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Molecular Endocrinology, doi:10.1210/me.2005-0145
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Molecular Endocrinology 20 (5): 1025-1034
Copyright © 2006 by The Endocrine Society

Role of Steroid Receptor Coactivators in Glucocorticoid and Transforming Growth Factor ß Regulation of Plasminogen Activator Inhibitor Gene Expression

Gangyong Li, Joanne H. Heaton and Thomas D. Gelehrter

Departments of Human Genetics (G.L., J.H.H., T.D.G.) and Internal Medicine (T.D.G.), University of Michigan Medical School, Ann Arbor, Michigan 48109-0618

Address all correspondence and requests for reprints to: Thomas D. Gelehrter, Department of Human Genetics, Box 0618, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618.E-mail: tdgum{at}umich.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TGFß is a major regulator of extracellular matrix deposition and a potent inducer of type-1 plasminogen activator inhibitor (PAI-1) gene expression. We have reported that liganded glucocorticoid receptor (GR) represses TGFß transactivation of PAI-1 in Hep3B human hepatoma cells and that it interacts functionally and physically with the C-terminal activation domain of Smad3, a mediator of TGFß signaling. The ligand binding domain of GR is required for GR-mediated transrepression, but the GR DNA binding domain and activation function 1 domains are not. We report here that overexpression of steroid receptor coactivator-1 (SRC-1) and GR-interacting protein-1 (GRIP-1) enhanced repression by liganded GR, and by a GR mutant defective in repression. Surprisingly, SRC-1 and GRIP-1 also enhanced TGFß-induced activation from the TGFß-responsive sequence of the PAI-1 gene by a GR-independent mechanism. Coimmunoprecipitation and mammalian one-hybrid experiments demonstrated that SRC-1 and GRIP-1 interact physically with endogenous Smad3 and functionally with the C-terminal domain of Smad3 to directly enhance transcription. Thus, the GR coactivators, SRC-1 and GRIP-1, act as both corepressors of the glucocorticoid repression of PAI-1 gene transcription, and coactivators of TGFß-induced activation of the PAI-1 promoter.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GLUCOCORTICOIDS AND TGFß have opposing actions on extracellular matrix deposition and wound healing (1, 2, 3, 4). TGFß is a major inducer of extracellular matrix deposition (1, 2, 4, 5) and a potent inducer of type-1 plasminogen activator inhibitor (PAI-1) gene expression (3, 6). Glucocorticoids are widely used as antiinflammatory and antifibrotic agents and are known to block the TGFß-induced expression of extracellular matrix proteins, including PAI-1 (3). The antiinflammatory actions of glucocorticoids are thought to be mediated primarily by repression of AP-1 and nuclear factor (NF)-{kappa}B action (7, 8, 9, 10). In both cases, liganded glucocorticoid receptor (GR) monomers interact directly with the transcriptional activators, fos-jun, and p65, respectively, tethered to their cognate DNA binding sites (7, 8, 11, 12, 13).

We have reported previously that liganded GR strongly represses TGFß activation of transcription from the PAI-1 promoter by interacting physically and functionally with Smad3, the major intracellular mediator of TGFß induction of PAI-1 (1, 3). Like the GR repression of AP-1 and NF-{kappa}B function (14, 15, 16, 17), repression is mediated by protein-protein interactions. Mutations in the D-loop of the GR DNA binding domain (DBD), which interfere with DNA binding by GR and gene transactivation, do not interfere with transrepression. Furthermore, the GR-antagonist RU486 acts as a partial agonist, and the GR ligand binding domain (LBD) is required for repression (18). However, unlike the glucocorticoid repression of AP-1 and NF-{kappa}B action, the glucocorticoid repression of TGFß activation is not reciprocal; TGFß does not repress GR transactivation from a GRE (3).

As a model system, we have used human Hep3B hepatoma cells, which have intact TGFß signaling pathways, and express endogenous PAI-1, but lack functional GRs, enabling us to study the role of mutant or wild-type GR transfected into these cells. As a reporter for TGFß action, we have used a luciferase reporter gene driven by a minimal E1b promoter element and six copies of a 12-bp Smad3/Smad4 binding sequence from the hPAI-1 gene that mediates strong induction by TGFß (3, 6). To map the domain(s) in the GR involved in repression of TGFß signaling, we examined the ability of various GR truncation, deletion, and substitution mutants to repress TGFß transactivation in Hep3B cells. We demonstrated that the LBD of GR is required for GR-mediated repression, but the GR DBD and activation function (AF) 1 ({tau}1) activation domains are not. Ligand binding to GR is necessary, but not sufficient, for repression of TGFß activation of PAI-1 gene expression (18).

We have suggested previously (18) that interaction of liganded GR with the C-terminal transactivation domain of Smad3 might result in recruitment of other activator or repressor molecules that repress TGFß transactivation. Relevant to this hypothesis, it has been reported that GR-interacting protein 1 (GRIP-1), but not steroid receptor coactivator 1 (SRC-1), acted as a corepressor of AP-1 induction of the human collagenase-3 gene, and of NF-{kappa}B induction of IL-8 gene expression by glucocorticoids (19, 20). To analyze the mechanisms of GR/TGFß cross talk, we have examined the effects of SRCs on GR repression.

The SRCs comprise a family of p160 proteins including SRC-1; GRIP-1, also known as transcriptional intermediary factor 2; and pCIP (p300/cAMP response element binding protein-binding protein-interacting protein), also known as activator of the thyroid and retinal receptors, receptor-associated coactivator 3, thyroid hormone receptor activator molecule 1, or amplified in breast cancer 1 (21, 22). These proteins interact with the AF2 ({tau} C) region of the steroid ligand binding domain in an agonist-dependent manner to enhance transcriptional activation from hormone response elements (23, 24). We report here that overexpression of the coactivators SRC-1 and GRIP-1 enhanced repression by wild-type GR, and by GR S561A, a mutant defective in transactivation and repression, but not by LBD deletion mutants unable to bind ligand. Surprisingly, we also found that both SRC-1 and GRIP-1 enhance TGFß-induced transactivation, and this enhancement is completely independent of GR. Coimmunoprecipitation and mammalian one-hybrid experiments demonstrated that SRC-1 and GRIP-1 physically interact with endogenous Smad3, and functionally with the C-terminal domain of Smad3, to enhance transcription from a TGFß-responsive region of the human PAI-1 gene.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Overexpression of the Steroid Receptor Coactivators, SRC-1 and GRIP-1, Enhances Repression by GR
To investigate the mechanisms of GR/TGFß cross talk, we first examined whether corepressors, which have histone deacetylase (HDAC) activity, might be involved in GR repression. Hep3B cells were transfected with the reporter plasmid pTRS6E1b-luc and GR expression plasmid pRShGR{alpha}. After 16 h, the cells were cultured in the presence or absence (control) of TGFß, and/or dexamethasone, and with or without 1 µM trichostatin A , an HDAC inhibitor (25). As reported for the dexamethasone repression of AP-1 and NF-{kappa}B (19, 20), trichostatin A inhibition of HDAC did not abrogate GR repression, suggesting that corepressors were not involved in GR repression of TGFß transactivation of the PAI-1 promoter (data not shown).

To examine whether coactivators act as corepressors of GR repression of TGFß transactivation, we overexpressed SRC-1 and GRIP-1 in Hep3B cells, which were cotransfected with the TRS6 reporter and GR-expressing plasmids. Cells were treated with TGFß in the presence or absence of dexamethasone. SRC-1 and GRIP-1 further increased repression of TGFß transactivation (from 82% to 93% and 98%, respectively) by maximally effective concentrations of GR (Fig. 1AGo). When Hep3B cells were transfected with suboptimal amounts of GR (5 ng GR expression plasmid per 22 mm well vs. 200 ng per well), GR repression of TGFß transactivation was reduced to 57%. Under these conditions, overexpression of SRC-1 or GRIP-1 could restore almost full GR repression (85% and 92% repression, respectively) (Fig. 1BGo). These observations suggest that the concentrations of coactivators SRC-1 and GRIP-1 in Hep3B cells are limiting for maximal GR repression of TGFß-activated transcription, most strikingly when the amount of GR is suboptimal.


Figure 1
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  		Fig. 1. SRC-1 and GRIP-1 Enhance the Ability of Liganded GR to Repress TGFß Transactivation of Human PAI-1 TRS

Hep3B cells cultured in 12-well plates were transiently transfected with 0.5 µg/well reporter plasmid pTRS6E1b-luc and either 200 ng/well (A) or 5 ng/well (B) GR expression plasmid pRShGR{alpha}, without or with the cotransfection of 300 ng/well SRC-1 or GRIP-1 expression plasmids. After 16 h, the cells were cultured in the presence or absence (control) of 50 pM TGFß, and/or 100 nM dexamethasone for 24 h. Luciferase activity was assayed as described in Materials and Methods. Data are shown as the mean ± SD relative light units of triplicate wells. The experiment was performed at least three times. Note the difference in scale of the y-axis in the different panels. Open bar, Control; shaded bar, TGFß; solid bar, TGFß + dexamethasone.

 
Overexpression of SRC-1 or GRIP-1 Enhances Repression by Some GR Mutants, But Not Those Affecting GR Ligand Binding
We have reported previously (18) that a point mutation, S561A, in the {tau}2 domain of the mouse GR [corresponding to S555A in the human GR (hGR)] has diminished repression activity (15–20% of wild type), although it binds dexamethasone as well as wild type (26). As shown in Fig. 2Go, overexpression of SRC-1 and GRIP-1 increased repression by this mutant from 15% to more than 50%. In contrast, SRC-1 or GRIP-1 overexpression did not substantially increase repression by GR mutants with deletions in the LBD region that are unable to bind glucocorticoids ({Delta}488–532, {Delta}532–697, and {Delta}{tau}C) and unable to repress TGFß transactivation (Fig. 2Go). By immunoblot assay, all GR mutants were expressed at approximately the same level as wild-type GR. When GR mutants were tested at lower concentrations (5–10 ng/well), we observed similar enhancement of repression by GR S561A (~3-fold), but none by the LBD mutants (data not shown). These observations confirm that ligand binding to GR is necessary for repression even in the presence of coactivators.


Figure 2
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  		Fig. 2. SRC-1 and GRIP-1 Enhance the Repression Function of the {tau}2 Mutant GR S561A, But Not by LBD Deletion Mutants

Hep3B cells in 12-well plates were transiently transfected with 0.5 µg/well pTRS6E1b-luc and plasmids containing wild-type GR (5 ng/well or 200 ng/well) or various GR mutants (all tested at 200 ng/well), with or without the cotransfection of 300 ng/well SRC-1 or GRIP-1 expressing plasmids. Incubation of cells with TGFß and/or dexamethasone and luciferase activity assays were performed as described in the Fig. 1Go. The percentage of repression is shown as the mean ± SEM. All experiments were performed at least three times.

 
SRC-1 and GRIP-1 Physically Interact with Wild-Type GR and GR S561A
Because overexpression of SRC-1 and GRIP-1 could increase repression, especially by suboptimal amounts of GR, and it could also enhance the repression function of GR S561A, we performed coimmunoprecipitation assays to examine whether this GR mutant was able to physically interact with SRC-1 and GRIP-1. As shown in Fig. 3Go, wild-type GR and GR S561A were able to physically interact with overexpressed SRC-1 and GRIP-1, suggesting that physical interaction might be necessary for repression function of GR S561A. Although GR{Delta}488–532 could physically interact with both SRC-1 and GRIP-1, its repression function was not enhanced by the overexpression of either of coactivator, suggesting that physical interaction alone was not sufficient, and other factors such as ligand binding ability were also necessary. Interestingly, GR{Delta}532–697 and GR{Delta}{tau}C interacted very little, if at all, with GRIP-1 although they did coimmunoprecipitate with SRC-1.


Figure 3
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  		Fig. 3. In Vivo Interaction between SRC-1/GRIP-1 and Wild-Type or Mutant GRs

Hep3B cells cultured in 100-mm dishes were transfected with 9 µg/dish wild-type (WT) or mutant GR expressing plasmids, and with 6 µg/dish GRIP-1 (panel A) or SRC-1 (panel B) expressing plasmids. After 16 h, the cells were cultured in the presence of dexamethasone for 24 h. The lysates were immunoprecipitated (IP) with anti-GR antibodies, and the immunoprecipitation complexes subjected to SDS-PAGE under reducing conditions. After blotting to a nitrocellulose membrane, mouse anti-GRIP-1 or anti-SRC-1 antibodies were used to probe GRIP-1 (A) or SRC-1 (B). Lysates were also immunoprecipitated with anti-GR antibodies and blots probed with anti-GR antibodies (C). The level of expression of mutant GRs was approximately the same as wild-type GR. The experiment was performed three times. IB, Immunoblotting.

 
Overexpression of SRC-1 or GRIP-1 Enhances Repression by Suboptimal Concentrations of Dexamethasone
Based on the above observations that coactivator concentrations are limiting for maximal GR repression, one might predict that the coactivators would also enhance repression by submaximal concentrations of glucocorticoid in the presence of maximally effective amounts of GR (200 ng/well). As predicted, and as shown in Fig. 4Go, the concentration of dexamethasone required for repression is 3-fold lower in the presence of SRC-1 and GRIP-1 (EC50: 0.7 nM vs. 2.5 nM). Thus, maximal glucocorticoid repression of TGFß-mediated transcriptional activation requires sufficient cellular concentrations of ligand, receptor, and coactivators. Binding of GR by agonist ligands and by coactivators is known to modulate the conformation of the GR, making it a more potent transcriptional activator (23, 24); similar mechanisms might be involved in transrepression.


Figure 4
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  		Fig. 4. SRC-1 and GRIP-1 Enhance Repression by Suboptimal Concentrations of Dexamethasone

Hep3B cells cultured in 12-well plates were transiently transfected with 0.5 µg/well reporter plasmid pTRS6E1b-luc, 200 ng/well pRShGR{alpha}, and without (open circles) or with 300 ng/well SRC-1 (closed squares) or GRIP-1 (closed triangles). After 16 h, the cells were cultured in various concentrations of dexamethasone for 24 h. Luciferase assay was performed and repression calculated as described. Data, based on the average of triplicate wells, are shown as a percentage of repression.

 
Overexpression of SRC-1 or GRIP-1 Enhances Repression by the Partial Antagonist RU486
Binding of SRCs is generally thought to be agonist dependent, although there is evidence that such interaction can also occur in the presence of antagonists (27, 28). We have reported previously (18) that RU486, an antagonist of GR transactivation from hormone response elements, acted as a partial agonist with respect to GR repression of transcription from TRS6 (40% repression). In the presence of overexpressed SRC-1 and GRIP-1, RU486 repression is significantly enhanced to 68% and 80%, respectively (Fig. 5Go).


Figure 5
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  		Fig. 5. SRC-1 and GRIP-1 Enhance Repression by the Partial Antagonist RU486

Hep3B cells were cultured as described in Fig. 1AGo, except that after 16 h the cells were incubated in the presence of 50 pM TGFß and the presence of either 100 nM dexamethasone or 1 µM RU486 for 24 h [the maximally effective concentration of each steroid) (18 )]. Luciferase activity was assayed and repression calculated as described. The experiment was performed three times.

 
In summary, we have shown that overexpression of the coactivators SRC-1 and GRIP-1 enhances repression of TGFß-mediated transactivation by liganded GR. This is particularly noted under conditions of suboptimal repression as in the case of 1) suboptimal concentration of transfected GR, 2) submaximal concentration of the agonist dexamethasone, 3) the partial agonist RU486, and 4) GR S561A, a GR mutant able to bind ligand but with impaired ability to repress.

SRC-1 and GRIP-1 Enhance Both Basal and TGFß-induced Transactivation from the TGFß-responsive Region of the PAI-1 Gene, and this Enhancement Does Not Require GR
In the experiments shown in Fig. 1Go, we noted that overexpression of SRC-1 and GRIP-1 resulted in increased TGFß activation of TRS6-luc transcription. As shown in Fig. 6AGo, overexpression of either SRC-1 or GRIP-1 dramatically enhanced TGFß-induced transcription (15- and 35-fold, respectively), and this effect was completely independent of GR expression. Even in the absence of TGFß treatment, SRC-1 and GRIP-1 could enhance basal transcription of TRS6-luc (~8-fold), again independent of GR expression (Fig. 6BGo). These results demonstrate for the first time that SRC-1 and GRIP-1 can act directly upon the TGFß signaling pathway to enhance transcriptional activation.


Figure 6
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  		Fig. 6. SRC-1 and GRIP-1 Enhance TGFß Transactivation of Human PAI-1 TRS Independent of GR

A, Hep3B cells in 12-well plates were transfected with 0.5 µg/well pTRS6E1b-luc, with or without transfection of 200 ng/well wild-type GR expressing plasmid, and with or without 300 ng/well SRC-1 or GRIP-1 expressing plasmids. After 16 h, the cells were treated with 50 pM TGFß. Twenty-four hours later, cells were lysed and luciferase activity assays were performed. Data are shown as the mean ± SD relative light units of triplicate wells. B, Hep3B cells in 12-well plates were transfected with 0.5 µg/well pTRS6E1b-luc, with or without transfection of 200 ng/well wild-type GR expressing plasmid, and with or without 300 ng/well SRC-1 or GRIP-1 expressing plasmids. After a total of 40 h incubation without additions, luciferase activity was assayed. Data are shown as the mean ± SD relative light units of triplicate wells. Note the different scales on the y-axis in parts A and B. These experiments were repeated three times.

 
SRC-1 and GRIP-1 Functionally Interact with the MH2 Domain of Smad3 to Enhance Transcription
To analyze how SRC-1 and GRIP-1 affect TGFß signaling, we transfected Hep3B cells with GAL4-Smad3C (3), containing the GAL4 DBD fused to the MH2 transcriptional activation domain of Smad3, and the reporter plasmid, pG5B, with or without cotransfection of SRC-1 or GRIP-1. As shown in Fig. 7Go, and as we have reported previously (3), GAL-4-Smad3C potently induced activation of this reporter (125-fold in this experiment); SRC-1 or GRIP-1 overexpression further enhanced GAL4-Smad3C-mediated activation of the reporter 16-fold, demonstrating that SRC-1 or GRIP-1 could act directly on the TGFß downstream signaling molecule Smad3, independent of GR function.


Figure 7
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  		Fig. 7. SRC-1 and GRIP-1 Directly Enhance the Ability of GAL-4-Smad3C to Induce Transcription

Hep3B cells cultured in 12-well plates were transiently transfected with 0.5 µg/well reporter plasmid pG5E1b-luc, and cotransfected with 0.5 µg/well GAL-4 alone, or 10 ng/well GAL-4-Smad3C alone, or GAL-4-Smad3C together with SRC-1 or GRIP-1 expressing plasmids. Gal4 contains only the DBD of Gal4 without a transactivation domain. After 16 h, luciferase activity was assayed as described before. Data are shown as the mean ± SD relative light units of triplicate wells. The experiment was repeated three times.

 
SRC-1 and GRIP-1 Physically Interact with Endogenous Smad3
Because SRC-1 and GRIP-1 could interact functionally with Smad3 independently of GR, we carried out coimmunoprecipitation assays to examine whether they interact with one another physically in Hep3B cells. Cultured Hep3B cells, which have an intact TGFß signaling pathway, were transfected with SRC-1 or GRIP-1 expressing plasmids and then treated with TGFß for 24 h, followed by immunoprecipitation of cell lysates with anti-Smad3 antibody. As shown in Fig. 8Go, overexpressed SRC-1 or GRIP-1 physically interacted with endogenous Smad3 in Hep3B cells. The sensitivity of the coimmunoprecipitation assays was not sufficient to allow detection of interaction of Smad3 with endogenous SRC-1 or GRIP-1. As shown in Fig. 8CGo, incubation with an irrelevant antibody, goat {alpha}-myc, did not immunoprecipitate Smad3 or GRIP-1.


Figure 8
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  		Fig. 8. In Vivo Interactions between Endogenous Smad3 and Transfected SRC-1 or GRIP-1

A, Hep3B cells cultured in 100-mm dishes were transiently transfected with (+) or without (–) 8 µg/dish SRC-1 or GRIP-1 expression plasmids. After 16 h, the cells were cultured in the presence of 50 pM TGFß. Cell lysates were immunoprecipitated (IP) with goat anti-Smad3 antibody, and the immunoprecipitation complexes were subjected to SDS-PAGE under reducing conditions. After blotting to a nitrocellulose membrane, mouse anti-SRC-1 or anti-GRIP-1 antibodies were used to detect SRC-1 or GRIP-1. After stripping the nitrocellulose membranes, a rabbit anti-Smad3 antibody was used to detect Smad3, to ensure equal amounts of endogenous Smad3. B, Cell lysates were also immunoblotted (IB) with anti-SRC-1 or anti-GRIP-1 to demonstrate expression in the transfected cultures. C, In a separate experiment, lysates from cells transfected with GRIP-1 and incubated with TGFß were immunoprecipitated with goat anti-Smad3 or an irrelevant antibody, goat anti-myc. Complexes were subjected to SDS-PAGE and immunoblotted with mouse anti-GRIP-1 or rabbit anti-Smad3. Note that the lower (faster migrating) band detected by anti-Smad3 is IgG, and the upper band is Smad3.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The p160 family of SRCs binds to the C-terminal ligand binding domain (AF2 or {tau}C) of steroid receptors in the presence of agonist ligands. This results in recruitment of histone acetyltransferases to enhancer/promoter regions to enhance gene transcription. In contrast, the corepressors SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) and NCoR (nuclear receptor corepressor) are approximately 270-kDa nuclear proteins that were discovered by their binding to unliganded heterodimeric nuclear receptors. They are thought to act by recruiting histone deacetylases and repressing transcription (see Ref. 29 for review). Although these corepressors were initially not thought to bind to steroid receptors, there is now evidence that they can bind to several steroid receptors in the presence of their respective antagonist ligands. Recent evidence suggests further that corepressors can interact with both agonist and antagonist complexes of GR, and that mutually antagonistic interactions between corepressors and coactivators can modulate several aspects of GR function, including dose response relationships and partial agonist activity of antagonist-bound receptors (27, 28, 30).

Rogatsky et al. (19, 20) have reported previously that GRIP-1, but not SRC-1, enhances the glucocorticoid-mediated repression of human collagenase 3 gene expression activated by AP1 in U2OS osteoclast cells and represses the activation of the IL8 gene by NF-{kappa}B. In neither case did NCoR or SMRT appear to be involved and repression was not trichostatin A sensitive. We report here that trichostatin A also fails to block the glucocorticoid repression of TGFß transactivation from a TGFß-responsive promoter in the human PAI-1 gene.

While our manuscript was under review, Rogatsky’s laboratory has reported that GRIP-1 physically interacts with interferon regulatory factor 3 (IRF3), a regulator of the innate immune response. GR competes with interferon 3 (IFN3) for binding to GRIP-1 in vitro, and antagonizes IRF3-mediated transcriptional activation of target genes; overexpression of GRIP-1 rescues glucocorticoid repression (31). This interaction is clearly different from the one described in our report, in that overexpression of SRC-1 and GRIP-1 enhances, rather than abrogates, glucocorticoid repression of TGFß induction of PAI-1 gene expression, and that overexpression of both SRC-1 and GRIP-1 enhanced repression.

SRC-1 and GRIP-1 can also enhance repression by the GR {tau}2 domain mutant, S561A, which binds dexamethasone like wild-type GR but has markedly diminished transactivation and repression functions (18, 26). However, coactivator overexpression was not able to significantly enhance repression by ligand binding domain mutants of the GR that fail to bind dexamethasone (Fig. 2Go). Thus, ligand binding to the GR LBD is essential for repression, even in the presence of coactivators. Coimmunoprecipitation experiments indicated that the coactivators interact in vivo with both wild-type and mutant GRs, including those incapable of repression. Thus, physical interaction of GR with SRC-1 and GRIP-1 may be necessary but is not sufficient for the enhancement of repression. Finally, SRC-1 and GRIP-1 could enhance repression by GR in the presence of submaximally effective concentrations of dexamethasone, or in the presence of the partial agonist ligand, RU486.

SRC-1 has been reported to interact with a number of transcription factors besides nuclear receptors, including NF-{kappa}B (32, 33), AP-1 (34), serum response factor (35), p53 (36), STAT6 (signal transducer and activator of transcription 6) (37) and STAT3 (38), and class II transactivator (39). These interactions enhance their transcriptional activity, possibly by helping to recruit other coactivators; however the exact mechanisms remain unknown. We have shown in mammalian one-hybrid experiments (Fig. 7Go) that SRC-1 and GRIP-1 can directly enhance the transcriptional activation activity of the C-terminal MH2 domain of Smad3, without needing to posit any effect on Smad binding to DNA.

Overexpression of SRC-1 in CV-1 cells was found to block the mutual repression by 12-O-tetradecanoylphorbol-13-acetate and 9-cis-retinoic acid of transfected target reporter constructs (34). In contrast, we report that SRC-1 did not relieve the dexamethasone repression of TGFß-induced transcription, but rather enhanced it.

Various nuclear receptors including androgen receptor, estrogen receptor, vitamin D receptor (VDR), and the orphan nuclear receptor hepatocyte nuclear factor 4, have been shown to interact with Smad signaling molecules, both activating and repressing TGFß function (40, 41, 42, 43, 44). In the case of VDR, Smad3 could act as a coactivator of VDR-mediated gene transcription by forming a complex with VDR and SRC-1. Although Smad3, via its MH1 domain, interacts physically and functionally with VDR, no interaction with SRC-1 or transcriptional intermediary factor 2 was observed (44).

Smads interact positively and negatively with multiple transcription factors in addition to nuclear receptors (45, 46, 47). In only one case, however, have SRCs been directly involved. It has been reported that GRIP-1 potentiates skeletal muscle differentiation by activating myocyte enhancer factor, MEF-2C (48) TGFß is a potent inhibitor of myoblast terminal differentiation, and Smad3 has been demonstrated to bind to MEF-2C, physically disrupting its interaction with GRIP-1 (49). We report here that the C-terminal domain of Smad3 interacts functionally and physically with the SRC-1 and GRIP-1 to directly enhance transcription from a TGFß-responsive Smad-binding element in the human PAI-1 gene, and that this enhancement is not dependent on the presence of GR.

We suggest that the SRC-1 and GRIP-1 act as amplifiers of both transactivation and repression of gene expression from the hPAI-1 promoter. In the absence of liganded GR, TGFß-induced transactivation via Smad3 is markedly enhanced. In the presence of liganded GR, repression of TGFß-mediated transactivation is enhanced. Thus, it is the presence of liganded GR that determines whether transactivation or repression will occur. We have shown that Smad3 and GR interact physically and functionally (3) and that SRC-1 and GRIP-1 can interact physically and functionally with both Smad3 and GR. We propose that binding of coactivators to GR alters the conformation of the receptor such that it becomes a more potent repressor, as is the case with respect to its ability to activate transcription. We hypothesize, but have not yet shown, that in the presence of ligand, a trimolecular complex involving Smad3, GR, and the coactivator form at the Smad-binding site of the hPAI-1 promoter to which Smad3 is tethered. Our data do not allow us to distinguish between a model in which coactivators bind directly to both Smad3 and liganded GR, from one in which the coactivators bind to liganded GR, which, in turn, is bound to Smad3. Further experiments with mutant forms of SRC-1 and GRIP-1 would be required to test this hypothesis.

In summary, we report that overexpression of the coactivators SRC-1 and GRIP-1 in Hep3B cells enhances the glucocorticoid repression of TGFß transactivation by liganded wild-type GR and by the GR S561A mutant defective in repression. Unexpectedly, both SRC-1 and GRIP-1 enhanced TGFß-induced transcription in these same cells by a mechanism completely independent of the presence of GR. Furthermore, we have shown that SRC-1 and GRIP-1 interact physically and functionally with endogenous Smad3 to enhance transcription from a TGFß-responsive promoter. Thus, SRC-1 and GRIP-1 can act both as corepressors, in the presence of liganded GR, and as coactivators, in the absence of liganded GR, on the same promoter.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture
Hep3B is a human hepatoma cell line that has intact TGFß signaling and expresses endogenous PAI-1, but has little or no functionally active endogenous GR (3, 18). Hep3B cells were cultured in DMEM (Invitrogen Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.5 µg/ml Fungizone in 95% air and 5% carbon dioxide at 37 C.

Plasmids
TRS6 contains six copies of the TRS at –732/–721 of the human PAI-1 promoter upstream of an E1B TATA box linked to a luciferase reporter gene (3, 6). The human GR expression plasmid pRShGR{alpha}, and GR deletion mutants, GR{Delta}488–532 and GR{Delta}532–697, were kindly provided by Dr. R. Evans (The Salk Institute, La Jolla, CA). The mouse GR {tau}2 mutant S561A (corresponding to the hGR S555A), SRC-1 expressing plasmid (pSG5.HA-SRC-1a), and GRIP-1 expressing plasmid (pSG5.HA-GRIP-1) were kindly provided by Dr. M. R. Stallcup (University of Southern California, Los Angeles, CA). GAL4 and GAL4-Smad3C expressing plasmids, and the pG5B-luc reporter plasmid, which has five copies of the GAL4 binding site upstream of the E1b-luciferase reporter, have been described previously (3). The {tau}c deletion mutant GR1–726 was constructed as described previously (18).

Antibodies
Rabbit antihuman GR antibodies (E-20, against N-terminal and P-20 against C-terminal), goat anti-Smad3 and goat anti-myc antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-SRC-1 and anti-GRIP-1 antibodies were purchased from Affinity Bioreagents (Golden, CO) and Lab Vision (Fremont, CA), respectively. The horseradish peroxidase conjugated secondary antimouse and antirabbit antibodies were purchased from Amersham Life Science (Piscataway, NJ).

Transient Transfection
Hep3B cells cultured in 12-well (22 mm) plates at 60–80% confluency were transfected, in triplicate, using Fugene 6 (Roche, Indianapolis, IN), with 0.5 µg/well pTRS6E1b-luc reporter and selected expression plasmids as indicated in the figure legends. Sixteen hours after transfection, the cells were cultured in the presence or absence of 50 pM TGFß, and/or 100 nM dexamethasone for an additional 24 h. For immunoblot analysis and coimmunoprecipitation assays, Hep3B cells cultured in 100-mm dishes were transiently transfected with pTRS6E1b-luc, wild-type, or mutant steroid receptor expressing plasmids, and/or SRC-1 or GRIP-1 expressing plasmids as indicated in the figure legends.

Luciferase Assays
Cells were washed twice with PBS, and luciferase assays were performed as described (18) using a Microlumat LB96P (PerkinElmer, Wellesley, MA) luminometer, kindly provided by Dr. James Baker Jr. (University of Michigan, Ann Arbor, MI). Repression, expressed as a percentage, was calculated from luciferase activity as follows:

Formula

Immunoblot Analysis
Lysis, electrophoretic analyses, and transfers were performed as described (18). After blocking the membrane with 5% nonfat milk for 1 h at room temperature, the membrane was incubated with anti-GR, anti-SRC-1, anti-GRIP-1, or anti-Smad3 antibodies for 1 h. After washes with Tween 20-PBS, the membrane was incubated with appropriate horseradish peroxidase conjugated antibodies. The proteins were detected using Amersham ECL detection reagents (Amersham) and x-ray films (Kodak) according to the manufacturer’s instructions.

Coimmunoprecipitation Assays
Coimmunoprecipitation assays were performed as described (18). GRs or Smad3 were immunoprecipitated by incubating a mixture of rabbit anti-GR antibodies (against both N and C terminal of GR), or a goat anti-Smad3 antibody (Santa Cruz), with protein G agarose beads (Invitrogen Life Technologies) at 4 C overnight. Immunoprecipitates were washed four times with ice-cold lysis buffer and resolved by reducing SDS-PAGE. SRC-1 and GRIP-1 were detected using mouse anti-SRC-1 and anti-GRIP-1 antibodies.


   ACKNOWLEDGMENTS
 
We thank M. R. Stallcup, R. Evans, and J. Massague for plasmids, H. Zang for helpful discussions, and K. Grahl for help with preparation of the manuscript.


   FOOTNOTES
 
This work was supported by an Arthritis Foundation Biomedical Science Grant.

G.L., J.H.H., and T.D.G. have nothing to declare.

First Published Online January 19, 2006

Abbreviations: DBD, DNA binding domain; GR, glucocorticoid receptor; GRIP-1, GR-interacting protein-1; HDAC, histone deacetylase; hGR, human GR; LBD, ligand binding domain; NF, nuclear factor; PAI-1, plasminogen activator inhibitor; SRC, steroid receptor coactivator; TRS6, reporter plasmid pTRS6E1b-luc; TRS, TGFß-responsive sequence; VDR, vitamin D receptor.

Received for publication April 5, 2005. Accepted for publication January 9, 2006.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   GR
Coregulators:   SRC-1  |  GRIP1
Ligands:   Dexamethasone  |  RU486



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