17-Epiestriol, an Estrogen Metabolite, Is More Potent Than Estradiol in Inhibiting Vascular Cell Adhesion Molecule 1 (VCAM-1) mRNA Expression*

Tapan K. MukherjeeDagger , Lauren Nathan§, Hillary DinhDagger ||, Srinivasa T. ReddyDagger **, and Gautam ChaudhuriDagger §DaggerDagger

From the Departments of § Obstetrics and Gynecology, Dagger  Molecular and Medical Pharmacology, and ** Medicine, University of California, Los Angeles, California 90095-1740

Received for publication, August 1, 2002, and in revised form, January 17, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

17-beta estradiol (17-beta E2) attenuates the expression of vascular cell adhesion molecule 1 (VCAM-1) in vivo at physiological levels (pg/ml), whereas supraphysiological concentrations of 17-beta E2 (ng/ml) are required in vitro. We assessed whether a metabolite of estrogen, which could only be generated in vivo, might be a more potent inhibitor of VCAM-1 expression and thereby explain this discrepancy. We report here that 17-epiestriol, an estrogen metabolite and a selective estrogen receptor (ER) beta  agonist, is ~400× more potent than 17-beta E2 in suppressing tumor necrosis factor (TNF) alpha -induced VCAM-1 mRNA as well as protein expression in human umbilical vein endothelial cells. Genistein, an ERbeta agonist, at low concentrations (1 and 10 nM) also suppressed TNFalpha -induced VCAM-1 mRNA expression. These actions of 17-epiestriol and genistein were significantly attenuated in the presence of the estrogen receptor antagonist ICI-182780. Other estrogenic compounds such as ethinyl estradiol and estrone did not have any effect on TNFalpha -induced VCAM-1 expression at the concentrations tested. We further show that, 1) 17-epiestriol induces the expression of endothelial nitric-oxide synthase mRNA and protein, 2) 17-epiestriol prevents TNFalpha -induced migration of NFkappa B into the nucleus, 3) NG-nitro-L-arginine methyl ester, an inhibitor of NO synthesis, abolishes 17-epiestriol-mediated inhibition of TNFalpha -induced VCAM-1 expression and migration of NFkappa B from the cytoplasm to the nucleus. Our results indicate that 17-epiestriol is more potent than 17-beta E2 in suppressing TNFalpha -induced VCAM-1 expression and that this action is modulated at least in part through NO.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The mechanism by which estrogens attenuate the development of atherosclerosis is not known, although various actions of estrogens have been suggested to mediate this effect (1). We have previously demonstrated that 17-beta estradiol (17-beta E2) in vivo inhibits the adhesion of monocytes to endothelial cells of the rabbit aorta (2). We also demonstrated that following a cholesterol-enriched diet to ovariectomized rabbits, expression of VCAM-11 protein was induced in the aorta, and this was attenuated by administration of 17-beta E2 (2). Treatment of cultured rabbit aortic endothelial cells with 17-beta E2 also attenuated the lysophosphatidylcholine-induced expression of VCAM-1 protein. However, the concentrations of 17-beta E2 required to suppress VCAM-1 expression in vitro were in the supraphysiological range when compared with the physiological levels required for in vivo studies. We therefore postulated that one mechanism by which only relatively low concentrations of 17-beta E2 were required for in vivo studies when compared with in vitro studies was that 17-beta E2 might be converted to a more potent metabolite in vivo (2).

Some effects of estrogens are mediated through non-genomic mechanisms, whereas others require transcriptional activation of genes (3). These latter actions usually require that estrogens combine with specific receptors. Two types of estrogen receptors (ER) have been described. They are the "classical" ER, ERalpha and the "novel" more recently described ERbeta (4). Both ERalpha and ERbeta have been detected in endothelial cells and in vascular smooth muscle cells (5, 6). The affinity of various estrogenic compounds for the two ER subtypes markedly differ. 17-beta E2 binds to ERalpha and ERbeta with similar affinity. 17-alpha ethinyl estradiol (EE), a synthetic estrogen widely used in oral contraceptive formulations, has an ERalpha -selective agonist potency, whereas 17-epiestriol, an estrogen metabolite, and genistein, a phytoestrogen, at nM concentrations have ERbeta -selective agonist potency (7).

The present work was therefore undertaken to test whether certain estrogenic compounds, including an estrogen metabolite, with different agonistic potencies for the two types of estrogen receptors were more potent than 17-beta E2 in suppressing VCAM-1 mRNA expression and the potential mechanism(s) involved. Information obtained from these studies could lead to the development of compounds that attenuate atherogenesis with fewer side effects than those observed with 17-beta E2.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Materials-- The culture medium M199, HEPES buffer, and collagenase-type 1 from porcine skin were obtained from Invitrogen. Gelatin, endothelial-derived growth factor, genistein, TNFalpha , NG-nitro-L-arginine methyl ester (L-NAME), diethyl pyrocarbonate, salmon testes DNA, Denhardt's solution, and SDS were obtained from Sigma; the steroids 17-beta E2, EE, and 17-epiestriol were also obtained from Sigma with ~99% purity as assessed by thin layer chromatography. Fetal bovine serum (FBS) charcoal/dextran-treated was obtained from HyClone Laboratories. FBS was obtained from Atlas, Fort Collins, CO, and ICI-182780 was obtained from Tocris Cookson Ltd., Ballwin, MO. The human NFkappa B p65 Nushift kit (2006760) was obtained from Geneka Biotechnology (Montreal, Canada).

Cell Culture-- For isolation of human umbilical vein endothelial cells (HUVEC), umbilical cords from female fetus were selected as we observed increased expression of ERalpha and ERbeta mRNA when compared with those obtained from male fetus. HUVEC were isolated from freshly collected umbilical cords (female fetuses) as previously described (8) and cultured on 0.1% gelatin-coated 75-mm flasks in M199 medium supplemented with 20% FBS, 5 µg/ml endothelial-derived growth factor, 100 µg/ml heparin, 100 units of penicillin G sodium, 100 µg/ml streptomycin sulfate, 0.25 µg/ml amphotericin-B and 10 mM HEPES buffer, pH approx  7.5. Prior to the experiments, cells were shifted to phenol red-free M199 medium supplemented with 2% charcoal/dextran-treated FBS and the antibiotics mentioned above. Only second or third passage cells were utilized in all the current studies.

Reverse Transcription-PCR for ERalpha and ERbeta mRNA-- RNA extraction and purification were done using RNeasy mini kit as described by the manufacturer's protocol (Qiagen, Chatsworth, CA). Total RNA (3 µg per sample) was subjected to reverse transcription reaction with 50 units of Moloney murine leukemia virus reverse transcriptase at 42 °C for 25 min as previously described (9). The resulting cDNA samples were PCR-amplified using Gene Amp RNA PCR kit (PerkinElmer Life Sciences) according to the manufacturer's protocol. Oligonucleotide primers were designed for simultaneous PCR amplification (9) of specific DNA fragments contained in both ERalpha and ERbeta using the following set of primers: forward primer sequence, 5'-AAG AGC TGC CAG GCC TGC CG-3'; reverse primer sequence, 5'-GCC CAG CTG ATC ATG TGA ACC A-3'.

Northern Blot Analysis for VCAM-1 and Endothelial Nitric-oxide Synthase (eNOS) mRNA-- 10 µg of total RNA was loaded on 1% agarose-formaldehyde gel, electrophoresed, and transferred to Hybond membrane (Amersham Biosciences) overnight by capillary action. The RNA was UV-cross-linked using GS Gene Linker (BioRad). VCAM-1 (Research Genetics) and eNOS (a gift from Dr. Thomas Michel, Brigham and Women's Hospital, Boston, MA) cDNAs were labeled with [alpha -32P]dCTP (ICN Biochemicals) using the random priming method. Membranes were prehybridized at 42 °C overnight followed by hybridization with respective labeled probes for another 24 h at 42 °C. The membranes were washed twice at room temperature with 2× sodium chloride and sodium citrate (SSC) buffer and 0.5% SDS followed by washing at 65 °C for 30 min twice with 0.5× SSC and 0.1% SDS, and respective bands were quantitated using a Phosphoimager (Molecular Dynamics). The VCAM-1 band was normalized with the GAPDH band as an internal control.

Western Blot Analysis for eNOS and ELISA for VCAM-1 Protein-- Following the treatments, cells were lysed in a buffer containing 50 mM HEPES (pH 7.5), 1 mM DTT, 150 mM NaCl, 1 mM EDTA, 0.1% Tween 20, 10% glycerol, 10 mM beta -glycerophosphate, 1 mM NaF, 0.1 mM orthovanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride (10). 10 µg of total protein were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membrane using a semi-dry transfer apparatus. The membranes were incubated at room temperature with eNOS monoclonal antibody (1:1000) or GAPDH (1:5000) antibody for 60 min. The membranes were then incubated in diluted (1:1000) horseradish peroxidase-linked secondary antibody (1:1000) for 60 min at room temperature. The proteins were detected using ECL Western blotting kit following exposure of the membrane to autoradiography film (Hyperfilm-ECL, Amersham Biosciences). The relative intensities were quantified by densitometric analysis (Personal Densitometer, SI, Molecular Dynamics). Membrane-bound VCAM-1 protein was measured by ELISA as described previously (11).

Electrophoretic Mobility Shift Assay for NFkappa B-- After appropriate treatments, 108 cells were washed twice with ice-cold phosphate-buffered saline, resuspended in 1 ml of ice-cold harvest buffer (10 mM HEPES, pH 7.9, 5 mM MgCl2, 10 mM NaCl, 0.3 M sucrose, 0.1 mM EGTA, O.5 mM DTT, 0.5 mM PMSF, 1 µg/ml each of antipain, aprotinin, and leupeptin), homogenized in a Dounce homogenizer (20-30 strokes), and centrifuged for 30 s at 21,000 × g. The pellet was resuspended in 400 µl of nuclear extraction buffer (20 mM HEPES, pH 7.9, 0.3 mM KCl, 0.2 mM EGTA, 25% glycerol, 0.5 mM DTT, 0.5 mM PMSF), rocked gently at 4 °C for 30 min, and centrifuged again for 30 min at 21,000 × g at 4 °C. Nuclear proteins in the supernatant were dialyzed (20 mM HEPES, pH 7.9, 0.1 M KCl, 0.2 mM EDTA, 20% glycerol, 0.5 mM DTT, 0.5 mM PMSF) overnight at 4 °C. The debris was pelleted by centrifugation, and nuclear proteins in the supernatant were quantitated by Bradford reagent (BioRad). Using 10 µg of nuclear extract from each condition, NFkappa B electrophoretic mobility shift assay was performed as described in the manufacturer's protocol utilizing NFkappa B p65 Nushift kit (Geneka Biotechnology).

Data Analysis-- Values are expressed as mean ± S.D. obtained from three separate experiments in each group. Differences between groups were assessed by one-way analysis of variance and Newman-Keuls multiple comparison test where appropriate. p values < 0.05 were considered as significant.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of TNFalpha on VCAM-1 mRNA Expression and the Modulating Role of ER Agonists-- Second and third passage HUVEC, which were utilized for all our studies, expressed both ERalpha and ERbeta mRNA (data not shown). No basal VCAM-1 mRNA expression was detected in the unstimulated cells or those treated with the different estrogenic compounds alone. The structures of the different estrogens studied are shown in Fig. 1. Preliminary experiments indicated that HUVEC when exposed to 10 ng/ml of TNFalpha for 4 h led to maximal VCAM-1 mRNA expression. This concentration and time period were utilized for all the experiments.


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Fig. 1.   Structure of various estrogen receptor ligands.

The effects of preincubation of HUVEC with different concentrations of 17-beta E2, EE (100 and 300 pM, 1, 3, 10, 30, 100, 300 nM), 17-epiestriol (10, 30, 100, 300 pM and 1, 3, 10 nM), and genistein (1 and 10 nM) on TNFalpha (10 ng/ml)-induced VCAM-1 mRNA expression are shown in Figs. 2-5. 17-beta E2 at concentrations ranging from 1 to 300 nM attenuated the TNFalpha -induced VCAM-1 expression, whereas lower concentrations did not have any significant effect when compared with that observed with TNFalpha alone (Figs. 2 and 3). EE did not have any significant effect on VCAM-1 mRNA expression (Figs. 2 and 3). 17-epiestriol showed a response that was different from that of 17-beta E2 and EE (Fig. 4). At lower concentrations (30-300 pM), there was significant attenuation of VCAM-1 mRNA expression when compared with that observed with TNFalpha alone. The maximal attenuation was observed with 100 and 300 pM. The calculated IC50 (mean ± S.D.) for 17-beta E2 and 17-epiestriol were 16.3 ± 6.8 nM and 36 ± 5.3 pM, respectively. Interestingly, at higher concentrations (1 and 10 nM), the effect of 17-epiestriol was gradually lost, and the VCAM-1 expression started returning to baseline values. However, the magnitude of VCAM-1 expression at 3 nM of 17-epiestriol was still significantly less than that observed with TNFalpha alone. Estriol at similar concentrations to that of 17-epiestriol had no effect (data not shown). Genistein (1 and 10 nM) attenuated VCAM-1 mRNA expression (Fig. 5). The calculated IC50 (mean ± S.D.) for genistein was 7.16 ± 3 nM. The effects of all these estrogen receptor agonists were significantly attenuated in the presence of the ER antagonist ICI-182780 (1 µM), and the results for 17-epiestriol are shown in Fig. 6.


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Fig. 2.   Representative pictures of Northern blot analysis showing the effects of different concentrations of 17-beta estradiol (17-beta E2) (panel A) and 17alpha ethinyl 17-beta estradiol (EE) (panel B) on TNFalpha -induced VCAM-1 mRNA expression. Human umbilical vein endothelial cells were preincubated with either 17-beta E2 or EE at various concentrations for 48 h, and TNFalpha (10 ng/ml) was added to the culture media for the last 4 h. Following this, the cells were collected and total RNA was extracted. RNA was loaded on 1% agarose-formaldehyde gel, electrophoresed, and visualized under UV light after ethidium bromide staining. GAPDH was used as internal control. The treatment regimens and the concentration of the steroids are also shown and are described in detail under "Experimental Procedures."


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Fig. 3.   Quantitation of VCAM-1 mRNA expression using the Phosphoimager of the various treatments shown in Fig. 2. Quantitation is expressed as mean ± S.D. arbitrary densitometric units. Panels A and B show the effects of 17-beta E2 and EE respectively on TNFalpha -induced VCAM-1 mRNA expression. * signifies significant difference (p < 0.05) from TNFalpha only (lane 2) -treated cells.


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Fig. 4.   Representative picture of a Northern blot analysis showing the effects of different concentrations of 17-epiestriol on TNFalpha -induced VCAM-1 mRNA expression (panel A) and quantitation by Phosphoimager (panel B). Quantitation is expressed as mean ± S.D. of arbitrary densitometric units obtained from three separate experiments. GAPDH was used as internal control. * signifies significant difference (p < 0.05) from TNFalpha only (lane 2) -treated cells.


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Fig. 5.   Representative picture of a Northern blot analysis showing the effects of different concentrations of genistein on TNFalpha -induced VCAM-1 mRNA expression (Panel A) and quantitation by Phosphoimager (Panel B). Quantitation is expressed as mean ± S.D. of arbitrary densitometric units obtained from three separate experiments. GAPDH was used as internal control. * signifies significant difference (p < 0.05) from TNFalpha (lane 2) -treated cells.


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Fig. 6.   Representative picture of Northern blot analysis showing the effect of 300 pM 17-epiestriol on TNFalpha -induced (10 ng/ml) VCAM-1 and GAPDH mRNA expression in the presence and absence of an ER antagonist ICI-182780 (1 µM). The ER antagonist was added to the culture medium containing HUVEC 60 min before the addition of 17-epiestriol.

Effect of 17-Epiestriol on TNFalpha -induced VCAM-1 Protein Expression-- We next examined the effect of 17-epiestriol on TNFalpha -induced VCAM-1 protein expression. HUVEC were preincubated with different concentrations of 17-epiestriol for 48 h. Six hours after the addition of TNFalpha , the cells were fixed and assayed for cell-bound VCAM-1 protein by ELISA as described previously (11). A significant reduction in VCAM-1 protein was observed at a 300-pM concentration of 17-epiestriol (Fig. 7). Consistent with the observation related to VCAM-1 mRNA, TNFalpha -induced VCAM-1 protein expression also showed a biphasic response to 17-epiestriol.


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Fig. 7.   Effect of different concentrations (30-300 pM and 10 nM) of 17-epiestriol on TNFalpha -stimulated (10 ng/ml) VCAM-1 protein expression as analyzed by ELISA. Values represent absorbance (OD) at 490 nM. Quantitation is expressed as mean ± S.E. obtained from three separate experiments. * significant difference (p < 0.05) from that observed with TNFalpha alone.

Intermediate Role of Nitric Oxide in the Action of 17-Epiestriol-- To determine whether NO played an intermediate role in attenuating TNFalpha -induced VCAM-1 expression, we first analyzed the effect of 17-epiestriol on eNOS expression. Treatment of HUVEC with 17-epiestriol showed a biphasic response on eNOS protein expression. The maximal increase (68 ± 8%) was observed at 300 pM for both eNOS protein (Fig. 8) and mRNA expression (35%, data not shown). We next assessed the effect of three different concentrations of 17-epiestriol on TNFalpha -induced VCAM-1 mRNA expression in the absence and presence of L-NAME (3 × 10-4 M), an inhibitor of NO synthesis. This concentration of L-NAME has been previously demonstrated by us to significantly attenuate basal- and agonist-stimulated NO release by endothelial cells (12). L-NAME either alone or along with TNFalpha did not affect VCAM-1 expression. 17-epiestriol (100 and 300 pM and 1 nM) attenuated VCAM-1 mRNA expression, and this effect was not observed in the presence of an inhibitor of NO synthesis (Fig. 9).


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Fig. 8.   Representative picture of a Western blot analysis performed eNOS (top) and GAPDH (bottom) in the absence and presence of varying concentrations (30-300 pM and 1 and 10 nM) of 17-epiestriol. HUVEC were incubated with 17-epiestriol for 48 h (at concentrations indicated). Ten micrograms of protein were loaded onto each lane. The proteins were separated on 7.5% polyacrylamide gel. eNOS and GAPDH were analyzed using monoclonal eNOS- and GAPDH-specific antibodies. The treatment regimen of the various groups of cells in each of the lanes is also indicated.


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Fig. 9.   Representative picture of a Northern blot analysis demonstrating the effects of different concentrations of 17-epiestriol (100 and 300 pM and 1 nM) on TNFalpha -induced VCAM-1 mRNA expression in the absence and presence of an inhibitor of nitric oxide synthase inhibitor L-NAME. The various treatment regimens in each of the lanes are also shown. GAPDH was used as the internal control.

Effect of 17-Epiestriol on TNFalpha -induced NFkappa B Activation-- To determine whether 17-epiestriol regulated TNFalpha -induced VCAM-1 expression by inhibiting NFkappa B migration to the nucleus, we performed gel-shift assays using oligonucleotides corresponding to the tandem-kappa B sites on the VCAM-1 promoter. We also assessed whether NO played an intermediate role in this action by 17-epiestriol. 17-epiestriol at concentrations of 100 pM (data not shown) and 300 pM (Fig. 10, lane 5) significantly decreased the intensity of the shifted band produced by nuclear extracts obtained from HUVEC treated with TNFalpha . However, 10 nM of 17-epiestriol was less effective in decreasing the intensity of the shifted band when compared with 300 pM (Fig. 10, lane 6). Specificity of the complexes was determined by competition with an excess of unlabelled oligonucleotide and with a labeled mutant oligonucleotide when no band was observed (Fig. 10, lane 4). The specificity was further confirmed by supershift analysis (Fig. 10, lane 3) with an affinity-purified polyclonal antiserum to p65. Nuclear translocation of NFkappa B following treatment with 17-epiestriol in the presence of L-NAME was similar to that observed in the absence of 17-epiestriol and in the presence of L-NAME alone (Fig. 10, lanes 7 and 8). This suggests that the inhibitory effect of 17-epiestriol on the translocation of NFkappa B to the nucleus following stimulation of HUVEC with TNFalpha was mainly dependent on NO synthesis.


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Fig. 10.   Representative picture of an electrophoretic mobility shift assay showing the effect of TNFalpha -induced NFkappa B activation in HUVEC and the modulating role of 17-epiestriol (300 pM and 10 nM) in the absence and presence of L-NAME (3 × 10-4 M), an inhibitor of nitric oxide synthase. Cells were incubated with 17-epiestriol (300 pM and 10 nM) or vehicle for 48 h. TNFalpha (10 ng/ml) was added to the HUVEC for the last 4 h of incubation with either vehicle or 17-epiestriol. L-NAME was added 30 min prior to the addition of TNFalpha . Experiments were also performed in the presence of either wild type oligonucleotide (Wt Oligo) or mutant oligonucleotide (Mut Oligo). This picture is representative of four such experiments. The specificity of the assay was evaluated by incubating the extracts with p65 antibody (lane 3).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The primary objective of this study was to assess whether some selected estrogenic compounds, including a phytoestrogen, a synthetic estrogen, and an estrogen metabolite with different agonistic potencies for ERalpha and ERbeta were more potent than 17-beta E2 in attenuating TNFalpha -induced VCAM-1 expression of HUVEC in vitro. 17-beta E2 is the major circulating unconjugated estrogen in premenopausal women. It binds to human ERalpha and ERbeta with approximately similar affinity (7) and was therefore used as the prototype with which the comparisons to the other estrogens were made. EE, a synthetic estrogen, was selected since it is widely used as the estrogenic component in oral contraceptives and has ERalpha -selective agonistic potency (7). 17alpha epiestriol was selected because it is an estrogen metabolite (13), which has an ERbeta -selective agonist potency (7). Estriol was selected as its concentration markedly increases during pregnancy and is structurally very similar to 17alpha epiestriol except that the 17-OH group in estriol is in the 17-beta position, whereas that of 17alpha epiestriol is in the alpha  position (Fig. 1). This difference makes it a very weak agonist for both ERalpha and ERbeta (7). The isoflavone phytoestrogen genistein binds to ERbeta almost as well as 17-beta E2, whereas its affinity to ERalpha is considerably lower than that of 17-beta E2 (14).

Our results indicate that 17-beta E2 did not affect VCAM-1 mRNA expression at lower concentrations (100 and 300 pM), whereas at higher concentrations (1-300 nM) it decreased VCAM-1 mRNA in a concentration-dependent manner (Figs. 2 and 3). This action of 17-beta E2 in vitro has been studied by other investigators; however, the results are controversial. In studies using HUVEC, 17-beta E2 suppressed the induction of VCAM-1 mRNA expression induced by either lipopolysaccharide (2, 15) or by interleukin-1beta (8, 16). In contrast, Cid et al. (18) observed that 17-beta E2 did not affect basal VCAM-1 expression but caused a 30-50% increase in the presence of TNFalpha and had no effect when HUVEC were stimulated with interleukin-1. It has also been reported that 17-beta E2 had no effect on the TNFalpha -induced expression of VCAM-1 in cultured endothelial cells (19). These contrasting observations may be due to differences in the extent to which the endothelial cells used in the different studies expressed the estrogen receptors, differences in the concentration of 17-beta E2 utilized, or to differences in the agents used to induce VCAM-1 expression.

In this regard, in both the present study and in those previously reported (8, 15, 16), the concentrations of 17-beta E2 required for suppression of VCAM-1 expression in vitro were in the supraphysiological range (1 nM-10 µM). The physiological concentration of 17-beta E2 is in the pM range, whereas that of 17-epiestriol has not been reported in humans.

In contrast, EE, which is a more potent and selective agonist for ERalpha when compared with 17-beta E2, did not attenuate TNFalpha -induced VCAM-1 mRNA expression at any of the concentrations studied (Figs. 2 and 3). One potential explanation for the absence of any effect of EE may be that ERbeta mediates the estrogen-induced decrease in VCAM-1 mRNA expression, whereas ERalpha has a negligible role. This may be the case as 17-epiestriol, which has an ERbeta -selective agonistic potency, decreased VCAM-1 mRNA expression and the IC50 was ~400× lower than that observed with 17-beta E2. At present, we cannot explain why higher concentrations of 17-epiestriol were gradually less effective. A similar biphasic effect of 17-epiestriol on VCAM-1 protein expression (Fig. 7) and a shift of NFkappa B to the nucleus (Fig. 10) was also observed. Estriol, which is a very weak agonist for both ERalpha and ERbeta (7), did not affect VCAM-1 mRNA expression. This indicates that the alignment of the 17-OH group in the alpha  position is an important requirement for 17-epiestriol for its selective ERbeta agonistic activity. The ability of the various estrogens to attenuate VCAM-1 mRNA expression was not observed in the presence of the anti-estrogen 1C1-182780. This confirmed that the action of the estrogens were receptor-mediated. Further and more specific assessment of the precise ER type involved in mediating the VCAM-1 mRNA expression by estrogens will have to await the discovery of specific ERalpha and ERbeta receptor antagonists. Genistein at nanomolar concentrations has binding affinity to ERbeta , which is similar to that of 17-beta E2, whereas its affinity to ERalpha is considerably less (14). Our observation that genistein at nM concentrations (1 and 10 nM) decreased VCAM-1 mRNA expression supports the concept that ERbeta is most likely involved in the attenuation of VCAM-1 expression in endothelial cells and that ERalpha probably has very little role in this phenomenon. At these concentrations, genistein has very little tyrosine kinase inhibitory activity (14) and, hence, the effects are likely due to its action on the ERbeta .

No estrogen-responsive element in the promoter region of the VCAM-1 gene has yet been identified (20), and other indirect mechanisms need to be considered. The VCAM-1 promoter contains consensus binding sites for the nuclear transcription factor NFkappa B, the GATA family of transcription factors, and an AP-1 site (15). NFkappa B is a proinflammatory transcription factor up-regulating several genes involved in endothelial activation (21). It has recently been demonstrated that a very high concentration of 17-beta E2 (10 µM) suppressed VCAM-1 expression indirectly by inhibiting the nuclear translocation and DNA binding of NFkappa B, and this was also associated with a reduction of AP-1 and GATA transcription factors binding to the VCAM-1 promoter (15). These investigators also demonstrated that in human endothelial cells, NFkappa B inhibition by E2 was associated with decreased Ikappa B-alpha degradation.

On this basis we wanted to assess the possibility that estrogens may have increased the synthesis of NO, which in turn may have led to the suppression of DNA binding of transcription factors, thereby leading to inhibition of VCAM-1 expression. There is a lot of similarity between the actions of estrogens and NO on various transcription factors that modulate VCAM-1 expression. NO donors inhibited cytokine-induced expression of VCAM-1 and repressed VCAM-1 gene transcription in part by inhibiting nuclear binding protein NFkappa B (22, 23). NO also stabilized the NFkappa B inhibitor, Ikappa B-alpha (24), and cellular treatment with NO donor compounds also inhibited AP-1 binding to DNA (25).

We previously demonstrated that estrogens can increase NO production (12), and subsequently others have demonstrated that it can occur through both genomic (26) and non-genomic (27, 28) mechanisms. We therefore decided to assess whether 17-epiestriol at concentrations that attenuated VCAM-1 mRNA expression also increased eNOS protein and whether this was at least in part due to increased eNOS mRNA. Our results indicated that 17-epiestriol also had a biphasic effect on eNOS protein expression. 17-Epiestriol at a concentration that maximally suppressed TNFalpha -induced VCAM-1 mRNA expression also increased eNOS protein (Fig. 8) and mRNA, suggesting that NO may play an intermediary role in this action of 17-epiestriol. This was confirmed by observing that the suppression of VCAM-1 mRNA expression by 17-epiestriol was not observed following inhibition of NO synthesis (Fig. 9). Our results also suggest that 17-epiestriol-induced NO production played an intermediate role in inhibition of the nuclear translocation and DNA binding of NFkappa B in HUVEC as this inhibitory effect of 17-epiestriol was significantly attenuated in the presence of an inhibitor of NO synthase. Higher concentrations (1 and 10 nM) of 17-epiestriol attenuated the eNOS protein expression when compared with 300 pM, and this may explain the biphasic response of 17-epiestriol on VCAM-1 mRNA and protein expression. Our study did not address the precise molecular mechanism(s) by which NO production stimulated by 17-epiestriol inhibited the translocation of NFkappa B to the nucleus. It is also possible that the differences in actions observed between estrogenic compounds with different affinities for ERalpha and ERbeta receptors could be mediated by interaction with the AP-1 site. The interactions of ERalpha and ERbeta with the AP-1 site could lead to signaling in opposite ways and thus may play opposite roles in gene regulation (29). It would also be interesting to assess whether the reduction of AP-1 and GATA binding to the VCAM-1 promoter by estrogens is also mediated by an increase in NO production, thereby indicating an important intermediary role of NO in the regulation of some transcription factors by estrogens.

In conclusion, our results indicate that estrogens most likely inhibited VCAM-1 mRNA and protein expression by an action mediated by ERbeta and not by ERalpha and that NO played an intermediate role in this process. Our results further suggest that 17-epiestriol, an estrogen metabolite, is more potent than 17-beta E2. This may be one potential explanation as to why much lower concentrations of 17-beta E2 are required to suppress VCAM-1 expression in vivo (where various metabolites of estrogens are formed) when compared with those required in vitro.

These results may have important clinical implications. An important area of research with relation to estrogen replacement therapy is identifying the role of ERalpha and ERbeta in mediating the antiatherosclerotic action of estrogens. It has been demonstrated that 17-beta E2 can inhibit responses to vascular injury in an acute carotid artery endothelium-denuded model in ovariectomized ERalpha -/- (30) and ERbeta -/- (31) animals to the same extent as in wild type animals. On this basis it has been suggested that ERalpha and ERbeta can complement each other in vivo to mediate the vasoprotective action of estrogens following vascular injury. Recently, it has been demonstrated that genistein, which has selective binding affinity to ERbeta , provided vasoprotective actions that were equally efficacious to that observed with 17-beta E2 in an acute carotid artery endothelium-denuded rat model and yet were devoid of effects on the uterus, which mainly have ERalpha receptors (32). In the acute carotid artery endothelium-denuded model, in contrast to the atherosclerosis model, the endothelial cells are denuded, and it would therefore be difficult to assess the actions of estrogens exerted specifically via the endothelial cells. It would therefore be interesting to assess whether 17-epiestriol is as effective or more potent than 17-beta E2 in attenuating atherogenesis and yet is devoid of effects on the reproductive system when compared with 17-beta E2. Further studies are in progress to assess this in vivo in an animal model. Our results demonstrating the biphasic action of 17-epiestriol in modulating VCAM-1 gene and protein expression indicate that estrogens may have paradoxical effects in inflammatory processes. This may be one potential explanation for the controversy in relation to the effects of estrogen replacement therapy on cardiovascular morbidity (17).

    ACKNOWLEDGEMENTS

We thank Srirupa Mukhopadhyay, Julie Gage, Janis Cuevas, and Svetlana Arutyunova for technical assistance.

    FOOTNOTES

* This work was supported, in part, by NIA, National Institutes of Health Grant AG-15857.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.

Recipient of the Pfizer/Society for Women's Health Research Scholars Grant for Faculty Development in Women's Health.

|| Recipient of a graduate scholarship from The American Heart Association Southern California Chapter.

Dagger Dagger To whom correspondence should be addressed: Depts. of Obstetrics & Gynecology and Molecular & Medical Pharmacology, David Geffen School of Medicine at UCLA, Center for Health Sciences, 10833 Le Conte Ave., Los Angeles, CA 90095-1740. Tel.: 310-206-6575; Fax: 310-206-2057; E-mail: gchaudhuri@mednet.ucla.edu.

Published, JBC Papers in Press, January 23, 2003, DOI 10.1074/jbc.M207800200

    ABBREVIATIONS

The abbreviations used are: VCAM, vascular cell adhesion molecule; ER, estrogen receptors; EE, ethinyl estradiol; L-NAME, NG-nitro-L-arginine methyl ester; TNF, tumor necrosis factor; FBS, fetal bovine serum; HUVEC, human umbilical vein endothelial cells; eNOS, endothelial nitric-oxide synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; ELISA, enzyme-linked immunosorbent assay.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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