Leptin Induces, via ERK1/ERK2 Signal, Functional Activation of Estrogen Receptor α in MCF-7 Cells

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.

Although leptin is mainly synthesized by breast adipose tissue, its expression has also been detected in normal and tumoral human mammary epithelial cells (12,13). In addition, it has been shown that leptin receptors (short and long isoforms) are expressed in normal mammary epithelial cells (14) as well as in human breast cancer cell lines (11,15). 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 (16). It is now well established that postmenopausal women with upper body fat predominance experience a higher risk of breast cancer (17,18). The association between obesity and breast carcinoma is usually ascribed to estrogen excess, derived from androgen aromatization in peripheral fat deposits (19,20). In our recent work we have demonstrated that leptin is able to stimulate, through mitogenactivated protein kinase (MAPK) 1 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 (21). 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 (22,23). 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 (24). Also, the T-47D cells, an estrogen receptor-positive cell line, evidenced a dramatic increase in anchorage-independent growth after treatment with leptin (25).
Estrogen receptors (ER␣ and ER␤) 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 (26). The estrogen receptor ␣ (ER␣) signaling plays an essential role in pro-motion and progression of steroid hormone-dependent breast cancer (27). In addition to mediating the classic transcriptional effect of estrogen, ER␣ can be transcriptionally activated in the absence of estrogen, a process referred to as ligand-independent activation (28). Ligand-independent activation of ER␣ has been reported in response to a variety of stimuli (e.g. serum (29), dopamine (30), cAMP (31), caveolin 1 (32), Akt kinase (33), epidermal growth factor (34), and specific cyclins (35,36)). The most completely studied pathway for ligand-independent ER␣ activation involves MAPK-mediated activation of ER␣ in tumor-derived cell lines (34,37). In COS-1 cells, for example, growth factor-induced activation of ER␣ results from MAPKmediated phosphorylation of serine 118 in the A/B domain of the ER (34).
Taking into account that unliganded ER␣ is an effector of MAPK signal and that leptin is able, via Janus kinase 2, to activate the Ras-dependent MAPK pathway (6), the aim of the present study was to investigate whether leptin is able to induce the functional transactivation of ER␣ 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 ER␣ nuclear localization together with the typical features of ER␣ functional transactivation in breast cancer cells.
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). ER␣ 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 3,3Ј-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.
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 MgCl 2 , 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 ER␣ or ␤-actin. The antigenantibody 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 l 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 g of reporter plasmid XETL. A set of experiments was performed cotransfecting XETL and pCMV5myc vector containing the cDNA encoding dominant negative ERK2 K52R (ERK2Ϫ, 0.5 g/well). HeLa cells were cotransfected with XETL, HEGO, and ERK2 (0.5 g/well). Another set of experiments was carried out by using 0.5 g/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 (E 2 ) for 48 h. A concentration, 10 M, of the pure anti-estrogen ICI 182,780 was used. In another set of experiments, after transfection, we added MAPK inhibitor PD 98059 (50 M) 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 (40). 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 l of cold buffer A (10 mM HEPES-KOH, pH 7.9, at 4°C, 1.5 mM MgCl 2 , 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 l of cold Buffer B (20 mM HEPES-KOH, pH 7.9, 25% glycerol, 1.5 mM MgCl 2 , 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 Ϫ70°C. The yield was determined by Bradford method (41). The probe was generated by annealing single-stranded oligonucleotides and labeled with [␥-32 P]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) 5Ј-TCCCCCTGCAAGGTCACGGTGGCCAC-CCCGTG-3Ј. Oligonucleotides were synthesized by Sigma Genosys. The protein binding reactions were carried out in 20 l of buffer (20 mM HEPES, pH 8, 1 mM EDTA, 50 mM KCl, 10 mM dithiothreitol, 10% glycerol, 1 mg/ml BSA, 50 g/ml poly(dI-dC) with 50000 cpm of labeled probe, 20 g of MCF-7 nuclear protein, and 5 g 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 0.25ϫ Tris borate-EDTA for 3 h at 150 V. Gel was dried and subjected to autoradiography at Ϫ70°C.
Statistical Analysis-Each datum point represents the mean Ϯ S.E. of three different experiments. Data were analyzed by analysis of variance test using the STATPAC computer program.

RESULTS
Leptin Modulates ER␣ Nuclear Immunoreactivity in MCF-7 Cells-It is well documented that ER␣ is predominantly localized in the nucleus (42-44) and, upon ligand activation, under-goes conformational changes leading to homodimerization and target gene regulation (45).
To provide evidence that leptin is able to modulate ER␣ 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, ER␣ immunoreactivity was well detectable in the nuclear compartment (Fig. 1A) and down-regulated in cells treated for 24 h with 100 nM E 2 (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 ER␣ immunoreactivity was no longer detectable in the control ( Fig. 2A), whereas, in the same experimental conditions, the treatment with either E 2 (Fig. 2B) or leptin (Fig. 2C) for 24 h induced a strong ER␣ immunoreactivity in the nuclear compartment. No immunoreactivity was observed either by replacing the anti-ER␣ 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 ER␣ Expression-E 2 is known to down-regulate the levels of ER␣ in breast cancer cell line through an increased turnover of the E 2 -activated ER␣ protein and a reduced transcription rate of its own gene (46). This down-regulation represents an additional hallmark of ER␣ activation by an agonist. To evaluate if leptin may exhibit a like-estrogen action, we investigated the down-regulatory effects of ER␣ mRNA and total protein levels in MCF-7 cells. A treatment of 24 h with either 1000 ng/ml leptin or E 2 100 nM displayed in both circumstances a similar pattern of response consistent with a down-regulation of both ER␣ mRNA (Fig. 3,  A and B) and protein content (Fig. 3, C and D). ER␣ 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 ER␣, we investigated upon leptin exposure the expression of a classic estrogendependent 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 antiestrogen ICI 182,780 (Fig. 4, A and B).
Leptin Induces Functional Activation of ER␣ in MCF-7 and HeLa Cells-To corroborate the specificity of leptin to transactivate the endogenous ER␣, 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 Ͻ 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 effi- ciently antagonize the stimulatory effect of leptin on ER␣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 ER␣ has been reasonably investigated, because the MAPK signal is generally involved in enhancing ER␣ functional activation in a ligandindependent 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 ER␣ activation were completely abrogated (Fig. 6).
Leptin Increases ER␣ Transcriptional Activation through the AF-1 Domain-To specify which functional domain of ER␣ was mainly involved in ER␣ 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 leptin for 48 h induced an increased transcriptional activation only in transfected cells bearing the plasmid codifying for AF-1 domain (p Ͻ 0.01) (Fig. 7B). Our results clearly demonstrate that the Nterminal AF-1, but not the C-terminal AF-2, is necessary for the leptin response.
Leptin Is Unable to Induce a Transcriptional Activation of ER␣ Mutated In Ser-104/106/118 or Lacking a Nuclear Local-ization Sequence-ER␣ is predominately phosphorylated on Ser-118 and to a lesser extent on Ser-104 and Ser-106. These serine residues, which are effectors of ERK1/ERK2 signal, are all located within the AF-1 region of the N-terminal domain of ER␣ (47). To confirm the involvement of AF-1 domain in the activation of ER␣ by leptin and to demonstrate that the activation of ER␣ occurs at the genomic level, HeLa cells were cotransfected with XETL and either HEGO or Ser-104/106/ 118A-ER or HE241G. As shown in Fig. 7C, we observed how in transfectants with mutants 1000 ng/ml leptin for 48 h was no longer able to elicit any substantial activation on ERE luciferase signal as compared with the cells bearing wild-type ER␣.
Leptin Amplifies ER␣ Activation by Estradiol-It is worth noting how the combined treatment of E 2 and leptin synergized in up-regulating transactivation of ER␣ in HeLa cells expressing ER␣ ectopically (Fig. 8). In the presence of ER␣ mutated in serine residues 104/106/118, the up-regulatory effects on ER␣ activation by E 2 still persisted, whereas the potentiating effect induced by the combined presence of E 2 and leptin was no longer noticeable (Fig. 8).

Effect of in Vitro Leptin Treatment on ERE DNA-binding Activity in MCF-7 Cells-
The results obtained with the functional studies were corroborated by electrophoresis mobility shift (EMSA). Nuclear extracts from MCF-7 cells were analyzed by EMSA using a 32-bp DNA probe containing an estrogen-responsive element (ERE) from the human pS2 gene. In the EMSA shown in Fig. 9 we obtained a specific protein-DNA complex using nuclear extracts prepared from MCF-7 cells (lane 1). The formation of this complex was abolished by the addition of a 200-fold molar excess of non-radiolabeled probe (lane 2). A treatment of 48 h with 1000 ng/ml leptin or 100 nM E 2 induced a strong increase in ERE DNA-binding activity ( lanes  3 and 4), which was reversed in the presence of the pure antiestrogen ICI 182,780 when compared with basal level (lane 5). Using nuclear extracts from MCF-7 cells either treated with MAPK inhibitor PD 98059 or transiently transfected with dominant negative ERK2, the ERE DNA-binding activity induced by leptin treatment was drastically reduced (lanes 6 and 7). DISCUSSION An association between breast cancer and obesity has been recognized for at least 40 years, even though the mechanism underlying such relationship remains to be fully elucidated (16). Although in situ estrogen production by adipocytes has been considered an important risk factor for breast cancer progression (16 -18), an additional candidate that may play a major role in the same scenario is leptin. Leptin is a hormone with multiple biological actions that is produced predominantly by adipose tissue and is present at high concentrations in obese women who are exposed to a higher risk in developing breast cancer (1-3). In the same vein several actions of leptin, including the stimulation of normal and tumor cell growth, migration and invasion, and enhancement of angiogenesis, suggest that this hormone is involved in breast cancer progression (5)(6)(7)(8)(9)(10)(11)48). This assumption is sustained by recent findings that show how normal mammary gland morphogenesis is impaired in both non-transgenic genetically obese leptin-deficient and genetically obese leptin receptor-deficient mice (25). Similar results were obtained for transgenic transforming growth factor-␣/lep ob lep ob mice, which did not develop mammary tumors, in contrast to transgenic transforming growth factor-␣ mice that were either homozygous lep ϩ lep ϩ or heterozygous lep ϩ lep ob lean for the leptin gene, which exhibited mammary tumor incidence rates of 50% and 60%, respectively, by 24 months of age (25). All these findings seem to underscore an important role for leptin on mammary gland development and mammary gland tumorigenesis giving new interpretative clues to under- stand the epidemiological relationship between obesity and breast carcinogenesis.
The two isoforms of leptin receptor are both present in different breast cancer cell lines (11,14,15). The short and long isoforms induce the activation of one or more members of the Janus (or JAK) family of tyrosine kinases, which form a complex with the cytokine receptor subunits, thereby inducing autophosphorylation as well as phosphorylation of the receptor. These phosphorylated tyrosines form binding sites for various signaling molecules, which are themselves thought to be phosphorylated by JAK kinases like STAT proteins. The same phosphorylated tyrosine sites bind SHP2 protein-containing phosphatase. SHP2 is proposed as a positive regulator of leptin signaling through MAPK activation by the recruitment of the adapter protein growth receptor bound 2 and the activation of the Ras/Raf pathway. A secondary pathway for leptin-induced MAPK signaling is mediated directly via JAK2 (49).
Stemming from the evidence that MAPK signal induces the functional activation of unliganded ER␣ (34,37), it was reasonable to investigate the potential role of leptin in stimulating ER␣. In the present study we have demonstrated for the first time that leptin was able to induce functional transactivation of ER␣ that was abrogated in the presence of either MAPK inhibitor or dominant negative ERK2. This addresses a crucial role of MAPK signal to stimulate ER␣ upon leptin exposure. In different breast cancer cell lines, it has been demonstrated how the interaction between insulin/insulin-like growth factor-1 and estradiol signaling occurs also through the direct transcription activation of ER␣ via MAPK (34). In this concern, it is well documented that the human ER␣ is phosphorylated by ERK on Ser-118 (34,37,50). The phosphorylation of this serine is required for full activity of the ER␣ AF-1 domain. Overexpression of active ERK kinase or the active p21 ras , resulting in the ERK1/ERK2 activation, enhances estrogen-induced transcriptional activity of the wild type ER␣ but not of a mutant ER␣ with an alanine in place of Ser-118 (34).
All these observations fit with our data demonstrating that: 1) only the construct bearing the N-terminal domain (AF-1) was able to activate ERE reporter gene; 2) ER␣ mutated in serine residues 104/106/118 is no longer stimulated by leptin, and it is still transactivated by E 2 even though to a lesser extent with respect to wild type. This finding recalls a recent report (51) evidencing how the full activation of liganded receptor requires the integrity of phosphorylated serine residues 104/106/118. In addition, it has been demonstrated, by the same authors, how the mutation of serine 104/106/118 affects the physical and functional interaction of full-length ER␣ with p160/SRC and CBP in the absence of ligand. Thus, it is reasonable to assume that the ER␣, mutated in Ser-104/106/118 and being unable to recruit cofactor, fails to activate cell transcription machinery upon leptin stimulation.
It emerges from recent findings that in human vascular smooth muscle cells MAPK activation per se results in a nuclear translocation of ER␣, which supports the MAPK-mediated phosphorylation of ER␣ as a prerequisite for its nuclear localization (50). Thus the sustained MAPK activation induced by leptin may explain why even after prolonged incubation of MCF-7 cells in serum-free medium, leptin treatment for 24 h is able to induce ER␣ nuclear localization as revealed by our immunostaining data, whereas ER␣ immunoreactivity was just scantily detectable in untreated cells. This finding appears to be unrelated to the cell type specificity, which we reproduced in HeLa cells, where leptin was able to induce ER␣ wild type nuclear localization but failed to do so in the presence of ER␣ lacking nuclear localization signal (NLS) (data not show).
Therefore, in MCF-7 cells, concomitantly with leptin treatment, the classic biological features of ER␣ wild type functional transactivation were observed: 1) a nuclear compartmentalization of ER␣; 2) the down-regulation of its mRNA and total protein content; and 3) the up-regulation of a classic estrogendependent gene such as pS2, which was inhibited by the pure anti-estrogen ICI 182,780.
These results fit well with the EMSA findings. In this assay using 32 P-ERE sequence of pS2 promoter in the presence of nuclear extracts from MCF-7 cells, we observed that, upon leptin treatment, a strong increase in DNA binding occurred in the same extent of that induced by estradiol and was markedly reduced in the presence of either MAPK inhibitor or dominant negative ERK2.
These data broaden further the potential relationship existing between leptin and estrogens. Indeed it has been reported that estrogens appear to modulate leptin gene expression in adipose tissue (22,23). On the other hand, we recently reported for the first time that leptin is able to induce the aromatase gene expression in MCF-7 cells via AP-1 (21), thus addressing clearly how leptin may be involved in the pathophysiology of breast, modulating in situ estrogen production also in epithelial cells. Now we are able to demonstrate that the potential amplification of estrogen signals induced by leptin has two active components: the enhanced aromatase activity and a direct activation of ER␣ in the absence of the natural ligand. In addition, the potentiating effects of leptin on E 2 -induced activation of ER␣ address how different functional domains as effectors of two distinct signals may cooperate in a synergistic way. This gives a great emphasis to the role of leptin in promoting breast cancer in obese women through the potential use of readily aromatizable androgens in breast tissue, by enhancing in situ estradiol production together with its ability to directly activate ER␣ in epithelial breast cancer.