CBP Is a Dosage-Dependent Regulator of Nuclear Factor- B ...

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Molecular Endocrinology 22(2):263–272 Copyright © 2008 by The Endocrine Society doi: 10.1210/me.2007-0324

CBP Is a Dosage-Dependent Regulator of Nuclear Factor-␬B Suppression by the Estrogen Receptor Kendall W. Nettles, German Gil, Jason Nowak, Raphae¨l Me´tivier, Vandana B. Sharma, and Geoffrey L. Greene Department of Cancer Biology (K.W.N., G.G., J.N.), The Scripps Research Institute, Jupiter, Florida 33458; Unite´ Mixte de Recherche, Centre National de la Recherche Scientifique 6026 (Inte´ractions Cellulaires et Mole´culaires) (R.M.), Equipe Spatio-Temporal Regulation of Transcription in Eukaryotes, Universite´ de Rennes, Campus de Beaulieu, 35042 Rennes Cedex, France; Division of Oncology (V.B.S.), Stanford University School of Medicine, Stanford, California 94305; Ben May Institute for Cancer Research and Department of Biochemistry (G.L.G.), University of Chicago, Chicago, Illinois 60637 The estrogen receptor (ER) protects against debilitating effects of the inflammatory response by inhibiting the proinflammatory transcription factor nuclear factor-␬B (NF␬B). Heretofore cAMP response element-binding protein (CREB)-binding protein (CBP) has been suggested to mediate inhibitory cross talk by functioning either as a scaffold that links ER and NF␬B or as a required cofactor that competitively binds to one or the other transcriptional factor. However, here we demonstrate that ER is recruited to the NF␬B response element of the MCP-1 (monocyte chemoattractant protein-1) and IL-8 promoters and displaces CBP,

but not p65, in the MCF-7 breast cancer cell line. In contrast, ER displaced p65 and associated coregulators from the IL-6 promoter, demonstrating a gene-specific role for CBP in integrating inflammatory and steroid signaling. Further, RNA interference and overexpression studies demonstrated that CBP dosage regulates estrogen-mediated suppression of MCP-1 and IL-8, but not IL-6, gene expression. This work further demonstrates that CBP dosage is a critical regulator of gene-specific signal integration between the ER- and NF␬B-signaling pathways. (Molecular Endocrinology 22: 263–272, 2008)

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diverse signals related to its primary role as a mediator of the inflammatory response (3). Thus infection, hypoxia, and a variety of cytokines, including TNF␣, induce phosphorylation of I␬B inhibitory complexes that normally sequester NF␬B in the cytoplasm (4). This then leads to ubiquitination and proteosomal degradation of I␬B, which unmasks the nuclear localization signal of NF␬B, directing its translocation to the nucleus where it modulates transcription (5). Transcriptional regulation by NF␬B has been widely studied and requires several transcriptional coactivators, including members of the steroid receptor coactivator (SRC)1–3 family, cAMP response element-binding protein (CREB)-binding protein (CBP), and p300 CBP-associated factor (pCAF) (6, 7). MCP-1 (monocyte chemoattractant protein-1) is stimulated by NF␬B through a well-defined NF␬B response element located within the upstream enhancer of the gene (8). MCP-1 protein is associated with a high macrophage burden in breast tumors, early relapse, and other angiogenic and tumor-promoting factors (9, 10). MCP-1 also recruits macrophages in inflammatory bowel disease (11) and to atherosclerotic lesions (12). Serum levels of MCP-1 are increased in postmenopausal women, where it is associated with increased atherosclerotic burden, a condition that is reduced by hormone replacement or selective ER modulator therapy (13). The mechanism(s) by which

STRADIOL (E2) HAS both beneficial and damaging physiological effects through its binding to the estrogen receptors (ERs) ER␣ and ER␤ (1). ER modulates transcription by binding to specific DNA sequences, yet in the absence of direct DNA binding also controls transcription through its interactions with other factors, including activator protein 1 (AP-1) and nuclear factor-␬B (NF␬B). Indeed, many of the beneficial effects of E2 occur through negative regulation of NF␬B (2). Among the Rel gene family members that make up NF␬B, the predominant form is a heterodimer composed of p50 and p65. Activation of NF␬B occurs via First Published Online October 11, 2007 Abbreviations: AP-1, Activator protein 1; CBP, cAMP response element-binding protein (CREB)-binding protein; ChIP, chromatin immunoprecipitation; E2, estradiol; ER␣, estrogen receptor-␣; Grip1, glucocorticoid receptor-interacting protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione-S-transferase; LBD, ligand-binding domain; MCP-1, monocyte chemoattractant protein-1. NF␬B, nuclear factor-␬B; qPCR, quantitative PCR; pCAF, p300 CBP-associated factor; SDS, sodium dodecyl sulfate; siRNA, small interfering RNA; SRC, steroid receptor coactivator; SSC, standard sodium citrate; TBS, Tris-buffered saline. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community. 263

264 Mol Endocrinol, February 2008, 22(2):263–272

Nettles et al. • CBP Dosage in ER␣-NF␬B Cross Talk

ER␣ suppresses NF␬B-mediated signaling have not been clearly defined and may vary by gene and cell type. Proposed mechanisms include competition for limiting coactivators (14), reductions in DNA binding activity (15), regulation of I␬B␣ mRNA expression (16), and direct interaction of ER with coactivators (17). Demonstrating the widespread importance of this pathway, ER␣ was recently proven as an effective therapeutic target in suppressing NF␬B in animal models of septic shock, inflammatory bowel disease, and arthritis (2, 18). Here we used the MCP-1 gene to explore the molecular features through which ER␣ suppresses NF␬Bdependent transcription. A combination of approaches demonstrates that ER␣ suppresses MCP-1 expression at the level of transcription, but not through modulation of NF␬B activation, nuclear translocation, or DNA binding. Rather, ER␣ displaces CBP from the NF␬B binding site in the MCP-1 and IL-8 genes, but not the IL-6 gene. Modulating CBP levels demonstrates a parallel sensitivity for CBP dosage in ERmediated suppression of the MCP-1 and IL-8 genes, but not the IL-6 gene. Along with known effects of CBP dosage on a number of physiological processes and diseases (19–21), these findings establish gene-specific patterns of ER regulation and CBP utilization in the integration of hormonal and inflammatory signals.

RESULTS ER␣ Suppresses MCP-1 Transcription MCP-1 gene transcription is induced by TNF␣, and this requires NF␬B (8), but the gene lacks a defined estrogen response element. To determine the role of ER and NF␬B in regulating MCP-1, MCF-7 cells were treated with TNF␣ and/or estradiol (E2). As expected TNF␣ induced high levels of MCP-1 mRNA, yet this response was suppressed by E2 (Fig. 1A), and in a dose-dependent fashion (Fig. 1B). By contrast, agonists of the androgen or progesterone receptors failed to suppress the induction of MCP-1 mRNA by TNF␣ (Fig. 1B). Nuclear run-on assays demonstrated that E2 treatment significantly reduced the magnitude of MCP-1 transcription induced by TNF␣ (Fig. 1C); therefore, ligand-activated ER impairs MCP-1 transcription. The MCP-1 gene could be regulated at a number of different levels by E2, including transcription, the turnover of MCP-1 transcripts, or activation, nuclear translocation, and/or DNA binding by NF␬B. To investigate the effects of E2 on the rate of MCP-1 mRNA turnover, MCF-7 cells were treated with actinomycin-D to block transcription. E2 did not affect the rates of MCP-1 mRNA degradation after TNF␣ treatment (Fig. 1D). Furthermore, the expression of the upstream inhibitor of NF␬B, I␬B␣, was not transcriptionally regulated by E2 treatment (Fig. 1E). To assess whether the repressive effects of E2 were due to disruption of NF␬B DNA binding, we initially performed EMSAs using a radio-

Fig. 1. ER␣ Suppresses NF␬B-Dependent MCP-1 Transcription A, MCF-7 cells were treated with TNF␣ and/or E2 for 2 h and then processed for qPCR analysis. The mRNA levels were normalized to 18S mRNA. B, Northern blot of MCP-1 and GAPDH mRNA expression. MCF-7 cells were cultured for 3 d in steroid-depleted media and stimulated for 2 h with 50 ng/ml TNF␣ (as indicated). Cultures were supplemented with steroid receptor ligands at 10 nM unless otherwise specified: E, estradiol; OHT, 4-hydroxytamoxifen; 100 nM 4-hydroxytamoxifen; DES, diethylstilbestrol; E1, estrone; ORG 2058, a synthetic progestin; DHT, dihydrotestosterone. These blots are representative of four independent experiments. C, Nuclei from MCF-7 cells were isolated and subject to run-on transcription assays to measure levels of transcription. D, Actinomycin was used to arrest transcription in MCF-7 cells. MCP-1 mRNA was normalized to GADPH mRNA. E, Northern blot of I␬B␣ and GAPDH mRNA from MCF-7 cells treated with the indicated ligands. PR, progesterone receptor; Veh, vehicle.


labeled oligonucleotide comprised of the MCP-1 NF␬B response element and nuclear extracts from TNF␣-stimulated MCF-7 cells. E2 did not affect p65 DNA binding that was induced by TNF␣ (Fig. 2). E2 treatment also did not block nuclear translocation of p65, as shown by immunofluorescence analyses of TNF␣-stimulated MCF-7 cells (Fig. 3). Combined with the nuclear run-on assays, these data demonstrate that E2 suppresses MCP-1 gene expression through regulating transcription. ER␣ and p65 Physically Associate. ER␣ might impair NF␬B activity by competing for limiting cofactors, or through physical associations, which could be either direct or indirect in a complex with other cofactors. Immunofluorescence analyses demonstrated that ER␣ and p65 colocalize in MCF-7 cells in a liganddependent fashion (Fig. 3). To initially test whether ER␣ and p65 physically interact, we performed coimmunoprecipitation analyses of endogenous p65 and ER␣ proteins from whole-cell extracts of MCF-7 cells, treated ⫹/⫺ TNF␣ or E2 for 30 min, which demonstrated a TNF␣-dependent interaction (Fig. 4A). To gain insights into the domains of ER␣ that were required for interactions with p65 we assessed the suppressive activity a series of ER␣ deletion mutants on NF␬B activity in MCP-1 promoter-luciferase reporter assays and also examined their ability to interact with endogenous p65 by coimmunoprecipitation. Western blot analyses indicated that all of the ER␣ mutants tested were expressed at levels comparable to those of wild-type ER␣. The N-terminal A/B domain, the DNA-binding domain, and the hinge domain of ER␣ were all dispensable for the suppressive effects of ER␣ in the luciferase assay (Fig. 4B). In contrast, deletion of the ligand-binding domain (LBD), or a mutation of helix 12 that destroys the activation function 2 surface for coactivator recruitment (data not shown),

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Fig. 3. Immunofluorescence Analysis of ER␣ and p65 MCF-7 cells were grown on coverslips, treated for 30 min with ligands, methanol fixed, and stained for immunofluorescent microscopy. The TNF␣ treatment induced a diffuse nuclear and cytoplasmic staining for p65, as previously reported for MCF-7 cells (31). Ab, Antibody.

completely abrogated the suppressive effects of ER␣. The ER␣ LBD was also absolutely required for interaction with endogenous p65, because there was no detectible interaction in its absence, whereas the deletion of the DNA-binding domain or of the hinge domain had no effect on the physical association of ER␣ with endogenous p65 (Fig. 4B). Although the LBD is necessary for transcriptional repression and association with p65, it is not, however, sufficient to direct the association with p65. Specifically, glutathione-S-transferase (GST) fused to the ER␣ LBD was not able to pull down in vitro-translated p65 (Fig. 4C), although it did efficiently associate with the glucocorticoid receptor interacting protein 1 (Grip1) coactivator, in a ligand-dependent fashion. Furthermore, full-length in vitro-translated ER␣ did not immunoprecipitate with either in vitro-translated p65 or p50 (data not shown). Because ER␣ was capable of associating with p65 in cell extracts, these findings suggested that the functional interaction between ER␣ and NF␬B might involve another protein cofactor. CBP Is Sufficient for ER␣ to Suppress NF␬B

Fig. 2. Gel Shift of Radiolabeled NF␬B Response Element Oligonucleotide from MCP-1 Gene MCF-7 Cells Were Treated for 30 or 60 min and Then Made into Nuclear Extracts, Demonstrating that E2 Has No Effect on DNA Binding by p65. Ab, Antibody.

To identify potential bridging factors between ER␣ and NF␬B, we used MCF-7-ES cells, a clone of MCF-7 cells that was selected for high estrogen sensitivity (ES) for growth and that fail to exhibit E2-mediated repression of MCP-1 expression (Fig. 5A). The MCF-ES cells show a blunted TNF␣ response in the MCP-1 luciferase reporter assay and little to no response to cotreatment with E2 (Fig. 5A). In a gain of function assay, increasing amounts of transcriptional coregulators known to interact with NF␬B and ER␣ were transfected into these cells to identify those that could restore the E2-mediated suppression. As expected, in the absence of added coregulators, E2 did not influence the transcriptional activity of the MCP-1 luciferase reporter in MCF-7-ES cells in response to TNF␣ (Fig. 5B). Cotransfection of increasing amounts


Fig. 4. Cellular Associations between ER␣ and p65 A, Immunoprecipitation of MCF-7 cell extracts, treated with ligands for 30 min before cell lysis. A polyclonal ER␣ antibody (ER21) was used for precipitation, followed by Western blotting for p65 (Santa Cruz anti-p65 A20). B (left panel), Cos-1 cells were transfected with MCP-1 luciferase reporter and ER␣ expression plasmids. The next day, cells were treated with TNF␣ ⫹ E2 for 6 h and processed for luciferase activity; B (right panel), Cos-1 cells were transfected with p65 and ER␣ expression plasmids as indicated. After 48 h, cells were treated for 30 min with TNF␣ ⫹ E2 and lysed for protein extraction. Only the LBD deletion construct was unable to interact with p65 (asterisk), although it was expressed at high levels in the whole-cell extract (arrow). C, A GST-ER␣ LBD fusion protein bound to glutathione-Sepharose beads was treated with vehicle or 1 mM E2 for 1 h and then used to pull down in vitro-translated Grip1 or p65. After extensive washing, the bound proteins were eluted with glutathione and visualized with SDS-PAGE and autoradiography. AF2, Activation function 2; IP, immunoprecipitation; DBD, DNA-binding domain; V, vehicle; T, TNF␣; E, estradiol.

of an expression plasmid for Grip1/SRC-2 showed a statistically significant, dose-dependent coactivation of transcription but did not rescue the E2 response. Increasing amounts of a plasmid encoding Rac3/amplified in breast cancer 1/SRC-3 showed a weak and variable coactivation of TNF␣ responses, which was prevented by E2 treatment. In contrast, there was a robust coactivation with increasing amounts of CBP expression vector, and this was substantially blocked by E2 treatment (Fig. 5B). Among the other coactivators tested, only the CBP homolog p300 showed the same effect (data not shown). Specifically, the SRC1–3 family, peroxisomal proliferator-activated receptor-␥ coactivator 1, thyroid hormone receptor-associated protein 220, nuclear receptor corepressor, silencing mediator of retinoid and thyroid hormone receptor,


Fig. 5. Role of CBP in ER␣-NF␬B Cross Talk A, An MCP-1-promoter-luciferase reporter demonstrates the repressive effects of E2 on the MCP-1 gene. Cells were transfected with the reporter and treated with Vehicle, TNF␣, or TNF␣ ⫹ E2. Shown is the fold induction of the MCP-1 promoter activity relative to vehicle. We identified a clonal variant of MCF-7 cells that do not show the suppressive effects of E2, termed MCF-7-ES cells. B, MCP-1 promoter activity was measured in MCF-7-ES cells, which showed no suppression by E2 in the absence of added coactivator. The luciferase activity is shown relative to vehicle control. This luciferase reporter was cotransfected with increasing amounts of coactivator expression vector, or pBluescript to normalize the total DNA transfected. The next day, cells were treated for 6 h with the indicated ligands. Each data point was performed in triplicate, showing mean ⫹ SEM, and the experiments were repeated four to five times. AIB1, Amplified in breast cancer 1.

BRCA1, and pCAF all failed to rescue the E2-dependent suppressive response. It is noteworthy that CBP displayed a dosage-dependent effect, facilitating initial increases in TNF␣ response, but no suppression by E2, whereas higher amounts of CBP conferred E2-mediated repression of the TNF␣ response. Thus, CBP is associated with the repression of the MCP-1 gene by E2, and CBP is sufficient to confer suppression in cells that lack the E2-mediated suppressive response. Components and Assembly of the ER␣Repressive Complex CBP has been suggested to mediate inhibitory cross talk by functioning either as a scaffold that links ER and NF␬B or as a required cofactor that competitively binds to one or the other transcriptional factor (14, 17). To define the role of CBP more broadly in ER cross talk with NF␬B, we performed chromatin immunoprecipitation on the NF␬B response elements in the MCP-1 enhancer and also the promoters of the IL-6 and IL-8


genes. E2 is equally effective in suppressing TNF␣ induction of the IL-6 and IL-8 genes (see Fig. 7A), which are both well characterized as dependent upon NF␬B. Treatment of MCF-7 cells with E2 induced a strong association of both ER␣ and CBP with the estrogen response element region in the ER-responsive pS2 promoter (Fig. 6A), as shown previously (22, 23). The p65 protein demonstrated a TNF␣-dependent association with the NF␬B enhancer in the MCP-1 gene and with the IL-8 promoter (Fig. 6A), which was not altered by E2, consistent with the immunofluorescence and gel shift data showing that E2 does not alter nuclear translocation or DNA binding of p65 in these cells (Figs. 3 and 4C). TNF␣ treatment led to a weak association of ER␣ with both the MCP-1 enhancer and the IL-8 promoter, which was greatly strengthened by a combined treatment with E2 (Fig. 6A). Surprisingly, the TNF␣-dependent mobilization of CBP onto both the MCP-1 enhancer and the IL-8 promoter was displaced by E2 treatment. Re-chromatin immunoprecipitation (ChIP) experiments further show that CBP is relieved from the MCP-1 enhancer and IL-8 promoter upon integration of E2-bound ER␣ in a p65/pCAF complex (Fig. 6B). Together with the observation that CBP enables ER␣ to repress NF␬B-mediated stimulation of MCP-1 in MCF-7-ES cells, these results suggest a displacement mechanism underlying E2-mediated re-

Fig. 6. CBP Displacement Mediates Estrogen-Dependent Gene Repression A, Confluent MCF-7 cells were switched to media with charcoal-stripped serum for 3 d. Cells were then treated with TNF␣ and E2, as indicated for 3 h before formaldehyde fixation, and subject to ChIP analysis. Engagement of the depicted proteins onto pS2 control promoter and MCP-1 enhancer was evaluated by using cognate antibodies and relative quantitative PCR. B, Re-ChIP analysis evaluating the simultaneous presence of the indicated proteins within complexes engaged onto the MCP-1 enhancer region. Unspec Ab, Unspecified antibody.


pression of NF␬B-dependent transcription of MCP-1, whereby E2-bound ER displaces CBP␣. The IL-6 promoter demonstrates a distinctive pattern of cofactor recruitment from the IL-8 and MCP-1 genes, suggesting a differential role for CBP in mediating gene-specific cross talk between ER- and NF␬Bsignaling pathways. Combined treatment with TNF␣ and E2 led to dismissal of p65 from the complex, as well as displacement of CBP and pCAF, presumably secondary to loss of p65. Surprisingly, ER␣ was recruited to the IL-6 promoter, providing strong evidence that ER␣ associates with the NF␬B transcriptional complex independently of direct interaction with p65. Thus ER does not displace only CBP from the IL-6 promoter, but rather displaces p65 and associated coregulators. These data demonstrate that ER suppresses NF␬B transcriptional activity through distinct mechanisms involving displacement of p65 or CBP from specific inflammatory genes. CBP Is a Dosage-Sensitive Regulator of E2Mediated Suppression of MCP-1 To test the role of CBP dosage on the suppression of endogenous NF␬B targets by ER␣, we used small interfering RNA (siRNA) and CBP overexpression and examined the effects on MCP-1, IL-6, and IL-8 transcription using quantitative PCR (qPCR). The CBP knockdown was highly effective and specific and resulted in a significant loss of CBP mRNA, but not for Lamin A/C mRNA (Fig. 7B). CBP knockdown blocked TNF␣-induced expression of MCP-1, IL-6, and IL-8, but had no effect on Lamin A/C mRNA levels (Fig. 7C). The CBP siRNA targets the 3⬘-untranslated region of the CBP mRNA, allowing us to readily vary CBP mRNA levels through cotransfection of an expression plasmid driving CBP, which lacks this untranslated region. MCF-7 cells were treated with the CBP-targeted siRNA and increasing amounts of CBP expression vector. The next day, cells were treated with TNF ⫹/⫺ E2 for 2 h, and processed for qPCR analysis. Figure 8A shows the induction of MCP-1, IL-8, and IL-6 by TNF␣ as a function of CBP expression. Remarkably, the effects of TNF␣ ⫹ E2 on MCP-1 gene expression mirrored the transient transfection data with the MCP-1 promoter luciferase reporter, where low levels of CBP allowed coactivation by TNF␣ and no suppression by E2, whereas higher levels of CBP conferred E2-dependent suppression. Similar findings were evident in analysis of the IL-8 gene, but not with IL-6, which rather showed strong suppression by E2 for all levels of CBP. These data closely mirror the ChIP analysis, demonstrating gene-specific roles for CBP in integrating the ER␣- and NF␬B-signaling pathways. Collectively, these data are consistent with a model whereby CBP first integrates into the MCP-1 transcriptional complex at a site that does not compete with ER, whereas higher doses of CBP allow binding to another site that does compete with the receptor (Fig. 8C). Further, because the response of the IL-6 gene



Fig. 7. RNA Interference Targeting CBP A, RT-qPCR analysis of gene expression. MCF-7 cells were treated for 2 h with the indicated ligands and processed for analysis of the indicated gene expression, which was normalized to expression of 18S mRNA. B, RT-qPCR analysis of CBP or lamin gene expression. MCF-7 cells were transfected for 40 h with the indicated siRNA and then processed for qPCR analysis of lamin A/C or CBP, which is shown normalized to 18S mRNA. C, Effects of siRNA targeting CBP on inflammatory gene expression. MCF-7 cells were transfected with the indicated siRNAs for 40 h and then treated with vehicle, TNF␣, or TNF␣⫹E2 for 2 h, and then processed for qPCR analysis of gene expression. NS, Normal serum; TE, Tris-EDTA; Veh, vehicle; V, vehicle; T, TNF␣.

was distinct, this suggests that CBP and ER interact differently with the assembled transcriptional complex at this gene, consistent with the ChIP data.

DISCUSSION Our studies establish an unexpected mechanism for suppression of the MCP-1 gene by estrogen signaling, involving displacement of the CBP coactivator by ER on the NF␬B enhancer. A variety of approaches established that ER␣ and p65 do indeed physically interact in cells, including ligand-dependent colocalization by immunofluorescence assays, coimmunoprecipitation of endogenous ER␣ and p65 proteins, and ChIP assays that show in vivo interactions of ER␣ and p65 on the NF␬B-responsive MCP-1 enhancer. A series of ER␣ deletion con-

structs demonstrated that the ER␣ LBD is absolutely required for both transcriptional repression and physical association with p65. However, the lack of direct interaction using in vitro assays suggests that their physical association is indirect. This is further supported by the displacement of p65 by ER␣ on the IL-6 gene, demonstrating that ER␣ interacts with the NF␬B response element complex independently of p65. Using our MCF-7-ES cells as a screening system, we have demonstrated that CBP contributes to cross talk between ER␣ and NF␬B, whereas members of the SRC family and other transcriptional coregulators do not confer ER-mediated suppression of MCP-1 transcription. Others have shown that CBP overexpression can relieve repression (14, 17), although not in all cell types (24). However, to our knowledge this is the first example of inducing a repressive complex


Fig. 8. CBP Is a Dosage-Sensitive Regulator of ER-NF␬B Cross Talk A, MCF-7 cells were transfected with siRNA targeting CBP and varying amounts of pRSV-CBP expression vector. The next day, cells were treated for 2 h with vehicle, TNF␣ (15 ng/ml), and/or E2 (100 nM), and then processed for qPCR analysis of gene expression. After normalizing all mRNA levels to 18S, the target gene expression was expressed relative to the mRNA levels of CBP. B, Model of CBP interactions with the NF␬B enhancer in the MCP-1 gene. CBP has well-characterized interactions with the N-terminal domain of CBP. ER does not interact directly with p65 and thus may compete with CBP binding through some other component of the assembled complex (see text for details). CoA, Coenzyme A; N.S., normal serum; ␣-Tub, ␣-tubulin.

through the addition of CBP. The requirement for CBP in NF␬B-dependent gene expression was demonstrated with siRNA targeting CBP, which blocked TNF␣ induction of the MCP-1, IL-6, and IL-8 genes.


This is consistent with the broad requirement and importance of CBP and its close homolog p300 in NF␬Bmediated gene expression (6, 7, 25). There are interesting differences between this work and a ChIP analysis of the TNF␣ gene performed in osteosarcoma cells stably transfected with an inducible ER␣ expression plasmid (26). The TNF␣ gene contains a composite c-Jun/NF␬B binding site and shows a different pattern of cofactor recruitment than the MCP-1, IL-6, and IL-8 genes. The TNF␣ gene demonstrated a strong estrogen-independent association of ER␣ and CBP upon TNF␣ treatment of the cells. In this context, the unliganded ER␣ acts as a transcriptional coactivator, further stimulating TNF␣ gene expression. The hinge domain of ER␣ located between the ligand and DNA-binding domains has been shown to interact directly with c-jun (27) and thus could contribute to the nucleation of distinct protein complexes. In this work, ER␣ recruitment is strongly enhanced by cotreatment with both E2 and TNF␣, as evaluated through ChIP analysis. This is consistent with an indirect association, likely E2 dependent, of ER␣ through some components of the NF␬B transcriptional complex. We show that ER displaces CBP from the MCP-1 and IL-8 genes but displaces p65 and associated coregulators from the IL-6 promoter. Thus, ER␣ and CBP show distinct patterns of association with a c-jun/NF␬B response element (26), and with the NF␬B response elements examined here. A second significant difference with our results is that E2 treatment induced a release of both ER␣ and CBP from the TNF␣ promoter and the recruitment of Grip1 (26). Grip1 was shown to act as a transcriptional corepressor for the TNF␣ gene, as was previously shown for GR-mediated suppression of AP-1 in regulating the collagenase-3 gene (28). In contrast, Grip1 stimulated MCP-1 -luciferase activity in our MCF7-ES cells, an effect that was unresponsive to E2 treatment. This also contrasts with similar experiments done with GR and AP-1 signaling, in which overexpression of Grip1 enabled glucocorticoid-associated repression (28). The differences between the TNF␣ and MCP-1 genes suggest that binding of ER␣ to one partner allows it to nucleate distinct signaling complexes and to specify different interactions with the CBP coactivator. The combined use of siRNA and overexpression allowed us to titrate intracellular CBP levels and to demonstrate a dosage-specific effect on ER suppression of inflammatory gene expression. For both the MCP-1 and IL-8 genes, low levels of CBP promoted TNF␣-responsive gene expression yet were not sufficient to direct E2-mediated suppression. However, higher levels of CBP allowed E2 to suppress transcription. This is consistent with a variety of genetic data demonstrating that a number of physiological and developmental parameters are exquisitely sensitive to CBP dosage, including embryonic development and differentiation (21), hematopoiesis and cancer (20), stem cell self-renewal (19), and Rubinstein-Taybi syndrome (29).


The dosage effect of CBP suggests a model whereby CBP binds to the MCP-1 gene via a higher affinity site, which is not ER competitive, and to a lower affinity site that binds to CBP or ER (Fig. 8C). CBP has been shown to interact directly with both N-terminal and C-terminal domains of p65 (7). However, we do not detect a direct in vitro interaction of ER with p65 or p50, suggesting that the in vivo interaction may involve another coregulator. Thus we propose a model whereby CBP binds directly to p65, and at higher doses, indirectly though another site in the assembled complex, which is competitive with ER (Fig. 8C). Whereas IL-8 gene expression showed a similar CBP dosage effect, the IL-6 gene allowed E2-mediated suppression at any dose of CBP, implying gene-specific patterns of CBP utilization in modulating inflammatory gene expression.

MATERIALS AND METHODS


plates. The next day, Cos-1 cells were transfected with 2 ␮g ER␣ expression plasmid and 2 ␮g p65 expression plasmid using Polyfect (QIAGEN, Chatsworth, CA). For siRNA experiments, MCF-7 cells were transfected with X-tremeGENE Transfection Reagent (Roche Applied Science, Indianapolis, IN) with siRNA targeting CBP, lamin, or nonspecific control. After 24 h, the cells were treated with vehicle, TNF (15 ng/ml), and/or E2 (100 ␮M) for 2 h before processing for qPCR analysis of gene expression. The siRNAs used include: CBP (Sigma-Genosys, antisense: 5⬘-gcg gcu guu gau ucc uca a-3⬘); Lamin A/C (QIAGEN, catalog no. 1022050); AllStars Negative Control siRNA (QIAGEN, catalog no. 1027281). Preparation of Whole-Cell Extracts and Immunoprecipitation Cos-1 cells were transfected overnight as described above. MCF-7 or Cos-1 cells were plated in 10-cm plates and left overnight, or transfected overnight, respectively. The next day, plates were washed with PBS and switched to charcoalstripped media. After another 24 h, ligands were added to the plates for 30–60 min and processed for immunoprecipitation, as described in Supplemental Methods published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org.

Plasmids and Mutagenesis Chromatin Immunoprecipitation The plasmids 3xERE luciferase, MCP-1 luciferase, and pRSV-CBP were kind gifts of Dr. D. McDonnell, Dr. A. Ueda, and Dr. R. Goodman, respectively. The various ER␣ deletion mutants and wild-type mammalian expression vectors were a gift of Dr. P. Chambon. These were subcloned into the PCR3.1 expression vector (Invitrogen, Carlsbad, CA) using the EcoRI site common to all of the constructs. Nuclear Run-On Transcription Assay Subconfluent cultures were stimulated for 2 h with vehicle, E2 (10 nM), TNF␣ (50 ng/ml), or TNF␣ ⫹ E2. Nuclei were isolated by detergent lysis. Run-on transcription was performed at 30 C for 15 min, and total RNA was isolated, and hybridized to cloned cDNAs immobilized to nitrocellulose filters (5 ␮g per slot). Hybridization was performed at 65 C for 36 h with constant shaking. The filters were washed as follows: 65 C, 1 h, 2⫻ standard sodium citrate (SSC) with constant shaking, twice; 37 C, 30 min, with 8 ml 2⫻ SSC and 8 ml 10 mg/ml RNase A; 37 C, 30 min, 2⫻ SSC with shaking. The filters were allowed to air dry and autoradiographic film was exposed. Specific transcription was quantified by scanning densitometry (AMBIS Optical Imaging System, San Diego, CA). Cell Culture and Transient Transfections Cells were maintained in DMEM (Invitrogen) supplemented with 10% fetal calf serum (Atlanta Biologicals, Norcross, GA), glutamine, and antibiotic/antimycotic. The MCF-7-ES cell line was generated by picking individual clones of the MCF-7 cell line. After growing individual clones in charcoal/dextranstripped serum for 3 d, proliferation in response to E2 was measured by uptake of tritiated thymidine. The MCF-7-ES clone was selected as one with the greatest increase in proliferation in response to E2. For luciferase assays, cells were plated into wells of 48well plates for transfection. At the time of transfection cells were switched to media containing charcoal/dextranstripped serum. After 16 h, cells were washed with PBS, and ligands were added. After 24 additional hours, cells were lysed for luciferase assay. Data points represent three to six separate wells and are representative of at least four experiments. For immunoprecipitations, cells were plated in 10-cm

After 3 h treatment with E2, TNF␣, or both ligands, MCF-7 cells were washed twice in ice-cold PBS, and cross-linked with 1.5% formaldehyde in PBS. Cells were scraped in collection buffer [100 mM Tris-HCl (pH 9.4), 100 nM dithiothreitol]. ChIP and Re-ChIP were then performed using 5.106 cells with minor modifications of the procedure described by Me´tivier et al. (22), after two quick washes in ice-cold PBS. For each ChIP sample we used 0.8 to 1 ␮g of antibodies raised against ER␣ (HC20), p65 (A), CBP (A22), p/CAF (H369), or the hemagglutinin epitope as a control (Y11) purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The primers used to amplify the MCP-1 NF␬B enhancer and pS2 promoter were: MCP-1 forward, 5⬘-ggggtaactgaggattctggacag3⬘; MCP-1 reverse, 5⬘-GTGAGAGAAGTGAGTGGAAATTC-3⬘; pS2 forward, 5⬘-gttgtcaggccaagcctttt-3⬘; and pS2 reverse, 5⬘-gagcgttagataacatttgcc-3⬘; IL-8 forward, 5⬘-AAATTACCTCCCCAATAAAATGA-3⬘; IL-8 reverse, 5⬘-CCCCCTACTAGAGAACTTATGCACC-3⬘; IL-6 forward, 5⬘-AGCACTGGCAGCACAAGGCAAAC-3⬘; IL-6 reverse, 5⬘-CAAGCCTGGGATTATGAAGAAG-3⬘. GST Pull Down The GST-ER␣ pull downs were performed as previously described (30). Proteins were eluted by boiling the beads for 10 min in sample buffer. Bound [35S]GRIP1 was visualized by autoradiography after SDS-PAGE. RNA Isolation and qPCR Total RNA was isolated from MCF-7 cells using RNeasy (QIAGEN), which was used to generate cDNA. PCR analysis was performed on an ABI PRISM 7900HT. Values are normalized with 18S or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA content. Immunofluorescence MCF7 cells were grown on coverslips in six-well plates. Cells were treated with ligands for 30 or 60 min, washed three times in ice-cold PBS, fixed in ⫺20 C methanol for 5 min, and washed again three times in PBS, as previously described for


MCF7 cells (31). Primary antibodies against ER␣ (rat: H222) and p65 (rabbit: Santa Cruz Biotechnology) were diluted 1:5000 in Tris-buffered saline (TBS)-Tween (0.02%), after which the cells were incubated with the antibodies overnight at 4 C. The cells were blocked with a 3% solution of carnation nonfat milk in TBS ⫹ 0.02% Tween at room temperature for 1.5 h. Primary antibodies were diluted (1:500) in 1% carnation nonfat dried milk TBS-Tween solution and incubated overnight at 4C. After three 5-min washes in TBS-Tween, the cells were incubated for 1 h in the secondary antibody, also diluted in 1% milk, TBS-Tween solution (1:200). The secondary antibodies were fluorescein isothiocyanate-conjugated antirat and tetramethylrhodamine isothiocyanate-conjugated antirabbit (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). The coverslips were then washed three times in TBS-Tween and mounted onto slides with Prolong Reagent (Molecular Probes, Inc., Eugene, OR). Image analysis was performed using Openlab software with a Zeiss Axioplan Microscope (Carl Zeiss, Thornwood, NY) and a Triple Fluorescence Emission Filter (4⬘,6-diamidino-2-phenylindole, fluorescein isothiocyanate, Texas Red). Gel Shift An oligonucleotide containing the MCP-1 A2 NF␬B response element sequence (5⬘-AGAGTGGGAATTTCCACTCA-3⬘) or a mutant oligo (5⬘-AGAGTGGGAATTcggACTCA-3⬘) was annealed with the complementary reverse oligo and labeled with [32P]ATP. The DNA-binding reaction was assembled with 10 ␮g protein extract, 2 ␮l labeled DNA, 5 ␮l 4⫻ binding buffer (40 mM HEPES, pH 7.6; 200 mM KCl; 0.4 mM EDTA; 20 mM MgCl2; 4 mM dithiothreitol; 40% glycerol), and water to 20 ␮l total volume. After 25 min on ice, 1.5 ␮l of loading dye was added, and the mixture was loaded onto an 18-cm 5% polyacrylamide gel and electrophoresed at 200 V for 1.5 h at 16 C using the Hoefer SE 600 Cooled Vertical Unit. The gel was dried and visualized using the STORM imaging system. Northern Blot Hybridization Total cellular RNA (10–30 ␮g), isolated from control and stimulated cells, was separated by electrophoresis through a 1% agarose gel containing 2.2 M formaldehyde and 1⫻ 3[Nmorpholino]propanesulfonic acid. RNA was transferred to Duralon-UV nylon membranes (Stratagene; La Jolla, CA) with 10⫻ SSC (1.5 M sodium chloride; 0.15 M sodium citrate, pH 7.0) and fixed to the membrane by cross-linking with UV light. Membranes were prehybridized for 2–4 h at 42 C in 25% formamide, 10⫻ Denhardt’s [2% ficoll; 2% polyvinylpyrrolidine; 2% BSA; 0.02% sodium dodecyl sulfate (SDS)], 5⫻ SSPE (0.75 M sodium chloride; 50 mM NaH2PO4; 5 mM EDTA, pH 7.4), 1% SDS, 100 ␮g/ml denatured salmon sperm DNA. Overnight hybridization was performed in the same buffer supplemented with 106 cpm/ml 32P-labeled cDNA probe. Blots were washed in: 2⫻ SSC-0.1% SDS, 42 C for 30 min, two times; and with 0.2⫻ SSC-0.1% SDS, 56 C for 30 min, two times. mRNA expression was quantified by autoradiography.

Acknowledgments We thank John Cleveland for comments on the manuscript. Received June 26, 2007. Accepted October 1, 2007. Address all correspondence and requests for reprints to: Kendall W. Nettles, Department of Cancer Biology, The Scripps Research Institute, Jupiter, Florida 33458. E-mail: [email protected]; or Geoffrey L. Greene, Ben May Institute for Cancer Research and Department of Biochemistry, University of Chicago, Chicago, Illinois 60637. E-mail: [email protected].


This work was supported by Department of Defense Grant DAMD17-01-1-0198 (to K.W.N. and G.L.G.) and by National Institutes of Health Grant 5R01 CA89489 (to G.L.G.). Disclosure Statement: The authors have nothing to disclose.

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