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Originally published In Press as doi:10.1074/jbc.M004691200 on September 11, 2000

J. Biol. Chem., Vol. 275, Issue 49, 38921-38928, December 8, 2000
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Estradiol Decreases IGF-1 and IGF-1 Receptor Expression in Rat Aortic Smooth Muscle Cells

MECHANISMS FOR ITS ATHEROPROTECTIVE EFFECTS*

Kathrin J. ScheideggerDagger , Bruno Cenni§, Didier Picard§, and Patrick DelafontaineDagger

From the Dagger  Division of Cardiology, University Hospital of Geneva, 1211 Geneva 14, Switzerland and the § Department of Cell Biology, University of Geneva, 1211 Geneva 4, Switzerland

Received for publication, May 31, 2000, and in revised form, August 22, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insulin-like growth factor (IGF-1) is a potent mitogen for vascular smooth muscle cells. Both IGF-1 and its receptor have been shown to be highly expressed in atherosclerotic lesions. Here we investigated whether part of the vasculoprotective properties of E2 may be mediated by its negative regulation of the IGF-1 system. HeLa cells, which do not contain endogenous estrogen receptors (ER), were transiently transfected with IGF-1R promoter constructs with or without a plasmid encoding human ERalpha or ERbeta and treated with 100 nM 17beta -estradiol (E2) for 24 h. E2 treatment decreased basal luciferase activity by 51%, and this effect was dependent on co-expression of ERalpha , whereas no repression was observed with ERbeta . A mutation within the DNA binding domain of the ERalpha abolished the repressor function of the ER receptor. Similarly, E2 decreased IGF-1R transcription by 21% in rat aortic smooth muscle cells (RASMC), which express endogenous ER. This effect was specific for E2, because it was inhibited by an antiestrogen and because progesterone did not have any effect on IGF-1R expression in HeLa or RASMC transfected with progesterone receptor. Accordingly, E2 decreased IGF-1R and IGF-1 mRNA in RASMC by 47% and 33%. Western blot analysis and radioligand binding studies showed that E2 also dose-dependently decreased IGF-1R protein expression in RASMC by 40% and 30%, respectively, and that IGF-1 protein was reduced by 43%. Repression of IGF-1R promoter activity by a combination of ERalpha and E2 did not appear to be mediated via direct binding of ER to the IGF-1R promoter but rather by inhibition of SP1 binding to the IGF-1R promoter. Thus, E2 down-regulates IGF-1R and IGF-1 expression in vascular smooth muscle cells. This may have important implications for the understanding of the beneficial effects of estrogen in the cardiovascular system.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Several studies, but not all, have suggested that estrogens are cardioprotective in postmenopausal women (1, 2). The mechanisms of estradiol-induced reduction in the risk of coronary artery disease remain unclear. Although these atheroprotective effects of estrogen were principally attributed to the hormone's effects on serum lipid concentrations (3-5), recent findings suggest that the majority of the vasculoprotective effects of estrogen are due to direct effects on the vasculature (6). Direct effects of estrogens have been demonstrated in vitro and in vivo both in animal and human models. These include effects on gene expression (7, 8), ion channel function (9, 10), response to vasoactive substances (11-14), as well as vascular smooth muscle cell proliferation and migration (14, 15).

The possible involvement of insulin-like growth factor-1 (IGF-1)1 and IGF-1 receptor (IGF-1R) in cardiovascular pathology has recently raised interest. In vitro data have shown that IGF-1 is a potent vascular smooth muscle cell (VSMC) mitogen (16, 17), and several reports have documented that VSMCs express IGF-1 and its receptor (18-20). We and others have shown that several growth factors up-regulate IGF-1R on VSMC and this ability of growth factors to increase the number of IGF-1R is likely critical for their mitogenic effects (17, 21-24). Furthermore, regulation of IGF-1 binding proteins (IGFBPs) by growth factors may be physiologically important (25).

Steroid receptors, including the estrogen receptors (ER) alpha  and beta , mediate the specific response of cells to their respective ligands by virtue of their ability to bind cis-acting regulatory sequences termed steroid response elements (for review see Ref. 26). Although much is known about mechanisms of gene activation by ER, less information exists about repression of gene expression by ER. Although activation of genes by estrogens is typically mediated by binding of the activated receptor to the respective response element(s) present upstream of or within target genes, negative regulation by these hormones cannot always be explained by receptor-DNA interaction (27, 28). To our knowledge, the inhibition of IL-6 in HeLa cells, lipoprotein lipase in 3T3-L1 cells, tumor necrosis factor alpha  in U937 cells, IGF-1 gene expression in primary rat osteoblasts, and the mannose-6 IGF-II receptor gene in breast cancer cells by estrogens are the only documented examples of repression by estrogens (29-33). It was therefore of interest to us to explore the effects of estradiol on IGF-1R and IGF-1 expression in vascular cells such as RASMC and to determine the molecular mechanisms of ER-mediated action on IGF-1R gene expression.

We show that E2 dose-dependently decreases IGF-1R and IGF-1 expression and that the antiproliferative activity of E2 involves a down-regulation of IGF-1R and IGF-1. However, we found no direct binding of ER to sequences in the IGF-1R promoter that were sufficient to confer repression by ER in functional experiments. Nevertheless, results obtained from bandshift experiments indicated that there was an interaction between SP1 and ER, because ER decreased SP1 binding to the IGF-1R promoter. These data indicate that ER can modulate transcription from promoters that lack classical estrogen response elements (ERE) and have important implications for understanding cardiovascular effects of estrogens.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Cell culture media and LipofectAMINE were purchased from Life Technologies (Basel, Switzerland). 4-Hydroxytamoxifen (OHT), 17beta -estradiol, and progesterone were from Sigma (St. Louis, MO). The antiestrogen ICI 164,384 was a kind gift from Dr. A. Wakeling (Zeneca Pharmaceuticals, Macclesfield, UK). The Dual-Luciferase reporter assay, the TNT-T7 Quick kit, and human recombinant SP1 were from Promega (Wallisellen, Switzerland). Recombinant ERalpha was from Panvera (Madison, WI), and anti-ERalpha (314) was from NeoMarkers (Fremont, CA). Antibodies against the beta -chain of the IGF-1 receptor and SP1 were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). Peroxidase-conjugated anti-mouse IgG was from Transduction Laboratories (Lexington, KY), and horseradish peroxidase-conjugated anti-rabbit immunoglobulin was from Amersham Pharmacia Biotech (Dübendorf, Switzerland). Iodinated IGF-1 was purchased from PerkinElmer Life Sciences (Boston, MA).

Cell Culture-- RASMC (kindly provided by Dr. K. Griendling, Emory University, Atlanta, GA) were grown in DMEM supplemented with 10% heat-inactivated calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin and incubated at 37 °C in a humidified 5% CO2 atmosphere. HeLa cells were cultured under similar conditions with 5% fetal bovine serum. The ER-expressing, human breast tumor cells, MCF-7 (34), were maintained in DMEM and 10% fetal bovine serum. Prior to experiments, cell media were changed to DMEM without Phenol Red, containing dextran-coated, charcoal-treated, heat-inactivated (DCT) fetal bovine serum.

Plasmids and Transfection-- The full-length promoter of the IGF-1R p(-2350/+640-Luc) and the shorter construct p(-476/+640-Luc) were a generous gift from Dr. H. Werner (National Institutes of Health, Bethesda, MD). Deletion fragments were made from the full-length promoter construct and subcloned upstream of the firefly luciferase cDNA (35). The plasmids encoding human ERalpha (HEG0), HE82, human ERbeta (pCMV-hERbeta ), and pCMV-SP1 were kind gifts from Dr. P. Chambon (Strasbourg, France), Dr. S. Mader (Montreal, Canada), Dr. J.-Å. Gustafsson (Huddinge, Sweden), and Dr. R. Tjian (University of California at Berkeley, CA), respectively. The following constructs have previously been described: the empty vector pSG5 (36), and XETL (37). In brief, HEG0 carries human wild-type ERalpha cDNA, HE82 contains human ERalpha cDNA with a mutation in the DNA binding domain resulting in the recognition of a glucocorticoid response element instead of an ERE (38), and the reporter plasmid XETL expresses firefly luciferase under control of one vitellogenin A2 ERE upstream of the herpes simplex virus thymidine kinase promoter. The coding sequence of the human progesterone receptor B (PRB) was subcloned into the unique EcoRI site of pSG5 resulting in pSG5/hPR.

HeLa cells were plated in 24-well and RASMC in 12-well plates and transfected with 1 µg of reporter plasmid and 5 ng of pRL-TK per well with or without 200 ng of HEG0, pSG5/hPR, HE82, pCMV-hERbeta , or pCMV-SP1 with LipofectAMINE reagent. 20 h after transfection, the DNA-containing medium was changed and the cells were treated with or without E2 (100 nM) or progesterone (100 nM) for 12-24 h. In some experiments transfected cells were incubated with OHT (1 µM) or ICI (0.1 µM) for 1 h prior to the addition of E2. Luciferase activity was measured with the Dual-Luciferase kit according to the manufacturer's recommendations. Firefly luciferase activity was normalized to the internal control Renilla luciferase (Luc/Ren).

RNase Protection Assays-- RNase protection assays were performed as described previously (18). In brief, 20 µg of total RNA was hybridized with [32P]UTP-labeled antisense IGF-1R and IGF-1 riboprobe and cohybridized with an 18 S probe (Ambion, Austin, TX). After overnight hybridization at 42 °C and RNase digestion, samples were proteinase K-treated, phenol-extracted, and analyzed by 6% polyacrylamide/8 M urea denaturing gel electrophoresis. Densitometric analyses were performed using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Radioligand Binding Studies-- Radioligand binding assays were performed as described previously (39). Briefly, RASMC cultured under DCT serum conditions treated with or without E2 in 24-well plates were incubated with 0.1 nM 125I-IGF-1 and 0-0.1 µM unlabeled IGF-1 for 90 min at room temperature. Cells were washed in ice-cold binding buffer and solubilized in 0.2 N NaOH before counting. All assays were performed in duplicate for each experimental point. Data were analyzed using the LIGAND program.

Western Blot Analysis-- Prior to the experiments, cultured RASMC were switched to Phenol Red-free DMEM containing DCT FBS for 48 h before adding E2 at various concentrations for 24 h. Cells were washed in ice-cold phosphate-buffered saline and lysed as previously published (35). Lysates were subjected to SDS-PAGE on 7.5% gels, and separated proteins were transferred to polyvinylidene difluoride membranes. Blots were blocked with 5% dry milk and incubated with anti-IGF-1Rbeta antibody and secondary peroxidase-conjugated donkey anti-rabbit antibody. Immunopositive bands were visualized by enhanced chemiluminescence.

IGF-1 Radioimmunoassay-- Specific IGF-1 immunoreactivity of cell-conditioned medium was determined as described previously (18). Briefly, cell medium was dialyzed, lyophilized, and chromatographed using Bio-Gel P-30 polyacrylamide columns (Bio-Rad Laboratories AG, Glattbrugg, Switzerland). IGF-1 fractions were assayed using a polyclonal anti-IGF-1 rabbit antiserum (kindly provided by Dr. A. F. Parlow, UCLA). Standard curves were generated using human recombinant IGF-1.

Thymidine Incorporation-- RASMC were plated in 24-well plates in DMEM without Phenol Red alone or containing DCT FBS. After 48 h cells were treated with or without E2, for 24 h in complete medium. 1 µCi/ml of [3H]thymidine was added during the last 2 h of the incubation period. Cells were washed three times with ice-cold phosphate-buffered saline, incubated for 30 min in 10% trichloroacetic acid on ice, washed two times in ice-cold 95% ethanol, and lysed in 0.2 N NaOH. Samples were measured by liquid scintillation spectrophotometry. All experiments were performed in quadruplicates.

Electrophoretic Mobility Shift Assay (EMSA)-- The human ER, PRB, and HE82 proteins were synthesized in vitro in TNT-T7-coupled rabbit reticulocyte lysates. Nuclear extracts from untransfected MCF-7 and RASMC, or HeLa cells transfected with HEG0, were incubated with recombinant human SP1, NFkappa B, or ERalpha proteins in binding buffer containing 10 mM HEPES, pH 7.9, 10% glycerol, 100 mM KCl, 2 mM dithiothreitol, 0.1 mM EDTA, 5 mM MgCl2, 2 µg of poly(dI-dC), 0.3 µg/µl bovine serum albumin, and 32P-labeled DNA in a final volume of 20 µl at room temperature. Preincubations containing ligand, antibody, and/or cold competitor (200-fold excess) as indicated were performed at room temperature for 15 min. After the incubation step the probe was added and binding conducted for additional 20 min. Reaction mixtures were loaded onto a 6% PAGE gel in 0.5 × Tris borate-EDTA (TBE). The following oligonucleotide and its complement were used as labeled probes and cold/unlabeled competitors: ERE, 5'-GATCTCTTTGATCAGGTCACTGTGACCTGACTTTG-3'. The probe for the IGF-1R promoter extended from nucleotides -476/+21.

Statistics-- All experiments were performed at least three times. Statistical significance was measured by Student's t test. A value of p < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of E2 on IGF-1R Promoter Activity in HeLa or Rat Aortic Smooth Muscle Cells-- To measure the effect of E2 on IGF-1R gene expression, HeLa cells, which do not contain endogenous ER, were transiently transfected with the full-length IGF-1R promoter reporter construct with or without cotransfecting HEG0, a plasmid encoding human ERalpha . In HeLa cells estradiol treatment (100 nM for 24 h) decreased basal luciferase activity to 49 ± 5% (Fig. 1A). This effect was ER-dependent, because E2 did not reduce basal IGF-1R expression in HeLa cells when human ER was not cotransfected. Similarly, E2 decreased by 21% IGF-1R transcription in RASMC-expressing endogenous ER (p = 0.005) (Fig. 1B). Using specific primers for rat ERalpha and rat ERbeta , we found both transcripts in RASMC (data not shown) as has been previously published by others (40-43). The fact that E2 stimulated transactivation of a minimal ERE promoter reporter construct in RASMC without transfecting HEG0 (data not shown) supports the notion that the endogenous ERs were functional as has been previously shown by others (41).



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  		Fig. 1.   Regulation of IGF-1R promoter activity by E2 is ER-dependent. A, HeLa cells were transiently transfected with 1 µg of the full-length IGF-1R promoter construct p(-2350/+640-Luc) with (lane 3) or without (lane 2) 0.2 µg of HEG0, encoding human ERalpha . Five nanograms of the internal control vector for Renilla luciferase was used. Control cells received the same volume of vehicle (ethanol). B, effect of E2 on IGF-1R promoter (p(-476/+21-Luc) in RASMC containing endogenous ER. C, effect of OHT or ICI on E2-induced repression of IGF-1R promoter activity in HeLa cells. D, E2 effect is dependent on ERalpha and on an intact ER DNA binding domain. Data is represented as percentage of control values (Luc/Ren), mean ± S.E. from at least three independent experiments. *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

Accordingly, these effects of E2 appeared to be specific, because progesterone did not have any effect on IGF-1R expression in HeLa, or RASMC transfected with progesterone receptor (data not shown). In addition, the E2 antagonist ICI 164,384 reversed the reduction in IGF-1R promoter activity induced by E2, demonstrating a specific ER-mediated effect, whereas the partial antagonist OHT acted in a synergistic way by further decreasing luciferase activity (Fig. 1C).

Interestingly, the repression of IGF-1R promoter activity by E2 was abrogated when smaller IGF-1R promoter deletion mutants were used. Indeed, the reduction in luciferase activity was maintained with the construct p(-476/+21) and p(-416/+21), however, the reduction disappeared with p(-330/+21), suggesting that the E2-responsive region was located 5' of base pair -330 in the IGF-1R promoter (data not shown). Importantly, HE82, an ER mutant carrying a mutation within the DNA binding domain and thus recognizing a glucocorticoid response element instead of an ERE, was unable to repress expression from the IGF-1R promoter, suggesting that an ER with an intact DNA binding domain is required (Fig. 1D).

Since the identification of a second ER subtype, termed the ERbeta (44, 45), much research has been focused on the potentially distinct role of ERalpha and ERbeta in vasculoprotection. It was therefore of interest to determine whether the reduction in IGF-1R transcription by the combination of E2 and ERalpha was subtype-specific or whether it could also be observed using ERbeta . Most interestingly, E2 did not repress IGF-1R transcription when HeLa cells were transfected with human ERbeta , which suggests a ERalpha subtype-specific effect (Fig. 1D).

E2 Decreases IGF-1 and IGF-1R mRNA Levels-- To confirm the results obtained in transfection studies, endogenous IGF-1 and IGF-1R mRNA levels were measured by RNase protection assay in RASMC treated with or without E2. In agreement with the transfection studies, E2 significantly and dose-dependently reduced basal levels of IGF-1 and IGF-1R by 47% and 33%, respectively (Fig. 2A), whereas OHT had similar effects as E2 (Fig. 2B). Similarly to the transcriptional assays, the antiestrogen ICI reversed the decreasing effect of E2 on IGF-1 and IGF-1R (Fig. 2B). A dose-response with ICI on IGF-1R mRNA showed that 10-7 M and 10-6 M ICI, but not 10-8 M ICI, blocked the effect of E2: control, 100 ± 0%; E2, 65 ± 11%; ICI 10-8 M/E2, 63 ± 12%; ICI 10-7 M/E2, 100 ± 17%; ICI 10-6 M/E2, 109 ± 37%; ICI 10-8 M, 88 ± 1%; ICI 10-7 M, 114 ± 19%; ICI 10-6 M, 104 ± 19%; n = 3. 



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  		Fig. 2.   E2 decreases IGF-1 and IGF-1R mRNA in a dose-dependent manner. A, representative autoradiograph of a RNase protection assay. Total RNA (20 µg/lane) was co-hybridized to 32P-labeled IGF-IR, IGF-1, and 18 S antisense riboprobes. After RNase digestion, products were analyzed by sequencing gel electrophoresis. The densitometric analysis of RNase protection assays is shown below the autoradiograph. Shown is the percentage over control of five separate experiments. Mean ± S.E. B, representative RNase protection assay showing effect of ICI (0.1 µM) and OHT (1 µM) on E2-induced effect (100 nM) on IGF-1R and IGF-1 mRNA expression in RASMC (n = 4 experiments, mean ± S.E.; IGF-1: control, 100 ± 0%; 100 nM E2, 53 ± 23%; OHT/E2, 56 ± 11%; ICI/E2, 96 ± 8%; OHT, 70 ± 23%; ICI, 135 ± 58%; IGF-1R: control, 100 ± 0%; 100 nM E2, 66 ± 18%; OHT/E2, 64 ± 13%; ICI/E2, 110 ± 26%; OHT, 84 ± 19%; ICI, 122 ± 31%).

E2 Reduces IGF-1R and IGF-1 Protein Levels-- To assess whether E2 decreased IGF-1R protein levels, cell lysates of RASMC treated with or without increasing doses of E2 were assayed for IGF-1R protein level by Western immunoblot and radioligand binding. E2 dose-dependently decreased IGF-1R protein expression after 24 h, starting with doses of 1 nM E2 and resulting in a 40% reduction with 100 nM E2 (Fig. 3A). Similarly, E2 (100 nM) reduced basal IGF-1 binding sites by approximately 30% as measured by radioligand binding studies, further confirming the results seen in Western blots (percentage change in IGF-1R number: control = 100 ± 0% and E2 = 71 ± 7%, respectively, n = 4). In addition, IGF-1 protein levels in RASMC were also significantly reduced by E2 (43% reduction with 100 nM E2) as measured by RIA of cell-conditioned medium (Fig. 3B).



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  		Fig. 3.   IGF-1R and IGF-1 protein levels are reduced in RASMC following E2 treatment. A, shown is a representative IGF-1R immunoblot. RASMC were treated with various E2 doses for 24 h. Total proteins from cell lysates were separated by SDS-PAGE under reducing conditions and transferred to polyvinylidene difluoride membranes. Membranes were then probed with an antibody recognizing the beta -subunit of the IGF-1R. After stripping the membrane, blots were incubated with an anti-beta -actin antibody for loading control (n = 3 experiments; control, 100 ± 0%; 1 nM E2, 83 ± 11%; 10 nM E2, 78 ± 14%; and 100 nM E2, 60 ± 13%, p = 0.01). B, RASMC were incubated with or without E2 (100 nM) for 24 h. IGF-1 protein was measured from supernatants by RIA. Data is represented as mean ± S.E. from four experiments.

Effect of Estradiol on Serum-induced DNA Synthesis-- IGF-1 is a potent mitogen, and a functional IGF-1R is required for the mitogenic effects of various growth factors (22, 24). To determine whether the reduced levels of IGF-1 and IGF-1R expression induced by E2 could explain the reduced DNA synthesis observed after E2 treatment (14, 46), we measured [3H]thymidine incorporation in confluent RASMC. As shown in Table I, E2 dose-dependently reduced DNA synthesis under serum conditions by approximately 50%. Exogenous addition of IGF-1 (50-100 ng/ml), however, was not able to reverse the E2-induced decrease in thymidine incorporation (data not shown).


                              
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Table I
E2 decreases serum-induced DNA synthesis in RASMC
DNA synthesis of RASMC treated with increasing doses of E2 was determined by measuring [3H]thymidine incorporation. Shown is the percentage change from control. Data are mean ± S.E. from four separate experiments performed in quadruplicates.

ERalpha Does Not Bind Directly to the IGF-1R Promoter-- The IGF-1R promoter contains multiple SP1 sites in both the 5'-flanking and 5'-untranslated regions (47). Consensus EREs consist of an inverted repeat of the palindrome GGTCA separated by a 3-base pair spacer (27, 48, 49). However, no evident ERE is present within the IGF-1R promoter. In preliminary experiments, we tested our EMSA conditions by determining classical ER binding to its consensus response element and concurrent supershift with anti-ER antibodies (Fig. 4A). To investigate the binding of ER to the IGF-1R promoter sequences, the ER-regulated promoter construct p(-476/+21) was used. Nuclear extracts from MCF-7 cells formed retarded bands with the 32P-labeled IGF-1R probe, which could be supershifted by anti-ERalpha antibody (data not shown). However, neither in vitro synthesized or purified ER bound the IGF-1R promoter probe, whereas SP1 and NFkappa B proteins formed a retarded band complex (data not shown). Interestingly, the intensity of the two main SP1-dependent bands was significantly reduced by co-incubation with in vitro synthesized ER or purified ER (Fig. 4B), whereas both ER preparations had no effect on the NFkappa B-dependent band (data not shown). Although ER protein diminished the SP1/probe band, in vitro translated PRB or HE82, human ERalpha carrying a mutation in the DNA binding domain, had no effect, indicating not only an ER-specific effect but also an effect dependent on a conserved ER DNA binding domain (Fig. 4B).



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  		Fig. 4.   Recombinant ERalpha does not bind to IGF-1R promoter sequence but reduces SP1 binding in EMSA. A, in vitro translated ER (HEG0) was allowed to interact with a 35-bp ERE DNA probe in the presence of 200× excess of unlabeled oligonucleotide (lane 3), anti-ER antiserum (lanes 4 and 5), or control IgG (lane 6). B, recombinant ERalpha , in vitro translated HEG0, pSG5/hPR, or HE82 were allowed to interact with a 0.497-kilobase DNA fragment from the IGF-1R promoter in the presence or absence of SP1 protein and analyzed by electrophoresis on a 6% polyacrylamide gel.

E2-induced Decrease in IGF-1R Transcription Is Blunted by Overexpressing SP1-- Because recent studies have demonstrated that physical and functional interactions exist between ERalpha and the transcription factor SP1 (50-54) and because we found a decrease of SP1 binding to the IGF-1R promoter in the presence of ER, we tested the possibility that the repression of IGF-1R transcription by E2 may be due to ER-SP1 protein-protein interaction. Indeed, transient overexpression of SP1 blunted the ER/E2-induced decrease in IGF-1R transcription using HeLa cells (Fig. 5).



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  		Fig. 5.   Overexpression of SP1 blunts the repressor effect of E2 on IGF-1R transcription. HeLa cells were transfected with p(-476/+640-Luc) IGF-1R promoter construct, HEG0, and increasing doses of pCMV-SP1. Total amount of transfected DNA was kept constant by adding empty pCMV vector DNA. After transfection, cells were then stimulated with 100 nM E2 for 12 h. Mean ± S.E. of five independent experiments.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Premenopausal women have less coronary artery disease than do men. However, the incidence of the disease rises markedly after menopause, and hormone replacement therapy may reduce the risks to premenopausal levels (55-58). Until recently, the atheroprotective effects of estrogen were attributed principally to the hormone's effects on serum lipid concentrations. However, it is now evident that E2 has other vasoprotective effects, such as increasing vasodilation via stimulation of nitric oxide synthase (59), decreasing plasma concentrations of renin and ACE (60), and decreasing vascular expression of the Ang II AT1 receptor gene (61). These vascular effects of estradiol are likely to play an important role in the atheroprotective effects of the hormone. In addition our studies demonstrate that 17beta -estradiol modulates IGF-1 and IGF-1R mRNA and protein levels in RASMC. Although previous reports have shown that estrogen may alter expression of IGFBPs and IGF-1R mRNA and IGF-1 binding sites in human breast cancer cells (33, 62, 63), we provide the first evidence that estradiol inhibits IGF-1 and IGF-1R expression in vascular smooth muscle cells. Together with the inhibiting effect of estrogen on vascular smooth muscle cell proliferation, these findings suggest a possible mechanism for the observation that estradiol has antiatherogenic properties in vivo and in vitro.

Because IGF-1 is an important migration factor and mitogen for smooth muscle cells (64-66) and because IGF-1 expression is increased after balloon injury (67) and in atherosclerotic lesions (68), it was of interest to us to study the effects of estradiol on IGF-1 and IGF-1R expression in vascular smooth muscle cells. Initially, we focused on transient transfection assays using IGF-1R promoter constructs (35). HeLa cells were initially chosen because of their lack of ER. Thus, these cells provided a useful model in which to study ER-independent and ER-dependent effects on the IGF-1R promoter. Our results show that E2 diminished IGF-1R transcription via an ER-dependent pathway. Similar results were also found in COS-1 cells (data not shown). Not only was ER required for the repressor effect of E2 on IGF-1R transcriptional activity, but the effect was also specific for E2, because the antiestrogen ICI 164,384 completely abrogated the repression and progesterone had no effect, in the presence of PRB, on IGF-1R transcription. Further evidence that a functional ER was necessary for the transrepression of E2 on the IGF-1R promoter, was provided by studies using the mutant ER construct HE82. This mutant consist of a wild-type ER in which three amino acids in the first zinc finger have been replaced by the equivalent amino acids of the first zinc finger of the glucocorticoid receptor changing the DNA binding specificity of HE82 to that of the glucocorticoid receptor (38).

In agreement with the above described findings, E2 reduced IGF-1R transcription in RASMC, which do express endogenous ER. Also E2 dose-dependently decreased IGF-1R mRNA and protein when compared with control. We have previously shown that small changes in the number of IGF-1R have major effects on cell growth (69). Thus, this reduction in IGF-1R may be physiologically relevant and may explain the decrease in DNA synthesis observed.

Little information is available regarding the relationship between E2 and IGF-1 in vascular smooth muscle cells. Most studies have focused on the reproductive organs and other estrogen-sensitive cells. In these cells E2 is often related to an increase in IGF-1 and IGF-1R signaling by sensitizing cells to the mitogenic effects of IGF-1 as shown in breast cancer cells (62) and in vivo in the uterus (70, 71). However, in RASMC we observed a reduction in IGF-1 mRNA and protein expression induced by E2. This is in agreement with reports where oral E2 replacement therapy in postmenopausal women induced a marked decrease in serum IGF-1 levels (72, 73). The observed decrease in IGF-1 mRNA is also in agreement with the study of McCarthy et al. (32), using primary rat osteoblasts, however, it is in contrast to the reports from Ernst and Rodan (74). Interestingly, OHT further potentiated the depressor effect of E2 on IGF-1 and IGF-1R mRNA in RASMC and in transient transfection studies using HeLa cells, whereas ICI blunted the E2 response. The finding that OHT similarly to E2 decreased IGF-1 and IGF-1R mRNA expression is in good agreement with reports that OHT can act as a partial agonist of ER, depending on cell context (75). However, E2 clearly down-regulated IGF-1 and IGF-1R protein expression, consistent with its effect on IGF-1 and IGF-1R mRNA levels. Because IGF-1 is a potent mitogen, the decrease in IGF-1 and IGF-1R could at least partially explain the inhibitory effect of E2 on DNA synthesis. Thus, although exogenous IGF-1 failed to reverse the inhibitory effect of E2 on DNA synthesis, this is quite possibly due to the persistent reduction in IGF-1R. Indeed, we have previously shown that IGF-1R density is a critical determinant of vascular smooth muscle cell growth responses (39).

Bioactivity of IGF-1 is modulated by several high affinity binding proteins (IGFBPs) present in the vasculature (76, 77). These IGFBPs control the distribution of IGF-1 between extracellular and cellular compartments and can also alter IGF-1 bioactivity by modulating its interaction with its receptor (78). The major two IGFBPs found to be secreted by RASMC were IGFBP-2 and IGFBP-4 (25). However, E2 had no effect on IGFBP secretion when compared with control, suggesting that the E2 inhibitory response in DNA synthesis is rather related to its depressor effect on IGF-1 and IGF-1R expression than on the expression of inhibitory binding proteins (data not shown).

ER-mediated transactivation is a complex process regulated by ligand-dependent or ligand-independent mechanisms (reviewed in Ref. 79) and by interactions with coactivators and/or co-repressors, by binding directly to various DNA elements or by indirectly enhancing DNA binding via protein-protein interaction (80). In our studies, ER-dependent repression of the IGF-1R promoter, unlike transactivation of ERE-containing reporter and EMSA probe by the same combination, did not appear to be mediated via high affinity binding of the ER to the IGF-1R promoter probe, because no direct binding of purified ER alone to the IGF-1R promoter was observed. Instead, our results suggested that ER inhibited SP1 binding to the IGF-1R promoter. This would suggest that protein-protein interactions between ER and SP are responsible for the inhibitory action of ER. Indeed, the IGF-1R promoter contains putative consensus sequences for SP1 (81) but also other regulatory elements like Egr-1 (82), AP-2 (83), platelet-derived growth factor-responsive element (84). This is somewhat in contrast to the previous finding of Porter et al. (51), who described an enhancement of SP1 binding to SP1 consensus sequence by co-incubating with ER protein. However, this could likely be promoter- and cell-specific. To confirm the inhibitory effect of ER on SP1 binding to IGF-1R promoter, we transiently overexpressed SP1 and showed that this blocked the ability of E2 to decrease IGF-1R transcription. In addition, an intact DNA binding domain of ER was required for the effects of SP1 binding to the IGF-1R promoter, even if direct binding of ER to the same promoter was not detected. The 3-amino acid mutation within the DNA binding domain of HE82 may thus be sufficient to prevent interaction with SP1 bound to the IGF-1R promoter.

Interactions between ligand-activated steroid hormone receptors and specific genomic sequences are well described and are believed to be the dominant mechanism whereby this class of hormones exerts its molecular effects (27). ERs preferentially bind to a 5-bp palindromic DNA sequence, the ERE. This element can be sufficient to enhance transcription, as can "imperfect" or half-EREs, which can be located a great distance from the transcription start site (85). Although traditionally thought to be enhancing, there are accumulating reports of ER negatively affecting transcription (29, 86, 87). As does ours, these reports speculate about ERE interactions. Ray et al. (86), studied the negative effect of E2 on IL-1-mediated IL-6 gene activation. Although inhibition involved the ER, high affinity binding of ER to the IL-6 promoter could not be demonstrated nor did recombinant ER bind to the promoter fragment in gel mobility shift assays. Likewise, there was no binding observed between ER and the promoter 1 of the rat IGF-1 gene (32). This could be explained by an interaction between ligand-activated ER and other trans-activating factors, as has been described for the transcription factor AP1 (88-90), SP1 (50-52, 91), and NFkB (86, 92, 93). It appears that in the absence of obvious and typical EREs in the promoters of negatively regulated target genes, the ER may function as repressor by antagonizing the activity of positively regulating transcription factors without direct ER-DNA contacts (29, 86).

Recently, a second ER, ERbeta , has been discovered (44, 45). Although ERalpha and ERbeta share a high degree of identity in their ligand binding and DNA binding domain, and although both have similar affinities for E2 and recognize the same consensus ERE, they do respond differentially to partial agonist antiestrogens in transactivation assays (44, 45, 94, 95). In addition, ERbeta has biological roles that are distinct from those of ERalpha as evidenced with the different phenotypes of the alpha ERKO and beta ERKO mice (96, 97). Expression of ERalpha has been reported to increase in rabbit cardiac allografts (98), whereas ERbeta is up-regulated, but not ERalpha , in rat endothelial cells after carotid artery injury (99). Our results showing that only ERalpha was able to repress IGF-1R transcription in HeLa cells, are among the first to show a differential effect of E2 via ERalpha or ERbeta . Indeed, Paech et al. (89), have shown a differential ligand activation of ERalpha and ERbeta at AP1 sites. However, at this point we cannot rule out that ERalpha /ERbeta heterodimers are involved in the repression of IGF-1 or IGF-1R in RASMC, because ERbeta transcripts have been found in these cells (41, 42, 44).

In summary, our results demonstrate that E2 inhibits IGF-1R and IGF-1 mRNA and protein expression in vascular smooth muscle cells. This decrease may explain the inhibitory effect of E2 on DNA synthesis and its antiproliferative effects on vascular cells and may, thus, offer one mechanism by which estradiol retards atherosclerosis in premenopausal women.


    ACKNOWLEDGEMENTS

We thank Drs. K. Griendling, R. Tjian, H. Werner, P. Chambon, S. Mader, J.-Å. Gustafsson, S. Shimasaki, and A. Wakeling for the generous gift of materials.


    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL47035 and HL45317, the Canton de Genève, the Swiss National Science Foundation (grant FNSR3100-050799.97), the Swiss Cardiology Foundation, and the Gerbex-Bourget Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Patrick Delafontaine, Div. of Cardiology, University Hospital of Geneva, Rue Micheli-du-Crest 24, 1211 Geneva 14, Switzerland. Tel.: 41-22-372-7192; Fax: 41-22-382-7245; E-mail: Patrice.Delafontaine@hcuge.ch.

Published, JBC Papers in Press, September 11, 2000, DOI 10.1074/jbc.M004691200


    ABBREVIATIONS

The abbreviations used are: IGF-1, insulin-like growth factor-1; IGF-1R, insulin-like growth factor-1 receptor; RASMC, rat aortic smooth muscle cells; ER, estrogen receptor; E2, 17beta -estradiol; PRB, progesterone receptor B; DCT, dextran-coated charcoal-treated; OHT, 4-hydroxytamoxifen; ICI, ICI 164,384; IGFBP, insulin-like growth factor binding protein; ERE, estrogen response element; EMSA, electrophoretic mobility shift assay; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); IL-1, interleukin 1.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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An Activator Protein 1-Like Motif Mediates 17{beta}-Estradiol Repression of Gonadotropin-Releasing Hormone Receptor Promoter via an Estrogen Receptor {alpha}-Dependent Mechanism in Ovarian and Breast Cancer Cells
Mol. Endocrinol., December 1, 2003; 17(12): 2613 - 2629.
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 EndocrinologyHome page
S. Zaina, L. Pettersson, A. B. Thomsen, C.-M. Chai, Z. Qi, J. Thyberg, and J. Nilsson
Shortened Life Span, Bradycardia, and Hypotension in Mice with Targeted Expression of an Igf2 Transgene in Smooth Muscle Cells
Endocrinology, June 1, 2003; 144(6): 2695 - 2703.
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 J. Biol. Chem.Home page
D. Kressler, S. N. Schreiber, D. Knutti, and A. Kralli
The PGC-1-related Protein PERC Is a Selective Coactivator of Estrogen Receptor alpha
J. Biol. Chem., April 12, 2002; 277(16): 13918 - 13925.
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 J. Appl. Physiol.Home page
R. V. Sharma, M. V. Gurjar, and R. C. Bhalla
Genome and Hormones: Gender Differences in Physiology: Selected Contribution: Estrogen receptor-alpha gene transfer inhibits proliferation and NF-kappa B activation in VSM cells from female rats
J Appl Physiol, November 1, 2001; 91(5): 2400 - 2406.
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