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Originally published In Press as doi:10.1074/jbc.M605947200 on October 30, 2006

J. Biol. Chem., Vol. 281, Issue 51, 39169-39178, December 22, 2006
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Orphan Nuclear Receptor Small Heterodimer Partner Inhibits Transforming Growth Factor-beta Signaling by Repressing SMAD3 Transactivation*

Ji Ho Suh{ddagger}1, Jiansheng Huang§, Yun-Yong Park{ddagger}1, Hyun-A Seong, Dongwook Kim||, Minho Shong||, Hyunjung Ha, In-Kyu Lee**, Keesook Lee{ddagger}, Li Wang§, and Hueng-Sik Choi{ddagger}2

From the {ddagger}Hormone Research Center, School of Biological Science and Technology, Chonnam National University, Kwangju 500-757, Republic of Korea, the Department of Biochemistry, School of Life Sciences, Chungbuk National University, Cheongju 361-763, Republic of Korea, ||Laboratory of Endocrine Cell Biology, the Department of Internal Medicine, Chungnam National University School of Medicine, Daejeon 301-721, Republic of Korea, the **Department of Internal Medicine, Kyungpook University School of Medicine, Daegu 702-701, Republic of Korea, and the §Department of Medicine and Pharmacology, the University of Kansas Medical Center, Kansas City, Kansas 66160

Received for publication, June 21, 2006 , and in revised form, October 2, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Orphan nuclear receptor small heterodimer partner (SHP) is an atypical member of the nuclear receptor superfamily; SHP regulates the nuclear receptor-mediated transcription of target genes but lacks a conventional DNA binding domain. In this study, we demonstrate that SHP represses transforming growth factor-beta (TGF-beta)-induced gene expression through a direct interaction with Smad, a transducer of TGF-beta signaling. Transient transfection studies demonstrate that SHP represses Smad3-induced transcription. In vivo and in vitro protein interaction assays revealed that SHP directly interacts with Smad2 and Smad3 but not with Smad4. Mapping of domains mediating the interaction between SHP and Smad3 showed that the entire N-terminal domain (1–159 amino acids) of SHP and the linker domain of Smad3 are involved in this interaction. In vitro glutathione S-transferase pulldown competition experiments revealed the SHP-mediated repression of Smad3 transactivation through competition with its co-activator p300. SHP also inhibits the activation of endogenous TGF-beta-responsive gene promoters, the p21, Smad7, and plasminogen activator inhibitor-1 (PAI-1) promoters. Moreover, adenovirus-mediated overexpression of SHP decreases PAI-1 mRNA levels, and down-regulation of SHP by a small interfering RNA increases both the transactivation of Smad3 and the PAI-1 mRNA levels. Finally, the PAI-1 gene is expressed in SHP–/– mouse hepatocytes at a higher level than in normal hepatocytes. Taken together, these data indicate that SHP is a novel co-regulator of Smad3, and this study provides new insights into regulation of TGF-beta signaling.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
SHP3 is an atypical member of the orphan nuclear receptor superfamily because its lacks a conventional DBD (1). SHP represses the transcriptional activity of various nuclear receptors, including the estrogen receptor (ER), constitutive androstane receptor, thyroid receptor, retinoic acid receptor, retinoid X receptor, hepatocyte nuclear factor 4{alpha}, androgen receptor (AR), liver receptor homolog-1, estrogen-related receptor {gamma} (ERR{gamma}), glucocorticoid receptor (GR), pregnane X receptor, hepatocyte nuclear factor 3, and Nur77 (1–14), via direct physical interactions. Moreover, SHP acts as a co-repressor of transcription factors, such as the basic helix-loop-helix-Per-Arnt-Sim domain protein, AhR nuclear translocator (ARNT), and basic helix-loop-helix protein BETA2 (15, 16). However, it remains to be determined whether SHP regulates cytokine-mediated cell signaling or other signaling pathways.

The mechanism of repression by SHP is not clearly understood. However, previous reports have suggested that SHP represses transcription factor-mediated transactivation by inhibition of DNA binding (1, 12, 13), recruitment of unknown co-repressors (5, 6, 9) or histone deacetylases (17), or through interactions with EID-1 (18). Moreover, several reports have demonstrated that repression by SHP may involve competition with co-activators, such as TIF2, PGC-1, and p300 (10–12, 14, 16). SHP is expressed in a variety of tissues, including liver, heart, spleen, adrenal gland, small intestine, and pancreas (4, 8, 16, 20). Although a detailed mechanism for regulation of SHP gene expression largely remains to be determined, several reports have demonstrated that SHP gene transcription is regulated by several members of the nuclear receptor superfamily, including the bile acid receptor FXR (21–23), steroidogenic factor-1 (SF-1) (24, 25), hepatocyte nuclear factor 4{alpha} (26), liver receptor homolog-1 (24), ERR{gamma} (8), ER{alpha} (27), LXR-{alpha} (28), E47 (25), and SREBP1 (29). SHP may also play a pivotal role in regulation of cholesterol homeostasis via a bile acid-activated regulatory cascade in the liver (21–23, 30, 31).

Transforming growth factor-beta (TGF-beta) plays diverse biological roles in a variety of cell types (32). TGF-beta regulates cell growth, differentiation, apoptosis, and embryonic development (33–36), and in the liver, TGF-beta plays an important role in the regulation of cell growth, apoptosis, carcinogenesis, and regeneration upon liver injury (37, 38). TGF-beta signals are mediated through activation of the TGF-beta type I and type II serine/threonine kinase receptors (39). Smad proteins have been identified as direct downstream targets of the TGF-beta receptors. Phosphorylated receptor-activated Smads (R-Smad), such as Smad2 and Smad3, can form heteromeric complexes with the common mediator Smad (Co-Smad), Smad4. Smad complexes translocate into the nucleus and bind to Smad-binding elements in TGF-beta-responsive genes (32, 39–41). The Smad complex also regulates transcription through interactions with transcription factors and co-regulators of its target genes (40, 42). The R-Smads contain two conserved domains as follows: the N-terminal Mad homology 1 (MH1) domain binds to DNA, and the C-terminal Mad homology 2 (MH2) domain binds to receptors, Smads, transcription factors, or co-regulators (32, 39, 40, 42). Previous reports have demonstrated that several nuclear receptors interact with Smad proteins and regulate TGF-beta signaling (43–49). These nuclear receptors, including AR (47), ER (48), and GR (49), interact with the MH2 domain of Smad3 and repress TGF-beta signaling. Smad3 also represses AR-mediated transcription, and the ligand binding domain of AR inhibits Smad3 binding to the Smad-binding element (47). ER-mediated transcriptional activation is enhanced by TGF-beta signaling (46, 48). Moreover, Smad3 and Smad4, but not Smad2, physically interact with HNF-4 via their MH1 domains. TGF-beta-regulated Smad3 and Smad4 transactivate the apolipoprotein C-III promoter in hepatic cells via a hormone-response element that binds HNF-4 (50, 51), and retinoic acid receptors interfere with Smad signaling in a ligand-dependent manner (52). PIASy, an inhibitor of activated STAT, can repress Smad transcriptional activity by interacting with histone deacetylase (53), and interferon-{gamma} can interfere with TGF-beta signaling through a direct interaction between YB-1, the Y box-binding protein, and Smad3, which interferes with the interaction between Smad3 and p300 (54).

TGF-beta regulates cell cycle-related genes and induces apoptosis by the activation of caspases in rat hepatocytes (55). In the FaO hepatoma cell line, TGF-beta induced apoptosis through Smad3-dependent cleavage of BAD (56) or phosphorylation of the retinoblastoma protein by TGF-beta-induced activation of Cdc2 and Cdk2 kinase (57). In HepG2 cells, Smad proteins (Smad3/4) up-regulate the p21 gene (58, 59), suggesting that TGF-beta-mediated activation of Smad plays a role in the regulation of p21 and cell growth in liver. Moreover, TGF-beta signaling mediates G1 arrest in HepG2 cells through inactivation of Cdk2 by inhibition of the Cdk2-activating kinase (60).

Here we investigated the role of the orphan nuclear receptor SHP in TGF-beta signaling. We have demonstrated that SHP inhibits TGF-beta signaling through a direct interaction with Smad3 and represses Smad3-mediated transactivation by competition with the co-activator p300. Moreover, TGF-beta-responsive gene promoters are inhibited by SHP expression, and overexpression or knockdown of SHP modulates the mRNA levels of TGF-beta-responsive genes. This study provides new insight into the cross-talk between SHP and the TGF-beta signaling pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagent and Animals—TGF-beta1 was purchased from Sigma. SHP–/– mice have been described previously (63, 65).

Plasmids and DNA Construction—The plasmids encoding LexA-hSHP and mSHP, mSHP deletion constructs (1, 2, 6, 9), and the Gal4tk luciferase reporter gene (9), have been described previously. (CAGA)9 MLP-luc (41) was a kind gift from Jean-Michel Gauthier (Laboratoire Glaxowellcome, France). The p21 promoter plasmid –2300/+8 p21-luc (59) was a kind gift from Aristidis Moustakas (Ludwig Institute for Cancer Research, Sweden). The pcDNA3-Smad7 and pcDNA3HA-ALK5TD (41) plasmids were kind gifts from Carl-Henrik Heldin (Ludwig Institute for Cancer Research, Sweden). The Smad7 promoter plasmid –339/641 Smad7-luc (61) was a kind gift from Fang Liu (University of Medicine and Dentistry of New Jersey Medical School). The pRK5-Smad2, pRK5-Smad3, and pRK5-Smad4 (62) plasmids were kind gifts from Rik Derynck (University of California, San Francisco). For expression in mammalian cells, pRK5-Smad2, pRK5-Smad3, and pRK5-Smad4 were re-cloned into the pCDNA3-FLAG vector by PCR. GST-Smad2 was constructed by inserting a SalI-digested DNA fragment from pRK-Smad2 into SalI-digested pGEX-4T-1 (Amersham Biosciences), and B42-Smad2 was constructed by inserting an EcoRI-XhoI-digested DNA fragment from GST-Smad2 into EcoRI-SalI digested pB42AD/pJG4-5 (Clontech). GST-Smad3 and B42-Smad3 were constructed by inserting an EcoRI-SalI-digested DNA fragment from pRK5-Smad3 into an EcoRI-SalI-digested pGEX-4T-1 vector and into an EcoRI-XhoI-digested pB42AD/pJG4–5 vector (Clontech), respectively. GST-Smad4 and B42-Smad4 were constructed by inserting an EcoRI-SalI-digested DNA fragment into EcoRI-XhoI-digested pGEX-4T-1 and pB42AD/pJG4–5 vectors, respectively. Smad3 deletion constructs were cloned into the B42 vector by PCR. The Gal4-Smad3 fusion protein was constructed by inserting an EcoRI-SalI-digested DNA fragment from GST-Smad3 into an EcoRI-SalI-digested pCMXGal4N vector. All plasmids were confirmed by sequencing.

Cell Culture and Transient Transfection Assays—HepG2, CV-1, and HeLa cells were maintained in Dulbecco's modified essential medium (DMEM; Invitrogen), supplemented with 10% fetal bovine serum (BioWhittaker) and antibiotics (Invitrogen). Cells were aliquoted into 24-well plates at a density of 2–20 x 104 cells/well the day before transfection. Cells were cultured for 24 h in serum-free conditions followed by treatment with TGF-beta1 (5 ng/ml). Transient transfections were performed using the SuperFect transfection reagent (Qiagen Inc.), according to the manufacturer's instructions. The total DNA used in each transfection was adjusted to 1 µg/well by adding an appropriate amount of pcDNA3 empty vector and 100 ng of cytomegalovirus-beta-galactosidase plasmid as an internal control. Cells were harvested ~40–48 h after transfection for luciferase and beta-galactosidase assays. The luciferase activity was normalized to the beta-galactosidase activity.

Yeast Two-hybrid and Liquid beta-Galactosidase Assays—Yeast two-hybrid interaction assays were performed as described previously (8). Briefly, the plasmid encoding the LexA fusion of SHP, the LexA-DBD fused to the full-length murine SHP, and plasmids for B42-AD or B42-AD fused to Smad2, Smad3, or Smad4 were co-transformed into yeast strain EGY48 cells. The transformants were selected on plates (Ura–, His–, and Trp–) with appropriate selection markers and assayed for beta-galactosidase activity. The liquid beta-galactosidase assay was carried out as described previously (8).

GST Pulldown and GST Pulldown Competition Assays—GST pulldown assays were performed according to a method described previously (8). Briefly, SHP was labeled with [35S]methionine using the TNT-coupled reticulocyte lysate system (Promega Corp.), according to the manufacturer's instructions. GST-fused proteins were expressed in Escherichia coli BL21 (DE3) pLys induced with 0.2 mM isopropyl beta-D-thiogalactopyranoside. The cells were lysed and GST fusion proteins were pre-bound to glutathione-Sepharose beads, washed, and then incubated with in vitro translated [35S]methionine-labeled SHP protein in binding buffer containing 25 mM Hepes (pH 7.6), 120 mM NaCl, 0.2 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 20% glycerol at 4 °C for 3–4 h. For in vitro competition assays, purified His-SHP protein was added to the binding reaction, and beads were washed three times with the binding buffer, resuspended in 2x SDS loading buffer, separated by SDS-PAGE, and visualized using a PhosphorImager (BAS-1500, Fuji). The GST-fused or His-tagged proteins used in each reaction were analyzed by SDS-PAGE and quantified by Coomassie Blue staining; 20% of the in vitro translated SHP and p300C used in each reaction was loaded in the input lanes of the gels.

In Vivo Interaction Assays—293T cells grown in DMEM supplemented with 10% fetal bovine serum were plated in 6-well flat-bottomed microplates (Nunc) at a concentration of 2 x 105 cells per well the day before transfection, as described previously (13). Briefly, 1 µg of each plasmid DNA was transfected into 293T cells using a calcium phosphate precipitation method. Forty eight hours after transfection, cells were solubilized with 100 µl of lysis buffer (20 mM Hepes (pH 7.9), 10 mM EDTA, 0.1 M KCl, and 0.3 M NaCl) containing 0.1% Nonidet P-40, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 mM sodium fluoride, 2 µg/ml {alpha}1-antitrypsin, 2 mM sodium pyrophosphate, 25 mM sodium beta-glycerophosphate, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride. The cleared lysates (80 µl) were mixed with 15 µl of glutathione-Sepharose beads (Amersham Biosciences) and rotated for 2 h at 4°C. The bound proteins were eluted by boiling in SDS sample buffer, separated by SDS-PAGE, and then transferred to polyvinylidene difluoride membranes (Millipore Corp.). The membranes were probed with an anti-FLAG monoclonal antibody (12CA5) (Roche Applied Science) and then developed by ECL (Amersham Biosciences), according to the manufacturer's instructions.

Preparation of Recombinant Adenovirus—For ectopic expression of SHP, adenoviral delivery systems were used as described previously (13, 64). Briefly, the cDNA encoding SHP was cloned into the pAd-YC2 shuttle vector. For homologous recombination, the pAd-YC2 shuttle vector (5 µg) and a rescue vector, pJM17 (5 µg), were co-transfected into 293 cells.

siRNA Expression—The siRNAs for SHP were prepared as described previously (13, 63). Briefly, the siRNAs for SHP (si-SHP/I and/II) (Fig. 4B) were chemically synthesized (Dharmacon Research, Lafayette, CO), deprotected, annealed, and transfected according to the manufacturer's instructions (13). SHP siRNAs (Fig. 7, A and B) were generated using the Dicer siRNA generation kit according to the manufacturer's instructions (Gene Therapy Systems, Inc.) (63). HepG2 cells and hepatocytes were transfected with siRNA using the Oligofectamine reagent (Qiagen).

Wild Type and SHP–/– Hepatocyte Cultures—Hepatocytes were isolated as described previously (65), using a modification of the collagenase perfusion method, and further purified by Percoll gradient centrifugation. The cells were suspended in DMEM plus 10% charcoal-stripped serum and plated in 10-cm plates coated with Matrigel (Discovery Labware, Bedford, MA) at a cell density of 2 x 106 viable cells. Two hours later, the medium was replaced with William's E medium (Invitrogen) containing 3 x 10–8 M selenium, 10 mg/liter insulin, 10 mg/liter transferrin, 1 x 10–7 M somatotropin, 1 x 10–6 M T3, 1 x 10–6 M dexamethasone, 50 µg/liter epidermal growth factor, and 25 mg/liter gentamycin, and the cells were then cultured overnight. The cells were harvested at the indicated times and used for RNA isolation.

Northern Blot Analysis—Total RNA was isolated using the TRI ReagentTM (Sigma). Total RNA (20 µg) was fractionated by electrophoresis on a 1% agarose gel containing formaldehyde and transferred to nylon membranes (Zeta-Probe, Bio-Rad) by capillary blotting with 10x sodium citrate/sodium chloride (SSC). After UV cross-linking and prehybridization, membranes were hybridized overnight at 42 °C in a solution containing 50% formamide, 10% dextran sulfate, 5x SSC, 1 mM EDTA, 10 mg/ml denatured salmon sperm DNA, and a total of 2–4 x 106 cpm of 32P-labeled probes. After hybridization, membranes were washed twice for 5 min at room temperature in 2x SSC and 0.1% SDS, followed by 30 min at 65 °C in 0.5x SSC and 0.1% SDS. Membranes were then exposed using Kodak RX film (Eastman Kodak Co., Rochester, NY) for 12–24 h at –70 °C. The signals were normalized to the 18 S ribosomal RNA internal control.

RT-PCR Analysis—RT-PCR analyses were performed as described previously (13). Briefly, total RNA was treated with DNase I (Invitrogen) for 1 h and then diluted to 0.1 µg/µl. Diluted RNA (10 µl) was used for reverse transcription at 42 °C for 1 h. The template was diluted 1:10, and 10 µl was added to each PCR, and the genes were amplified for 30–35 cycles. PCR product (3 µl) was separated on a 1% agarose gel. PCRs were carried out with the Smad3 primer set (forward primer, 5'-GCTGGGTTGGAAGAAGGG-3', and reverse primer, 5'-TTGTGTGCTGGGGACATCGG-3'), Smad4 primer set (forward primer, 5'-TGTGACAGTGTCTGTGTGA-3', and reverse primer, 5'-CCTACCTGAACGTCCATTTC-3'), and beta-actin primer set (forward primer, 5'-GTGTGATGGTGGGAATGGGT-3', and reverse primer, 5'-GGATTCCATACCCAAGAAG-3'). The primer sequence used for human SHP has been described previously (13).


Figure 1
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  		FIGURE 1.
SHP represses TGF-beta signaling through Smad3. HepG2 cells were co-transfected with 150 ng of the reporter gene (CAGA)9 MLP-luc and the SHP expression plasmid pcDNA3HA-SHP. A, after HepG2 cells were co-transfected with the indicated amounts of pcDNA3HA-SHP, cells were preincubated in serum-free medium for 24 h. These quiescent cells were then treated with 5 ng/ml TGF-beta1 and assayed for luciferase activity after 12 h. B, HepG2 cells were co-transfected with 200 ng of expression vectors for pcDNA3HA-ALK5TD and the indicated amounts of pcDNA3HA-SHP. C and D, HepG2 cells and CV-1 cells were co-transfected with 200 ng of expression vectors for pcDNA3FLAG-Smad2, pcDNA3FLAG-Smad3, and the indicated amounts of pcDNA3HA-SHP. The empty pcDNA3 vector was used to adjust the total DNA amounts, and 1 µg of pCMV-beta-galactosidase expression plasmid was used as an internal control. The data are representative of at least three independent experiments.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
SHP Represses TGF-beta Signaling—Several nuclear receptors have been reported to interact with Smad3 and regulate TGF-beta1-induced Smad signaling (43, 45–49, 50–52). To examine the role of SHP in TGF-beta1-mediated transcriptional activity of Smad in hepatocytes, we performed reporter assays in HepG2 cells using the TGF-beta1-responsive (CAGA)9 MLP-luc reporter, which contains nine copies of the Smad-binding element (41). When HepG2 cells transfected with (CAGA)9 MLP-luc were exposed to TGF-beta1, expression of the reporter construct increased by ~50-fold. ALK5TD, a constitutively active form of the TGF-beta1 receptor (41), also strongly induced transcriptional activation of this reporter construct (Fig. 1, A and B). When HepG2 cells co-transfected with the SHP expression vector and (CAGA)9 MLP-luc were incubated in the presence of TGF-beta1, TGF-beta1-induced (CAGA)9 MLP-luc activity was suppressed in an SHP dose-dependent manner (Fig. 1A). Moreover, SHP also strongly repressed ALK5TD-induced transcriptional activity (Fig. 1B).


Figure 2
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  		FIGURE 2.
SHP directly interacts with Smad3. A, GST pulldown assay. SHP was labeled with [35S]methionine by in vitro translation and incubated with glutathione S-Sepharose beads containing bacterially expressed GST alone, GST-Smad2, GST-Smad3, and GST-Smad4 fusion proteins. The input lane represents 20% of the total volume of GST constructs used in the binding assays. B, yeast two-hybrid interaction assay. LexA-DBD or LexA-SHP with plasmids encoding B42-AD fusions of Smad2, Smad3, or Smad4 was transformed into yeast strain EGY48. C, in vivo interaction assay. 293T cells were co-transfected with pcDNA3/FLAG-Smad3 (FLAG-Smad3) or pcDNA3/FLAG-Smad4 (FLAG-Smad4) and pEBG-SHP (GST-SHP) or GST alone (pEBG) as a control. After 48 h, cells were lysed in a buffer containing 0.1% Nonidet P-40. GST fusion proteins were purified using glutathione-Sepharose beads (GST purification) and analyzed by SDS-PAGE. Complex formation (upper, GST purification) and the amounts of FLAG-Smad3 or FLAG-Smad4 used for the in vivo binding assays (lower, Lysate) were determined by anti-FLAG antibody immunoblot (WB). The same blot was stripped and re-probed with an anti-GST antibody (middle) to confirm the expression levels of the GST fusion protein (GST-SHP) and the GST control (GST).

 


Figure 3
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  		FIGURE 3.
Mapping of the SHP and Smad3 interaction domains. A, schematic structures of mSHP wild type and deletion constructs described previously (5). INT domain and REP indicate the nuclear receptor interaction domain and the repression domain of SHP, respectively. Plasmids encoding LexA-mSHP wild type (WT) or LexA-mSHP deletion mutants were co-transformed with plasmids for B42AD and B42-Smad3 into the yeast strain EGY48. B, the schematic structures of Smad3 deletion constructs are presented as described previously (40). Plasmids encoding B42-Smad3 deletion mutants were co-transformed with plasmids for LexA and LexA-SHP into the yeast strain EGY48.

 
Overexpression of constitutively active Smad3 strongly activated the (CAGA)9 MLP-luc reporter, whereas overexpression of constitutively active Smad2 did not significantly affect (CAGA)9 MLP-luc expression in HepG2 cells (24). To determine the effect of SHP on Smad-induced (CAGA)9 MLP-luc activity, we performed transient co-transfections of constitutively active Smad2, Smad3, and SHP expression vectors in both HepG2 and CV-1 cells (Fig. 1, C and D). The induction of (CAGA)9 MLP-luc expression by Smad3 was significantly blocked in an SHP dose-dependent manner in both HepG2 cells (Fig. 1C) and CV-1 cells (Fig. 1D). Taken together, these results demonstrate that SHP inhibits TGF-beta1 induced transcriptional activity mediated by Smad3.

SHP Physically Interacts with Smad3 but Not with Smad4–To determine whether the SHP repression was mediated through direct physical interactions with Smads, we performed in vitro GST pulldown assays. [35S]Methionine-labeled SHP was synthesized by in vitro translation. GST alone, GST-Smad2, GST-Smad3, and GST-Smad4 were expressed in bacteria. [35S]Methionine-labeled SHP was pulled down by GST-Smad3 and GST-Smad2 but not GST-Smad4 or GST alone (Fig. 2A), demonstrating that SHP directly interacts with Smad2 and Smad3 in vitro. To confirm the interaction between SHP and Smad family members, we performed yeast two-hybrid interaction assays. LexA fused to human SHP strongly interacted with the B42 activation domain fused to Smad3 or Smad2, whereas B42-Smad4 did not show any interaction with LexA-SHP (Fig. 2B). The interaction between SHP and Smad2/3 is consistent with the results of the in vitro GST pulldown assays. To confirm an in vivo interaction between SHP and Smad family proteins, we performed an in vivo interaction assay by co-transfection of mammalian expression vectors encoding either GST alone or GST-SHP together with FLAG-Smad3 or FLAG-Smad4 in 293T cells. As shown in Fig. 2C (upper panel), Smad3 was detected in the co-precipitate only when co-expressed with GST-SHP, not with the control GST alone. In contrast, interaction between SHP and Smad4 was not observed, although the expression level of Smad4 was similar to that of Smad3. The amount of GST- and FLAG-tagged proteins was confirmed by Western blot analysis of cell lysates with antibodies against GST and FLAG (Fig. 2C, middle and lower panels, respectively). These results demonstrate that SHP physically interacts with Smad3 but not with Smad4.

Interaction Domains of SHP and Smad3—To map the domains within SHP required for interaction with Smad3, we used a number of deletion constructs (2, 8) in yeast two-hybrid interaction assays. The W160X (1–159 amino acids) construct was reported previously to interact with BETA2/NeuroD (16). The LexA-W160X (1–159 amino acids) construct, which contains the entire N terminus and the nuclear receptor interaction domain, interacted with B42-Smad3. Deletion constructs, which lacked part or all of the W160X domain, showed little or no significant interaction with Smad3 (Fig. 3A). These data indicate that, in addition to the nuclear receptor interaction domain, the N terminus of SHP is required for the Smad3 interaction. These results suggest that the entire N-terminal domain (1–159 amino acids) of SHP is required for the Smad3 interaction.

To map the domains within Smad3 required for the interaction with SHP, we performed yeast two-hybrid interaction assays. Smad proteins consist of two conserved domains, the N-terminal (MH1) and the C-terminal (MH2) domain, and a linker region. The MH1 domain regulates transcription by binding to DNA, and the MH2 domain regulates Smad oligomerization. Moreover, the MH2 domain is recognized by TGF-beta receptors and interacts with several transcription factors. The linker region connects MH1 and MH2 and regulates ubiquitination (40). B42-MH1+Linker and B42-MH2+Linker interacted with LexA-SHP. However, the B42-MH1 construct and the B42-MH2 construct, which contains the interaction domain for nuclear receptors, including AR, GR, and ER, did not show any significant interaction with SHP. Furthermore, SHP interacted with MH2+Linker more strongly than with MH1+Linker (Fig. 3B). These results suggest that the linker region of Smad3 is required for the SHP interaction.

SHP Inhibits Smad3 Transactivation by Competition with the Co-activator p300—To examine whether SHP is directly involved in Smad3-mediated transcriptional activation, we performed transient transfections with the Gal4-Smad3 expression vector and a heterologous reporter containing the Gal4 DNA-binding site in HepG2 cells. In the presence of ALK5TD, Gal4-Smad3 increased transcriptional activity of the reporter gene ~700-fold. Overexpression of SHP produced a dose-dependent decrease in ALK5TD-dependent transactivation by Gal4-Smad3 (Fig. 4A). To investigate the role of endogenous SHP in regulating Smad3 transactivation, we examined the effect of SHP siRNAs (si-SHP/I or II) in HepG2 cells. Endogenous SHP gene expression was significantly decreased by si-SHP/II, but not by si-SHP/I, in HepG2 cells (Fig. 4B) (16). Gal4-Smad3-mediated transactivation increased upon down-regulation of SHP by si-SHP/II, but no changes were detected in si-SHP/I-treated cells (Fig. 4B). These results indicate that SHP may directly regulate the transcriptional activity of Smad3.


Figure 4
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  		FIGURE 4.
SHP represses p300-enhanced transactivation of Smad3 by interfering with p300 binding to Smad3. A, SHP inhibits TGF-beta1-induced Gal4-Smad3 transactivation. HepG2 cells were co-transfected with 100 ng of Gal4-tk-luc reporter plasmid, 200 ng of expression vectors for Gal4-Smad3 and pcDNA3HA-ALK5TD, and the indicated amounts of pcDNA3HA-SHP. B, knockdown of endogenous SHP by siRNA SHP increases transactivation of Gal4-Smad3 in HepG2 cells. HepG2 cells were co-transfected with 100 ng of the Gal4-tk-luc reporter plasmid, 200 ng of the Gal4-Smad3 expression vector, and si-SHP/II or si-SHP/I. SHP and beta-actin mRNA levels were determined by RT-PCR. C and D, HeLa cells were co-transfected with 100 ng of the Gal4-tk-luc reporter plasmid, and 200 ng of expression vectors for Gal4-Smad3, pcDNA3-p300 (C and D), and the indicated amounts of pcDNA3HA-SHP. The pcDNA3 empty vector was used to adjust the total amount of DNA, and 1 µg of the pCMV-beta-galactosidase expression plasmid was used as an internal control. The data are representative of at least three independent experiments. E, SHP interferes with the interaction between Smad3 and p300. p300C is the C-terminal region of p300 (1254–2141 amino acids), including the C/H3 and Q-rich domains. p300C labeled with [35S]methionine was incubated with GST alone or the GST-Smad3 fusion protein bound to GSH-Sepharose beads. Increasing amounts of purified His-SHP proteins were then added to the binding reaction and analyzed by SDS-PAGE. The input lanes represent 20% of the total volume of 35S-labeled p300C used in the assay.

 
The transcriptional activity of Smad3 is enhanced by p300/CBP binding to the MH2 domain of Smad (62, 67). In addition, SHP inhibits the transcriptional activity of nuclear receptors by competing with co-activators binding to the AF-2 surface of nuclear receptors (5, 6, 9). To determine whether SHP can regulate the p300-enhanced transcriptional activity of Gal4-Smad3, we co-transfected SHP, p300, and Gal4-Smad3 expression vectors into HeLa cells. Overexpression of p300 enhanced the transcriptional activity of Gal4-Smad3 ~3-fold, and SHP caused a dose-dependent decrease in the p300-enhanced transcriptional activity of Gal4-Smad3 (Fig. 4C). In addition, SHP repression of Smad3-induced transactivation was blocked by p300 overexpression in a dose-dependent manner (Fig. 4D), suggesting that SHP may interfere directly with p300-regulated Smad3 transactivation.

To determine whether SHP physically competes with p300 for Smad3 binding, we used in vitro GST pulldown competition assays using the 35S-labeled C-terminal amino acid residues 1254–2141 of p300 (p300C), which includes the Smad3 binding region (62, 67). 35S-Labeled p300C interacted with GST-Smad3, and SHP blocked the p300C and GST-Smad3 interaction in a dose-dependent manner (Fig. 4E). Taken together, these results demonstrate that SHP specifically inhibits the transcriptional activity of Smad3 by interfering with p300 binding to Smad3.


Figure 5
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  		FIGURE 5.
SHP inhibits the Smad3-induced transcription of p21, Smad7, and PAI-1. HepG2 cells were co-transfected with 100 ng of the reporter gene. A, the p21 promoter plasmid (–2300/+8); B, the Smad7 promoter plasmid (–339/+641); C, the PAI-1 promoter plasmid (–800); and D, the glucose-6-phosphatase promoter plasmid (–1227/+57) and 200 ng of expression vectors for pcDNA3FLAG-Smad3, pcDNA3FLAG-Smad4, pcDNA3HA-ALK5TD, and pcDNA3HA-SHP. The pcDNA3 empty vector was used to adjust the total amount of DNA, and 1 µg of pCMV-beta-galactosidase expression plasmid was used as an internal control. The data are representative of at least three independent experiments.

 
Effect of SHP on TGFbeta-induced Target Gene Expression–Downstream targets of TGF-beta1 signaling include the p21, SMAD7, and PAI-1 genes. Smad3 can activate the promoters of these three genes in HepG2 cells (41, 58, 59, 61, 68, 69). To examine whether SHP significantly affects Smad3-induced transactivation of endogenous gene promoters, we performed co-transfection assays with the p21 gene promoter in the presence of constitutively active Smad3 and ALK5TD. Induction of the p21-luc reporter activity by Smad3 was greatly increased by ALK5TD and significantly repressed by SHP (Fig. 5A). Additionally, we performed co-transfection assays with the Smad7 or PAI-1 promoters and constitutively active Smad3, Smad4, and ALK5TD expression vectors. Smad3 increased the transcriptional activity of Smad7-luc or PAI-1-luc when co-transfected with ALK5TD, whereas SHP strongly repressed the Smad3-induced transcription activity of Smad7-luc and PAI-1-luc. When we performed co-transfections with both Smad3 and Smad4 expression vectors, the Smad3/4-mediated transactivation of Smad7-luc or PAI-1-luc in the presence of ALK5TD was significantly repressed by SHP (Fig. 5, B and C). As a control, we examined the effects of SHP on a promoter that is not responsive to TGF-beta1. Overexpression of SHP did not affect the transactivation of the glucose-6-phosphatase promoter, which is not responsive to TGF-beta1 (Fig. 5D). Taken together, these results demonstrate that SHP inhibits the transcriptional activation of TGF-beta1 target genes.


Figure 6
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  		FIGURE 6.
Effects of SHP overexpression on the expression of TGF-beta-mediated target genes. A and B, adenovirus-mediated overexpression of SHP represses PAI-1 mRNA levels. HepG2 cells (A) and mouse primary hepatocytes (B) were infected with adenovirus expressing SHP (multiplicity of infection = 50) or null adenovirus (multiplicity of infection = 50) in the presence or absence of TGF-beta1. Three mice per genotype were used for hepatocyte isolation, and duplicate plates were used for each treatment. Total RNAs were pooled from each treatment (B). Total RNA was isolated from cells, and the PAI-1 and SHP mRNA levels were determined by Northern blot analysis and quantified using a PhosphorImager. 18 S ribosomal RNA was used as an internal control. Expression levels for PAI-1 and SHP were normalized to the 18 S ribosomal RNA levels. The basal levels of expression for PAI-1 and SHP in cells were defined as 1.0. The data are representative of at least two independent experiments. The error bars indicate standard deviations.

 
To confirm whether SHP can repress TGF-beta1 target gene expression, we performed Northern blot analysis to measure the PAI-1 mRNA level in HepG2 cells after adenovirus-mediated overexpression of SHP. PAI-1 mRNA increased in response to TGF-beta1 treatment in cells infected with a null adenovirus. Adenovirus-mediated overexpression of SHP significantly decreased the PAI-1 mRNA level in the presence TGF-beta1 (Fig. 6A). In addition, the steady-state PAI-1 mRNA level was also reduced by SHP overexpression in the absence TGF-beta1 (Fig. 6A). The SHP mRNA level was strongly increased by adenovirus (data not shown). Moreover, adenovirus-mediated overexpression of SHP also decreased the PAI-1 mRNA level in mouse primary hepatocytes (Fig. 6B). These data demonstrate that SHP represses PAI-1 gene expression in HepG2 cells and hepatocytes, and these results were consistent with the repression of TGF-beta1-induced transactivation of the PAI-1 promoter (Fig. 5C).

We showed that siRNA-mediated knockdown of SHP enhanced Smad3 transactivation (Fig. 4B). To investigate the role of endogenous SHP in the regulation of PAI-1 mRNA levels, we examined the effect of SHP siRNA in HepG2 cells and hepatocytes. The steady-state PAI-1 mRNA level was increased when SHP was down-regulated using SHP siRNA in HepG2 cells (Fig. 7A) and in mouse primary hepatocytes (Fig. 7B). The PAI-1 mRNA level was increased in response to TGF-beta1 treatment in cells transfected with control siRNA. In the presence of TGF-beta1, the PAI-1 mRNA level was slightly increased by the SHP knockdown. PAI-1 mRNA was normalized to 18 S rRNA levels (Fig. 7A). To further confirm the effect of down-regulation of endogenous SHP on the TGF-beta1 target gene, we examined PAI-1 mRNA levels in SHP–/– hepatocytes. Hepatocytes were isolated from SHP–/– and wild type mice and Northern blot analysis was performed. The steady-state PAI-1 mRNA level in SHP–/– hepatocytes was higher than the level in wild type hepatocytes (Fig. 7C). Moreover, knockdown of endogenous SHP did not affect the mRNA levels of Smad3 or Smad4 (Fig. 7D). However, in the presence of TGF-beta1, PAI-1 mRNA was not increased in SHP–/– hepatocytes, compared with the increase in PAI-1 mRNA following TGF-beta1 treatment of wild type hepatocytes (Fig. 7C). The PAI-1 mRNA levels may have reached saturation in the SHP–/– hepatocytes due to the removal of the negative regulator. Taken together, these results suggest that endogenous SHP can inhibit the expression of TGF-beta1-mediated target genes.


Figure 7
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  		FIGURE 7.
Effects of SHP knockdown on the expression of TGF-beta-mediated target genes. A and B, knockdown of the endogenous SHP by siRNA SHP increases the mRNA level of PAI-1 in HepG2 cells (A) and mouse primary hepatocytes (B). HepG2 cells and mouse primary hepatocytes were transfected with control siRNA or SHP siRNA in the presence or absence of TGF-beta1. PAI-1 and SHP mRNA levels were determined by Northern blot analysis and quantified using a PhosphorImager. C and D, hepatocytes were isolated from SHP–/– and wild type mice and cultured in the presence or absence of TGF-beta1. Three mice per genotype were used for hepatocyte isolation, and duplicate plates were used for each treatment. Total RNAs were pooled from each treatment (B–D). The PAI-1 and SHP mRNA levels were determined by Northern blot analysis and quantified using a PhosphorImager. The mRNA levels of Smad3 and Smad4 were determined by RT-PCR analysis. 18 S ribosomal RNA and beta-actin were used as internal controls. PAI-1 and SHP expression levels were normalized to the 18 S ribosomal RNA levels. The basal PAI-1 and SHP expression levels in cells were defined as 1.0. The data are representative of at least two independent experiments. The error bars indicate standard deviations.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrated that SHP represses TGF-beta signaling through inhibition of transactivation by Smad. SHP directly interacts with Smad3 and inhibits Smad3-mediated transactivation of the p21, SMAD7, and PAI-1 genes by competing for Smad3 binding to p300. Moreover, knockdown of the endogenous SHP gene increases the transactivation of Smad3 and increases PAI-1 mRNA levels. These current observations suggest that the orphan nuclear receptor SHP plays a physiological role in TGF-beta signaling.

Several nuclear receptors have been reported to regulate TGF-beta signaling through direct interactions with Smad3 (43–49). For example, the ligand binding domain of AR inhibits Smad3 binding to the Smad-binding element (47). However, the mechanism of repression by other nuclear receptors, such as ER, GR, and HNF-4, has not yet been characterized. Previous reports have suggested that SHP competes with co-activators and nuclear receptors (10–12, 14, 16). Here we showed that the orphan nuclear receptor SHP interferes with Smad3 binding to p300. The MH2 domain of Smad3 interacts with the C-terminal domain of p300. However, neither the Smad MH1 nor MH2 alone interacted with SHP; the presence of the linker region was required for interactions with SHP. YB-1 can repress TGF-beta signaling by interfering with the interaction of Smad3 and p300, although YB-1 interacts with the MH1 domain of Smad3 (54). Although the MH2 domain of Smad3 has been shown to interact with several nuclear receptors, including AR (47), ER (48), and GR (49), our results demonstrated that the Smad linker region plays an important role in the interaction between Smad and SHP. These data suggest that the linker region may be important for cross-talk between Smad and nuclear receptors.

The interaction of SHP with Smad3 (Fig. 3A) is similar to the SHP interaction with BETA2/NeuroD (16) but differs from SHP interactions with retinoid X receptor (6) and ERR{gamma} (8). These results suggest that the SHP domains interacting with nuclear receptors may be different from regions of the protein that interact with other transcription factors. Our result shows that SHP interacts with Smad2 and Smad3 but not with Smad4 (Fig. 2, A–C). However, we focused on SHP repression of TGF-beta signaling by repression of Smad3 transactivation, because overexpression of Smad2 did not produce any significant effects on endogenous TGF-beta-responsive gene promoter activity (19, 59). Moreover, Smad2 is not required for TGF-beta-stimulated apoptosis and inhibition of cell growth in hepatocytes (66).

Finally, overexpression of the SHP gene represses TGF-beta-induced increases in PAI-1 mRNA as well as Smad3 transactivation (Fig. 5 and Fig. 6). Conversely, steady-state PAI-1 mRNA levels are increased by SHP siRNA (Fig. 7, A and B). Interestingly, the steady-state PAI-1 mRNA levels of SHP–/– mice were higher than wild type mice (Fig. 7C), suggesting that SHP can act as a repressor of TGF-beta-responsive target gene expression in vivo. TGF-beta induces apoptosis via increased Smad3 transactivation in hepatoma cells (56). In HepG2 cells, TGF-beta up-regulated the expression of p21 via Smad protein (Smad3/4) (58, 59), suggesting that the TGF-beta-mediated activation of Smad may play a role in apoptosis regulating liver growth. SHP is highly expressed in the liver and plays a crucial role in negative feedback regulation of bile acid biosynthesis. Therefore, under conditions of high SHP gene expression induced by bile acid, or by other inducers of SHP gene expression, SHP may inhibit TGF-beta-mediated apoptosis and cell cycle arrest. Interestingly, SHP mRNA was decreased by TGF-beta1 (data not shown), and this result suggests that a negative feedback regulation exists between SHP and TGF-beta signaling.

In conclusion, we have demonstrated that SHP represses TGF-beta signaling by directly binding to Smad3. SHP also inhibits the Smad-induced transactivation of TGF-beta-responsive gene promoters, suggesting that SHP is involved in regulation of TGF-beta-induced gene expression. Finally, our data suggest that the orphan nuclear receptor SHP is a novel co-regulator of Smad and that SHP gene expression modulates the TGF-beta signaling pathway.


   FOOTNOTES
 
* This work was supported by National Research Laboratory Grant M1-0500-4705J-4710, KRF Grant C00126 [GenBank] , a Marine Bio21 grant (to H.-S. C.), by funds from the Kansas Masonic Cancer Research Institute, National Institutes of Health Grant P20 RR016475 from the INBRE Program of the NCRR, a Liver Scholar Award from ALF/AASLD, and a BGIA Award from the American Heart Association (to L. W.). 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

1 Members of the post Brain Korea 21 program. Back

2 To whom correspondence and reprint requests should be addressed. Tel.: 82-62-530-0503; Fax: 82-62-530-0500; E-mail: hsc{at}chonnam.ac.kr.

3 The abbreviations used are: SHP, small heterodimer partner; ALK5TD, constitutively active TGF-beta receptor I; DBD, DNA binding domain; GST, glutathione S-transferase; MH1, Mad homology 1; MH2, Mad homology 2; SHP–/–, SHP null; mSHP, mouse SHP; PAI-1, plasminogen activator inhibitor-1; TGF-beta, transforming growth factor-beta; GR, glucocorticoid receptor; ER, estrogen receptor; ERR{gamma}, estrogen-related receptor {gamma}; AR, androgen receptor; RT, reverse transcription; DMEM, Dulbecco's modified essential medium; siRNA, small interfering RNA. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Jean-Michel Gauthier, Aristidis Moustakas, Carl-Henrik Heldin, Fang Liu, and Rik Derynck for their kind gifts of plasmids used in this study. We also thank Dr. S. Paul Oh and Dr. Seong-Jin Kim for helpful discussions and suggestions.



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