Orphan Nuclear Receptor Small Heterodimer Partner Inhibits Transforming Growth Factor-β Signaling by Repressing SMAD3 Transactivation*

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-β (TGF-β)-induced gene expression through a direct interaction with Smad, a transducer of TGF-β 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-β-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-β signaling.


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-␤ (TGF-␤)-induced gene expression through a direct interaction with Smad, a transducer of TGF-␤ 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-␤-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-␤ signaling.
TGF-␤ regulates cell cycle-related genes and induces apoptosis by the activation of caspases in rat hepatocytes (55). In the FaO hepatoma cell line, TGF-␤ induced apoptosis through Smad3-dependent cleavage of BAD (56) or phosphorylation of the retinoblastoma protein by TGF-␤-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-␤-mediated activation of Smad plays a role in the regulation of p21 and cell growth in liver. Moreover, TGF-␤ signaling mediates G 1 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-␤ signaling. We have demonstrated that SHP inhibits TGF-␤ signaling through a direct interaction with Smad3 and represses Smad3-mediated transactivation by com-petition with the co-activator p300. Moreover, TGF-␤-responsive gene promoters are inhibited by SHP expression, and overexpression or knockdown of SHP modulates the mRNA levels of TGF-␤-responsive genes. This study provides new insight into the cross-talk between SHP and the TGF-␤ signaling pathway.
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 ϫ 10 4 cells/well the day before transfection. Cells were cultured for 24 h in serum-free conditions followed by treatment with TGF-␤1 (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 g/well by adding an appropriate amount of pcDNA3 empty vector and 100 ng of cytomegalovirus-␤-galactosidase plasmid as an internal control. Cells were harvested ϳ40 -48 h after transfection for luciferase and ␤-galactosidase assays. The luciferase activity was normalized to the ␤-galactosidase activity.
Yeast Two-hybrid and Liquid ␤-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 (UraϪ, HisϪ, and TrpϪ) with appropriate selection markers and assayed for ␤-galactosidase activity. The liquid ␤-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 [ 35 S]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 ␤-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 [ 35 S]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 2ϫ SDS loading buffer, separated by SDS-PAGE, and visualized using a Phos-phorImager (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 ϫ 10 5 cells per well the day before transfection, as described previously (13). Briefly, 1 g 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 l 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 g/ml aprotinin, 10 g/ml leupeptin, 10 mM sodium fluoride, 2 g/ml ␣ 1 -antitrypsin, 2 mM sodium pyrophosphate, 25 mM sodium ␤-glycerophosphate, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride. The cleared lysates (80 l) were mixed with 15 l 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 g) and a rescue vector, pJM17 (5 g), were co-transfected into 293 cells.
Wild Type and SHP Ϫ/Ϫ 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 ϫ 10 6 viable cells. Two hours later, the medium was replaced with William's E medium (Invitrogen) containing 3 ϫ 10 Ϫ8 M selenium, 10 mg/liter insulin, 10 mg/liter transferrin, 1 ϫ 10 Ϫ7 M somatotropin, 1 ϫ 10 Ϫ6 M T3, 1 ϫ 10 Ϫ6 M dexamethasone, 50 g/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 Reagent TM (Sigma). Total RNA (20 g) was fractionated by electrophoresis on a 1% agarose gel containing formaldehyde and transferred to nylon membranes (Zeta-Probe, Bio-Rad) by capillary blotting with 10ϫ 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, 5ϫ SSC, 1 mM EDTA, 10 mg/ml denatured salmon sperm DNA, and a total of 2-4 ϫ 10 6 cpm of 32 P-labeled probes. After hybridization, membranes were washed twice for 5 min at room temperature in 2ϫ SSC and 0.1% SDS, followed by 30 min at 65°C in 0.5ϫ SSC and 0.1% SDS. Membranes were then exposed using Kodak RX film (Eastman Kodak Co., Rochester, NY) for 12-24 h at Ϫ70°C. The signals were normalized to the 18 S ribosomal RNA internal control.
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. [ 35 S]Methionine-labeled SHP was synthesized by in vitro translation. GST alone, GST-Smad2, GST-Smad3, and GST-Smad4 were expressed in bacteria. [ 35 S]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-␤ receptors and interacts with several transcription factors. The linker region connects MH1 and MH2 and regulates ubiquitination (40). B42-MH1ϩLinker and B42-MH2ϩLinker 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 MH2ϩLinker more strongly than with MH1ϩLinker (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 expres- 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. DECEMBER 22, 2006 • VOLUME 281 • NUMBER 51

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sion 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 ϳ700-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 siR-NAs (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.
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 ϳ3-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 dosedependent 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 35 S-labeled C-terminal amino acid residues 1254 -2141 of p300 (p300C), which includes the Smad3 binding region (62,67). 35 S-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.  D), and the indicated amounts of pcDNA3HA-SHP. The pcDNA3 empty vector was used to adjust the total amount of DNA, and 1 g of the pCMV-␤-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 [ 35 S]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 35 S-labeled p300C used in the assay.

Effect of SHP on TGF␤-induced Target Gene Expression-
Downstream targets of TGF-␤1 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-1luc. 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-␤1. Overexpression of SHP did not affect the transactivation of the glucose-6-phosphatase promoter, which is not responsive to TGF-␤1 (Fig. 5D). Taken together, these results demonstrate that SHP inhibits the transcriptional activation of TGF-␤1 target genes.
To confirm whether SHP can repress TGF-␤1 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-␤1 treatment in cells infected with a null adenovirus. Adenovirus-mediated overexpression of SHP significantly decreased the PAI-1 mRNA level in the presence TGF-␤1 (Fig. 6A). In addition, the steady-state PAI-1 mRNA level was also reduced by SHP overexpression in the absence TGF-␤1 (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-␤1-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-␤1 treatment in cells transfected with control siRNA. In the presence of TGF-␤1, the PAI-1 mRNA level was slightly increased by the 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 (Ϫ2300/ϩ8); B, the Smad7 promoter plasmid (Ϫ339/ϩ641); C, the PAI-1 promoter plasmid (Ϫ800); and D, the glucose-6phosphatase promoter plasmid (Ϫ1227/ϩ57) 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 g of pCMV-␤-galactosidase expression plasmid was used as an internal control. The data are representative of at least three independent experiments. 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. DECEMBER 22, 2006 • VOLUME 281 • NUMBER 51 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-␤1 target gene, we examined PAI-1 mRNA levels in SHP Ϫ/Ϫ hepatocytes. Hepatocytes were isolated from SHP Ϫ/Ϫ and wild type mice and Northern blot analysis was performed. The steady-state PAI-1 mRNA level in SHP Ϫ/Ϫ 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-␤1, PAI-1 mRNA was not increased in SHP Ϫ/Ϫ hepatocytes, compared with the increase in PAI-1 mRNA following TGF-␤1 treatment of wild type hepatocytes (Fig. 7C). The PAI-1 mRNA levels may have reached saturation in the SHP Ϫ/Ϫ hepatocytes due to the removal of the negative regulator. Taken together, these results suggest that endogenous SHP can inhibit the expression of TGF-␤1-mediated target genes.

DISCUSSION
In this study, we demonstrated that SHP represses TGF-␤ 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-␤ signaling.
Several nuclear receptors have been reported to regulate TGF-␤ signaling through direct interactions with Smad3 (43)(44)(45)(46)(47)(48)(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-␤ 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␥ (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-␤ signaling by repression of Smad3 transactivation, because overexpression of Smad2 did not produce any significant effects on endogenous TGF-␤-responsive gene promoter activity (19,59). Moreover, Smad2 is not required for TGF-␤-stimulated apoptosis and inhibition of cell growth in hepatocytes (66).
Finally, overexpression of the SHP gene represses TGF-␤induced increases in PAI-1 mRNA as well as Smad3 transactivation ( Fig. 5 and Fig. 6). Conversely, steady-state PAI-1 mRNA FIGURE 7. Effects of SHP knockdown on the expression of TGF-␤-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-␤1. 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 Ϫ/Ϫ and wild type mice and cultured in the presence or absence of TGF-␤1. 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 ␤-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. levels are increased by SHP siRNA (Fig. 7, A and B). Interestingly, the steady-state PAI-1 mRNA levels of SHP Ϫ/Ϫ mice were higher than wild type mice (Fig. 7C), suggesting that SHP can act as a repressor of TGF-␤-responsive target gene expression in vivo. TGF-␤ induces apoptosis via increased Smad3 transactivation in hepatoma cells (56). In HepG2 cells, TGF-␤ up-regulated the expression of p21 via Smad protein (Smad3/4) (58,59), suggesting that the TGF-␤-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-␤-mediated apoptosis and cell cycle arrest. Interestingly, SHP mRNA was decreased by TGF-␤1 (data not shown), and this result suggests that a negative feedback regulation exists between SHP and TGF-␤ signaling.
In conclusion, we have demonstrated that SHP represses TGF-␤ signaling by directly binding to Smad3. SHP also inhibits the Smad-induced transactivation of TGF-␤-responsive gene promoters, suggesting that SHP is involved in regulation of TGF-␤-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-␤ signaling pathway.