Advertisement

Journal of Endocrinological Investigation

, Volume 38, Issue 8, pp 875–884 | Cite as

Effects of monobutyl phthalate on steroidogenesis through steroidogenic acute regulatory protein regulated by transcription factors in mouse Leydig tumor cells

  • Y. Hu
  • C. Dong
  • M. Chen
  • Y. Chen
  • A. Gu
  • Y. Xia
  • H. Sun
  • Z. Li
  • Y. Wang
Original Article

Abstract

Objective

Dibutyl phthalate (DBP) is one of the most widely used phthalate esters, and it is ubiquitous in the environment. DBP and its major metabolite, monobutyl phthalate (MBP), change steroid biosynthesis and impair male reproductive function. However, the regulatory mechanism underlying the steroid biosynthesis disruption by MBP is still unclear.

Methods

We analyzed the progesterone production, steroidogenic acute regulatory protein (StAR) mRNA, protein expression, and DNA-binding affinity of transcription factors (SF-1 and GATA-4).

Results

Our results reveal that MBP inhibited progesterone production. At the same time, StAR mRNA and protein were decreased after MBP exposure. Furthermore, electrophoretic mobility shift assay showed that DNA-binding affinity of transcription factors (SF-1 and GATA-4) was decreased in a dose-dependent manner after MBP treatments. Western blot tests next confirmed that protein of SF-1 was decreased, but GATA-4 protein was unchanged. However, phosphorylated GATA-4 protein was decreased with 800 μM of MBP.

Conclusions

This study reveals an important and novel mechanism whereby SF-1 and GATA-4 may regulate StAR during MBP-induced steroidogenesis disruption.

Keywords

Monobutyl phthalate Progesterone Steroidogenic acute regulatory protein Transcription factors Mouse Leydig tumor cells-1 

Abbreviations

BSA

Bovine serum albumin

C/EBP-β

CCAAT/enhancer binding protein-β

DAX-1

Dosage-sensitive sex reversal adrenal hypoplasia critical region on chromosome X gene 1

DBP

Dibutyl phthalate

DEPC

Diethylpyrocarbonate

FBS

Fetal bovine serum

MBP

Monobutyl phthalate

MLTC-1

Mouse Leydig tumor cells-1

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

PAEs

Phthalate esters

SF-1

Steroidogenic factor 1

SDS

Sodium dodecyl sulfate

SREBP

Sterol regulatory element-binding protein

SFM

Serum-free medium

StAR

Steroidogenic acute regulatory protein

Notes

Acknowledgments

YH YW conceived and designed the experiments. CD MC YC performed the experiments. YH MC XH analyzed the data. AG YK HS contributed reagents/materials/analysis tools. YH wrote the paper. All authors read and approved the final manuscript. Research was funded by National Natural Science Foundation (81373041).

Conflict of interest

All the authors state that they have no conflicts of interest.

Ethical approval

Our research only used cell mode without Human Participants and/or Animals.

Informed consent

Not required.

References

  1. 1.
    Kobrosly RW, Parlett LE, Stahlhut RW, Barrett ES, Swan SH (2012) Socioeconomic factors and phthalate metabolite concentrations among United States women of reproductive age. Environ Res 115:11–17PubMedCrossRefGoogle Scholar
  2. 2.
    Prasanth GK, Divya LM, Sadasivan C (2009) Effects of mono and di(n-butyl) phthalate on superoxide dismutase. Toxicology 262:38–42PubMedCrossRefGoogle Scholar
  3. 3.
    Niu L, Xu Y, Xu C, Yun L, Liu W (2014) Status of phthalate esters contamination in agricultural soils across China and associated health risks. Environ Pollut 195C:16–23CrossRefGoogle Scholar
  4. 4.
    Meeker JD, Ferguson KK (2011) Relationship between urinary phthalate and bisphenol A concentrations and serum thyroid measures in US adults and adolescents from the National Health and Nutrition Examination Survey (NHANES) 2007–2008. Environ Health Perspect 119:1396–1402PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Meeker JD, Ferguson KK (2014) Urinary phthalate metabolites are associated with decreased serum testosterone in men, women, and children from NHANES 2011–2012. J Clin Endocrinol Metab 99:4346–4352PubMedCrossRefGoogle Scholar
  6. 6.
    Scott HM, Mason JI, Sharpe RM (2009) Steroidogenesis in the fetal testis and its susceptibility to disruption by exogenous compounds. Endocr Rev 30:883–925PubMedCrossRefGoogle Scholar
  7. 7.
    Manikkam M, Tracey R, Guerrero-Bosagna C, Skinner MK (2013) Plastics derived endocrine disruptors (BPA, DEHP and DBP) induce epigenetic transgenerational inheritance of obesity, reproductive disease and sperm epimutations. PLoS One 8:e55387PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Kuhl AJ, Ross SM, Gaido KW (2007) Using a comparative in vivo DNase I footprinting technique to analyze changes in protein-DNA interactions following phthalate exposure. J Biochem Mol Toxicol 21:312–322PubMedCrossRefGoogle Scholar
  9. 9.
    Giribabu N, Sainath SB, Sreenivasula Reddy P (2014) Prenatal di-n-butyl phthalate exposure alters reproductive functions at adulthood in male rats. Environ Toxicol 29:534–544PubMedCrossRefGoogle Scholar
  10. 10.
    Chen X, Zhou QH, Leng L, Sun ZR, Tang NJ (2013) Effects of di(n-butyl) and monobutyl phthalate on steroidogenesis pathways in the murine Leydig tumor cell line MLTC-1. Environ Toxicol Pharmacol 36:332–338PubMedCrossRefGoogle Scholar
  11. 11.
    Mylchreest E, Cattley RC, Foster PM (1998) Male reproductive tract malformations in rats following gestational and lactational exposure to di(n-butyl) phthalate: an antiandrogenic mechanism? Toxicol Sci 43:47–60PubMedCrossRefGoogle Scholar
  12. 12.
    Hannas BR, Lambright CS, Furr J, Howdeshell KL, Wilson VS, Gray LE Jr (2011) Dose-response assessment of fetal testosterone production and gene expression levels in rat testes following in utero exposure to diethylhexyl phthalate, diisobutyl phthalate, diisoheptyl phthalate, and diisononyl phthalate. Toxicol Sci 123:206–216PubMedCrossRefGoogle Scholar
  13. 13.
    Ishii T, Mitsui T, Suzuki S, Matsuzaki Y, Hasegawa T (2012) A genome-wide expression profile of adrenocortical cells in knockout mice lacking steroidogenic acute regulatory protein. Endocrinology 153:2714–2723PubMedCrossRefGoogle Scholar
  14. 14.
    Manna PR, Dyson MT, Stocco DM (2009) Regulation of the steroidogenic acute regulatory protein gene expression: present and future perspectives. Mol Hum Reprod 15:321–333PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Bao AM, Man XM, Guo XJ, Dong HB, Wang FQ, Sun H, Wang YB, Zhou ZM, Sha JH (2011) Effects of di-n-butyl phthalate on male rat reproduction following pubertal exposure. Asian J Androl 13:702–709PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Dube C, Bergeron F, Vaillant MJ, Robert NM, Brousseau C, Tremblay JJ (2009) The nuclear receptors SF1 and LRH1 are expressed in endometrial cancer cells and regulate steroidogenic gene transcription by cooperating with AP-1 factors. Cancer Lett 275:127–138PubMedCrossRefGoogle Scholar
  17. 17.
    Sugawara T, Holt JA, Kiriakidou M, Strauss JF 3rd (1996) Steroidogenic factor 1-dependent promoter activity of the human steroidogenic acute regulatory protein (StAR) gene. Biochemistry 35:9052–9059PubMedCrossRefGoogle Scholar
  18. 18.
    Caron KM, Ikeda Y, Soo SC, Stocco DM, Parker KL, Clark BJ (1997) Characterization of the promoter region of the mouse gene encoding the steroidogenic acute regulatory protein. Mol Endocrinol 11:138–147PubMedCrossRefGoogle Scholar
  19. 19.
    Mizutani T, Ju Y, Imamichi Y, Osaki T, Yazawa T, Kawabe S, Ishikane S, Matsumura T, Kanno M, Kamiki Y, Kimura K, Minamino N, Miyamoto K (2014) C/EBPbeta (CCAAT/enhancer-binding protein beta) mediates progesterone production through transcriptional regulation in co-operation with SF-1 (steroidogenic factor-1). Biochem J 460:459–471PubMedCrossRefGoogle Scholar
  20. 20.
    Tremblay JJ, Hamel F, Viger RS (2002) Protein kinase A-dependent cooperation between GATA and CCAAT/enhancer-binding protein transcription factors regulates steroidogenic acute regulatory protein promoter activity. Endocrinology 143:3935–3945PubMedCrossRefGoogle Scholar
  21. 21.
    Martin LJ, Bergeron F, Viger RS, Tremblay JJ (2012) Functional cooperation between GATA factors and cJUN on the star promoter in MA-10 Leydig cells. J Androl 33:81–87PubMedCrossRefGoogle Scholar
  22. 22.
    Manna PR, Wang XJ, Stocco DM (2003) Involvement of multiple transcription factors in the regulation of steroidogenic acute regulatory protein gene expression. Steroids 68:1125–1134PubMedCrossRefGoogle Scholar
  23. 23.
    Caballero F, Fernandez A, De Lacy AM, Fernandez-Checa JC, Caballeria J, Garcia-Ruiz C (2009) Enhanced free cholesterol, SREBP-2 and StAR expression in human NASH. J Hepatol 50:789–796PubMedCrossRefGoogle Scholar
  24. 24.
    Okuhara K, Abe S, Kondo T, Fujita K, Koda N, Mochizuki H, Fujieda K, Tajima T (2008) Four Japanese patients with adrenal hypoplasia congenita and hypogonadotropic hypogonadism caused by DAX-1 gene mutations: mutant DAX-1 failed to repress steroidogenic acute regulatory protein (StAR) and luteinizing hormone beta-subunit gene promoter activity. Endocr J 55:97–103PubMedCrossRefGoogle Scholar
  25. 25.
    Ikeda Y, Swain A, Weber TJ, Hentges KE, Zanaria E, Lalli E, Tamai KT, Sassone-Corsi P, Lovell-Badge R, Camerino G, Parker KL (1996) Steroidogenic factor 1 and Dax-1 colocalize in multiple cell lineages: potential links in endocrine development. Mol Endocrinol 10:1261–1272PubMedGoogle Scholar
  26. 26.
    Malikova J, Camats N, Fernandez-Cancio M, Heath K, Gonzalez I, Caimari M, Campo MD, Albisu M, Kolouskova S, Audi L, Fluck CE (2014) Human NR5A1/SF-1 mutations show decreased activity on BDNF (brain-derived neurotrophic factor), an important regulator of energy balance: testing impact of novel SF-1 mutations beyond steroidogenesis. PLoS One 9:e104838PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Sandhoff TW, Hales DB, Hales KH, McLean MP (1998) Transcriptional regulation of the rat steroidogenic acute regulatory protein gene by steroidogenic factor 1. Endocrinology 139:4820–4831PubMedGoogle Scholar
  28. 28.
    Hui YY, Lavoie HA (2008) GATA4 reduction enhances 3’,5’-cyclic adenosine 5′-monophosphate-stimulated steroidogenic acute regulatory protein messenger ribonucleic acid and progesterone production in luteinized porcine granulosa cells. Endocrinology 149:5557–5567PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Silverman E, Yivgi-Ohana N, Sher N, Bell M, Eimerl S, Orly J (2006) Transcriptional activation of the steroidogenic acute regulatory protein (StAR) gene: GATA-4 and CCAAT/enhancer-binding protein beta confer synergistic responsiveness in hormone-treated rat granulosa and HEK293 cell models. Mol Cell Endocrinol 252:92–101PubMedCrossRefGoogle Scholar
  30. 30.
    LaVoie HA, Singh D, Hui YY (2004) Concerted regulation of the porcine steroidogenic acute regulatory protein gene promoter activity by follicle-stimulating hormone and insulin-like growth factor I in granulosa cells involves GATA-4 and CCAAT/enhancer binding protein beta. Endocrinology 145:3122–3134PubMedCrossRefGoogle Scholar
  31. 31.
    Shi ZM, Feng YX, Wang JS, Zhang HX, Ding L, Dai JY (2010) Perfluorododecanoic acid-induced steroidogenic inhibition is associated with steroidogenic acute regulatory protein and reactive oxygen species in cAMP-stimulated Leydig cells. Toxicol Sci 114:285–294PubMedCrossRefGoogle Scholar
  32. 32.
    Han XM, Tang R, Chen XJ, Xu B, Qin YF, Wu W, Hu YH, Song L, Xia YK, Wang XR (2012) 2,2′,4,4′-Tetrabromodiphenyl ether (BDE-47) decreases progesterone synthesis through cAMP-PKA pathway and P450scc downregulation in mouse Leydig tumor cells. Toxicology 302:44–50PubMedCrossRefGoogle Scholar
  33. 33.
    Shelby MD (2006) NTP-CERHR monograph on the potential human reproductive and developmental effects of di (2-ethylhexyl) phthalate (DEHP). NTP CERHR MON: v, vii-7, II-iii-xiii passimGoogle Scholar
  34. 34.
    Heudorf U, Mersch-Sundermann V, Angerer J (2007) Phthalates: toxicology and exposure. Int J Hyg Environ Health 210:623–634PubMedCrossRefGoogle Scholar
  35. 35.
    Koch HM, Preuss R, Angerer J (2006) Di(2-ethylhexyl) phthalate (DEHP): human metabolism and internal exposure—an update and latest results. Int J Androl 29:155–165 (discussion 181–5)PubMedCrossRefGoogle Scholar
  36. 36.
    Manna PR, Chandrala SP, Jo Y, Stocco DM (2006) cAMP-independent signaling regulates steroidogenesis in mouse Leydig cells in the absence of StAR phosphorylation. J Mol Endocrinol 37:81–95PubMedCrossRefGoogle Scholar
  37. 37.
    Xiao YC, Hardy DO, Sottas CM, Li XK, Ge RS (2010) Inhibition of LH-stimulated androgen production in rat immature Leydig cells: effects on nuclear receptor steroidogenic factor 1 by FGF2. Growth Factors 28:1–9PubMedCrossRefGoogle Scholar
  38. 38.
    Kuhl AJ, Ross SM, Gaido KW (2007) CCAAT/enhancer binding protein beta, but not steroidogenic factor-1, modulates the phthalate-induced dysregulation of rat fetal testicular steroidogenesis. Endocrinology 148:5851–5864PubMedCrossRefGoogle Scholar
  39. 39.
    El-Khairi R, Martinez-Aguayo A, Ferraz-de-Souza B, Lin L, Achermann JC (2011) Role of DAX-1 (NR0B1) and steroidogenic factor-1 (NR5A1) in human adrenal function. Endocr Dev 20:38–46PubMedGoogle Scholar
  40. 40.
    Jameson JL (2004) Of mice and men: the tale of steroidogenic factor-1. J Clin Endocrinol Metab 89:5927–5929PubMedCrossRefGoogle Scholar
  41. 41.
    Lala DS, Rice DA, Parker KL (1992) Steroidogenic factor I, a key regulator of steroidogenic enzyme expression, is the mouse homolog of fushi tarazu-factor I. Mol Endocrinol 6:1249–1258PubMedGoogle Scholar
  42. 42.
    Luo X, Ikeda Y, Parker KL (1994) A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77:481–490PubMedCrossRefGoogle Scholar
  43. 43.
    Huang CC, Yao HH (2010) Inactivation of Dicer1 in steroidogenic factor 1-positive cells reveals tissue-specific requirement for Dicer1 in adrenal, testis, and ovary. BMC Dev Biol 10:66PubMedCentralPubMedCrossRefGoogle Scholar
  44. 44.
    Sadovsky Y, Crawford PA, Woodson KG, Polish JA, Clements MA, Tourtellotte LM, Simburger K, Milbrandt J (1995) Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids. Proc Natl Acad Sci USA 92:10939–10943PubMedCentralPubMedCrossRefGoogle Scholar
  45. 45.
    Wooton-Kee CR, Clark BJ (2000) Steroidogenic factor-1 influences protein-deoxyribonucleic acid interactions within the cyclic adenosine 3,5-monophosphate-responsive regions of the murine steroidogenic acute regulatory protein gene. Endocrinology 141:1345–1355PubMedGoogle Scholar
  46. 46.
    Reinhart AJ, Williams SC, Clark BJ, Stocco DM (1999) SF-1 (steroidogenic factor-1) and C/EBP beta (CCAAT/enhancer binding protein-beta) cooperate to regulate the murine StAR (steroidogenic acute regulatory) promoter. Mol Endocrinol 13:729–741PubMedGoogle Scholar
  47. 47.
    Christenson LK, Johnson PF, McAllister JM, Strauss JF 3rd (1999) CCAAT/enhancer-binding proteins regulate expression of the human steroidogenic acute regulatory protein (StAR) gene. J Biol Chem 274:26591–26598PubMedCrossRefGoogle Scholar
  48. 48.
    Borch J, Metzdorff SB, Vinggaard AM, Brokken L, Dalgaard M (2006) Mechanisms underlying the anti-androgenic effects of diethylhexyl phthalate in fetal rat testis. Toxicology 223:144–155PubMedCrossRefGoogle Scholar
  49. 49.
    Sugawara T, Kiriakidou M, McAllister JM, Holt JA, Arakane F et al (1997) Regulation of expression of the steroidogenic acute regulatory protein (StAR) gene: a central role for steroidogenic factor 1. Steroids 62:5–9PubMedCrossRefGoogle Scholar
  50. 50.
    Ito E, Toki T, Ishihara H, Ohtani H, Gu L, Yokoyama M, Engel JD, Yamamoto M (1993) Erythroid transcription factor GATA-1 is abundantly transcribed in mouse testis. Nature 362:466–468PubMedCrossRefGoogle Scholar
  51. 51.
    Nishida H, Miyagawa S, Vieux-Rochas M, Morini M, Ogino Y, Suzuki K, Nakagata N, Choi HS, Levi G, Yamada G (2008) Positive regulation of steroidogenic acute regulatory protein gene expression through the interaction between Dlx and GATA-4 for testicular steroidogenesis. Endocrinology 149:2090–2097PubMedCrossRefGoogle Scholar
  52. 52.
    Tremblay JJ, Viger RS (2003) Novel roles for GATA transcription factors in the regulation of steroidogenesis. J Steroid Biochem Mol Biol 85:291–298PubMedCrossRefGoogle Scholar
  53. 53.
    Murayama C, Miyazaki H, Miyamoto A, Shimizu T (2012) Luteinizing hormone (LH) regulates production of androstenedione and progesterone via control of histone acetylation of StAR and CYP17 promoters in ovarian theca cells. Mol Cell Endocrinol 350:1–9PubMedCrossRefGoogle Scholar
  54. 54.
    Hiroi H, Christenson LK, Chang L, Sammel MD, Berger SL, Strauss JF 3rd (2004) Temporal and spatial changes in transcription factor binding and histone modifications at the steroidogenic acute regulatory protein (stAR) locus associated with stAR transcription. Mol Endocrinol 18:791–806PubMedCrossRefGoogle Scholar
  55. 55.
    Tremblay JJ, Viger RS (2003) Transcription factor GATA-4 is activated by phosphorylation of serine 261 via the cAMP/protein kinase a signaling pathway in gonadal cells. J Biol Chem 278:22128–22135PubMedCrossRefGoogle Scholar
  56. 56.
    Nalbant D, Williams SC, Stocco DM, Khan SA (1998) Luteinizing hormone-dependent gene regulation in Leydig cells may be mediated by CCAAT/enhancer-binding protein-beta. Endocrinology 139:272–279PubMedGoogle Scholar
  57. 57.
    Piontkewitz Y, Enerback S, Hedin L (1996) Expression of CCAAT enhancer binding protein-alpha (C/EBP alpha) in the rat ovary: implications for follicular development and ovulation. Dev Biol 179:288–296PubMedCrossRefGoogle Scholar

Copyright information

© Italian Society of Endocrinology (SIE) 2015

Authors and Affiliations

  • Y. Hu
    • 1
    • 2
  • C. Dong
    • 1
    • 2
  • M. Chen
    • 1
    • 2
  • Y. Chen
    • 3
  • A. Gu
    • 1
    • 2
  • Y. Xia
    • 1
    • 2
  • H. Sun
    • 1
    • 2
    • 4
  • Z. Li
    • 5
  • Y. Wang
    • 1
    • 6
  1. 1.State Key Laboratory of Reproductive Medicine, Institute of Toxicology, School of Public HealthNanjing Medical UniversityNanjingChina
  2. 2.Key Laboratory of Modern Toxicology of Ministry of Education, School of Public HealthNanjing Medical UniversityNanjingChina
  3. 3.Department of Molecular Cell Biology and Toxicology, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Cancer Center, School of Public HealthNanjing Medical UniversityNanjingChina
  4. 4.Jiangsu Provincial Center for Disease Control and PreventionNanjingChina
  5. 5.Department of Nutrition and Food Hygiene, School of Public HealthNanjing Medical UniversityNanjingChina
  6. 6.Safety Assessment and Research Center for Drug, Pesticide and Veterinary Drug of Jiangsu ProvinceNanjing Medical UniversityNanjingChina

Personalised recommendations