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Molecular Endocrinology 12 (1): 45-56
Copyright © 1998 by The Endocrine Society

Cross-Talk between Nuclear Factor-{kappa}B and the Steroid Hormone Receptors: Mechanisms of Mutual Antagonism

Lorraine I. McKay and John A. Cidlowski

Laboratory of Signal Transduction National Institutes of Environmental Health Sciences National Institutes of Health Research Triangle Park, North Carolina 27709


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear factor {kappa}B (NF-{kappa}B) is an inducible transcription factor that positively regulates the expression of proimmune and proinflammatory genes, while glucocorticoids are potent suppressors of immune and inflammatory responses. NF-{kappa}B and the glucocorticoid receptor (GR) physically interact, resulting in repression of NF-{kappa}B transactivation. In transient cotransfection experiments, we demonstrate a dose-dependent, mutual antagonism between NF-{kappa}B and GR. Functional dissection of the NF-{kappa}B p50 and p65 subunits and deletion mutants of GR indicate that the GR antagonism is specific to the p65 subunit of NF-{kappa}B heterodimer, whereas multiple domains of GR are essential to repress p65-mediated transactivation. Despite its repression of GR transactivation, p65 failed to block the transrepressive GR homologous down-regulation function. We also demonstrate that negative interactions between p65 and GR are not selective for GR, but also occur between NF-{kappa}B and androgen, progesterone B, and estrogen receptors. However, although each of these members of the steroid hormone receptor family is repressed by NF-{kappa}B, only GR effectively inhibits p65 transactivation. Further, in cotransfections using a chimeric estrogen-GR, the presence of the GR DNA-binding domain is insufficient to confer mutual antagonism to the p65-estrogen receptor interaction. Selectivity of p65 repression for each steroid receptor is demonstrated by I{kappa}B rescue from NF-{kappa}B-mediated inhibition. Together these data suggest that NF-{kappa}B p65 physically interacts with multiple steroid hormone receptors, and this interaction is sufficient to transrepress each steroid receptor. Further, the NF-{kappa}B status of a cell has the potential to significantly alter multiple steroid signaling pathways within that cell.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear factor {kappa}B (NF-{kappa}B) is a widely expressed, inducible transcription factor of particular importance to cells of the immune system. Originally identified as an enhancer binding protein for the Ig {kappa}-light chain gene in B cells (1), NF-{kappa}B is now known to positively regulate the expression of many genes involved in mammalian immune and inflammatory responses, including cytokines, cell adhesion molecules, complement factors, and a variety of immunoreceptors (for a detailed list of NF-{kappa}B-regulated immune and inflammatory genes, see Ref.2). The NF-{kappa}B transcription factor is a heterodimeric protein that comprises the p50 and p65 (Rel A) subunits. These subunits are proteins of the Rel family of transcriptional activators. Members of the Rel family share a conserved 300-amino acid Rel homology domain responsible for DNA binding, dimerization, and nuclear localization. While transcriptionally active homodimers of both p50 and p65 can form, the p50/65 heterodimer is preferentially formed in most cell types (3).

In the absence of stimulatory signals, the NF-{kappa}B heterodimer is retained in the cytoplasm by its physical association with an inhibitory phosphoprotein, I{kappa}B. Multiple forms of I{kappa}B have been identified (4). Two of these forms, I{kappa}B{alpha} and I{kappa}Bß, have been shown to modulate the function of the NF-{kappa}B heterodimer, and these two I{kappa}Bs are phosphorylated in response to different extracellular stimuli (4, 5). Recent studies indicate that the catalytic subunit of protein kinase A (PKAC) is associated with the NF-{kappa}B/I{kappa}B{alpha} complex (6). In this p50/p65/I{kappa}B{alpha}/PKAC tetrameric configuration, I{kappa}B{alpha} renders PKAC inactive and masks the nuclear localization signal on NF-{kappa}B. A variety of extracellular stimulatory signals, such as cytokines, viruses, and oxidative stressors (2), can activate kinases that phosphorylate I{kappa}B. [A cytokine-activated I{kappa}B kinase termed IKK was recently isolated and identified as the key regulatory kinase for I{kappa}B{alpha} (5).] Phosphorylation at serines 32 and 36 targets I{kappa}B{alpha} for ubiquitination and subsequent rapid proteolysis via a proteasome-mediated pathway (7, 8, 9, 10), resulting in the release of NF-{kappa}B/PKAC. The now active PKAC subunit dissociates and phosphorylates the p65 subunit of NF-{kappa}B. Phosphorylated NF-{kappa}B then translocates to the cell nucleus, where it binds to target sequences in the chromatin and activates specific gene subsets, particularly those important to immune and inflammatory function (4).

Glucocorticoids are steroid hormones whose effects are mediated by the glucocorticoid receptor (GR), a member of the steroid/thyroid/retinoid receptor superfamily of nuclear receptors. GR binds glucocorticoid in the cytoplasm, which results in the receptor undergoing a conformational change. The ligand-activated receptor then 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. Synthetic glucocorticoids that act via these pathways are widely employed as clinical immunosuppressive/antiinflammatory agents. However, the widespread use of these steroid hormones in the treatment of a broad range of conditions including chronic asthma (11, 12), rheumatoid arthritis (13), systemic lupus erythematosus (11), and tissue/organ transplantation (14) is based on empirical evidence of their efficacy, while surprisingly little is understood concerning the mechanisms by which glucocorticoids suppress immune function. Recent studies indicate that NF-{kappa}B and GR physically interact, resulting in a mutual transcriptional antagonism (4, 15, 16). We have shown that the GR DNA-binding domain (DBD) is required for this interaction. GR is also reported to increase the expression of the NF-{kappa}B-inhibitory subunit, I{kappa}B{alpha} (17). These findings suggest that an additional mechanism of glucocorticoid- induced repression of cytokine transcription involves GR-mediated inhibition of NF-{kappa}B transactivation. To clarify the mechanism(s) by which GR and NF-{kappa}B interactions cause the antagonism of both transcription factors, we examined the specificity of the interaction between GR and NF-{kappa}B. We show that the observed dose-dependent mutual antagonism is mediated by the p65, but not the p50, subunit of NF-{kappa}B. Further, we identify multiple regions of the GR as essential for mutual antagonism with p65.

Interestingly, despite the potent repressive effect of NF-{kappa}B on GR-mediated transactivation, we show for the first time that NF-{kappa}B p65 is incapable of interfering with homologous transcriptional down-regulation of the GR. Since these results indicate that NF-{kappa}B does not prevent all nuclear effects of GR, it is unlikely that the mechanism of p65 repression of GR involves sequestration of GR in the cytoplasm of the cell.

Our data demonstrate that p65 inhibition of GR transactivation is not specific to GR, but occurs with the structurally and functionally related androgen, progesterone B, and estrogen receptors (AR, PRB, and ER) as well. Interestingly, these data also indicate that the mechanisms of mutual transcriptional antagonism are more specific. While all steroid receptors examined here were repressed by p65, presumably through direct physical interaction, only the GR, which has an immune suppressive function, effectively antagonized NF-{kappa}B transactivation. Examination of the repressive effects of a chimeric ER with a GR DBD provided further new evidence that the DBD of GR, while required for negative interaction with NF-{kappa}B, is not sufficient for mutual antagonism. Taken together, these data indicate that a simple physical interaction between activated NF-{kappa}B and the steroid receptors is sufficient to repress steroid receptor-mediated transactivation, but the potent immunosuppressive effects of glucocorticoids rely on a more specific, multidomain interaction between p65 and GR to repress NF-{kappa}B transactivation.

We also provide new data, which indicate that overexpression of I{kappa}B{alpha} blocks p65-mediated transrepression of GRs, ARs, PRBs, and ERs. This ability of I{kappa}B{alpha} to rescue p65-repressed steroid receptor function demonstrates the specificity of the p65-steroid receptor interaction. Further, it suggests that NF-{kappa}B p65 interferes with steroid hormone receptor-mediated signaling only in its transcriptionally active state.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Coexpression of NF-{kappa}B and GR Results in Mutual Transcriptional Inhibition
Previous studies have demonstrated that NF-{kappa}B and the GR mutually repress each other’s transactivation function (15, 18). To further characterize the interaction between these two transcription factors, we transiently cotransfected COS-1 cells with NF-{kappa}B, GR, and chloramphenicol acetyl transferase (CAT) reporter. Since the p65 subunit of NF-{kappa}B has been shown to be the transcriptionally active and inducible subunit in most cell types (Refs. 3 and 19 and our unpublished observations), we employed the p65 subunit expression vector for these studies. The data in Fig. 1Go demonstrate that when a constant amount of p65 plasmid DNA is cotransfected with increasing amounts of GR plasmid DNA (Fig. 1AGo), p65-mediated transactivation of the NF-{kappa}B-responsive 3XMHCCAT reporter is inhibited by ligand-activated GR in a dose-dependent manner. Similarly, dexamethasone-mediated GR transactivation of the glucocorticoid-responsive GRECAT reporter is dose-dependently inhibited by p65 (Fig. 1CGo). Western analyses indicate that the observed repression of p65 and GR transactivation function is not associated with an alteration of p65 (Fig. 1BGo) or GR (Fig. 1DGo) protein expression in these cell cultures.



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  		Figure 1. GR and NF-{kappa}B Interactions Result in Mutual Transcriptional Antagonism

COS-1 cells were transiently transfected with 12 µg total DNA, including 5 µg of the appropriate CAT reporter construct (3XMHCCAT or GRECAT), the p65 subunit of NF-{kappa}B (pCMVp65), hGR (pYChGR), and empty expression vector (pCMV5) to maintain a constant 12 µg DNA per transfection. After transfection, the appropriate cultures were treated with 10-7 M dexamethasone, and all cultures were incubated at 37 C in 5% CO2 humidified air for 18 h before harvest. Cell extracts were assayed for CAT activity. CAT activities per µg protein are expressed as percent control activity (control = p65, 0 µg GR, and no dexamethasone for panel A; GR, 0 µg p65, and no dexamethasone for panel C) for plotting. For Western blots, cells were transfected as for the corresponding CAT assays, and 100 µg protein from whole cell extracts were loaded per lane and then detected as described in Materials and Methods. A, p65 transactivation vs. amount of transfected GR. Cells were transfected with 0.1 µg pCMVp65, 5 µg 3XMHCCAT. B, p65 protein levels corresponding to 0, 1, 2.5, and 5 µg cotransfected GR. C, GR transactivation vs. amount of transfected p65. Cells were transfected with 5 µg pYChGR, 5 µg GRECAT. D, GR protein levels corresponding to 0, 0.5, 1, and 2.5 µg cotransfected p65. The CAT data represent the mean ± SEM of three independent experiments. Western blots shown are representative examples of three independent experiments.

 
To eliminate the possibility that the observed transrepression of GR by p65 might be specific to the minimal promoter reporter construct GRECAT, we also performed the GR transactivation assay using two other GR-responsive reporter constructs with more complex promoter regions: GRE2CAT (data not shown) and MMTVCAT (Fig. 2Go). The same dose-dependent repression of GR transactivation by p65 was observed regardless of which reporter construct was employed. To confirm that the observed reduction in CAT activity was not due to some direct inhibition of the reporter gene product by GR or NF-{kappa}B, we performed control experiments using a constitutively active thymidine kinase-CAT reporter, PBL2CAT, which is not responsive to NF-{kappa}B or GR (data not shown). Increasing amounts of either transcription factor had no effect on the level of CAT activity in these samples.



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  		Figure 2. NF-{kappa}B p65 Represses GR Transactivation of the MMTVCAT Reporter

GR transactivation of the complex-promoter construct MMTVCAT was determined in the presence of increasing amounts of p65. Transfections and CAT assays were performed as in Fig. 1Go, using 5 µg MMTVCAT, 5 µg pCYhGR, 0–2.5 µg p65, and pCMV5 for a constant 12 µg per transfection. Data are presented as fold induction over control transactivation (control = 0 µg p65, no dexamethasone) and represent the mean ± SEM of three independent experiments.

 
The p65 Subunit of NF{kappa}B Is Required for Inhibition of GR Transactivation
Having established that the p65 subunit of NF-{kappa}B negatively interacts with the GR, we wished to examine whether the p50 subunit of the NF-{kappa}B heterodimer could also antagonize GR transactivation. As Fig. 3Go demonstrates, p65 homodimers (Fig. 3AGo), as well as equimolar amounts of cotransfected p65 and 50 (Fig. 3BGo), are capable of repressing GR transactivation. (Since p65/p50 heterodimers are preferentially formed when both p65 and p50 subunits are present (19), the effect in Fig. 2BGo is presumably mediated by the heterodimer.) In contrast, p50 homodimers have no effect on GR-mediated transcription, regardless of the amount of p50 transfected (Fig. 3CGo). This indicates that negative regulation of GR by the NF-{kappa}B heterodimer is mediated solely through the p65 subunit. Since the p50 subunit of NF-{kappa}B is expressed at similar levels to p65, but not transcriptionally active in this cotransfection system (as assessed by Western analysis and CAT assay, data not shown), these data also suggest that the mechanisms of reciprocal transcriptional repression of NF-{kappa}B by GR are mediated solely through the p65 subunit.



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  		Figure 3. The p65 Subunit of NF-{kappa}B Is Required for Inhibition of GR Transactivation

GR activation of the GRECAT reporter was determined in the presence of increasing amounts of NF-{kappa}B dimer. Transfections and CAT assays were performed as in Fig. 1Go, using 5 µg GRECAT, 5 µg pCYhGR per transfection. From 0–2.5 µg of expression vector for NF-{kappa}B, subunits p65, p50 or equimolar amounts of p65 and p50 were used per transfection. GR transactivation is presented as percent of control CAT activity (for each panel, control = no NF-{kappa}B, no dexamethasone). A, p65 only; B, equimolar p65 and p50; C, p50 only.

 
The Mutual Antagonism of GR and NF-{kappa}B Is Mediated by Multiple Domains of the GR
To identify regions of the GR that might be involved in the mutual transrepression with NF-{kappa}B, a series of GR deletion mutants were assessed for their ability to repress and be repressed by NF-{kappa}B p65. Each of these mutant GRs has been previously demonstrated to be efficiently expressed and capable of hormone-mediated nuclear translocation in transfected COS-1 cells (with the exception of the I550 steroid-binding domain mutant, which is localized to the nucleus both in the presence and absence of hormone) (20).

In terms of GR’s ability to inhibit p65 transactivation, three separate regions of the receptor appear to play a role (Fig. 4AGo). As previously demonstrated (15, 21), the DNA binding domain (DBD) of GR is essential to this repressive function (see {Delta} 428–490). More specifically, ablation of either of the two zinc finger domains ({Delta} 420–451, {Delta}450–487) of this region completely releases the inhibition of NF-{kappa}B. The mutant GR I550 demonstrates the importance of the steroid-binding domain (SBD) to p65 transrepression. This receptor, which lacks the SBD, is incapable of NF-{kappa}B transrepression despite its nuclear localization (20) and the presence of an intact DBD.



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  		Figure 4. Multiple Domains of GR Are Necessary for Mutual Antagonism with the p65 Subunit of NF-{kappa}B

Deletion mutants of hGR were assessed both for their ability to repress p65 transactivation and for the ability of p65 to repress their ligand- dependent transactivation function. Cotransfections contained 5 µg of the appropriate reporter construct, 0.1 µg of the p65 construct, and 5 µg of GR mutant construct per assay. CAT activities were determined as described in Fig. 1Go. and plotted as percent of control CAT activity. A, Effect of GR on p65 transactivation. Control = p65 transactivation, no GR or dexamethasone (data not shown). B, Effect of p65 on GR transactivation. Control = wild-type GR transactivation, no dexamethasone or p65. C, Schematic diagram of GR deletion mutants used in A and B.

 
Interestingly, the GR amino-terminal mutant {Delta} 9–385 was also incapable of transrepression. However, GR {Delta} 77–262, which contains a smaller deletion of the transactivation domain nested within the region missing from {Delta} 9–385, transrepresses p65. One possible interpretation of this result is that a small region of the GR present in {Delta} 77–262, but missing from {Delta} 9–385, functions as a third site on the receptor involved in repression of p65 transactivation. An alternative interpretation is that the deletion from 9–385 of the GR is sufficiently adjacent to the DBD of GR to affect the function of that region. As we have shown, a functional DBD is essential for transrepression of NF-{kappa}B p65 by GR. The current experiments do not enable us to distinguish between these alternative interpretations.

These data demonstrate that multiple regions of the GR are involved in the repression of p65 transactivation. In contrast, inhibition of GR-mediated transcription by p65 is a more general phenomenon. As shown in Fig. 4BGo, any mutant that is transcriptionally active can have that activity repressed by p65. Of particular interest is the ability of p65 to repress the function of GR I550. This receptor mutant, because it lacks a steroid-dependent component, is nuclear and constitutively transcriptionally active. That p65 inhibits the function of this mutant GR suggests that the mechanism of p65 action involves something other than a simple sequestering of GR in the cytoplasm, where it cannot interact with chromatin.

p65 Does Not Inhibit Homologous Down-Regulation of the GR
While widely known to be a transcriptional activator, GR also functions as a negative modulator of gene transcription. A special case of negative regulation of gene expression by GR is the homologous down-regulation of the GR, both at the message and protein levels, via direct binding of intragenic elements within the GR DNA (22). Given the generally repressive effects of NF-{kappa}B p65 on steroid hormone receptor transactivation function, we sought to determine whether NF-{kappa}B also negatively impacted this transrepressive function of GR. To address this previously unexamined question, we determined whether cotransfected p65 could block the dexamethasone-mediated down-regulation of both GR mRNA and protein by GR in transiently transfected COS cells. Figure 5Go shows that cotransfection of p65, at a dose that caused almost complete repression of GR transactivation (see Fig. 1CGo, 2Go.5 µg p65), cannot prevent GR-mediated repression of GR message (Fig. 5AGo) or GR protein (Fig. 5BGo) expression. The inability of p65 to block this nuclear effect of GR indicates that the mechanism by which NF-{kappa}B represses GR does not involve cytoplasmic sequestration of the GR by activated NF-{kappa}B. It also argues against the hypothesis that NF-{kappa}B interaction with GR completely blocks GR DNA binding.



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  		Figure 5. NF-{kappa}B p65 Does Not Inhibit Homologous Down-Regulation of the GR

T-75 flasks of COS-1 cells were transiently cotransfected with a total of 35 µg DNA, containing 25 µg pCYhGR and 10 µg of either pCMVp65 of pCMV5 backbone vector. After an overnight incubation, cells were treated with 10-7 M dexamethasone as appropriate and incubated for an additional 24 h before harvest. For Northern blots, 30 µg total RNA were loaded per lane. For Western blots, 25 µg of protein from whole-cell extracts were loaded per lane. Blots shown are representative of at least three independent experiments. A, GR mRNA. B, GR protein from a parallel cotransfection experiment to that shown in panel A.

 
p65 Antagonizes Multiple Members of the Steroid Hormone Receptor Family, but Not All These Receptors Reciprocally Repress p65
The difference between p65 inhibition of GR and the reciprocal inhibition of p65 by GR was intriguing and prompted us to question the specificity of the p65 interaction with other closely related steroid hormone receptors. Figures 6Go and 7Go address the interaction of p65 and human (h) ARs, PRBs, and ERs. (The A form of PR was found to be transcriptionally inactive in our system and, therefore, was not examined in detail.) Figure 6AGo demonstrates that AR inhibits p65 transactivation in a dose-dependent manner. This effect is similar to the GR-mediated inhibition of p65 observed in Fig. 1AGo, but is much weaker, requiring 2.5–5 µg of transfected plasmid hAR to achieve the same 50% reduction in maximal transactivation observed with 1 µg of plasmid GR. PRB and ER, however, have little or no inhibitory effect on p65 transactivation.



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  		Figure 6. AR, but Not PR or ER, Can Repress Transactivation by NF-{kappa}B p65

Transient cotransfection assays were performed as in Fig. 1AGo, using varying amounts of human AR, PRB, or ER expression vector in place of hGR. Data show p65 transactivation of 3XMHCCAT in the presence of increasing amounts of ligand-activated steroid receptor. A, AR ± 10-6 M R1881 B, PR ± 10-7 M progesterone. C, ER ± 10-6 M estradiol.

 


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  		Figure 7. NF-{kappa}B p65 Represses Ligand-Dependent Transactivation by AR, PR, and ER

Transient cotransfection assays were performed as in Fig. 1BGo, using 5 µg human AR, PRB, or ER expression with 5 µg of the corresponding CAT reporter construct in place of hGR and GRECAT. Data show the ligand-dependent steroid receptor transactivation with increasing amounts of p65 (0–2.5 µg). A, AR transactivation. Ligand = 10-6 M R1881, reporter construct = MMTVCAT. B, PR B transactivation. Ligand = 10-7 M progesterone, reporter construct = MMTVCAT. C, ER transactivation. Ligand = 10-6 M estradiol, reporter construct = vit-tk-CAT.

 
While the steroid receptors clearly differ in their ability to repress p65 transactivation (Fig. 6Go), no differences are observed in terms of their repression by p65. As depicted in Fig. 7Go, p65 strongly and dose-dependently transrepresses each of the steroid hormone receptors tested, much like it represses GR function. (Control Western blots, not shown, confirm that p65 has no effect on the levels of AR, ER, or PR expression in this cotransfection system). These data suggest that a similar physical interaction underlies the repression in each case. While this interaction between p65 and steroid hormone receptors is sufficient to inhibit the function of each receptor, it is apparently insufficient for reciprocal repression of p65. Taken together, the data in Figs. 6Go and 7Go support the conclusions in Fig. 4Go: the mechanism of mutual antagonism between p65 and GR is highly specific, whereas the one-way antagonism of all steroid receptors by p65 is of a more general nature.

The DBD of GR Cannot, by Itself, Confer Reciprocal Transrepression to the p65-ER Interaction
We have established that the DBD of GR is essential to mutual functional antagonism with p65. In addition, regions of the SBD and possibly the amino terminus of GR are involved in this specific inhibitory interaction (Fig. 4AGo). These observations led us to consider whether the DBD of GR is responsible for the specific interaction with p65, which results in mutual antagonism, while the SBD and/or amino-terminal regions of the receptor are simply necessary to stabilize the interaction.

To address this issue, we employed a chimeric ER/GR construct. The construct expresses a hER that has a GR DBD substituted for its own. The chimeric receptor binds and is activated by estradiol but recognizes and binds a glucocorticoid-response element in the chromatin. If the DBD of the GR were the only region of the receptor responsible for mutual antagonism with p65, an intact chimeric ER/GR would be able to suppress p65 transactivation in our system. Figure 8Go demonstrates that this is not the case. ER/GR exhibits the same pattern of repression as ER, not GR. Specifically, p65 represses ER/GR transactivation of the GRE2CAT reporter (Fig. 8AGo), but ER/GR has no inhibitory effect on p65 transactivation (Fig. 8BGo).



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  		Figure 8. Substitution of the DBD of ER with That of GR Does Not Make ER-p65 Transrepression Reciprocal

ER-GR, an ER construct with a GR DNA binding domain, was cotransfected into COS-1 cells with p65 and the appropriate reporter constructs to assess both steroid receptor and p65 transactivation. Transfections and CAT assays were performed as described in Fig. 1AGo, Ligand-activated ER-GR transactivation in the presence of increasing amounts of p65 expression vector; 10-6 M estradiol (E2) was used as ligand, and the CAT activity was determined with a GRE2CAT reporter construct. B, p65 transactivation in the presence of increasing amounts of ER-GR expression vector.

 
These data indicate that other regions of the GR, in addition to the DBD, are required for specific interactions with p65 and do not act simply as stabilizers of the NF-{kappa}B p65-receptor interaction.

I{kappa}B{alpha} Blocks p65-Mediated Repression Transactivation by Multiple Steroid Receptors
Although our (unpublished) observation that transcription of constitutively expressed PBL2CAT reporter was unaffected in our cotransfection system argued against general transcriptional repression, we sought to verify the specificity of p65 as the mediator of steroid receptor transrepression. We approached this question by exploiting the interaction of NF-{kappa}B with the inhibitory rel subunit I{kappa}B{alpha}. Increased expression of this inhibitory NF-{kappa}B subunit causes an increased binding of nuclear NF-{kappa}B and attenuates NF-{kappa}B transactivation by sequestering the transcription factor in the cell cytoplasm. Glucocorticoids have also been shown to modulate NF-{kappa}B function indirectly by increasing expression of I{kappa}B{alpha} (17). We sought to determine whether overexpression of I{kappa}B{alpha} could interfere with NF-{kappa}B-mediated repression of steroid-dependent GR transactivation in this cotransfection system. Figure 9AGo indicates that p65-mediated repression of GR transactivation of the GRE2CAT reporter is completely blocked by cotransfection of an equal amount of I{kappa}B{alpha}. Interestingly, steroid-mediated GR transactivation function is actually considerably enhanced compared with control in the presence of transfected I{kappa}B{alpha} (Fig. 9AGo, I{kappa}B, I{kappa}B/p65). Since I{kappa}B has no effect on the reporter construct in the absence of steroid receptor (data not shown), we interpret this to be the result of I{kappa}B{alpha} sequestration of the endogenous NF-{kappa}B present in COS-1 cells and subsequent release of the basal GR inhibition by NF-{kappa}B.



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  		Figure 9. I{kappa}B Blocks p65 Inhibition of Steroid Hormone Receptor Action

Ligand-activated steroid hormone receptor transactivation was assessed in the presence of cotransfected NF-{kappa}B p65, I{kappa}B, or both I{kappa}B and p65. In each experiment, 3 µg each of the appropriate steroid hormone receptor expression vector (for GR, AR, PRB, or ER) and 2 µg of the appropriate reporter construct were cotransfected into COS-1 cells. pCMVp65 (3 µg) and pCMVI{kappa}B{alpha} (3 µg) were cotransfected as indicated in each figure. Transfected DNA was held constant at 12 µg by the addition of pCMV5 backbone vector DNA. Cells were treated with ligand (as in Figs. 1Go and 6Go) then harvested for CAT assay. Results represent the mean ± SEM of three experiments and are presented as percent of control transactivation. (Control = ligand-dependent transactivation in the absence of p65 or I{kappa}B, represented by the first bar of each panel, and set to 100%). A, Dexamethasone-activated GR transactivation of GRE2CAT. B, R1881-activated AR transactivation of MMTVCAT. C, Progesterone-activated PRB transactivation of MMTVCAT. D, Estradiol activated ER transactivation of vit-tk-CAT.

 
The ability of I{kappa}B{alpha} to block the p65 repression of other steroid hormone receptors was also examined. Figure 9Go, B-D, shows that p65 repression of ARs, PRBs, and ERs, respectively, is similarly blocked by overexpression of I{kappa}B{alpha}. However, some differences in efficacy were observed with the different receptors. For GRs, PRs, and ERs, I{kappa}B{alpha} completely restored the receptor transactivation function in the presence of p65, while for AR, I{kappa}B{alpha} could only restore approximately 50% of control transactivation. It is also intriguing that I{kappa}B{alpha} overexpression in the absence of p65 resulted in an approximate 5-fold enhancement of steroid-dependent PR transactivation and 3-fold enhancement of GR transactivation, but no enhancement of AR or ER transactivation. These data suggest that I{kappa}B{alpha} and its induction by GR may play an important role in enhancing and sustaining the immunosuppressive effects of glucocorticoids: ligand-activated GR simultaneously represses NF-{kappa}B transactivation and enhances I{kappa}B{alpha} expression. I{kappa}B{alpha} then further represses p65 and enhances GR function, possibly resulting in a feed-forward mechanism of GR repression of NF-{kappa}B.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Negative cross-talk between NF-{kappa}B and the GR has recently gained attention as a potentially important general mechanism by which glucocorticoids exert their potent antiinflammatory and immunosuppressive actions (15, 16, 21). Because the processes that underlie glucocorticoid-mediated immunosuppression are of clinical importance, but are as yet poorly understood, the work we present here focuses on the mutual antagonism between NF-{kappa}B and the steroid hormone receptors that transduce endocrine signals. In particular, we begin to examine the mechanisms that underlie the negative cross-talk between these transcription factors.

We have shown that the transrepression of NF-{kappa}B by GR is both reciprocal and dose-dependent. Further, we show for the first time that this mutual antagonism is mediated solely through the p65 subunit of NF-{kappa}B. The data presented here support the possibility of two mechanisms by which this mutual antagonism might occur. We propose that one mechanism of mutual repression involves direct physical interactions between p65 and GR and that this interaction prevents both transcription factors from binding to their DNA-responsive elements and activating transcription. The idea that this dose-dependent pattern of mutual inhibition is due to a physical interaction between NF-{kappa}B and the GR is supported by work from both our own and other laboratories (15, 16, 21). Specifically, these studies have demonstrated by coimmunoprecipitation that p65 and the GR physically associate with each other. In addition, electrophoretic mobility shift assays (15, 16) show that GR can inhibit the binding of p65 to its response element. A recent report demonstrating coimmunoprecipitation of the progesterone receptor and p65 (18) lends further support to the idea of a direct steroid receptor-p65 interaction as the basis of our observed transrepression.

A second potential mechanism by which GR and NF-{kappa}B might function as mutual antagonists is competition between these two transcription factors for a common transcriptional cofactor(s). The dose-dependent nature of the observed antagonism, as well as the attenuating effect of I{kappa}B{alpha} on p65 transrepression of each steroid receptor, demonstrated here for the first time, would also be consistent with such a mechanism. In addition, the fact that p65 cannot interfere with homologous down-regulation of the GR, a process that involves GR binding to intragenic elements of GR but is independent of the promoter region of the GR gene, suggests that p65 interactions with GR do not completely block GR binding to DNA.

We evaluated the effect of increasing amounts of p65 on transactivation by PRBs, ARs, and ERs. These data prove that each of these steroid hormone receptors is dose-dependently inhibited by p65 in a manner similar to the inhibition of GR by p65. From this observation, we conclude that a similar direct physical interaction or competition for cofactors underlies the transrepression in each case. Surprisingly, although this interaction between NF-{kappa}B and the steroid receptors represses receptor-mediated transactivation in all cases, it is insufficient for the reciprocal inhibition of p65 function. While for GR and AR mutual antagonism with p65 was observed, PRB and ER had no effect on p65 activation. These findings contradict a recently published study that suggests that PRB can repress p65 (21). Although that study did not present data to experimentally document this repression, our own work indicates only a very small repression of p65 at very high levels of transfected PRB plasmid. Also, in contrast to our current findings, recent publications provide evidence for ER effects on p65 function (23, 24, 25); however, these findings appear to be dependent on the presence of cell-type/tissue-specific cooperating factors. The existence of cell type-specific differences in steroid receptor/p65 interactions is consistent with the hypothesis that cofactor competition is involved in NF-{kappa}B/steroid receptor antagonism.

The observation that, in our experimental system, some steroid receptors are capable of mutual antagonism with NF-{kappa}B, while others cannot reciprocate, fits well with the conclusions from our experiments with deletion mutants of the GR. Taken together, Fig. 4Go, A and B, shows that the mutual antagonism of p65 and GR is complex and requires multiple domains of the GR. In contrast, the one-way inhibition of active GR by p65 is more general, occurring regardless of what region of GR has been deleted. These data indicate that all transcriptionally active deletion mutants of the GR tested are transrepressed by p65. However, we were able to identify at least two, and possibly three, regions of the GR that play a role in the transrepression of p65. We interpret these data to mean that the mechanisms of transrepression of p65 and NF-{kappa}B are distinct. While both inhibitory functions require physical interactions between the transcription factors, inhibition of p65 requires the presence of multiple domains of the GR, including the DBD, the SBD, and possibly small regions of the amino terminus. No specific regions of the GR necessary for its repression by p65 could be definitively identified.

Further evidence supporting the importance of multiple domains of the GR in p65 transrepression came from our examination of the chimeric ER. The DBD of GR has been identified both here and in previous studies (15, 21) as an essential region of the receptor for the antagonism of p65. In addition, DBD of GR has been identified as the region through which cross-talk with another transcription factor, AP-1, occurs (26). For this reason, we speculated as to whether the DBD of GR was the only specific region of the receptor for p65-GR cross-talk, while the SBD and amino-terminal regions of the receptor were merely stabilizers of the physical interaction. If this were true, then replacement of the DBD of ER with one from GR should confer mutual antagonism to the ER interaction with p65. The data presented here indicate that this is not the case. Chimeric ER does not have the ability to repress p65 despite the presence of a GR DBD, indicating that specificity lies in multiple regions of the GR.

Glucocorticoids are known to negatively impact many aspects of immune and inflammatory response. They have been shown to repress the expression of interleukin 6, CINC/gro, and other cytokines that cause inflammation (16, 27). They also suppress the cytokine-induced expression of NOS II, an important mediator of macrophage activity and other immune and inflammatory responses (28). This GR-mediated inhibition of cytokines occurs despite the lack of any identifiable glucocorticoid-responsive elements in the promoter regions of these genes. Glucocorticoids have also been shown to interfere with immune function by altering the distribution pattern of lymphocytes (28) and decreasing the number of lymphocytes by both inhibiting cell growth and inducing apoptosis (Ref. 30 and unpublished observations). GR is also known to positively regulate the expression of I{kappa}B, a powerful inhibitor of NF-{kappa}B transactivation function (17). The functions of NF-{kappa}B in the immune system, on the other hand, include the up-regulation of cytokine expression and the regulation of cell adhesion molecules that are involved in lymphocyte infiltration during inflammation (2, 15, 27, 28). The ability of glucocorticoids to suppress immune function is closely related to the ability of GR to inhibit NF-{kappa}B transcriptional activity.

We maintain that the direct physical association of NF-{kappa}B and GR is one important mechanism of NF-{kappa}B repression in immune cells, and consequently, a key factor in suppression of immune and inflammatory response. We further propose that additional mechanisms of NF-{kappa}B-GR antagonism may also contribute to modulation of the immune response. Specifically, our data are also consistent with a mechanism of antagonism involving competition for common transcriptional cofactors. Our observations concerning the specific nature of NF-{kappa}B antagonism by activated GR speak to the physiological importance of this mechanism. We have shown that NF-{kappa}B is a ubiquitously expressed transcription factor that interacts with a variety of steroid hormone receptors. Inhibition of NF-{kappa}B by any activated steroid hormone receptor with which it interacts would reduce the value of GR as a specific inhibitor of immune response. Instead, we demonstrate that multiple regions of GR are required for this inhibition, ensuring that immune suppression occurs in those cells in which glucocorticoids, GR, and NF-{kappa}B are all present. Other steroid hormone receptors, such as estrogen or progesterone receptors, while under the conditions evaluated here are not specific repressors of NF-{kappa}B, may specifically interact with NF-{kappa}B in a cell type-specific manner due to the presence of specific transcriptional cofactors in that cell. The identity of cofactors that might modulate the interactions between NF-{kappa}B and the steroid hormone receptors remains to be elucidated and is likely to become a prime focus of future investigation.


   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 (Irvine Scientific, Santa Ana, CA), 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells were passaged every 3–4 days. One hour before each transfection experiment, culture media were changed to a supplemented 3% FCS/CS formulation and maintained in this media for the duration of transfection.

Steroids
R1881 (methyltrienolone) was obtained from NEN Research Products (Boston, MA). Progesterone (4-pregnen-3, 20-dione), dexamethasone (1, 4-pregnadien-9{alpha}-fluoro-16{alpha}-methyl-11ß, 17, 21-triol-3, 20-dione), and 17ß- estradiol (1, 3, 5(10)-estratrien-3,17-ß-diol) were purchased from Steraloids, Inc. (Wilton, NH).

Recombinant Expression Vectors
Vectors containing the human transcription factor NF-{kappa}B subunits p65 (pCMVp65) and p50 (pCMVp50) in a pCMV4T backbone, the inhibitory {kappa}B subunit I{kappa}B in the pCMV4T backbone (I{kappa}B{alpha}), as well as 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 (15, 31, 32). 3XMHCCAT has three copies of a major histocompatibility complex class I NF-{kappa}B site cloned upstream of a minimal promoter CAT expression vector (15). The GR expression vector pCYGR contains hGR cDNA cloned into the pCMV5 plasmid. For determination of GR transactivation, GRECAT and GRE2CAT reporter constructs were employed. These constructs are described in detail elsewhere (33). Briefly, GRECAT and GRE2CAT contain one or two copies, respectively, of the GRE sequence from the tyrosine aminotransferase gene and an adenoviral TATA box upstream from the chloramphenicol acetyl transferase gene. hAR cloned into the CMV3 expression vector was obtained from Dr. M. McPhaul, University of Texas Southwestern Medical Center (Dallas, TX) (34). hPRB in a pRST7 (RSV promoter) expression vector backbone was a gift from Dr. D. McDonnell, Duke University (Durham, NC) (35). For both hAR and hPRB transactivation studies the reporter construct pGMCS, which contains mouse mammary tumor virus promoter sequences upstream of the CAT gene, was used. This reporter was obtained from Dr. K. Yamamoto, University of California (San Francisco, CA) (36). The hER cloned into a pKCR2 backbone (HEO), an ER/GR cassette (ER/GR) construct containing the hER with an hGR DNA binding domain, and the vit-tk-CAT (estrogen responsive) reporter construct were obtained from Dr. P. Chambon, INSERM (Paris, France) (37, 38). Expression vectors containing deletion mutants of the hGR ({Delta}428–490, {Delta}420–451, {Delta}450–487, {Delta}77–262, {Delta}9–385, and I550) were obtained from Dr. R. Evans (Salk Institute, San Diego, CA) and are described elsewhere (39, 40, 41).

Transient Transfections
COS-1 cells were transfected with vector DNA constructs using a standard calcium phosphate method (42) or by SuperFect (Qiagen, Inc. Santa Clarita, CA) cationic transfection reagent. The cells were exposed to calcium phosphate-DNA of SuperFect-DNA precipitates for 4 h. Calcium phosphate- transfected cells were then subjected to osmotic shock for 30 sec in a 15% glycerol/3% FCS/CS supplemented DMEM-H solution. Fresh 10% FCS/CS supplemented media were applied to all cells, and steroid hormone (10-7 M dexamethasone or progesterone, or 10-6 M R1881 or estradiol) was added to the media as appropriate. After transfection, cells were incubated in the presence or absence of hormone for 18–20 h before harvest in ice-cold PBS (1x PBS).

CAT Assays
Transcriptional activities were determined by standard CAT assay (42). 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 (43), using a commercially available reagent kit (Bio-Rad, Hercules, CA). Volumes of extract containing known amounts of total protein from 1–150 µg were then incubated for 18 h with 14C-labeled chloramphenicol (NEN Research Products) in the presence of 1 mM acetyl coenzyme A (Boehringer Mannheim, 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 quantitated by liquid scintillation counting. CAT activities are measured as percent substrate converted to acetylated products. Average percent acetylation in triplicate control samples is set equal to 1, and all other values are presented in terms of fold increase over average control.

Isolation of Total RNA and Northern Hybridization
Total RNA was isolated from transiently transfected COS-1 cells with TRIzol Reagent (GIBCO, Grand Island, NY), and RNA integrity was assessed by visual inspection of ribosomal RNA bands after agarose gel electrophoresis and ethidium bromide staining.

For Northern hybridization, RNA samples were denatured by treatment in glyoxal/dimethylsulfoxide and electrophoresed on a 1% agarose/10 mM NaHPO4 (pH 7) gel. After transfer to 0.2-µm Biotrans nylon membrane (ICN, Irvine, CA) and UV cross-link, RNA was hybridized at 65 C in 50% formamide with an antisense riboprobe synthesized from the pT3/T7 hGR cDNA vector as previously described (22).

Western Blot Analyses
Transiently transfected COS-1 cells were incubated in the absence or presence of hormone as for CAT assay. Cells were then washed in ice-cold PBS, then harvested by scraping with a rubber spatula into cold PBS containing a protease inhibitor cocktail (containing phenylmethylsulfonyl fluoride, pepstatin, aprotinin, leupeptin, antipain) at manufacturer-specified concentrations (Boehringer Mannheim, Indianapolis, IN). Cells were homogenized on ice using three 10-sec bursts with a Tekmar Tissuemizer (Cincinnati, OH) with cooling for 30 sec between bursts, after which the whole-cell lysates were cleared by ultracentrifugation at 45,000 x g for 45 min. Resulting supernatant cell extracts were mixed with 6x Fairbanks gel loading buffer (6 mM EDTA, 6% SDS, 30% sucrose, 60 µg/ml Pyronin Y, 36 mg/ml dithiothreitol), boiled for 4 min, and then electrophoresed on 7.5% polyacrylamide gels with 3% polyacrylamide stack gel. Electrophoretically separated proteins were electroblotted onto nitrocellulose filters. Quality of transfer and consistency of loading were visually assessed using Ponceau S staining. Filters were blocked in 10 mM Tris/15 mM NaCl, pH 7.4 buffer (CTT) containing 10% nonfat dry milk and 0.1% Tween-20.

Primary antibody incubations were performed in CTT buffer plus 1% nonfat dry milk for 1 h at room temperature. [Primary antibodies: GR = antibody 57, epitope-specific rabbit polyclonal antibody, described previously (44); p65= NF-{kappa}B p65 (A) rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA); p50 = anti-human NF-KB p50 rabbit monospecific antibody (Rockland, Gilbertsville, PA).] Secondary antibody incubations and subsequent chemiluminescent detection with the ECL detection kit (Amersham, Buckinghamshire, U.K.) were performed in accordance with kit recommendations.


   FOOTNOTES
 
Address requests for reprints to: Dr. John A. Cidlowski, National Institutes of Environmental Health Sciences, P.O. Box 12233, MD E2–02, Research Triangle Park, North Carolina 27709.

Received for publication August 28, 1997. Accepted for publication October 3, 1997.


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 INTRODUCTION
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 DISCUSSION
 MATERIALS AND METHODS
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