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(Circulation Research. 2000;87:1006.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Competition for p300 Regulates Transcription by Estrogen Receptors and Nuclear Factor-B in Human Coronary Smooth Muscle Cells

Edith Speir, Zu-Xi Yu, Kazuyo Takeda, Victor J. Ferrans, Richard O. Cannon, III

From the Cardiology Branch (E.S., R.O.C.) and Pathology Section (Z-X.Y., K.T., V.J.F.), National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md.

Correspondence to Edith Speir, National Institutes of Health, Building 10, Room 7B15, 10 Center Dr, Bethesda, MD 20892-1650. E-mail speire{at}nih.gov

	Abstract

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Abstract—Previous studies suggest that estrogen may prevent expression of cell adhesion molecules implicated in vascular inflammation associated with atherosclerosis. We demonstrate the interaction and reciprocal interference of estrogen receptors (ERs) with p65, the nuclear factor-B component, in smooth muscle cells that express ER and ERß after exposure to 17ß-estradiol for 48 to 72 hours. ER and p65 do not associate directly, as shown by lack of coprecipitation, but instead compete for limiting amounts of p300, a close relative of the CREB-binding protein. Overexpressed p300 significantly reduced the inhibitory effect of ER on p65-dependent transcription as well as the inhibitory effect of p65 on ER-dependent transcription. These actions were ligand-dependent. The expression of both ER and nuclear factor-B–dependent reporter genes was partially rescued from ER/p65 mutual inhibition by transient transfection of smooth muscle cells with a p300 expression vector. These actions of 17ß-estradiol may play an important role in the cytokine-induced expression of immune and inflammatory genes implicated in atherogenesis.

Key Words: estrogen receptors /ß • intercellular adhesion molecule-1 • nuclear factor-B • transcriptional coactivator p300 • vascular smooth muscle cells

	Introduction

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Estrogen replacement therapy in postmenopausal women is associated with a 40% to 50% reduction in the risk of coronary artery disease.¹ Although a substantial portion of these effects may be mediated through changes in serum lipids, other mechanisms also are likely to be involved, as suggested by inhibition of expression of cell adhesion molecules implicated in atherosclerosis.² Estrogen receptors (ERs) are sequence-specific DNA-binding transcription factors that alter patterns of gene expression in response to their ligand.³ Their transcriptional activity can be stimulated or inhibited by interaction of steroid receptors with other transcription factors. In particular, glucocorticoid receptors have recently been shown to interact with p65/RelA, a component of nuclear factor-B (NF-B), which in turn promotes transcription of genes that encode inflammatory proteins.⁴ In most cells, including vascular smooth muscle cells (SMCs), NF-B is sequestered in the cytoplasm and is activated by a variety of stimuli, including viral infection and cytokines such as tumor necrosis factor (TNF).⁵ NF-B is important in cytomegalovirus (CMV)-induced viral and cellular gene expression, and CMV nucleic acid sequences are frequently found in arterial SMCs in situ.^{6 7 8}

We have recently shown that estradiol has antioxidant effects that interfere with CMV-induced signaling of viral and cellular gene expression in infected SMCs. These beneficial effects were probably attributable to a direct action, because ER subtypes were undetectable in these SMCs. We now report that coronary SMCs express functional estrogen receptors (ER and ERß) after exposure to 17ß-estradiol (E₂) for 72 hours and that these receptors affect transcriptional activation of inflammatory genes by NF-B.

	Materials and Methods

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Cells and Reagents
Human coronary SMCs (Biowhittaker) from a female (age 38) and male (age 58) donor were grown as described.⁷ COS-1 cells were from American Type Culture Collection (CRL-1650). TNF and the TNF Direct kit were from Roche and Intergen, respectively. ER antibodies were from Santa Cruz Biotechnology. The following plasmids have been described before: 3XB-CAT and 3XB mutant-CAT.⁷ The following plasmids were gifts: pMT2T65 and pMT2Tp50 (U. Siebenlist, National Institute of Allergy and Infectious Diseases, NIH), ERE2-TATA-CAT and pER (B. Katzenellenbogen, University of Illinois, Champaign-Urbana, Ill), and human ERß plasmid (S. Ali, Imperial College of Medicine, London, UK). The intercellular adhesion molecule-1 (ICAM-1)–CAT constructs (-277/+1 and -182/+1) were from S.W. Caughman,⁹ (Emory University, Atlanta, Ga), and the p300 coactivator was from G. Nabel (Vaccine Research Center, National Institutes of Health, Bethesda, Md). E₂ was obtained from Sigma, and ICI 782,780 was from Zeneca Pharmaceuticals.

Immunocytochemistry
SMCs were grown in 4-chamber coverslips for 6 hours; every 24 hours thereafter, the medium was changed to phenol red-free EBM (Clonetics) containing 2.5% charcoal-filtered serum and E₂ (10 nmol/L). At 72 hours, SMCs were fixed with methanol at -10°C and treated overnight at 4°C with antibodies against ER (sc-8002 and sc-8005) 20 µg/mL or ERß (sc-6822 and sc-6820) 10 µg/mL and proliferating cell nuclear antigen (PCNA) 5 µg/mL followed by the appropriate secondary antibodies and FITC-conjugated or Texas red–conjugated streptavidin. The experiments were repeated 5 times. For controls, we used antibodies preadsorbed with their immunizing peptides or nonimmune IgG. Nuclei were counterstained with 4,[prime]6-diamidino-2-phenylindole dihydrochloride (DAPI) 0.1 µg/mL (Sigma) (blue) for 15 minutes. MCF-7 cells from American Type Culture Collection were grown in phenol-red free medium and served as positive controls. We also performed immunostaining for ICAM-1 using biotinylated anti-ICAM antibodies (R&D Systems) and fluorescein-conjugated streptavidin.

Immunoblots
Immunoblots for ER were performed with lysates of whole SMCs pretreated with E₂ (10 nmol/L for 72 hours). Nuclear extracts of SMCs treated with TNF and with or without E₂ (1 or 10 nmol/L) were immunoblotted to determine the effect of E₂ on p65 and p50 translocation (antibodies sc-109 and sc-7178 at 2 µg/mL, Santa Cruz). Nuclear extracts were prepared as described elsewhere.⁷ Two separate experiments were performed.

Transfections and CAT Assays
SMCs were grown to 90% confluence in 100-mm dishes and transfected 20 hours later with reporter constructs ICAM-1-CAT, B-deleted ICAM-1-CAT, 3XB-CAT, 3XmutB-CAT, and ERE2-CAT or cotransfected with plasmids expressing the B proteins p50 (0.1 µg) and p65 (0.01 to 1 µg), the ER plasmids (ER and ERß) 0.01 to 0.5 µg, and the p300 cofactor plasmid (1 to 2 µg). DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethyl-sulfate; Roche) lipofection agent was used as described previosuly.⁷ Three experiments were performed with each combination.

Statistical Analysis
All CAT assays were performed in triplicate with separate cell cultures. Data are presented as mean±SD. One way ANOVA analysis with Tukey-Kramer multiple comparisons test was performed using Instat 3 Software.

	Results

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Immunocytochemistry and Immunoblotting for ER and ERß Expression
ERs were not detectable in these SMCs (at passage 4, the lowest passage available). In SMCs kept in medium containing charcoal-filtered serum (2.5%) and E₂ (10 nmol/L) or vehicle (0.1% ethanol) for 48 to 72 hours, ERs were detectable in E₂-treated (but not in vehicle-treated) cells by both immunocytochemistry and immunoblotting. ER and ERß were immunolocalized to the cytoplasm and perinuclear area of SMCs from both the female (Figures 1a through 1d) and male donor. In both cell types, ERß was expressed in higher amounts than ER, and exposure of cells to antibodies preadsorbed with their immunizing peptides abolished the staining. Single staining of SMCs with antibodies to ER and PCNA and dual staining for ERß and PCNA showed localization of ER to the cytosol and of PCNA to the nuclei (Figures 1e through 1h). MCF-7 breast cancer cells, which express both ER and ERß,^{10 11} served as positive controls (Figures 1i through 1l). SMC lysates were immunoblotted with anti-ER and anti-ERß antibodies after exposure of cells to E₂ for 72 hours (Figure 2).

View larger version (44K): [in this window] [in a new window]   Figure 1. Figure 1. SMCs express ER and ERß (green). Nuclei are stained with DAPI (blue). a through d, SMCs from female donor. There are small amounts of ER (a), whereas ERß is prevalent (b). ER and ERß are immunolocalized to the cytoplasm and perinuclear area. Nonimmune IgG (c) and preincubation of antibodies with the immunizing peptide (d) served as negative controls. P indicates peptide. e through h, Staining for ER receptors (green) and PCNA (red). e, Cytoplasmic staining for ER. f, Nuclear staining for PCNA (purple, because it is colocalized with DAPI). Dual staining (g) shows nuclear staining for PCNA (red) and cytoplasmic staining for ERß (green). IgG control (h) shows only blue staining. i through l, Staining for ER and ERß in MCF-7 breast cancer cells. The reaction for ER is mostly nuclear but also cytoplasmic (i) (green). ERß staining (j) (red) is mostly cytoplasmic but also nuclear. Colocalization of the signals for ER and ERß (k) is shown in yellow and is observed mainly in the nuclei. Cells stained with normal IgG are negative (l). Bars for each row=20 µm.

View larger version (69K): [in this window] [in a new window]   Figure 2. Figure 2. Immunoblots for ER expression in SMCs. Lane 1, SMCs exposed to vehicle (0.1% ethanol); lanes 2 and 3, SMCs exposed to E2 (1 and 10 nmol/L, respectively) for 72 hours; and lane 4, lysates of MCF-7 breast cancer cells were used as positive controls. Exposure to antibodies against ERß or ER resulted in bands migrating at 55 kDa and 66 kDa and comigration with bands from the lysates of MCF-7 cells.

SMCs Express Functional ERs
To determine if these receptors (above) are competent, we transfected SMCs with a reporter construct containing 2 estrogen receptor–responsive elements (ERE2-CAT). Cells transfected with ERE2-CAT had background CAT activity, but treatment with E₂ (10 nmol/L and 100 nmol/L) caused a dose-dependent increase in CAT activity (Figure 3).

View larger version (10K): [in this window] [in a new window]   Figure 3. Figure 3. Bar graphs of ERE-CAT activity in SMCs. Lane 1, Treatment with vehicle had little effect on reporter activity. Lanes 2 and 3, SMCs pretreated for 72 hours with E2 (10 nmol/L) and then transfected with an ER-responsive reporter (ERE2-CAT) were exposed (40 hours later) to E2 (10 nmol/L or 100 nmol/L). E2 treatment stimulated transcriptional activity 2- or 3-fold. *P<0.01.

Effect of E₂ on NF-B Translocation
Pretreatment with E₂ (1 or 10 nmol/L) for 72 hours and then with TNF for 1 hour resulted in translocation of NF-B to the nucleus. E₂ treatment did not interfere with NF-B nuclear translocation, as shown by immunoblotting of nuclear extracts (Figure 4).

View larger version (47K): [in this window] [in a new window]   Figure 4. Figure 4. Immunoblots for p65 and p50 expression in SMCs. Estrogen does not interfere with TNF-induced translocation of NF-B to the nucleus. SMCs were treated with E2 for 72 hours and then with TNF (500 U/mL) for 1 hour. Nuclear extracts were immunoblotted with anti-p65 or p50 antibodies. A and B, Untreated controls (lane 1), TNF (lane 2), E2 (1 nmol/L) and TNF (lane 3), and E2 (10 nmol/L) and TNF (lane 4).

Effect of E₂ on Activation of TNF-Dependent ICAM-1-CAT
SMCs exposed to TNF (2 ng/mL) for 1 hour express ICAM-1 mRNA, and E₂ treatment inhibits ICAM-1 mRNA at 3 hours.¹² We examined whether E₂/ER can affect ICAM-1 message via transcriptional interference. In cells transfected with the reporter ICAM-1-CAT (-277/+1; 2 µg), treatment with TNF (2 ng/mL) for 1 hour caused a 4-fold increase in ICAM-CAT activity. This was attenuated dose-dependently by pretreatment with E₂ for 48 hours and an additional dose of E₂ for 24 hours after TNF exposure (Figure 5).

View larger version (15K): [in this window] [in a new window]   Figure 5. Figure 5. Bar graph showing activation of SMCs pretreated with E2 and transfected with an ICAM-1-CAT (-277/+1) reporter with an essential NF-B site. Bar 1, Vehicle; bars 2 and 3, E2 (10 or 100 nmol/L); bars 4 through 7, TNF (2 ng/mL) for 1 hour and TNF plus E2 (1, 10, and 100 nmol/L, respectively). TNF caused 4-fold activation of ICAM-CAT; pretreatment for 48 hours with E2 dose-dependently inhibited transcriptional activation of the ICAM promoter. *P<0.01.

ER Interferes with p65-Dependent Activation of ICAM-1-CAT
To determine whether E₂ inhibits NF-B and whether this effect of E₂ is ER-dependent, we cotransfected SMCs with ICAM-1-CAT, p65 or p50, and ER expression vectors (Figure 6A). ICAM-1-CAT activity was increased 5.5-fold by p65 but not by p50. ER at 0.5 µg completely blocked p65 (0.1 µg) transcriptional activity but only partially blocked activity (3-fold) of 10-fold higher p65 concentrations (1 µg). CAT activity was dependent on the intact NF-B–sequence, because SMCs cotransfected with p65 and NF-B–deleted ICAM-1-CAT lacked activity. These effects were similar in SMCs from both a female (Figure 6B) and male donor and were ligand-dependent: ER did not block p65-dependent activation in the absence of E₂ (not shown). Furthermore, repression by E₂ /ER was rescued by the ER blocker ICI 782,780. SMCs were treated with E₂ (10 nmol/L) for 48 hours before transfection and for 24 hours at 15 hours after transfection.

View larger version (18K): [in this window] [in a new window]   Figure 6. Figure 6. Bar graphs of ICAM-CAT activation in SMCs. A, SMCs were cotransfected with 2 µg ICAM-1-CAT and with expression plasmids for the NF-B components p65 or p50 and with increasing amounts of the human ER plasmid. Cells were treated with E2 (10 nmol/L). Bars 1 through 4, ICAM-CAT activity was not influenced by ER overexpression; bars 5 to 10, p65 increased ICAM-CAT 5.5-fold and this was inhibited dose-dependently by cotransfected ER. Repression of CAT-activity by ER was relieved by transfecting 10-fold higher amounts of p65. Bars 11 through 14, Overexpression of p50 had little effect on transcription. *P<0.01. SMCs transfected with the 6B-deleted reporter ICAM-1-CAT (-182/+1) had little or no response (data not shown). B, SMCs were transfected with ICAM-1-CAT, ER, and p65 and treated with E2 and ICI 182,780 (10 nmol/L and 1 µmol/L, respectively). Results are similar to those shown in Figure 5A: p65 transactivates ICAM, and ER dose-dependently interferes with this activation in SMCs from a female and a male donor. Treatment with 100-fold excess of the complete ER blocker ICI 182,780 relieves interference of ER with p65-induced transcription. *P<0.01.

Mutual Repression of ER and ERß and p65-Dependent Transcription
The ICAM-1 promoter has binding sites for transcription factors other than NF-B,⁹ which could influence the interaction of ERs with p65. Therefore, we cotransfected SMCs with a reporter containing 3 NF-B–binding sites only or with a reporter with mutated NF-B elements (3XB-CAT and 3XmB-CAT) and with p65 and ER expression vectors. CAT activity was increased 6- to 10-fold by p65, and this was dependent on intact NF-B sites. This was inhibited by 64% by 0.1 µg of ER and by 90% by 0.5 µg of ER. Again, transfection of 10-fold more p65 partially rescued p65-induced transcription. SMCs transfected with the mutated reporter did not respond; cotransfection of 3XmB-CAT with p65, p65, and ER or with p65 and ERß did not yield any transcriptional activity. Because SMCs seem to express more ERß than ER, the experiment was repeated using an ERß expression vector. As expected, p65 activated its cognate promoter, and overexpressed ERß (0.1 and 0.5 µg) attenuated p65-induced CAT activity by 50% and 80%, respectively, whereas cells with the mutated reporter lacked any response. SMCs were treated with E₂ (10 nmol/L) for 48 hours before transfection and for 15 hours after transfection. We also transfected SMCs with an ER-responsive ERE-CAT reporter and with ER or p65 expression vectors. Cotransfection of the reporter with ER (200 ng) caused a small increase in CAT activity (2.1-fold), whereas addition of E₂ (10 nmol/L) resulted in a 9-fold activation. When ER was cotransfected with p65 in the presence of E₂, the ER-induced CAT transcription was reduced by 40%. Addition of ICI 182,780 together with E₂ abrogated this interaction. ERß cotransfection and E₂ treatment caused 4-fold transcriptional activation, and cotransfection with p65 and E₂ treatment caused a 50% repression of ERß-dependent transcription. These results suggest that both transcriptional activation by ER and repression by p65 are ligand-dependent, as shown by the lack of activation in the absence of E₂ and by the reversal of the effect of E₂/ER in the presence of the complete ER blocker ICI 182, 780 (not shown).

Immunoprecipitation of ER and p65 (COS-1)
To examine a possible direct protein-protein interaction between ER and p65, which could explain mutual transcriptional interference, we performed immunoprecipitation of SMCs transfected ER and p65. Several experiments and subsequent immunoblotting gave variable and unsatisfactory results because of very low protein expression in SMCs. However, we found that COS-1 cells, when transfected with ER or ERß, consistently expressed detectable amounts of ER and p65. We therefore cotransfected COS-1 cells with both p65 and ER and precipitated the cell lysates with anti-p65 antibodies, which were positive when blotted with p65 antibodies but negative when blotted with anti-ER antibodies. Lysates precipitated with anti-ER antibody were positive when blotted for ER but negative when blotted with anti-p65 antibodies. The experiment was repeated 3 times with the same results. This indicates that direct protein-protein interaction between p65 and ER is not likely in COS-1 cells.

Role of Coactivator p300 in the Interaction of ER and p65
Both ER and p65 recruit and interact with the p300 protein coactivator.^{13 14} To examine whether mutual transcriptional repression by ER and p65 might be attributable to competition for limited amounts of p300, we cotransfected SMCs (pretreated for 48 hours with E₂) with ERE-CAT and ER-, ERß-, p65-, or p300-expression vectors. Cotransfection with p300 did not modify CAT activity (Figure 7, bar 2) beyond the 2-fold increase in activity noted with endogenous ER and estradiol (Figure 3). Furthermore, ER or ERß caused 6- or 5-fold activation (Figure 7, bars 3 and 7), exogenous p300 had an inhibitory effect on ER-dependent transcription by 10% and 34% (bars 4 and 8), and p65 inhibited ER activity by 73% and 67% (bars 5 and 9). The inhibitory effect of p65 was reduced 50% and 33% by cotransfection of p300 (bars 6 and 10). Both ER and p65 recruit p300, and by cotransfecting SMCs with the p65-responsive 3XB-CAT reporter and vectors for p65, ER, and p300, we determined that the negative regulation of ER/p65 is reciprocal. P300 overexpression can significantly reduce repression of p65 transcriptional activity by ER (Figure 8). Experiments performed in the absence of E₂ pretreatment showed a lack of adequate ER/ERE-CAT activation, minimal p300 interaction with ER/p65/ERE-CAT, and inconsistent interaction of p65/ER/3XB-CAT. In many experiments, exogenous p300 had little effect on ERE-CAT or 3XB-CAT activation. However, in a few experiments, transfected p300 caused a 2-fold increase in CAT activity.

View larger version (20K): [in this window] [in a new window]   Figure 7. Figure 7. Coprecipitation indicated that there is no direct interaction of p65 with ER in this model, but both proteins interacted with p300 coactivator. To examine the possibility of competition for p300, SMCs were pretreated for 48 hours with 10 nmol/L E2 and then transfected with ERE2-CAT, ER, p65, and p300 plasmids. The graph shows that overexpressed p300 did not modify CAT activity, which was increased 2-fold by endogenous ER (bar 2). Exogenous (and endogenous) ER caused a 6-fold increase in reporter activity. Cotransfection of ER and p300 caused a 10% and 34% decrease in ER- and ERß-induced transcription (bars 4 and 8), p65 inhibited ER-dependent CAT activity (73% and 67%, bars 5 and 9), and cotransfection of ER, p65, and p300 partially rescued ER-dependent transcription (bars 6 and 10). Results were similar with ERß (bars 7 to 10). This is not surprising, because p300 interacts with the AF-2 region of the ER, which is homologous in both ER isotypes. *P<0.01.

View larger version (20K): [in this window] [in a new window]   Figure 8. Figure 8. Partial rescue by p300 is reciprocal. The transfection experiment in Figure 7 was set up in reverse: SMCs were transfected with 3XB-CAT with or without 1 µg or 2 µg of p300 (bars 1 through 3). As shown above, cotransfection of p65 activates its cognate promoter more than 12-fold (bar 4,*P<0.01). ER at 0.5 µg and 1 µg represses p65-induced CAT activity by 20% and 56%, respectively (bars 6 and 7), and p300 (1 to 2 µg) partially rescues p65-dependent CAT activity (bars 9 and 11 vs bar 7). *P<0.01. The mutated B-reporter did not respond to cotransfection with p65 or p300 (data not shown).

	Discussion

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We have recently shown by electrophoretic mobility shift assay that treatment for 1 hour with E₂ inhibits TNF-induced NF-B–binding to its cognate DNA.¹² We now demonstrate by immunoblotting nuclear lysates from SMCs exposed to E₂ for 72 hours that nuclear translocation of the NF-B subunits p65 and p50 is not inhibited by pretreatment with E₂ for 72 hours. These findings suggest that E₂ and ER affect NF-B–DNA binding. We additionally found that estrogen inhibits TNF-induced ICAM-1 mRNA expression in SMCs at 3 hours (not shown). In this study, we asked whether there might also be ER-dependent effects. We detected both ER and ERß (Figure 1); in 5 experiments, both receptors immunolocalized to the cytoplasm. Clearly, at least some of the ERs were localized to the nucleus at some point, perhaps transiently, because SMCs transfected with an ER-responsive reporter (ERE-CAT) responded to E₂ treatment by increased CAT activity. A positive nuclear reaction for PCNA was found in 60% of SMCs, indicating that the negative nuclear staining for ER was not attributable to lack of antibody penetration. Recent reports by other laboratories also show immunolocalization of functional ERs to the cytosol of vascular SMCs.^{15 16} Of note, in ER-transfected SMCs and COS-1 cells, receptors immunolocalized to both nuclei and cytoplasm. It is possible that the conditions of the transfection experiments (overexpression of ER) may lead to suppression of the cytosolic retention mechanism.

The mutual transcriptional repression of ER and p65 toward the ICAM-1, ERE, and NF-B reporters can be reduced by transfecting with 10-fold excess of the activating transcription factor (Figure 6A, bar 10). The same is true for the ER/ERE and p65 interaction (not shown). For 17ß-estradiol–induced transactivation, the N-terminal A/B domain (AF-1) and the C-terminal E/F domain of ERs are required. Ligand binding induces functional synergism between AF-1 and AF-2; the molecular mechanism was only recently elucidated. Ligand-induced functional synergism between AF-1 and AF-2 is mediated through p300 by its direct binding to both ER and ERß.¹⁷

Estrogen has been implicated not only in vasculoprotective¹⁸ but also in proinflammatory processes.¹⁹ The particular effect is dependent on tissue and cell type, cell-associated factors and cofactors, ER isotype and number, and gender. Clearly, the modifying cellular factors are different in cells activated by cytokines or in transformed cells.^{2 19 20}

The ER belongs to the nuclear receptor superfamily, and members of this family are known to exist in a complex of chaperones in the cytosol. Once activated by the respective ligand, ERs translocate to the nucleus, where they associate with multiple cofactors. These cofactors determine the potential transcriptional activation or repression of target genes by nuclear receptors.^{3 21} P300, a histone acetyltransferase, is a close relative of the CREB-binding protein and has been shown to be a component of the coactivator complex of nuclear receptors as well as of NF-B.^{13 14} Some interactions of nuclear receptors with other nuclear proteins, such as p300 and NF-B, seem to be cell type–specific but are generally ligand-dependent.^{13 21} E₂ is essential for activation of endogenous and transfected ERE (Figure 7) and is necessary for maximal inhibitory effect on p65-dependent activation (Figures 6 and 8). This indicates that p300 interacts with the ER ligand–binding domain.

Our findings suggest that interaction between ER and ERß and NF-B may depend on the state of activation of SMCs. In the presence of stressors such as CMV and TNF, activated NF-B may suppress E₂- and ER-dependent transcription by binding limiting amounts of p300. In the absence of potent and prolonged NF-B activators, ER and ERß have the potential of inhibiting NF-B by binding the available p300. This has important implications for the role of estrogen in processes such as vasculitis and atherogenesis in female patients.

Received August 18, 2000; revision received September 29, 2000; accepted September 29, 2000.

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				O. Bukulmez, D. B. Hardy, B. R. Carr, R. A. Word, and C. R. Mendelson Inflammatory Status Influences Aromatase and Steroid Receptor Expression in Endometriosis Endocrinology, March 1, 2008; 149(3): 1190 - 1204. [Abstract] [Full Text] [PDF]

				K. W. Nettles, G. Gil, J. Nowak, R. Metivier, V. B. Sharma, and G. L. Greene CBP Is a Dosage-Dependent Regulator of Nuclear Factor-{kappa}B Suppression by the Estrogen Receptor Mol. Endocrinol., February 1, 2008; 22(2): 263 - 272. [Abstract] [Full Text] [PDF]

				L. Sunday, C. Osuna, D. N. Krause, and S. P. Duckles Age alters cerebrovascular inflammation and effects of estrogen Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2333 - H2340. [Abstract] [Full Text] [PDF]

				H. A. Harris Estrogen Receptor-{beta}: Recent Lessons from in Vivo Studies Mol. Endocrinol., January 1, 2007; 21(1): 1 - 13. [Abstract] [Full Text] [PDF]

				C. R. Montague, M. G. Hunter, M. A. Gavrilin, G. S. Phillips, P. J. Goldschmidt-Clermont, and C. B. Marsh Activation of Estrogen Receptor-{alpha} Reduces Aortic Smooth Muscle Differentiation Circ. Res., September 1, 2006; 99(5): 477 - 484. [Abstract] [Full Text] [PDF]

				M. J. Hardman, A. Waite, L. Zeef, M. Burow, T. Nakayama, and G. S. Ashcroft Macrophage Migration Inhibitory Factor: A Central Regulator of Wound Healing Am. J. Pathol., December 1, 2005; 167(6): 1561 - 1574. [Abstract] [Full Text] [PDF]

				H. Grasberger, U. Ringkananont, P. LeFrancois, M. Abramowicz, G. Vassart, and S. Refetoff Thyroid Transcription Factor 1 Rescues PAX8/p300 Synergism Impaired by a Natural PAX8 Paired Domain Mutation with Dominant Negative Activity Mol. Endocrinol., July 1, 2005; 19(7): 1779 - 1791. [Abstract] [Full Text] [PDF]

				Y Zhou, S Eppenberger-Castori, U Eppenberger, and C C Benz The NF{kappa}B pathway and endocrine-resistant breast cancer Endocr. Relat. Cancer, July 1, 2005; 12(Supplement_1): S37 - S46. [Abstract] [Full Text] [PDF]

				R. H. Sohn, C. B. Deming, D. C. Johns, H. C. Champion, C. Bian, K. Gardner, and J. J. Rade Regulation of endothelial thrombomodulin expression by inflammatory cytokines is mediated by activation of nuclear factor-kappa B Blood, May 15, 2005; 105(10): 3910 - 3917. [Abstract] [Full Text] [PDF]

				C. C. Chadwick, S. Chippari, E. Matelan, L. Borges-Marcucci, A. M. Eckert, J. C. Keith Jr., L. M. Albert, Y. Leathurby, H. A. Harris, R. A. Bhat, et al. Identification of pathway-selective estrogen receptor ligands that inhibit NF-{kappa}B transcriptional activity PNAS, February 15, 2005; 102(7): 2543 - 2548. [Abstract] [Full Text] [PDF]

				V. X. Jin, Y.-W. Leu, S. Liyanarachchi, H. Sun, M. Fan, K. P. Nephew, T. H.-M. Huang, and R. V. Davuluri Identifying estrogen receptor {alpha} target genes using integrated computational genomics and chromatin immunoprecipitation microarray Nucleic Acids Res., December 17, 2004; 32(22): 6627 - 6635. [Abstract] [Full Text] [PDF]

				M. F. McCarty Targeting Multiple Signaling Pathways as a Strategy for Managing Prostate Cancer: Multifocal Signal Modulation Therapy Integr Cancer Ther, December 1, 2004; 3(4): 349 - 380. [Abstract] [PDF]

				K. H. Seo, H.-S. Lee, B. Jung, H.-M. Ko, J.-H. Choi, S. J. Park, I.-H. Choi, H.-K. Lee, and S.-Y. Im Estrogen Enhances Angiogenesis through a Pathway Involving Platelet-Activating Factor-Mediated Nuclear Factor-{kappa}B Activation Cancer Res., September 15, 2004; 64(18): 6482 - 6488. [Abstract] [Full Text] [PDF]

				K. L. Hamilton, F.N. Mbai, S. Gupta, and A.A. Knowlton Estrogen, Heat Shock Proteins, and NF{kappa}B in Human Vascular Endothelium Arterioscler. Thromb. Vasc. Biol., September 1, 2004; 24(9): 1628 - 1633. [Abstract] [Full Text] [PDF]

				J. A. Ospina, H. N. Brevig, D. N. Krause, and S. P. Duckles Estrogen suppresses IL-1{beta}-mediated induction of COX-2 pathway in rat cerebral blood vessels Am J Physiol Heart Circ Physiol, May 1, 2004; 286(5): H2010 - H2019. [Abstract] [Full Text] [PDF]

				D. C. Harnish, L. M. Albert, Y. Leathurby, A. M. Eckert, A. Ciarletta, M. Kasaian, and J. C. Keith Jr. Beneficial effects of estrogen treatment in the HLA-B27 transgenic rat model of inflammatory bowel disease Am J Physiol Gastrointest Liver Physiol, January 1, 2004; 286(1): G118 - G125. [Abstract] [Full Text] [PDF]

				H. Liu, E.-S. Lee, C. Gajdos, S. T. Pearce, B. Chen, C. Osipo, J. Loweth, K. McKian, A. De Los Reyes, L. Wing, et al. Apoptotic Action of 17{beta}-Estradiol in Raloxifene-Resistant MCF-7 Cells In Vitro and In Vivo J Natl Cancer Inst, November 5, 2003; 95(21): 1586 - 1597. [Abstract] [Full Text] [PDF]

				J. Frasor, J. M. Danes, B. Komm, K. C. N. Chang, C. R. Lyttle, and B. S. Katzenellenbogen Profiling of Estrogen Up- and Down-Regulated Gene Expression in Human Breast Cancer Cells: Insights into Gene Networks and Pathways Underlying Estrogenic Control of Proliferation and Cell Phenotype Endocrinology, October 1, 2003; 144(10): 4562 - 4574. [Abstract] [Full Text] [PDF]

				B. Martin-McNulty, D. M. Tham, V. da Cunha, J. J. Ho, D. W. Wilson, J. C. Rutledge, G. G. Deng, R. Vergona, M. E. Sullivan, and Y.-X. Wang 17{beta}-Estradiol Attenuates Development of Angiotensin II-Induced Aortic Abdominal Aneurysm in Apolipoprotein E-Deficient Mice Arterioscler. Thromb. Vasc. Biol., September 1, 2003; 23(9): 1627 - 1632. [Abstract] [Full Text] [PDF]

				M. R. Adams, D. L. Golden, T. C. Register, M. S. Anthony, J. B. Hodgin, N. Maeda, and J.K. Williams The Atheroprotective Effect of Dietary Soy Isoflavones in Apolipoprotein E-/- Mice Requires the Presence of Estrogen Receptor-{alpha} Arterioscler. Thromb. Vasc. Biol., November 1, 2002; 22(11): 1859 - 1864. [Abstract] [Full Text] [PDF]

				M. J. Evans, K. Lai, L. J. Shaw, D. C. Harnish, and C. C. Chadwick Estrogen Receptor {alpha} Inhibits IL-1{beta} Induction of Gene Expression in the Mouse Liver Endocrinology, July 1, 2002; 143(7): 2559 - 2570. [Abstract] [Full Text] [PDF]

				J. Pfeilschifter, R. Koditz, M. Pfohl, and H. Schatz Changes in Proinflammatory Cytokine Activity after Menopause Endocr. Rev., February 1, 2002; 23(1): 90 - 119. [Abstract] [Full Text] [PDF]