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Proc Natl Acad Sci U S A. 2006 June 13; 103(24): 9063–9068. Published online 2006 June 5. doi: 10.1073/pnas.0603339103. | PMCID: PMC1482566 |
Copyright © 2006 by The National Academy of Sciences of the USA Cell Biology A variant of estrogen receptor-α, hER-α36: Transduction of estrogen- and antiestrogen-dependent membrane-initiated mitogenic signaling ZhaoYi Wang,*† XinTian Zhang,* Peng Shen,*‡ Brian W. Loggie,* YunChao Chang,§ and Thomas F. Deuel§ *Cancer Center, Creighton University, 2500 California Plaza, Omaha, NE 68178; ‡First Affiliated Hospital, Medical College of Zhejiang University, 79 QingChun Road, Hangzhou 310003, People's Republic of China; and §Departments of Molecular and Experimental Medicine and Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 Received October 20, 2005. The status of the 66-kDa human estrogen receptor-α (hER-α66) is a critical determinant in the assessment of the prognosis and in the design of treatment strategies of human breast cancer. Recently, we cloned the cDNA of an alternatively spliced variant of hER-α66, termed hER-α36; the predicted protein lacks both transcriptional activation domains of hER-α66 but retains its DNA-binding domain, partial dimerization, and ligand-binding domains and three potential myristoylation sites located near the N terminus. These findings thus predict that hER-α36 functions very differently from hER-α66 in response to estrogen signaling. We now demonstrate that hER-α36 inhibits the estrogen-dependent and estrogen-independent transactivation activities of hER-α66 and hER-β. We further demonstrate that hER-α36 is predominantly associated with the plasma membrane where it transduces both estrogen- and antiestrogen-dependent activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and stimulates cell growth. We conclude that hER-α36 is a predominantly membrane-based, unique alternatively spliced variant of hER-α66 that acts as a dominant-negative effector of both estrogen-dependent and estrogen-independent transactivation functions signaled through hER-α66 and ER-β; it also transduces membrane-initiated estrogen-dependent activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase mitogenic signaling pathway. The estrogen and antiestrogen signaling pathways mediated by hER-α36 provide an alternative explanation for why some human breast cancers are resistant to and others are worsened by antiestrogen therapy; the data suggest that hER-α36 also may be an important marker to direct therapy in human breast cancers, and perhaps hER-α36 also may transduce estrogen-dependent signaling in other estrogen target tissues. Keywords: breast cancer, mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), membrane antiestrogen signaling, membrane estrogen signaling Breast cancer is the second-most-common cause of mortality in women in the United States; the toll in human life is particularly devastating because breast cancer strikes women at times of highest productivity and in the years of childbearing and rearing of family. This toll on morbidity and human life continues despite great progress in the diagnosis and development of treatment strategies in human breast cancer, leaving as a high priority the solution to the many remaining questions in understanding the mechanisms that drive breast cancer. A landmark in breast cancer research and in understanding mechanisms of how hormones signal their many functions in hormone-responsive tissues was the result of early efforts that led to the identification of the human estrogen receptor (hER) ( 1– 8). Estrogen signaling is mediated by specific nuclear receptors designated as hER-α66, hER-α46, and hER-β. hER-α66 and hER-β share a common structural architecture ( 1, 2), which includes three independent but interacting functional domains, the N-terminal A/B domains, the C domain or the DNA-binding domain, and the D/E/F domains, collectively known as the ligand-binding domains. The N-terminal domain of hER-α66 also has a ligand-independent activation function (AF-1); this region is involved in the interactions of hER-α66 with coactivators and transcriptional activation of target genes. The DNA-binding domain, or C domain, contains a two-zinc-finger-like structure, which is important in receptor dimerization and the binding of hER-α66 to specific DNA sequences. The C-terminal E/F domain is a ligand-binding domain that mediates ligand binding, receptor dimerization, nuclear localization, and a ligand-dependent transactivation function (AF-2). hER-α46 lacks the first 173 aa (A/B, or AF-1 domain) of hER-α66. It is derived from alternative splicing of the hER-α66 gene by skipping exon 1 ( 9). This alternative splicing event generates a mRNA that has an AUG in a favorable Kozak sequence for translation initiation in frame with the remainder of the original ORF ( 9). The identification of the differences in domain structures between hER-α66 and -α46 provides the basis to better understand how the estrogen receptors (ERs) work and for the striking diversity of estrogen-signaled responses in different cellular contexts. Estrogen-stimulated cell proliferation by 17β-estradiol (E2β) is believed to be largely mediated by activation of hER-α66 localized in the nucleus. However, earlier studies also reported that E2β binds to a cell surface receptor and stimulates a rapid generation of cAMP ( 10). Subsequently, other reports of a plasma membrane-localized ER that transduces membrane-initiated estrogen signaling appeared (reviewed in refs. 11– 14); this membrane-initiated pathway, the “nonclassic,” “nongenomic,” or “membrane signaling” pathway, was found to activate different cytoplasmic signaling proteins and other membrane-initiated signaling pathways ( 11– 14), and later pathways initiated through membrane estrogen signaling included the adenylate cyclase ( 15), the phospholipase C ( 16), G protein-coupled receptor-activated ( 17), and the mitogen-activated protein kinase (MAPK) ( 17– 19) pathways. Furthermore, limited amounts of hER-α66 and -α46 protein copurified with 5′ nucleotidase and membrane-initiated signaling were found to stimulate the synthesis of NO ( 20, 21). In other studies, more than one functionally distinct membrane-associated pathway could be identified in cells stimulated by the membrane-impermeable estradiol (E2)–BSA; one pathway was sensitive to antiestrogens, and the second pathway was resistant to antiestrogens ( 22). In another study, it was demonstrated in ER-α66-deficient mice that the mice retained rapid estrogen-stimulated membrane effects in neurons that were not blocked by ICI-182,780, a potent estrogen antagonist ( 22), and recently, Toran-Allerand et al. ( 23) reported the existence of a novel plasma membrane-associated ER (ER-X) with an estimated molecular mass of 63–65 kDa. It reacts with antibodies (Abs) to the ligand-binding domain of ER-α66 and responds equally to 17α-estradiol (E2α) and E2β ( 23). The molecular identification of ER-X awaits its cloning and sequencing. The different studies collectively lead to the conclusion that membrane-initiated estrogen signaling is a physiological event and is likely to be of major biological importance. However, the studies leave open the questions of the identity of the membrane-based ER in different cellular contexts and the functional significance of membrane-initiated estrogen signaling in human breast cancer and in other estrogen-responsive tissues. We recently reported the identification, cloning, and expression of a 36-kDa novel isoform of hER-α66, which we designated hER-α36 to distinguish it from hER-α66 and -α46 ( 24). It is generated from a promoter located in the first intron of the hER-α66 gene. hER-α36 differs from hER-α66 by lacking both transcriptional activation domains (AF-1 and AF-2), but it retains the DNA-binding domain and partial dimerization and ligand-binding domains. It possesses an extra, unique 27-aa domain to replace the last 138 aa encoded by exons 7 and 8 of the hER-α66 gene. The cDNA was found to express a protein of molecular mass ≈ 36-kDa recognized by the H222 Ab in hER-α36 expression vector transfected HEK293 cells. Northern blot analysis of total RNA from ER-positive breast cancer MCF7 cells using a DNA probe that was synthesized from a unique 3′ UTR of hER-α36 identified a single mRNA with estimated size of 5.6 kb, the predicted size of the hER-α36 mRNA. The previous study thus identified a unique variant of hER-α66 that is expressed in the human breast cancer cell line MCF7 ( 24). The predicted properties of hER-α36 suggest that it is a dominant-negative effector of estrogen-stimulated activation of estrogen-responsive genes through hER-α66, and, because the potential myristoylation sites are found far closer to the N-terminal of hER-α36 than they are found in hER-α66, it was suggested that hER-α36 had greater potential to signal estrogen-stimulated membrane responses than hER-α66. hER-α36 Is Expressed in both ER (hER-α66)-Positive and -Negative Human Breast Cancer Cells. To further characterize hER-α36, we developed polyclonal anti-ER-α36 Abs raised against the C-terminal 20 aa of hER-α36 that are unique to hER-α36 as described in ref. 24. The anti-hER-α36-specific Abs were used as a probe in Western blot analysis of cell extracts from MCF10A (“normal”) mammary epithelial cells; HEK293 cells transfected with the hER-α36 expression vector and with the empty expression vector control; ER-positive breast cancer MCF7, HB3396, and T47D cells; and the ER-negative breast cancer MDA-MB-231 and MDA-MB-436 cells. The blots were stripped and probed with anti-actin Abs to ensure equal loading. demonstrates that hER-α36 is expressed in both MDA-MB-231 and MDA-MB-436 cells, two well known hER-α66-negative breast cancer cell lines. It also is shown that hER-α36 is expressed in the hER-α66-positive MCF7, T47D, and H3396 breast cancer cells, a result consistent with the Northern blot data reported in ref. 24. MCF10A mammary epithelial cells and HEK293 cells transfected with the empty expression vector exhibit undetectable levels of hER-α36, whereas HEK293 cells transfected with hER-α36 expression vector show expression of hER-α36 at levels comparable with other breast cancer cells (). hER-α36 Inhibits Transactivation of both Estrogen-Stimulated and Nonstimulated hER-α66 and hER-β. hER-α36 is devoid of both the AF-1 and AF-2 transactivation domains of hER-α66 but retains the DNA-binding domain, predicting, as cited earlier, that hER-α36 lacks transactivation functions. To test this hypothesis, HEK293 cells were transiently transfected with a luciferase-expressing reporter gene construct that contains two estrogen response elements (EREs) placed upstream of the thymidine kinase promoter (2× ERE-tk-Luc), together with an empty expression vector or the hER-α36 expression vector. The HEK293 cell line was selected because it was reported that the AF-1 and -2 domains of hER-α66 function well in HEK293 cells ( 25), and, in preliminary experiments, it was determined that ERs are not expressed in these cells (data not shown). The HEK293 cells transfected with 2× ERE-tk-Luc reporter were treated with E2β (10 nM) or, in the control studies, were not treated with E2β. The cells were incubated for 12 h, and luciferase activity was measured in five separate experiments. We found that hER-α36 lacks detectable levels of intrinsic transcriptional activity in the presence or absence of E2β (), consistent with the absence of both transcription activation domains in hER-α36. The ability of hER-α36 to inhibit the transactivation functions mediated by the AF-1 and -2 domains of hER-α66 then was tested in HEK293 cells expressing hER-α36 and the expression vectors pSG, pSG-hERα66, or pSG-hERβ either in the presence or absence of E2β (10 nM). The cells were incubated for 12 h before the luciferase activity was assayed. The results of five separate experiments demonstrate that the presence of hER-α36 strikingly inhibits E2β-dependent and -independent transactivation functions mediated by the AF-1 and -2 domains of ER-α66 (). The results also demonstrate that hER-α36 inhibits E2β-dependent and -independent transactivation activities of hER-β (). These results may be highly significant, because they establish that hER-α36 blocks genomic estrogen signaling mediated by both hER-α66 and hER-β; hER-α36 thus appears to effectively compete with hER-α66 and hER-β for the DNA-binding elements (EREs) in estrogen-responsive genes. These results suggest that hER-α36 also may suppress the estrogen-dependent transactivation functions of hER-α66 in hER-α66-positive breast cancer cells, and it is anticipated that hER-α36 itself may not function as a transcription factor to regulate genomic estrogen signaling in ER-negative breast cancer cells. hER-α36 Is Predominantly a Membrane-Based ER. To pursue the possibility that hER-α36 may transmit estrogen-dependent signaling through a membrane-based locus, we examined stable HEK293 cell lines that express exogenous hER-α36. Nuclear, plasma membrane, and cytosolic fractions were isolated from hER-α36-expressing HEK293 cells. Western blots were prepared from lysates of the different fractions isolated and probed with the different Abs indicated in that recognize the marker proteins of the different fractions and with the anti-hER-α36 Abs described above. The data make clear that a high percentage of hER-α36 (≈50%) fractionates with the plasma membrane and lesser, but significant, amounts fractionate with cytosol (≈40%) and with nuclei (≈10%) (). There was little contamination among different fractions, as confirmed by Western blots of different marker proteins (mSin3A, the nucleus; GDP dissociation inhibitor, the cytosol; 5′ nucleotidase, the plasma membrane; and β-COP, Golgi). These results thus demonstrate that hER-α36 is predominantly a membrane-based ER; they support the conclusion that hER-α36 not only functions as a dominant-negative effector of hER-α66 transactivation functions in the nucleus, presumably through competitive binding with hER-α66 and -β for the EREs of target genes, but ER-α36 also may function to mediate estrogen-dependent membrane-initiated signaling. hER-α36 Mediates Membrane-Initiated Estrogen Signaling Pathway. Previous reports have indicated that E2β stimulates a rapid activation of the MAPK/extracellular signal-regulated kinase (ERK) pathway ( 17– 19), raising the possibility that hER-α36 is involved in this signaling pathway as well. To test this hypothesis, cell lysates were prepared from quiescent HEK293 cells expressing recombinant hER-α36 that were either untreated or treated with E2β for various lengths of time. ERK activation was measured in Western blots prepared from lysates of treated and nontreated cells probed with phosphorylation-state-dependent and -independent anti-ERK Abs. The results demonstrate a remarkable ≈10-fold increase in the ERK1/2 phosphorylation within 5 min in the cells transfected with hER-α36 expression vector ( A) but not in the control cells transfected with the empty vector ( B). The increase in phosphorylation of ERK1/2 remained for ≈45 min before declining to levels of untreated cells. To establish the specificity of the response to E2β, HEK293 cells not expressing hER-α36 were treated with serum; serum activated ERK1/2 in the control cells not expressing hER-α36 ( B), indicating that there is not a global defect in the MAPK signaling pathway in the control cells and confirming the specificity of the response mediated by hER-α36. In addition, the ethanol vehicle has no effect on the MAPK/ERK activation (data not shown). To more conclusively demonstrate the ERK1/2 activation is initiated by a membrane-initiated estrogen-signaling pathway, hER-α36-transfected 293 cells also were treated with E2β–BSA, a membrane-impermeable form of E2β. E2β–BSA was found to also strongly activate ERK1/2 phosphorylation in ER-α36-transfected HEK293 cells (A). Finally, to further clarify that hER-α36 activates the MAPK signaling pathway, we tested the ability of E2β to stimulate Mek1 in hER-α36-transfected HEK293 cells; Mek1 is the kinase that phosphorylates and activates ERK1/2. E2β was found to increase phosphorylation of Mek1 within 5 min of stimulation with E2β similar in time course found with ERK1/2 in cells stimulated with E2β. It also was found that, like ERK1/2, Mek1 maintains a high level of phosphorylation for up to ≈45 min after stimulation with E2β (A). We also treated hER-α36-transfected HEK293 cells with estrone (E1), E2β, E2α, estriol (E3), and estetrol (E4) and found that each of these estrogens, with the single exception of E1, activated ERK1/2 phosphorylation to a very similar level, demonstrating that hER-α36 equally recognizes these estrogens at a level sufficient to stimulate full activation of ERK1/2 phosphorylation (C). Antiestrogens Stimulate ERK1/2 Activation Through Membrane-Associated hERα-36. The antiestrogens tamoxifen, 4OH-tamoxifen, and ICI-182,780 also were tested to determine whether hER-α36-mediated estrogen signaling is sensitive to antiestrogens. Tamoxifen, 4OH-tamoxifen, and the “pure” antiestrogen ICI-182,780 failed to block the estrogen-stimulated ERK1/2 activation mediated by hER-α36. Remarkably, and to the contrary of expectations, activation of ERK1/2 was seen after the addition of antiestrogens tamoxifen, 4OH-tamoxifen, and ICI-182,780; the level of stimulation was found to be higher than that with E2β alone (C). Furthermore, hER-α36 transfected cells treated with 1 μM tamoxifen alone, a concentration that reproducibly blunts the functions of both hER-α66 and -β in nuclear signaling, had a strong and persistent activation of ERK1/2 that lasted >8 h (D). In the control experiment, tamoxifen, at the same concentration, had no effect in HEK293 cells transfected with the empty expression vector (data not shown), making it clear that the tamoxifen effect is specific to hER-α36. These results support the potentially very significant conclusions that hER-α36 mediates membrane-initiated estrogen signaling, that antiestrogens equally or perhaps even more potently potentiate the estrogen signaling through hER-α36 to activate ERK1/2, and that the antiestrogen tamoxifen alone fully activates ERK1/2; the effect of tamoxifen in the activation of ERK1/2 is significantly prolonged. ER-α36 Mediates E2β-Stimulated Cell Proliferation. To further determine whether the estrogen-activated MAPK pathway mediated by hER-α36 leads to activated transcriptional signaling, we examined the ability of membrane-initiated estrogen signaling to activate the transcription factor Elk, a downstream effector of the MAPK/ERK signaling pathway. The hER-α36-transfected 293 cells were transiently transfected with the ERK-responsive GAL-Elk chimeric transcription factor, consisting of the DNA-binding domain of the yeast transcription factor GAL4 fused to the ERK-responsive transactivation domain of human Elk1, and the activity of a luciferase reporter gene containing GAL4-binding sites in the presence of E2β was measured. E2β treatment of hER-α36-transfected cells consistently induced an ≈2-fold increase of Elk/Gal4 fusion protein-mediated transactivation of the reporter gene, whereas E2β had no effect on the transcription activity of the Elk/Gal4 fusion protein in the control cells transfected with empty vector (A). The data thus strongly support the conclusion that the estrogen-dependent activation of the MAPK/ERK signaling pathway is transduced into the nucleus to initiate the activation of the ELK1 transcriptional activities through the estrogen-stimulated, membrane-initiated hER-α36-mediated pathway. To further pursue the significance of membrane estrogen signaling through ER-α36, we tested whether hER-α36 mediates estrogen-stimulated cell proliferation using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. hER-α36 transfected HEK293 cells and control HEK293 cells were maintained in charcoal-stripped serum for 2–3 days and incubated in serum-free medium for 12 h. Medium containing 10 nM E2β (E2) alone or together with 10 nM tamoxifen (TAM), 4OH-tamoxifen (OHT), or 7.2 nM UO126, or with 10 nM tamoxifen or 4OH-tamoxifen alone were added to each well for 48 h. The MTT assay was performed, the results were measured at 490 nm, the results of five independent experiments were averaged, and the mean and SEM were determined with the paired t test. The statistical analysis of the data made clear the striking significance of the results in both ER36–293 and vector–293 cells and the clear ability of estrogens and antiestrogens to stimulate proliferation through membrane-localized hER-α36 as measured in the MTT test. Thus, the results established that E2β stimulated proliferation of hER-α36-transfected 293 cells, whereas E2β had no effect on the growth of the control 293 cells transfected with empty expression vector (B). The inclusion of the antiestrogens, including tamoxifen and 4OH-tamoxifen, did not block E2β-stimulated cell growth (B). Surprisingly, and potentially of major significance, tamoxifen or 4OH-tamoxifen alone strongly stimulated growth of the hER-α36-transfected cells, whereas the specific inhibitor of MAPK pathway, UO126, strongly inhibited E2β-stimulated cell growth. In our previous study, a novel variant of hER-α, hER-α36, was identified, cloned and characterized ( 24). This ER-α isoform is the product of a transcript initiated from a previous unidentified promoter in the first intron of hER-α66 gene. The hER-α36 protein is identical to the hER-α66 protein encoded by exons 2–6 of the hER-α66 gene. This isoform is devoid of the domains that were previously identified to have transactivation activities, AF-1 and -2. In this study, it was found that hER-α36 lacks intrinsic transcriptional activity, but it efficiently suppresses the transactivation activities mediated by the AF-1 and -2 domains of liganded and unliganded hER-α66 and hER-β, indicating that hER-α36 is a potent inhibitor of genomic estrogen signaling. This finding thus parallels a previous report that hER-α46 that lacks the AF-1 domain functions as a powerful competitor to suppress the AF-1 activity of hER-α66 ( 9). As described in the introduction, the presence and significance of a plasma-membrane-based ER that triggers estrogen signaling has been a point of controversy for a long time. In our experiments, we demonstrate that a unique ER-α variant, hER-α36, is predominantly localized on the plasma membrane and that stimulation of growth by estrogens and antiestrogens is through activation of the MAPK/ERK signaling pathway. This finding leads to the unanticipated, but likely to be critically important, conclusion that estrogens and antiestrogens can both stimulate cell proliferation through membrane-associated hER-α36. The precise mechanisms by which liganded ER-α36 enhanced the phosphorylation of ERK1/2 are currently unclear. It was reported that estrogens activate the MAPK/ERK signaling pathway through interaction of hER-α66 with c-Src protein ( 26). It is possible that hER-α36 also mediates activation of the MAPK/ERK pathway through direct interaction with c-Src. It also was reported that estrogen rapidly down-regulates MAPK phosphatase 1 activity, which in turn activate ERK activity ( 27). In addition, it is reasonable to speculate that hER-α36 also may influence the MAPK phosphatase activity. It will be interesting to study the mechanisms by which hER-α36 mediates membrane-initiated estrogen and antiestrogen signaling. It was surprising that antiestrogens such as tamoxifen activate the MAPK/ERK signaling and stimulate cell growth and, furthermore, that the antiestrogens appear to have a stronger and a more prolonged activation of the MAPK/ERK signaling pathway than the estrogens tested. These findings thus support the conclusion that tamoxifen functions as both agonist and antagonist of estrogen signaling; the results are consistent with the possibility that expression of hER-α36 may be involved in the development of tamoxifen-resistant human breast cancer and raises the possibility that antiestrogen activated, hER-α36 membrane-initiated signaling may accelerate the course of tamoxifen-resistant human breast cancer. We demonstrate that antiestrogens did not block hER-α36-mediated MAPK/ERK activation, thus suggesting that ER-α36 is involved in the antiestrogen-insensitive signaling pathway in ER-α66 −/− mice as cited above ( 22). In that study, the ER-α66 −/− mice were created by an insertional disruption of the first coding exon of the mouse ER-α66 gene, the exon that is skipped in the generation of transcripts of hER-α36. It is thus possible that the production of the mouse counterpart of hER-α36 remains normal in these ER-α66 −/− mice. The mechanism by which hER-α36 is localized on the plasma membrane is not clear. hER-α36 has no N-terminal signal peptide; it retains, however, the two nuclear localization signals found in hER-α66 but has, in addition, three potential myristoylation sites at amino acid residues 25–30 (GVWSCE), 76–81 (GMMKGG), and 171–176 (GLLTNL). Because it has been reported that the membrane-localized hER-α46 is posttranslationally modified by the palmitoylation that is required for its membrane localization ( 21), it is reasonable to assume, but not to conclude, that hER-α36 also may be localized to the plasma membrane by posttranslational modification. hER-α36 has a unique ligand-binding domain that replaces the last 5 helixes (helix 8–12) of the 12 helixes in the hER-α66 with a unique 27-aa domain. This 27-aa domain may change the ligand-binding specificity of hER-α36. In these studies, it was found that hER-α36 elicited membrane-initiated signaling equally well in response to E2α, E2β, E3, and E4, as well as to tamoxifen, indicating that hER-α36 possess a broader ligand-binding spectrum than hER-α66, making hER-α36 a potentially more potent mediator of mitogenic estrogen signaling. The ability of hER-α36 to trigger membrane-initiated estrogen and antiestrogen signaling that leads to cell growth makes it an important member of the estrogen signaling pathway. Its potential to antagonize estrogen-stimulated transactivational functions transduced by hER-α66 also is an important feature of hER-α36 functional responses to estrogens. Further studies of this potentially very important protein are likely to significantly advance our understanding of the diverse physiologic and pathologic effects of estrogen action. Cell Culture and Stable Cell Lines. MCF10A cells were obtained from the Karmanos Cancer Institute (Detroit). HEK293 cells, MCF-7 cells, T47D, MDA-MB-231, MDA-MB-436, and HB3396 human breast cancer cells were obtained from American Type Culture Collection. All cells were maintained at 37°C in a 5% CO 2 atmosphere in appropriate tissue culture medium. To express recombinant hER-α36, HEK293 cells were plated at a density of 1 × 10 5 cells per 60-mm dish and were transfected 24 h later with the hER-α36 expression vector driven by the cytomagalovirus (CMV) promoter described in ref. 24 using the FuGene6 transfection reagent (Roche Molecular Biochemicals). An “empty vector” also was transfected into cells to serve as a control. Forty-eight hours after transfection, the cells were replated and selected with 500 μg/ml G418 (Invitrogen) for 2 weeks. The resulting G418-resistant cells were expanded for further analysis. Estrogen and Antiestrogen Treatment of Cells and MTT Assay. Before treatment, cells were cultured in phenol-red-free medium with 2.5% dextran-coated charcoal-stripped FCS for 48–72 h, washed with PBS, and placed in fresh phenol-red-free, serum-free medium containing 0.1 μg/ml BSA and 5 μg/ml insulin for 12 h. Quiescent cells were stimulated at 37°C in serum-free medium for different periods of time. Estrogens and antiestrogens were purchased from Steraloids (Newport, RI). E2β-BSA was purchased from Sigma with 30 mol of E2β per mol of BSA. E2β-BSA free of E2β was prepared according to the protocol described by Taguchi et al. ( 28). Briefly, 400 μl of E2β-BSA (10 −5 M in estrogen) dissolved in 50 mM Tris (pH 8.5) was added to a centrifugal filter unit with a molecular weight cutoff at 3,000 (Millipore) and centrifuged at 14,000 × g until 50 μl of retentate remained. The retentate was washed three times with 350 μl of buffer, recovered, and volume adjusted to 400 μl. The purified E2β-BSA then was tested in transfection assays with hER-α66 expression vector and 2× ERE reporter plasmid. No activation of nuclear signaling mediated by ER-α66 was observed in cells treated with up to 0.5 μM E2β–BSA in estrogen (data not shown), indicating the prepared E2β–BSA contains undetectable levels of free E2β. For MTT assays, cells were added to each well of a 96-well culture plate to a final concentration of 1 × 103 cells per well and incubated in fresh phenol-red-free, serum-free medium containing 0.1 μg/ml BSA and 5 μg/ml insulin for 12 h at 37°C in a CO2 incubator. Medium containing 10 nM E2β, 10 nM tamoxifen or 4OH-tamoxifen, or 7.2 nM UO126 (Calbiochem) was added to each well for 48 h. The MTT assay was performed with the CellTiter96 Aqueous One Solution Cell Proliferation Assay Kit (Promega) according to the manufacturer's recommendation. A microplate reader (Bio-Rad) was used to measure absorbance at 490 nm. Cell Fractionation. Cell fractionations were performed as described by Marquez and Pietras ( 18). Western Blot Analysis and Abs Used. For Western blot analyses, cells were disrupted with RIPA buffer (150 mM NaCl/50 mM Tris·HCl, pH 7.4/1% Triton X-100/1% sodium deoxycholate/0.1% SDS proteinase inhibitor cocktail), and the cell lysates were boiled in a gel-loading buffer and separated by SDS/PAGE in 10% gels. After PAGE, the proteins were transferred to a poly(vinylidene difluoride) membrane (Millipore); the membrane was probed with the indicated Abs and visualized with the appropriate species-specific horseradish peroxidase-conjugated secondary Abs (Santa Cruz Biotechnology) and enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia). The anti-ERK1/2 (K-23) Abs were purchased from Santa Cruz Biotechnology. The MAPK pathway Abs were purchased from Cell Signaling Technology (Beverly, MA). Rat anti-ER-α Abs (H222) were purchased from Research Diagnostics (Flanders, NJ). Abs directed against COPB (Y-20), mSin3A (AK-11), and 5′ nucleotidase (H-300) were purchased from Santa Cruz Biotechnology. The D4-GDI Ab (clone 97A1015) was obtained from Upstate Biotechnology. We developed affinity-purified rabbit polyclonal anti-hER-α36 Abs as a custom service from Alpha Diagnostic (San Antonio, TX). The Abs were raised against a synthetic peptide antigen corresponding to the unique C-terminal 20 aa of hER-α36. The specificity of the Ab was tested in hER-α36 expression vector transfected HEK293 cells that do not express endogenous hER-α. Immunofluorescence was used to demonstrate immunoreactive signals only in transfectants with the hER-α36-expressing vectors but not in transfectants harboring an empty expression vector (data not shown). DNA Transfection and Luciferase Assays. For transient transfection assays, HEK293 cells were seeded in six-well dishes and grown to 60–70% confluence in phenol-red-free medium plus 2.5% steroid-free FCS. Cells were washed and transiently transfected with a total of 5 μg of plasmid with the FuGene6 reagent (Roche Molecular Biochemicals). A reporter plasmid containing two EREs (sequence from −331 to −289 of the chicken Vitellogenin A2 gene) placed upstream of the thymidine kinase promoter was used (2× ERE-tk-Luc; a kind gift of Katarine Pettersson, Karolinska Institute, Stockholm). Expression vectors containing hER-α66 and -β were obtained from K. Pettersson. Amounts of the expression vectors used in each transfection assay were normalized by the empty expression vector. The luciferase assays were performed by using the Luciferase Assay kit from Promega according to the manufacturer's recommendations. The Gal4 L-ELK plasmid was purchased from Stratagene. We thank Dr. Katarine Pettersson for the expression and reporter plasmids and Megan Coleman for technical assistance. This work was supported by National Institutes of Health Grants R01 CA84328 (to Z.W.) and R01 CA84400 (to T.F.D.) and Nebraska Tobacco Settlement Biomedical Research Program Award LB-692. This is manuscript 17432-MEM from The Scripps Research Institute. Abbreviations: | | ER | estrogen receptor | | | hER | human ER | | | AF | activation function | | | E2 | estradiol | | | E2β | 17β-estradiol | | | E2α | 17α-estradiol | | | MAPK | mitogen-activated protein kinase | | | ERK | extracellular signal-regulated kinase | | | ERE | estrogen response element | | | MTT | 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. |
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