Title: Leptin Induces, via ERK1/ERK2 Signal, Functional Activation of Estrogen Receptor α in MCF-7 Cells
Abstract: Leptin is a hormone with multiple biological actions, produced predominantly by adipose tissue. In humans, plasma levels correlate with total body fat, and high concentrations occur in obese women. Among its functions, leptin is able to stimulate normal and tumor cell growth. We demonstrated that leptin induces aromatase activity in MCF-7 cells evidencing its important role in enhancing in situ estradiol production and promoting estrogen-dependent breast cancer progression. Estrogen receptor α (ERα), which plays an essential role in breast cancer development, can be transcriptionally activated in a ligand-independent manner. Taking into account that unliganded ERα is an effector of mitogen-activated protein kinase (MAPK) signal and that leptin is able, via Janus kinase, to activate the Ras-dependent MAPK pathway, in the present study we investigate the ability of leptin to transactivate ERα. We provided evidence that leptin is able to reproduce the classic features of ERα transactivation in a breast cancer cell line: nuclear localization, down-regulation of its mRNA and protein levels, and up-regulation of a classic estrogen-dependent gene such as pS2. Transactivation experiments with a transfected reporter gene for nuclear ER showed an activation of ERα either in MCF-7 or in HeLa cells. Using a dominant negative ERK2 or the MAPK inhibitor PD 98059, we showed that leptin activates the ERα through the MAPK pathway. The N-terminal transcriptional activation function 1 appears essential for the leptin response. Finally, it is worth noting that leptin exposure potentates also the estradiol-induced activation of ERα. Thus, we are able to demonstrate that the amplification of estrogen signal induced by leptin occurs through an enhancing in situ E2 production as well as a direct functional activation of ERα. Leptin is a hormone with multiple biological actions, produced predominantly by adipose tissue. In humans, plasma levels correlate with total body fat, and high concentrations occur in obese women. Among its functions, leptin is able to stimulate normal and tumor cell growth. We demonstrated that leptin induces aromatase activity in MCF-7 cells evidencing its important role in enhancing in situ estradiol production and promoting estrogen-dependent breast cancer progression. Estrogen receptor α (ERα), which plays an essential role in breast cancer development, can be transcriptionally activated in a ligand-independent manner. Taking into account that unliganded ERα is an effector of mitogen-activated protein kinase (MAPK) signal and that leptin is able, via Janus kinase, to activate the Ras-dependent MAPK pathway, in the present study we investigate the ability of leptin to transactivate ERα. We provided evidence that leptin is able to reproduce the classic features of ERα transactivation in a breast cancer cell line: nuclear localization, down-regulation of its mRNA and protein levels, and up-regulation of a classic estrogen-dependent gene such as pS2. Transactivation experiments with a transfected reporter gene for nuclear ER showed an activation of ERα either in MCF-7 or in HeLa cells. Using a dominant negative ERK2 or the MAPK inhibitor PD 98059, we showed that leptin activates the ERα through the MAPK pathway. The N-terminal transcriptional activation function 1 appears essential for the leptin response. Finally, it is worth noting that leptin exposure potentates also the estradiol-induced activation of ERα. Thus, we are able to demonstrate that the amplification of estrogen signal induced by leptin occurs through an enhancing in situ E2 production as well as a direct functional activation of ERα. Leptin, the product of the ob gene, mainly secreted by adipocytes, is involved in the control of body weight and results strongly correlated to the body fat mass (1Zhang Y. Proenca R. Maffei M. Barone M. Leopold L. Friedman J.M. Nature. 1994; 372: 425-432Crossref PubMed Scopus (11940) Google Scholar, 2Caro J.F. Sinha M.K. Kolaczynski J.W. Zhang P.L. Considine R.V. Diabetes. 1996; 45: 1455-1462Crossref PubMed Scopus (931) Google Scholar, 3Friedman J.M. Halaas J.L. Nature. 1998; 395: 763-770Crossref PubMed Scopus (4603) Google Scholar, 4Lonnqvist F. Arner P. Nordfors L. Schalling M. Nat. Med. 1995; 1: 950-9553Crossref PubMed Scopus (701) Google Scholar). Recently, leptin was reported to stimulate the proliferation of various cell types (5Islam M.S. Morton N.M. Hansson A. Emilsson V. Biochem. Biophys. Res. Commun. 1997; 238: 851-855Crossref PubMed Scopus (72) Google Scholar, 6Machinal-Quelin F. Dieudonne M.N. Leneveu M.C. Pecquery R. Giudicelli Y. Am. J. Cell. Physiol. 2002; 282: C853-C863Crossref PubMed Scopus (111) Google Scholar, 7Sierra-honigmann M.R. Nath A.K. Murakami C. Garcia-cardena G. Papapetropoulos A. Sessa W.C. Madge L.A. Schehner J.S. Schwabb M.B. Polverini P.J. Floresriveros J.R. Science. 1998; 281: 1683-1686Crossref PubMed Scopus (1287) Google Scholar, 8Tsuchiya T. Shimizu H. Horie T. Mori M. Eur. J. Pharmacol. 1999; 365: 273-279Crossref PubMed Scopus (213) Google Scholar, 9Schneider R. Bornstein S.R. Chrousos G.P. Boxberger S. Ehninger G. Breidert M. Hormon. Metab. Res. 2001; 33: 1-6Crossref PubMed Scopus (59) Google Scholar, 10Stallmeyer B. Kampfer H. Podda M. Kaufmann R. Pfeilschifter J. Frank S. J. Invest. Dermatol. 2001; 117: 98-105Abstract Full Text Full Text PDF PubMed Google Scholar, 11Dieudonne M.N. Machinal-Quelin F. Serazin-Leroy V. Leneveu M.C. Pecquery R. Giudicelli Y. Biochem. Biophys. Res. Commun. 2002; 293: 622-628Crossref PubMed Scopus (261) Google Scholar) leading to consider leptin as a novel growth factor. Indeed several studies have shown how leptin is able to activate the proliferation of pancreatic β cells (5Islam M.S. Morton N.M. Hansson A. Emilsson V. Biochem. Biophys. Res. Commun. 1997; 238: 851-855Crossref PubMed Scopus (72) Google Scholar), vascular endothelium (7Sierra-honigmann M.R. Nath A.K. Murakami C. Garcia-cardena G. Papapetropoulos A. Sessa W.C. Madge L.A. Schehner J.S. Schwabb M.B. Polverini P.J. Floresriveros J.R. Science. 1998; 281: 1683-1686Crossref PubMed Scopus (1287) Google Scholar), lung (8Tsuchiya T. Shimizu H. Horie T. Mori M. Eur. J. Pharmacol. 1999; 365: 273-279Crossref PubMed Scopus (213) Google Scholar), gastric mucosa (9Schneider R. Bornstein S.R. Chrousos G.P. Boxberger S. Ehninger G. Breidert M. Hormon. Metab. Res. 2001; 33: 1-6Crossref PubMed Scopus (59) Google Scholar), keratinocytes cells (10Stallmeyer B. Kampfer H. Podda M. Kaufmann R. Pfeilschifter J. Frank S. J. Invest. Dermatol. 2001; 117: 98-105Abstract Full Text Full Text PDF PubMed Google Scholar), and, recently, breast cancer cells (11Dieudonne M.N. Machinal-Quelin F. Serazin-Leroy V. Leneveu M.C. Pecquery R. Giudicelli Y. Biochem. Biophys. Res. Commun. 2002; 293: 622-628Crossref PubMed Scopus (261) Google Scholar). Although leptin is mainly synthesized by breast adipose tissue, its expression has also been detected in normal and tumoral human mammary epithelial cells (12O'Brien S.N. Welter B.H. Price T.M. Clemson U. Glemson S.G. Biochem. Biophys. Res. Commun. 1999; 259: 695-698Crossref PubMed Scopus (115) Google Scholar, 13Smith-Kirwin S.M. O'Connor D.M. Johnston J. De Lancey E. Hassink S.G. Funanage V.L. J. Clin. Endocrinol. Met. 1998; 83: 1810-1813Crossref PubMed Scopus (0) Google Scholar). In addition, it has been shown that leptin receptors (short and long isoforms) are expressed in normal mammary epithelial cells (14Laud K. Gourdou I. Belair L. Keisler D.H. Djiane J. FEBS Lett. 1999; 463: 194-198Crossref PubMed Scopus (94) Google Scholar) as well as in human breast cancer cell lines (11Dieudonne M.N. Machinal-Quelin F. Serazin-Leroy V. Leneveu M.C. Pecquery R. Giudicelli Y. Biochem. Biophys. Res. Commun. 2002; 293: 622-628Crossref PubMed Scopus (261) Google Scholar, 15Laud K. Gourdou I. Pessemesse L. Peyrat J.P. Djiane J. Mol. Cell. Endocrinol. 2002; 188: 219-226Crossref PubMed Scopus (178) Google Scholar). These data suggest an important role of leptin on mammary gland development and tumorigenesis, giving more emphasis to the epidemiological studies that evidence a relationship between obesity and breast carcinogenesis. Obesity is an important health concern, because it is associated with a variety of metabolic disorders and an increased risk of developing cancer (16Bray G.A. J. Nutr. 2002; 132: 3451S-3455SCrossref PubMed Google Scholar). It is now well established that post-menopausal women with upper body fat predominance experience a higher risk of breast cancer (17Stoll B.A. Breast. Cancer Res. Treat. 1998; 49: 187-193Crossref PubMed Scopus (102) Google Scholar, 18van den Brandt P.A. Spiegelman D. Yaun S.S. Adami H. Beeson L. Folsom A.R. Fraser G. Goldbohm R.A. Graham S. Kushi L. Marshall J.R. Miller A.B. Rohan T. Smith-Warner S.A. Speizer F.E. Willet W.C. Wolk A. Hunter D.J. Am. J. Epidemiol. 2000; 152: 514-527Crossref PubMed Scopus (805) Google Scholar). The association between obesity and breast carcinoma is usually ascribed to estrogen excess, derived from androgen aromatization in peripheral fat deposits (19Chen S. Front. Biosci. 1998; 3: d922-d933Crossref PubMed Scopus (99) Google Scholar, 20Reed M.J. Purohit A. Clin. Endocrinol. 2001; 54: 563-571Crossref PubMed Scopus (55) Google Scholar). In our recent work we have demonstrated that leptin is able to stimulate, through mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; STAT, signal transducers and activators of transcription; ER, estrogen receptor; DMEM, Dulbecco's modified Eagle's medium; CS, calf serum; BSA, bovine serum albumin; PMSF, phenylmethylsulfonyl fluoride; HEGO, human ERα expression vector; NLS, nuclear localization signal; RT, reverse transcription; E2, estradiol; JAK2, Janus kinase 2; ERK, extracellular signal-regulated kinase; EMSA, electrophoretic mobility shift assay; ERE, estrogen-responsive element. and signal transducers and activators of transcription (STAT) signals, aromatase expression in the MCF-7 cell line evidencing its important role in enhancing in situ estradiol production and promoting cell proliferation (21Catalano S. Marsico S. Giordano C. Mauro L. Rizza P. Panno M.L. Andò S. J. Biol. Chem. 2003; 278: 28668-28676Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). In addition, a potential relationship between leptin and estrogens stems also from the evidence that estrogens appear to modulate leptin gene expression in adipose tissue (22Shimuzu H. Shimomura Y. Nakanishi Y. Futawatari T. Ohtani K. Sato N. Mori M. J. Endocrinol. 1997; 154: 285-292Crossref PubMed Scopus (447) Google Scholar, 23Casabiell X. Pineiro V. Peino R. Lage M. Camina J. Gallego R. Vallejo L.G. Dieguez C. Casanueva F.F. J. Clin. Endocrinol. Metab. 1998; 83: 2149-2155Crossref PubMed Scopus (229) Google Scholar). Although estrogen receptor-positive breast tumors are usually more responsive to therapy than estrogen receptor-negative tumors, there is a report demonstrating that estrogen receptor-positive breast tumor status in obese women is actually associated with a poorer prognosis than estrogen receptor-negative status (24Maehle B. Tretli S. Breast. Cancer Res. Treat. 1996; 41: 123-130Crossref PubMed Scopus (73) Google Scholar). Also, the T-47D cells, an estrogen receptor-positive cell line, evidenced a dramatic increase in anchorage-independent growth after treatment with leptin (25Hu X. Juneja S.C. Maihle N.J. Cleary M.P. J. Natl. Cancer. Inst. 2002; 94: 1704-1711Crossref PubMed Scopus (408) Google Scholar). Estrogen receptors (ERα and ERβ) are members of the superfamily of nuclear steroid hormone receptors, which are able to regulate the transcriptional activity of target genes by interacting with different DNA response elements (26Jensen E.V. Ann. N. Y. Acad. Sci. 1995; 761: 1-17Crossref PubMed Scopus (16) Google Scholar). The estrogen receptor α (ERα) signaling plays an essential role in promotion and progression of steroid hormone-dependent breast cancer (27Lopez-Otin C. Diamandis E.P. Endocrinol. Rev. 1998; 19: 365-396Crossref PubMed Scopus (185) Google Scholar). In addition to mediating the classic transcriptional effect of estrogen, ERα can be transcriptionally activated in the absence of estrogen, a process referred to as ligand-independent activation (28Cenni B. Picard D. Trends Endocrinol. Metab. 1999; 10: 41-46Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Ligand-independent activation of ERα has been reported in response to a variety of stimuli (e.g. serum (29Karas R.H. Gauer E.A. Bieber H.E. Baur W.E. Mendelsohn M.E. J. Clin. Invest. 1998; 101: 2851-2861Crossref PubMed Scopus (66) Google Scholar), dopamine (30Power R.F. Mani S.K. Codina J. Conneely O.M. O'Malley B.W. Science. 1991; 254: 1636-1639Crossref PubMed Scopus (510) Google Scholar), cAMP (31Ine B.A. Montano M.M. Katzenellenbogen B.S. Mol. Endocrinol. 1994; 8: 1397-1406PubMed Google Scholar), caveolin 1 (32Schlegel A. Wang C.G. Katzenellenbogen B.S. Pestell R.G. Lisanti M.P. J. Biol. Chem. 1999; 274: 33551-33556Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), Akt kinase (33Campbell R.A. Bhat-Nakshatri P. Patel N.M. Constantinidou D. Ali S. Nakshatri H. J. Biol. Chem. 2001; 276: 9817-9824Abstract Full Text Full Text PDF PubMed Scopus (832) Google Scholar), epidermal growth factor (34Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Sasaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1731) Google Scholar), and specific cyclins (35Neuman E. Ladha M.H. Lin N. Upton T.M. Miller S.J. Direnzo J. Pestell R.G. Hinds P.W. Dowdy S.F. Brown M. Ewen M.E. Mol. Cell. Biol. 1997; 17: 5338-5347Crossref PubMed Scopus (347) Google Scholar, 36Trowbridge J.M. Rogatsky I. Garabedian M.J. Proc. Natl. Acad. Sci. 1997; 94: 10132-10137Crossref PubMed Scopus (79) Google Scholar)). The most completely studied pathway for ligand-independent ERα activation involves MAPK-mediated activation of ERα in tumor-derived cell lines (34Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Sasaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1731) Google Scholar, 37Bunone G. Briand P.A. Miksicek R.J. Picard D. EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (858) Google Scholar). In COS-1 cells, for example, growth factor-induced activation of ERα results from MAPK-mediated phosphorylation of serine 118 in the A/B domain of the ER (34Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Sasaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1731) Google Scholar). Taking into account that unliganded ERα is an effector of MAPK signal and that leptin is able, via Janus kinase 2, to activate the Ras-dependent MAPK pathway (6Machinal-Quelin F. Dieudonne M.N. Leneveu M.C. Pecquery R. Giudicelli Y. Am. J. Cell. Physiol. 2002; 282: C853-C863Crossref PubMed Scopus (111) Google Scholar), the aim of the present study was to investigate whether leptin is able to induce the functional transactivation of ERα using as model systems the estrogen-dependent MCF-7 breast cancer cells and steroid receptor-negative HeLa cells. Our results have demonstrated, for the first time, the ability of leptin to induce ERα nuclear localization together with the typical features of ERα functional transactivation in breast cancer cells. Materials—Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham (DMEM/F-12), l-glutamine, Eagle's non-essential amino acids, penicillin, streptomycin, calf serum (CS), bovine serum albumin (BSA), phosphate-buffered saline were purchased from Eurobio (Les Ullis Cedex, France). TRIzol reagent by Invitrogen (Carlsbad, CA), FuGENE 6 by Roche Applied Science (Indianapolis, IN). TaqDNA polymerase, 50-bp DNA ladder, Dual Luciferase kit, and TK Renilla luciferase plasmid were provided by Promega (Madison, WI). Aprotinin, leupeptin, phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate, and recombinant human leptin were purchased by Sigma (Milan, Italy). MAPK inhibitor PD98059 was provided by Calbiochem (San Diego, CA). Antibodies against ERα and β-actin were provided by Santa Cruz Biotechnology (Santa Cruz, CA). Biotinylated horse-anti-mouse IgG and ABC complex/horseradish peroxidase were provided by Vector Laboratories (Burlingame, CA). Chromogen 3-di-aminobenzidine tetrachloride dihydrate was purchased by Bio-Optica. An ECL system, [γ32P]ATP, and Sephadex G-50 spin columns were purchased from Amersham Biosciences (Buckinghamshire, UK). Plasmids—Firefly luciferase reporter plasmid is XETL, a construct containing an estrogen-responsive element (37Bunone G. Briand P.A. Miksicek R.J. Picard D. EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (858) Google Scholar). The wild type human ERα expression vector (HEGO) consists of the full-length ERα cDNA fused with the SV40 early promoter and expressed in the pSG5 vector (38Tora L. Mullick A. Metger D. Ponglikitmongkol M. Park I. Chambon P. EMBO J. 1989; 8: 1981-1986Crossref PubMed Scopus (388) Google Scholar). pSG5/HE15 and pSG5/HE19 plasmids codify for N-terminal ERα (AF-1, amino acid 1–281) and for C-terminal ERα (AF-2, amino acids 179–595), respectively (a gift from Dr. D. Picard, University of Geneve, Switzerland). S104/106/118A-ER plasmid was mutated in serine residues 104, 106, and 118 to Ala (a gift from Dr. D. A. Lannigan, University of Virginia, Charlottesville, VA); HE241G ERα plasmid mutant that lacks a nuclear translocation signal (NLS) (Δ250–303) (kindly provided by Dr. P. Chambon, CNRS-INSERM, University of Louis Pasteur, Strasbourg, France). pCMV5myc vector containing the c-DNA encoding dominant negative ERK2 K52R (ERK2-; gift from Dr. M. Cobb, Department of Pharmacology, Southwestern Medical Center, Dallas, TX). Cell Cultures—Wild-type human breast cancer (MCF-7) cells were a gift from E. Surmacz (Philadelphia, PA). Human uterin cervix adenocarcinoma (HeLa) cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA). MCF-7 and HeLa cells were maintained in DMEM/F-12 containing 5% CS, 1% l-glutamine, 1% Eagle's non-essential amino acids, and 1 mg/ml penicillin-streptomycin. Cells were cultured in Phenol red-free DMEM containing 5% charcoal-stripped fetal calf serum (CS-fetal calf serum), 0.5% BSA, and 2 mm l-glutamine, for 24 h before each experiment. Immunocytochemical Staining—Paraformaldehyde-fixed MCF-7 and HeLa cells (2% paraformaldehyde A for 30 min) were used for immunocytochemical staining. Endogenous peroxidase activity was inhibited by hydrogen peroxide (3% in absolute methanol for 30 min), and nonspecific sites were blocked by normal horse serum (10% for 30 min). ERα immunostaining was then performed using as primary antibody a mouse monoclonal antiserum (1:40, overnight at 4 °C), whereas a biotinylated horse-anti-mouse IgG (1:600, for 1 h at room temperature) was utilized as secondary antibody. Avidin-biotin-horseradish peroxidase complex (ABC complex/horseradish peroxidase) was applied (30 min), and the chromogen 3,3′-diaminobenzidine tetrachloride dihydrate was used as detection system (5 min). TBS-T (0.05 m Tris-HCl plus 0.15 m NaCl, pH 7.6 containing 0.05% Triton X-100) served as washing buffer. The primary antibody was replaced by normal mouse serum at the same concentration in control experiments on MCF-7 cultured cells. RNA Isolation—Total cellular RNA was extracted from MCF-7 cells using TRIzol reagent as suggested by the manufacturer. The purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis before carrying out the analytical procedures. RT-PCR Assay—The evaluation of ERα and pS2 mRNA expression was performed by semi-quantitative RT-PCR (39Maggiolini M. Donzé O. Picard D. Biol. Chem. 1999; 380: 695-697Crossref PubMed Scopus (35) Google Scholar). For ERα, pS2, and the internal control gene 36B4, the primers were: 5′-GTGTACAACTACCCCGAGG-3′ (ERα forward) and 5′-CAGATTCATCATGCGGAACCGAATG-3′ (ERα reverse), 5′-TTCTATCCTAATACCATCGACG-3′ (pS2 forward) and 5′-TTTGAGTAGTCAAAGTCAGAGC-3′ (pS2 reverse), and 5′-CTCAACATCTCCCCCTTCTC-3′ (36B4 forward) and 5′-CAAATCCCATATCCTCGT-3′ (36B4 reverse) to yield products of 1172, 210, and 408 bp, with 20, 15, and 15 PCR cycles, respectively. Western Blot Analysis—MCF-7 cells were grown in 100-mm dishes up to 70–80% confluence and then lysed. Protein lysates were obtained with a buffer containing 50 mm HEPES (pH 7.5), 150 mm NaCl, 1.5 mm MgCl2, 1 mm EGTA, 10% glycerol, 1% Triton X-100, a mixture of protease inhibitors (aprotinin, PMSF, and sodium orthovanadate). Equal amounts of total protein were resolved on an 11% SDS-polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane, probed with the antibody F-10 against ERα or β-actin. The antigen-antibody complex was detected by incubation of the membrane at room temperature with a peroxidase-coupled goat anti-mouse IgG and revealed using the ECL system. Transfection Assay—MCF-7 cells were transferred into 24-well plates with 500 μl of regular growth medium/well the day before transfection. The medium was replaced with DMEM lacking phenol red as well as serum on the day of transfection, which was performed using the FuGENE 6 reagent as recommended by the manufacturer with the mixture containing 0.5 μg of reporter plasmid XETL. A set of experiments was performed cotransfecting XETL and pCMV5myc vector containing the cDNA encoding dominant negative ERK2 K52R (ERK2-, 0.5 μg/well). HeLa cells were cotransfected with XETL, HEGO, and ERK2 (0.5 μg/well). Another set of experiments was carried out by using 0.5 μg/well pSG5/HE15, pSG5/HE19, S104/106/118A-ER, and HE241G plasmids. Six hours after transfection, the medium was changed and the cells were treated in DMEM/F-12 in the presence of 100 and 1000 ng/ml leptin or 100 nm estradiol (E2) for 48 h. A concentration, 10 μm, of the pure anti-estrogen ICI 182,780 was used. In another set of experiments, after transfection, we added MAPK inhibitor PD 98059 (50 μm) overnight in the medium before starting the treatment with leptin. TK Renilla luciferase plasmid (25 ng/well) was used to normalize the efficiency of the transfection. Firefly and Renilla luciferase activities were measured using a Dual Luciferase kit. The firefly luciferase data for each sample were normalized on the basis of transfection efficiency measured by Renilla luciferase activity. Gel Mobility Shift Assay—Nuclear extracts were prepared from MCF-7 as previously described (40Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2237) Google Scholar). Briefly, MCF-7 cells plated into 60-mm dishes were scraped into 1.5 ml of cold phosphate-buffered saline. Cells were pelleted for 10 s and resuspended in 400 μl of cold buffer A (10 mm HEPES-KOH, pH 7.9, at 4 °C, 1.5 mm MgCl2, 10 mm KCl, 0.5 mm dithiothreitol, 0.2 mm PMSF, 1 mm leupeptin) by flicking the tube. The cells were allowed to swell on ice for 10 min and then vortexed for 10 s. Samples were then centrifuged for 10 s, and the supernatant fraction was discarded. The pellet was resuspended in 50 μl of cold Buffer B (20 mm HEPES-KOH, pH 7.9, 25% glycerol, 1.5 mm MgCl2, 420 mm NaCl, 0.2 mm EDTA, 0.5 mm dithiothreitol, 0.2 mm PMSF, 1 mm leupeptin) and incubated on ice for 20 min for high salt extraction. Cellular debris was removed by centrifugation for 2 min at 4 °C, and the supernatant fraction (containing DNA-binding proteins) was stored at -70 °C. The yield was determined by Bradford method (41Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (223028) Google Scholar). The probe was generated by annealing single-stranded oligonucleotides and labeled with [γ-32P]ATP and T4 polynucleotide kinase and then purified using Sephadex G50 spin columns. The DNA sequences used as probe or as cold competitor is the following (the nucleotide motif of interest is underlined) 5′-TCCCCCTGCAAGGTCACGGTGGCCACCCCGTG-3′. Oligonucleotides were synthesized by Sigma Genosys. The protein binding reactions were carried out in 20 μl of buffer (20 mm HEPES, pH 8, 1 mm EDTA, 50 mm KCl, 10 mm dithiothreitol, 10% glycerol, 1 mg/ml BSA, 50 μg/ml poly(dI-dC) with 50000 cpm of labeled probe, 20 μg of MCF-7 nuclear protein, and 5 μg of poly(dI-dC)). The above-mentioned mixture was incubated at room temperature for 20 min in the presence or absence of unlabeled competitor oligonucleotide. The entire reaction mixture was electrophoresed through a 6% polyacrylamide gel in 0.25× Tris borate-EDTA for 3 h at 150 V. Gel was dried and subjected to autoradiography at -70 °C. Statistical Analysis—Each datum point represents the mean ± S.E. of three different experiments. Data were analyzed by analysis of variance test using the STATPAC computer program. Leptin Modulates ERα Nuclear Immunoreactivity in MCF-7 Cells—It is well documented that ERα is predominantly localized in the nucleus (42King W.J. Greene G.L. Nature. 1984; 307: 745-747Crossref PubMed Scopus (1140) Google Scholar, 43Welshons W.V. Lieberman M.E. Gorski J. Nature. 1984; 307: 747-749Crossref PubMed Scopus (723) Google Scholar, 44Htun H. Holth L.T. Walker D. Davie J.R. Hager G.L. Mol. Biol. Cell. 1999; 10: 471-486Crossref PubMed Scopus (223) Google Scholar) and, upon ligand activation, undergoes conformational changes leading to homodimerization and target gene regulation (45Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6166) Google Scholar). To provide evidence that leptin is able to modulate ERα nuclear localization in MCF-7 cells, we performed two sets of immunostaining experiments using different culture conditions. Fig. 1 shows that, in MCF-7 cells maintained in medium without serum for 24 h, ERα immunoreactivity was well detectable in the nuclear compartment (Fig. 1A) and down-regulated in cells treated for 24 h with 100 nm E2 (Fig. 1B) and 1000 ng/ml leptin (Fig. 1C). In the other set of experiments, MCF-7 cells were cultured in serum deprivation conditions for 96 h (Fig. 2). We observed that ERα immunoreactivity was no longer detectable in the control (Fig. 2A), whereas, in the same experimental conditions, the treatment with either E2 (Fig. 2B) or leptin (Fig. 2C) for 24 h induced a strong ERα immunoreactivity in the nuclear compartment. No immunoreactivity was observed either by replacing the anti-ERα antibody by irrelevant mouse IgG (insets in Figs. 1 and 2) or by using the primary antibody pre-absorbed with an excess of receptor protein (data not shown). Leptin Down-regulates ERα Expression—E2 is known to down-regulate the levels of ERα in breast cancer cell line through an increased turnover of the E2-activated ERα protein and a reduced transcription rate of its own gene (46Santagati S. Gianazza E. Agrati P. Vegeto E. Patrone C. Pollio G. Maggi A. Mol. Endocrinol. 1997; 11: 938-949Crossref PubMed Scopus (45) Google Scholar). This down-regulation represents an additional hallmark of ERα activation by an agonist. To evaluate if leptin may exhibit a like-estrogen action, we investigated the down-regulatory effects of ERα mRNA and total protein levels in MCF-7 cells. A treatment of 24 h with either 1000 ng/ml leptin or E2 100 nm displayed in both circumstances a similar pattern of response consistent with a down-regulation of both ERα mRNA (Fig. 3, A and B) and protein content (Fig. 3, C and D). ERα mRNA levels were compared by semiquantitative RT-PCR and standardized on the mRNA levels of the housekeeping gene 36B4 (Fig. 3, A and B). Leptin Up-regulates pS2 mRNA—To provide further evidence for the ability of leptin to activate per se ERα, we investigated upon leptin exposure the expression of a classic estrogen-dependent gene, pS2. We observed, by RT-PCR, in MCF-7 cells treated with 1000 ng/ml leptin for 24 h, a strong increase of pS2 mRNA that was inhibited by the addition of the pure anti-estrogen ICI 182,780 (Fig. 4, A and B). Leptin Induces Functional Activation of ERα in MCF-7 and HeLa Cells—To corroborate the specificity of leptin to transactivate the endogenous ERα, we transiently transfected MCF-7 cells with the gene reporter XETL, which carries firefly luciferase sequences under the control of an estrogen response element upstream of the thymidine kinase promoter. A significant enhancement of XETL expression was observed in the transfected cells exposed to 1000 ng/ml leptin for 48 h (p < 0.01) (Fig. 5). Similar results were obtained in estrogen receptor-negative HeLa cells cotransfected with HEGO and XETL plasmids tested in the same experimental conditions (Fig. 5). Remarkably the anti-estrogen ICI 182,780 was shown to efficiently antagonize the stimulatory effect of leptin on ERα-regulated transactivation in MCF-7 and HeLa cells (Fig. 5). Leptin is able, via JAK2, to activate the Ras-dependent MAPK pathway. Thus, a potential role of ERK1/ERK2 pathway in mediating the stimulatory effects of leptin on ERα has been reasonably investigated, because the MAPK signal is generally involved in enhancing ERα functional activation in a ligand-independent manner. In the presence of MAPK inhibitor PD 98059 or in the cells transiently transfected with ERK2 dominant negative plasmid in MCF-7 and HeLa cells, the up-regulatory effects induced by leptin on XETL luciferase activity through ERα activation were completely abrogated (Fig. 6). Leptin Increases ERα Transcriptional Activation through the AF-1 Domain—To specify which functional domain of ERα was mainly involved in ERα transactivation, HeLa cells were cotransfected with the XETL reporter gene and PSG5/HE15 or PSG5/HE19 plasmids codifying for AF-1 and AF-2 domains, respectively. The treatment with 1000 ng/ml lept