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17β-Estradiol Rapidly Activates Calcium Release from Intracellular Stores via the GPR30 Pathway and MAPK Phosphorylation in Osteocyte-Like MLO-Y4 Cells

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Abstract

Estrogen regulates critical cellular functions, and its deficiency initiates bone turnover and the development of bone mass loss in menopausal females. Recent studies have demonstrated that 17β-estradiol (E2) induces rapid non-genomic responses that activate downstream signaling molecules, thus providing a new perspective to understand the relationship between estrogen and bone metabolism. In this study, we investigated rapid estrogen responses, including calcium release and MAPK phosphorylation, in osteocyte-like MLO-Y4 cells. E2 elevated [Ca2+] i and increased Ca2+ oscillation frequency in a dose-dependent manner. Immunolabeling confirmed the expression of three estrogen receptors (ERα, ERβ, and G protein-coupled receptor 30 [GPR30]) in MLO-Y4 cells and localized GPR30 predominantly to the plasma membrane. E2 mobilized calcium from intracellular stores, and the use of selective agonist(s) for each ER showed that this was mediated mainly through the GPR30 pathway. MAPK phosphorylation increased in a biphasic manner, with peaks occurring after 7 and 60 min. GPR30 and classical ERs showed different temporal effects on MAPK phosphorylation and contributed to MAPK phosphorylation sequentially. ICI182,780 inhibited E2 activation of MAPK at 7 min, while the GPR30 agonist G-1 and antagonist G-15 failed to affect MAPK phosphorylation levels. G-1-mediated MAPK phosphorylation at 60 min was prevented by prior depletion of calcium stores. Our data suggest that E2 induces the non-genomic responses Ca2+ release and MAPK phosphorylation to regulate osteocyte function and indicate that multiple receptors mediate rapid E2 responses.

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References

  1. Gallagher JC, Levine JP (2011) Preventing osteoporosis in symptomatic postmenopausal women. Menopause 18(1):109–118

    Article  PubMed  Google Scholar 

  2. Emerton KB, Hu B, Woo AA et al (2010) Osteocyte apoptosis and control of bone resorption following ovariectomy in mice. Bone 46(43):577–583

    Article  PubMed  CAS  Google Scholar 

  3. Prossnitz ER, Jeffrey BA, Harriet OS (2008) Estrogen signaling through the transmembrane G protein-coupled receptor GPR30. Annu Rev Physiol 70:165–190

    Article  PubMed  CAS  Google Scholar 

  4. Wu TW, Wang JM, Chen S, Brinton RD (2005) 17β-Estradiol induced Ca2+ influx via L-type calcium channel activates the SRC/ERK/c-AMP response element binding protein signal pathway and BCL-2 expression in rat hippocampal neurons: a potential initiation mechanism for estrogen-induced neuroprotection. Neuroscience 135:159–172

    Article  Google Scholar 

  5. Kuo J, Hamid N, Bondar G, Prossnitz ER, Micevych P (2010) Membrane estrogen receptors stimulate intracellular calcium release and progesterone synthesis in hypothalamic astrocytes. J Neurosci 30(39):12950–12957

    Article  PubMed  CAS  Google Scholar 

  6. Muchekehu RW, Brain JH (2008) 17β-Estradiol rapidly mobilizes intracellular calcium from ryanodine-receptor-gated stores via a PKC-PKA-Erk-dependent pathway in the human eccrine sweat gland cell line NCL-SG3. Cell Calcium 44:276–288

    Article  PubMed  CAS  Google Scholar 

  7. Huo B, Lu XL, Costa KD et al (2010) An ATP-dependent mechanism mediates intercellular calcium signaling in bone cell network under single cell nanoindentation. Cell Calcium 47(43):234–241

    Article  PubMed  CAS  Google Scholar 

  8. Marcello M, Didier P (2010) The unfolding stories of GPR30, a new membrane-bound estrogen receptor. J Endocrinol 204:105–114

    Article  Google Scholar 

  9. Prossnitz ER, Maggiolini M (2009) Mechanisms of estrogen signaling and gene expression via GPR30. Mol Cell Endocrinol 308(1–2):32–38

    Article  PubMed  CAS  Google Scholar 

  10. Wu TW, Chen S, Brinton RD (2011) Membrane estrogen receptors mediate calcium signaling and MAP kinase activation in individual hippocampal neurons. Brain Res 1379:1334–1343

    Article  Google Scholar 

  11. Albanito L, Madeo A, Lappano R et al (2007) G protein-coupled receptor 30 (GPR30) mediates gene expression changes and growth response to 17beta-estradiol and selective GPR30 ligand G-1 in ovarian cancer cells. Cancer Res 67(4):1859–1866

    Article  PubMed  CAS  Google Scholar 

  12. Hsieh YC, Yu HP, Frink M et al (2007) G protein-coupled receptor 30-dependent protein kinase A pathway is critical in non-genomic effects of estrogen in attenuating liver injury after trauma-hemorrhage. Am J Pathol 170(4):1210–1218

    Article  PubMed  CAS  Google Scholar 

  13. Mann V, Huber C, Kogianni G et al (2007) The antioxidant effect of estrogen and selective estrogen receptor modulators in the inhibition of osteocyte apoptosis in vitro. Bone 40(43):674–684

    Article  PubMed  CAS  Google Scholar 

  14. Heino TJ, Chagin AS, Sävendahl L (2008) The novel estrogen receptor G-protein-coupled receptor 30 is expressed in human bone. J Endocrinol 197(2):R1–R6

    Article  PubMed  CAS  Google Scholar 

  15. Chin ER (2005) Role of Ca2+/calmodulin-dependent kinases in skeletal muscle plasticity. J Appl Physiol 99(2):414–423

    Article  PubMed  CAS  Google Scholar 

  16. Hewitt SC, Deroo BJ, Korach KS (2005) A new mediator for an old hormone? Science 307(5715):1572–1573

    Article  PubMed  CAS  Google Scholar 

  17. Cheskis BJ, Greger JG, Nagpal S, Freedman LP (2007) Signaling by estrogens. J Cell Physiol 213(213):610–611

    Article  PubMed  CAS  Google Scholar 

  18. Bologa CG, Revankar CM, Young SM et al (2006) Virtual and biomolecular screening converge on a selective agonist for GPR30. Nat Chem Biol 2(4):207–212

    Article  PubMed  CAS  Google Scholar 

  19. Dennis MK, Burai R, Ramesh C et al (2009) In vivo effects of a GPR30 antagonist. Nat Chem Biol 5(6):421–427

    Article  PubMed  CAS  Google Scholar 

  20. Henriksen Z, Hiken JF, Steinberg TH, Jørgensen NR (2006) The predominant mechanism of intercellular calcium wave propagation changes during long-term culture of human osteoblast-like cells. Cell Calcium 39(35):435–444

    Article  PubMed  CAS  Google Scholar 

  21. Jessop HL, Sjoberg M, Cheng MZ et al (2001) Mechanical strain and estrogen activate estrogen receptor alpha in bone cells. J Bone Miner Res 16(6):1045–1055

    Article  PubMed  CAS  Google Scholar 

  22. Lucas TF, Siu ER, Esteves CA et al (2008) 17β-Estradiol induces the translocation of the estrogen receptors ESR1 and ESR2 to the cell membrane, MAPK3/1 phosphorylation and proliferation of cultured immature rat sertoli cells. Biol Reprod 78(71):101–114

    Article  PubMed  CAS  Google Scholar 

  23. Maiti K, Paul J, Read M et al (2011) G-1-activated membrane estrogen receptors mediate increased contractility of the human myometrium. Endocrinology 152(6):2448–2455

    Article  PubMed  CAS  Google Scholar 

  24. Cheskis BJ, Greger J, Cooch N et al (2008) MNAR plays an important role in ERa activation of Src/MAPK and PI3 K/Akt signaling pathways. Steroids 73(79–10):901–905

    Article  PubMed  CAS  Google Scholar 

  25. Tomida T, Hirose K, Takizawa A et al (2003) NFAT functions as a working memory of Ca2+ signals in decoding Ca2+ oscillation. EMBO J 22(15):3825–3832

    Article  PubMed  CAS  Google Scholar 

  26. Terasawa E, Noel S, Keen KL (2009) Rapid action of oestrogen in luteinising hormone-releasing hormone neurones: the role of GPR30. J Neuroendocrinol 21(24):316–321

    Article  PubMed  CAS  Google Scholar 

  27. Gingerich S, Kim GL, Chalmers JA et al (2010) Estrogen receptor α and G-protein coupled receptor 30 mediate the neuroprotective effects of 17β-estradiol in novel murine hippocampal cell models. Neuroscience 170(171):154–166

    Google Scholar 

  28. Clapham DE (2007) Calcium signaling. Cell 131(136):1047–1058

    Article  PubMed  CAS  Google Scholar 

  29. Pearson G, Robinson F, Gibson BT et al (2001) Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22(2):153–183

    Article  PubMed  CAS  Google Scholar 

  30. Thompson WR, Majid AS, Czymmek KJ et al (2011) Association of the α2δ1 subunit with Ca(v)3.2 enhances membrane expression and regulates mechanically induced ATP release in MLO-Y4 osteocytes. J Bone Miner Res 26(9):2125–2139

    Article  PubMed  CAS  Google Scholar 

  31. Johnston SR (2010) New strategies in estrogen receptor-positive breast cancer. Clin Cancer Res 16(7):1979–1987

    Article  PubMed  CAS  Google Scholar 

  32. Burness ML, Grushko TA, Olopade OI (2010) Epidermal growth factor receptor in triple-negative and basal-like breast cancer: promising clinical target or only a marker? Cancer J 16(1):23–32

    Article  PubMed  CAS  Google Scholar 

  33. Avruch J, Khokhlatchev A, Kyriakis JM et al (2001) Ras activation of the Raf kinase: tyrosine kinase recruitment of the MAP kinase cascade. Recent Prog Horm Res 56:127–155

    Article  PubMed  CAS  Google Scholar 

  34. Bonewald LF (1999) Establishment and characterization of an osteocyte-like cell line, MLO-Y4. J Bone Miner Metab 17(1):61–65

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We are indebted to Dr. Lynda Bonewald for her kind gift of MLO Y4 cells and thank Ms. Jennifer Rosser for help in arranging the transportation and logistics. This work was supported by the Nature Science Foundation of Shanghai, China (Grant 11ZR1440700).

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Authors and Affiliations

  1. Imaging and Characterization Lab, King Abdullah University of Science and Technology, Thuwal, 23955-6900, Saudi Arabia

    Jian Ren

  2. School of Stomatology, Tongji University, No. 399, Yanchang Middle Road, Shanghai, 200072, People’s Republic of China

    Jun Hua Wu

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  1. Jian Ren
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  2. Jun Hua Wu
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Correspondence to Jun Hua Wu.

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The authors have stated that they have no conflict of interest.

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Ren, J., Wu, J.H. 17β-Estradiol Rapidly Activates Calcium Release from Intracellular Stores via the GPR30 Pathway and MAPK Phosphorylation in Osteocyte-Like MLO-Y4 Cells. Calcif Tissue Int 90, 411–419 (2012). https://doi.org/10.1007/s00223-012-9581-x

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  • DOI: https://doi.org/10.1007/s00223-012-9581-x

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