Membrane estrogen receptor-α levels predict estrogen-induced ERK1/2 activation in MCF-7 cells

Breast Cancer Research

Cheryl S Watson¹  Dragoslava Zivadinovic¹ 

[1] Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas, USA

关键词: extracellular regulated protein kinase;    MCF-7 human breast cancer cells;    membrane estrogen receptor-α;   

Others  :  1115050
DOI  :  10.1186/bcr959

 received in 2004-04-16, accepted in 2004-10-07,  发布年份 2004

【 摘 要 】

Introduction

We examined the participation of a membrane form of estrogen receptor (mER)-α in the activation of mitogen-activated protein kinases (extracellular signal-regulated kinase [ERK]1 and ERK2) related to cell growth responses in MCF-7 cells.

Methods

We immunopanned and subsequently separated MCF-7 cells (using fluorescence-activated cell sorting) into mER-α-enriched (mER^high) and mER-α-depleted (mER^low) populations. We then measured the expression levels of mER-α on the surface of these separated cell populations by immunocytochemical analysis and by a quantitative 96-well plate immunoassay that distinguished between mER-α and intracellular ER-α. Western analysis was used to determine colocalized estrogen receptor (ER)-α and caveolins in membrane subfractions. The levels of activated ERK1 and ERK2 were determined using a fixed cell-based enzyme-linked immunosorbent assay developed in our laboratory.

Results

Immunocytochemical studies revealed punctate ER-α antibody staining of the surface of nonpermeabilized mER^high cells, whereas the majority of mER^low cells exhibited little or no staining. Western analysis demonstrated that mER^high cells expressed caveolin-1 and caveolin-2, and that ER-α was contained in the same gradient-separated membrane fractions. The quantitative immunoassay for ER-α detected a significant difference in mER-α levels between mER^high and mER^low cells when cells were grown at a sufficiently low cell density, but equivalent levels of total ER-α (membrane plus intracellular receptors). These two separated cell subpopulations also exhibited different kinetics of ERK1/2 activation with 1 pmol/l 17β-estradiol (E₂), as well as different patterns of E₂ dose-dependent responsiveness. The maximal kinase activation was achieved after 10 min versus 6 min in mER^high versus mER^low cells, respectively. After a decline in the level of phosphorylated ERKs, a reactivation was seen at 60 min in mER^high cells but not in mER^low cells. Both 1A and 2B protein phosphatases participated in dephosphorylation of ERKs, as demonstrated by efficient reversal of ERK1/2 inactivation with okadaic acid and cyclosporin A.

Conclusion

Our results suggest that the levels of mER-α play a role in the temporal coordination of phosphorylation/dephosphorylation events for the ERKs in breast cancer cells, and that these signaling differences can be correlated to previously demonstrated differences in E₂-induced cell proliferation outcomes in these cell types.

【 授权许可】

   
2004 Zivadinovic and Watson, licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Tsai MJ, O'Malley BW: Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 1994, 63:451-486.
• [2]Watson CS, Gametchu B: Proteins of multiple classes participate in nongenomic steroid actions. Exp Biol Med (Maywood) 2003, 228:1272-1281.
• [3]Watson CS, Gametchu B: Membrane-initiated steroid actions and the proteins that mediate them. Proc Soc Exp Biol Med 1999, 220:9-19.
• [4]Farach-Carson MC, Davis PJ: Steroid hormone interactions with target cells: cross talk between membrane and nuclear pathways. J Pharmacol Exp Ther 2003, 307:839-845.
• [5]Watson CS: The Identities of Membrane Steroid Receptors ... and Other Proteins Mediating Nongenomic Steroid Action. Boston: Kluwer Academic Publishers; 2003.
• [6]Shaul PW: Regulation of endothelial nitric oxide synthase: location, location, location. Annu Rev Physiol 2002, 64:749-774.
• [7]Razandi M, Oh P, Pedram A, Schnitzer J, Levin ER: ERs associate with and regulate the production of caveolin: implications for signaling and cellular actions. Mol Endocrinol 2002, 16:100-115.
• [8]Watson CS, Gametchu B: Membrane-initiated steroid actions and the proteins which mediate them [review]. Proc Soc Exp Biol Med 1999, 220:9-19.
• [9]Russell KS, Haynes MP, Sinha D, Clerisme E, Bender JR: Human vascular endothelial cells contain membrane binding sites for estradiol, which mediate rapid intracellular signaling. Proc Natl Acad Sci USA 2000, 97:5930-5935.
• [10]Endoh H, Sasaki H, Maruyama K, Takeyama K, Waga I, Shimizu T, Kato S, Kawashima H: Rapid activation of MAP kinase by estrogen in a bone cell line. Biochem Biophys Res Commun 1997, 235:99-102.
• [11]Watters JJ, Campbell JS, Cunningham MJ, Krebs EG, Dorsa DM: Rapid membrane effects of steroids in neuroblastoma cells: effects of estrogen and mitogen activated protein kinase signalling cascade and C-FOS immediate early gene transcription. Endocrinology 1997, 138:4030-4033.
• [12]Migliaccio A, Di Domenico M, Castoria G, De Falco A, Bontempo P, Nola E, Auricchio F: Tyrosine kinase/P21(RAS)/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells. EMBO J 1996, 15:1292-1300.
• [13]Razandi M, Pedram A, Park ST, Levin ER: Proximal events in signaling by plasma membrane estrogen receptors. J Biol Chem 2003, 278:2701-2712.
• [14]Zhang Z, Maier B, Santen RJ, Song RX: Membrane association of estrogen receptor alpha mediates estrogen effect on MAPK activation. Biochem Biophys Res Commun 2002, 294:926-933.
• [15]Pappas TC, Gametchu B, Yannariello-Brown J, Collins TJ, Watson CS: Membrane estrogen receptors in GH3/B6 cells are associated with rapid estrogen-induced release of prolactin. Endocrine 1994, 2:813-822.
• [16]Pappas TC, Gametchu B, Watson CS: Membrane estrogen receptor-enriched GH3/B6 cells have an enhanced non-genomic response to estrogen. Endocrine 1995, 3:743-749.
• [17]Pappas TC, Gametchu B, Watson CS: Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand binding. FASEB J 1995, 9:404-410.
• [18]Norfleet AM, Clarke C, Gametchu B, Watson CS: Antibodies to the estrogen receptor-a modulate prolactin release from rat pituitary tumor cells through plasma membrane estrogen receptors. FASEB J 2000, 14:157-165.
• [19]English J, Pearson G, Wilsbacher J, Swantek J, Karandikar M, Xu S, Cobb MH: New insights into the control of MAP kinase pathways. Exp Cell Res 1999, 253:255-270.
• [20]Gametchu B: Glucocorticoid receptor-like antigen in lymphoma cell membranes: correlation to cell lysis. Science 1987, 236:456-461.
• [21]Gametchu B, Watson C, Shih C, Dashew B: Studies on the arrangement of glucocorticoid receptors in the plasma membrane of S-49 lymphoma cells. Steroids 1991, 56:411-419.
• [22]Norfleet AM, Thomas ML, Gametchu B, Watson CS: Estrogen receptor-a detected on the plasma membrane of aldehyde-fixed GH3/B6/F10 rat pituitary tumor cells by enzyme-linked immunocytochemistry. Endocrinology 1999, 140:3805-3814.
• [23]Campbell CH, Watson CS: A comparison of membrane vs. intracellular estrogen receptor-alpha in GH3/B6 pituitary tumor cells using a quantitative plate immunoassay. Steroids 2001, 66:727-736.
• [24]Campbell CH, Bulayeva N, Brown DB, Gametchu B, Watson CS: Regulation of the membrane estrogen receptor-α: role of cell density, serum, cell passage number, and estradiol. FASEB J 2002, 16:1917-1927.
• [25]Song KS, Li SW, Okamoto T, Quilliam LA, Sargiacomo M, Lisanti MP: Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains: detergent-free purification of caveolae membranes. J Biol Chem 1996, 271:9690-9697.
• [26]Bulayeva NN, Gametchu B, Watson CS: Quantitative measurement of estrogen-induced ERK 1 and 2 activation via multiple membrane-initiated signaling pathways. Steroids 2004, 69:181-192.
• [27]Lottering M-L, Haag M, Seegers JC: Effects of 17β-estradiol metabolites on cell cycle events in MCF-7 cells. Cancer Res 1992, 52:5926-5932.
• [28]Zivadinovic D, Gametchu B, Watson CS: Membrane estrogen receptor-α levels in MCF-7 breast cancer cells predict cAMP and proliferation responses. Breast Cancer Res 2004, 7:R101-R112.
• [29]Lobenhofer EK, Marks JR: Estrogen-induced mitogenesis of MCF-7 cells does not require the induction of mitogen-activated protein kinase activity. J Steroid Biochem Mol Biol 2000, 75:11-20.
• [30]Wera S, Hemmings BA: Serine/threonine protein phosphatases. Biochem J 1995, 311:17-29.
• [31]Boe R, Gjertsen BT, Vintermyr OK, Houge G, Lanotte M, Doskeland SO: The protein phosphatase inhibitor okadaic acid induces morphological-changes typical of apoptosis in mammalian-cells. Exp Cell Res 1991, 195:237-246.
• [32]Cohen P, Holmes CF, Tsukitani Y: Okadaic acid: a new probe for the study of cellular regulation. Trends Biochem Sci 1990, 15:98-102.
• [33]Pietras RJ, Szego CM: Specific binding sites for oestrogen at the outer surfaces of isolated endometrial cells. Nature 1977, 265:69-72.
• [34]Berthois Y, Pourreau-Schneider N, Gandilhon P, Mittre H, Tubiana N, Martin PM: Estradiol membrane binding sites on human breast cancer cell lines. Use of a fluorescent estradiol conjugate to demonstrate plasma membrane binding systems. J Steroid Biochem 1986, 25:963-972.
• [35]Germain PS, Metezeau P, Tiefenauer LX, Kiefer H, Ratinaud MH, Habrioux G: Use of a biotinyl-estradiol derivative to demonstrate estradiol-membrane binding sites on adherent human breast cancer MCF-7 cells. Anticancer Res 1993, 13:2347-2354.
• [36]Sager R, Sheng S, Anisowicz A, Sotiropoulou G, Zou Z, Stenman G, Swisshelm K, Chen Z, Hendrix MJ, Pemberton P: RNA genetics of breast cancer: Maspin as paradigm. Cold Spring Harb Symp Quant Biol 1994, LIX:537-546.
• [37]Engelman JA, Zhang X, Galbiati F, Volonte D, Sotgia F, Pestell RG, Minetti C, Scherer PE, Okamoto T, Lisanti MP: Molecular genetics of the caveolin gene family: implications for human cancers, diabetes, Alzheimer disease, and muscular dystrophy. Am J Hum Genet 1998, 63:1578-1587.
• [38]Lavie Y, Fiucci G, Liscovitch M: Up-regulation of caveolae and caveolar constituents in multidrug-resistant cancer cells. J Biol Chem 1998, 273:32380-32383.
• [39]Scherer PE, Lewis RY, Volonte D, Engelman JA, Galbiati F, Couet J, Kohtz DS, van Donselaar E, Peters P, Lisanti MP: Cell-type and tissue-specific expression of caveolin-2. Caveolins 1 and 2 co-localize and form a stable hetero-oligomeric complex in vivo. J Biol Chem 1997, 272:29337-29346.
• [40]Jensen J, Kitlen JW, Briand P, Labrie F, Lykkesfeldt AE: Effect of antiestrogens and aromatase inhibitor on basal growth of the human breast cancer cell line MCF-7 in serum-free medium. J Steroid Biochem Mol Biol 2003, 84:469-478.
• [41]Santen R, Jeng MH, Wang JP, Song R, Masamura S, McPherson R, Santner S, Yue W, Shim WS: Adaptive hypersensitivity to estradiol: potential mechanism for secondary hormonal responses in breast cancer patients. J Steroid Biochem Mol Biol 2001, 79:115-125.
• [42]Poola I, Koduri S, Chatra S, Clarke R: Identification of twenty alternatively spliced estrogen receptor alpha mRNAs in breast cancer cell lines and tumors using splice targeted primer approach. J Steroid Biochem Mol Biol 2000, 72:249-258.
• [43]Fasco MJ, Keyomarsi K, Arcaro KF, Gierthy JF: Expression of an estrogen receptor alpha variant protein in cell lines and tumors. Mol Cell Endocrinol 2000, 162:167-180.
• [44]Li L, Haynes MP, Bender JR: Plasma membrane localization and function of the estrogen receptor alpha variant (ER46) in human endothelial cells. Proc Natl Acad Sci USA 2003, 100:4807-4812.
• [45]Flouriot G, Brand H, Denger S, Metivier R, Kos M, Reid G, Sonntag-Buck V, Gannon F: Identification of a new isoform of the human estrogen receptor-alpha (hER-alpha) that is encoded by distinct transcripts and that is able to repress hER-alpha activation function 1. EMBO J 2000, 19:4688-4700.
• [46]Marquez DC, Pietras RJ: Membrane-associated binding sites for estrogen contribute to growth regulation of human breast cancer cells. Oncogene 2001, 20:5420-5430.
• [47]Bulayeva N, Gametchu B, Watson CS: Quantitative measurement of MAP kinase activation by estrogens via multiple signaling pathways. Steroids 2004, in press.
• [48]Filardo EJ, Quinn JA, Bland KI, Frackelton AR: 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 2000, 14:1649-1660.
• [49]Ahola TM, Manninen T, Alkio N, Ylikomi T: G protein-coupled receptor 30 is critical for a progestin-induced growth inhibition in MCF-7 breast cancer cells. Endocrinology 2002, 143:3376-3384.
• [50]Ahola TM, Purmonen S, Pennanen P, Zhuang YH, Tuohimaa P, Ylikomi T: Progestin upregulates G-protein-coupled receptor 30 in breast cancer cells. Eur J Biochem 2002, 269:2485-2490.
• [51]Chang FM, Steelman LS, Shelton JG, Lee JT, Navolanic PM, Blalock WL, Franklin R, McCubrey JA: Regulation of cell cycle progression and apoptosis by the Ras/Raf/MEK/ERK pathway. Int J Oncol 2003, 22:469-480.
• [52]Pink JJ, Jordan VC: Models of estrogen receptor regulation by estrogens and antiestrogens in breast cancer cell lines. Cancer Res 1996, 56:2321-2330.
• [53]Dudley MW, Sheeler CQ, Wang H, Khan S: Activation of the human estrogen receptor by the antiestrogens ICI 182,780 and tamoxifen in yeast genetic systems: implications for their mechanism of action. Proc Natl Acad Sci USA 2000, 97:3696-3701.
• [54]Aronica SM, Kraus WL, Katzenellenbogen BS: Estrogen action via the cAMP signaling pathway: stimulation of adenylate cyclase and cAMP-regulated gene transcription. Proc Natl Acad Sci USA 1994, 91:8517-8521.
• [55]Kiefer T, Ram PT, Yuan L, Hill SM: Melatonin inhibits estrogen receptor transactivation and cAMP levels in breast cancer cells. Breast Cancer Res Treat 2002, 71:37-45.
• [56]Keshamouni VG, Mattingly RR, Reddy KB: Mechanism of 17-beta-estradiol-induced Erk1/2 activation in breast cancer cells. A role for HER2 AND PKC-delta. J Biol Chem 2002, 277:22558-22565.
• [57]Improta-Brears T, Whorton AR, Codazzi F, York JD, Meyer T, McDonnell DP: Estrogen-induced activation of mitogen-activated protein kinase requires mobilization of intracellular calcium. Proc Natl Acad Sci USA 1999, 96:4686-4691.
• [58]Varma H, Conrad SE: Antiestrogen ICI 182,780 decreases proliferation of insulin-like growth factor I (IGF-I)-treated MCF-7 cells without inhibiting IGF-I signaling. Cancer Res 2002, 62:3985-3991.
• [59]Yue W, Wang JP, Conaway M, Masamura S, Li Y, Santen RJ: Activation of the MAPK pathway enhances sensitivity of MCF-7 breast cancer cells to the mitogenic effect of estradiol. Endocrinology 2002, 143:3221-3229.
• [60]Wong JK, Le HH, Zsarnovszky A, Belcher SM: Estrogens and ICI182,780 (Faslodex) modulate mitosis and cell death in immature cerebellar neurons via rapid activation of p44/p42 mitogen-activated protein kinase. J Neurosci 2003, 23:4984-4995.
• [61]Watson CS, Pappas TC, Gametchu B: The other estrogen receptor in the plasma membrane: implications for the actions of environmental estrogens. Environ Health Perspect 1995, 103(Suppl 7):41-50.
• [62]Robertson JA, Zhang Y, Ing NH: ICI 182,780 acts as a partial agonist and antagonist of estradiol effects in specific cells of the sheep uterus. J Steroid Biochem Mol Biol 2001, 77:281-287.
• [63]Manole D, Schildknecht B, Gosnell B, Adams E, Derwahl M: Estrogen promotes growth of human thyroid tumor cells by different molecular mechanisms. J Clin Endocrinol Metab 2001, 86:1072-1077.
• [64]Cohen P: The regulation of protein function by multisite phosphorylation: a 25 year update. Trends Biochem Sci 2000, 25:596-601.
• [65]Carr BI, Wang Z, Kar S: K vitamins, PTP antagonism, and cell growth arrest. J Cell Physiol 2002, 193:263-274.
• [66]Pawlowski M, Ragab A, Rosa JP, Bryckaert M: Selective dephosphorylation of the threonine(183) residue of ERK2 upon (alpha)llb(beta)3 engagement in platelets. FEBS Lett 2002, 521:145-151.
• [67]Camps M, Nichols A, Arkinstall S: Dual specificity phosphatases: a gene family for control of MAP kinase function. FASEB J 2000, 14:6-16.
• [68]Zhang ZY, Zhou B, Xie L: Modulation of protein kinase signaling by protein phosphatases and inhibitors. Pharmacol Ther 2002, 93:307-317.