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(Circulation Research. 2000;87:19.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Estrogens and Glucocorticoids Inhibit Endothelial Vascular Cell Adhesion Molecule-1 Expression by Different Transcriptional Mechanisms

Tommaso Simoncini, Silvia Maffei, Giuseppina Basta, Giuseppina Barsacchi, Andrea R. Genazzani, James K. Liao, Raffaele De Caterina

From the Scuola Superiore di Studi e di Perfezionamento "S. Anna" (T.S.), Pisa; Department of Reproductive Medicine and Child Development (T.S., A.R.G.) and Cellular and Developmental Biology Division (G. Barsacchi), University of Pisa, Italy; and CNR Institute of Clinical Physiology (S.M., G. Basta, R.D.C.), Pisa, Italy, and Cardiovascular Division (T.S., J.K.L.), Brigham and Women’s Hospital, Boston, Mass.

Correspondence to James K. Liao, MD, Vascular Medicine Unit, Department of Medicine, 221 Longwood Ave, LMRC-322, Boston, MA 02115. E-mail jliao{at}rics.bwh.harvard.edu

	Abstract

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Abstract—The antiatherogenic effect of estrogen is mediated, in part, by inhibitory effects on endothelial vascular cell adhesion molecule-1 (VCAM-1) expression. To determine the mechanism by which estrogen regulates VCAM-1 expression, we compared the effect of 17ß-estradiol (E₂) and of the glucocorticoid dexamethasone (Dex) on lipopolysaccharide (LPS)–induced VCAM-1 expression in human endothelial cells. E₂ decreased LPS-induced VCAM-1 mRNA and protein expression to a greater extent than Dex. Dex, but not E₂, stabilized VCAM-1 mRNA. This correlated with inhibition of monocytoid U937 cell adhesion to endothelial cells. Transfection of endothelial cells with a functional VCAM-1 promoter construct showed that E₂ inhibited LPS-induced VCAM-1 gene transcription more potently than did Dex. However, using a truncated construct containing only the nuclear factor-B (NF-B)–responsive elements but lacking the consensus sequences for activator protein-1 (AP-1) and GATA, E₂ and Dex had similar inhibitory effects. Consistently, gel-shift assays showed that E₂ and Dex comparably inhibit LPS-induced activation of NF-B, whereas E₂ inhibited LPS-induced activation of AP-1 and GATA to a greater extent than Dex. E₂ inhibition of NF-B after LPS treatment was associated with decreased inhibitor B (IB) kinase activity and with a stabilization of the NF-B inhibitor IB. These results indicate that E₂ decreases VCAM-1 gene expression through the inhibition of NF-B, AP-1, and GATA and suggest novel mechanisms for the antiatherogenic effect of estrogen on the vascular wall. (Circ Res. 2000;87:19-25.)

Key Words: estrogen • glucocorticoid • endothelial-leukocyte adhesion molecules • nuclear factor-B • endothelial cells

	Introduction

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The molecular pathways by which estrogen acts on the cardiovascular system remain largely unknown despite recent interest in using these agents in postmenopausal women for prevention of cardiovascular diseases.¹ The cardioprotective effect of estrogen is only partially explained by favorable lipid profile modifications,² and recent evidence suggests that the direct vascular effects of estrogen may actually account for most of their protective action.³

Atherosclerosis is considered a chronic inflammatory process,⁴ and the endothelium plays an important role in its initiation.⁵ Various atherogenic stimuli, such as modified LDLs, oxidative free radicals, homocysteine, and infectious agents, lead to the development of endothelial dysfunction.⁴ Many of these stimuli cause endothelial cell activation, which is defined as functional and antigenic changes in endothelial cells promoting monocyte adhesion.⁶ Endothelial activation involves the coordinated induction of genes encoding for leukocyte adhesion molecules (such as vascular cell adhesion molecule-1 [VCAM-1] and intercellular adhesion molecule-1), chemotactic factors (such as monocyte chemoattractant protein-1 [MCP-1]), and growth factors (such as macrophage colony-stimulating factor).⁶ Endothelial VCAM-1 is an important mediator of mononuclear cell adhesion, given that its cognate ligand, the integrin very late antigen-4 (VLA₄), is selectively expressed on monocytes (and on some T lymphocytes) but not on neutrophils.⁷

In different animal models of atherosclerosis,^{8 9} 17ß-estradiol (E₂) protects endothelial integrity and function and reduces leukocyte adhesion and intimal accumulation.¹⁰ In vitro, E₂¹¹ and other estrogenic compounds¹² decrease cytokine-induced expression of adhesion molecules and of monocyte chemoattractants.¹³ Glucocorticoids also inhibit the expression of endothelial cell adhesion molecules.¹⁴ However, the mechanism for these effects of estrogen, and whether estrogen and glucocorticoid have a common mechanism of action, is still not known. The purpose of this study, therefore, is to determine and compare the mechanism by which estrogen and glucocorticoid regulate endothelial cell adhesion molecule expression.

	Materials and Methods

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Cell Cultures
Human saphenous vein endothelial cells (HSVECs) and bovine aortic endothelial cells (BAECs) were harvested and maintained as described.¹⁵ HSVECs were obtained by aortocoronary grafts from both male and female donors with an age range from 36 to 87 years. In preliminary experiments, no differences were noted regarding the investigated parameters. HSVECs were grown in the presence of 5% FBS, whereas for BAECs 10% FBS was used. All media were phenol red–free. Before treatments, cells were shifted to media containing FBS treated with charcoal stripping to remove steroid hormones.

Monocytoid Cell Adhesion Assays
Endothelial cells were pretreated with steroids for 48 hours, after which lipopolysaccharide (LPS; 100 ng/mL) was added for 18 hours. In some experiments, endothelial cells were treated with a VCAM-1 blocking antibody (E1/6). The adhesion assay was performed by adding U937 cells (10⁶ cells) to each monolayer under rotating conditions (63 rpm) at 21°C, as described.¹⁵

Cell Surface Enzyme Immunoassays
Endothelial cells were grown on 96-well culture dishes and pretreated for 48 hours before the addition of LPS (100 ng/mL for 18 hours), interleukin-1 (1 ng/mL), or tumor necrosis factor- (TNF-) (1 ng/mL) (Hoffmann-La Roche). Cell surface VCAM-1 was assayed using the monoclonal antibody E1/6. The antibody E1/1, recognizing a constitutive, non–cytokine-inducible endothelial cell antigen,¹⁶ was used in control studies. Enzyme immunoassays were carried out as described.¹⁵ At least 8 wells were used for each condition.

Northern Analysis
Endothelial cells were pretreated for 48 hours with E₂ or dexamethasone (Dex) followed by a 4-hour stimulation with LPS (100 ng/mL). Northern analysis was performed as described.¹⁵

Western Blotting
Proteins (40 µg/lane) were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore Corp). Membranes were hybridized with standard technique. Immunodetection was accomplished with enhanced chemiluminescence (Amersham).

Transfection Assays
Human VCAM-1 promoter constructs have been described.¹⁷ BAECs were used because of their higher transfection efficiency (15% versus 1% for human cells) with the calcium phosphate precipitation method. Transfection, chloramphenicol acetyltransferase (CAT), and ß-galactosidase assays were performed as described.¹⁵

Electrophoretic Mobility Shift Assays
Nuclear and cytosolic extracts were prepared as described.¹⁵ Oligonucleotide probes corresponding to the nuclear factor-B (NF-B) (5'-TGCCCTGGGTTTCCCCTTGAAGGGATTTCCCTCC-3'), activator protein-1 (AP-1) (5'-CGCTTGATGACTCAGCCGGAA-3'), and GATA (5'-CACTTGATAACAGAAAGTGATAACTCT-3') binding sites on VCAM-1 promoter¹⁷ were labeled, and DNA binding reactions were performed as described.¹⁵

Immunofluorescence Analysis
Endothelial cells were grown on glass slides precoated with 2% gelatin. Cells were fixed with acetone and blocked with 3% normal goat serum. Cells were incubated with an antibody directed against the NF-B subunit Rel A (Santa Cruz Biotechnology). A biotinylated antibody was used as secondary antibody. Streptavidin-FITC was added, and immunofluorescence was analyzed and photographed by an investigator blinded as to the experimental conditions.

Inhibitor B (IB) Kinase Assay
After preincubation, endothelial cells were treated with LPS, and cell lysates were prepared as described.¹⁸ After immunoprecipitation with an antibody versus IB kinase (Santa Cruz Biotechnology), 1 µg of glutathione S-transferase (GST)–fusion IB was added, and [³²P]dATP was used as phosphate donor.¹⁸ The reaction products were run on SDS-PAGE, which was dried and exposed for autoradiography.

Statistical Analysis
Multiple comparisons were performed by 1-way ANOVA,¹⁹ and individual differences were tested by the Fisher protected least significance difference test¹⁹ after the demonstration of significant intergroup differences by ANOVA. Two-group comparisons were performed by the unpaired Student t test.¹⁹

	Results

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Effect of E₂ and Dex on Monocyte Adhesion
To determine the effect of estrogen on endothelial cell–monocyte interactions, we performed adhesion assays. LPS treatment increased U937 cell adherence to endothelial surface (Figure 1). Pretreatment with E₂ (10 µmol/L, 48 hours) decreased U937 cell adhesion by >50%, whereas Dex (10 µmol/L, 48 hours) reduced adhesion to a lesser degree. A blocking anti–VCAM-1 monoclonal antibody produced a 37% decrease in cell adhesion (not shown), suggesting that some of the E₂ effect may be mediated by factors other than VCAM-1.

View larger version (138K): [in this window] [in a new window]   Figure 1. E2 and Dex reduce U937 monocytoid cell adhesion to activated endothelial cells. Data are mean±SD of adherent cells counted in 8 different microscopic fields. E2 and Dex concentration was 10 µmol/L. Pictures are representative optical fields. The experiment was repeated 3 times with equal results.

Effect of E₂ and Dex on LPS-Induced VCAM-1 Expression
In a concentration-dependent manner, treatment with E₂ and Dex inhibited LPS-induced VCAM-1 expression (Figure 2A). The steroids also inhibited VCAM-1 expression induced by interleukin-1 or TNF- (not shown). Consistent with monocyte adhesion findings, E₂ was more potent than Dex. The 2 compounds did not produce cellular toxicity, as assessed by cell number and viability (ie, morphology and trypan blue exclusion) and were specific, because they did not affect the expression of the constitutive surface antigen E1/1¹⁶ (not shown). VCAM-1 protein reduction corresponded to a comparable decrease in mRNA levels (Figure 2B).

View larger version (48K): [in this window] [in a new window]   Figure 2. A, E2 and Dex decrease LPS-induced VCAM-1 endothelial cell–surface expression. All data have been plotted as percentage of LPS-treated cells values. n8; *P<0.05. Experiments were repeated 5 times with equal results. B, E2 and Dex decrease LPS-induced VCAM-1 mRNA. Upper panel shows VCAM-1 mRNA. Lower panel shows the corresponding 18S ribosomal RNA, as loading control. Blot is representative of 3 different experiments. E2 and Dex concentration was 10 µmol/L. C, Cultured HSVECs express and ß estrogen receptor (ER) subtypes. Different lanes show samples from different cell lines. The experiment was repeated twice with equal results.

E₂ inhibition of VCAM-1 expression was competitively blocked by the pure estrogen receptor antagonist Imperial Chemical Industries (ICI) 182,780, suggesting that the effect is mediated through 1 or both estrogen receptors (Figure 2A), which are both expressed in our culture conditions (Figure 2C). In addition, the inactive E₂ stereoisomer, E₂, had no effect on VCAM-1 expression (data not shown), indicating that the estrogen effect is not mediated by intrinsic chemical properties (eg, antioxidant effects).

Effect of E₂ and Dex on VCAM-1 mRNA Stability
To determine whether E₂ or Dex may regulate VCAM-1 through posttranscriptional effects, we pretreated endothelial cells for 48 hours with E₂, Dex, or vehicle, and then we stimulated them with LPS for 4 hours to induce a maximal synthesis of VCAM-1 mRNA. Thereafter, we added the RNA polymerase inhibitor actinomycin-D. With Northern analysis, we studied VCAM-1 mRNA stability, examining samples harvested at 1-hour intervals. E₂ did not affect VCAM-1 mRNA half-life (t_1/2=164±21 minutes versus 183±26 minutes). Instead, Dex prolonged VCAM-1 mRNA half-life (t_1/2=248±37 minutes) (Figures 3A and 3B), thus potentially posttranscriptionally increasing VCAM-1 production.

View larger version (48K): [in this window] [in a new window]   Figure 3. Dex stabilizes VCAM-1 mRNA. A, Upper panel shows VCAM-1 mRNA. Lower panel shows corresponding 18S ribosomal RNA bands as loading control. E2 and Dex concentration was 10 µmol/L. B, mRNA decay curves obtained by measuring optical densities of the autoradiographic bands and by plotting the values on a semilogarithmic scale. To show differences in slope of decay curves (indicating rate of mRNA decay), all values for each treatment group have been normalized to the corresponding 0-hour time point, taking this point as 100%. The experiment was repeated twice with comparable results.

Effect of E₂ and Dex on VCAM-1 Gene Promoter Region
Steroid hormones usually act as transcription factors, but VCAM-1 promoter contains no estrogen or glucocorticoid response elements.¹⁷ To determine whether E₂ and Dex regulate VCAM-1 promoter and to identify which regions are involved, we transfected deletional VCAM-1 promoter constructs linked to the chloramphenicol acetyl transferase reporter gene¹⁷ into BAECs. F0.CAT is the VCAM-1 promoter construct containing the AP-1, GATA, and tandem -B sites (Figure 4A). F3.CAT contains the -B sites, and F4.CAT contains only a TATA box (Figure 4A). LPS increased F0.CAT activity by 5-fold compared with unstimulated cells (Figure 4B). Pretreatment with E₂ or Dex (10 µmol/L, 48 hours) inhibited LPS induction by 60% and 30%, respectively (Figure 4B). In cells transfected with F3.CAT, however, E₂ and Dex produced similar decreases in promoter activity (Figure 4B). F4.CAT was only minimally induced by LPS (Figure 4B). These findings suggest that E₂ and Dex inhibition of VCAM-1 expression is transcriptional and potentially due to regulation of different transcription factors and that the different steroid potencies may be the consequence of distinct modulations of AP-1 and/or GATA.

View larger version (40K): [in this window] [in a new window]   Figure 4. E2 and Dex differential inhibitions of VCAM-1 promoter constructs activity. A, Structure of the used constructs. B, Data are mean±SD of the CAT/ß-galactosidase ratio of 3 replicates for each condition. *P<0.05 vs LPS. **P<0.05 vs both F0.CAT LPS bar and F0.CAT LPS+Dex bar. E2 and Dex concentration was 10 µmol/L. The experiment was repeated 3 times with equal results.

Effect of E₂ and Dex on NF-B Activation
To determine whether E₂ and Dex regulate VCAM-1 expression by inhibiting NF-B, we performed gel-shift assays using an oligonucleotide corresponding to the tandem -B sites on VCAM-1 promoter. Both steroids (10 µmol/L, 48 hours) markedly decreased the intensity of the shifted band produced by nuclear extracts treated with LPS (Figure 5). Specificity of the complexes was determined with antibodies versus NF-B subunits Rel A (p65), p50, and c-Rel, which supershifted the complexes, and by competition with an excess of unlabeled oligonucleotide. Because cytoplasmic extracts from the same cells showed no differences, the changes in nuclear NF-B binding are probably due to inhibition of NF-B nuclear translocation. In agreement with transfection studies, the 2 steroids had similar potency with respect to NF-B inhibition, furthermore suggesting that other cis-acting elements within the VCAM-1 promoter mediate the differential inhibitory effect of E₂ versus Dex.

View larger version (81K): [in this window] [in a new window]   Figure 5. LPS-induced NF-B nuclear translocation and DNA binding activity are inhibited by E2 and Dex. Upper panel, Bands shifted by cytoplasmic extracts. Lower panel, Bands shifted by nuclear protein extracts. E2 and Dex concentration was 10 µmol/L. The experiment was repeated 4 times, with the same results. cRel indicates the NF-B subunit c-Rel.

Immunoblot of nuclear and cytoplasmic extracts confirmed that E₂ and Dex decrease nuclear localization of both p50 and Rel A after LPS treatment (Figure 6A). Immunofluorescence staining of Rel A also showed that E₂ and Dex, but not E₂, prevent LPS-induced NF-B nuclear translocation (Figure 6B).

View larger version (67K): [in this window] [in a new window]   Figure 6. E2 and Dex inhibition of p50 and p65 NF-B subunit nuclear translocation. A, Lanes show protein extracts obtained from purified nuclear or cytoplasmic fractions. Panels show p50 (upper) and p65 (lower) immunoblots and are representative of 3 separate experiments. E2 and Dex concentration was 10 µmol/L. B, Immunofluorescent staining for p65 NF-B subunit. Pictures are representative microscopic fields at different magnifications. E2 and Dex concentration was 10 µmol/L. The experiment was repeated 2 times with equal results.

Effect of E₂ on IB Degradation and IB Kinase Activity
To clarify whether the E₂ effect on NF-B nuclear translocation is due to regulation of the NF-B inhibitor IB, we pretreated endothelial cells with E₂ and then added LPS for different times to follow LPS-induced IB degradation. E₂ slightly increased basal IB levels and markedly reduced IB degradation after LPS treatment, whereas E₂ had no effect (Figure 7A).

View larger version (57K): [in this window] [in a new window]   Figure 7. E2 inhibits IB degradation and IKK activity induced by LPS. A, Immunoblot shows IB levels in endothelial cells. E2 and Dex concentration was 10 µmol/L. Blot is representative of 3 separate experiments. B, Autoradiography shows phosphorylation of recombinant glutathione S-transferase–fusion IB by immunoprecipitated IKK (see details in text). E2 and Dex concentration was 10 µmol/L. The experiment was repeated 3 times with comparable results.

Because IB degradation is triggered by phosphorylation by IB kinase (IKK), we studied whether E₂ treatment was associated with inhibition of IKK. LPS markedly increased IKK activity (Figure 7B). Pretreatment with E₂, but not with E₂, decreased LPS-induced IKK kinase activity nearly to baseline level. As controls, samples were immunoprecipitated with an irrelevant antibody (versus endothelial NO synthase), and others received a mutated IB protein that cannot be phosphorylated. These results suggest that E₂ inhibition of NF-B nuclear translocation may be mediated by a blockade of IKK activation, leading to decreased IB degradation.

Effect of E₂ and Dex on AP-1 and GATA Activation
To determine whether the steroids differentially regulate other transcription factors, as suggested by transfection studies, we performed gel shifts using oligonucleotides corresponding to AP-1 and GATA response elements on VCAM-1 promoter. E₂ markedly decreased LPS-induced AP-1 activation, whereas Dex produced a much weaker inhibition (Figure 8A). Additionally, both E₂ and Dex strongly inhibited LPS-induced nuclear binding to GATA consensus element, but E₂ was significantly more effective (Figure 8B). Thus, the different inhibitions of AP-1 and GATA binding may explain the different potencies of the 2 steroids on VCAM-1 expression.

View larger version (75K): [in this window] [in a new window]   Figure 8. E2 inhibits more than Dex LPS-triggered AP-1 and GATA nuclear DNA binding activity. A, E2 and Dex differently decrease LPS-induced AP-1 nuclear binding activity. The specificity of the shifted band is confirmed by the supershift with anti–c-Jun and anti–c-Fos antibodies, as well as by competition with a 5-fold excess of cold oligonucleotide. E2 and Dex concentration was 10 µmol/L. The experiment was repeated 4 times with equal results. B, E2 inhibits LPS-dependent GATA binding activity, with a greater potency than Dex. The specificity of the shifted band is confirmed by competition with increasing fold excesses of cold oligonucleotide. E2 and Dex concentration was 10 µmol/L. The experiment was repeated 3 times, with the same results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our results demonstrate that estrogen and Dex inhibit VCAM-1 expression by transcriptional and posttranscriptional mechanisms. Treatment with E₂ and Dex is associated with differential inhibition of NF-B, AP-1, and GATA transcription factors, resulting in reduced VCAM-1 promoter activity, mRNA, and protein synthesis. This leads to reduced endothelial adhesiveness toward monocytoid cells and may be the mechanism for some of the antiatherogenic effects of estrogen.

VCAM-1 regulation by E₂ has been described with contrasting results,^{11 20} and none of the previous reports clarified the underlying molecular mechanisms. Our findings confirm that E₂ decreases VCAM-1 expression in human endothelial cells. E₂ and Dex decreased LPS-induced endothelial VCAM-1 protein and mRNA levels, as well as monocytoid cell adhesion. E₂, however, was consistently more potent than Dex, suggesting distinct mechanisms. Indeed, the 2 steroids differentially modulate NF-B, AP-1, and GATA, reducing their ability to activate VCAM-1 promoter.

This study provides a comparison of the effects of pharmacological concentrations of estrogen and Dex. The effects of estrogen on VCAM-1 surface expression were apparent already at physiological (nanomolar range) concentrations.²¹ Most of our other studies, however, were performed using higher concentrations (10 µmol/L), at which inhibitory effects were more evident. This discrepancy between in vitro and in vivo concentrations of estrogen has been noted previously.^{11 22} This suggests the relevance of an intact vascular microenvironment, or of the prolonged endothelial exposure to estrogens, as occurring in chronic replacement studies, in decreasing the concentration requirement for the inhibitory effect on adhesion molecule expression. Concentrations of Dex during anti-inflammatory dosing are in this concentration range, for which inhibitory effects on inflammatory transcription factors have been reported.²³

The inhibition of nuclear translocation and DNA binding of NF-B by E₂ represents a potentially important mechanism for the vascular protective effects of estrogens. NF-B is a proinflammatory transcription factor upregulating several genes involved in endothelial activation.⁶ The action of E₂ and Dex on NF-B may thus explain their effects on adhesion molecule expression^{11 14} and on other mediators implicated in monocyte adhesion, such as MCP-1.¹³ Furthermore, because NF-B activation is a common pathway for many atherogenic stimuli (such as oxidized LDL, reactive oxygen species, and cytokines),⁶ NF-B inhibition by E₂ may further explain its antiatherogenic properties. Indeed, a recent in vivo study confirms that estrogens reverse aortic VCAM-1 upregulation and leukocyte adhesion and intimal accumulation in hypercholesterolemic rabbits.¹⁰

Glucocorticoids inhibit NF-B, inducing the synthesis of NF-B inhibitor IB,²³ as well as through protein-protein interactions between the ligand-bound glucocorticoid receptor and NF-B subunit Rel A.²⁴ E₂ blocks NF-B activation in an osteoblast cell line through binding of the estrogen receptor with Rel A and p50 subunits.²⁵ Our results indicate that in human endothelial cells, NF-B inhibition by E₂ is associated with decreased IB degradation, possibly as a result of inhibition of LPS-induced IKK, proposing a novel mechanism for regulation of the -B transcription factor family by estrogens.

Our results demonstrate that inhibition of VCAM-1 by E₂ and Dex is associated with a reduction of AP-1 and GATA binding to VCAM-1 promoter and that differences in the potency of these steroids correlate with differences in the inhibition of these transcription factors.

Glucocorticoid receptor interacts with the c-Jun/c-Fos heterodimer, resulting in a mutual inhibition of DNA binding activity.²⁶ Less is known about estrogen, but, consistent with our findings, E₂ regulates AP-1 nuclear binding in a tissue-selective way.²⁷ Moreover, E₂ downregulates cytokine-induced TNF expression by inhibiting Jun NH₂-terminal kinase (JNK), resulting in reduced c-Jun and JunD expression and binding on TNF gene promoter.²⁸ In endothelial cells, AP-1 is activated by various proinflammatory stimuli, such as LDL²⁹ and cytokines,³⁰ and induces several genes involved in monocyte adhesion, such as MCP-1.³⁰ The simultaneous inhibition of AP-1 and NF-B by estrogens may thus result in a synergic endothelial atheroprotective effect, further enhanced by likely decreased interaction of AP-1 with NF-B, which increases NF-B activation of VCAM-1 promoter in endothelial cells.³¹

A new and intriguing finding of our study is E₂ inhibition of GATA binding to its consensus motif. Recent evidence suggests that some of the GATA transcription factors (GATA-4 and GATA-6) may be under hormonal control in reproductive tissues.³² Moreover, in erythroid precursor cells, estrogen receptor inhibits GATA-1 by ligand-dependent protein-protein interaction, downregulating several erythroid-specific genes.³³ GATA-2 may represent a potential target for E₂ in endothelial cells. In fact, endothelin-1 gene promoter is driven by GATA-2 binding on 2 GATA sites,³⁴ and estrogen has been shown to repress endothelin-1 production from endothelial cells.³⁵ Glucocorticoid interactions with GATA transcription factors have been reported in different systems, in which interactions of the glucocorticoid receptor with GATA-1 have been documented.³⁶

Interestingly, Dex, but not estrogen, prolongs VCAM-1 mRNA half-life. Dex would therefore exert 2 opposing actions on VCAM-1 expression, reducing transcription through inhibition of NF-B– and GATA-dependent promoter activation on the one hand and acting at the posttranscriptional level increasing VCAM-1 mRNA stability on the other. These data agree with other reports describing posttranscriptional regulations of the expression of various genes by glucocorticoids in mammalian cells,³⁷ although no studies are available on the molecular mechanisms of these actions. The lack of such an effect for E₂ may contribute, in part, to its greater inhibitory effect on VCAM-1 expression compared with Dex.

In conclusion, our findings characterize the mechanisms through which estrogen and glucocorticoid decrease cytokine-induced endothelial activation, demonstrating differential inhibitions of NF-B, AP-1, and GATA transcription factors, and help explain the molecular basis of some of the antiatherogenic effects of estrogen on the vascular wall.

	Acknowledgments

 
This study was supported in part by NIH Grant HL-52233 and by the Italian National Research Council. J.K.L is an Established Investigator of the American Heart Association. T.S. is a recipient of a Travel Grant by Scuola Superiore di Studi e di Perfezionamento "S. Anna." We thank Guido Lazzerini, Robert Vignali, and Martin Spiecker for their helpful advice; Alan E. Wakeling (AstraZeneca) for ICI 182,780; Michael A. Gimbrone, Jr, for E1/6 and E1/1 antibodies; Tucker Collins for VCAM-1 promoter constructs; and Michael Karin for the mutated IB.

Received January 27, 2000; accepted May 4, 2000.

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