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Originally published In Press as doi:10.1074/jbc.M707203200 on February 7, 2008

J. Biol. Chem., Vol. 283, Issue 14, 8995-9001, April 4, 2008
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RAP250 Is a Coactivator in the Transforming Growth Factor β Signaling Pathway That Interacts with Smad2 and Smad3*Formula

Per Antonson1, Tomas Jakobsson, Tova Almlöf, Karolin Guldevall, Knut R. Steffensen, and Jan-Åke Gustafsson

From the Department of Biosciences and Nutrition, Karolinska Institutet, Novum, S-14157 Huddinge, Sweden

Received for publication, August 28, 2007 , and in revised form, February 4, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
RAP250 is a coactivator for nuclear receptors as well as other transcription factors. Recent studies have established RAP250 as an essential coactivator for many important biological processes, but its exact mechanism of action is not fully understood. To identify novel proteins that can associate with RAP250, we used a yeast two-hybrid system to screen cDNA libraries and identified the intracellular mediators of transforming growth factor-β (TGF-β) response Smad2 and Smad3 as direct interacting proteins. We show that the interaction between RAP250 and Smad2/3 is dependent upon the second LXXLL interaction motif in RAP250 and the MH2 domain in Smad2 and Smad3. Mouse embryonic fibroblasts lacking RAP250 have reduced expression of the TGF-β target gene PAI-1 after stimulation by TGF-β when compared with wild type cells. Furthermore, we demonstrate a cross-talk between TGF-β and liver X receptors (LXR) signaling pathways and show that stimulation of cells with TGF-β and LXR agonists have a synergistic effect on the expression of the LXR target gene ABCG1. Our data identify RAP250 as a new coactivator in the TGF-β signaling pathway that binds Smad2 and Smad3. Our data also suggest that the interaction between RAP250, Smad2, and Smad3 constitutes an important bridging mechanism linking LXR and TGF-β signaling pathways.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transcription factors play an important role in regulation of gene expression where they bind to specific DNA sequences in promoter and enhancer regions and pass on signals that either repress or enhance transcription. The importance of transcriptional coregulators in this process is well established. Coregulators are auxiliary transcription modulators that can both induce (coactivators) and suppress (corepressors) the transcription potential of a given transcription factor. Today more than 200 different coactivators have been identified (1). Coactivators recruit multiprotein complexes that stimulate the process of transcription initiation. The accessibility for complete transcriptional networks to promoter regions is regulated by ATP-dependent chromatin remodeling by SWI/SNF family complexes that results in unwinding of DNA (2). Coactivators with intrinsic histone acetyltransferase activity covalently modify histones so that additional regulatory proteins are allowed to bind to DNA; examples are the general coactivators CBP2 and p300 (3). The mediator/TRAP/DRIP/ARC multiprotein complex has a central role in recruitment of RNA polymerase II by making contacts between transcriptional activators and the RNA polymerase II transcription machinery (4).

The three members of the SRC-1 family are among the most studied coactivators for nuclear receptors. These coactivators bind to nuclear receptors through LXXLL interaction motifs, also called NR box motifs (5). The LXXLL motif is a short hydrophobic domain that is sufficient for ligand-dependent interactions with nuclear receptors. Other LXXLL-containing coactivators that are important for nuclear receptor signaling are CBP/p300 (3), PGC-1 (6), MED1 (TRAP220/DRIP205/PBP) (7), and RAP250 (8).

RAP250 (9), also known as NcoA6/ASC-2/AIB3/PRIP/TRBP/NRC, was isolated as a nuclear receptor coactivator, but it also acts on several other transcription factors and is considered as a general coactivator. It has an intrinsic glutamine-rich activation domain and two LXXLL interaction motifs. LXXLL-1 (amino acids 887–891) is located in the middle of RAP250 and binds to most nuclear receptors, but LXXLL-2 (amino acids 1491–1495) located in the C-terminal region is very selective in its binding and binds only liver X receptors (LXR{alpha} and LXRβ) (10). RAP250 has no intrinsic enzymatic activity but may recruit proteins with histone acetyltransferase (11, 12), methylase (13), and helicase activity (14). Knock-out studies in mice indicate an essential role for RAP250 since lack of RAP250 results in embryonic lethality (15–18). Transgene expression of a dominant negative form of RAP250 that includes the LXXLL-2 interaction domain results in a phenotype similar to that seen in LXR{alpha} knock-out mice with accumulation of cholesterol in the liver (19), suggesting that RAP250 could be an important factor in LXR signaling.

LXR{alpha} and LXRβ promote reverse cholesterol transport. Decreased atherosclerotic lesions were observed in the aorta of LDLR–/– and apoE–/– mice after activation of LXR where expression of both ABCA1 and ABCG1 was induced in the macrophages in the lesions. Furthermore, transplantation of macrophages from LXR{alpha}/β–/– mice into the same mouse models increased atherosclerosis in both recipient strains (reviewed in Ref. 20). Recent studies have shown that transforming growth factor-β (TGF-β) limits atherosclerosis by modulating a number of processes, including the accumulation of lipids in the vessel wall. This effect is probably mediated by induced expression of ABCA1, ABCG1, and apoE in macrophages (21–23) that are also known LXR target genes.

In this study, we describe a novel function of RAP250 as a coactivator in TGF-β signaling. We show that RAP250 can bind directly to Smad2 and Smad3 through a short domain consisting of LXXLL interaction domain 2 and its C-terminal-flanking region. Our results suggest that RAP250 is a coactivator for Smad2 and Smad3 on TGF-β regulated genes and that RAP250, together with Smad2 and Smad3, can act as a coactivator for LXRs.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—pGBT9-RAP250 plasmids were generated using PCR and standard cloning methods. The bait plasmid used in the yeast two-hybrid screen was made with primers F6 and R7 (9), and shorter forms of this plasmid were made either with PCR or with restriction enzyme digests. The LXXLL to AXXAL mutation was generated with PCR using the following primers: 5'-TCGGCAAGTCAAGCTCTTGACAACTCTGGAGCTCCCA-3' and 5'-GTCAAGAGCTTGACTTGCCGATGTTGGTGCTTCCCTCA-3'. Smad plasmids for the mammalian two-hybrid system were generated by PCR with pcDNA3-FLAG-Smad2 or pcDNA3-FLAG-Smad4 (24) as template using the following primers: Smad2-MH2-F, 5'-GATCGAATTCGAACTATCTCCTACTACTCT-3'; Smad2-MH2-R, 5'-GATCTCTAGATTATGACATGCTTGAGCAAC-3'; Smad4-MH2-F, 5'-GATCGAATTCCCCGGACATTACTGGCCTGT-3'; Smad4-MH2-R: 5'-GATCTCTAGATCAGTCTAAAGGTTGTGGGT-3'.

The PCR products were cloned into the EcoRI and XbaI sites of pM and pVP16 (Clontech). The expression vectors pM-NR2 and pVP16-NR2 were made by cloning of EcoRI/XbaI fragments coding for amino acids 1467–1512 of RAP250 into pM and pVP16. The ALK5, ca-ALK5, and F-Smad2/3/4 (24) expression vectors were a generous gift from C.-H. Heldin (Ludwig Institute for Cancer Research, Uppsala, Sweden). The reporter plasmids used were UAS-tk-LUC (9) and pCMVβ (Clontech).

Yeast Two-hybrid Screening and Yeast β-Galactosidase Assay—Gal4-RAP250-del6/7 (amino acids 1300–1771 of RAP250) was used to screen a human ovary cDNA library in pACT2 vector (Clontech), using the lithium-acetate method and the yeast strain AH109 according to the manufacturer's protocols. High stringency selection, i.e. trp–, leu–, his–, adenine– selection agar, was used, and positive clones were assayed for β-galactosidase activity in a filter assay. 500,000 colony-forming units were screened, and nine positive clones were isolated. Plasmids from positive clones were isolated and transformed into Escherichia coli and prepared and sequenced. Six of the positive clones contained cDNA from either Smad2 or Smad3. For the liquid β-galactosidase assay, the yeast strain Y187 was transformed with bait and prey plasmids, and interactions were monitored as β-galactosidase activity.

Cell Culture and Transient Transfections—RAP250 knockout and wild type mouse embryonic fibroblasts (MEFs) (16), LXR{alpha}/β knock-out and wild type MEFs (25), and Huh7 cells were cultured in Dulbecco's modified Eagle's medium high glucose medium supplemented with 10% fetal bovine serum, 100 µg/ml penicillin, and 100 µg/ml streptomycin (Invitrogen) and 2 mM L-glutamine. U937 cells were grown in RPMI 1640 medium with the same supplements as above. Transient transfections were done in 24-well plates with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cell extracts were prepared 48 h after transfection and assayed for luciferase and β-galactosidase activities. Transfections of siRNA were done with Dharmafect 4 (Dharmacon), and the siRNA against Smad4 was a validated RNA interference with the target sequence 5'-CCUGAGUAUUGGUGUUCCAUUGCUU-3' (Invitrogen). TGF-β was purchased from Peprotech and was used at a final concentration of 5 ng/ml when indicated, and LXR agonists GW3965 (26) (a gift from GlaxoSmithKline Research) and T0901317 (27) were used at a final concentration of 2 µM and 1 µM, respectively.

RNA Extraction, cDNA Synthesis, and Real-time PCR Analysis of mRNA Expression Levels—RNA from cell lines was isolated using RNeasy mini kit (Qiagen) according to the manufacturer's protocol. The concentration and quality of the purified total RNA were determined spectrophotometrically. Synthesis of single-stranded cDNA was carried out on 0.1 µgof RNA using the SuperScript II reverse transcriptase kit (Invitrogen) following standard protocol. Real-time reverse transcription-PCR assay on the basis of SYBR Green I technology was performed with ABI 7500 fast real-time PCR system (Applied Biosystems). All primer pairs span intron-exon boundaries, and for each pair of primers, a dissociation curve analysis was conducted to validate the specificity of the PCR amplification. 100 nM primer concentrations were used in all reverse transcription-PCR analyses. We calculated relative changes employing the comparative method using 18 S as the reference gene and controls as calibrators. Primer sequences used are as follows: RAP250-F, 5'-GCTCATGGGAACAGAGCAGTTAT-3'; RAP250-R, 5'-GCACCGCCAGGTAAGCTG-3'; PAI1-F, 5'-CACAACCCCACAGGAACAGTC-3'; PAI1-R, 5'-AGGGTCAGGGTTCCATCACTT-3'; hABCG1-F, 5'-TGTTCGCGGCCCTCAT-3'; hABCG1-R, 5'-CCTTCAGGCTGTACCAGTAGTTC-3'; mCTGF-F, 5'-CATTAAGAAGGGCAAAAAGTGCA-3'; mCTGF-R, 5'-ACAGGCTTGGCGATTTTAGGT-3'; mABCA1-F, 5'-CGTTTCCGGGAAGTGTCCTA-3'; mABCA1-R, 5'-GCTAGAGATGACAAGGAGGATGGA-3'; mSREBP1c-F, 5'-GGAGCCATGGATTGCACATT-3'; mSREBP1c-R, 5'-GCTTCCAGAGAGGAGGCCAG-3'.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Smad2 and Smad3 as RAP250-interacting Proteins—To identify proteins that can interact with RAP250, we used the yeast two-hybrid system with RAP250 (amino acids 1300–1771) as bait. The bait does not contain the RAP250 transactivation domain, which is active in yeast, or the well characterized LXXLL-1 interaction domain. In a screen of a human ovary cDNA library under high stringency conditions, we isolated several clones that encoded Smad2 and its homolog Smad3. Smad2 and Smad3 are intracellular mediators of TGF-β response and have a similar structure consisting of an N-terminal mad homology (MH)1 domain and a C-terminal MH2 domain separated by a linker region, as reviewed in Refs. 28 and 29. All Smad2 clones encoded amino acids 272–467, including the entire MH2 domain, and all Smad3 encoded amino acids 22–425, including most of the MH1 and the entire linker and MH2 domains. These results suggest that it is the highly conserved MH2 domain that is responsible for the interaction with RAP250. The MH2 domain is a known protein-protein interaction domain and interacts with many different proteins including TGF-β type 1 receptors, transcription factors, nuclear receptors, and transcriptional coactivators, and this domain is also responsible for oligomerization of Smads.

To confirm the interaction between RAP250 and Smad2/3 and to map the interaction domain in RAP250, the liquid β-galactosidase assay was used. Fig. 1A shows that when Y187 yeast was transformed with bait and prey plasmids, the bait used in the screen did not interact with the empty prey vector but only with the Smad2 clone. The Smad3 clone was also positive in the liquid β-galactosidase assay (data not shown), and the strength of the interaction between Smad2 and 3 and RAP250 was high, about the same as between the known RAP250 interaction protein peroxisome proliferator-activated receptor-{gamma} and LXXLL-1, as determined by yeast two-hybrid assays (Fig. 1, b and c). Deletion mutants of the RAP250 bait showed that the interaction domain in RAP250 could be localized to a region between amino acids 1467 and 1512 (RAP250 1467–1512 in Fig. 1A). Interestingly, this interaction domain contains RAP250 LXXLL-2, the unusual LXXLL interaction motif that selectively binds LXR; alanine substitutions of the LXXLL interaction motif core positions +1 and +4(LXXLL to AXXAL) abolished the interaction with Smad2. Fig. 1B shows that the LXXLL-1 interaction motif in RAP250 can interact with a range of nuclear receptors but not with Smad2, which is in contrast to LXXLL-2 that only interacts with LXRs and Smad2 (Fig. 1C). This result suggests selectivity in the interaction between Smad2 and LXXLL interaction motifs.

To further investigate the interaction between Smad2 and RAP250 LXXLL-2, we made N- and C-terminal deletions of the RAP250 1467–1512 bait and found that the interaction domain could be narrowed down to 27 amino acids (RAP250 1485–1512) (Fig. 1D). The C-terminal region flanking the LXXLL interaction domain seems to be a part of the Smad interaction domain since a deletion in this region (RAP250 1467–1502) prevents the binding to Smad2. However, this bait can still interact with LXRs (Fig. 1D). Next, we used baits that were chimeras of RAP250 LXXLL-1 and LXXLL-2 interaction motifs in the yeast two-hybrid system (Fig. 1E). A construct that contained LXXLL-1 in its normal context (Del4) did not interact with the Smad2 clone, and replacing LXXLL-1 with LXXLL-2 (Del4-NR2) did not affect binding (Fig. 1F). The chimeric bait D4-NR2-D6, which contains the N-terminal-flanking region from LXXLL-1, LXXLL-2, and its C-terminal region, interacted as strongly with Smad2 as LXXLL-2 in its normal context (Del6). However, the construct with LXXLL-2 and the C-terminal region from LXXLL-1 (D6-NR2-D4) did not interact with Smad2. These results indicate that the Smad2/3 interaction domain in RAP250 consists of the LXXLL-2 motif and its C-terminal-flanking region.

Smad2 MH2 Interacts with RAP250 LXXLL-2 in Mammalian Cells—To examine whether Smad2 and RAP250 can interact in mammalian cells, we used the mammalian two-hybrid system. First, we confirmed that the RAP250 LXXLL-2 interaction motif could interact with LXR{alpha} and LXRβ as described previously (10) (supplemental Fig. S1, A and B). Next, RAP250-NR2 (amino acids 1467–1512 containing LXXLL-2), Smad2-MH2, and Smad4-MH2 (the dimerization partner of Smad2) were cloned into the mammalian expression vectors as fusions to GAL4-DBD (pM) and VP16-activation domain (pVP16) and assayed on a GAL4-responsive reporter gene in the presence of TGF-β. With RAP250-NR2 fused to GAL4-DBD (pM-NR2), enhanced reporter activity was detected together with VP16-Smad2-MH2, and to a certain extent, also with VP16-Smad4-MH2 but not with other tested constructs (Fig. 2A). These results confirm the yeast two-hybrid data that Smad2-MH2 can interact with the RAP250 LXXLL-2 interaction domain. Smad2-MH2 fused to Gal4-DBD (pM-Smad2) enhanced expression together with VP16-NR2 and VP16-Smad4-MH2 (Fig. 2B). The -fold change was lower in this experiment, perhaps due to the higher transcription activation activity of the Smad2-MH2 domain that harbors an intrinsic transcription activation domain. Gal4-Smad4-MH2 (pM-Smad4) interacted as expected with VP16-Smad2 but not with the other constructs (Fig. 2C). Similar interaction data were obtained when a transfected constitutively active TGF-β receptor was used in the assay (supplemental Fig. S2, A–C). Together, these results demonstrate that RAP250 LXXLL-2 can interact with the MH2 domain of Smad2 in mammalian cells. Fig. 2D shows that the Smad interaction domain in RAP250 when fused to Gal4-DBD (pM-NR2) can act as a TGF-β-induced activation domain since it specifically conferred TGF-β-induced transcription of the reporter gene, suggesting that it can recruit endogenous Smad proteins. To further examine the mechanism of the TGF-β action on LXXLL-2, we used siRNA against Smad4 (siSmad4). Transfected siSmad4 reduced the mRNA levels of Smad4 by more than 90% (supplemental Fig. S3A) and reduced the induction of plasminogen activator inhibitor-1 (PAI-1) by TGF-β to about 50% (supplemental Fig. S3B). Transfected siSmad4 enhanced the TGF-β-stimulated transcriptional activity of pM-NR2 (Fig. 2E), further suggesting a regulatory role of endogenous Smad proteins on the activity of this motif.

RAP250 Is Required for Maximum Induction of PAI-1 after TGF-β Stimulation—The observed interaction between Smad2 and Smad3 with RAP250 suggested that RAP250 could be a coactivator for Smad2 and Smad3. To test this hypothesis, we used MEFs from wild type and RAP250 knock-out embryos and monitored the expression of the TGF-β target gene PAI-1, which is regulated by direct binding of Smad proteins to the promoter region (30, 31). As seen in Fig. 3A, PAI-1 is induced 12-fold in wild type cells but only 6-fold in RAP250 knock-out cells, suggesting that RAP250 is a coactivator in the TGF-β signaling pathway. The connective tissue growth factor gene (CTGF) was also less induced by TGF-β in RAP250 knock-out cells (5-fold) when compared with wild type cells (10-fold) (Fig. 3B). We also assayed the TGF-β target genes Smad7 and p21, but no major regulation by TGF-β was monitored (data not shown). The fact that the activity in the RAP250 knock-out cells is not completely abolished probably reflects coactivator redundancy. It is well known that Smad proteins are able to interact with a number of coactivators including CBP/p300 and Mediator (32–34); hence, a completely blunted activity was not expected in the RAP250 knock-out cells.


Figure 1
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  		FIGURE 1.
RAP250 LXXLL-2 and its C-terminal-flanking region are necessary for interactions between RAP250 and Smad2. A, in a yeast two-hybrid system, Y187 yeast was transformed with different Gal4-DBD-RAP250 fusions and Gal4-AD-Smad2 (amino acids 272–467) and assayed for β-galactosidase activity. The numbering indicates the amino acids of RAP250 present in the bait constructs. The construct RAP250 1467–1512mut contains a mutation of the LXXLL interaction motif from LXXLL to AXXAL. pACT2 is the parental prey vector, and del4(LXXLL-1) contains the RAP250 interaction motif LXXLL-1. All constructs were tested at least three times, and values shown are the means of experiments performed in triplicate. β-gal, β-galactosidase. B, yeast two-hybrid system experiment using a bait containing RAP250 LXXLL-1 (amino acids 819–1096 (del4)) together with LXRs, RXRs, peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}), and Smad2 as prey. Black bars indicate the presence of ligand (2 µM GW3965 for LXRs or 1 µM 9-cis retinoic acid for RXRs). C, same as B but with a bait containing RAP250 LXXLL-2 (amino acids 1467–1512). D, yeast two-hybrid system experiment using N- and C-terminal deletions of the flanking regions of LXXLL-2. Black bars indicate the presence of 2 µM GW. E, Illustration of the different bait constructs used in F. F, yeast two-hybrid system experiment with constructs that contain swaps between protein interaction motifs LXXLL-1 and LXXLL-2 as baits with Gal4-AD-Smad2 (amino acids 272–467) as prey. Values shown are the means of experiments performed in triplicate, and the error bars represent standard deviation.

 
TGF-β Stimulates the Expression of LXR Reporter Genes and ABCG1—Since RAP250 is known to be a coactivator for LXR and in view of our finding that Smad2 and Smad3 can interact directly with RAP250, we wanted to investigate whether TGF-β could influence the expression of LXR target genes. As seen in Fig. 4A, TGF-β alone had a moderate effect on an LXR reporter gene, but in combination with the synthetic LXR agonist GW3965, there was a significant increase in reporter gene expression. Next, we investigated the effect of TGF-β stimulation on the common LXR and TGF-β target gene ABCG1 using quantitative PCR. We found that TGF-β strongly induced the expression of ABCG1 in combination with GW3965 in Huh7 cells, which express low basal levels of ABCG1 (Fig. 4B), as well as in the macrophage cell line U937, which expresses higher basal levels of ABCG1 (Fig. 4C). However, ABCA1 and SREBP-1c were not affected by stimulation by TGF-β (supplemental Fig. S4, A and B), suggesting that the synergistic effects of TGF-β and LXR agonists are promoter-specific. PAI-1 was induced by TGF-β in both the U937 and the Huh7 cells, and the addition of LXR agonists had little effect on the expression of this gene (supplemental Fig. S4, C and D). To further investigate the mechanism for the synergistic activation by LXR agonists and TGF-β, we used LXR{alpha}/β double knock-out MEFs. As seen in Fig. 4D, there is a synergistic effect of GW3965 and TGF-β on ABCG1 in wild type MEFs. However, in LXR double knock-out cells, TGF-β had no effect on ABCG1 expression levels (Fig. 4E), although PAI-1 mRNA was induced by TGF-β stimulation (supplemental Fig. S5, A and B), suggesting that the positive effect of TGF-β stimulation is mediated through LXRs.


Figure 2
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  		FIGURE 2.
RAP250 interacts with Smad2 but not Smad4 in mammalian cells. A, mammalian two-hybrid assay in Huh7 cells using Gal4-DBD-tagged RAP250, pM-NR2, (amino acids 1467–1512), together with pVP16, pVP16-RAP250-NR2, pVP16-Smad2-MH2, and pVP16-Smad4-MH2. The assay was performed with TGF-β and the reporter plasmids UAS-tk-Luc and pCMVβ. Normalized luciferase activity is shown with the activity of pVP16 together with respective pM-fusion set to 1.0. RLU, relative light units. B, as in A but with Gal4-DBD-tagged Smad2-MH2. C, as in A but with Gal4-DBD-tagged Smad4-MH2. D, luciferase reporter assay in Huh7 cells transfected with either pM (Gal4-DBD) or pM-NR2 with UAS-tk-Luc and CMVβ reporter plasmids and with or without TGF-β. E, luciferase reporter assay in Huh7 cells transfected with pM-NR2 and the reporter plasmids UAS-tk-Luc and CMVβ. The cells were also transfected with siRNA against Smad4 and stimulated with TGF-β as indicated. All experiments were performed at least three times. Values shown are the means of experiments performed in triplicate, and error bars indicate standard deviation.

 


Figure 3
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  		FIGURE 3.
RAP250 is necessary for maximum TGF-β induction of PAI-1 and CTGF. Wild type MEFs (WT-MEFs) and RAP250 knock-out MEFs (RAP250-KO-MEFs) were grown in Dulbecco's modified Eagle's medium and stimulated with TGF-β for 16 h when indicated. Total RNA was extracted, and the expression of PAI-1 (A) or CTGF (B) was monitored by real-time PCR analysis. The values shown are the means of experiments performed in triplicate where the expression of PAI-1 or CTGF in untreated cells was set to 1. The experiment was performed three times, and the error bars indicate standard deviation.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have used the yeast two-hybrid system to identify proteins that directly interact with RAP250, and we identify the intracellular mediators of TGF-β signaling as RAP250-interacting proteins. Our results indicate that RAP250 is a new transcriptional coactivator for Smad2 and Smad3 and that the interaction between Smad2/3 and RAP250 influences the activity of Smad proteins both as transcription factors and as coactivators. The Smad interaction domain in RAP250 was mapped to a short unique domain containing LXXLL interaction domain 2, and we also show that it is the MH2 domain in Smad2 and Smad3 that is responsible for the interaction. MH2 domains are known to interact with a variety of different proteins (28, 29), but the LXXLL interaction motif is mainly associated with protein-protein interactions between nuclear receptors and their coactivators (35, 36). There are now a few reports of LXXLL interaction motifs involved in other protein-protein interactions (37), but so far, no interactions with Smad proteins have been described. In addition to LXXLL-2, its flanking C-terminal domain is required for the interaction between RAP250, Smad2, and Smad3. This is similar to the interactions between SRC-1 and nuclear receptors where the specificity of protein-protein interactions is determined by amino acids C-terminally to the LXXLL interaction motif (38).


Figure 4
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  		FIGURE 4.
TGF-β and LXR have additive effects on ABCG1 expression. A, transient transfection of an LXR reporter gene in Huh7 cells. Cells were stimulated with LXR agonist (GW3965 (GW)) and TGF-β as indicated. Normalized luciferase activity is shown from a representative experiment performed in triplicate, and the error bars indicate standard deviation. The experiment was performed three times. B–E, expression levels of ABCG1 in Huh7 cells (B), U937 cells (C), wild type MEFs (WT MEFs)(D), and LXR{alpha}/β knock-out MEFs (LXR{alpha}/β KO MEFs)(E). Cells were stimulated with an LXR agonist (GW3965) and TGF-β for 18 h as indicated, and total RNA was extracted. Expression of LXR-responsive gene ABCG1 was determined by real-time PCR. The values shown are the means of experiments performed in triplicate where the expression of ABCG1 in untreated cells was set to 1. The experiment was performed three times, and the error bars indicate standard deviation.

 


Figure 5
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  		FIGURE 5.
Model of RAP250- and Smad-binding structures. Smad proteins have DNA binding capacity and bind to Smad-binding elements (SBE) after stimulation with TGF-β. Our results indicate that RAP250 can act as a coactivator on TGF-β-responsive genes that contain an Smad-binding element. LXR forms dimers with RXR and binds to LXR-response elements (LXRE). Stimulation of cells with TGF-β and LXR agonists may recruit a complex that contains RAP250 that can interact with both LXR/RXR and Smad2/3 using LXXLL interaction motifs. Possible other coactivators that have the capacity to bind both LXR/RXR and Smad protein, such as CBP, may be a part of this complex.

 
The Smad interaction domain in RAP250 shows no strong similarity to other Smad interaction domains such as the Smad-binding domains in Smad anchor for receptor activation (SARA) or the Smad-interacting motifs in FoxH1 (39, 40) or other proteins in the National Center for Biotechnology Information (NCBI) data base and appears to be selective in its binding since Smad2, Smad3, and LXRs are the only proteins that are known to bind to this domain. This domain may therefore represent a good target for inhibition of TGF-β activity in diseases where TGF-β signaling is unwanted such as fibrosis and cancer.

The role of RAP250 as a coactivator in the TGF-β signaling pathway was demonstrated using RAP250 knock-out MEFs. We show that the TGF-β target genes PAI-1 and CTGF are less induced in MEFs from RAP250 knock-out mice, indicating a role for RAP250 in its regulation. PAI-1 is a direct TGF-β target gene and binds Smad proteins in its promoter region after TGF-β stimulation. A possible role for RAP250 in the regulation of PAI-1 could be as a linker between CBP/p300 and mediator complexes since both RAP250 and Smad proteins are known to associate with CBP, p300, and the mediator components (11, 12, 34, 41), but other effects of RAP250 recruitment cannot be ruled out since RAP250 is known to associate with proteins that affect processes such as protein methylation and RNA processing (8). Our study also shows that costimulation by TGF-β and LXR agonists results in a dramatic induction of the ABCG1 expression. Since the role of ABCG1 in reverse cholesterol transport is well known and it is believed to have important anti-atherogenic properties, simultaneous stimulation of these signaling pathways might be a way to reduce cholesterol accumulation. TGF-β has previously been reported to inhibit macrophage cholesterol ester accumulation by enhancing cholesterol efflux by up-regulation of ABCG1 expression (21), and the latter observation is in agreement with our findings here. However, the synergistic effect of LXR agonists and TGF-β on ABCG1 expression is a novel finding. In this case, we believe that RAP250 affects the role of Smad proteins as coactivators. Smad proteins have been shown to bind directly to nuclear receptors such as estrogen receptors (42) and vitamin D receptor (43), and it is interesting to speculate that RAP250 with its capacity to bind both Smad2/3 and nuclear receptors would stabilize such interactions. The importance of RAP250 LXXLL-2 is supported by the study by Li et al. (44) that showed that mice with disrupted NcoA6 LXXLL-2 exhibited impaired LXR{alpha}-regulated lipogenesis and cholesterol/bile acid homeostasis.

In our model, Fig. 5, we suggest that RAP250 can bind to DNA-bound Smad proteins on TGF-β target genes such as PAI-1 and act as a coactivator after stimulation by TGF-β. We also show that TGF-β and LXR agonists synergistically activate LXR reporter genes and the LXR target gene ABCG1. In this case, we find it likely that RAP250 and Smad proteins and possibly other Smad-interacting coactivators such as CBP would form a coactivator complex on the DNA-bound LXR-RXR heterodimer. In such a multiprotein complex, both RAP250 and CBP would bind to the LXR-RXR heterodimer through LXXLL interaction motifs (LXXLL-1 in RAP250), and the Smads would act as adaptors that interact with RAP250 LXXLL-2 and the C-terminal domain of CBP. Taken together, our studies identify RAP250 as a novel coactivator in the TGF-β signaling pathway through direct interactions with Smad2/3.


   FOOTNOTES
 
* This study was supported by grants from the Swedish Science Council. J.-Å. G. is a shareholder, research grant recipient, and consultant of Karo Bio AB. 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

Formula The on-line version of this article (available at http://www.jbc.org) contains five supplemental figures. Back

1 To whom correspondence should be addressed. Tel.: 46-8-6089147; Fax: 46-8-7745538; E-mail: per.antonson{at}biosci.ki.se.

2 The abbreviations used are: CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; TGF, transforming growth factor; LXR, liver X receptor; RXR, retinoid X receptor; MEF, mouse embryonic fibroblasts MH, mad homology; PAI-1, plasminogen activator inhibitor-1; CTGF, connective tissue growth factor gene; siRNA, small interfering RNA; siSmad4, siRNA against Smad4. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Carl-Henrik Heldin for providing plasmids and Patricia Humire for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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