CBP (CREB Binding Protein) Integrates NF-{kappa}B (Nuclear Factor-{kappa}B) and Glucocorticoid Receptor Physical Interactions and Antagonism

Lorraine I. McKay and John A. Cidlowski

Molecular Endocrinology Group Laboratory of Signal Transduction National Institute of Environmental Health Sciences National Institutes of Health Research Triangle Park, North Carolina 27709-2233


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear factor–{kappa}B (NF-{kappa}B) and the glucocorticoid receptor (GR) are transcription factors with opposing actions in the modulation of immune/inflammatory responses. NF-{kappa}B induces the expression of proinflammatory genes, while GR suppresses immune function in part by suppressing expression of the same genes. Previously, we demonstrated that physiological antagonism between NF-{kappa}B and GR is due to a mutual transcriptional antagonism that requires the p65 subunit of NF-{kappa}B and multiple domains of GR (1). To elucidate the mechanism(s) of NF-{kappa}B p65 and GR transcriptional antagonism, we analyzed the interactions of wild-type p65 and p65 RHD (rel homology domain, a dominant negative mutant of p65 which lacks a transactivation domain) with GR. We show that p65RHD blocks p65-mediated transactivation, yet does not block the repression of GR transactivation by p65, indicating that transcriptional activity by p65 is not required to repress GR function. Both p65 and p65 RHD physically interact with GR, but only intact p65 represses GR-mediated signaling, implicating the p65 transactivation domain in the transcriptional repression of GR. To further characterize p65-GR interactions, we examined the role of the transcriptional co-integrator CREB binding protein (CBP) in their mutual antagonism. GR-mediated repression of p65 transactivation and p65-mediated repression of GR transactivation, as well as the physical interaction between NF-{kappa}B and GR, are enhanced by CBP. GR bound to the antagonist RU 486, although transcriptionally inactive, retains the ability to repress p65 transactivation. However, CBP does not physically interact with antagonist-bound GR and does not enhance its repressive effect on p65. These data suggest that CBP functions as an integrator of p65/GR physical interaction, rather than as a limiting cofactor for which p65 and GR compete.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The inflammatory response is a highly regulated physiological process that is critically important to homeostasis. Precise physiological control of inflammation allows a timely reaction to invading pathogens or other insult without an overreaction that might itself cause damage to the host. Two cellular signaling pathways that have been identified as important regulators of inflammation are the nuclear factor-{kappa}B (NF-{kappa}B) and glucocorticoid-mediated signal transduction cascades.

NF-{kappa}B is a widely expressed transcription factor that positively regulates the expression of genes involved in immune responses and inflammation, including cytokines and cell adhesion molecules (for a detailed list of NF-{kappa}B-regulated immune and inflammatory genes, see Ref. 2). NF-{kappa}B transcription factors are dimers whose subunits comprise the Rel family of transcriptional activators. Rel proteins share a conserved, 300-amino acid Rel homology domain that contains the DNA binding, dimerization, and nuclear localization functions of NF-{kappa}B. The preferentially formed NF-{kappa}B transcription factor in most cell types is the p50/p65 (Rel A) heterodimer (3). Unactivated NF-{kappa}B is retained in the cytoplasm by the inhibitory protein I{kappa}B. Multiple forms of I{kappa}B have been identified (4), and two of these forms (I{kappa}B{alpha} and I{kappa}Bß) have been shown to modulate the function of the NF-{kappa}B p65/p50 heterodimer. When a stimulus such as a proinflammatory cytokine or an oxidative stressor activates the NF-{kappa}B signaling pathway, I{kappa}B is phosphorylated (5) and consequently targeted for ubiquitination (6, 7, 8) and removal via the proteosome. I{kappa}B{alpha} and I{kappa}Bß are phosphorylated in response to different extracellular stimuli (4, 9). Degradation of I{kappa}B allows NF-{kappa}B to migrate to the nucleus (10, 11). There are two different but related ser/thr kinases, IKK-1 and IKK-2, which are generally held responsible for the inducible phosphorylation of I{kappa}B (9, 12, 13, 14).

Activation of NF-{kappa}B also involves phosphorylation of the p65 subunit by the catalytic subunit of protein kinase A (PKAc). Dissociation of I{kappa}B allows PKAc to phosphorylate p65 on serine 276, dramatically increasing the transcriptional activity of NF-{kappa}B (15). Recent studies show that this PKA-mediated phosphorylation of p65 is required for recruitment of the transcriptional cofactor CREB-binding protein (CBP)/p300 by NF-{kappa}B p65 (16). In the nucleus, activated NF-{kappa}B binds to consensus sequences in the chromatin and activates specific gene subsets, particularly those important to immune and inflammatory function (4).

Glucocorticoid effects are mediated primarily by the {alpha}-form of the glucocorticoid receptor (GR{alpha} or simply GR), a member of the steroid/thyroid/retinoid receptor superfamily of nuclear receptors. GR binds glucocorticoid in the cytoplasm, resulting in receptor activation. Activation of GR involves a conformational change, dissociation from cytoplasmic regulatory proteins, and increased receptor phosphorylation. Ligand-activated receptor translocates to the nucleus, where it functions as a modulator of gene transcription, activating the transcription of specific sets of genes while repressing the expression of others. Endogenous and exogenous glucocorticoids, presumably acting via the GR, are potent immunosuppressive/antiinflammatory agents. However, the widespread clinical use of these steroid hormones to treat a broad range of inflammatory disorders including chronic asthma (17, 18), rheumatoid arthritis (19), and systemic lupus erythematosus (17) has been based largely on the empirical evidence of their efficacy, while until recently, little was understood concerning the mechanisms by which glucocorticoids suppress the inflammatory response. Recent attention, however, has turned to the opposing roles that NF-{kappa}B and GR play in regulating the immune system as an important mechanism for glucocorticoid suppression of immunity and inflammation. The mechanisms by which NF-{kappa}B and GR antagonize each other’s function is now the subject of intensive study (Refs. 1, 20, 21, 22, 23, 24, 25, 26 ; reviewed in Ref. 2).

Several studies have demonstrated that NF-{kappa}B and GR physically interact (4, 27, 28), which presumably mediates their functional antagonism. We have also demonstrated that GR and NF-{kappa}B are mutual transcriptional antagonists. This mutual transcriptional antagonism requires the p65 subunit of NF-{kappa}B and multiple domains of GR. We have now expanded our examination of the mechanisms by which NF-{kappa}B/GR mutual antagonism occurs. Using functional dissection of the p65 subunit of NF-{kappa}B, we demonstrate that the transactivation domain of p65 is required to repress GR function but not for physical interaction between NF-{kappa}B and GR. Thus, physical interaction alone is insufficient to drive p65/GR antagonism. We show that CBP enhances the physical interaction of GR and p65 and plays a role in their mutual transcriptional antagonism. Based on these data, we propose a model for NF-{kappa}B/GR antagonism that involves a physical stabilization mediated by CBP.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Transactivation Domain of p65 Mediates Inhibition of GR Transactivation
Previous studies have demonstrated that the p65 subunit of the NF-{kappa}B heterodimer inhibits GR transactivation function (1, 24). To determine whether specific subdomains of p65 could be identified as essential for the observed repression of GR function, we performed a functional dissection of p65 using a C-terminal truncation of p65, termed p65 RHD (Rel homology domain), in transient cotransfection/CAT reporter assays as described in Fig. 1Go. p65 RHD (completely described in Ref. 29 and diagrammed in Fig. 1AGo) contains the highly conserved RHD found in all members of the Rel family of transcriptional activators (reviewed in Refs. 2, 4), including sequences that drive the nuclear localization of p65, DNA binding, and homo-/heterodimerization. However, it lacks the C-terminal transactivation domains that are present in wild-type p65 and is therefore transcriptionally inactive. Figure 1BGo demonstrates the transcriptional inactivity of p65RHD as compared with wild-type p65 (control Western blots, not shown, confirm that the truncated form of p65 is efficiently expressed, at levels comparable to intact p65, in our transfection system). These data also indicate that p65RHD is a dominant negative regulator of p65-mediated transactivation in our model system as in others. Anrather et al. (29) have demonstrated that this dominant negative function of p65RHD is probably due to p65 RHD homodimers competing for NF-{kappa}B binding sites in the DNA. Although other possibilities for the mechanism of dominant negative activity cannot be ruled out, p65 RHD does not interfere with the expression of the p65 protein (data not shown).



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Figure 1. p65 RHD Is a Dominant Negative Regulator of p65-Mediated Transactivation, but Not the p65-Mediated Blockage of GR Transactivation

COS-1 cells were transiently transfected with 2.5 µg total DNA, including 0.7 µg of the appropriate CAT reporter construct (3XMHCCAT for p65 transactivation, GRE2CAT for GR transactivation), 0.7 µg GR or 0.03 µg p65 and/or p65 RHD as transactivator, 0.7 µg GR or 0.3 µg p65 or p65RHD as a repressor, and empty backbone vector (pCMV5) to maintain a constant 2.5 µg DNA per transfection. Eighteen hours after transfection, cells were harvested and subjected to CAT assay and data analysis as described in Materials and Methods. Data represent the mean ± SEM of three to four independent experiments. A, Schematic representation of the NF-{kappa}B p65 subunit and the NF-{kappa}B p65 RHD truncation mutant. Both p65 and p65 RHD contain an N-terminal region of approximately 300 amino acids termed the Rel homology domain (RHD), which includes the nuclear localization sequence (NLS), as well as dimerization and DNA binding functions. p65 also contains a C-terminal transactivation domain (containing transactivation functions 1 and 2) which is missing in p65 RHD. B, Transactivation of the p65-responsive reporter 3XMHCCAT. p65 transactivates efficiently, while p65 RHD is transcriptionally inactive on this reporter. When coexpressed, p65 RHD blocks the ability of p65 to transactivate this reporter. C, Transactivation of the GR-responsive reporter GRE2CAT. The first two bars represent GR-mediated transcriptional activity in the absence and presence of ligand, respectively. p65 RHD alone (third bar) has no effect on ligand-activated GR transactivation, while p65 (fourth bar) antagonizes this GR-mediated transactivation. Cotransfected p65 RHD does not rescue p65-mediated antagonism of GR transactivation (bars 5 and 6).

 
The ability of p65RHD to interfere with the transactivation function of ligand-activated GR was then assessed, as shown in Fig. 1CGo. In contrast to wild-type p65, p65RHD is unable to block GR-mediated transactivation, indicating that the transactivation domain of p65 contains a function that mediates the repression of GR by p65. Interestingly, when the truncated and full-length constructs of p65 are coexpressed, p65RHD does not block the ability of p65 to repress GR function, despite the fact that under these same conditions, p65RHD is a dominant negative for p65 transactivation function (Fig. 1BGo). These data suggest that, while the transactivation domain of p65 is required to block the transactivating function of GR, transcriptional activity by p65 is not essential.

Physical Interaction of GR and p65 Is Independent of the p65 Transactivation Domain
To further understand the role of p65/GR physical interactions in the repression of GR transactivation by NF-{kappa}B, we assessed the ability of GR to physically interact with both intact p65 and the truncated p65RHD (which lacks the LXXLL protein-protein interaction motif found in the C terminus of intact p65) in an in vitro coimmunoprecipitation assay. Confirming previous observations that p65 and GR physically interact (24, 27, 28), 35S-labeled in vitro translated GR weakly coimmunoprecipitates with antisera recognizing p65. Surprisingly, GR coimmunoprecipitates with p65 RHD more efficiently than it does with intact p65. Similar results (not shown) were obtained when 35S-labeled p65 or p65RHD were pulled down in immune complexes with unlabeled GR using antisera recognizing GR. These data indicate that the LXXLL motif is not necessary for the physical interaction of p65 and GR in vitro. Taken together, the data from Figs. 1Go and 2Go demonstrate that a physical interaction alone is insufficient to explain the antagonism of GR by p65 and suggests that a more complex function mediates mutual p65-GR transcriptional antagonism.



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Figure 2. GR Physically Interacts with Both p65 and p65 RHD

IVT 35S-labeled GR was incubated with unlabeled, IVT p65 or p65 RHD and then immunoprecipitated with antisera against the N terminus of the p65 subunit. The labeled GR coimmunoprecipitates with both intact (left panel) and truncated (center panel) forms of p65. As an immunoprecipitation control and to confirm GR integrity, labeled GR was also immunoprecipitated with antisera recognizing GR (right panel) and HSP 90 (data not shown).

 
CBP Enhances the Mutual Transcriptional Antagonism of GR and p65
Given that physical interactions between GR and p65 are insufficient to explain the mutual transcriptional antagonism of GR and NF-{kappa}B, the potential role of transcriptional cofactors as mediators of the antagonism was considered. The transcriptional cointegrator molecule CBP has been reported to bind, and in some cases to coactivate, both GR (30, 31) and NF-{kappa}B p65 (32, 33). We considered that CBP might modulate p65-GR antagonism by serving as a limiting cofactor required by both NF-{kappa}B and GR. Therefore, using transient cotransfection/CAT reporter assays, we examined whether overexpression of CBP influences p65/GR antagonism. [A similar approach has recently been reported by Sheppard et al. (30). However, the ratios of transfected components in that manuscript differed significantly from those presented here.] Western blot analyses showed that COS-1 cells have some detectable endogenous CBP, and that CBP is overexpressed under the conditions used for the transactivation assay (Fig. 3AGo, top panel, 3-fold induction over mock transfected cells). These analyses also demonstrate that CBP cotransfection has little effect on the expression of p65 and significantly influences neither the expression nor the characteristic ligand-induced homologous down-regulation of GR (Fig. 3AGo, center and lower panels). Transactivation of an NF-{kappa}B responsive reporter by low levels of cotransfected p65, shown in Fig. 3BGo, is coactivated by coexpressed CBP. Surprisingly, coexpression of CBP under these submaximal transactivation conditions significantly enhances the repressive effect of GR on p65 signaling (Fig. 3CGo). Specifically, coexpression of CBP allowed full repression of p65 transactivation with only 100 ng of transfected GR, while in the absence of CBP, 250 ng of GR are required for full repression. When larger amounts of p65 are transfected (Fig. 3DGo) the p65 signaling is not enhanced by overexpression of CBP, presumably because there is already a maximal transcriptional response. However, even under these conditions, GR-mediated repression of p65 is enhanced by coexpressed CBP. Dexamethasone has been shown to increase expression of the NF-{kappa}B inhibitory subunit I{kappa}B{alpha} in some cell types (21). We therefore considered whether the observed repression of p65 by GR might be due, in part, to induction of I{kappa}B{alpha} levels, an effect that could be stimulated by overexpression of CBP. Western analyses of the cotransfected cells 6 and 18 h after addition of dexamethasone indicated that neither GR activation nor overexpression of CBP alters I{kappa}B{alpha} levels in these cells (18 h, shown in Fig. 3CGo).



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Figure 3. CBP Enhances p65-Mediated Transactivation and Transcriptional Antagonism of NF-{kappa}B p65 by GR

COS-1 cells were transiently transfected with 5 µg total DNA as described in Fig. 1Go. DNA transfected included: .7 µg 3XMHCCAT reporter; 0–0.7 µg GR as indicated; 0.03 µg (for A and D) or 4 ng (for B and C) p65 as a transactivator; 0.35, 0.7, or 3.5 µg CBP; and empty backbone vector (pCMV5) to maintain a constant amount of DNA per transfection. All data points represent the mean ± SEM of three to four independent experiments. Western blots are representative samples of two to three independent experiments. A, Western analysis of CBP overexpression in transfected COS-1 cells (3.5 µg, i.e. 10X, CBP DNA transfected) (top panel), and the inability of CBP coexpression to alter p65 (center panel) or the expression or ligand-induced down-regulation of GR (lower panel). B, Coactivation of p65-mediated transactivation by overexpressed CBP. Suboptimal p65-mediated transactivation of 3XMHCCAT (4 ng of p65) is increased by 64% in the presence of 3.5 µg cotransfected CBP. C, Submaximal p65 transactivation (4 ng p65). CBP (3.5 µg) significantly enhances repression of p65 transactivation by ligand-bound GR (compare third and fourth bars). D, Maximal transactivation of the p65-responsive reporter 3XMHCCAT (0.03 µg p65; 0.7 µg GR; 0.35 or 0.7 µg CBP). Ligand-activated GR (third bar) antagonizes p65 transactivation, while CBP alone (fourth bar) does not affect p65 transactivation. CBP cotransfected with GR (bars 5 and 6) enhances the GR-mediated antagonism of p65 transactivation. (* = significant enhancement of repression). E, Western blot of I{kappa}B{alpha} in COS-1 cotransfected with 0.7 µg GR or GR plus 3.5 µg CBP and subjected to 18 h of dexamethasone stimulation. Neither activation of GR nor overexpression of CBP alter I{kappa}B levels.

 
We then considered the effect of overexpressed CBP on the transactivation potential of GR and its repression by p65. We first performed a GR transactivation assay using a submaximal concentration of glucocorticoid (10-9 M dexamethasone) as shown in Fig. 4AGo. When assessed under these conditions, GR transactivation is significantly enhanced by coexpression of CBP. Although CBP coactivates GR transcriptional activity under these conditions, GR function is not fully rescued from p65-mediated repression by CBP (Fig. 4BGo). Rather, the partial repression of GR by p65 (Fig. 4BGo, fourth set of bars) is significantly enhanced to full repression by coexpressed CBP. Figure 4CGo shows that, under maximal stimulation (i.e. 10-7 M dexamethasone), GR transactivation of the GRE2CAT reporter is not enhanced by overexpressed CBP, but CBP slightly enhances the repressive effect of p65 on GR function. We attribute the lack of GR coactivation by overexpressed CBP to the fact that the saturating dexamethasone concentration fully activated the GR in our transactivation assay, leaving no room for transcriptional enhancement by CBP.



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Figure 4. CBP Enhances Transcriptional Activation by GR and the Antagonism of GR by NF-{kappa}B p65

COS-1 cells were transiently transfected as in Fig. 1Go, with the exception that in the GR transactivation studies, 3.5 µg CBP were included where indicated, while p65 was ramped from 0–0.25 µg. For panel D., 0.7 µg CBP and 0.35 µg p65RHD or p65 were used. All data points represent the mean ± SEM of three to four independent experiments. A. Coactivation of GR transcriptional activity by CBP. At 10–9 M dex, GR transactivation of GRE2CAT is significantly enhanced by coexpression of 10X CBP. B, Transactivation of the reporter GRE2CAT by GR (10-9 M dex). Cotransfection of CBP (black bars) significantly enhances p65-mediated antagonism of ligand-dependent GR transactivation. (* = significant coactivation of GR, = significantly enhanced p65-mediated repression of GR) C, Transactivation of the reporter GRE2CAT by GR (10-7 M dex). Cotransfection of CBP (black bars) weakly enhances p65-mediated antagonism of ligand-dependent GR transactivation. D, Transactivation of GRE2CAT by GR (10-7 M dex). CBP (third bar) has no significant effect on GR transactivation. p65 represses transactivation by 87% (fourth bar), while coexpression of CBP with p65 increases this repression to 94% (fifth bar). p65RHD does not significantly repress GR-mediated transactivation (sixth bar), while coexpression of CBP with p65RHD (seventh bar) represses GR function by 28%.

 
Together, the data presented in Figs. 3Go and 4AGo–C support the idea that CBP is an important modulator of NF-{kappa}B/GR interactions and antagonism. However, since overexpressed CBP enhances NF-{kappa}B/GR antagonism rather than rescuing their transcriptional activation, these data argue that CBP does not function as a limiting factor for which these transcription factors compete, as previously proposed (30). Since CBP enhanced the mutual antagonism of p65 and GR, we also considered the possibility that CBP might facilitate the ability of p65 RHD to repress GR signaling. As shown in Fig. 4DGo, overexpression of CBP enables p65 RHD to partially repress GR transactivation. However, although this repression is statistically significant, it is very weak when compared with the repressive effect of the wild-type p65. Together, the results of these functional studies indicate that CBP is an integrator of GR/NF{kappa}B antagonism.

CBP Enhances the Physical Interaction and Function of p65 with Agonist-Bound, but Not Antagonist-Bound, GR
To further elucidate the mechanism(s) by which CBP enhances p65-GR antagonism, we examined the role of CBP in p65-GR physical interaction by in vitro coimmunoprecipitation assay. Addition of in vitro translated CBP enhanced the physical interaction of p65 with GR bound to the glucocorticoid agonist dexamethasone (Fig. 5AGo), suggesting that CBP enhances p65-GR transcriptional antagonism by facilitating their proper physical interaction. When GR was treated with the glucocorticoid antagonist RU 486 (Fig. 5BGo), GR/p65 interactions in the absence of transfected CBP were similar to those seen for agonist bound GR. However, addition of CBP had no enhancing effect on the physical interaction of antagonist-bound GR and p65. These data suggested that antagonist-bound GR possesses a conformation sufficiently similar to the agonist-bound GR to allow physical interaction with p65, yet sufficiently different to block the GR interaction with CBP. To test this hypothesis, we assessed the ability of CBP to coimmunoprecipitate GR in the presence of dexamethasone as compared with RU 486. Figure 5CGo shows that dexamethasone-bound GR precipitates in immune complexes with CBP, whereas RU 486-bound GR does not coimmunoprecipitate with CBP (similar results were seen when an antibody against unlabeled GR was used to coimmunoprecipitate labeled CBP, data not shown).



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Figure 5. CBP Differentially Affects the Physical Interaction of Agonist- or Antagonist-Bound GR with p65

In vitro coimmunoprecipitation assays were performed as described in Materials and Methods and Fig. 2Go. For panels A and B, labeled GR was immunoprecipitated with antiserum recognizing the unlabeled p65. For panel C, antiserum recognizing the unlabeled IVT CBP was used. Graphs represent the results of five to seven independent coimmunoprecipitations expressed as the mean ± SEM. A typical autoradiogram is shown for each data set. A, CBP enhances physical interaction of dexamethasone-bound GR with p65. B, CBP does not enhance physical interaction of RU-486-bound GR with p65. C, Dexamethasone-bound GR physically interacts with CBP, while RU-486-bound GR does not.

 
Since we observed that RU 486-bound GR is capable of repressing p65-mediated transactivation (27), it became of considerable interest to determine whether CBP overexpression also enhances the ability of this RU 486/GR complex to repress p65 transactivation. To address this question, we performed a p65 transactivation assay identical to the assay depicted in Fig. 3BGo, with the exception that RU 486 replaced dexamethasone as the ligand for GR. The results, shown in Fig. 6Go, confirm that RU 486-bound GR is capable of repressing p65-mediated transactivation (the level of repression is ~50% of that seen with dexamethasone-bound GR, Figs. 6AGo and 3BGo), but CBP coexpression does not enhance the repressive effect of antagonist-bound GR.



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Figure 6. Repression of p65 Transactivation by Antagonist-Bound GR is Not Enhanced by CBP

Transactivation assay was performed as described in Fig. 3BGo. A, A representative experiment showing the effect of dexamethasone-bound GR and CBP on p65 transactivation. B, Antagonism of p65 function by RU 486-bound GR (third bar) is not affected by cotransfected CBP (bars 4 and 5).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The regulation of immune and inflammatory responses is of profound importance to the survival of an organism. Without inflammation and activation of the immune system, an organism could not survive the insult of injury or invading pathogens, yet without mechanisms to shut down prolonged, inappropriate, or excessive immune response and inflammation, the organism would die from damage caused by these physiological responses. Given the critical nature of immune homeostasis, understanding the mechanisms by which NF-{kappa}B and GR, transcription factors with prominent and opposing roles in the regulation of inflammation, interact to regulate these physiological processes has become a major focus of research over the past decade.

We have focused on understanding the nature of the mutual transcriptional antagonism that occurs between NF-{kappa}B and GR, the mechanism of which is understood to involve a physical interaction between the p65 subunit of NF-{kappa}B and multiple domains of GR (1, 20, 24, 27, 28). The data presented here more clearly define the mechanisms of interaction between p65 and GR that result in their mutual antagonism, both by identifying the transactivation domain of p65 as an essential element for transrepression of GR and by identifying a potential role for transcriptional cofactors such as CBP as mediators of the NF-{kappa}B/GR physiological antagonism.

We demonstrate that the p65 transactivation domain is required to block GR transactivation (Fig. 1CGo), but not for efficient physical interactions between GR and p65 (Fig. 2Go). These data argue that, while physical interactions between NF-{kappa}B and GR probably mediate their mutual antagonism, some function beyond the physical interaction must occur to elicit efficient transrepression. Therefore, we considered the role of the transcriptional cointegrator molecule CBP as a mediator of the observed mutual antagonism. CBP is known to interact with a wide variety of basal transcription factors, inducible transcription factors (including NF-{kappa}B and GR), and cofactors, serving in some instances as a coactivator and in others as a central "integrator/adapter" that assembles multiple transcription regulatory proteins together on DNA (30, 31, 32, 33, 34, 35, 36). Given the apparent dual nature of CBP function, we considered two possible ways that CBP might modulate the interactions of p65 and GR: first, as a limiting, mutually required transcriptional cofactor for which GR and p65 compete [a mechanism of action previously described for CBP as a modulator of GR/AP-1 antagonism (31)] or second, as an adapter which brings p65 and GR into a specific physical association on the DNA. A recently published study by Sheppard et al. (30), using in vitro transactivation assays similar to those employed here, concludes that CBP is indeed a limiting cofactor for p65 and GR. The data shown here also indicate a role for CBP in p65-GR antagonism but do not support the same proposed mechanism of action. Given that our experiments showed overexpression of CBP to enhance both the mutual antagonism of p65 and GR (Figs. 3Go and 4Go) and the physical interaction between these two transcription factors (Fig. 5AGo), our data clearly argue against a competition mechanism and support a mechanism whereby CBP integrates p65 and GR on the DNA. Another possible mechanism for CBP involvement in NF-{kappa}B/GR antagonism is hinted at in studies by Lopez et al. (37) concerning the role of CBP and other coactivators in the cross-talk between estrogen (ER) and thyroid hormone (TR) receptors. These investigators also found that overexpression of the coactivators CBP and SRC-1A exacerbated, rather than abolished, nuclear receptor negative cross-talk. Interestingly, unpublished data from our laboratory also suggest a role for SRC-1A in mediating NF-{kappa}B/GR interactions.

Some important differences exist between the CBP overexpression studies shown here and those of Sheppard et al., which may explain the lack of agreement concerning the role of CBP in p65-GR interactions. For example, we used an NF-{kappa}B reporter construct containing three copies of the major histocompatibility complex (MHC) class I NF-{kappa}B site (27), which results in robust transactivation in response to p65 even in the absence of cotransfected CBP; in contrast, those investigators used a reporter construct containing a portion of the E-selectin promoter, which apparently requires the presence of CBP, SRC-1, or other coactivator to strongly activate transcription. While the GR reporter construct and cell type we used for GR transactivation studies did not differ from those reported by Sheppard et al., CBP or SRC-1 was required for strong transactivation by GR under their experimental conditions; under the conditions reported here, however, GR exhibits a strong ligand-dependent transactivation function in the absence of cotransfected coactivators. Perhaps most importantly, we find that at submaximal dexamethasone concentrations we get significant coactivation of GR transactivation function with a 10-fold excess of transfected CBP DNA, while Sheppard et al. demonstrate coactivation using as much as 1000-fold excesses of CBP DNA.

Previous work from our laboratory showed that RU-486 (antagonist)-bound GR, which is localized in the nucleus and binds DNA but is transcriptionally inactive, represses p65-mediated transactivation (27). However, as confirmed in Fig. 6Go, the repression of p65 transactivation by antagonist-bound GR is only about 50% as efficient as that observed with agonist-bound GR. These data led us to hypothesize that antagonist-bound GR attains a conformation that allows it to physically interact with both DNA and p65 in a manner similar to agonist-bound GR, allowing partial repression of p65 transactivation, yet differs in its interactions with CBP, therefore lacking the efficiency of repression observed with the agonist-bound conformation. Figure 6Go shows that, unlike the situation for dexamethasone-bound GR (Figs. 3BGo and 6AGo), CBP does not enhance the repression of p65 transactivation by antagonist-bound GR. Taken together with the data in Fig. 5Go, B and C, showing that 1) RU-486-bound GR coimmunoprecipitates with p65, 2) the coimmunoprecipitation is not enhanced in the presence of CBP, and 3) this antagonist-bound form of GR does not co-immunoprecipitate with CBP whereas the agonist-bound form of GR does, these data make a strong case for the validity of our hypothesis.

As diagrammed in Fig. 7Go, we propose a working model for the mechanisms of NF-{kappa}B/GR interactions and transcriptional antagonism that might explain, in part, their physiological antagonism. Figure 7AGo represents two alternative models for the role of CBP in p65/GR interaction. As shown, activated NF-{kappa}B and GR physically interact, and both transcription factors are capable of binding to CBP. These two transcription factors may compete for binding to available CBP, a mechanism that we term the "mutually exclusive" model. Alternatively, as we propose, CBP might bind to both factors simultaneously, resulting in the ternary complex diagrammed in the bottom panel and termed the "integrative model." This schematic may help to explain the apparent inconsistencies in the observed role of CBP in p65-GR antagonism between our studies and those of Sheppard et al. (30), since vast overexpression of CBP would greatly increase the likelihood of having each transcription factor bound to a separate molecule of CBP, in effect driving the integrated complex of NF-{kappa}B, GR, and CBP to the separate entities diagrammed in the mutually exclusive model. Figure 7BGo summarizes the p65- and GR-mediated signaling pathways and the proposed role of CBP both as a coactivator and as an integrator of transcription factor cross-talk. Transactivation of a glucocorticoid-responsive gene is mediated by binding of activated GR homodimer to glucocorticoidresponsive elements in the promoter. The DNA-bound GR then interacts, directly, and indirectly via CBP, with the general transcriptional machinery to activate transcription. Similarly, transactivation of an NF-{kappa}B-responsive gene is mediated by binding of activated NF-{kappa}B p65/p50 heterodimer to {kappa}B-sites in the gene promoter and subsequent interaction of this DNA-bound heterodimer with CBP and the general transcriptional machinery. The simultaneous activation of GR and NF-{kappa}B results in both factors being present in the same nucleus. We propose that the interaction of these factors in the nucleus causes mutual transcriptional antagonism because it results in a transcription factor/cofactor complex that holds both GR and NF-{kappa}B in a conformation that is incapable of forming correct contacts with the general transcriptional machinery and/or other transcriptional cofactors. Since p65 and GR interact both directly and through the integrator CBP (reviewed in Ref. 2), the theorized function of CBP in this model is to enhance or stabilize the interaction that occurs between p65 and GR. The finding that antagonist-bound GR does not interact with CBP, yet partially represses p65-mediated transcription, suggests, however, that CBP integration is not the sole mediator of p65-GR interaction. It is likely that direct transcription factor interaction or the actions of other transcriptional cofactors also contribute to the observed mutual antagonism.



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Figure 7. Transcriptional Regulation by NF-{kappa}B and GR: Mechanisms of Action, Cross-Talk, and the Role of CBP

A model for transcriptional regulation of NF-{kappa}B and GR-responsive genes. In response to an extracellular signal or ligand, cytoplasmic NF-{kappa}B and GR shed regulatory proteins and translocate to the nucleus, where they bind to cognate respons elements in the chromatin (GRE, glucocorticoid-responsive element; NF-{kappa}B, NF-{kappa}B-responsive element) and to transcriptional cofactors (here, CBP), ultimately interacting with the general transcription machinery (GTM) to activate transcription. Direct and indirect physical interactions between activated p65 and GR block the interaction of both transcription factors with the GTM, resulting in decreased transactivation.

 
The data presented here support an important role for CBP in modulating the NF-{kappa}B/GR interactions and suggest that the relative abundance of p65, GR, and CBP may be an important factor in the transcriptional outcome of these interactions. For transcriptional activation of the GR- and NF-{kappa}B responsive reporter genes examined in this report, the result of NF-{kappa}B interacting with GR is mutual transcriptional antagonism. Future study into the mechanisms of NF-{kappa}B/GR interaction may also reveal other outcomes of this transcription factor cross-talk, which are modulated by additional, cell type-specific transcriptional cofactors. However, mutual antagonism, mediated by the action of CBP and probably other transcriptional cofactors [e.g. reports by Sheppard et al. (30), Lopez et al. (37), and our unpublished results suggest a role for SRC-1A in NF-{kappa}B/GR antagonism as well], has been well studied and is very likely the predominant outcome of NF-{kappa}B/GR interactions in immune cells in which both these signaling pathways are activated.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
COS-1 (African Green Monkey kidney) cells were maintained at 37 C in a 5% CO2 humidified atmosphere in DMEM with high glucose (DMEM-H), supplemented with 2 mM glutamine, 10% FCS-calf serum (CS) 1:1 (Summit Biotechnology, Ft. Collins, CO), 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells were passaged every 3 to 4 days. JEG-3 (human choriocarcinoma) cells obtained from ATCC (Manassas, VA) were maintained at 37 C in a 5% CO2 humidified atmosphere in Eagle’s MEM with Earle’s salts, 2 mM glutamine, 10% FCS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Two hours before each transfection experiment, culture media were changed to formulations using sera stripped of endogenous steroids using dextran-coated charcoal. Cells were maintained in this charcoal-stripped media until cells were harvested for reporter assays after transfection.

Steroids
Dexamethasone (1,4-pregnadien-9{alpha}-fluoro-16{alpha}-methyl-11ß, 17, 21-triol-3, 20-dione) was purchased from Steraloids, Inc. (Wilton, NH). RU 38486 (RU 486) was obtained from Roussel Uclaf (Hoescht Marion Roussel, Inc., Kansas City, MO).

Recombinant Expression Vectors
Human transcription factor NF-{kappa}B p65 subunit (pCMVp65) in a pCMV4T backbone and the NF-{kappa}B reporter 3XMHCCAT were obtained from Dr. A. S. Baldwin (University of North Carolina, Chapel Hill, NC). Detailed descriptions for these constructs are available elsewhere (27, 38, 39). 3XMHCCAT has three copies of a major histocompatibility complex class I NF-{kappa}B site cloned upstream of a minimal promoter CAT (chloramphenicol acetyl transferase) expression vector (27). The GR expression vector pCYGR contains human GR cDNA cloned into the pCMV5 plasmid. For determination of GR transactivation, the GRE2CAT reporter was used. This construct, described in detail elsewhere (40) contains two copies of the GRE sequence from the tyrosine aminotransferase gene and an adenoviral TATA box upstream from the CAT gene. p65 RHD was obtained from Dr. H. Winkler (Sandoz Center for Immunobiology, Harvard Medical School, Boston, MA). This truncated human p65 construct, generated by PCR from the parent pCMV4Tp65 vector, was completely described by Anrather et al. (29). The murine CBP expression construct pcDNA3-CBP-FLAG2X, obtained from Dr. M. G. Rosenfeld (University of California, San Diego, La Jolla, CA) was described by Kamei et al. (31).

Transient Transfections
COS-1 cells were transfected with vector DNA constructs using SuperFect (QIAGEN, Inc. Santa Clarita, CA) cationic transfection reagent after a 2-h incubation in medium stripped of endogenous steroids. SuperFect-DNA precipitates were applied to cells for 1.5 h, after which the transfection solution was replaced with fresh supplemented, charcoal-stripped growth medium. Where appropriate, 10-7 M dexamethasone or 10-6 M RU 486 was added to the medium after transfection, and cells were allowed to grow in the presence or absence of hormone for 18–20 h before harvest for CAT assay. Parallel transfections to assess protein expression by Western blot were processed as described previously (1).

CAT Assays
Transcriptional activities were determined by standard CAT assay (41). Briefly, 18–20 h after transfection, monolayer cells were harvested by scraping from culture dish into cold PBS. Cells were then pelleted, resuspended in 0.25 M Tris buffer (pH 8), and lysed by tip sonication. Cellular debris was pelleted at 14,000 x g, and the supernatant was incubated at 65 C for 10 min to inactivate endogenous inhibitors of CAT. Activated cell extracts were assayed for protein content by the method of Bradford (42), using a commercially available reagent kit (Bio-Rad Laboratories, Inc. Hercules, CA). Volumes of extract containing known amounts of total protein from 1–150 µg were then incubated for 18 h with 14C-radiolabeled chloramphenicol (NEN Life Science Products, Boston, MA) in the presence of 1 mM acetyl-CoA (Roche Molecular Biochemicals, Indianapolis, IN). Reactions were stopped by ethyl acetate extraction, and CAT activities were determined by separation of acetylated products from substrate by TLC on silica gel 60 plates in 1:19 methanol-chloroform. Acetylated product formed was detected by exposing silica plates to an imaging plate and quantitated using PhosphorImager technology (Molecular Dynamics, Inc., Sunnyvale, CA). CAT activities are measured as percent substrate converted to acetylated products. Average percent acetylation in control samples is set equal to 1, and all other values are presented in terms of percent average control transactivation (acetylation). Variations in fold induction of dexamethasone samples over control samples between GR transactivation experiments are attributable to fluctuations in absolute levels of background GR transactivation/CAT activity, which we have observed to vary somewhat with batch of serum and cell passage number. In all cases, the data represent the mean ± SEM of at least three independent experiments.

In Vitro Coimmunoprecipitation Assays
Proteins to be assessed were in vitro translated (IVT) using the TnT Reticulocyte Lysate System from Promega Corp. (Madison, WI). When a labeled product was desired, Tran35S-label 70% L-methionine (ICN Biochemicals, Inc., Irvine, CA) was included with the programmed lysate. In vitro translated GR was treated with 10-7 M dexamethasone or 10-6 M RU 486 for 2 h at 0 C and then activated by incubation at room temperature for 30 min. IVT products were incubated together in cross-link buffer (20 mM HEPES, 50 mM KCl, 2.5 mM MgCl2 and 1 mM dithiothreitol) at room temperature and then cross-linked with 2.5 mM DSP (dithiobis succinmidyl propionate, Pierce Chemical Co., Rockford, IL). Cross-link reagent was quenched with 0.1 M ethanolamine, and samples were then diluted in IP buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.5% Na+ deoxycholate, 0.5% NP40, 0.1 M ethanolamine) for subsequent immunoprecipitation. A protease inhibitor cocktail (containing phenylmethylsulfonyl fluoride, pefabloc, pepstatin, aprotinin, leupeptin, antipain) at manufacturer-specified concentrations (Roche Molecular Biochemicals) was included in all buffers. Reactions were precleared for 20 min at room temperature with normal rabbit serum and protein A sepharose to reduce nonspecific binding to the sepharose beads. Antisera (described below) to target proteins and protein A sepharose were then added to the cleared reactions, and samples were rotated at 4 C overnight. Sepharose was pelleted at 14,000 x g for 1 min and then washed extensively in cold IP buffer. Pellets were resuspended in Laemmli sample buffer boiled for 5 min to dissociate immune complexes from the sepharose. Samples were subjected to SDS-PAGE and autoradiography to detect the amount of labeled protein that was coimmunoprecipitated with the unlabeled target protein. Amount of labeled protein immunoprecipitated was quantitated (in pixels) by densitometry using NIH Image software and was expressed as percent above background (amount pulled down with nonspecific serum, i.e. preimmune or normal rabbit serum, present). For each coimmunoprecipitation shown, a converse control experiment was also performed where the opposite component of the complex was radiolabeled and pulled down with antisera against the unlabeled component. These data, not shown, were used to confirm that precipitation results were not peculiar to one antiserum but could be reproduced with antibodies against any component of the immune complex.

Antibodies
To immunoprecipitate GR, a 1:1 mixture of antibodies 57 and 59 [epitope-specific rabbit polyclonal antibodies, described previously (43)] were used. For p65 immunoprecipitation, a 1:1 mixture of NF-{kappa}B p65 (A) and NF-{kappa}B p65 (C) rabbit polyclonal antibodies that recognize, respectively, the amino- and carboxy-terminal regions of p65, (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used. To immunoprecipitate CBP, anti-CBP CT, a rabbit polyclonal IgG raised against the C terminus of murine CBP, was used (Upstate Biotechnology, Inc. Lake Placid, NY). For detection of proteins by Western blot (some data not shown), antibody 57, NF-{kappa}B p65 (N), anti- I{kappa}B{alpha} (Santa Cruz Biotechnology, Inc.), and anti-CBP CT were used.

Statistical Analyses
All data indicated as significant were analyzed using a t test at the 0.05 confidence level. Western blot data were analyzed for significance by performing densitometry using NIH Image and performing a t test on the pixel data from each lane.

Other
All other reagents were obtained from Sigma (St. Louis, MO).


    FOOTNOTES
 
Address requests for reprints to: Dr. John A. Cidlowski, National Institute of Environmental Health Sciences, P.O. Box 12233, MD E2–02, Research Triangle Park, North Carolina 27709. E-mail: Cidlowski{at}NIEHS.NIH.GOV

Received for publication October 22, 1999. Revision received April 20, 2000. Accepted for publication May 1, 2000.


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