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Originally published In Press as doi:10.1074/jbc.M305350200 on August 5, 2003

J. Biol. Chem., Vol. 278, Issue 43, 41779-41788, October 24, 2003
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Identification of Glucocorticoid Receptor Domains Involved in Transrepression of Transforming Growth Factor-{beta} Action*

Gangyong Li{ddagger}, Shengfu Wang{ddagger}, and Thomas D. Gelehrter{ddagger}§

From the Departments of {ddagger}Human Genetics and §Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109

Received for publication, May 21, 2003 , and in revised form, July 14, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The transforming growth factor-{beta} (TGF-{beta}) and glucocorticoid signaling pathways interact both positively and negatively in regulating a variety of physiological and pathologic processes. We previously reported that liganded glucocorticoid receptor (GR) repressed TGF-{beta} induction of human plasminogen activator inhibitor-1 gene transcription by directly targeting the transcriptional activation function of Smad3. To identify the domain(s) in the glucocorticoid receptor involved in this repression, we have examined the ability of various GR truncation, deletion, and substitution mutants to repress TGF-{beta} transactivation in Hep3B human hepatoma cells that lack functional endogenous GR. Partial deletions in the ligand-binding domain (LBD), including the {tau}2 and {tau}c regions, greatly reduced or eliminated GR repression, whereas deletion of the N-terminal AF1 ({tau}1) domain and substitution mutations in the DNA-binding domain had little or no effect. Liganded androgen receptor repressed TGF-{beta} transactivation, whereas mineralocorticoid receptor did not, and studies with rat GR-mineralocorticoid receptor chimeras confirmed that the GR C-terminal domains were required for repression. RU486, a strong antagonist of transactivation by GR, partially reversed repression by wild type GR. Co-immunoprecipitation experiments in Hep3B cells indicated that physical interaction between GR and Smad3 is necessary but not sufficient for repression. Physical interaction required activation of Smad3 by TGF-{beta} but not dexamethasone binding to GR. Glutathione S-transferase pull-down assays demonstrated that several regions of the LBD could mediate GR-Smad3 physical interaction. We conclude that the LBD of GR, but not the DNA-binding domain or the N-terminal activation domain, is required for GR-mediated transrepression of TGF-{beta} transactivation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The biological effects of glucocorticoids are mediated by intracellular glucocorticoid receptor (GR),1 a member of the nuclear steroid hormone receptor family (1–3). GR can modulate gene expression through at least two distinct pathways: one through binding as a homodimer to specific DNA sequences, termed glucocorticoid-responsive elements (GREs) (4–6), and the other through a GRE-independent pathway, in which GR interacts as a monomer with other transcription factors bound to DNA (7–11). The fact that GR-deficient mutant mice died shortly after birth, but GR dimerization-deficient mice survive to adulthood, indicates that the GRE-independent pathway may be critically important in survival (12, 13). The domain structure of GR has been characterized. GR is divided into several functional domains and subdomains. Located N-terminally, amino acid residues 77–262 of GR comprise the ligand-independent activation function domain, AF-1, or {tau}1 (5, 14). Amino acid residues 418–488 comprise the DNA-binding domain (DBD) containing two zinc fingers (5). Residues 490–515 are a hinge region that contains a nuclear localization signal. C-terminal residues 526–777 make up the ligand-binding domain (LBD) of GR, which also contains two subdomains, {tau}2 (residues 526–556) (15) and AF-2 domain (residues 727–763, also called the {tau}c domain), which are ligand-dependent activation domains (16). Like the hinge region, the LBD of GR also contains a nuclear localization signal. The GR LBD interacts with various co-activator proteins including p300/CREB-binding protein, co-integrator-associated protein, steroid receptor co-activator (17, 18), and transcription factor intermediary factor-2 or its mouse homolog glucocorticoid receptor interacting protein (16, 19–21).

TGF-{beta} regulates cell growth, cell differentiation, immune and inflammatory responses, extracellular matrix production, and apoptosis (22, 23). TGF-{beta} signaling is mediated by transmembrane serine/threonine kinase receptors that phosphorylate the receptor-regulated Smads, Smad2 and Smad3. These phosphorylated Smads form heteromeric complexes with the common Smad, Smad4, and translocate into the nucleus to regulate gene expression (24, 25). TGF-{beta} actions can be antagonized by a TGF-{beta}-inducible inhibitory Smad, Smad7 (26). An 8-base pair palindromic DNA sequence, GTCTAGAC, was identified as an optimal Smad-binding element (27). Our laboratory, and others, have demonstrated sequence-specific Smad binding by Smad3 and Smad4 to the human type 1 plasminogen activator inhibitor (PAI-1) promoter (28, 29). Mutation of these Smad-binding sequences resulted in the loss of both specific Smad binding and transactivation by TGF-{beta} (28–31). Smads can also interact with co-activators and co-repressors (32–34).

TGF-{beta} and glucocorticoids interact both positively and negatively in regulating the expression of various genes (35, 36). Glucocorticoids inhibit the TGF-{beta}-induced expression of extracellular matrix proteins including fibronectin, collagen, tissue inhibitors of metalloproteinases, and human PAI-1 (37–40). Hence, glucocorticoids and TGF-{beta} may be important opposing physiological regulators of wound healing and fibrosis. Our laboratory has been investigating the molecular mechanism(s) by which liganded GR inhibits TGF-{beta} transactivation of the PAI-1 gene in human Hep3B cells. We have previously reported that liganded GR repressed TGF-{beta} transcriptional activation of both the PAI-1 promoter and a multimerized TGF-{beta}-responsive sequence (TRS) of the PAI-1 promoter containing Smad3/4 binding sites. We also showed that liganded GR repressed transactivation by the Smad3 and Smad4 C-terminal activation domains, and that GR physically interacted with Smad3 both in vitro and in vivo (37). In this report, we characterize the GR domain(s) responsible for the repression of TGF-{beta} transactivation of the human PAI-1 gene and further investigate the GR domain(s) involved in the physical interaction between GR and Smad3 and the correlation between physical interaction and transrepression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Hep3B is a human hepatoma cell line that has intact TGF-{beta} signaling and expresses endogenous PAI-1. However, Hep3B cells have little or no functional endogenous GR activity and thus allow us to study the function of various GR mutants transiently transfected into these cells. Hep3B cells are cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.5 µg/ml Fungizone in 95% air and 5% carbon dioxide at 37 °C.

Plasmids—The TRS reporter plasmid, pTRS6E1B-luc, 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 (28, 37). The human GR expression plasmid (pRShGR{alpha}) and GR deletion mutants were kindly provided by Dr. R. Evans (Salk Institute) (41). GR mutants GR1–488, GR1–515, GR1–550, GR418–777, and GR-Gal4-GR were kindly provided by Dr. F. Lemaigre (Louvain University Medical School, Brussels, Belgium). Mouse GR {tau}2 mutant S561A (42) was kindly provided by Dr. M. R. Stallcup (University of Southern California, Los Angeles, CA). GR DBD mutants GR D4X and N454D/A458T (43) were kindly provided by Dr. A. C. B. Cato (Institute of Genetics, Karlsruhe, Germany). The human AR expression plasmid was kindly provided by Dr. E. Keller (University of Michigan). Rat MR and GR-MR chimera expression plasmids (44) were kindly provided by Dr. K. Yamamoto (University of California, San Francisco, CA). The {tau}c deletion mutant GR1–726 was constructed by digesting pZP-hGR with NsiI, followed by self-ligation of the 6342-bp fragment. The GRE expressing reporter plasmid pGRE4E1b-luc was constructed by inserting four copies of a 15-bp GRE from the rat tyrosine aminotransferase gene upstream of the E1bTATA box of pE1b-luc (37).

For GST pull-down assays, wild type and mutant GR cDNA sequences were subcloned into the expression plasmid pcDNA3.1/Zeo(+) (Invitrogen). pRSV-hGR{alpha} (5) was digested with DraI and KpnI. The 2.5-kb hGR cDNA was inserted into KpnI and EcoRV of pcDNA3.1/Zeo(+). Likewise, GR418–777, GR{Delta}488–532, GR{Delta}490–515, GR{Delta}532–697, GR{Delta}550–600, GR{Delta}589–697 (41), GRD4X, and GRN454D/A458T (43) were subcloned into pcDNA3.1/Zeo(+) in a similar manner. To make the deletion mutant pZPGR{Delta}420–499, the 295-bp EcoRI/PstI from pZPhGR fragment was ligated with a 7007-bp XcmI/PstI fragment, blunting the XcmI and PstI sites. pZpGR1–420 was constructed by digesting pZPhGR with BxtXI/T4 blunting, XcmI, EcoRI, or ClaI/NotI blunting, followed by self-ligation of the larger fragment. p6RMR and p6RMR604.GR (44) were digested with KpnI and XbaI, whereas p6RGR524.MR and p6RGR438.MR, were digested with XbaI; the resulting fragments containing the nuclear receptor or chimeras were subcloned into the corresponding sites in pcDNA3.1/Zeo(+). Ultraspiracle expressed with pcDNA3.1/Zeo(+) is as described before (45).

Antibodies—Rabbit anti-human GR antibodies (E-20 against N-terminal and P-20 against C-terminal), goat and rabbit anti-Smad2 and anti-Smad3 antibodies, and rabbit and mouse anti-human AR were purchased from Santa Cruz Biotechnology. Mouse anti-MR antibodies were purchased from Zymed Laboratories Inc.and Affinity Bioreagents, Inc. The horseradish peroxidase-conjugated secondary anti-mouse and anti-rabbit antibodies were purchased from Amersham Biosciences.

Transient Transfection—Hep3B cells cultured in 12-well (22-mm) plates at 60–80% confluency were transiently transfected in triplicate with 0.5 µg/well pTRS6E1B-luc and 0.2 µg/well GR expression plasmid (pRShGR{alpha}), mutant GR expression plasmids, or AR, MR, or GR-MR chimera expression plasmids, using FuGENE 6 (Roche Applied Science). Sixteen hours after transfection, the cells were cultured in the presence or absence of 50 pM TGF-{beta}, and/or 100 nM dexamethasone, or 10–100 nM dihydrotestosterone, or 100–500 nM aldosterone for 24 h. For immunoblot analysis and co-immunoprecipitation assays, Hep3B cells cultured in 100-mm dishes were transiently transfected with 8 µg/dish pTRS6E1B-luc and 4 µg/dish wild type or mutant steroid receptor expressing plasmids.

Luciferase Assays—The cells were washed twice with phosphate-buffered saline and then lysed in a luciferase cell lysis buffer (Promega). Luciferase assays were done according to the Promega standard manual using a Microlumat LB96P luminometer (Wallac) kindly provided by Dr. J. Baker, Jr. (University of Michigan). Repression was calculated as follows. Luciferase activity in control (untreated) cells was first subtracted from both the TGF-{beta}-treated and TGF-{beta} plus dexamethasone-treated values; then TGF-{beta} plus Dex luciferase activity was subtracted from the TGF-{beta}-treated value and divided by the TGF-{beta}-treated value.

Immunoblot Analysis—Cell monolayers in 100-mm dishes were lysed in 1.5 ml of cell lysis buffer (2% SDS, 125 mM Tris-HCl, pH 6.8, and 20% glycerol). The cell lysates were boiled for 5 min and then centrifuged at 1000 x g for 10 min to remove debris. The protein concentrations were measured using BCA protein assay reagent (Pierce). Equal amounts of protein samples with freshly added {beta}-mercaptoethanol (5%) and bromphenol blue (0.05%) were boiled for 5 min and subjected to reducing SDS-PAGE. After electrophoresis, the proteins were transferred to a nitrocellulose membrane. Following blocking the membrane with 5% nonfat milk for 1 h at room temperature, the membrane was incubated with anti-GR, anti-MR, anti-AR, or anti-Smad3 antibodies for 1 h at 37 °C. After three washes with Tween-phosphate-buffered saline, the membrane was incubated with appropriate horseradish peroxidase-conjugated secondary antibodies. The proteins were detected using the ECL detection reagents (Pierce) and x-ray films (Kodak) according to the manufacturer's instructions.

Co-immunoprecipitation Assays—Hep3B cells cultured in 100-mm dishes were transiently transfected with 8 µg/dish pTRS6E1B-luc and 4 µg/dish wild type or mutant GR expression plasmids. Sixteen hours after transfection, the cells were cultured in the presence or absence of TGF-{beta} and/or steroids for 24 h. The cells were lysed in ice-cold RIPA buffer (phosphate-buffered saline, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS) containing freshly added protease inhibitors (Roche Applied Science). The lysates were centrifuged for 15 min at 12,000 x g to remove debris, and Smad3 was immunoprecipitated by incubating with a goat anti-Smad3 antibody (Santa Cruz) and protein G-agarose beads (Invitrogen) at 4 °C overnight. Immunoprecipitates were washed four times with ice-cold RIPA buffer and resolved by reducing SDS-PAGE. Wild type and mutant GRs were detected using a mixture of rabbit anti-GR antibodies that react with both N- and C-terminal amino acid residues. MR, GR-MR chimeras, and AR were detected using respective anti-MR, anti-GR, and anti-AR antibodies or antibody mixtures.

In Vitro Protein Synthesis and GST Pull-down Assay—[35S]Methionine-labeled protein was produced with a rabbit reticulocyte lysate coupled transcription/translation system (TNT; Promega), utilizing the T7 promoter preceding cDNA sequences in the vector pcDNA3.1/Zeo(+) as described above. The protein was first analyzed by SDS-PAGE and quantified with a PhosphorImager. For each pull-down assay, ~1.5 fmol of labeled protein was used. GST-Smad3C fusion protein preparation and pull-down assays were conducted as described previously (37).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Liganded GR Represses TGF-{beta} Transactivation of Human PAI-1 in Hep3B Cells—To examine the effect of the liganded GR on TGF-{beta} transactivation of PAI-1 gene expression, Hep3B cells, which express no functionally active endogenous GR but have intact endogenous TGF-{beta} signaling, were transiently transfected with 0.2 µg/well GR expressing plasmid pRShGR{alpha} and 0.5 µg/well reporter plasmid pTRS6E1B-luc. Subsequent treatment of the cells with 50 pM TGF-{beta} resulted in ~30-fold increase in luciferase activity. Dex treatment (100 nM), which by itself had little effect on luciferase activity in transfected Hep3B cells, resulted in more than 80% inhibition of TGF-{beta} induction of PAI-1 TRS (Fig. 1). The Dex inhibition of TGF-{beta} transactivation was GR-dependent, because Dex showed no inhibitory effect on transactivation by TGF-{beta} in Hep3B cells in the absence of co-transfected GR or in the presence of the empty control plasmid for GR (data not shown).



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  		FIG. 1.
Liganded GR represses TGF-{beta} transactivation of human PAI-1 TRS in Hep3B cells. Hep3B cells cultured in 12-well plates were transfected with 0.5 µg/well reporter plasmid pTRS6E1b-luc and 0.2 µg/well GR expression plasmid pRShGR{alpha}. After 16 h, the cells were cultured in the presence or absence (Control) of 50 pM TGF-{beta} and/or 100 nM dexamethasone for 24 h. Luciferase activity was assayed as described as under "Materials and Methods." The data are shown as the means ± S.D. relative light units of triplicate wells. The experiment was repeated at least three times.

 

The Ligand-binding Domain of GR and Its Subdomains, {tau}2 and {tau}c, Are Necessary for GR Repression of TGF-{beta} Transactivation of PAI-1 TRS—Our previous studies suggested that the C-terminal half of GR (GR amino acids 418–777) was responsible for repression of TGF-{beta} transactivation of PAI-1 TRS (37). To define the exact domain(s) of GR that is/are required for repression of TGF-{beta} transactivation, we have examined the ability of various GR truncation, deletion, and substitution mutants to repress TGF-{beta} transactivation. As shown in Fig. 2A, deletion of the N-terminal half of GR (GR418–777), which abrogates transactivation, did not affect GR repression of TGF-{beta} transactivation. The repression by this mutant was almost as strong as that of wild type GR. However, a longer N-terminal deletion in which the DBD, the hinge region, and the {tau}2 subdomain of the LBD were deleted (GR550–777) abolished GR transrepression. Truncation deletions of the C-terminal half of GR including GR LBD and hinge region (GR1–488, GR 1–515 and GR1–550), which confer constitutive transactivation activity, completely nullify their ability to repress (Fig. 2A).



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  		FIG. 2.
Mapping repression domains of GR. A–C, Hep3B cells were transfected with pTRS6E1B-luc and plasmids containing wild type GR or various GR mutants. The cell treatment with TGF-{beta} and dexamethasone and luciferase activity assay was done as described under "Materials and Methods" and in the legend to Fig. 1. The percentage of repression is shown as the mean ± S.E. N indicates the number of experiments. D, the expression levels of wild type and mutant GRs were evaluated by immunoprecipitation and immunoblot analysis. Lysates of Hep3B cells transfected with wild type and mutant GR were immunoprecipitated with goat anti-GR antibodies. Immunoblotting was carried out using rabbit anti-GR antibodies. Control, cells not transfected with GR.

 

To further define the domains and/or subdomains in the C-terminal half of GR that is/are responsible for transrepression function, we tested the ability of a series of LBD and hinge region interstitial deletion mutants. As shown in Fig. 2B, deletions of the hinge region and the N-terminal portion of the {tau}2 subdomain in GR LBD (GR{Delta}488–532) almost completely eliminated its repression of TGF-{beta} transactivation, whereas deletion of the hinge region alone (GR{Delta}490–515) had little effect on GR transrepression, implying that the hinge region may not be required. Interstitial deletion of the central part of the GR LBD (GR{Delta}550–600 and GR{Delta}589–697) also abolished GR transrepression, as did deletion of both the GR {tau}2 region and the N-terminal half of the GR LBD (GR{Delta}532–697) or the {tau}c subdomain (GR1–726) (Fig. 2B). All of these mutations, except GR{Delta}490–515, have previously been reported to abolish ligand binding (41). Thus ligand binding appears to be essential for transrepression. However, a substitution mutation of a conserved serine in the mouse GR {tau}2 subdomain (S561A), corresponding to S556A in the human GR, significantly reduced GR transrepression, although ligand binding ability is not affected (42). This suggests an important role of the {tau}2 domain in GR transrepression independent of or in addition to its role in hormone binding (Fig. 2C).

Substitution mutations in the dimerization domain of the GR DBD (N454D/A458T and D4X) had little or no effect on GR repression of TGF-{beta} transactivation. In fact, replacement of the entire GR DNA-binding domain (418–488) with the DBD (amino acids 1–147) of Gal4 (GR-Gal4-GR) did not affect GR transrepression either (Fig. 2C), suggesting that the DBD of GR may not be required in the transrepression.

To exclude the possibility that the failure of the LBD deletion mutants to transrepress might be explained by a failure to be expressed in transfected cells, the expression levels of wild type and mutant GRs in the transfected Hep3B cells were evaluated by immunoprecipitation and immunoblot analysis. Lysates of Hep3B cells transfected with wild type and mutant GRs were immunoprecipitated with goat anti-GR antibodies and immunoblotted with rabbit anti-GR antibodies. As shown in Fig. 2D, mutant GRs were all expressed at approximately the same levels as wild type GR.

Taken together, these findings suggest that the GR LBD and its subdomains {tau}2 and {tau}c are critically important for GR repression of TGF-{beta} transactivation of the TRS from human PAI-1 promoter, whereas the N-terminal half of GR including the {tau} 1 domain, the DBD, and the hinge region are not required.

Liganded AR Represses TGF-{beta} Transactivation of Human PAI-1, whereas Liganded MR Does Not—To examine whether other closely related steroid nuclear receptors such as AR and MR also repress TGF-{beta} transactivation of PAI-1 TRS, Hep3B cells were transfected with TRS expression plasmid (pTRS6E1B-luc) and GR expression plasmid (pRShGR{alpha}), a human AR expression plasmid (pSG5AR), or a rat MR expression plasmid (p6RMR). Sixteen hours after transfection, the cells were treated with either 50 pM TGF-{beta} alone, with 100 nM steroid alone, or with both for 24 h. The results showed that liganded AR repressed as strongly as wild type GR (Fig. 3A), whereas liganded MR did not repress (Fig. 3B).



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  		FIG. 3.
A, liganded AR represses TGF-{beta} transactivation of human PAI-1 TRS as well as liganded GR. Hep3B cells were transfected with TRS expression plasmid pTRS6E1B-luc and GR expression plasmid pRShGR{alpha} or a human AR expression plasmid pSG5AR. After 16 h, the transfected cells were treated with either TGF-{beta} alone or TGF-{beta} plus 100 nM dexamethasone or 100 nM dihydrotestosterone. The control was without TGF-{beta} or steroid treatment. The data are shown as the means ± S.D. relative light units of triplicate wells. The experiment was repeated at least three times. B, liganded MR does not repress TGF-{beta} transactivation of human PAI-1 TRS. Hep3B cells were transfected with TRS expression plasmid pTRS6E1B-luc and GR expression plasmid pRShGR{alpha} or a rat MR expression plasmid p6RMR. The transfected cells were treated with either TGF-{beta} alone or TGF-{beta} plus 100 nM dexamethasone or 200 nM aldosterone. The control was without TGF-{beta} or steroid treatment. The data are shown as the means ± S.D. relative light units of triplicate wells. The experiment was repeated three times. C, expression of GR, AR, and MR in transfected Hep3B Cells. The cell lysates of Hep3B cells transfected with GR, AR, and MR expressing plasmids were immunoprecipitated with goat anti-GR, rabbit anti-AR, and mouse anti-MR antibodies. GR, AR, and MR expression levels were detected using rabbit anti-GR, mouse anti-AR, and anti-MR antibodies. IP, immunoprecipitation; IB, immunoblot.

 

The expression levels of GR, AR, and MR were evaluated by immunoprecipitation and immunoblot analysis. The lysates of Hep3B cells transfected with GR, AR, and MR were immunoprecipitated with anti-GR, -AR, and -MR antibodies. The immune complexes were assayed using immunoblot analysis. As shown in Fig. 3C, GR, AR, and MR were expressed at approximately the same levels. The results indicate that different steroid nuclear receptors behave differently in repression of TGF-{beta} transactivation of PAI-1 TRS in Hep3B cells.

Repression by GR-MR Chimeras Confirms That the GR LBD Confers Repression of TGF-{beta} Transactivation of PAI-1 Gene Expression—We have taken advantage of the difference between GR and MR to further define the role of LBD by examining the ability of several rat GR-MR chimeras, GR438.MR, GR524.MR, and MR604.GR shown in Fig. 4, to repress TGF-{beta} transactivation. All of these constructs are in the same parent plasmid and are expressed at similar levels (data not shown). As shown in Fig. 4, MR604.GR (including the LBD and the {tau}2 and {tau}c subdomains of GR) repressed almost as strongly as wild type GR. In contrast, GR438.MR and GR524.MR with MR LBD did not repress as strongly, confirming that it is the GR LBD that confers strong repression of TGF-{beta} transactivation of PAI-1.



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  		FIG. 4.
Repression of TGF-{beta} transactivation of PAI-1 TRS by GR-MR chimeras. GR-MR chimeras are named according to the amino acid residues of the first receptor at which the upstream portion ends followed by the second receptor. For example, GR438.MR has GR amino acids 1–438 fused at the homologous site to MR. The latter half contains MR amino acid residues. Hep3B cells were transfected with pTRS6E1B-luc and pRShGR{alpha} or GR-MR chimera expression plasmids. The transfected cells were treated with either TGF-{beta} alone or TGF-{beta} plus steroids. The control was without either TGF-{beta} or steroid treatment. The data are shown as the means ± S.E. relative light units of three independent experiments.

 

RU486 Is a Partial Antagonist and Partial Agonist of GR Repression of TGF-{beta} Transactivation of Human PAI-1—Liganded GR is known to induce transcriptional activation of GRE. RU486 is a strong glucocorticoid antagonist and weak agonist. To examine the effect of RU486 on GR repression of TGF-{beta} transactivation, Hep3B cells transfected with pTRS6E1b-luc and pRShGR{alpha} were cultured in the presence or absence of 50 pM TGF-{beta}, and/or 100 nM Dex, and/or various concentrations of RU486 for 24 h. As shown in Fig. 5A, RU486 partially reversed GR repression of TGF-{beta} transactivation of PAI-1 gene expression. In addition, RU486 alone also partially repressed TGF-{beta} transactivation of PAI-1, although less strongly than Dex, indicating that RU486 is a partial GR agonist and partial antagonist with respect to repression of TGF-{beta} transactivation.



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  		FIG. 5.
A, the effect of RU486 on GR repression of TGF-{beta} transactivation of human PAI-1 TRS. Hep3B cells cultured in 12-well plates were transfected with pTRS6E1b-luc and pRShGR{alpha}. After 16 h, the cells were cultured in the presence or absence of TGF-{beta}, dexamethasone, and/or various concentrations of RU486 for 24 h. The data are shown as the means ± S.D. relative light units of triplicate wells. The experiment was repeated three times. B, the effect of RU486 on GR induction of GREs. Hep3B cells cultured in 12-well plates were transfected with 0.5 µg/well GRE expression plasmid pGRE4-luc and 0.2 µg/well pRShGR{alpha}. After 16 h, the cells were cultured in the presence or absence of 100 nM dexamethasone and/or RU486 for 24 h. The data are shown as the means ± S.D. relative light units of triplicate wells. The experiment was repeated three times.

 

The glucocorticoid antagonist nature of RU486 was demonstrated in Fig. 5B. Liganded GR induced activation from GRE more than 1000-fold in Hep3B cells. With both Dex and RU486 treatment, RU486 strongly repressed GR induction of GRE in a dose-dependent manner. RU486 alone also weakly induced GR activation of GRE, indicating its weak agonistic nature. The activation effect of Dex and RU486 was GR-dependent, because neither Dex nor RU486 was able to induce activation from GRE in Hep3B cells without GR transfection (data not shown). These results suggest that RU486 is a partial antagonist and partial agonist of GR repression of TGF-{beta} transactivation of the PAI-1 gene. In contrast, RU486 is a strong antagonist and weak agonist of GR induction of activation from GRE.

Physical Interaction of Liganded Wild type GR and Mutant GRs with Smad3 in Vivo in Hep3B Cells—Previous studies in our laboratory using the mammalian one-hybrid approach established a functional interaction between GR and Smad3/4. Using GST pull-downs and co-immunoprecipitation assays in COS cells that have no endogenous GR or Smad3 expression, we demonstrated that wild type GR physically interacted both in vitro and in vivo with Smad3 (37). To study the relationship between the GR transrepression and its interaction with Smad3, we conducted co-immunoprecipitation assays to examine endogenous Smad3 interaction with both wild type GR and various GR mutants in vivo, using Hep3B cells that expressed no functional endogenous GR but preserved intact endogenous TGF-{beta} signaling, including endogenous Smad2 and Smad3 expression. We expected that the presence or absence of physical interaction between wild type or mutant GRs and Smad3 would correlate with repression. Hep3B cells cultured in 100-mm dishes were transiently transfected with 8 µg of pTRS6E1b-luc and 4 µg of wild type GR or mutant GR expression plasmids. Co-immunoprecipitation assay revealed that Smad3 interacted with liganded wild type GR and all mutant GRs that repressed TGF-{beta} transactivation of PAI-1 gene. However, Smad3 also interacted with mutant GRs, including GR{Delta}488–532, GR{Delta}550–600, GR{Delta}589–697, GR1–488, GR1–515, and GR1–550 that did not repress TGF-{beta} transactivation (Fig. 6). These results suggest that physical interaction between GR and Smad3, although it may be necessary for GR repression of TGF-{beta} transactivation of human PAI-1 TRS, is not sufficient to bring about repression.



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  		FIG. 6.
In vivo interactions between endogenous Smad3 and transfected wild type GR or GR mutants using co-immunoprecipitation analysis. Hep3B cells cultured in 100-mm dishes were transfected with 8 µg/dish pTRS6E1b-luc and 4 µg/dish pRShGR{alpha} or 4 µg/dish mutant GR plasmids. After 16 h, the cells were cultured in the presence of 50 pM TGF-{beta} and 100 nM dexamethasone for 24 h. The cells were lysed in ice-cold RIPA buffer. The lysates were immunoprecipitated with an anti-Smad3 antibody together with 30 µl of protein G-agarose beads. The immunoprecipitation complexes were subjected to SDS-PAGE under reducing condition. After blotting to a nitrocellulose membrane, rabbit anti-GR antibodies were used to probe GR. GR Control, no transfection with GR. IP, immunoprecipitation; IB, immunoblot.

 

The Interaction of GR with Smad3 Requires TGF-{beta} but Not Dex—Co-immunoprecipitation assays demonstrated that Smad3 interacted not only with wild type GR and mutant GRs that could repress but also with mutant GRs lacking intact LBDs that were necessary for ligand binding and transrepression. These findings prompted us to further investigate the conditions in which wild type GR and Smad3 would interact. Hep3B cells were transfected with pTRS6E1b-luc and wild type pRShGR{alpha}. The cells were then treated with either TGF-{beta} alone, Dex alone, or TGF-{beta} plus Dex. As shown in Fig. 7, cells treated with TGF-{beta} alone or TGF-{beta} plus Dex showed interaction between GR and Smad3. In contrast, cells treated with Dex alone did not show GR-Smad3 interaction. Thus physical interaction requires TGF-{beta} activation of Smad3 but not Dex binding to GR.



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  		FIG. 7.
TGF-{beta}, but not dexamethasone, is required for in vivo physical interaction between endogenous Smad3 and transfected GR. Hep3B cells cultured in 100-mm dishes were transfected with pTRS6E1b-luc and pRShGR{alpha}. After 16 h, the cells were cultured in the presence or absence of TGF-{beta} and/or dexamethasone for 24 h. The cells were lysed in ice-cold RIPA buffer. The lysates were immunoprecipitated with a goat anti-Smad3 antibody. The immunoprecipitation complexes were subjected to SDS-PAGE under reducing conditions. After blotting to a nitrocellulose membrane, rabbit anti-GR antibodies were used to probe GR.

 

Because Smad3 and Smad2 are specific for TGF-{beta} signaling and can be activated by TGF-{beta}, we also examined whether Smad3 and Smad2 expression was up-regulated by TGF-{beta} by performing immunoprecipitation assays. TGF-{beta} treatment markedly increased Smad3 expression, but Smad2 expression remained unchanged. We have previously reported that Smad2 does not bind to the TRS (28). Dex had no effects on Smad3 or Smad2 expression (Fig. 8).



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  		FIG. 8.
TGF-{beta} induces Smad3 expression, but not Smad2 expression. Hep3B cells cultured in 100-mm dishes were transfected with pTRS6E1b-luc and pRShGR{alpha}. After 16 h, the cells were cultured in the presence or absence of TGF-{beta} and/or dexamethasone for 24 h. The cells were lysed in ice-cold RIPA buffer. The lysates were immunoprecipitated with a goat anti-Smad3 antibody. The immunoprecipitation complexes were subjected to SDS-PAGE under reducing conditions. After blotting to a nitrocellulose membrane, mouse anti-Smad3 and Smad2 antibodies were used to detect Smad3 and Smad2. IP, immunoprecipitation; IB, immunoblot.

 

Glucocorticoid Receptor Ligand-binding Domain Is Required for in Vitro Interaction with the Smad3 C-terminal Activation Domain—We used GST pull-down assays to analyze the physical interaction of the GR and Smad3 in vitro. In these experiments, wild type or mutant GR proteins were expressed using the TNT system and labeled with [35S]methionine, whereas the GST or GST-Smad3C proteins were expressed in bacteria. As reported previously (37), wild type GR binds Smad3C efficiently, whereas GST proteins alone did not exhibit any detectable level of binding to GR (Fig. 9A, lanes 1–3). As a negative control, we tested binding of Smad3C to the mosquito Ultraspiracle protein, the insect homolog of retinoid X receptor (45), which displayed virtually no binding to either GST-mad3C or GST (Fig. 9A, lanes 4–6).



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  		FIG. 9.
Glucocorticoid receptor domains involved in in vitro interaction with Smad3 C-terminal activation domain. Bacterially expressed GST protein or GST-Smad3C was incubated with 35S-labeled TNT-expressed wild type GR, Ultraspiracle, or GR N-terminal and C-terminal domain mutants (A), GR DBD mutants (B), GR LBD mutants (C), Gal4-GR LBD fusion proteins (D), or GR/MR chimeras (E). The GST (lanes 2, 5, 8, 11, and 14) or GST-Smad3C complexes (lanes 3, 6, 9, 12, and 15) were purified with glutathione beads and subjected to SDS-PAGE followed by autoradiography. Input (lanes 1, 4, 7, 10, and 13) contained 5% 35S-labeled proteins. The molecular weight markers are indicated on the left.

 

We then investigated whether the N-terminal domain of GR was involved with direct binding to Smad3C. Deletion of the entire N-terminal domain (GR418–777) did not affect GR-Smad interaction (Fig. 9A, lanes 10–12). In confirmation, we found no detectable binding when the N-terminal domain was expressed independently (GR1–420) and incubated with Smad3, indicating that the N-terminal domain did not directly bind Smad3 at all (Fig. 9A, lanes 7–9).

Next, we tested the potential interaction of GR DBD with Smad3C. The GR DBD D-loop mutants GRD4X, showed robust binding to Smad3C (Fig. 9B, lanes 1–3). Deletion of the entire DBD (GR{Delta}420–499) did not significantly alter the GR binding to Smad3C (Fig. 9B, lanes 4–6) either, suggesting that the LBD in GR was mainly responsible for physical interaction with Smad3. To further define the regions in LBD in GR that were involved with binding to Smad3C, we tested a variety of LBD mutants. Deletion of part of N-terminal region of the GR LBD (GR{Delta}490–515 and GR{Delta}488–532) did not show any reduced binding activity to Smad3C (Fig. 9C, lanes 1–6), and neither did deleting of the middle of the LBD (GR{Delta}532–697, GR{Delta}550–600, and GR{Delta}589–697; Fig. 9C, lanes 7–15). We then tested the direct binding of LBD alone with Smad3C. Surprisingly, neither the entire LBD (GR486–777) nor the C-terminal of LBD exhibits any strong binding activity to Smad3C (data not shown). In fact, we observed the increased intensity of a lower band, which was most likely due to the instability of LBD when expressed alone (46). To increase the stability of the LBD, we tested GR LBDs fused with GAL4. These fusion proteins Gal4-GR550–777 and GAL4-GR532–777, bound strongly with Smad3C (Fig. 9D, lanes 1–6). These results indicated that multiple regions in the LBD participated in GR physical interaction with Samd3C.

Finally, we tested physical interaction of GR-MR fusion proteins with Smad3C. The rat GR-MR fusion proteins MR604GR, which harbor the GR LBD, displayed strong binding to Smad3C (Fig. 9E, lanes 10–12). However, MR, GR438MR, and GR524MR (with MR LBD) also showed strong binding to Smad3C (Fig. 9E, lanes 1–9), although these constructs did not display any significant functional repression (Fig. 4).

Taken together, our GST pull-down assay results confirm that the LBD was mainly responsible for physical interaction with Smad3C and that multiple regions in the LBD bound Smad3C. Furthermore, the physical interaction is apparently not sufficient to mediate functional repression.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have previously reported that liganded GR represses TGF-{beta} transactivation of both the PAI-1 promoter and a multimerized TGF-{beta}-responsive sequence from this promoter. Using GST pull-down and co-immunoprecipitation assays, we demonstrated that GR and Smad3 physically interacted in vitro and in vivo (37). In the present study, we have identified the GR domains that are responsible for this transrepression.

We have utilized Hep3B cells, a line of human hepatoma cells that have an intact TGF-{beta} signaling pathway and express PAI-1. However, Hep3B cells lack functional endogenous GR, enabling us to test the effects of transfected wild type and mutant GRs on repression. As a reporter, we have utilized a multimerized TGF-{beta}-responsive sequence (TRS6) containing six copies of a 12-bp TGF-responsive sequence that binds Smad3 and Smad4 (28). This reporter has a very low background activity and is highly responsive to TGF-{beta}, and we have shown that GR dramatically represses TGF-{beta} gene activation, reflecting its actions on the full-length PAI-1 promoter. We report here that the GR LBD, which includes subdomains {tau}2 and {tau}c, is essential for transrepression, whereas the {tau}1 domain, the DBD, and the hinge region appear to be dispensable. Binding of ligand to GR, although necessary for transrepression, is not sufficient, as demonstrated by failure of the GR mutant S561A to repress. Repression is not absolutely dependent on agonist binding, as is the transactivation of gene expression by GR, because the steroid analog, RU486, acts as a partial agonist as well as partial antagonist of GR transrepression. We have extended our studies on the physical interaction of GR with endogenous Smad3 in vivo in Hep3B cells using co-immunoprecipitation assays and have identified the domains of GR necessary for this interaction by GST pull-down experiments. These latter studies indicated that under the conditions of the GST pull-down assays, the GR LBD was required for interaction. Although physical interaction between GR and Smad3 may be necessary for GR repression of TGF-{beta} transactivation, our results indicate that interaction per se was not sufficient for transrepression to occur. Interestingly, the physical interaction between GR and Smad3 requires TGF-{beta} activation of Smad3 but not dexamethasone binding to GR.

The GR transrepression of TGF-{beta} action has similarities with, but important differences from, the well studied interactions between GR and two families of transcriptional factors, AP-1 and NF-{kappa}B (47–49). Like the latter, transrepression is mediated by protein-protein interactions rather than DNA binding and gene transactivation. As is true for transrepression of AP-1 and NF-{kappa}B function (43, 50), mutations in the D-loop of the GR DBD, which prevent dimerization and thus transactivation from a GRE, did not interfere with GR transrepression of TGF-{beta} action. Thus the repressive molecule does not bind directly to DNA but is tethered to the repressed transcriptional activator. Consistent with this view is our earlier observation that liganded GR can directly repress transactivation by the C-terminal transactivation domain of Smad3 fused to the GAL4 DNA-binding domain (37). Second, the ability of RU486 to act as a partial agonist of transrepression has also been described for AP-1 and NF-{kappa}B (43, 50). Finally, the ligand-binding domain of GR was shown to be important in transrepression of both AP-1 and NF-{kappa}B (7, 8).

In contrast to repression of AP-1 and NF-{kappa}B, however, the transrepression by GR of TGF-{beta} transactivation is different in two important respects. First, the transrepression is not mutual; TGF-{beta} has no effect on transcriptional activation of a GRE by liganded GR (37). Second, as shown in this study, the DNA-binding domain, required for the effects of GR on both AP-1 and NF-{kappa}B (7, 8, 11, 50), appears to be dispensable for the repression of TGF-{beta} transactivation. Mutations that block the ability of GR to dimerize have no effect on repression, and replacement of the entire GR DBD with an unrelated DBD, that of GAL4, does not impair transrepression at all. In contrast, replacement of the GR DBD with the DBD of TR{beta} results in the loss of its repressive function for NF-{kappa}B (50). GR DBD was reported to interact directly with the bZip domain of AP-1 (7) and with the REL A subunit of NF-{kappa}B (51). In contrast, as shown in our GST pull-down experiments, the GR DBD is not required for interaction of GR with Smad3.

Smad proteins have been reported to interact with other nuclear receptors. Smad3 acts as a co-activator of vitamin D receptor gene activation; the MH1 domain of Smad3 and the ligand-binding domain of vitamin D receptor appear to be necessary for an interaction that also involves the steroid receptor co-activator 1 protein (52, 53). Smads have also been reported to synergistically activate transcription with orphan nuclear receptor hepatocyte nuclear factor 4 (54). In this case, the N-terminal activation function of hepatocyte nuclear factor 4 and the MH1 domain of Smad3 appear to be required for this interaction. The ligand-binding domain of the androgen receptor AR has been reported to inhibit the binding of Smad3 to Smad-binding elements (55), and TGF-{beta} and Smad3 have been reported to inhibit transcriptional activation by AR on two androgen-responsive promoters. The N-terminal activation domain of AR and the MH2 domain of Smad3 were found to be involved in physical interactions in this case (56). Finally, Smad3 and ER were reported to transrepress each other's transcriptional activation functions; this interaction required the MH2 domain of Smad3 (57).

It is noteworthy that AR is fully capable of repressing TGF-{beta} activation from the TRS6 promoter; the domains of the AR that are active on TGF-{beta} transactivation in this system have not been defined. It is clear, however, that it is the LBD of GR that interacts with the MH2 C-terminal transcriptional activation domain of Smad3 to repress its function. Whether there is any effect of GR on Smad3 binding to the promoter has not been evaluated, but there is no necessity to posit such an action given the effective transrepression of the Smad3 C-terminal transactivation domain by GR (37).

The consequences of cross-talk between transcriptional pathways can be complex. The cross-talk between GR and AP-1 on the composite response element of the proliferin gene promoter is such an example in which the outcome, transactivation or transrepression, depends on the composition of the AP-1 subunits (58). On a different promoter, the collagenase 3 response element, liganded GR repressed AP-1-mediated activation regardless of the composition of AP-1 (59).

A number of mechanisms have been proposed to explain the consequences of transcription factor cross-talk. These include 1) masking of transactivation domains, 2) interference with binding to target DNA, 3) competition for necessary co-activator molecules, 4) post-translational modification of transcription factors altering their activity, and 5) alteration of the conformation of a transcription factor such that interaction with co-repressors might be favored. As noted above, all of these mechanisms have been suggested, and some evidence has been found in support (49, 60–62). Although it has been suggested that GR might repress NF-{kappa}B-mediated transcription by up-regulation of a specific inhibitor, I{kappa}B (63), this explanation is inconsistent with the observation that dimerization-defective GR mutants are capable of repression (43, 62). Furthermore, the suggestion that GR might block AP-1 or NF-{kappa}B binding to their target genes has also been excluded (62, 64). Although there are reports that GR and AP-1 might compete for the common co-activator CREB-binding protein or p300 (65, 66), other studies have suggested that this is not the case (47, 51). Our own data also suggest that competition for CREB-binding protein/p300 does not account for the GR transrepression of TGF-{beta} activation.2 Several studies have suggested that GR can prevent post-translational modification of transcription factors, thus repressing their activity; GR has been found to prevent phosphorylation of c-Jun by blocking the JNK pathway (67, 68). It has also been reported that GR interferes with serine phosphorylation on RNA polymerase II induced by NF-{kappa}B (69).

We have previously reported, using mammalian one-hybrid analyses, that liganded GR can interact directly with the C-terminal MH2 domain of Smad3 and repress its transactivation activity. In the current studies, we provide evidence for the direct physical interaction of GR with endogenous Smad3 by co-immunoprecipitation experiments. GST pull-down experiments indicate that it is the GR LBD that is directly involved in this interaction and that the N-terminal activation domain and GR DBD are not required. As expected, wild type GR and mutant GRs capable of repressing TGF-{beta} activity interacted physically with Smad3. Rather unexpectedly, however, GR mutants that fail to repress also interacted with Smad3. These mutants generally were incapable of binding ligand, suggesting that the physical interaction did not require dexamethasone binding. The fact that these interactions could be detected in the nucleus3 prompted us to examine the conditions in which GR and Smad3 would interact. Co-immunoprecipitation assays demonstrated that TGF-{beta} was required for this physical interaction, whereas the presence or absence of Dex did not affect the interactions. How unliganded GR gets to the nucleus is not yet clear. In addition, we have observed that TGF-{beta} induces Smad3 expression in Hep3B cells, as indicated by both Western blot and Northern blot analysis.3 It is possible that the overexpressed Smad3 in some way carries unliganded GR into the nucleus.

Studies with the mouse GR mutant S561A suggest that although ligand binding is necessary for transrepression, it is not sufficient. This mutant GR is capable of binding Dex but is a weak activator of transcription (42); our studies indicate that it is similarly a weak transrepressor. It seems likely that the interaction of GR bound to an agonist ligand with the transactivation domain of Smad3 results in recruitment of other activator or repressor molecules that repress TGF-{beta} transactivation. Relevant to this possibility are the interesting observations by Rogatsky et al. (59, 70) demonstrating that transcription factor intermediary factor-2/glucocorticoid receptor interacting protein 1, a steroid receptor co-activator, can act as a co-repressor with respect to glucocorticoid transrepression of AP-1 activity on a collagenase 3 promoter. Studies are currently underway in our laboratory to pursue these possibilities.

Glucocorticoids continue to be widely used as anti-inflammatory and immunosuppressive therapeutic agents, although their long term usage frequently causes severe adverse effects. Their immunosuppressive and anti-inflammatory functions are generally thought to be the result of the ability of GR to repress AP-1, NF-{kappa}B, and other important transcription factors, whereas the adverse effects of glucocorticoids are believed to be due mainly to DNA binding-dependent GR induction of transcription from GREs (60, 71). Therefore, a better understanding of GR cross-talk could help in the design of dissociated steroids that could mediate strong GR-dependent repression yet trigger minimal GR transactivation (43, 72, 73).

In conclusion, we have demonstrated that the GR LBD, which includes subdomains {tau}2 and {tau}c, is responsible for GR transrepression of TGF-{beta} transactivation of the PAI-1 gene. Physical interaction between GR and Smad3 is necessary but not sufficient for this transrepression. Similarly, ligand binding is essential but not sufficient to explain this repression.


   FOOTNOTES
 
* This work was supported by an Arthritis Foundation Biomedical Science grant and, in part, by pilot/feasibility study grants from the University of Michigan Multipurpose Arthritis Center Grant P60 AR20557 and the Michigan Diabetes Research and Training Center Grant P60 DK20572 from the National Institutes of Health (to T. D. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Human Genetics, 4909 Buhl Bldg., Box 0618, 1241 East Catherine St., Ann Arbor, MI 48109-0618. Tel.: 734-764-5491; Fax: 734-763-5831; E-mail: tdgum{at}umich.edu.

1 The abbreviations used are: GR, glucocorticoid receptor; GRE, glucocorticoid-responsive element; DBD, DNA-binding domain; LBD, ligand-binding domain; CREB, cAMP-responsive element-binding protein; TGF-{beta}, transforming growth factor-{beta}; PAI-1, type 1 plasminogen activator inhibitor; TRS, TGF-{beta}-responsive sequence; AR, androgen receptor; MR, mineralocorticoid receptor; GST, glutathione S-transferase; Dex, dexamethasone. Back

2 C.-Z. Song and T. D. Gelehrter, unpublished data. Back

3 G. Li and T. D. Gelehrter, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Joanne Heaton for helpful discussions and Karen Grahl for expert help in preparation of the manuscript.



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