Glucocorticoid Repression of AP-1 Is Not Mediated by Competition for Nuclear Coactivators

Karolien De Bosscher, Wim Vanden Berghe and Guy Haegeman1

Department of Molecular Biology University of Gent-VIB 9000 Gent, Belgium


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Interleukin-6 (IL-6) is a pleiotropic cytokine that is involved in many autoimmune and inflammatory diseases. Transcriptional control of IL-6 gene expression is exerted by various compounds, among which glucocorticoids are the most potent antiinflammatory and immunosuppressive agents currently in use. Glucocorticoids exert their transrepressive actions by negatively interfering with transcription factors, such as nuclear factor-{kappa}B (NF-{kappa}B) and AP-1. Both factors make use of the coactivator cAMP response element-binding protein (CREB)-binding protein (CBP) to enhance their transcriptional activities, which led to the hypothesis that a mutual antagonism between p65 or c-Jun and activated glucocorticoid receptor (GR) results from a limited amount of CBP. Recently, we showed that glucocorticoid repression of NF-{kappa}B-driven gene expression occurs irrespective of the amount of coactivator levels in the cell. In the current study, we extend this observation and demonstrate that also AP-1-targeted gene repression by glucocorticoids is refractory to increased amounts of nuclear coactivators. From results with Gal4 chimeric proteins we conclude that glucocorticoid repression occurs by a promoter-independent mechanism involving a nuclear interplay between activated GR and AP-1, independently of CBP levels in the cell.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The transcription factor AP-1 is encoded by protooncogenes and regulates various aspects of cell proliferation and differentiation. AP-1 can be composed of either homo- or heterodimers among members of the Jun family (c-Jun, junB, and JunD) or among proteins of the Jun and Fos (c-Fos, FosB, Fra1, and Fra2) families, respectively (1); they all belong to the class of the basic zipper (bZIP) family of sequence-specific dimeric DNA-binding proteins (2). The AP-1 binding site is most commonly recognized by c-Jun homodimers or c-Jun/c-Fos heterodimers. AP-1 was originally identified to interact with the control regions of genes, which contain TPA (12-O-tetradecanoyl phorbol 13-acetate)-responsive promoter elements (TRE) and become activated by mitogens, oncoproteins, and UV light. In addition to positive regulatory effects, the AP-1 complex has also been shown to confer negative regulation (3). The transcriptional activity of c-Jun is enhanced by amino-terminal phosphorylation at serine 63 and 73 by Jun amino-terminal kinase (JNK). This inducible phosphorylation step is required to recruit the transcriptional coactivator cAMP response element-binding protein (CREB)-binding protein (CBP), which leads to transcriptional enhancement (4, 5). CBP and its homolog p300 are large cointegrator proteins that provide a docking platform for many members from diverging transcription factor families and contain an enzymatic histone acetyltransferase (HAT) activity (6, 7). This HAT activity functions to shift the chromatin structure into a looser configuration, thereby facilitating the access of specific and basal transcription factors and subsequently gene transcription. Other coactivators, belonging to the p160 family, such as steroid coactivator-1 (SRC-1), are suggested to increase the specificity and strength of the interaction of nuclear receptors with members of the CBP family. Interestingly, some of these coactivators, including SRC-1 and its homolog, activator of retinoic acid receptor (ACTR), were recently shown to also contain HAT activities and to associate, similarly as CBP and p300, with another HAT protein, p/CAF (8, 9, 10); all together, they give rise to a functional coactivator complex. This additional interaction platform could then potentially provide a link to the core transcriptional machinery (11).

Interleukin-6 (IL-6) is a pleiotropic cytokine that is implicated in endocrine and metabolic actions, as well as in immune regulation and aging. IL-6 is thought to play a key role in a number of inflammatory processes, such as rheumatoid arthritis, trauma, and stress, and is also involved in the pathogenesis of osteoporosis, HIV infection, sepsis, and progression of cancer (12, 13). Understanding the regulation of this gene may therefore lead to a controlled and tissue-restricted modulation of its pleiotropic actions.

Glucocorticoids not only inhibit proliferation by suppressing AP-1 activity of genes involved in proliferation, such as c-Jun (14), but they can also mediate a strong suppression of AP-1-driven genes involved in inflammation and immune dysregulation, including IL-6. Glucocorticoid action is mediated by binding to the glucocorticoid receptor (GR), which belongs to the family of nuclear hormone receptors. These ligand-regulated sequence-specific transcription factors may activate or repress gene expression. Whereas gene activation is generally mediated by binding of homodimeric GR subunits to their cognate DNA elements, experiments with mice expressing a dimerization-defective GR demonstrated that gene repression is mainly conducted by interference of the GR monomer with the activities of other transcription factors, including AP-1 (15). Recently, the negative interference between GR and AP-1 was demonstrated in an in vivo model system of TPA-induced expression of collagenase and stromelysin in skin, and GC repression of these genes was also shown to involve the DNA-binding independent function of GR (16).

As CBP can enhance transcriptional activation of AP-1 as well as of nuclear receptors (reviewed in Ref. 17), it was proposed that mutual antagonism between these different signal transduction pathways could be explained by the mutual competition for limiting amounts of CBP within the cell (11). Recent reports also showed that SRC-1 can functionally interact and enhance AP-1-mediated gene expression (18). SRC-1 was originally identified as a coactivator for the nuclear receptor superfamily (19), prompting a role for SRC-1 also in mediating nuclear receptor-dependent gene repression of AP-1-driven genes and vice versa (18). The idea behind a limitation in the amount of coactivator protein such as CBP arose from the observation that a single mutated CBP allele, leading to a heterozygous phenotype, already results in severe developmental defects. This mutation correlates with a disorder called the Rubinstein-Taybi syndrome and includes facial distortions, broadening of thumbs and toes, and mental retardation (20).

In a previous study we demonstrated that glucocorticoid repression of various nuclear factor (NF)-{kappa}B-driven genes occurs independently of coactivator levels in the cell (21). In the present study we demonstrate that glucocorticoids can mediate suppression of the AP-1-driven IL-6 promoter, independently of the levels of coexpressed coactivators. Our data further rule out a direct involvement of CBP in transrepression of GR on other AP-1-driven genes as well.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoids Target AP-1-Induced IL-6 Promoter Activity
Previously we have demonstrated that the IL-6 promoter activity results from a concerted cooperation between NF-{kappa}B, AP-1, CREB, and C/EBP transcription factors. However, depending on the stimulus, the signaling pathways leading to the two most important players, NF-{kappa}B and AP-1, are clearly distinguishable. Indeed, tumor necrosis factor (TNF) induction of the IL-6 promoter almost exclusively triggers NF-{kappa}B activity, while induction of the IL-6 promoter by staurosporine (STS), a protein kinase inhibitor, is predominantly mediated by activation of AP-1, CREB, and C/EBP (22).

We were interested to determine whether activated GR was able to repress TNF-{alpha}- or STS-dependent pathways to a similar extent. Figure 1AGo shows the regulation by TNF, STS, and dexamethasone (DEX) of the wild-type IL-6 promoter and two crucial point-mutated variants, stably transfected in L929sA cells. With the construction p1168hu.IL6P-luc+, glucocorticoids can repress the TNF-induced as well as the STS-induced IL-6-promoter activity (lanes 3 vs. 4 and lanes 5 vs. 6, respectively). The synergism between TNF and STS is also efficiently inhibited by DEX (lane 7 vs. 8). A point-mutated variant of the NF-{kappa}B response element abrogates inducibility by TNF (lane 11), but retains STS-induced IL-6 promoter activity, which is also clearly repressed by DEX to background levels (lanes 13 vs. 14 and 15 vs. 16).



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Figure 1. Effect of DEX on TNF- and STS-Induced IL-6 Promoter Variants

A, Confluent L929sA cell monolayers of a stable pool of the indicated promoter reporter gene constructs were untreated or treated either with 2500 IU/ml TNF or with 60 nM STS, for 6 h, in the presence or not of 1 µM DEX, starting at –2 h. At the end of the induction, cell lysates were assayed for reporter gene activities. The experiment is carried out in triplicate, and the results are representative of at least five independent experiments. B, Similar to panel A, but with inductions performed on the various point-mutated IL-6 promoter reporter constructs stably integrated in L929sA cells. Point-mutated variants are indicated by their respective mutated transcription factor-binding site. C, Similar to panel A, but with inductions performed on the pTRE-luc+ reporter construct stably integrated in L929sA cells.

 
As mentioned above, STS has been demonstrated to stimulate AP-1, CREB, and C/EBP activities within the IL-6 promoter context (22). Mutation of the AP-1 response element in the IL-6 promoter gives rise to a construct, p1168(AP-1 mut).IL6P-luc+, that can still be significantly induced by STS (lane 21). Glucocorticoid repression has been reported not only to occur via AP-1, but also via CREB (23). Accordingly, we observe that glucocorticoid repression is also directed to the other transcription factor activities that contribute to IL-6 induction by STS (lane 22). As a matter of fact, it may be seen from Fig. 1BGo that mutation of the AP-1 or the CRE site greatly abolishes inducibility by STS, whereas mutation of the C/EBP site was only marginally effective. Moreover, a double mutant AP-1/CRE is even no longer responsive at all to STS, demonstrating a more prominent role for both AP-1 and CREB as compared with C/EBP for induction by STS and repression by DEX. This is also evidenced by the fact that the synergism between STS and TNF is less outspoken in the AP-1 response element-mutated promoter construct (Fig. 1AGo, lane 7 vs. lane 23). Consequently, the AP-1 activity may represent an as likely target for glucocorticoid action, almost as effective for IL-6 gene repression as NF-{kappa}B.

Figure 1CGo demonstrates that repression is also apparent on an STS-induced recombinant AP-1-driven promoter construct pTRE-Luc+. TNF cannot stimulate this promoter variant, which is in agreement with the results obtained with the NF-{kappa}B response element-mutated variant of the IL-6 promoter (Fig. 1AGo, lane 11). Background levels of pTRE-luc+ promoter activity can also be repressed by DEX, most probably by antagonizing endogenously activated, DNA-bound AP-1.

Glucocorticoid Repression Acts on AP-1-Driven IL-6 Gene Expression, Irrespective of Coactivator Levels in the Cell
We set out to explore how glucocorticoids suppress the AP-1-driven IL-6 gene. The IL-6 promoter construct p1168hu.IL6P-luc+ can be activated through its NF-{kappa}B element, as well as via its AP-1-element, and this promoter activity can be effectively inhibited by glucocorticoids. Both transcription factors have been reported to enhance their transcriptional activities via recruitment of the coactivator CBP (4, 24, 25, 26). Within the IL-6 promoter context, activation of NF-{kappa}B is necessary and sufficient to engage this coactivator for transcriptional stimulation (22). Here, we demonstrate a significant cooperative enhancement of coexpressed CBP with c-Jun-driven IL-6 promoter activity, indicating that CBP also mediates amplification of the AP-1 response. Furthermore, we investigated whether glucocorticoid repression of c-Jun-induced IL-6 promoter activity still occurs in the presence of increasing amounts of CBP. Figure 2AGo shows that CBP alone is able to slightly enhance background promoter activity (lane 3), most probably via endogenous transcription factors that are constitutively bound to the DNA (i.e. AP-1, CREB, NF-IL6). Consistent herewith, DEX can suppress the background level as well as the activity of the promoter induced by CBP alone (lanes 2 and 4). Furthermore and as expected, activated GR also represses c-Jun transactivation to background levels (lanes 6 and 7 vs. lane 5). Additional CBP stimulates the c-Jun-induced promoter activity 2- to almost 4-fold (lanes 8 and 11 vs. lane 5). Most importantly, we observe that the synergistic activation by CBP and c-Jun is also inhibited by glucocorticoids to almost background levels (lanes 10 and 13) irrespective of the amounts added of CBP. Moreover, cotransfection of the coactivators SRC-1 and p/CAF further contributes to c-Jun transactivation, but does not relieve glucocorticoid-mediated repression (Fig. 2BGo). This extends our observations beyond CBP and indicates that glucocorticoid repression works independently of coactivator complexes present in the cell.



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Figure 2. Effect of CBP on Glucocorticoid Repression of c-Jun-Driven IL-6 Promoter Activity

A, Hek293T cells were transiently transfected with 80 ng of the reporter plasmid p1168hu.IL6P-luc+ and various expression plasmids pRSV-c-Jun (40 ng), pSVhGR{alpha} (50 or 100 ng), pCMV-CBP (as indicated), or empty vector plasmids pcDNA3 or pRSV, keeping the total amount of DNA constant at 600 ng per 24-well plate. Treatment with 1 µM of DEX was started the day after transfection for a period of 24 h. In control lanes, + represents the maximally used concentration of expression plasmid. Cell lysates were assayed for luciferase activities and normalized for protein content. Promoter activities are expressed as relative induction factor, i.e. the ratio of the expression levels of induced vs. noninduced state, the latter being regarded as 1. Assays were performed in triplicate and are representative of two independent experiments. B, The same transfection conditions as in panel A, except that 40 ng of pRSV-c-Jun, 100 ng of pSVhGR{alpha}, 100 ng of pCMVCBP, 100 ng of PCR3.1 SRC-1a, and 100 ng of pCX-p/CAF were used. Cell lysates were assayed and plotted as described in the legend to panel A. C, The same transfection conditions as in panel A, except that 80 ng of 1168({kappa}Bmut)IL6P-luc+, 40 ng of pRSV-c-Jun, 200 ng pCMV-CBP, and 50 or 100 ng of pSVhGR{alpha} were used. Cell lysates were assayed and plotted as described in the legend for panel A.

 
NF-{kappa}B has previously been designated to be the most important transcription factor for IL-6 promoter regulation, at least in response to TNF. Nevertheless, we also tested the point-mutated variant 1168({kappa}Bmut).IL6P-luc+, in which the {kappa}B site is abolished, leaving an almost exclusive c-Jun-driven regulation by the AP-1 site. Figure 2CGo shows that, although the level of c-Jun activation is similar as compared with the wild-type promoter, the cooperation with CBP is less. Because stress is one of the inducing agents of NF-{kappa}B, it may well be that the activity of the c-Jun-induced wild-type IL-6 promoter is a combination of the activity of transfected c-Jun and of endogenous NF-{kappa}B, which is synergistically enhanced by administering extra amounts of CBP. Since glucocorticoids strongly repress NF-{kappa}B activity, this hypothesis could also explain why the relative repression of the {kappa}B-mutated IL-6 promoter variant is also less than repression of the wild-type promoter (lanes 7 and 9 of Fig. 2CGo vs. lanes 11 and 13 of Fig. 2AGo). Nevertheless, the relative repression level by glucocorticoids of the combined activity by c-Jun and CBP is comparable to that exerted by c-Jun alone (Fig. 2CGo, lanes 4 and 6 vs. lanes 7 and 9), ruling out competition for CBP as a way to mediate glucocorticoid repression.

We conclude that glucocorticoids effectively repress AP-1-driven gene expression of the IL-6 wild-type promoter, as well as of the variant with the NF-{kappa}B site abolished, irrespective of the amount of CBP present in the cell.

Glucocorticoid Repression Is Maintained on a Recombinant AP-1-Driven Promoter Construct, Irrespective of CBP Levels in the Cell
To investigate the general applicability of these findings, we tested the regulation by glucocorticoids on a c-Jun-induced recombinant promoter construct pAP-1-luc+, containing three AP-1 sites followed by a viral E1B TATA box. Figure 3Go shows that CBP stimulates this promoter by means of endogenously present AP-1 (lane 2 vs. lane 1) and that glucocorticoids repress this activity even below background levels (lane 3). As expected, c-Jun activity is abolished by treatment with glucocorticoids (lane 4 vs. lanes 5 and 6). CBP costimulates the c-Jun-activated recombinant construct, similarly as observed for the {kappa}B-mutated IL-6 promoter construct (lane 4 vs. lane 7). Most importantly, activated GR efficiently transrepresses the cooperative potential of c-Jun with CBP, regardless of the presence of extra CBP in the cells (lane 7 vs. lane 8).



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Figure 3. Effect of CBP on Glucocorticoid Repression of a Recombinant AP-1-Driven Reporter Gene

HEK293T cells were transiently transfected with 80 ng pAP-1-luc+, the expression plasmids pRSV-c-Jun (40 ng), pSVhGR{alpha} (50 to 100 ng) and/or pCMV-CBP (200 ng), and/or the empty vectors pRSV or pcDNA3. The total amount of DNA was fixed at 600 ng. Cell lysates were assayed and plotted as described in the legend to Fig. 2Go.

 
Glucocorticoid Repression in the Gal4-One-Hybrid System Works Independently of Coexpressed CBP
To make abstraction of the specific promoter context, we tested the effect of glucocorticoids and coexpressed CBP on the activity of a Gal4-c-Jun chimera. This fusion protein, composed of the DNA-binding domain of the yeast nuclear protein Gal4 and the transcription factor c-Jun, stimulates p(Gal)2–50 hu.IL6P-luc+, a luciferase reporter gene preceded by two Gal4-binding DNA sequence elements and the minimal IL-6 promoter. In this particular nuclear set-up, no direct influence of other responsive element-bound transcription factors, which are normally present in the IL-6 promoter context, or interference of cytoplasmic events needs to be taken into account.

Gal4 alone or Gal4 combined with CBP do not activate the reporter (Fig. 4AGo, lanes 1 and 2). Gal4-c-Jun induces a slight but significant activation (lane 3). We further demonstrate that glucocorticoids repress the Gal4-c-Jun activity to background levels, suggesting that glucocorticoid repression of AP-1 is mediated by a direct interference between activated GR and the transactivation function of c-Jun (lane 3 vs. lane 4). Increasing amounts of CBP stepwise increase Gal4-c-Jun-dependent transcription from the Gal4-driven reporter (lanes 5 and 6), but transrepression still occurs under conditions of maximal cooperation between c-Jun and CBP (lanes 7 and 8).



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Figure 4. Specificity of Glucocorticoid Repression, as Demonstrated in the Gal4 One-Hybrid System

HEK293T cells were transiently transfected with 80 ng of p(Gal)2-50-luc+ and cotransfected with the various expression plasmids, pGal4 or pGal4-c-Jun (40 ng) (A) or pGal4-VP16 (40 ng) (B), with or without pCMV-CBP (25 ng or 100 ng) and/or pSVhGR{alpha} (25 ng to 100 ng), as indicated. The total amount of DNA was fixed at 400 ng. Cell lysates were assayed and plotted as described in the legend to Fig. 2Go.

 
As a control, the Gal4-VP16 expression plasmid was also cotransfected with the p(Gal)2–50 hu.IL6P-luc+ reporter plasmid. Figure 4BGo shows that CBP can also stimulate this strong viral transactivator by a factor 2 in agreement with data from Utley and co-workers (27), demonstrating that the enzymatic HAT activity of CBP cooperates with the neighboring acidic transactivation domains of VP16. Interestingly, specific glucocorticoid repression, as observed for Gal4-c-Jun-driven Gal4-dependent reporter gene expression (Fig. 4AGo), does not occur with reporter stimulation by Gal4-VP16 (Fig. 4BGo, lanes 4, 6, and 7), whether or not in the presence of CBP. This result provides additional proof that GC repression of Gal4-c-Jun, after overexpression and subsequent activation of GR, is not caused by nonspecific squelching effects or by affecting CBP activity in a transcriptionally active promoter complex, but is highly specific for Gal4-c-Jun-driven transactivation, irrespective of the amount of CBP present in the cell.

Inhibition of JNK Activation by Glucocorticoids May Contribute to Their Repressive Effects on AP-1-Driven Genes
We tested the effect of DEX on different phosphorylated, activated mitogen-activated protein kinases (MAPKs), since the antagonism between c-Jun and the glucocorticoid receptor in HeLa cells was reported to result from an inhibition of the JNK pathway (28). Figure 5AGo demonstrates that this is also the case in L929sA cells, which contain endogenous GR. The amount of phosphorylated p46/p54 protein is reduced upon cotreatment of TNF and DEX (Fig. 5AGo, compare lane 7 to lane 8). In contrast, in untransfected HEK293T cells, the amount of GR is negligible, which may explain the lack of effect of DEX on the amount of TNF-activated JNK kinases (Fig. 5AGo, compare lane 3 to lane 4). In comparison, the amount of phosphorylated ERK and p38 MAPK (Fig. 5Go, B and C) is unaffected by DEX treatment in both cell lines. Similar results were obtained in L929sA cells for the synthetic glucocorticoids RU24782 and RU24858, which dissociate transactivation from AP-1 transrepression (Fig. 5DGo).



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Figure 5. Effect of Glucocorticoids on Activated MAPKs

HEK293T cells were treated with 1 µM DEX and/or 2,000 IU/ml TNF; L929sA cells were treated with 1 µM DEX, or 1 µM RU24782, or 1 µM RU24858, and/or 2,000 IU/ml TNF. Cell lysates were made and activated JNK, ERK, or p38 were detected using the corresponding phospho-specific MAPK antibodies. The slight reduction of lane 8 vs. lane 7 in panel C is not representative.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoids counteract the expression of AP-1- and NF-{kappa}B-driven proinflammatory genes. Even before the actual target was known at the molecular level, glucocorticoids were widely acknowledged as powerful antiinflammatory and immunosuppressive agents on a purely empirical basis. Studies aimed at elucidating their mechanism of action could therefore contribute to the design of antiinflammatory compounds devoid of side effects.

We demonstrated earlier that the signaling pathways induced by TNF and STS, which lead to activation of the IL-6 promoter, could be discriminated at the transcription factor level. TNF almost exclusively leads to NF-{kappa}B activity, while STS focuses on activation of AP-1, CREB, and C/EBP (22). Thus, in the IL-6 promoter context, NF-{kappa}B and other functional elements, such as AP-1, do not compete with each other for limiting amounts of CBP, but instead cooperate to establish a functional enhanceosome-like structure, comparable to a model that has been reported earlier for the interferon-ß promoter (29). Previous work from our hands focused on the mechanism of GC-mediated repression of various NF-{kappa}B-driven genes (21). We could clearly demonstrate that cofactor squelching is not a general mechanism by which activated GR inhibits NF-{kappa}B activity and vice versa. This finding urged us to also closely investigate the mechanism of GC-mediated suppression of AP-1-driven gene expression. Depending on the investigated cell type or promoter, different results are apparent, making the mechanism by which glucocorticoids repress AP-1-driven genes a controversial issue (reviewed in Refs. 30, 31). One hypothesis proposes that competition between nuclear factors for limited amounts of coactivator molecule accounts for the observed inhibition of AP-1 activity by glucocorticoids (11, 18). Our data present conclusive evidence that glucocorticoid repression of c-Jun-mediated activation of the IL-6 promoter is not relieved by overexpression of coactivator molecules in the cell. These results are in contradiction with conclusions made by Kamei et al. (11), and Lee et al. (18), which suggest that glucocorticoid repression of AP-1 activity can be abolished by adding extra amounts of CBP or SRC-1. We noticed that both reports failed to demonstrate the necessary controls showing the induction level of CBP or SRC-1 together with c-Jun or c-fos in the absence of repression, and therefore do not allow to compare the repression of AP-1 alone vs. the repression of AP-1 with CBP or SRC-1 together. In contrast, our data show that repression is maintained under conditions of cooperativity of c-Jun with CBP. From these results we conclude that repression does not result from limiting the amount of CBP, which would favor a competition model as a means to explain transrepression. A competition model has also been proposed for androgen receptor-mediated repression (32, 33), although Aarnisalo et al. (32) did not find evidence to support this model for glucocorticoid repression of AP-1-activity, which is in agreement with the data presented here.

A valid alternative mechanism concerns a direct interaction between AP-1 and GR. Direct interference was proposed to mediate GC repression of the AP-1-driven collagenase gene (3, 34, 35). However, for some genes containing a composite element, such as the proliferin gene, the transcriptional outcome of glucocorticoid treatment highly depends on the composition of AP-1 and can be inhibitory for the Jun/Fos pair, but stimulatory for the Jun homodimer (36, 37, 38). Inversely, transactivation by nuclear receptors is either negatively or positively influenced by AP-1 and appears to be a cell-specific phenomenon (39). The findings that describe the conditional occurrence of costimulatory effects between AP-1 and GR cannot be reconciled with a general competition model. Moreover, as various transcription factors converge at the level of CBP/p300 for their transcriptional activity, pure competition for cofactors cannot account for the strict specificity of repression phenomena.

Furthermore, the existence of dissociating ligands and of various receptor point-mutants of GR (40, 41, 42, 43), which separate transactivation and transrepression, disfavors a competition model as well. According to this model GR is supposed to attract equally well coactivators in its repressive state, while a separation of transactivation and transrepression functions indirectly implies that GR in its repressive state might no longer be able to attract coactivators and perhaps only recruits corepressor molecules. This idea is supported, although indirectly, by the observation that retinoids that are only involved in transrepression (44) no longer recruit coactivators (Dr. H. Gronemeyer, personal communication).

In further contradiction to the coactivator competition model is the fact that Gal4-VP16 activity, although enhanced by overexpressed CBP, cannot be repressed by activated GR, whereas the Gal4-c-Jun activity distinctly is. This rules out that the mechanism of gene repression relies on a general and aspecific squelching of and competition for common cofactors.

Displacement of an NF-{kappa}B- or AP-1-specific coactivating complex by a so-far-unidentified GR-specific silencing corepressor complex, as identified for unliganded retinoic acid receptor/retinoid X receptor (RAR/RXR) and thyroid hormone receptor (TR) (45, 46), might also be hypothesized, but is not supported by any experimental evidence. Alternatively, glucocorticoid transrepression might also be achieved by a GR-mediated posttranslational modification of coactivators or associated transcription factors (17). In this respect, glucocorticoids have been shown to inhibit AP-1 transactivation by interference with the upstream JNK pathway (28). Our data can indeed substantiate an inhibitory effect of DEX pretreatment on TNF-induced activation of JNK kinases, but not of phosphorylated p38 or ERK kinases. Moreover, the dissociated compounds RU24782 and RU24858, which only exhibit transrepressive capacities, are also able to inhibit JNK activation. The inhibitory role of GR on the upstream JNK kinases and the direct link with transrepression by inactivating transcriptional complexes therefore represent plausible alternative mechanisms that may partially help to explain transrepression by nuclear receptors of AP-1, but not of NF-{kappa}B.

In conclusion, our data do not support the involvement of competition as a mechanism of transrepression of AP-1-driven genes by glucocorticoids. Rather, a recently described allosteric model, a variant of the direct interaction model, which links differential actions of GR in transactivation and transrepression to a different conformational state of the DNA-binding domain, together with the inhibition of incoming signals from the JNK pathway, may provide answers to many observations, previously considered bottlenecks. This model is currently the only one that also provides an explanation for the differential interplay between GR and AP-1 and is, finally, in agreement with our results (47).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The full size IL-6 promoter reporter gene construct p1168hu.IL6P-luc+ and the point-mutated variants p1168({kappa}Bmut).IL6P-luc+, p1168(AP-1 mut).IL6P-luc+, p1168(CREmut).IL6P-luc+, p1168(AP-1-CREmut).IL6P-luc+, and p1168(C/EBPmut).IL6P-luc+ were described previously (22, 48). The reporter gene plasmid pAP1-luc+ was purchased from Stratagene Cloning Systems (La Jolla, CA) and the TK-promoter containing TRE-driven reporter gene, pTRE-luc+, was kindly donated by Dr. Resche-Rigon (Marion Hoechst Roussel-Uclaf, Paris).

Construction of the pRSV-c-Jun expression vector was described previously (22). The expression plasmids pCMV-CBP, pRSV and pRSV-p65, and pSVhGR{alpha} were kind gifts from Dr. R. Eckner (Institute for Molecular Biology, Zurich, Switzerland), Dr. G. Manfioletti (University of Trieste, Trieste, Italy), and Dr. W. Rombauts (University of Leuven, Leuven, Belgium), respectively. The pcDNA3 vector, used as an empty control vector for the CBP-expressing plasmid, was purchased from Invitrogen (San Diego, CA). The expression plasmids coding for SRC-1 (PCR3.1 SCR-1a) and p/CAF (pCX-p/CAF) were kind gifts from Dr. M. Tsai (Department of Cell Biology, Baylor College of Medicine, Houston, TX) and Dr. Nakatani (Laboratory of Molecular Growth Regulation, Bethesda, MD), respectively. The plasmids pGal4, pGal4-p65, and pGal4-VP16 were generously provided by Dr. M. L. Schmitz (German Cancer Research Center, Heidelberg, Germany). p(Gal)2–50 hu.IL6P-luc+ and pGal4-c-Jun were previously described (49, 50).

Cytokines and Reagents
DEX was purchased from Sigma-Aldrich Corp. (Irvine, UK). The dissociated glucocorticoids RU24782 and RU24858 were kindly donated by Dr. M. Resche-Rigon and previously described (40, 41). The origin and activity of TNF, as well as the preparation of luciferase (luc) reagent, were described previously (48). STS was purchased from Calbiochem-Novabiochem International (San Diego, CA) and was stored as a 2 mM solution in dimethyl sulfoxide at –20 C. Luciferase (luc) assays were carried out according to the manufacturer’s instructions (Promega Corp., Madison, WI). Control experiments showed that the final quantities of organic solvent used did not interfere with any of the assays. Normalization of luc activity, expressed as arbitrary light units, was performed by measurement of ß-galactosidase (ß-gal) levels in a chemiluminescent reporter assay Galacto-Light kit (Tropix, Inc., Bedford, MA) or according to Bradford’s protein determination (51). Light emission was measured in a luminescence microplate counter (Topcount; Packard Instruments, Meriden, CT).

The phospho-specific p38 (Thr-180/Tyr-182), p42/p44 (Thr-202/Tyr-204) and SAPK/JNK (Thr-183/Tyr-185) MAPK polyclonal rabbit antibodies detect only the dual phosphorylated form of MAPK. They were purchased from New England Biolabs, Inc. (Beverly, MA) as part of a kit, which also includes antirabbit IgG coupled to horseradish peroxidase, used as a second antibody for Western blotting.

Transfections
Stable transfections of L929sA cells were described previously (48). HEK293T cells were transiently transfected by the calcium phosphate coprecipitation protocol (52). Briefly, 105 actively growing cells were seeded in a 24-well plate 24 h before transfection and either 400 or 600 ng of total DNA were transfected. Sixteen hours post-transfection the medium was replaced with fresh medium, containing 10-6 M DEX where appropriate for another 24 h. Cells were lysed with lysis buffer (Tropix, Inc.), and samples were assayed for their protein or ß-gal content and luciferase activity.

MAPK Activation Assay
The assay was performed essentially as described by Boone et al. (53). Briefly, HEK293T or L929sA cells were seeded at 250,000 cells per well in six-well plates. After 24 h, cells were either left untreated, or treated with 1 µM DEX or with dissociated compounds (RU24858 or RU24782) for 2 h and/or 2,000 IU/ml TNF for 15 min. At the end of the incubation period, cells were washed in PBS. Cell extracts were essentially prepared as described in the protocol of a PhosphoPlus p38 MAPK antibody kit (New England Biolabs, Inc.). One fifth of the total cell lysate (20 µl) was separated by 12% SDS-PAGE and blotted onto a nitrocellulose membrane. Western blot analysis was performed to detect phosphorylated MAPK proteins.


    ACKNOWLEDGMENTS
 
We thank K. Van Wesemael for excellent technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Dr. Guy Haegeman, Department of Molecular Biology, University of Gent-VIB, K. L. Ledeganckstraat 35, 9000 Gent, Belgium. E-mail: Guy.Haegeman{at}dmb.rug.ac.be

Research was supported by the Interuniversitaire Attractiepolen.

1 Research Director with the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. Back

Received for publication March 16, 2000. Revision received October 12, 2000. Accepted for publication October 13, 2000.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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