Melatonin, an Endogenous-specific Inhibitor of Estrogen Receptor α via Calmodulin
2004; Elsevier BV; Volume: 279; Issue: 37 Linguagem: Inglês
10.1074/jbc.m403140200
ISSN1083-351X
AutoresBeatriz del Río, Juana M. García‐Pedrero, Carlos Martínez‐Campa, Pedro Zuazua, Pedro S. Lazo, Sofı́a Ramos,
Tópico(s)Circadian rhythm and melatonin
ResumoMelatonin is an indole hormone produced mainly by the pineal gland. We have previously demonstrated that melatonin interferes with estrogen (E2) signaling in MCF7 cells by impairing estrogen receptor (ER) pathways. Here we present the characterization of its mechanism of action showing that melatonin is a specific inhibitor of E2-induced ERα-mediated transcription in both estrogen response element- and AP1-containing promoters, whereas ERβ-mediated transactivation is not inhibited or even activated at certain promoters. We show that the sensitivity of MCF-7 cells to melatonin depends on the ERα/ERβ ratio, and ectopic expression of ERβ results in MCF-7 cells becoming insensitive to this hormone. Melatonin acts as a calmodulin antagonist inducing conformational changes in the ERα-calmodulin (CaM) complex, thus impairing the binding of E2·ERα·CaM complex to DNA and, therefore, preventing ERα-dependent transcription. Moreover the mutant ERα (K302G,K303G), unable to bind calmodulin, becomes insensitive to melatonin. The effect of melatonin is specific since other related indoles neither interact with CaM nor inhibit ERα-mediated transactivation. Interestingly, melatonin does not affect the binding of coactivators to ERα, indicating that melatonin action is different from that of current therapeutic anti-estrogens used in breast cancer therapy. Thus, they target ERα at different levels, representing two independent ways to control ERα activity. It is, therefore, conceivably a synergistic pharmacological effect of melatonin and current anti-estrogen drugs. Melatonin is an indole hormone produced mainly by the pineal gland. We have previously demonstrated that melatonin interferes with estrogen (E2) signaling in MCF7 cells by impairing estrogen receptor (ER) pathways. Here we present the characterization of its mechanism of action showing that melatonin is a specific inhibitor of E2-induced ERα-mediated transcription in both estrogen response element- and AP1-containing promoters, whereas ERβ-mediated transactivation is not inhibited or even activated at certain promoters. We show that the sensitivity of MCF-7 cells to melatonin depends on the ERα/ERβ ratio, and ectopic expression of ERβ results in MCF-7 cells becoming insensitive to this hormone. Melatonin acts as a calmodulin antagonist inducing conformational changes in the ERα-calmodulin (CaM) complex, thus impairing the binding of E2·ERα·CaM complex to DNA and, therefore, preventing ERα-dependent transcription. Moreover the mutant ERα (K302G,K303G), unable to bind calmodulin, becomes insensitive to melatonin. The effect of melatonin is specific since other related indoles neither interact with CaM nor inhibit ERα-mediated transactivation. Interestingly, melatonin does not affect the binding of coactivators to ERα, indicating that melatonin action is different from that of current therapeutic anti-estrogens used in breast cancer therapy. Thus, they target ERα at different levels, representing two independent ways to control ERα activity. It is, therefore, conceivably a synergistic pharmacological effect of melatonin and current anti-estrogen drugs. Melatonin is an indole hormone that is the major secretory product of the pineal gland. The most clearly defined actions of melatonin have been demonstrated on the reproductive system of seasonally breeding animals and on circadian rhythms and sleep. A rapidly emerging avenue of investigation is the oncostatic and anti-proliferative effects of melatonin on endocrine-responsive neoplasms, especially in those concerning the mammary gland (1Sánchez-Barceló E. Cos S. Mediavilla M.D. Gupta D. Attanasio A. Reiter R.J. The Pineal Gland and Cancer. Brain Research Promotion, Tübingen, Germany1988: 221-232Google Scholar). The most common conclusion in animal models of tumorigenesis is that either experimental manipulations that activate the pineal gland or the administration of melatonin reduces the incidence and development of chemically induced mammary tumors, whereas pinealectomy usually stimulates breast cancer growth (2Blask D.E. Reiter R.J. The Pineal Gland. Raven Press, New York1984: 253-284Google Scholar, 3Blask D.E. Hill S.M. Miles A. Philbrick D.R.S. Thompson C. Melatonin Clinical Perspectives. Oxford University Press, New York1988: 128-173Google Scholar, 4Sánchez-Barceló E. Mediavilla M.D. Cos S. Webb S.M. Puig-Domingo M. Moller M. Pevet P. Pineal Update: From Molecular Mechanisms to Clinical Implications. PJD Publications Ltd., New York1997: 361-368Google Scholar). Epidemiological studies have shown a low incidence of breast tumors in blind women as well as an inverse relationship between breast cancer incidence and the degree of visual impairment. Because light inhibits melatonin secretion, the increase in melatonin-circulating levels might be interpreted as proof of the protective role of this hormone on mammary carcinogenesis (5Coleman M.P. Reiter R.J. Eur. J. Cancer. 1992; 28: 501-503Abstract Full Text PDF PubMed Scopus (54) Google Scholar). A moderate increase in breast cancer risk among women who worked extended periods of rotating night shifts (light exposure during night suppresses melatonin production) has also been described (6Schernhammer E.S. Laden F. Speizer F.E. Willett W.C. Hunter D.J. Kawachi I. Colditz G.A. J. Natl. Cancer Inst. 2001; 93: 1563-1568Crossref PubMed Scopus (907) Google Scholar). Different mechanisms have been proposed to explain how melatonin could reduce the development of mammary tumors; they are indirect neuroendocrine mechanisms such as melatonin regulation of some pituitary and gonadal hormones that control tumor growth (1Sánchez-Barceló E. Cos S. Mediavilla M.D. Gupta D. Attanasio A. Reiter R.J. The Pineal Gland and Cancer. Brain Research Promotion, Tübingen, Germany1988: 221-232Google Scholar, 4Sánchez-Barceló E. Mediavilla M.D. Cos S. Webb S.M. Puig-Domingo M. Moller M. Pevet P. Pineal Update: From Molecular Mechanisms to Clinical Implications. PJD Publications Ltd., New York1997: 361-368Google Scholar, 5Coleman M.P. Reiter R.J. Eur. J. Cancer. 1992; 28: 501-503Abstract Full Text PDF PubMed Scopus (54) Google Scholar, 6Schernhammer E.S. Laden F. Speizer F.E. Willett W.C. Hunter D.J. Kawachi I. Colditz G.A. J. Natl. Cancer Inst. 2001; 93: 1563-1568Crossref PubMed Scopus (907) Google Scholar, 7Blask D.E. Hill S.M. Pelletier D.B. Anderson J.M. Lemus-Wilson A. Reiter R.J. Pang S.F. Advances in Pineal Research. 3. John Libbey & Co. Ltd., London1989: 259-263Google Scholar) and the direct effects of melatonin as an endogenous hydroxyl radical scavenger (8Reiter R.J. Poeggeler B. Tan D.X. Chen L.D. Mancherter L.C. Guerrero J.L. Neuroendocrinol. Lett. 1993; 15: 103-116Google Scholar) or as a modulator of the immune response to the presence of a malignant neoplasm (9Maestroni J.M. Conti A. Lissoni P. Cancer Res. 1994; 54: 4740-4743PubMed Google Scholar, 10Morrey K.M. Mclachlan J.A. Serkin C.D. Baouche O. J. Immunol. 1994; 153: 2671-2679PubMed Google Scholar). On the other hand, direct anti-estrogenic melatonin actions at the cellular level have been proposed (11Hill S.M. Blask D.E. Cancer Res. 1988; 48: 6121-6126PubMed Google Scholar, 12Cos S. Blask D.E. Lemus-Wilson A. Hill A.B. J. Pineal. Res. 1991; 10: 36-42Crossref PubMed Scopus (152) Google Scholar). Studies using MCF-7 human breast cancer cells (ER+) 1The abbreviations used are: ER, estrogen receptor; ERE, estrogen response element; E2, estrogen; CaM, calmodulin; GST, glutathione S-transferase; MOPS, 4-morpholinepropanesulfonic acid; EGF, epidermal growth factor; TFP, trifluoperazine; OHT, 4-hydroxytamoxifen. (an estrogen-dependent model system, as is the case for more than 60% of primary breast tumors) demonstrate that physiological concentrations of melatonin (1 nm to 1 pm) exert a direct anti-proliferative effect on estrogen-induced proliferation of these cells (11Hill S.M. Blask D.E. Cancer Res. 1988; 48: 6121-6126PubMed Google Scholar, 12Cos S. Blask D.E. Lemus-Wilson A. Hill A.B. J. Pineal. Res. 1991; 10: 36-42Crossref PubMed Scopus (152) Google Scholar) and reduce their invasiveness, causing a decrease in cell attachment and cell motility, probably by interacting with estrogen-mediated mechanisms (13Cos S. Fernandez R. Guezmes A. Sanchez-Barcelo E.J. Cancer Res. 1998; 58: 4383-4390PubMed Google Scholar). However, the molecular basis of melatonin action remains largely unknown. In a previous report (14Rato A.G. Pedrero J.G. Martinez M.A. del Rio B. Lazo P.S. Ramos S. FASEB J. 1999; 13: 857-868Crossref PubMed Scopus (117) Google Scholar), we presented evidence that melatonin interferes with estrogen-signaling pathways. We demonstrated that melatonin acts as anti-estrogen by preventing the estrogen-dependent transcriptional activation in MCF-7 cells through destabilization of the E2·ER complex from binding to DNA, and we proposed calmodulin (CaM) as a potential candidate for mediating the anti-estrogenic effects of melatonin. Several lines of evidence support this hypothesis; the interaction of this calcium-regulated protein with ER has been known for several years, and a number of CaM antagonists exhibit anti-estrogenic activity and decrease the affinity of ERα for its ligand as well as the stability of E2·ER binding to DNA (15Hardcastle I.R. Rowlands M.G. Houghton J. Jarman M. J. Med. Chem. 1996; 39: 999-1004Crossref PubMed Scopus (22) Google Scholar). In addition, it has been shown that melatonin binds to calmodulin in a Ca2+-dependent fashion, resulting in the inhibition of calmodulin (16Benitez-King G. Huerto-Delgadillo L. Anton-Tay F. Brain Res. 1991; 557: 289-292Crossref PubMed Scopus (94) Google Scholar, 17Romero M.P. García-Pergadeña A. Guerrero J.M. Osuna C. FASEB J. 1998; 12: 1401-1408Crossref PubMed Scopus (61) Google Scholar). In the search for differences between ERα and the most recently described ERβ, we analyzed the interaction of both receptors with calmodulin, and we demonstrated that ERα but not ERβ directly interacts with calmodulin. Consequently, CaM antagonists are selective modulators of ERα-mediated transcription (18Garcia Pedrero J.M. Rio B. Martinez-Campa C. Muramatsu M. Lazo P.S. Ramos S. Mol. Endocrinol. 2002; 16: 947-960Crossref PubMed Google Scholar). In the present study, we have investigated whether calmodulin could be a link between melatonin and the estrogen-signaling pathway. Our results indicate that melatonin acts as specific inhibitor of ERα at physiological doses, and therefore, clinical studies on the possible therapeutic value of melatonin on breast cancer should be considered. Materials—Melatonin, 17β-estradiol, 4-hydroxytamoxifen, and other chemicals were purchased from Sigma. ICI 182,780 was provided by Dr. A. E. Wakeling (Zeneca Pharmaceuticals, Macckesfield, Cheshire, UK). [35S]Methionine (Pro-mix; 14.3 mCi/ml; >1000 Ci/mmol) was from Amersham Biosciences. Plasmids—The expression vector pcDNA-ERα and the recombinant plasmid GST-ERα-(280–595) have been previously described (18Garcia Pedrero J.M. Rio B. Martinez-Campa C. Muramatsu M. Lazo P.S. Ramos S. Mol. Endocrinol. 2002; 16: 947-960Crossref PubMed Google Scholar, 19Garcia Pedrero J.M. Zuazua P. Martinez-Campa C. Lazo P.S. Ramos S. Endocrinology. 2003; 144: 2967-2976Crossref PubMed Scopus (52) Google Scholar). pERE-TK-Luc, pS2-Luc, and pCMX-mERβ were kindly provided by Dr. V. Giguère from the R. W. Johnson Pharmaceutical Research Institute, Don Mills, Ontario, Canada. pCXN2-hERβ-(1–530), GST-hERα-(117–595) (20Ogawa S. Inoue S. Orimo A. Hosoi T. Ouchi Y. Muramatsu M. FEBS Lett. 1998; 423: 129-132Crossref PubMed Scopus (37) Google Scholar), and pRL-TK (Promega Corp., Madison, WI) were also used in this work. The plasmid 3x-ERE-TATA-Luc was kindly provided by Dr. S. Safe from the Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX. Δcoll-73 was kindly provided by Dr. A. Aranda from Instituto de Investigaciones Biomédicas "Alberto Sols" Consejo Superior de Investigaciones Científicas, Madrid, Spain. Cell Culture and Transient Transfection Assays—HeLa cells were propagated as previously described (18Garcia Pedrero J.M. Rio B. Martinez-Campa C. Muramatsu M. Lazo P.S. Ramos S. Mol. Endocrinol. 2002; 16: 947-960Crossref PubMed Google Scholar). Before transfection, HeLa cells were seeded in 12-well plates and incubated for 12–18hat37 °C. Then cells were transferred to phenol red-free Dulbecco's modified Eagle's medium containing 0.5% charcoal, dextran-treated fetal calf serum and maintained for 3 days. At 60–80% confluency, cells were transfected with 0.5 μg of estrogen response element (ERE)-driven or AP1-driven reporter plasmids, 0.1 μg of ER expression vector, and 50 ng of an internal control Renilla luciferase plasmid, pRL-TK (Promega), using FuGENE 6 transfection reagent from Roche Applied Science following the manufacturer's protocols. After 18–24 h, medium was renewed, and cells were stimulated for 24 h with different chemicals as indicated. Luciferase was assayed with the dual luciferase system (Promega). Luciferase activities were normalized to Renilla luciferase activity to correct for differences in transfection efficiency. The results represent the means ± S.D. of three independent experiments performed at least in duplicate. Transactivation experiments were performed with both mouse and human ERβ, and identical trends in ligand behavior were observed in both ERβs in HeLa cells. MCF-7 cells were propagated in RPMI 1640 medium containing 25 mm HEPES, NaOH, pH 7.3, and synchronized cells were transfected as above. When indicated, ERβ expression vector or the empty vector was included in the transfection. Electrophoretic Mobility Shift Assay—Binding of the E2·ER to ERE was performed as previously described (14Rato A.G. Pedrero J.G. Martinez M.A. del Rio B. Lazo P.S. Ramos S. FASEB J. 1999; 13: 857-868Crossref PubMed Scopus (117) Google Scholar). Five to ten microliters of nuclear extracts of transient transfections were mixed with buffer B (20 mm HEPES-KOH, pH 7.9, 10 mm MgCl2, 1 mm EDTA, 10% (v/v) glycerol, 100 mm KCl, 0.2 mm phenylmethylsulfonyl fluoride, 0.2 mm dithiothreitol, 0.5% Nonidet P-40, and protease inhibitors) and incubated with 1 μg of poly(dI·dC) in a total volume of 40 μl. Mixtures were preincubated at 0 °C for 15 min followed by incubation with the indicated hormones at 0 °C for 10 min. 32P-Labeled probe (10 fmol containing 3–5 × 104 dpm) corresponding to the ERE from Xenopus vitellogenin A2 gene was added to the reaction and allowed to proceed for 1 h at 0 °C followed by 30 min at room temperature. The samples were loaded onto a pre-electrophoresed (10 mA) 5% polyacrylamide gel (acrylamide to bisacrylamide ratio of 40:1) in TBE (45 mm Tris borate, 1 mm EDTA) at 11 mV/cm. Gels were vacuum-dried and exposed at –80 °C to obtain the autoradiography. For specificity assays, a 100-fold excess of unlabeled oligonucleotide was used as competitor before adding the probe to the binding reaction. Proteolysis Assays—The pcDNA-ERα plasmid (1 μg) containing full-length cDNA of the wild-type human ERα was used to produce 35S-radiolabeled ERα using 40 μl of a coupled transcription-translation system according to the manufacturer's instructions (Promega). The protease digestion was performed essentially as described by McDonnell et al. (21McDonnell D.P. Clemm D.L. Hermann T. Golman M.E. Pike J.W. Mol Endocrinol. 1995; 9: 659-669Crossref PubMed Google Scholar). An aliquot (4.5 μl) of reticulocyte lysate was incubated for 20 min in the absence or presence of 1 μm 17β-estradiol, 1 nm melatonin, and 1 μm W7 as indicated. Equal aliquots of the untreated or the hormone-treated receptor were subsequently incubated with a trypsin solution (Roche Applied Science), giving final enzyme concentration of 25 μg/ml. After 10 min of incubation at room temperature, the digestion reaction was terminated by the addition of gel-denaturing buffer and boiling for 5 min. The products of the digestion procedure were separated on a 12% polyacrylamide-SDS gel. After electrophoresis the gel was treated with a 0.5 m sodium salicylate solution for 15 min. The gel was dried under vacuum, and the radiolabeled products were visualized by autoradiography. When indicated, 1 μg of goat polyclonal anti-CaM antibodies (SC-1988, Santa Cruz Biotechnology, Inc.) was added before the treatment with hormones. In Vitro Protein-Protein Interaction Assays—GST fusion proteins were expressed and purified essentially as described by Frangioni and Neel (22Frangioni J.V. Neel B.G. Anal. Biochem. 1993; 210: 179-187Crossref PubMed Scopus (833) Google Scholar). GST pull-down experiments were performed as previously described by Cavailles et al. (23Cavailles V. Dauvois S. Danielian P.S. Parker M.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10009-10013Crossref PubMed Scopus (341) Google Scholar). 35S-Labeled SRC-1a coactivator was synthesized by in vitro transcription-translation (Promega) using pCR-SRC-1a as template. The GST fusion proteins loaded on glutathione-Sepharose beads (25 μl) were preincubated with 1 μm concentrations of ligands (17β-estradiol, 4-hydroxytamoxifen, or ICI 182,780) or 1 nm melatonin for 30 min at 4 °C followed by incubation with 35S-labeled proteins for 1.5 h at 4 °C in a total volume of 150 μl of IPAB buffer (20 mm HEPES-KOH, pH 7.9, 5 mm MgCl2, 150 mm KCl, 0.02 mg/ml bovine serum albumin, 0.1% (v/v) Triton X-100, 0.1% Nonidet P-40, and protease inhibitors). Beads were washed 4–5 times with IPAB without bovine serum albumin, collected by centrifugation, and resuspended in 20 μl of loading buffer for SDS-PAGE analysis. The gel was vacuum-dried, and the radiolabeled products were visualized by autoradiography. In Vitro Interaction with Dansyl-CaM—Fluorescence experiments were performed in a PerkinElmer Life Sciences fluorimeter using a 100-μl cuvette. 2.5 nmol of dansyl-CaM (Sigma) were dissolved in 100 μl of 10 mm MOPS, pH 7.2, 1 mm MgCl2, 100 mm KCl, and 1 mm CaCl2. Emission fluorescent spectra were obtained (λEx 333 nm) before and after the addition of 1 nm melatonin or the indole derivatives. Equivalent amounts of buffer were added in the controls. Melatonin Is a Specific Inhibitor of ERα-mediated Transcription—We have previously demonstrated that melatonin is able to inhibit estrogen-dependent transcription and proliferation in MCF-7 cells (14Rato A.G. Pedrero J.G. Martinez M.A. del Rio B. Lazo P.S. Ramos S. FASEB J. 1999; 13: 857-868Crossref PubMed Scopus (117) Google Scholar). Because MCF-7 is a carcinoma-derived cell line (ER+) that endogenously expresses both ERα and ERβ, we further investigated the inhibitory effect of melatonin on E2-dependent transactivation mediated by each receptor isoform independently. For this purpose, we transiently transfected HeLa cells with either ERα or ERβ expression vectors along with the ERE-driven reporter plasmids pEREtkLuc (Fig. 1A), pS2Luc (Fig. 1B), or 3xERELuc (Fig. 1C). In all cases 10 nm E2 stimulated transcription for both ERα- and ERβ-transfected cells. As expected, the highest E2 stimulation was obtained using a strong promoter containing three ERE sites (Fig. 1C). Physiological concentrations of melatonin (1 nm) inhibited ERα-mediated transactivation by 45–60% depending on the promoter tested. In contrast, ERβ-mediated transcription was not affected (Fig. 1, B and C) or even potentiated (Fig. 1A) by this concentration of melatonin. In titration experiments we observed that melatonin inhibited ERα-mediated transcription in a dose-dependent manner, whereas ERβ activity was unaffected by the different concentrations of melatonin assayed (Fig. 1D). These results indicate that melatonin is a selective modulator of ERα, as we have recently described for CaM antagonists (18Garcia Pedrero J.M. Rio B. Martinez-Campa C. Muramatsu M. Lazo P.S. Ramos S. Mol. Endocrinol. 2002; 16: 947-960Crossref PubMed Google Scholar). An ERα Mutant Unable to Bind CaM Is Insensitive to Melatonin—In a previous report we demonstrated that residues Lys-302 and Lys-303 of hERα are essential for CaM binding. Although the wild-type ERα normally binds to CaM, substitution of lysines 302 and 303 by glycine abolished the interaction of ERα (K302G,K303G) with CaM (18Garcia Pedrero J.M. Rio B. Martinez-Campa C. Muramatsu M. Lazo P.S. Ramos S. Mol. Endocrinol. 2002; 16: 947-960Crossref PubMed Google Scholar). Transcriptional activation studies further demonstrated that these two critical residues for ERα binding to CaM are not essential for ERα transcriptional activation. Thus, when HeLa cells were transiently transfected with wild-type ERα and compared with those transfected with ERα (K302G,K303G), both showed similar levels of basal and E2-induced transcriptional activation (18Garcia Pedrero J.M. Rio B. Martinez-Campa C. Muramatsu M. Lazo P.S. Ramos S. Mol. Endocrinol. 2002; 16: 947-960Crossref PubMed Google Scholar). However, ERα transactivation was 80% inhibited by 10–6m W7, whereas transcription mediated by ERα (K302G,K303G) was completely insensitive to this calmodulin antagonist. If melatonin acts as a calmodulin antagonist on ERα-mediated transcription, we could predict no inhibitory effect of the pineal hormone on ERα (K302G,K303G)-mediated transactivation. Indeed, when HeLa cells were transiently transfected with ERα (K302G,K303G) along with the ERE-driven reporter plasmid 3xERELuc (Fig. 1E) we observed that ERα (K302G, K303G) transcription was unaffected by the different concentrations of melatonin assayed. The Sensitivity of MCF7 Cells to Melatonin Depends on the ERα/ERβ Ratio—In MCF7 cells increasing concentrations of melatonin resulted in the progressive inhibition of the E2-dependent transcription, reaching nearly 100% of inhibition at pharmacological concentrations of melatonin (Fig. 2A). The IC50m was obtained at 1.26 × 10–11 as determined with GraphPad Prism. We next analyzed whether the sensitivity of MCF7 cells to inhibition by melatonin was associated with the high ERα/ERβ ratio present in these cells, as we have previously reported for CaM antagonists (18Garcia Pedrero J.M. Rio B. Martinez-Campa C. Muramatsu M. Lazo P.S. Ramos S. Mol. Endocrinol. 2002; 16: 947-960Crossref PubMed Google Scholar). To test this hypothesis, MCF7 cells were transfected with the 3xERELuc reporter plasmid in the absence or presence of an ERβ expression vector. We then determined whether ERβ overexpression affects the sensitivity of these cells to melatonin and compared its effect to those of the CaM antagonists W7 and calmidazolium. As expected, both melatonin and CaM antagonists inhibited E2-dependent transcriptional activation in MCF7 cells (Fig. 2B). Interestingly, the inhibitory effects of both melatonin and CaM antagonists were abolished by ERβ overexpression (Fig. 2B). These results imply that the sensitivity to melatonin of E2-induced transcription in MCF7 cells depends on the presence of ERα. Inhibition by melatonin correlates with a high ERα/ERβ ratio, whereas an increased expression of ERβ impairs the effect of the hormone. Melatonin Inhibits E2·ERα-mediated Transcription in AP1-containing Promoters—We have previously demonstrated that CaM is a regulator of ERα-mediated transcription in both ERE- and AP1-containing promoters since transcription mediated by both complexes is sensitive to CaM antagonists (18Garcia Pedrero J.M. Rio B. Martinez-Campa C. Muramatsu M. Lazo P.S. Ramos S. Mol. Endocrinol. 2002; 16: 947-960Crossref PubMed Google Scholar). Therefore, we decided to test the ability of melatonin to inhibit transcription on ERα/AP1 pathways. For that purpose, HeLa cells were transfected with either ERα or ERβ along with the reporter plasmid Δcoll-73-Luc (containing an AP1 binding site). Even though E2·ERα-mediated AP1 activation in HeLa cells and other cell lines have been described (24Paech K. Webb P. Kuiper G.G. Nilsson S. Gustafsson J. Kushner P.J. Scanlan T.S. Science. 1997; 277: 1508-1510Crossref PubMed Scopus (2074) Google Scholar, 25Bouhoute A. Leclercq G. Biochem. Biophys. Res. Commun. 1995; 208: 748-755Crossref PubMed Scopus (45) Google Scholar), we and other authors found it necessary to prime the cells with EGF to observe this effect. EGF stabilizes the levels of c-Jun and c-Fos family proteins, allowing a synergistic effect between these factors and ERα on AP1 transcription (26Philips A. Teyssier C. Galtier F. Rivier-Covas C. Rey J.M. Rochefort H. Chalbos D. Mol. Endocrinol. 1998; 7: 973-985Crossref Scopus (77) Google Scholar, 27Bollig A. Miksicek R.J. Mol. Endocrinol. 2000; 14: 634-649Crossref PubMed Scopus (113) Google Scholar). We found that AP1 activity was increased by EGF in cells expressing either ERα or ERβ (Fig. 3). E2 significantly potentiated AP1 activity in ERα-transfected cells but diminished AP1 activity in ERβ-transfected cells. These results agree with previous reports (26Philips A. Teyssier C. Galtier F. Rivier-Covas C. Rey J.M. Rochefort H. Chalbos D. Mol. Endocrinol. 1998; 7: 973-985Crossref Scopus (77) Google Scholar, 27Bollig A. Miksicek R.J. Mol. Endocrinol. 2000; 14: 634-649Crossref PubMed Scopus (113) Google Scholar) indicating that EGF synergizes with E2. Very importantly, the synergistic effect of EGF and E2 in cells expressing ERα was sensitive to melatonin, whereas no effect was observed in cells expressing ERβ. Both the activation by E2 and the inhibition by melatonin were statistically significant. We can infer from these experiments that melatonin, as other CaM antagonists, regulates ERα-mediated transcription not only in ERE-dependent pathways but also in AP1 pathways. Melatonin but Not Other Indole Derivatives Interact with CaM—We have previously demonstrated that melatonin blocks the binding of the E2·ER complex to ERE in vitro and that this effect is dose-dependent, saturable, and specific, since different methoxy- and hydroxyindoles have no effect on binding to DNA (14Rato A.G. Pedrero J.G. Martinez M.A. del Rio B. Lazo P.S. Ramos S. FASEB J. 1999; 13: 857-868Crossref PubMed Scopus (117) Google Scholar). Therefore, we expected that other indole derivatives would have no effect on E2·ERα-mediated transcription. To analyze this possibility we carried out transient transfections in MCF-7 cells using 3xERE-Luc as reporter plasmid. As shown in Fig. 4B, melatonin effectively inhibited (60%) E2-induced transactivation, whereas treatment with other indole metabolites resulted in no significant decrease on the E2-mediated transcription, indicating that the inhibitory effect of melatonin on estrogen response is specific. To further investigate the basis for the specific inhibition exhibited by melatonin, we examined the ability of the indole derivatives to bind to dansyl-CaM. Changes on emission fluorescence intensity of dansyl-CaM reflect conformational/structural changes, suggesting interaction with CaM. As observed in Fig. 4C, melatonin specifically decreased the fluorescence of dansyl-CaM, whereas the other indoles tested did not modify the fluorescence of dansyl-CaM, indicating that only melatonin is able to interact with this protein. We hypothesize that melatonin acts as a CaM antagonist, interfering with the ERα-CaM complex and that this is the underlying basis by which melatonin specifically inhibits ERα-mediated transcription. Melatonin Induces Conformational Changes on ERα Structure via CaM—Conformational changes on ERα structure can be shown by using a protease digestion assay as previously described by McDonnell et al. (21McDonnell D.P. Clemm D.L. Hermann T. Golman M.E. Pike J.W. Mol Endocrinol. 1995; 9: 659-669Crossref PubMed Google Scholar). Also, W7 induces CaM to form a globular structure (29Osawa M. Kuwamoto S. Izumi Y. Yap K.L. Ikura M. Shibanuma T. Yokokura H. Hidaka H. Matsushima N. FEBS Lett. 1999; 442: 173-177Crossref PubMed Scopus (47) Google Scholar). We have determined the effects of melatonin and W7 on ERα structure on the basis of the differential susceptibility of the receptor to proteolysis by trypsin. 35S-Labeled ERα was synthesized in vitro and preincubated with vehicle, E2, melatonin, W7, or combinations of these compounds. The resulting complexes were then subjected to limited digestion with trypsin, and the products were resolved by SDS-PAGE (Fig. 5). ERα was highly sensitive to trypsin degradation in the absence of ligand (Fig. 5, lane 2), whereas in the presence of E2, a trypsin-resistant 32-kDa fragment was observed (Fig. 5, lane 4), in agreement with results previously published (21McDonnell D.P. Clemm D.L. Hermann T. Golman M.E. Pike J.W. Mol Endocrinol. 1995; 9: 659-669Crossref PubMed Google Scholar). Incubation of the labeled receptor in the presence of E2 plus either melatonin (Fig. 5, lane 6) or W7 (Fig. 5, lane 8) yielded a distinct digestion patron as compared with E2 alone. Under these conditions, ERα becomes highly sensitive to protease digestion. Therefore, treatment with melatonin or CaM antagonists abolished the protective effect of E2 on limited trypsin digestion. Interestingly, the effects of melatonin (Fig. 5, lane 5) and W7 (Fig. 5, lane 7) were reverted in the presence of anti-CaM antibodies. Taken together, our data indicate that melatonin, similar to W7 through the interaction with CaM, induce conformational changes on ERα that also affect the stability of ERα against proteolysis. ERα Stability Is Not Altered by Melatonin—It has been reported that the inhibition of the interaction between CaM and ER reduces the total cellular content of estrogen receptor (28Hall J.M. Chang C.Y. McDonnell D.P. Mol. Endocrinol. 2000; 12: 2010-2023Google Scholar). Thus, treatment of MCF7 cells with calmodulin antagonists such as trifluoperazine (TFP) or CGS9343B reduced the number of estrogen receptors in the cells (30Li Z. Joyal J.L. Sacks D.B. J. Biol. Chem. 2001; 276: 17354-17360Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). We have addre
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