Competition for p300 Regulates Transcription by Estrogen Receptors and Nuclear Factor-κB in Human Coronary Smooth Muscle Cells
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-κB component, in smooth muscle cells that express ERα and ERβ after exposure to 17β-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-κB–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 17β-estradiol may play an important role in the cytokine-induced expression of immune and inflammatory genes implicated in atherogenesis.
- estrogen receptors α/β
- intercellular adhesion molecule-1
- nuclear factor-κB
- transcriptional coactivator p300
- vascular smooth muscle cells
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-κB (NF-κB), which in turn promotes transcription of genes that encode inflammatory proteins.4 In most cells, including vascular smooth muscle cells (SMCs), NF-κB is sequestered in the cytoplasm and is activated by a variety of stimuli, including viral infection and cytokines such as tumor necrosis factor α (TNFα).5 NF-κB 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 (ERα and ERβ) after exposure to 17β-estradiol (E2) for 72 hours and that these receptors affect transcriptional activation of inflammatory genes by NF-κB.
Materials and Methods
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). TNFα and the TNFα Direct kit were from Roche and Intergen, respectively. ER antibodies were from Santa Cruz Biotechnology. The following plasmids have been described before: 3XκB-CAT and 3XκB 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 pERα (B. Katzenellenbogen, University of Illinois, Champaign-Urbana, Ill), and human ERβ 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.
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 ERα (sc-8002 and sc-8005) 20 μg/mL or ERβ (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 for ER were performed with lysates of whole SMCs pretreated with E2 (10 nmol/L for 72 hours). Nuclear extracts of SMCs treated with TNFα 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, κB-deleted ICAM-1-CAT, 3XκB-CAT, 3XmutκB-CAT, and ERE2-CAT or cotransfected with plasmids expressing the κB proteins p50 (0.1 μg) and p65 (0.01 to 1 μg), the ER plasmids (ERα and ERβ) 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.
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.
Immunocytochemistry and Immunoblotting for ERα and ERβ 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. ERα and ERβ 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, ERβ was expressed in higher amounts than ERα, and exposure of cells to antibodies preadsorbed with their immunizing peptides abolished the staining. Single staining of SMCs with antibodies to ERα and PCNA and dual staining for ERβ 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 ERα and ERβ,10 11 served as positive controls (Figures 1i⇓ through 1l). SMC lysates were immunoblotted with anti-ERα and anti-ERβ antibodies after exposure of cells to E2 for 72 hours (Figure 2⇓).
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⇓).
Effect of E2 on NF-κB Translocation
Pretreatment with E2 (1 or 10 nmol/L) for 72 hours and then with TNFα for 1 hour resulted in translocation of NF-κB to the nucleus. E2 treatment did not interfere with NF-κB nuclear translocation, as shown by immunoblotting of nuclear extracts (Figure 4⇓).
Effect of E2 on Activation of TNFα-Dependent ICAM-1-CAT
SMCs exposed to TNFα (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 TNFα (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⇓).
ERα Interferes with p65-Dependent Activation of ICAM-1-CAT
To determine whether E2 inhibits NF-κB and whether this effect of E2 is ER-dependent, we cotransfected SMCs with ICAM-1-CAT, p65 or p50, and ERα expression vectors (Figure 6A⇓). ICAM-1-CAT activity was increased 5.5-fold by p65 but not by p50. ERα 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-κB–sequence, because SMCs cotransfected with p65 and NF-κB–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: ERα did not block p65-dependent activation in the absence of E2 (not shown). Furthermore, repression by E2 /ERα 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.
Mutual Repression of ERα and ERβ and p65-Dependent Transcription
The ICAM-1 promoter has binding sites for transcription factors other than NF-κB,9 which could influence the interaction of ERs with p65. Therefore, we cotransfected SMCs with a reporter containing 3 NF-κB–binding sites only or with a reporter with mutated NF-κB elements (3XκB-CAT and 3XmκB-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-κB sites. This was inhibited by 64% by 0.1 μg of ERα and by 90% by 0.5 μg of ERα. Again, transfection of 10-fold more p65 partially rescued p65-induced transcription. SMCs transfected with the mutated reporter did not respond; cotransfection of 3XmκB-CAT with p65, p65, and ERα or with p65 and ERβ did not yield any transcriptional activity. Because SMCs seem to express more ERβ than ERα, the experiment was repeated using an ERβ expression vector. As expected, p65 activated its cognate promoter, and overexpressed ERβ (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 ERα (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 ERα 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. ERβ cotransfection and E2 treatment caused 4-fold transcriptional activation, and cotransfection with p65 and E2 treatment caused a 50% repression of ERβ-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 ERα and p65 (COS-1)
To examine a possible direct protein-protein interaction between ERα 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 ERα or ERβ, 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 ERα-, ERβ-, 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, ERα or ERβ 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 3XκB-CAT reporter and vectors for p65, ERα, and p300, we determined that the negative regulation of ER/p65 is reciprocal. P300 overexpression can significantly reduce repression of p65 transcriptional activity by ERα (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/3XκB-CAT. In many experiments, exogenous p300 had little effect on ERE-CAT or 3XκB-CAT activation. However, in a few experiments, transfected p300 caused a 2-fold increase in CAT activity.
We have recently shown by electrophoretic mobility shift assay that treatment for 1 hour with E2 inhibits TNFα-induced NF-κB–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-κB subunits p65 and p50 is not inhibited by pretreatment with E2 for 72 hours. These findings suggest that E2 and ER affect NF-κB–DNA binding. We additionally found that estrogen inhibits TNFα-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 ERα and ERβ (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-κB 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 ERα/ERE and p65 interaction (not shown). For 17β-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 ERα and ERβ.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-κB.13 14 Some interactions of nuclear receptors with other nuclear proteins, such as p300 and NF-κB, 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 ERα and ERβ and NF-κB may depend on the state of activation of SMCs. In the presence of stressors such as CMV and TNFα, activated NF-κB may suppress E2- and ER-dependent transcription by binding limiting amounts of p300. In the absence of potent and prolonged NF-κB activators, ERα and ERβ have the potential of inhibiting NF-κB 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.
- © 2000 American Heart Association, Inc.
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