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(Circulation. 1998;98:413-421.)
© 1998 American Heart Association, Inc.

Clinical Investigation and Reports

Estrogen Stimulates Delayed Mitogen-Activated Protein Kinase Activity in Human Endothelial Cells via an Autocrine Loop That Involves Basic Fibroblast Growth Factor

Seunghee Kim-Schulze, PhD; William L. Lowe, Jr, MD; ; H. William Schnaper, MD

From the Division of Nephrology, Department of Pediatrics, Northwestern University Medical School and the Children's Memorial Institute for Education and Research (S.K.-S., H.W.S.), and the Department of Medicine (W.L.L.), VA Chicago Healthcare System, Northwestern University Medical School, Chicago, Ill.

Correspondence to Seunghee Kim-Schulze, PhD, Pediatrics W-140, 303 E Chicago Ave, Chicago, IL 60611-3008. E-mail ski057{at}nwu.edu

	Abstract

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Background—Estrogen plays a significant role in protecting premenopausal women from cardiovascular disease. We have found that estradiol augments endothelial cell activities related to vascular healing and that human coronary artery and umbilical vein endothelial cells express estrogen receptors (ERs). Classically, the ER functions as a transcription factor, but the cytoplasmic targets of this genomic effect have not been defined for endothelial cells. In the present study, we examined the potential role of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 as mediators of estrogen action.

Methods and Results—Human umbilical vein endothelial cells were estrogen depleted by culturing in hormone-free medium for 48 hours before experiments. 17ß-Estradiol (E2) stimulated a delayed (3 hours) 5- to 7-fold induction of ERK1/2 activity requiring activation of ER and new transcription/translation. Conditioned media from cells stimulated for 3 hours with E2 induced immediate ERK1/2 activation and phosphorylation of the basic fibroblast growth factor (bFGF) receptor. Moreover, ERK1/2 activation by E2 or by conditioned media was abrogated by treatment with neutralizing anti-bFGF antibody.

Conclusions—These data describe an autocrine mechanism for E2 induction of ERK1/2 in HUVEC. Because our previous studies suggested that certain cardioprotective effects of estrogen are genomic in nature, the results are consistent with the hypothesis that autocrine stimulation of endothelial ERK1/2 activity by bFGF may play a role in the beneficial effects of estrogen on cardiovascular biology.

Key Words: fibroblast growth factor, basic • endothelium • signal transduction • p 42 (MAP K) kinase

	Introduction

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Estrogen appears to play an important role in vascular regulation, protecting premenopausal women from cardiovascular disease.^{1 2 3} This gonadal steroid directly affects blood vessels by increasing the secretion of endothelium-derived relaxing factor (nitric oxide).^{4 5} Estradiol also enhances endothelial cell attachment, proliferation, migration, and differentiation in vitro and markedly increases the angiogenic effect of bFGF in mice in vivo.⁶ Moreover, it enhances cytokine-stimulated adhesion molecule and integrin expression.^{7 8} These effects, combined with differing effects on vascular smooth muscle cells, could promote vessel wall healing and thereby decrease atherosclerosis.⁹ The cardioprotective effects are likely mediated through an ER. Several investigators have described ERs in vascular smooth muscle cells^10,11; we and others have identified a functional ER in cultured human endothelial cells.^{12 13}

Classically, ERs are intracellular receptors that serve as transcription factors. In many cases, the activated ER binds to an estrogen response element, leading to altered target gene expression.^{14 15} Evidence from in vitro and in vivo studies strongly implicates estrogen as a modulator of GFs and their receptors. Estrogen regulates cell growth and the secretion of polypeptide GFs in human breast carcinomas.¹⁶ Estrogen also induces high levels of bFGF mRNA in the rat ovary and induces epidermal GF mRNA, precursor protein, and receptor in the mouse uterus. The actions of ER and GFs converge in a synergistic manner via a mechanism that is poorly understood.^{17 18 19 20} ER activation can induce expression of other proteins that interact with and/or activate GFs, GF receptors, or GF signaling pathways.²¹ Conversely, activation of intracellular kinases of the epidermal GF signal transduction pathway can phosphorylate ER and change its activity.²²

In our previous studies,^{6 8} we found that estrogen augments a variety of endothelial cell functions rather than directly stimulating these cell activities. Moreover, studies by others²³ indicate that ER may play an important role in bFGF-stimulated angiogenesis in vivo. These observations suggest that estrogen facilitates or amplifies endothelial responses to cytokines and GFs. We therefore examined the effect of E2 on MAP kinase activity. MAP kinases are central participants in a signaling cascade that is ubiquitous and is used by many GFs and peptide hormones to regulate physiological responses.^{24 25}

Herein, we describe the ability of estrogen to increase the activity of the MAP kinases, ERK1^mapk44 and ERK2^mapk42. Estrogen treatment induced a delayed (3 hours) increase in ERK1/2 activity by a mechanism that requires ER and de novo protein synthesis, suggesting a classic genomic effect. In contrast, media conditioned by estrogen-treated cells rapidly activated ERK1/2. Induction of ERK1/2 activity by E2 or by conditioned media correlates with bFGF receptor phosphorylation. Furthermore, both ERK1/2 activation and bFGF receptor phosphorylation are decreased by treatment with neutralizing anti-bFGF antibody. These results suggest the existence of an extracellular bFGF autocrine loop for ERK1/2 activation that is upregulated by ER activation.

	Methods

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Growth Factors, Antibodies, and Other Reagents
Recombinant human bFGF and polyclonal anti–human bFGF neutralizing antibodies were purchased from R&D Systems. Neutralizing activity of anti-bFGF antibody was confirmed by its ability to inhibit bFGF-stimulated HUVEC proliferation (data not shown). Monoclonal anti–bFGF receptor antibody was purchased from Oncogene Research. Rabbit anti-ERK1^mapk44 (K-23) and -ERK2^mapk42 (C-14) antibodies were purchased from Santa Cruz Biotechnology Inc. Monoclonal anti-phosphotyrosine antibody (IgG_bk) was purchased from Upstate Biotechnology, Inc. ICI 182,780 was kindly provided by Zeneca Pharmaceuticals (Macclesfield, England). Heparin sephadex (6B) was purchased from Pharmacia. E2, actinomycin D, cycloheximide, MBP, EDTA, EGTA, ß-glycerophosphate, aprotinin, leupeptin, pepstatin A, PMSF, sodium orthovanadate, and protein A-sepharose were purchased from Sigma Chemical Co. P-81 phosphocellulose filter paper was purchased from Whatman. [-³³P]ATP (5000 Ci/mL) was obtained from Amersham Corp. Prestained molecular weight standards for polyacrylamide gels were purchased from Bio-Rad.

Cell Culture
We isolated HUVEC from anonymous pathological specimens by collagenase digestion using standard techniques.²⁶ Cultured cells stained positively for von Willebrand factor and were negative for -smooth muscle actin and mycoplasma. They were grown in a standard cell culture medium consisting of RPMI 1640 medium (Gibco BRL) supplemented with 20% bovine calf serum (Hyclone Laboratory Inc), 100 U/mL penicillin/streptomycin, 50 µg/mL gentamycin, 2 mmol/L glutamine, 5 U/mL heparin, and 200 µg/mL endothelial cell growth supplement (Collaborative Research) and used between passages 3 and 7. Breast cancer cell lines MCF-7 (ER-positive) and MDA-MB-231 (ER-negative), provided by S. Rosen (Lurie Cancer Center, Northwestern University, Chicago, Ill), were grown in medium consisting of RPMI 1640 supplemented with 10% FCS and 2 mmol/L glutamine.

Cell Stimulation and Preparation of Cell-Free Extract
For experiments, cells were estrogen depleted for 48 hours by switching to phenol red–free RPMI 1640 medium containing 20% charcoal-stripped serum, followed by growth arrest by incubation for 24 hours in RPMI 1640 containing 2% hormone-free fetal bovine serum. The absence of phenol red avoids potential estrogen-like effects of this compound.²⁷ Cells were then switched to RPMI 1640 containing only 1% BSA before being stimulated with E2. Cells were washed twice with PBS before being lysed on ice in lysis buffer (20 mmol/L Tris HCl, pH 7.5, 10 mmol/L EGTA, 60 mmol/L ß-glycerophosphate, pH 7.3, 10 mmol/L MgCl₂, 1% Triton X-100, 1 mmol/L Na₃VO₄, 2 mmol/L DTT, 1 mmol/L PMSF, and a mixture of protease inhibitors [10 µg/mL leupeptin, 10 µg/mL pepstatin, and 2 µg/mL aprotinin]). After transfer to microcentrifuge tubes, cell lysates were spun at 10 000g for 20 minutes at 4°C. Supernatant was collected, and protein content was determined by Bradford protein assay.²⁸

Immunoprecipitation and In Vitro Kinase Assay of ERK1/2
Immune complex kinase reactions were performed as previously described with minor modifications.²⁹ Cells were grown and treated as described above. Hormone-depleted cells were treated with either E2 or GFs for the indicated time periods. Cells were then washed and lysed. For immunoprecipitation of MAP kinase, equal aliquots of 1 to 2 mg of total cellular protein were incubated at 4°C for 2 hours with 25 µL of protein A sepharose (50% slurry with lysis buffer, 50 mmol/L ß-glycerophosphate, pH 7.3, 1.5 mmol/L EGTA, 0.1 mmol/L Na₃VO₄, 1 mmol/L DTT, and mixture of protease inhibitors) and 2.5 µg/mL of a mixture (50:50) of anti-ERK1^mapk44 and -ERK2^mapk42 antibodies. Immunoprecipitates were washed twice with a solution containing 20 mmol/L Tris-HCl, pH 7.5, 500 mmol/L NaCl, and 2 mmol/L DTT and twice with kinase buffer (50 mmol/L ß-glycerophosphate, pH 7.3, 5 mmol/L MgCl₂, 2.5 mmol/L EGTA, 1 mmol/L DTT, 0.2 mmol/L Na₃VO₄, mixture of protease inhibitors, and 0.1 mmol/L ATP). We measured ERK1/2 activity by performing an in vitro kinase reaction with immunoprecipitated MAP kinase, using MBP (12.5 µg/50 µL reaction) as the substrate. The reactions contained 10 µCi of [-³³P]ATP (specific activity 2000 cpm/pmol) and were carried out at 30°C for 10 minutes. The reaction was stopped by the addition of 15 µL 2x SDS sample buffer (100 mmol/L Tris, pH 6.8, 200 mmol/L DTT, 4% SDS, 0.2% bromophenol blue, and 20% glycerol), and the mixture was boiled for 5 minutes. Samples were then resolved by 13% SDS-PAGE. The gels were dried and exposed to x-ray film. Quantification of phosphorylated bands (MBP) on the autoradiographs was performed by use of densitometric scanning analysis. Simultaneously, an aliquot of the assay mixture was spotted on P-81 phosphocellulose filter paper, which was subsequently washed with 3 changes of 0.75% phosphoric acid and 2 changes of 100% acetone. The amount of radioactivity retained on the P-81 paper was determined by liquid scintillation counting. As controls, several reactions were simultaneously performed without immunoprecipitated ERK1/2 or MBP. Final ERK1/2 activity was determined by correction for control reactions.

Immunoblot Analysis
We assayed the expression of ERK1/2 by immunoblotting immunoprecipitated ERK1/2 using standard methods with a mixture of anti-ERK1 and -ERK2 antibodies (1:2000) or with anti-phosphotyrosine antibody (1:1000) to detect tyrosine phosphorylated ERK1/2. bFGF expression and its receptor tyrosine phosphorylation were analyzed with anti-bFGF antibody (1:500) and with anti-phosphotyrosine antibody (1:1000). Immune complexes were visualized by the enhanced chemiluminescence Western blotting procedure (ECL; Amersham, Inc). The reaction was examined in the linear range of its development as suggested by the manufacturer. Quantification was achieved by densitometric analysis of the results obtained from 3 separate experiments.

Heparin-Sepharose Chromatography for Purification of bFGF in E2-Treated Conditioned Media
Media conditioned by E2-treated HUVEC or by control cells, along with the cell lysates, were centrifuged at 43 000g for 45 minutes at 4°C to collect a supernatant that was free of cell debris or protein aggregates. Subsequent steps in the isolation were also performed at 4°C. The cleared sample was applied to a 8x2.5-cm heparin-sephadex (6B) column equilibrated with 50 mmol/L phosphate buffer, pH 7.5, containing 150 mmol/L NaCl and 1 mmol/L DTT. The bound proteins were eluted with increasing concentrations of NaCl (0.5 to 3 mol/L) in phosphate buffer. Fractions of 7 mL were collected, and their protein concentrations were determined by Bradford assay.²⁸ The 5 fractions containing protein from the chromatography of each sample were pooled and dialyzed against 20 mmol/L Tris buffer, pH 7.5, containing 1 mmol/L DTT, 100 µmol/L PMSF, and 0.01% sodium azide. The dialysate was concentrated 100-fold on ultrafiltration membranes (YM10; 10 000 M_r exclusion; Amicon Inc), and final protein concentrations were determined by Bradford assay. Aliquots of each elution were subjected to immunodetection of bFGF by Western blot.

Ribonuclease Protection Assay
Total cellular RNA was isolated by homogenization in 4 mol/L guanidinium isothiocyanate, 25 mmol/L sodium acetate, pH 5.2, with 10 mmol/L ß-mercaptoethanol.³⁰ Fluid-phase hybridization was performed by use of a Ribonuclease Protection Assay Kit II (Ambion) according to the manufacturer's instructions. Homogeneously [³²P]-labeled antisense cRNA probes were synthesized in vitro from pbFGF (a 488-bp segment of bFGF cDNA sequence), kindly provided by Volkhard Lindner, Maine Medical Center Research Institute, South Portland, Maine, and from pActin (a 250-bp segment of ß-actin cDNA sequence) with the use of T7 polymerase. The ³²P-labeled antisense RNAs (specific activity >1.0x10⁸ cpm/µL) were hybridized with 50 µg of total RNA at 42°C for 48 hours. Samples were subjected to RNase digestion at 37°C for 30 minutes. They were then electrophoresed on an 8 mol/L urea, 5% polyacrylamide gel at 200 V and visualized by autoradiography.

	Results

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Estradiol Activates ERK1/2 in HUVEC
To determine whether E2 activates ERK1/2 in HUVEC, cells were treated with 3 nmol/L E2, and ERK1/2 was immunoprecipitated from cell-free extracts for an in vitro kinase assay with MBP used as a substrate. This dose of estradiol was chosen because it had optimal effects on assays of biological function of endothelial cells.^{6 12} Estrogen stimulated a gradual increase in ERK1/2 activity, with a peak at 3 hours, as reflected by increased phosphorylation of MBP (Figure 1A, top). By 5 hours, ERK1/2 activity was decreased to 20% of the maximum induction observed at 3 hours. In the absence of immunoprecipitated MAP kinase, no ³³P incorporation into MBP was observed (data not shown). ERK1/2 activity was quantified either by densitometric scanning of the autoradiogram shown in Figure 1A or by determining ³³P incorporation into MBP, as measured by its retention on P81 phosphocellulose paper (Figure 1B); results were essentially identical using both methods. ERK1/2 activity was increased 5- to 7-fold over basal activity after 3 hours of E2 treatment. The degree of tyrosine phosphorylation of ERK1/2 also was determined by Western blot analysis (Figure 1A, middle). Phosphorylation of ERK1/2 was most prominent after 3 hours of E2 treatment, demonstrating that ERK1/2 phosphorylation paralleled ERK1/2 activity. In contrast, Western blot analysis revealed that total ERK1/2 protein levels in immunoprecipitated samples were not affected by E2 treatment (Figure 1A, bottom). Similar results were obtained in 5 different experiments regardless of whether cells were derived from male or female donors. In addition, separate immunoprecipitation of ERK1 or ERK2 by use of specific antibodies indicated that each isoform was phosphorylated and activated after the cells were treated with E2 (data not shown). These findings suggest that E2 stimulates a delayed peak of ERK1/2 activity in human endothelial cells by a mechanism that regulates ERK1/2 phosphorylation rather than by increasing protein levels.

View larger version (35K): [in this window] [in a new window]   Figure 1. Time course of E2-stimulated induction of ERK1/2 activity in HUVEC. A, Hormone-depleted cells were treated with E2 (3 nmol/L) for different time periods. Cell-free lysates were used for ERK1/2 immunoprecipitation with a mixture of anti-ERK1 and -ERK2 (K-23 and C-14) antibodies to determine activation of MAP kinase using an in vitro kinase assay and to determine phosphorylation of ERK1/2. Reaction mixtures were resolved by 15% PAGE and revealed by autoradiography. Top, 33P incorporation into MBP by immunoprecipitated ERK1/2. Middle, ERK1/2 immunoprecipitated with a mixture of anti-ERK1 and -ERK2 antibodies and analyzed by Western blot analysis with a monoclonal anti-phosphotyrosine antibody (IgG2bk). Bottom, Expression of ERK1/2 proteins from cell-free lysates was demonstrated by immunoblot using a mixture of anti-ERK1 and -ERK2 antibodies. P indicates phosphorylation. Similar results were obtained in 5 separate experiments using HUVEC derived from either male or female donors. B, Incorporation of 33P into MBP · min-1 · mg-1 protein was determined by scintillation counting of retained radioactivity from phosphorylated protein reaction mix on P-81 phosphocellulose filter paper (n=5, mean±SE). Overall effect of E2 was statistically significant (P<0.0001; 1-way ANOVA). **Significantly different from control cells (P<0.01) by Dunnett post hoc test. *Significantly different from control cells (P<0.05).

Inhibition of ERK1/2 Activation by an ER-Specific Antagonist
To determine whether E2 activation of ERK1/2 is mediated by ERs, cells were treated with the specific ER antagonist ICI 182,780 (10 nmol/L) 1 hour before or simultaneously with E2. Lysates from ICI 182,780–treated cells were analyzed for activity (Figure 2, top). E2 induction of ERK1/2 activity was inhibited completely by the ER antagonist. Consistent with this result, the ER antagonist also prevented E2-stimulated tyrosine phosphorylation of ERK1/2 (Figure 2, middle). In contrast, total ERK1/2 proteins levels were not affected by ICI 182,780 treatment (Figure 2, bottom). These data demonstrate that delayed induction of ERK1/2 activity by E2 is mediated by the ER.

View larger version (71K): [in this window] [in a new window]   Figure 2. ER-specific antagonist inhibits estrogen activation of ERK1/2 in HUVEC. Cells were preincubated in the presence (+) or absence (-) of the specific ER antagonist ICI 182,780 (10 nmol/L) and then treated with E2 for 3 hours. ERK1/2 was immunoprecipitated to determine kinase activity (top). ERK1/2 from cell-free lysates was analyzed with an anti-phosphotyrosine antibody by Western blot analysis (middle). Expression of ERK1/2 proteins in cell-free lysates was analyzed with a mixture of anti-ERK1 and -ERK2 antibodies (bottom). These results are representative of 4 independent experiments.

Different Kinetics of E2-Induced ERK1/2 Activity in MCF-7
Because an ER antagonist inhibits E2-stimulated ERK1/2 activity in human endothelial cells, the human ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cell lines were used to determine more directly whether ER mediates the E2 effect on ERK1/2 activation. Results of a representative experiment are shown as an autoradiogram of ³³P-phosphorylated MBP (Figure 3A) and graphic representation of the mean data obtained from the densitometric scanning of the autoradiographic films from 3 independent experiments (Figure 3B). For comparison, induction of ERK1/2 activity by E2 in HUVEC is also shown. The timing of ERK1/2 activation by E2 in MCF-7 cells was strikingly different from that in endothelial cells. Whereas induction of ERK1/2 activity in HUVEC peaked at 3 hours, E2 stimulated ERK1/2 activity in MCF-7 cells as early as 2 minutes after E2 treatment; activity was maximal at 5 minutes (9-fold induction) as measured by ³³P-incorporation into MBP. Activation of ERK1/2 in MCF-7 cells was transient and returned to near-basal levels after 30 minutes. Of note, in 50% of experiments, a small increase in HUVEC ERK1/2 activity was seen at 2 minutes. This immediate increase was not blocked by ICI 182,780, indicating that the effect was not mediated by ER. Moreover, because a similar increase was seen in some experiments in which HUVEC were treated with control vehicle (data not shown), this small, early increase was not specific for E2. Rapid induction of ERK1/2 activity in MCF-7 cells was prevented by the specific ER antagonist ICI 182,780. A slight induction of ERK1/2 activity at 5 to 30 minutes that was apparent in MCF-7 cells, despite ICI 182,780 treatment, was significantly lower than induction without the inhibitor and was not seen in all experiments. The ER-negative MDA-MB-231 cell line showed neither an immediate or delayed increase in ERK1/2 activity after E2 treatment. Immediate induction of ERK1/2 by E2 in a breast cancer cell line that expresses ER is consistent with the data of others³¹ and is in marked contrast to the delayed induction seen in HUVEC.

View larger version (36K): [in this window] [in a new window]   Figure 3. Comparison of time course for induction of ERK1/2 activity by E2 in HUVEC and breast cancer cell lines. Cells were treated with E2 (1 ng/mL) for time periods noted. Cell-free lysates were used for ERK1/2 immunoprecipitation with anti-ERK1 and -ERK2 antibodies to determine ERK1/2 kinase activity by phosphorylation of MBP. A, Autoradiograms of ERK1/2 activity in HUVEC, MCF-7 cells, MCF-7 cells treated with ICI 182,780 (10 nmol/L), and MDA-MB-231 cells. B, Graphic representation of data obtained by quantitative densitometric scanning of autoradiographic signals for ERK1/2 activity obtained in 3 independent experiments. indicates HUVEC; , MCF-7; , MCF-7 cells treated with ICI 182,780; and , MDA-MB-231.

New Transcription and Translation Are Required for E2 Activation of ERK1/2
Delayed activation of ERK1/2 by E2 in endothelial cells suggested an indirect effect of E2 on ERK1/2 activity. A possible explanation for this delay is a requirement for new protein synthesis. To evaluate whether ERK1/2 induction requires mRNA and/or protein synthesis, hormone-depleted cells were incubated with 3 µg/mL actinomycin D or 100 µmol/L cycloheximide. Pretreatment of cells with actinomycin D blocked both E2-induced ERK1/2 activity (Figure 4A, top) and tyrosine phosphorylation of ERK1/2 (middle). The total amount of ERK1/2, however, was not affected by actinomycin D treatment (bottom). Pretreatment with 100 µmol/L cycloheximide 1 hour before treatment with 3 nmol/L E2 completely suppressed stimulation of ERK1/2 activity by E2 (Figure 4B, top) with much less inhibition when cycloheximide was added simultaneously or 1 hour after E2 treatment. Again, ERK1/2 protein levels were unchanged by cycloheximide (Figure 4B, bottom), indicating that the increase in ERK1/2 activity was not secondary to increased ERK1/2 synthesis. These data demonstrate that E2 activates ERK1/2 indirectly through a mechanism that requires new mRNA and protein synthesis.

View larger version (53K): [in this window] [in a new window]   Figure 4. Actinomycin D (Act D; A) and cycloheximide (CHX; B) inhibit activation of ERK1/2 by E2. A, Cells were incubated with 3 µg/mL actinomycin D for 1 hour before E2 treatment for indicated time periods, and ERK1/2 was immunoprecipitated with a mixture of anti-ERK1 and -2 antibodies. ERK1/2 activity was determined by phosphorylation of MBP (top). Immunoprecipitated ERK1/2 was analyzed by Western blot analysis using either anti-phosphotyrosine antibody (middle) or a mixture of anti-ERK1 and -ERK2 antibodies (bottom). B, Cells were treated with 100 µmol/L CHX 1 hour before (T-1), simultaneously with (T0), or 1 hour after (T+1) addition of E2 (+) or control vehicle (-). After 3 hours, ERK1/2 was immunoprecipitated with a mixture of anti-ERK1 and -ERK2 antibodies, and ERK1/2 activity was determined by phosphorylation of MBP (top). Expression of ERK1/2 proteins was analyzed by Western blot analysis using a mixture of anti-ERK1 and -ERK2 antibodies (bottom). Results are representative of 3 independent experiments.

Time- and Concentration-Dependent Activation of ERK1/2 by Media Conditioned by E2-Stimulated HUVEC
To determine whether E2 stimulates release of factor(s) responsible for the delayed induction of ERK1/2 activity, conditioned media from hormone-depleted cells (control) or from E2-stimulated cells were added to other hormone-depleted cells. Medium harvested from endothelial cells treated with E2 for 3 hours induced ERK1/2 activity within 5 minutes (Figure 5A), suggesting a relatively direct mechanism of activation. ERK1/2 activity returned to basal levels by 1 to 2 hours (data not shown). In contrast, cells treated with control conditioned medium did not show induction of ERK1/2 activity. To determine whether rapid ERK1/2 activation was concentration dependent, cells were treated with various concentrations of conditioned medium for 30 minutes (Figure 5B). ERK1/2 activity was maximal in cultures treated with 100% conditioned medium and declined proportionally with decreasing concentrations of conditioned medium added to the culture. These results support the hypothesis that E2 stimulates the release of an autocrine factor that mediates ERK1/2 activity.

View larger version (25K): [in this window] [in a new window]   Figure 5. Time- and concentration-dependent activation of ERK1/2 by conditioned media (CM) from HUVEC cells stimulated with E2 for 3 hours. A, Conditioned medium samples were obtained from cells cultured without (-E2 CM) or with E2 (+E2 CM) for 3 hours. These samples were then added to hormone-depleted cells for indicated time periods (5 to 30 minutes). ERK1/2 was immunoprecipitated with a mixture of anti-ERK1 and -ERK2 antibodies, and kinase activity was determined by phosphorylation of MBP. First 2 columns (-E2, +E2) represent ERK1/2 activity from control cells and E2-stimulated cells after 3 hours, respectively. Inset shows autoradiogram depicting MAP kinase activity in 1 experiment, from which data were taken. Lanes are numbered in the same order as the treatments are shown in the graph. Bars represent mean±SD of ERK1/2 activity from 3 independent experiments. Differences in MAP kinase activity comparing all groups treated with CM from E2-treated cells with all groups treated with control CM are statistically significant (P<0.0001 by 1-way ANOVA; followed by Tukey-Kramer post hoc test as follows: -E2/CM 5, 10, and 30 minutes versus +E2/CM 30 minutes, P<0.001, and -E2/CM 5, 10, and 30 minutes versus +E2/CM 10 minutes, P<0.05). B, Hormone-depleted cells were treated for 30 minutes with decreasing amounts of CM collected from cells treated with E2 for 3 hours in serum-free condition. ERK1/2 was immunoprecipitated with a mixture of anti-ERK1 and -ERK2 antibodies, and its kinase activity was determined by phosphorylation of MBP. First 2 columns (-E2, +E2) represent ERK1/2 activity from control cells and E2-stimulated cells after 3 hours, respectively. Differences in MAP kinase activity in all CM-treated cells are statistically significant (P<0.0001 by 1-way ANOVA; followed by Tukey-Kramer post hoc test comparing 100% CM-treated cells with all others, P<0.001).

Phosphorylation of bFGF Receptor by E2 Treatment
Several autocrine GFs could be involved in the delayed induction of ERK1/2 activity. A likely candidate is the endothelial cell mitogen and angiogenic factor bFGF. Preincubation of cells with neutralizing anti-bFGF antibody prevented ERK1/2 activity induction by E2, suggesting that bFGF mediates stimulation of ERK1/2 activity by E2 (data not shown). The bFGF receptor is a tyrosine kinase that is activated via ligand-induced autophosphorylation.³² To determine whether ERK1/2 activation correlates with bFGF receptor activation, cells were treated with E2, then bFGF receptors were immunoprecipitated from the cell-free lysates for subsequent Western blot analysis with anti-phosphotyrosine antibody. Maximal tyrosine phosphorylation of the bFGF receptor was seen after treatment with E2 for 3 hours (Figure 6A). Total receptor protein expression levels were not changed by E2 treatment. Thus, peak ERK1/2 activity coincides with peak bFGF receptor phosphorylation in HUVEC.

View larger version (64K): [in this window] [in a new window]   Figure 6. Phosphorylation of bFGF receptor (bFGFR-P) in HUVEC treated with either E2 or E2-stimulated cell conditioned medium. A, Hormone-depleted cells were treated with E2 for indicated time periods. Cells were collected, and bFGF receptor was immunoprecipitated from cell-free lysates with monoclonal anti-bFGF receptor antibody (Ab-1). bFGF receptor was immunoprecipitated, and tyrosine phosphorylation was determined by Western blot analysis using anti-phosphotyrosine antibody. The major band corresponds to an apparent molecular weight of 125 kDa, the size of the bFGF receptor. B, Cells were treated for 2 and 5 minutes with conditioned media (CM) obtained from cells cultured without (-E2 CM) or with (+E2 CM) E2 for 3 hours. First 2 lanes on the left represent analysis of bFGF receptors from control cell lysate and E2-stimulated (3 hours) cell lysate, respectively. Results are representative of 3 independent experiments.

We next investigated whether conditioned medium from E2-treated cells increased bFGF receptor phosphorylation. The conditioned medium induced endothelial cell bFGF receptor tyrosine phosphorylation within 5 minutes (Figure 6B). In contrast, control medium was not able to induce bFGF receptor phosphorylation. These results suggest that E2-treated cells release bFGF, causing their bFGF receptors to be phosphorylated on tyrosine residues.

Neutralizing Antibody Directed Against bFGF Decreases Induction of ERK1/2 Activity by E2-Stimulated Conditioned Medium
Further analysis of a potential autocrine loop involving bFGF in E2-treated HUVEC was accomplished by examining the effect of neutralizing anti-bFGF antibody on the ability of conditioned medium to activate ERK1/2. Conditioned medium incubated with anti-bFGF antibody (10 µg/mL) blocked induction of ERK1/2 activity (Figure 7, lane 4). Furthermore, the antibody inhibited kinase activity stimulated by control medium to which exogenous bFGF was added, confirming that ERK1/2 is activated by bFGF in E2-treated HUVEC (Figure 7, lane 7). In 3 of the 5 experiments we performed, including the one shown in Figure 7, not all of the E2-induced increase in ERK1/2 activity was blocked by the antibody, raising the possibility that >1 GF may contribute to the autocrine effects of the conditioned medium.

View larger version (32K): [in this window] [in a new window]   Figure 7. Neutralizing antibody directed against bFGF blocks induction of ERK1/2 activity by conditioned media from HUVEC stimulated with E2. First 2 lanes represent ERK1/2 activity in control cells (-E2) and cells after stimulation with E2 for 3 hours (+E2), respectively. Cells were treated with conditioned media (CM) obtained from cultures treated either with E2 for 3 hours (+E2 CM) or without E2 (-E2 CM). CM were preincubated with bFGF antibody before treatment of the cells. Inset shows autoradiogram depicting MAP kinase activity in 1 experiment, from which data were taken. Lanes are numbered in the same order as the treatments shown in the graph. Bars represent mean±SD of ERK1/2 activity from 3 independent experiments. Differences in MAP kinase activity comparing E2-treated and control cells and comparing groups with and without anti-bFGF antibody treatment are significant (P<0.005 by Student's t test). bFGF Ab indicates anti-bFGF antibody.

E2 Treatment Increases bFGF Production
To determine whether treatment of cells with E2 increases bFGF mRNA expression, cells were treated with E2 and total cellular RNA was isolated for ribonuclease protection assay. Expression of bFGF mRNA increased within 90 minutes after E2 treatment (Figure 8A). In contrast, no change was detected in expression of ß-actin mRNA. These data demonstrate that E2 increases bFGF production by HUVEC. We determined bFGF protein levels by collecting the cell-free lysate and conditioned media from cells treated with E2 or control vehicle and then subjecting these samples to heparin-affinity chromatography. Heparin-bound proteins were eluted and analyzed by Western blot analysis. The anti-bFGF antibody detected a protein with an apparent molecular weight of 18 kDa in both cell lysate and conditioned medium. Expression was increased 2-fold in cell lysate and 2.5-fold in conditioned medium (Figure 8B). These results indicate that E2 increases endothelial cell bFGF production.

View larger version (51K): [in this window] [in a new window]   Figure 8. E2 upregulates bFGF mRNA and protein in HUVEC. A, Expression of bFGF mRNA by ribonuclease protection assay. Antisense RNAs used in these assays correspond to 488 bases including the 3' end of the coding region and part of the 3' UTR for bFGF and 250 bases of sequence from the ß-actin as a control. RNase-treated bFGF-riboprobe and ß-actin riboprobe were completely digested, whereas undigested bFGF (lane 1) and control (lane 3) antisense RNAs indicate the full-length protected segments. Results were representative of 2 independent experiments. B, Western blot analysis of bFGF purified from cell lysates (CL) and conditioned media from E2-treated cells (CM) by heparin-sepharose chromatography. Cell-free lysate and conditioned media were collected from cells treated with E2 for 0 and 180 minutes in serum-free media and were subjected to heparin-affinity chromatography. Proteins eluted with 2.0 mol/L NaCl were concentrated and analyzed by Western blot using an anti-bFGF antibody. Results are representative of 2 independent experiments.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Previous work from our laboratory^{6 12} indicated that estrogen augments certain endothelial cell activities through a genomic mechanism. The present series of experiments suggests 1 potential action of estrogen. Treatment of HUVEC with E2 results in delayed induction of ERK1/2 activity (peak at 3 hour). This activity is completely inhibited by an ER-specific antagonist, suggesting that the effect of E2 on ERK1/2 activity is mediated by the ER. Actinomycin D or cycloheximide added into culture before E2 treatment inhibits activation, indicating that stimulation of endothelial cell ERK1/2 activity does not represent a direct effect of the ER complex on MAP kinase; rather, it is an indirect effect that requires new protein synthesis. These data are consistent with our previous observation that estrogen accelerates endothelial cell proliferation only after a lag phase, suggesting that time is required for new protein(s) to be synthesized.¹² The findings in the present study indicate that these newly synthesized proteins include bFGF; however, we cannot rule out the possible synthesis of additional proteins that either regulate bFGF production or release or mediate the effect of E2 on MAP kinase activity.

In contrast to delayed ERK1/2 induction in HUVEC, the major peak of E2-induced activation of ERK1/2 in MCF-7 cells occurred at 5 minutes, consistent with a recent report by Migliaccio et al.³¹ This result suggests that the mechanism of ERK1/2 activation in human endothelial cells is distinct from that in MCF-7 cells. In our own studies of breast cancer cells, the effect of E2 on ERK1/2 activity was immediate and was not inhibited by cycloheximide (data not shown). Because induction was inhibited by ICI 182,780, our results suggest a nongenomic mechanism of ER action on MAP kinase signaling pathways in MCF-7 cells. The response in MCF-7 cells reported by Migliaccio et al was demonstrated to occur by a direct interaction of ER with a p21^ras/c-Src–activated protein kinase signal transduction pathway. In some experiments, we noted a small increase in endothelial cell ERK activity just after we changed the medium to add E2 or control vehicle. Because this response is not specific for E2, we suspect that it is related to shear-stress effects on endothelial MAP kinase, as reported previously.^{33 34}

Two ERs have been described. ER-ß is highly homologous to conventional ER- and has similar transcription-regulatory activity.^{35 36} ICI 182,780 blocks both ER- and ER-ß.^{37 38} Therefore, the present studies cannot distinguish whether E2 induces endothelial ERK activity through the or ß receptors.

Conditioned media from E2-treated cells rapidly induced ERK1/2 activity in a time- and concentration-dependent manner. This induction was abolished by preincubation of cells or medium with neutralizing anti-bFGF antibody. Treatment with estradiol induced phosphorylation of bFGF receptor at 3 hours in HUVEC, whereas 3-hour–conditioned medium induced similar phosphorylation in 5 minutes, suggesting that the bFGF receptor was activated via an extracellular autocrine loop. These results support a model in which HUVEC synthesize and release bFGF after exposure to E2. Although it is possible that release of previously produced bFGF could stimulate additional bFGF production,³⁹ our observation that cycloheximide prevents ERK activation suggests that autocrine effects in our model are mediated by newly synthesized bFGF. This hypothesis is further supported by our finding that E2 increased bFGF mRNA levels and, subsequently, the level of bFGF in HUVEC culture. Taken together, our findings describe a system in which E2 treatment of HUVEC results in the synthesis and release of bFGF, phosphorylation of bFGF receptor, and subsequent activation of ERK1/2. These results are consistent with previous studies that demonstrated activation of ERK1/2 by bFGF in several cell types.^{40 41 42 43}

Little is known about the mechanism of bFGF release from cells because all of the isoforms of bFGF lack the leader sequence that is required for release through the classic ER-Golgi pathway.^{44 45} Recently, heat shock protein 27 has been proposed to facilitate the release of bFGF.⁴⁶ However, an autocrine mechanism of bFGF action has been found to be important in endothelial cell growth and function.⁴⁷ Media conditioned by mechanically injured aortic tissue contain bFGF that promotes microvessel sprouting, demonstrating that bFGF released by endothelial cells stimulates angiogenesis after injury.⁴⁸ In another model, tumor cells release a factor that rapidly upregulates an endothelial cell autocrine loop by which expression of bFGF induces capillary tube formation.⁴⁹ Thus, endothelial cell activities contributing to angiogenesis may be mediated by bFGF via an autocrine mechanism.

The findings described in the present report support the concept that ER- and GF-mediated pathways may act synergistically in tissues that are not classically defined as targets of estrogen. On the basis of the known effects of estrogen in classic target tissues such as breast, ovary, and uterus, a number of mechanisms might singly or in concert contribute to estrogenic modulation of endothelial cell function. First, estrogen-ER complexes may act directly as a transcription factor that enhances production of new proteins.^{50 51} Our results are consistent with this proposed mechanism. However, although we have provided evidence that bFGF production is increased, we have not addressed whether this increase is a direct effect of ER. Indeed, no data are available demonstrating a consensus binding site for ER in the bFGF gene promoter. Therefore, a second possible mechanism is that the ER may act indirectly by enhancing the activation of other transcription factors,⁵² which in turn regulate bFGF production. A third mechanism could involve interaction between estrogen and GF effects through genomic or nongenomic means.^{53 54} In the present study, we have described a system in which endothelial cell signaling is augmented by E2, at least in part by an autocrine mechanism involving bFGF. Thus, ER and GF signaling cascades may interact to amplify cellular responses.

	Selected Abbreviations and Acronyms

 

bFGF = basic fibroblast growth factor E2 = 17ß-estradiol ER = estrogen receptor ERK = extracellular signal-regulated protein kinase GF = growth factor HUVEC = human umbilical vein endothelial cells MAP = mitogen-activated protein MBP = myelin basic protein

	Acknowledgments

 
This study was supported by grants HL-53918 from the National Heart, Lung, and Blood Institute and DK49362 from the National Institute of Diabetes and Digestive and Kidney Diseases, as well as by the Children's Memorial Institute for Education and Research. Dr Kim-Schulze is the recipient of a Senior Fellowship from the American Heart Association of Metropolitan Chicago. The following provided reagents: Y.-K. Ho, equipment for heparin-sepharose chromatography; V. Lindner, bFGF plasmids; S. Vose, ICI 182,780; and S. Rosen, breast cancer cell lines. Susan Hubchak provided the HUVEC.

Received September 22, 1997; revision received March 18, 1998; accepted March 26, 1998.

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