Advertisement

GPER Mediates Non-Genomic Effects of Estrogen

  • Marco Pupo
  • Marcello Maggiolini
  • Anna Maria Musti
Part of the Methods in Molecular Biology book series (MIMB, volume 1366)

Abstract

Estrogens are important modulators of a broad spectrum of physiological functions in humans. However, despite their beneficial actions, a number of lines of evidence correlate the sustained exposure to exogenous estrogen with increased risk of the onset of various cancers. Mainly these steroid hormones induce their effects by binding and activating estrogen receptors (ERα and ERβ). These receptors belong to the family of ligand-regulated transcription factors, and upon activation they regulate the expression of different target genes by binding directly to specific DNA sequences. On the other hand, in recent years it has become clear that the G protein-coupled estrogen receptor 30 (GPR30/GPER) is able to mediate non-genomic action of estrogens in different cell contexts. In particular, GPER has been shown to specifically bind estrogens, and in turn to functionally cross-react with diverse cell signaling systems such as the epidermal growth factor receptor (EGFR) pathway, the Notch signaling pathway and the mitogen-activated protein kinases (MAPK) pathway. In this chapter we will present some of the different experimental techniques currently used to demonstrate the functional role of GPER in mediating non-genomic actions of estrogens, such as the dual luciferase assay, assessment of the involvement of GPER in the stimulation of cell migration in breast cancer cell lines and in cancer-associated fibroblasts, and chromatin immunoprecipitation assay. Overall, the experimental procedures described herein represent key instruments for assessing the biological role of GPER in mediating non-genomic signals of estrogen.

Key words

Estrogen Breast cancer GPR30/GPER Non-genomic signaling MAPK phosphorylation 

References

  1. 1.
    Liang J, Shang Y (2013) Estrogen and cancer. Annu Rev Physiol 75:225–240CrossRefGoogle Scholar
  2. 2.
    Ascenzi P, Bocedi A, Marino M (2006) Structure–function relationship of estrogen receptoralpha and beta: impact on human health. Mol Aspects Med 27:299–402CrossRefGoogle Scholar
  3. 3.
    Carmeci C, Thompson DA, Ring HZ, Francke U, Weigel RJ (1997) Identification of a gene (GPR30) with homology to the G-protein-coupled receptor superfamily associated with estrogen receptor expression in breast cancer. Genomics 45:607–617CrossRefGoogle Scholar
  4. 4.
    Maggiolini M, Picard D (2010) The unfolding stories of GPR30, a new membrane-bound estrogen receptor. J Endocrinol 204:105–114CrossRefGoogle Scholar
  5. 5.
    Albanito L, Madeo A, Lappano R et al (2007) G protein-coupled receptor 30 (GPR30) mediates gene expression changes and growthresponse to 17β-estradiol and selective GPR30 ligand G-1 in ovarian cancer cells. Cancer Res 67:1859–1866CrossRefGoogle Scholar
  6. 6.
    Albanito L, Sisci D, Aquila S et al (2008) EGF induces GPR30 expression in estrogen receptor-negative breast cancer cells. Endocrinology 149:3799–3808CrossRefGoogle Scholar
  7. 7.
    Chimento A, Sirianni R, Casaburi I et al (2012) 17β-Estradiol activates GPER- and ESR1-dependent pathways inducing apoptosis in GC-2 cells, a mouse spermatocyte-derived cell line. Mol Cell Endocrinol 355:49–59CrossRefGoogle Scholar
  8. 8.
    De Marco P, Bartella V, Vivacqua A et al (2013) Insulin-like growth factor-I regulates GPER expression and function in cancer cells. Oncogene 32:678–688CrossRefGoogle Scholar
  9. 9.
    Filice E, Recchia AG, Pellegrino D et al (2009) A new membrane G protein-coupled receptor (GPR30) is involved in the cardiac effects of17beta-estradiol in the male rat. J Physiol Pharmacol 60:3–10PubMedGoogle Scholar
  10. 10.
    Lappano R, Rosano C, De Marco P et al (2010) Estriolacts as a GPR30 antagonist in estrogen receptor-negative breast cancer cells. Mol Cell Endocrinol 320:162–170CrossRefGoogle Scholar
  11. 11.
    Maggiolini M, Vivacqua A, Fasanella G et al (2004) The G protein-coupled receptor GPR30 mediates c-fos up-regulation by17beta-estradiol and phytoestrogens in breast cancer cells. J Biol Chem 279:27008–27016CrossRefGoogle Scholar
  12. 12.
    Pupo M, Pisano A, Lappano R et al (2012) Bisphenol A induces gene expression changes and proliferative effects through GPER in breast cancer cells and cancer-associated fibroblasts. Environ Health Perspect 120:1177–1182CrossRefGoogle Scholar
  13. 13.
    Recchia AG, De Francesco EM, Vivacqua A et al (2011) The G protein-coupled receptor 30 is up-regulated by hypoxia inducible factor-1α (HIF-1α) in breast cancer cells and cardiomyocytes. J Biol Chem 286:10773–10782CrossRefGoogle Scholar
  14. 14.
    Thomas P, Pang Y, Filardo EJ et al (2005) Identity of an estrogen membrane receptor coupled to a G protein in human breast cancer cells. Endocrinology 146:624–632CrossRefGoogle Scholar
  15. 15.
    Vivacqua A, Bonofiglio D, Albanito L et al (2006) 17β-Estradiol, genistein, and 4-hydroxytamoxifen induce the proliferation of thyroid cancer cells through the G protein coupled-receptor GPR30. Mol Pharmacol 70:1414–1423CrossRefGoogle Scholar
  16. 16.
    Vivacqua A, Bonofiglio D, Recchia AG et al (2006) The G protein-coupled receptor GPR30 mediates the proliferative effects inducedby 17β-estradiol and hydroxytamoxifen in endometrial cancer cells. Mol Endocrinol 20:631–646CrossRefGoogle Scholar
  17. 17.
    Vivacqua A, Romeo E, De Marco P et al (2012) GPER mediates the Egr-1 expression induced by 17β-estradiol and 4-hydroxitamoxifen in breast and endometrial cancer cells. Breast Cancer Res Treat 133:1025–1035CrossRefGoogle Scholar
  18. 18.
    Prossnitz ER, Maggiolini M (2009) Mechanisms of estrogen signaling and gene expression via GPR30. Mol Cell Endocrinol 308:32–38CrossRefGoogle Scholar
  19. 19.
    Filardo EJ, Quinn JA, Bland KI (2000) Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Mol Endocrinol 14:1649–1660CrossRefGoogle Scholar
  20. 20.
    Lappano R, Pisano A, Maggiolini M (2014) GPER function in breast cancer: an overview. Front Endocrinol (Lausanne) 5:66Google Scholar
  21. 21.
    Pupo M, Pisano A, Abonante S et al (2014) GPER activates notch signaling in breast cancer cells and cancer-associated fibroblasts (CAFs). Int J Biochem Cell Biol 46:56–67CrossRefGoogle Scholar
  22. 22.
    Quail DF, Joyce JA (2013) Microenvironmental regulation of tumor progression and metastasis. Nat Med 11:1423–1437CrossRefGoogle Scholar
  23. 23.
    Dittmer J, Leyh B (2015) The impact of tumor stroma on drug response in breast cancer. Semin Cancer Biol 31:3–15CrossRefGoogle Scholar
  24. 24.
    Kalluri R, Zeisberg M (2006) Fibroblasts in cancer. Nat Rev Cancer 5:392–401CrossRefGoogle Scholar
  25. 25.
    Luo H, Yang G, Yu T et al (2014) GPER-mediated proliferation and estradiol production in breast cancer-associated fibroblasts. Endocr Relat Cancer 21:355–369CrossRefGoogle Scholar
  26. 26.
    Madeo A, Maggiolini M (2010) Nuclear alternate estrogen receptor GPR30 mediates17βestradiol-induced gene expression and migration in breast cancer-associated fibroblasts. Cancer Res 70:6036–6046CrossRefGoogle Scholar
  27. 27.
    Pupo M, Vivacqua A, Perrotta I et al (2013) The nuclear localization signal is required for nuclear GPER translocation and function in breast Cancer-Associated Fibroblasts (CAFs). Mol Cell Endocrinol 376:23–32CrossRefGoogle Scholar
  28. 28.
    Pandey DP, Lappano R, Albanito L et al (2009) Estrogenic GPR30 signaling induces proliferation and migration of breast cancer cells through CTGF. EMBO J 28:523–532CrossRefGoogle Scholar
  29. 29.
    Maggiolini M, Donzé O, Picard D (1999) A non-radioactive method for inexpensive quantitative RT-PCR. Biol Chem 380:695–697CrossRefGoogle Scholar
  30. 30.
    Chaqour B, Yang R, Sha Q (2006) Mechanical stretch modulates the promoter activity of the profibrotic factor CCN2 through increased actin polymerization and NF-kappaB activation. J Biol Chem 281:20608–20622CrossRefGoogle Scholar
  31. 31.
    Kaneyama JK, Shibanuma M, Nose K (2002) Transcriptional activation of the c-fos gene by a LIM protein, Hic-5. Biochem Biophys Res Commun 299:360–365CrossRefGoogle Scholar
  32. 32.
    Chen CC, Lee WR, Safe S (2004) Egr-1 is activated by 17beta-estradiol in MCF-7 cells by mitogen-activated protein kinase-dependent phosphorylation of ELK-1. J Cell Biochem 93:1063–1107CrossRefGoogle Scholar
  33. 33.
    Dennis MK, Field AS, Burai R et al (2011) Identification of a GPER/GPR30 antagonist with improved estrogen receptor counterselectivity. J Steroid Biochem Mol Biol 127:358–366CrossRefGoogle Scholar
  34. 34.
    DeRose YS, Gligorich KM, Wang G et al (2013) Patient-derived models of human breast cancer: protocols for in vitro and in vivo applications in tumor biology and translational medicine. Curr Protoc Pharmacol 14:14–23Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Marco Pupo
    • 1
  • Marcello Maggiolini
    • 1
  • Anna Maria Musti
    • 1
    • 2
  1. 1.Department of Pharmacy, Health and Nutritional SciencesUniversity of CalabriaRendeItaly
  2. 2.Institute for Clinical NeurobiologyUniversity of WürzburgWürzburgGermany

Personalised recommendations