Tumor Biology

, Volume 37, Issue 1, pp 455–462 | Cite as

Transforming growth factor-β signaling pathway cross-talking with ERα signaling pathway on regulating the growth of uterine leiomyoma activated by phenolic environmental estrogens in vitro

  • Yang Shen
  • Yanting Wu
  • Qing Lu
  • Peili Zhang
  • Mulan Ren
Original Article


The aim of this paper is to study the participation of transforming growth factor-β (TGF-β) signaling pathway in mediating the growth of human uterine leiomyoma (UL) activated by phenolic environmental estrogens (EEs), via the interaction between TGF-β and ER signaling pathways. The UL cells were prepared by primary culture and subculture methods. To validate the role of TGF-β3 (5 ng/ml) for the viability of human uterine leiomyoma cells, CCK-8 assay was performed in each of five treatment groups including E2 group (E2 109 mol/l), BPA group (bisphenol A 10 μmol/l), NP group (nonylphenol 32 μmol/l), OP group (octylphenol 8 μmol/l), or control group (DMSO only). Subsequently, qRT-PCR was applied to detect mRNA expressions of ERα and c-fos, while western blot assay was used to test the expressions of p-Smad3, SnoN, and c-fos proteins in all settings mentioned above; the expressions were compared among different groups, and also in settings with and without synchronous treatment of ICI 182,780. Primarily cultured UL cells were successfully established. Compared with the control group, there were statistically significant increases in the proliferation rate of the UL cells in all EE groups or treated with TGF-β3 only (p < 0.05). Nevertheless, a slight decrease in proliferation rate of UL was detected in coexistence with TGF-β3 in all EE groups (p > 0.05). Interestingly, mRNA expressions of ERα and c-fos reduced in the setting of coexistence of TGF-β3 and EEs compared to isolated EE treatment (p < 0.05). Compared with the control group, the expression of p-Smad3 and c-fos proteins significantly decreased (p < 0.05) in each of E2, BPA, NP, and OP group, and the expression of SnoN protein also significantly reduced only in BPA and NP groups (p < 0.05), followed by TGF-β3 treatment. When adding ICI 182,780, the expression of p-Smad3 protein significantly increased in OP group (p < 0.05), but slightly increased in E2, BPA, NP, and OP groups (p > 0.05). However, compared with the control group, the expressions of SnoN and c-fos proteins significantly decreased (p < 0.05) after adding ICI182,780. Moreover, there was a significant statistical difference in the expression of p-Smad3, SnoN, and c-fos proteins between pre- and post-treatment of ICI 182,780 in all groups (p < 0.05). The ERα signaling pathway and TGF-β signaling pathway have different roles in the control of UL cell proliferation. The phenolic EEs can be a promoter of UL cell proliferation, which is mediated by ERα signaling pathway and its cross-talking with TGF-β signaling pathway. Both less exposure to EEs and blockade of TGF signaling pathway are necessary strategies to prevent UL.


Uterine leiomyoma (UL) Environmental estrogens (EEs) Bisphenol A Octylphenol (OP) Nonylphenol (NP) Estrogen receptor (ER) Transforming growth factor-β (TGF-β) 



This work was supported by Maternal and Child Healthcare Project of Jiangsu Province Health Department (F201407), Youth Fund Project of Jiangsu Province Health Department (Q201305), Science and Technology Project of Nanjing City (201201054), National Natural Science Pre-research Project Funds of Southeast University (3290001102), and SRTP Project of Southeast University (T11431001).

Conflicts of interest



  1. 1.
    Levy G, Hill MJ, Beall S, et al. Leiomyoma: genetics, assisted reproduction, pregnancy and therapeutic advances. J Assist Reprod Genet. 2012;29(8):703–12.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Shen Y, Ren ML, Xu J, Xu Q, Ding YQ, Wu ZC, et al. A multicenter case-control study on screening of single nucleotide polymorphisms in estrogen-metabolizing enzymes and susceptibility to uterine leiomyoma in Han Chinese. Gynecol Obstet Invest. 2014;77(4):224–30.CrossRefPubMedGoogle Scholar
  3. 3.
    Shen Y, Xu Q, Xu J, Ren ML, Cai YL. Environmental exposure and risk of uterine leiomyoma: an epidemiologic survey. Eur Rev Med Pharmacol Sci. 2013;17(23):3249–56.PubMedGoogle Scholar
  4. 4.
    Shen Y, Xu Q, Ren M, Feng X, Cai Y, Gao Y. Measurement of phenolic environmental estrogens in women with uterine leiomyoma. PLoS One. 2013;8(11), e79838.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Shen Y, Ren ML, Feng X, Gao YX, Xu Q, Cai YL. Does nonylphenol promote the growth of uterine fibroids? Eur J Obstet Gynecol Reprod Biol. 2014;178:134–7.CrossRefPubMedGoogle Scholar
  6. 6.
    Shen Y, Ren ML, Feng X, Cai YL, Gao YX, Xu Q. An evidence in vitro for the influence of bisphenol A on uterine leiomyoma. Eur J Obstet Gynecol Reprod Biol. 2014;178:80–3.CrossRefPubMedGoogle Scholar
  7. 7.
    Chegini N. Proinflammatory and profibrotic mediators: principal effectors of leiomyoma development as a fibrotic disorder. Semin Reprod Med. 2010;28(3):180–203.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Zhao Y, Wen Y, Polan ML, Qiao J, Chen BH. Increased expression of latent TGF-beta binding protein-1 and fibrillin-1 in human uterine leiomyomata. Mol Hum Reprod. 2007;13(5):343–9.CrossRefPubMedGoogle Scholar
  9. 9.
    Norian JM, Malik M, Parker CY, Joseph D, Leppert PC, Segars JH, et al. Transforming growth factor beta3 regulates the versican variants in the extracellular matrix-rich uterine leiomyomas. Reprod Sci. 2009;16:1153–64.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Halder SK, Goodwin JS, Al-Hendy A. 1,25-Dihydroxyvitamin D3 reduces TGF-beta3-induced fibrosis-related gene expression in human uterine leiomyoma cells. J Clin Endocrinol Metab. 2011;96(4):E754–62.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ewan KB, Oketch-Rabah HA, Ravani SA, Shyamala G, Moses HL, Barcellos-Hoff MH. Proliferation of estrogen receptor-alpha-positive mammary epithelial cells is restrained by transforming growth factor-beta1 in adult mice. Am J Pathol. 2005;167(2):409–17.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Figueroa JD, Flanders KC, Garcia-Closas M, Anderson WF, Yang XR, Matsuno RK, et al. Expression of TGF-beta signaling factors in invasive breast cancers: relationships with age at diagnosis and tumor characteristics. Breast Cancer Res Treat. 2010;121(3):727–35.CrossRefPubMedGoogle Scholar
  13. 13.
    Wang J, Ohara N, Takekida S, et al. Comparative effects of heparin-binding epidermal growth factor-like growth factor on the growth of cultured human uterine leiomyoma cells and myometrial cells. Hum Reprod. 2005;20(6):1456–65.CrossRefPubMedGoogle Scholar
  14. 14.
    Skor MN, Wonder EL, Kocherginsky M, Goyal A, Hall BA, Cai Y, et al. Glucocorticoid receptor antagonism as a novel therapy for triple-negative breast cancer. Clin Cancer Res. 2013;19(22):6163–72.CrossRefPubMedGoogle Scholar
  15. 15.
    Stark K, Burger A, Wu J, Shelton P, Polin L, Li J. Reactivation of estrogen receptor a by vorinostat sensitizes mesenchymal-like triple-negative breast cancer to aminoflavone, a ligand of the aryl hydrocarbon receptor. PLoS One. 2013;8(9), e74525.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Islam MS, Protic O, Giannubilo SR, Toti P, Tranquilli AL, Petraglia F, et al. Uterine leiomyoma: available medical treatments and new possible therapeutic options. J Clin Endocrinol Metab. 2013;98(3):921–34.CrossRefPubMedGoogle Scholar
  17. 17.
    Maruo T, Ohara N, Wang J, Matsuo H. Sex steroidal regulation of uterine leiomyoma growth and apoptosis. Hum Reprod Update. 2004;10(3):207–20.CrossRefPubMedGoogle Scholar
  18. 18.
    Ishikawa H, Ishi K, Serna VA, Kakazu R, Bulun SE, Kurita T. Progesterone is essential for maintenance and growth of uterine leiomyoma. Endocrinology. 2010;151(6):2433–42.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Zhou F, Zhang L, Liu A, Shen Y, Yuan J, Yu X, et al. Measurement of phenolic environmental estrogens in human urine samples by HPLC-MS/MS and primary discussion the possible linkage with uterine leiomyoma. J Chromatogr B Analyt Technol Biomed Life Sci. 2013;938:80–5.CrossRefPubMedGoogle Scholar
  20. 20.
    Di X, Andrews DM, Tucker CJ, Yu L, Moore AB, Zheng X, et al. A high concentration of genistein down-regulates activin A, Smad3 and other TGF-β pathway genes in human uterine leiomyoma cells. Exp Mol Med. 2012;44(4):281–92.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Lee BS, Nowak RA. Human leiomyoma smooth muscle cells show increased expression of transforming growth factor-beta 3 (TGF beta 3) and altered responses to the antiproliferative effects of TGF beta. J Clin Endocrinol Metab. 2001;86(2):913–20.PubMedGoogle Scholar
  22. 22.
    Arici A, Sozen I. Transforming growth factor-beta3 is expressed at high levels in leiomyoma where it stimulates fibronectin expression and cell proliferation. Fertil Steril. 2000;73(5):1006–11.CrossRefPubMedGoogle Scholar
  23. 23.
    Borahay MA, Al-Hendy A, Kilic GS, Boehning D. Signaling pathways in leiomyoma: understanding pathobiology and implications for therapy. Mol Med. 2015 Apr 13. [Epub ahead of print].Google Scholar
  24. 24.
    Zhou Z, Qiao JX, Shetty A, Wu G, Huang Y, Davidson NE, et al. Regulation of estrogen receptor signaling in breast carcinogenesis and breast cancer therapy. Cell Mol Life Sci. 2014;71(8):1549.CrossRefPubMedGoogle Scholar
  25. 25.
    Chegini N, Luo X, Ding L, Ripley D. The expression of Smads and transforming growth factor beta receptors in leiomyoma and myometrium and the effect of gonadotropin releasing hormone analogue therapy. Mol Cell Endocrinol. 2003;209(1-2):9–16.CrossRefPubMedGoogle Scholar
  26. 26.
    Salama SA, Diaz-Arrastia CR, Kilic GS, Kamel MW. 2-Methoxyestradiol causes functional repression of transforming growth factor β3 signaling by ameliorating Smad and non-Smad signaling pathways in immortalized uterine fibroid cells. Fertil Steril. 2012;98(1):178–84.CrossRefPubMedGoogle Scholar
  27. 27.
    Tulchinsky E. Fos family members: regulation, structure and role in oncogenic transformation. Histol Histopathol. 2000;15(3):921–8.PubMedGoogle Scholar
  28. 28.
    Band AM, Laiho M. Crosstalk of TGF-β and estrogen receptor signaling in breast cancer. J Mammary Gland Biol Neoplasia. 2011;16(2):109–15.CrossRefPubMedGoogle Scholar
  29. 29.
    Goto N, Hiyoshi H, Ito I, Tsuchiya M, Nakajima Y, Yanagisawa J. Estrogen and antiestrogens alter breast cancer invasiveness by modulating the transforming growth factor-β signaling pathway. Cancer Sci. 2011;102(8):1501–8.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2015

Authors and Affiliations

  • Yang Shen
    • 1
  • Yanting Wu
    • 1
  • Qing Lu
    • 1
  • Peili Zhang
    • 1
  • Mulan Ren
    • 1
  1. 1.Department of Obstetrics and Gynecology, Zhongda Hospital, School of MedicineSoutheast UniversityNanjingChina

Personalised recommendations