulletin （Biological and Pharmaceutical Bulletin）  34 （ 10 ） 1624-1627 - 7 2011年10月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0918-6158

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Coffee is a beverage that is consumed world-wide on a daily basis and is known to induce a series of metabolic and pharmacological effects, especially in the digestive tract. However, little is known concerning the effects of coffee on transporters in the gastrointestinal tract. To elucidate the effect of coffee on intestinal transporters, we investigated its effect on expression of the breast cancer resistance protein (BCRP/ABCG2) in a human colorectal cancer cell line, Caco-2. Coffee induced BCRP gene expression in Caco-2 cells in a coffee-dose dependent manner. Coffee treatment of Caco-2 cells also increased the level of BCRP protein, which corresponded to induction of gene expression, and also increased cellular efflux activity, as judged by Hoechst33342 accumulation. None of the major constituents of coffee tested could induce BCRP gene expression. The constituent of coffee that mediated this induction was extractable with ethyl acetate and was produced during the roasting process. Dehydromethylepoxyquinomicin (DHMEQ), an inhibitor of nuclear factor (NF)-κB, inhibited coffee-mediated induction of BCRP gene expression, suggesting involvement of NF-κB in this induction. Our data suggest that daily consumption of coffee might induce BCRP expression in the gastrointestinal tract and may affect the bioavailability of BCRP substrates. © 2011 Pharmaceutical Society of Japan.

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Sulfation of estradiol in human epidermal keratinocyte.

Kushida A, Hattori K, Yamaguchi N, Kobayashi T, Date A, Tamura H

Biological & pharmaceutical bulletin （Biological and Pharmaceutical Bulletin）  34(7) （ 7 ） 1147-1151 - 51 2011年07月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0918-6158

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Epidermis is one of the well-known estrogen target tissues. Information regarding estrogen metabolism in epidermis is still very limited compared to that of estrogen action. In the breast cancer tissue, 17β-estradiol (E 2) is inactivated by sulfation and the expression level of estrogen sulfotransferase (SULT1E1) is inversely correlated with its malignancy. However, there is little datum about inactivation of estradiol in skin. In order to detect and measure E 2 and its metabolites simultaneously, we established an assay method with radio HPLC. A majority of [ 3H] labeled E 2 was converted to E 2 sulfate in normal human epidermal keratinocyte (NHEK) cells. The estimated activity of sulfotransferase toward E 2 at 20 nM was 0.11±0.01 (pmol/min/mg protein). Significant induction of estrogen sulfotransferase activity was observed in calcium-differentiated NHEK cells (0.58±0.07 (pmol/min/mg protein)). The gene expression of SULT1E1 was fifteen-fold higher in differentiated keratinocyte than in proliferating keratinocyte, whereas that of steroid sulfatase was reduced. These results suggest that E 2 inactivation is primarily mediated by SULT1E1 in keratinocyte and E 2 action is likely suppressed in epidermal differentiation. © 2011 Pharmaceutical Society of Japan.

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ブラックボックスから玉手箱へ.

田村悦臣.

塾 （慶應義塾）  48   28 2011年04月

研究論文（学術雑誌）, 単著

Megadose vitamin C suppresses sulfoconjugation in human colon carcinoma cell line Caco-2.

Tsuruta H, Yagishita T, Shimizu M, Tamura H

Toxicology in vitro : an international journal published in association with BIBRA （Toxicology in Vitro）  25 （ 2 ） 500-504 - 4 2011年03月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0887-2333

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Intake of high doses of vitamin C has known to modulate sulfoconjugation of drugs in the intestine, but the underlying mechanisms for this effect remain to be elucidated. In the present study, we investigated the effects of vitamin C (l-ascorbic acid (AA)) on sulfation of 1-naphthol using Caco-2 cells, a model of human intestinal cells. We found that high dose of AA inhibited the accumulation of 1-naphthyl sulfate in Caco-2 culture medium within 24h in a dose-dependent manner (IC 50=42mM). Dehydroascorbic acid (DA), an oxidized form of AA, showed no inhibition. AA did not inhibit the in vitro sulfotransferase (SULT) activity toward 1-naphthol, whereas it reduced the expression of genes belonging to SULT1A family, SULT1A1 and SULT1A3. DA showed no effect on SULT1A gene expression. Consistent with the reduction in gene expression, AA reduced the cytosolic SULT activity towards 1-naphthol in the AA-treated Caco-2 cells. In addition, cAMP exerted an additive effect on AA-mediated repression of SULT1A gene expression. Our results suggest that megadose AA suppresses sulfoconjugation in the intestine mainly by downregulating the expression of SULT1A genes. © 2010 Elsevier Ltd.

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Effects of coffee on the metabolism of glucocorticoid in human syncytiotrophoblast JEG-3 cells.

Arao T, Tamura H.

Journal of Health Science （日本薬学会）  56   131 2010年10月

研究論文（学術雑誌）, 共著, 査読有り

Effects of cytochrome P450 inducers on the gene expression of ocular xenobiotic metabolizing enzymes in rats

Sugamo T., Nakamura K., Tamura H.

Journal of Health Science （Journal of Health Science）  55(6) （ 6 ） 923-929 - 929 2009年12月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  13449702

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In our current study, we investigated the expression profiles of the cytochromes P450 (CYPs) and transporters of the ocular tissues in Sprague-Dawley (SD) rats. Extensive expression of CYP1A1 in the cornea and CYP2E1 in the iris was observed whereas the expression of CYP2B1 and transporters was mostly ubiquitous throughout the ocular tissues. To further understand the regulation of these genes in ocular tissues, we investigated the effects of CYP inducers on the expression of the CYP and other xenobiotic-metabolizing enzyme genes in the cornea and lens. The administration of β-naphthoflavone (BNF), an agonist for aryl hydrocarbon receptor (AhR), induced CYP1A1 gene expression in the cornea, lens and liver of the rats, although the levels of induction were greatest in the liver. An AhR-sensitive UDP-glucuronosyl transferase (UGT) 1A6 gene was also induced in the cornea and the lens by BNF. Phenobarbital (PB) is a known inducer of the CYP2B genes, the expression of which is mediated by constitutive activated receptor (CAR), but did not induce CYP2B1 in the cornea or lens. This insensitivity to PB may be due to the lack of CAR expression in the ocular tissues as revealed in our present study. Pregnenolone-16α- carbonitrile (PCN) is known to induce CYP3A gene expression in the liver via the activation of pregnane X receptor (PXR). However, although PCN was found to induce the CYP3A1 gene in the rat cornea and liver, it failed to do so in the lens. In addition, another of the PXR-mediated genes, multidrug resistance-associated protein 3 (Mrp3), was not induced by PCN in either ocular region. Since the expression of the PXR gene was not detected in the rat ocular tissues, an unknown mechanism for the inducible regulation of CYP3A1 gene expression by PCN in the cornea is suggested. ©2009 The Pharmaceutical Society of Japan.

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Retinoic acids induce neurosteroid biosynthesis in human glial GI-1 cells via the induction of steroidgenic genes.

Kushida A, Tamura H*.

J Biochem 146(6)   917-923 2009年12月

研究論文（学術雑誌）, 共著, 査読有り

Retinoic acids induce neurosteroid biosynthesis in human glial GI-1 Cells via the induction of steroidogenic genes.

Kushida A, Tamura H

Journal of biochemistry （Journal of Biochemistry）  146 （ 6 ） 917 - 23 2009年12月

研究論文（学術雑誌）,  ISSN  0021-924X

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The steroids synthesized in the central nervous system (CNS) are the neurosteroids. Since little information is currently available concerning the roles of the retinoic acids (RAs) during steroidogenesis in the CNS, we investigated the effects of RAs upon their synthesis in our current study. Specifically, we analyzed the effects of all-trans-retinoic acid (ATRA) upon the expression of neurosteroid biosynthesis genes in the human glial cell line GI-1, in which the major steroidogenic genes are expressed. Treatment with ATRA (10 μM) induced a 4.9-fold increase in the expression of the cytochrome P450scc (CYP11A1) gene, the product of which cleaves the cholesterol side chain, a rate-limiting step during steroidogenesis. ATRA also strongly induced the expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) (an increase of 5- and 50-fold, respectively). A retinoic acid receptor (RAR)-specific agonist, TTNPB, was unable to mimic this induction whereas a retinoid X receptor (RXR)-specific agonist, methoprene acid, in addition to 9-cis-RA, could do so. These data indicate that ATRA is isomerized to 9-cis-RA in the culture medium, as reported previously, and that 9-cis-RA activates the RXR. In addition, ATRA also induced the de novo synthesis of neurosteroids such as pregnenolone and progesterone. These results suggest that ATRA might induce the de novo neurosteroid synthesis via the induction of steroidogenic genes in human glial cells. The multiple effects of vitamin A upon CNS functions might therefore be partly explained by the induction of neurosteroidogenesis by RAs, since neurosteroids have also been reported to have multiple effects in the CNS.

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