title: Low-dose monobutyl phthalate stimulates steroidogenesis through steroidogenic acute regulatory protein regulated by SF-1, GATA-4 and C/EBP-beta in mouse Leydig tumor cells.
abstract: BACKGROUND
  The ubiquitous use of dibutyl phthalate (DBP), one of the most widely used plasticizers, results in extensive exposure to humans and the environment. DBP and its major metabolite, monobutyl phthalate (MBP), may alter steroid biosynthesis and their exposure may lead to damage to male reproductive function. Low-doses of DBP/MBP may result in increased steroidogenesis in vitro and in vivo. However, the mechanisms of possible effects of low-dose MBP on steroidogenesis remain unclear. The aim of present study was to elaborate the role of transcription factors and steroidogenic acute regulatory protein in low-dose MBP-induced distruption of steroidogenesis in mouse Leydig tumor cells (MLTC-1 cells).
  
  
  METHODS
  In the present study, MLTC-1 cells were cultured in RPMI 1640 medium supplemented with 2 g/L sodium bicarbonate. Progesterone level was examined by I125-pregesterone Coat-A-Count radioimmunoassay (RIA) kits. mRNA and protein levels were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and western blot, respectively. DNA-binding of several transcription factors was examined by electrophoretic mobility shift assay (EMSA).
  
  
  RESULTS
  In this study, various doses of MBP (0, 10(-9), 10(-8), 10(-7), or 10(-6) M) were added to the medium followed by stimulation of MLTC-1 cells with human chorionic gonadotrophin (hCG). The results showed that MBP increased progesterone production and steroidogenic acute regulatory protein (StAR) mRNA and protein levels. However, the protein levels of cytochrome P450scc and 3 beta-hydroxy-steroid dehydrogenase (3 beta-HSD) were unchanged after MBP treatment. EMSA assay showed that DNA-binding of steroidogenic factors 1(SF-1), GATA-4 and CCAAT/enhancer binding protein-beta (C/EBP-beta) was increased in a dose-dependent manner after MBP exposure. Western blot tests were next employed and confirmed that the protein levels of SF-1, GATA-4 and C/EBP-beta were also increased. Additionally, western blot tests confirmed the expression of DAX-1, negative factor of SF-1, was dose-dependently down regulated after MBP exposure, which further confirmed the role of SF-1 in MBP-stimulated steroid biosynthesis.
  
  
  CONCLUSIONS
  In conclusion, we firstly delineated the regulation of StAR by transcription factors including SF-1, GATA-4 and C/EBP-beta maybe critical mechanism involved in low-dose MBP-stimulated steroidogenesis.
authors.affiliation: State Key Laboratory of Reproductive Medicine, Institute of Toxicology, School of Public Health, Nanjing Medical University, Nanjing 211166, China.
authors.forename: Yanhui
authors.initials: Y
authors.lastname: Hu
authors.forename: Congcong
authors.initials: C
authors.lastname: Dong
authors.forename: Minjian
authors.initials: M
authors.lastname: Chen
authors.forename: Jing
authors.initials: J
authors.lastname: Lu
authors.forename: Xiumei
authors.initials: X
authors.lastname: Han
authors.forename: Lianglin
authors.initials: L
authors.lastname: Qiu
authors.forename: Yansu
authors.initials: Y
authors.lastname: Chen
authors.forename: Jingjing
authors.initials: J
authors.lastname: Qin
authors.forename: Xiaocheng
authors.initials: X
authors.lastname: Li
authors.forename: Aihua
authors.initials: A
authors.lastname: Gu
authors.forename: Yankai
authors.initials: Y
authors.lastname: Xia
authors.forename: Hong
authors.initials: H
authors.lastname: Sun
authors.forename: Zhong
authors.initials: Z
authors.lastname: Li
authors.forename: Yubang
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000818:Animals; D015153:Blotting, Western; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D057137:DAX-1 Orphan Nuclear Receptor / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D050991:GATA4 Transcription Factor / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D007984:Leydig Cell Tumor / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D010795:Phthalic Acids / Q000494:pharmacology*; D011374:Progesterone / Q000096:biosynthesis; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D054339:Steroidogenic Factor 1 / Q000378:metabolism*; D013256:Steroids / Q000096:biosynthesis; D000098682:Steroidogenic Acute Regulatory Protein
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D006063:Chorionic Gonadotropin; D057137:DAX-1 Orphan Nuclear Receptor; D050991:GATA4 Transcription Factor; C539966:Nr0b1 protein, mouse; D010750:Phosphoproteins; D010795:Phthalic Acids; D054339:Steroidogenic Factor 1; D013256:Steroids; D000098682:Steroidogenic Acute Regulatory Protein; D011374:Progesterone; C028577:monobutyl phthalate
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