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Molecular Endocrinology 13 (2): 276-285
Copyright © 1999 by The Endocrine Society

Ligand-Activated Retinoic Acid Receptor Inhibits AP-1 Transactivation by Disrupting c-Jun/c-Fos Dimerization

Xiao-Feng Zhou, Xi-Qiang Shen and Lirim Shemshedini

Department of Biology University of Toledo Toledo, Ohio 43606


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the presence of retinoic acid (RA), the retinoid receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), are able to up-regulate transcription directly by binding to RA-responsive elements on the promoters of responsive genes. Liganded RARs and RXRs are also capable of down-regulating transcription, but, by contrast, this is an indirect effect, mediated by the interaction of these nuclear receptors not with DNA but the transcription factor activating protein-1 (AP-1). AP-1 is a dimeric complex of the protooncoproteins c-Jun and c-Fos and directly regulates transcription of genes important for cellular growth. Previous in vitro results have suggested that RARs can block AP-1 DNA binding. Using a mammalian two-hybrid system, we report here that human RAR{alpha} (hRAR{alpha}) can disrupt in a RA-dependent manner the homo- and heterodimerization properties of c-Jun and c-Fos. This inhibition of dimerization is cell specific, occurring only in those cells that exhibit RA-induced repression of AP-1 transcriptional activity. Furthermore, this mechanism appears to be specific for the RARs, since another potent inhibitor of AP-1 activity, the glucocorticoid receptor, does not affect AP-1 dimerization. Our data argue for a novel mechanism by which RARs can repress AP-1 DNA binding, in which liganded RARs are able to interfere with c-Jun/c-Jun homodimerization and c-Jun/c-Fos heterodimerization and, in this way, may prevent the formation of AP-1 complexes capable of DNA binding.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Retinoic acid (RA), the most biologically active natural metabolite of vitamin A (retinol), exerts profound effects on vertebrate development, cellular differentiation, and homeostasis (reviewed in Ref. 1). RA is involved in epithelial differentiation (2), has a central role as a tissue-specific morphogen during embryogenesis (3), and represses malignant transformation of epithelial cells both in vitro (4) and in vivo (5). These diverse and pleiotropic effects of RA are mediated through the binding of RA to a family of nuclear receptors, which, together with the receptors for steroid and thyroid hormones and vitamin D, form the nuclear receptor superfamily (reviewed in Refs. 6, 7, 8, 9, 10). Nuclear receptors comprise the largest family of transcription factors, which, upon binding of their cognate ligands, modulate transcription initiated from promoters of target genes by interacting with specific cis-acting, DNA response elements.

In contrast to this positive effect of nuclear receptors on transcription, which requires receptor-DNA interactions, the retinoid receptors and other nuclear receptors can negatively affect gene expression without binding to DNA, via their ability to functionally interact with the transcription factor AP-1 (activating protein-1) (reviewed in Refs. 11, 12, 13, 14). AP-1 consists of homodimers and heterodimers of Jun (c-Jun, v-Jun, JunB, and JunD), Fos (c-Fos, v-Fos, FosB, Fra1, and Fra2), or activating transcription factor (ATF2, ATF3/LRF1, B-ATF) bZIP (basic region leucine zipper) proteins (15, 16, 17). The transcription of the c-jun and c-fos genes, encoding the major components of AP-1, is rapidly induced upon stimulation of cellular proliferation (37, 38). Like nuclear receptors, AP-1 activates transcription of target genes by binding to specific promoter elements, called TREs (TPA-responsive elements) (17, 18). TPA (12-O-tetra-decanoyl-phorbol-13-acetate) is a tumor promoter that induces the expression of the c-fos and c-jun genes (19, 20) and thereby indirectly stimulates the expression of AP-1 target genes.

Both positive and negative regulatory interactions between nuclear receptors and c-Jun/c-Fos have been reported (reviewed in Refs. 11, 12, 13, 14). The first results showed an inhibition of glucocorticoid receptor (GR)-induced transcription by either c-Fos or c-Jun (21, 22, 23, 24). We (25) and others (26, 27, 28, 29, 30, 31) have shown that this type of interference is not restricted to the GR, but seems to be a common characteristic of nuclear receptors, including the receptors for the hormones progesterone (PR), estrogen (ER), androgen (AR), and thyroid (TRs), and the RA (RARs, RXRs). Conversely, the activation of the collagenase and stromelysin genes by AP-1 is repressed in a ligand-dependent manner by several receptors, including GR (21, 22, 23, 24), PR (25), AR (25), ER (25), TR (28), and RARs/RXRs (26, 27, 29). By contrast, coexpression of c-Jun, c-Fos, and ER causes synergistic activation of the ovalbumin gene (32). GR has been shown to potentiate c-Jun-activated transcription from the proliferin- regulatory element (33). Similarly, transfected c-Jun enhances AR-induced transactivation, but it does so independently of promoter or cell type specificity (25, 34, 35).

In contrast to the interaction between AP-1 and AR, PR, or GR, which is nonmutual and can be either negative or positive (25, 32, 33), the interaction between AP-1 and the retinoid receptors is mutual and solely inhibitory. c-Jun and c-Fos, either individually or together, have been shown to repress the transcriptional activity of RAR and/or RXR (27). Conversely, both RAR/RXR heterodimers or homodimers of either can inhibit AP-1 transactivation of several AP-1-responsive promoters (27, 29). Indeed, the RAR/RXR antagonism of AP-1 has been directly implicated in the regulation of collagenase (27, 29) and stromelysin (26), two genes that play key roles in tumor potential and invasiveness.

While the molecular bases of these diverse regulatory interactions between nuclear receptors and AP-1 are not known, recent studies provide several attractive models. Based on the demonstration that CREB-binding protein (CBP) and the related p300 can act as transcriptional coactivators for both nuclear receptors (36, 37, 38) and AP-1 (39, 40), it has been proposed that the nuclear receptor-AP-1 antagonism depends on competition for limiting amounts of these two coactivator proteins (36). While this model may explain some of the observations made, it is not able to explain all of the cell, promoter, and receptor specificity that has been observed in the nuclear receptor-AP-1 interactions (25). More recently, it has been suggested that GR, RARs, and TRs can block AP-1 activity by inhibiting the activity of Jun amino-terminal kinase (JNK) (41), which enhances c-Jun transcriptional activity by phosphorylating Ser63/73 (42, 43). However, this model also is unable to account for the diverse nature of the interactions between nuclear receptors and AP-1. Others have argued, based on in vitro results, that AP-1 and receptors mutually inhibit each other’s DNA-binding ability (21, 24, 33). However, Konig et al. (44) have provided strong evidence that in vivo DNA binding, at least for GR and AP-1, is not affected, since the in vivo footprint of either of these transcriptional activators in the presence of the other did not change. Thus, the nuclear receptor-AP-1 antagonism may depend on multiple mechanisms with the involvement of cell- and receptor-specific factors.

Using a mammalian two-hybrid system, we provide evidence here that RAR, but not GR, is able to disrupt in vivo c-Jun/c-Fos dimerization in a ligand-dependent manner. This effect is not only receptor specific, but also cell specific, paralleling what has been reported previously with RA-induced inhibition of AP-1 transcriptional activity (27). Our results suggest that c-Jun/c-Fos dimerization may be a third target of nuclear receptor-mediated repression of AP-1 that may be specific for the transrepression activity of RARs and may partially explain the receptor- and cell-specific nature of this repression.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RA-Bound human RAR{alpha} (hRAR{alpha}) Inhibits AP-1 Activity in a Cell-Specific Manner
The interaction between nuclear receptors and AP-1 has been shown previously to be dependent on cell type (Refs. 25, 27 ; reviewed in Ref. 11). To test for cell specificity in RARs’ ability to inhibit AP-1 transcriptional activity, cells were transiently transfected with expression plasmids for c-Jun and hRAR{alpha} and the AP-1-inducible reporter TRE-tk-CAT (34). In keeping with previously published data (27), hRAR{alpha} inhibited in a ligand- and dose-dependent manner exogenous c-Jun activity in HeLa cells (Fig. 1AGo), but not in Cos cells (Fig. 1BGo). The same difference in activity was observed on endogenous AP-1 activity (data not shown).



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  		Figure 1. Ligand-Bound RAR Inhibits AP-1 Activity in HeLa Cells, but Not Cos Cells

HeLa (A) and Cos (B) cells were transfected with 1 µg of the TRE-tk-CAT reporter plasmid together with 1 µg of c-Jun and 1, 3, or 5 µg of hRAR{alpha} expression plasmids. Cells receiving hRAR{alpha} were treated with 10-7 M AT-RA as indicated. Note that CAT activity is represented relative to activity of first condition, which was set to 1.

 
Mammalian Two-Hybrid System Can Be Used to Measure c-Jun/c-Fos Dimerization in Vivo
The yeast two-hybrid system has been used previously to measure in vivo protein-protein interactions (45). In the current study, we have used a similar system to measure c-Jun/c-Fos heterodimerization and c-Jun/c-Jun homodimerization in cultured mammalian cells. In our system, two fusion proteins are expressed, one containing the GAL4 DNA-binding domain (DBD) fused to either full-length c-Jun or only its bZIP region and the other containing the VP16 transactivation domain fused to either full-length c-Fos or only its bZIP region (Fig. 2AGo). HeLa cells were transfected with expression plasmids for these different fusion proteins, and dimerization was monitored with the GAL4-inducible reporter 17M-tk-CAT. While GAL-cJun had a weak activity and VP16-cFos had no measurable activity, these two fusion proteins together resulted in a 14-fold stimulation in transcription (Fig. 2BGo), demonstrating a strong in vivo interaction between transfected c-Jun and c-Fos. If either c-Fos or c-Jun or both were absent from the fusion proteins, no interaction was detected (Fig. 2BGo). Since earlier work (46) has shown that the respective bZIP regions of these two protooncoproteins are sufficient for heterodimerization, we tested these same regions in our system. Indeed, GAL-cFos(137–216) and VP16-cJun(237–331) exhibited a dimerization capacity that is comparable to that observed with the full-length proteins (Fig. 2BGo). As further evidence, we measured c-Jun/c-Jun homodimerization by coexpressing GAL-cJun and VP16-cJun(237–331). In agreement with previous work (47), c-Jun/c-Jun homodimerization was significantly weaker than c-Jun/c-Fos heterodimerization, even with higher (~3-fold) amounts of c-Jun fusion proteins expressed (Fig. 2BGo). Identical results have been obtained in Cos cells (data not shown). These results together indicate that our mammalian two-hybrid system is faithfully measuring the ability of c-Jun and c-Fos to dimerize in vivo.



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  		Figure 2. c-Jun/c-Fos Heterodimerization Is More Efficient Than Is c-Jun-c-Jun Homodimerization in Vivo

A, A schematic representation showing full-length c-Jun and c-Fos and truncations of these two proteins, which were used as fusion proteins with GAL4 or VP16 in the mammalian two-hybrid screen. Numbers represent amino acid residues. B, HeLa cells were transfected with 1 µg of the 17 M-tk-CAT reporter plasmid together with 1 or 3 µg each of expression plasmids for GAL-cJun or VP16-cJun or 1 µg each of expression plasmids for VP16-cFos, GAL-cFos(137–216), VP16-cJun(237–331), GAL-DBD, or VP16. Note that CAT activity is represented relative to activity of GAL-cJun, which was set to 1.

 
hRAR{alpha} Disrupts c-Jun/c-Fos Dimerization in Vivo in a Ligand-Dependent Manner
To determine whether RAR can affect the in vivo dimerization between c-Jun and c-Fos, HeLa cells were transfected with an expression plasmid for hRAR{alpha} and treated with 10-7 M all-trans-retinoic acid (AT-RA). hRAR{alpha} was able to severely block dimerization between full-length c-Jun and c-Fos (Fig. 3AGo). This negative effect occurred only in the presence of AT-RA, since there was no effect by hRAR{alpha} in the absence of AT-RA (Fig. 3AGo). A similar ligand-dependent RAR{alpha}-induced inhibition was observed on c-Jun/c-Jun homodimerization (Fig. 3BGo). Since the bZIP regions of c-Jun and c-Fos are sufficient for dimerization (see Fig. 2BGo), we wanted to determine whether these regions are also sufficient for the negative effect of hRAR{alpha} on AP-1 dimerization. Liganded hRAR{alpha} was also able to repress the dimerization between GAL-cFos(137–216) and VP16-cJun (Fig. 4AGo) and that between GAL-cFos(137–216) and VP16-cJun(237–331) (Fig. 4BGo). Note that hRAR{alpha}, with or without AT-RA, did not affect the activity of GAL-VP16 (Fig. 3CGo), excluding a possible hRAR{alpha} interference of either GAL(DBD) or VP16 function. These results clearly show that hRAR{alpha} is able to block in a ligand-dependent manner both hetero- and homodimerization of AP-1, and thereby blocking in vivo DNA binding, by possibly targeting the respective bZIP regions of c-Jun and c-Fos.



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  		Figure 3. Ligand-Bound RAR Disrupts Both AP-1 Homodimerization and Heterodimerization

HeLa cells were transfected with 1 µg of the 17M-tk-CAT reporter plasmid and 1 µg of hRAR{alpha} expression plasmid together with 1 µg each of expression plasmids for GAL-cJun and VP-cFos for heterodimerization (A), 1 µg each of expression plasmids for GAL-cJun and VP16-cJun for homodimerization (B), or 1 µg GAL-cJun and 0.5 µg of GAL-VP16 expression plasmids (C). Cells receiving hRAR{alpha} were treated with 10-7 M AT-RA as indicated. Note that CAT activity is represented relative to activity in the absence of activator, which was set to 1.

 


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  		Figure 4. RAR Inhibition of c-Jun/c-Fos Dimerization Is Targeted to the bZIP Regions of These Protooncoproteins

HeLa cells were transfected with 1 µg of the 17M-tk-CAT reporter plasmid and 1 µg of hRAR{alpha} expression plasmid together with 1 µg each of expression plasmids for GAL-cFos(137–216) and either VP16-cJun (A) or VP16-cJun(237–331) (B). Cells receiving hRAR{alpha} were treated with 10-7 M AT-RA as indicated. Note that CAT activity is represented relative to activity of GAL-cFos(137–216), which was set to 1.

 
hRAR{alpha} Disruption of c-Jun/c-Fos Dimerization Is Cell Specific
The cell-specific nature of nuclear receptor-induced inhibition of AP-1 transcriptional activity prompted us to study hRAR{alpha}-induced inhibition of AP-1 dimerization in several different cells. As observed before (see Fig. 4BGo), liganded hRAR{alpha} is able to block the dimerization between the bZIP regions of c-Jun and c-Fos in HeLa cells (Fig. 5AGo); note that the ligand-dependent activity observed in the absence of transfected hRAR{alpha} is likely due to endogenous receptor. Importantly, however, when the same experiment was repeated in Cos cells, there was no detectable effect of hRAR{alpha}, either in the absence or presence of AT-RA, on c-Jun/c-Fos dimerization (Fig. 5BGo). This lack of RAR activity in Cos cells on AP-1 dimerization correlates with the lack of RAR activity on AP-1 transcriptional activity in these same cells (see Fig. 1BGo). To further examine the cell specificity, we used the yeast two-hybrid system to test the activity of hRAR{alpha} on AP-1 dimerization. In this system, we analyzed the interaction between c-Jun(237–331) fused to the B42 (acid blob) transcriptional activation domain and the LexA DBD joined to either c-Fos(137–216) for heterodimerization or full-length c-Jun for homodimerization. B42-cJun(237–331) interacted strongly with both LexA-cFos(137–216) and LexA-cJun, but cotransformed hRAR{alpha}, either in the absence or presence of AT-RA, had no significant effect (Fig. 6AGo). Accordingly, liganded hRAR{alpha} was unable to repress the transcriptional activity of LexA-cJun (Fig. 6BGo). These results together suggest that the ability of hRAR{alpha} to repress AP-1 dimerization is dependent on cell-specific factors that may not be found in either Cos or yeast cells.



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  		Figure 5. RAR Disrupts c-Jun/c-Fos Dimerization in a Cell-Specific Manner

HeLa (A) and Cos (B) cells were transfected with 1 µg of the TRE-tk-CAT reporter plasmid together with 1 µg each of expression plasmids for GAL-cFos(137–216), VP16-cJun(237–331), or hRAR{alpha}. Cells receiving hRAR{alpha} were treated with 10-7 M AT-RA as indicated. Note that CAT activity is represented relative to activity of GAL-cFos(137–231), which was set to 1.

 


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  		Figure 6. RAR Is Unable to Disrupt c-Jun/c-Fos Dimerization or LexA-cJun Activity in Yeast

Yeast cells EGY48 were transformed with 1 µg of the pSH18–34 reporter plasmid and 1 µg each of expression plasmids for hRAR{alpha}, LexA-cJun, LexA-cFos(137–216), and VP16-cJun (A) or hRAR{alpha}, LexA-cJun, and LexA (B). Cells receiving hRAR{alpha} were treated with 10-6 M AT-RA as indicated. Bars in panel A represent the following: open, B42; black, B42-cJun(237–331); gray, B42-cJun(237–331) + hRAR{alpha}; stippled, B42-cJun(237–331) + hRAR{alpha} + AT-RA.

 
hRAR{alpha} Disruption of AP-1 in Vitro DNA Binding Is Also Cell Specific
Our transfection results above show that liganded hRAR{alpha} can block AP-1 dimerization in a cell-specific manner (see Fig. 5Go). Disruption of AP-1 dimerization should lead to abolishment of AP-1 sequence-specific DNA binding. To analyze this, we measured the in vitro DNA-binding ability of endogenous AP-1 from either HeLa or Cos cells, which had been transfected with hRAR{alpha}, in the absence or presence of RA. Extracts from both cells exhibited significant AP-1 DNA-binding activity (Fig. 7Go, lanes 1 and 3). This AP-1 acitivity was confirmed by both addition of antibody, which disrupts c-Jun DNA binding, (compare lanes 5 and 6) and competition with unlabeled DNA (compare lanes 7 and 8). Importantly, RA-bound hRAR{alpha} was able to repress AP-1 DNA binding in HeLa cells (compare lanes 1 and 2), but not Cos cells (compare lanes 3 and 4), paralleling what was observed with the mammalian two-hybrid system (see Fig. 5Go).



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  		Figure 7. RAR Disrupts AP-1 in Vitro DNA Binding in a Cell-Specific Manner

HeLa and Cos cells were transfected with 10 µg of hRAR{alpha}. These cells were treated with 10-7 M AT-RA as indicated. Nuclear extracts were tested for AP-1 DNA binding using a gel mobility shift assay. Antibody disruption analysis was done with either an anti-c-Jun or antiandrogen receptor (AR) antibody. Note that the anti-c-Jun antibody is directed against the bZIP region and thus disrupts c-Jun DNA binding; therefore, no supershift is detectable. DNA competition was carried out with a 50-fold excess of unlabeled DNA elements (TRE, ARE). The arrow points to the AP-1-TRE complex, and FP represents the free probe. The protein amount (in micrograms) used in each reaction is the following: lane 1, 11.5; lane 2, 11.8; lane 3, 12.8; lane 4, 14.1; lane 5, 11.4; lane 6, 11.5; lane 7, 11.5; lane 8, 11.5.

 
hRAR{alpha} Disruption of c-Jun/c-Fos Dimerization Is Receptor Specific
Several nuclear receptors, including that for glucocorticoids (GR), have been shown to block AP-1 transcriptional activity in a ligand-dependent manner. Interestingly, however, GR has been shown to have no effect on in vivo AP-1 DNA binding, as measured by in vivo footprint analysis (44). Therefore, we tested the activity of GR in our mammalian dimerization assay in HeLa cells, which have previously been shown to exhibit dexamethasone (Dex)-induced repression of AP-1 transcriptional activity (Refs. 20, 21, 22, 23, 24 and data not shown). Importantly, GR, with or without Dex, had no significant influence on dimerization between GAL-cFos(137–216) and VP16-cJun(237–331) in HeLa cells (Fig. 8Go) Thus, inhibition of AP-1 dimerization in vivo is not only cell specific, but also receptor specific.



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  		Figure 8. GR Is Unable to Disrupt c-Fos/c-Jun Dimerization

HeLa cells were transfected with 1 µg of the 17M-tk-CAT reporter plasmid and 1 µg each of expression plasmids GAL-cFos(137–216) and/or VP16-cJun(237–331), and 1, 3, or 5 µg of hGR expression plasmid. Cells receiving hGR were treated with 10-7 M Dex as indicated. Note that CAT activity is represented relative to activity of GAL-cFos(137–216), which was set to 1.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Previous in vitro DNA binding studies suggested that RARs are able to antagonize AP-1 activity by blocking its DNA-binding ability (21, 23, 24, 27, 28, 29). We show in this paper that this interference is cell specific, occurring in HeLa but not Cos cells, and, significantly, provide the first in vivo evidence supporting this mechanism of transcriptional interference. Moreover, these data provide a potential basis for some of the cell- and receptor-specific effects that have been reported (25, 27). Our results indicate that hRAR{alpha} is able to disrupt in a RA-dependent manner the in vivo dimerization capacity of c-Jun with either itself or c-Fos, which would preclude the formation of DNA-binding-competent AP-1 complexes. In support of previous work (27), this RA-dependent effect on dimerization is cell specific, paralleling the previously observed activity of RARs on AP-1 transcriptional activity.

How might RAR prevent the formation of AP-1 homo- and heterodimers? AP-1 dimerization is mediated via the conserved bZIP regions found within c-Jun, c-Fos, and their protein families (reviewed in Ref. 16). Our transfection studies show that the bZIP regions of c-Jun and c-Fos are sufficient for the ligand-dependent RAR inhibition of dimerization. Interestingly, the bZIP regions of c-Jun and c-Fos have been previously shown to be essential for the transcriptional interactions between these protooncoproteins and nuclear receptors (23, 25). Thus, the bZIP regions of c-Jun and c-Fos may provide a common surface through which nuclear receptors can engage in a protein-protein interaction with c-Jun and c-Fos.

Several studies (21, 24, 27, 33, 48), using chemical cross-linking and coprecipitation approaches, have suggested that nuclear receptors can physically associate with both c-Jun and c-Fos, and thus block their ability as AP-1 to bind to DNA. However, these protein-protein interactions appear to be weak or indirect, since we and others (22, 23, 25, 26, 34) have been unable to detect a stable interaction between RAR and the AP-1 components. In fact, our current data support the model proposed by Pfahl (11), that additional factors, which appear to be expressed in a cell-specific manner, are essential for the antagonism between receptors and AP-1. Our previous results showed that the direction and magnitude of the nuclear receptor-AP-1 interaction is dependent on the type of cell, promoter, receptor, and AP-1 component (25). We now provide evidence that liganded hRAR{alpha} is able to inhibit AP-1 dimerization in cell-specific manner, occurring in HeLa cells, in which this receptor can inhibit AP-1 transcriptional activity, but not in Cos cells, where it is has no effect on AP-1 transcriptional activity. Further, liganded hRAR{alpha} does not disrupt c-Jun/c-Fos homo- and heterodimerization reconstituted in yeast, and, accordingly, as would be predicted, it is unable to repress LexA-cJun transcriptional activity in yeast. Thus, it is possible that RARs, and other nuclear receptors, can directly associate with c-Jun and/or c-Fos, but the affinity of this direct interaction is not sufficient in vivo to modulate transcription. Additional factors, expressed in a tissue-specific manner, may be needed to stabilize the RAR-AP-1 interaction and thus prevent AP-1 dimerization (see Fig. 9Go for a scheme). An example of a nonreceptor, cell-specific factor mediating the interaction of a nuclear receptor with another transcription factor comes from a study on the transactivation of the RARß2 promoter. Berkenstam et al. (49) have found that this promoter is synergistically activated by RAR and the TATA box-binding protein (TBP) in embryonal carcinoma (EC) cells but not Cos, and that this synergy can be restored in Cos cells by ectopically expressing E1A (50). It is also possible that RAR has a secondary effect, by inducing the expression of a protein that is directly involved in inhibiting AP-1 dimerization.



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  		Figure 9. A Model of How Ligand-Bound RAR Can Inhibit AP-1 Transcriptional Activity

In the absence of RA-activated RAR, c-Jun and c-Fos are able to dimerize to form AP-1, and this dimeric complex can bind to promoter elements (AP-1 elements) of AP-1-responsive genes. When RA is bound to RAR and the necessary cell-specific factor(s) (factor x) is present, the c-Jun/c-Fos heterodimer does not form; thus, the AP-1 element is vacant and the gene remains transcriptionally silent.

 
In the case of nuclear receptor- and AP-1-mediated transcription, there is compelling evidence that CBP and p300 can act as coactivators for both pathways (36, 37, 38, 39, 40). It was recently reported that CBP, and perhaps p300, can also facilitate the homodimerization of AR (51). Since CBP and p300 can associate with both c-Jun and c-Fos, it is possible that they are serving a similar role in AP-1 dimerization. However, our mammalian two-hybrid assay detected no p300 influence on either c-Jun/c-Fos dimerization or RA-induced disruption of dimerization (data not shown). It has been suggested that RARs and other nuclear receptors can compete with AP-1 for limiting amounts of CBP and p300 (36), thereby resulting in transcriptional efficacy going to one activator at the expense of the other. Since CBP and p300 are known to interact with and mediate the activities of several nuclear receptors, including TR (36, 52), GR (53), ER (54), PR (53, 55), and AR (51), then these same receptors should be in a competitive and mutually inhibitory interaction with the AP-1 components c-Jun and c-Fos. Although this is generally the case, there are several exceptions. First, the AP-1 interaction with AR can be either negative or positive, depending on the AP-1 component. Indeed, our laboratory has shown that c-Jun strongly enhances AR-induced transcription and c-Fos can inhibit this activity, independent of cell or promoter specificity (25, 34, 35). On GR-inducible promoters, c-Jun generally blocks GR activity except in several T cell lines (56). On the AP-1-inducible proliferin promoter, which contains a composite response element, GR and mineralocorticoid receptor (MR) can act either cooperatively or antagonistically with AP-1, depending on the identity of receptor (GR, MR) and AP-1 component (c-Jun/c-Jun or c-Jun/c-Fos) (33). In view of this complexity, it is likely that additional modes of interaction occur between nuclear receptors and AP-1. Recently, it was reported that several nuclear receptors, including RARs, are able to repress JNK activation of c-Jun, providing a second mechanism of nuclear receptor-induced inhibition (41). Our data suggest that an additional mode of action exists, in which RARs can disrupt the ability of c-Jun to homodimerize with itself and heterodimerize with c-Fos. In contrast to the other mechanisms, the last one closely parallels the cell-specific nature of RAR-induced repression of AP-1 transcriptional activity, appears to be specific for RARs, and may depend on involvement of cell-specific factors. Thus, it appears that RARs can use multiple mechanisms by which to blunt the transcriptional activity of AP-1. It is possible that one of these proposed mechanisms is the preferred choice in vivo or that these different modes of actions may cooperate to ensure the appropriate expression of AP-1-responsive genes.

RARs’ effect on AP-1 activity has been proposed to be responsible for the clinical effects of retinoids as antineoplastic, antiinflammatory, and immunosuppressive agents (26, 27, 29). In this regard, it is noteworthy that selective retinoids have been reported that allow a separation of the transactivation and transrepression activities of RARs (57, 58). Future work will be needed to determine whether some of these anti-AP-1-selective retinoids act by inducing RAR-mediated disruption of c-Jun/c-Fos dimerization.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
For mammalian expression, hRAR{alpha} (59), hGR (59), GAL-VP16 (59), and c-Jun (25) in pSG5 have been described. GAL-c-Jun and GAL-c-Fos were also expressed from the mammalian expression plasmid pGAL0 (60). GAL-cFos(137–216) was constructed by PCR amplification of c-Fos amino acids 137–216 using the upstream oligo 5'-GATCGAATTCATGGAAGAGAAACGGAGA-3' and the downstream oligo 5'-GATCGGATCCTCACATCTCCTCTGGGAA-3' and inserting into pGAL0. VP16-cJun was constructed by inserting full-length c-Jun into the BamHI/BglII sites of VP16/pTL1, pTL1 (34) containing the activation domain of VP16. VP16-cJun(237–331) was constructed by PCR amplification of c-Jun amino acids 237–331 using the upstream oligo 5'-GATCGAATTCCTCGAGATGGGCGAGACACCGCCC-3' and the downstream oligo 5'-GATCGGATCCTCAAAATGTTTGCAA-3' and inserting into VP16/pTL1.

For yeast expression, the expression plasmids pEG202 (45), pJG4–5 (45), and pYE10 (61) were used. LexA-cFos(NOREF>137–231) was constructed by digestion of cFos(137–231) from pGAL0 and insertion into pEG202. LexA-cJun was constructed by digestion of c-Jun from pTL1 with BglII, extension with klenow, and cutting with BamHI. This fragment was inserted into pEG202. B42-cJun(237–331) was constructed by inserting the PCR fragment encoding c-Jun amino acids 237–331 into pJG4–5. hRAR{alpha} was expressed from the plasmid pYE10 (61).

For mammalian cells, the reporter plasmids have the gene for chloramphenicol acetyl transferase (CAT) driven by the RA-inducible RARE-tk, AP-1-inducible TRE-tk, or GAL4-inducible 17M-tk promoters (34). Transfection efficiency was standardized by measuring the ß-galactosidase (ß-gal) activity, originating from the cotransfected plasmid pCH110 or CMV-LacZ (25). For yeast cells, the reporter was pSH18–34 (45), which has the LacZ gene under the control of a LexA-inducible promoter.

Cell Transfections and CAT Assays
HeLa and Cos cells were grown and transfected as described previously (34). hRAR{alpha} and GR were activated by the addition of 10-7 M of either AT-RA and Dex, respectively. CAT assays were performed and standardized according to the measured ß-gal activity as previously described (25). For all transfections, we used different amounts of expression plasmid, 1 µg of reporter plasmid (RARE-tk-CAT, 17 M-tk-CAT, or TRE-tk-CAT), 2 µg of pCH110 for Cos cells, and 0.5 µg CMV-LacZ for HeLa cells, and enough carrier DNA (Bluescript) to bring the final plasmid amount to 9 µg per dish. CAT assay results were quantified by densitometric scanning of autoradiograms of at least three repeats for each transfection, and each value represents the average of three to four repetitions plus standard deviation.

Gel Mobility Shift Assay
HeLa and Cos cells were transfected with 10 µg of hRAR{alpha} and 2 µg of pCH110. Cells receiving ligand were treated with 100 nM RA 24 h before harvesting. Cells were harvested in ice-cold PBS and spun at 5000 rpm for 5 min. Ten percent of the cells were used to perform a ß-gal assay for quantification of transfection efficiency. The remainder of the cells were resuspended in buffer I (10 mM Tris-HCl, pH 7.5; 10 mM NaCl; 5 mM MgCl2) and incubated at 4 C for 5 min. Sucrose (0.3 M) was then added and cells were lysed with a dounce homogenizer. Nuclei were pelleted by centrifuging lysed cells at 2500 rpm (600 x g) for 10 min. The nuclear pellet was washed once with buffer II (buffer I containing 0.3 M sucrose). Then, the nuclear pellet was resuspended in buffer III (50 mM Tris-HCl, pH 8; 150 mM NaCl; 5 mM EDTA; 0.1% Nonidet P-40) with protease inhibitors and incubated with shaking at 4 C for 30 min. The lysed nuclei were centrifuged at 15,000 rpm for 15 min, and the supernatant, constituting the nuclear extract, was saved. The amount of extract used was standardized according to ß-gal activity.

Gel mobility shift assays were performed with nuclear extracts containing the same amount of ß-gal activity. These reactions were performed in a final volume of 20 µl in DNA-binding buffer (10 mM Tris, pH 8; 0.1 mM EDTA; 4 mM dithiothreitol) which also contained 1 µg of poly(dI-dC), 100 mM KCl, and 150,000 cpm of 32P-labeled probe (5'-TCGAGTTGCATGAGTCAGACATCGATTGCA-3'). After the addition of x ß-gal units of nuclear extract, the reactions were gently vortexed and incubated for 15 min at 25 C. The samples were run on a 6% polyacrylamide gel for 1.5 h at room temperature, after which the gel was dried and exposed to autoradiography. In some reactions, 1 µl of either anti-c-Jun (sc-44, Santa Cruz Biotechnology, Santa Cruz, CA) or anti-hAR (sc-815, Santa Cruz Biotechnology) were added before addition of probe. Other reactions received a 50-fold excess of either unlabeled AP-1 element (given above) or androgen-response element (ARE) (5'-GATCCAAAGTCAGAACACAGTGTTCT-GATCAAAGA-3').

Yeast Two-Hybrid System
Yeast two-hybrid analysis and LexA-cJun activity were measured by quantifying ß-gal activity using o-nitrophenyl-ß-D-galactoside as a substrate as described (62). AT-RA was added to a final concentration of 10-6 M.


   ACKNOWLEDGMENTS
 
We would like to thank Roger Brent, Barak Cohen, and Lauren Ha for providing the materials for the yeast two-hybrid system, Gordon Tomaselli for GAL-cFos and GAL-cJun, Pierre Chambon and Hinrich Gronemeyer for the hRAR{alpha} plasmid, Athanasios Bubulya for helpful discussions, Yun Zhou for technical support, and Scott Leisner for critical reading of the manuscript.


   FOOTNOTES
 
Address requests for reprints to: Dr. L. Shemshedini, Department of Biology, University of Toledo, Toledo, Ohio 43606. Email: lshemsh@uoft02.utoledo.edu.

This work was supported in part by American Heart Association Grant NW-95–16-YI and NIH Grant DK-51274 to L.S.

Received for publication July 9, 1998. Revision received November 2, 1998. Accepted for publication November 4, 1998.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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