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Originally published In Press as doi:10.1074/jbc.M412275200 on March 21, 2005

J. Biol. Chem., Vol. 280, Issue 21, 20842-20850, May 27, 2005
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CHIP Controls the Sensitivity of Transforming Growth Factor-{beta} Signaling by Modulating the Basal Level of Smad3 through Ubiquitin-mediated Degradation*

Hong Xin{ddagger}§, Xialian Xu{ddagger}§, Linyu Li{ddagger}, Hongxiu Ning{ddagger}, Yu Rong{ddagger}, Yu Shang{ddagger}, Yinyin Wang{ddagger}, Xin-Yuan Fu{ddagger}, and Zhijie Chang{ddagger}||

From the {ddagger}Department of Biological Sciences and Biotechnology, and Institute of Biomedicine, Tsinghua University, Beijing 100084, China and the Department of Microbiology and Immunology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202-5120

Received for publication, October 29, 2004 , and in revised form, March 3, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transforming growth factor-{beta} (TGF-{beta}) signaling is critical in a variety of biological processes such as cell proliferation, differentiation, and apoptosis. TGF-{beta} signaling is mediated by a group of proteins including TGF-{beta} receptors and Smads. It is known that different cells can exhibit different sensitivities to TGF-{beta}. Several molecular mechanisms, such as the differential expression of the receptor levels, have been suggested as contributing to these differences. Here, we report evidence for a novel mechanism of regulating TGF-{beta} sensitivity that depends on the role of CHIP (carboxyl terminus of Hsc70-interacting protein) in regulating the basal level of Smad3 via the ubiquitin-dependent degradation pathway. First, using a luciferase assay we found that overexpression of CHIP inhibited TGF-{beta} signaling, whereas silencing CHIP expression by small interfering RNAs led to increased TGF-{beta} signaling sensitivity. Second, based on the results of cell proliferation assays and JunB expression, we found that TGF-{beta} signaling could be abolished by stably overexpressing CHIP. Third, in those cell lines with stably expressed CHIP, we observed that the Smad3 protein level was dramatically decreased. Finally, we demonstrated that CHIP served as a U-box dependent E3 ligase that can directly mediate ubiquitination and degradation of Smad3 and that this action of CHIP was independent of TGF-{beta} signaling. Taken together, these findings suggest that CHIP can modulate the sensitivity of the TGF-{beta} signaling by controlling the basal level of Smad3 through ubiquitin-mediated degradation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transforming growth factor-{beta} (TGF-{beta})1 superfamily cytokines play pivotal roles in a variety of bioprocesses including cell proliferation, differentiation, and apoptosis (1–3). Abnormal response to TGF-{beta} signaling has been reported to relate to many diseases such as tumors, chronic diseases associated with tissue fibrosis, inflammatory diseases, and developmental diseases (4). TGF-{beta} signaling is known to be mediated by many different proteins including the Smad family proteins (5), which have been classified into three different functional groups as follows: (a) receptor-regulated Smads (R-Smads; Smad1, 2, 3, 5, and 8); (b) the common Smad (Co-Smad; Smad4); and (c) the inhibitory Smads (I-Smads; Smad6 and Smad7) (1, 2, 4, 6–8). TGF-{beta} signaling has been shown to be regulated at different levels (for reviews see Refs. 2, 3, 9–12), including ligand activation, ligand binding to TGF-{beta} receptors, R-Smad phosphorylation (5), translocation of R-Smads and the common Smad into the nucleus (2, 3, 6, 7, 10, 13–15), Smad transcriptional activity controlled by co-activators (8, 16–18) or co-repressors (19–22), and proteasomal degradation via ubiquitination-dependent and independent pathways (11, 15, 23–27).

It has been reported that degradation of Smad proteins through ubiquitination is an important mechanism for regulating cellular responsiveness to TGF-{beta} family ligands (11, 12, 15). The effects of most of the known ubiquitination ligases, however, are mainly for terminating the TGF-{beta} signaling. For example, several E3 ligases, such as SCF/Roc1 (28) and Smurf1/2 (24, 25, 29–32), are thought to mediate ubiquitination and degradation of the activated form of Smad proteins. Roc1 is a Cullin-binding protein with RING finger domain and has been reported to associate with Smad3 in a Smad3-SCF/Roc1 complex in the nucleus, which is then transported into the cytoplasm for degradation (28). Smurf1 and Smurf2 are HECT (homologous to E6-AP C terminus) domain-containing E3 ligases (24, 25, 29–32). Smurf1 selectively interacts with receptor-regulated Smads specifically in the BMP (bone morphogenitic protein) pathway to trigger Smad1 ubiquitination and degradation (32). Smurf1 has also been reported to interact with TGF-{beta} type I receptor through Smad7 to regulate receptor degradation (30). It has also been shown that TGF-{beta}-induced Smad2-Smurf2 association with the transcriptional co-repressor SnoN leads to Smurf2 targeting SnoN for ubiquitin-mediated degradation by proteasome (29). These findings provided a mechanism for the down-regulation of TGF-{beta} signaling via degradation of activated R-Smads (15).

It will be interesting to investigate whether some of the E3 ligases may also mediate the degradation of Smads in the absence of TGF-{beta} signaling and, thus, could regulate the basal state of Smads. At present, whether Smurf2 could target unstimulated Smads is still unclear (25). Recently, based on a yeast two-hybrid study, we found that CHIP interacted with Smad1/4 and regulated the bone morphogenetic protein signal pathway (23). CHIP was originally identified as a novel tetratricopeptide repeat-containing protein and was confirmed to interact with heat shock proteins (33). This protein has been reported to inhibit Hsp40-stimulated ATPase activity of Hsc70. It may also block the Hsc70-substrate-binding cycle to reduce chaperon efficiency (33) or enhance Hsp70-dependent folding activity (34). It has been demonstrated that CHIP interacts directly with a tetratricopeptide repeat domain in Hsp90 to induce ubiquitination and degradation of the glucocorticoid receptor through the proteasome (35). Also, CHIP was identified as mediating the degradation of the cystic fibrosis transmembrane conductance regulator (36), Parkin-associated endothelin-receptor-like receptor (Pael-R) (37), the androgen receptor (38), and ErbB2 (39). In these previous studies, CHIP was considered as a novel E3 ligase to change the function of Hsp70/Hsp90 chaperons from a protein-folding machine into a degradation factor (35, 36, 39–42). We would like to explore whether CHIP may have a functional role in the TGF-{beta} signaling mechanism. Here, we present evidence that CHIP can be an important regulator in controlling the sensitivity of the TGF-{beta} signaling by reducing the basal level of Smad3 through ubiquitination.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids and Constructs—The Myc-tagged CHIP expression vector pRK1M/Myc-CHIP was constructed by PCR based on pACT2-CHIP (23) and the mammalian expression vector pRK1M (a gift from Dr. Ying Zhang). HA-tagged CHIP constructs (pcDNA6/V5/HA-CHIP and pEFNeo/HA-CHIP) were generated using the same method by subcloning into the pcDNA6/V5 (Invitrogen) and pEFNeo (from Dr. Xin-yuan Fu) vectors, respectively. The Myc-tagged CHIP mutant H260Q (histidine to glutamine at position 260) was generated using the primers 5'-attgaggagcagctgcagcgtg-3' and 5'-gtccttgcggtcataggtgat-3' based on Myc-CHIP with the site-directed mutagenesis MutanBEST Kit (Takara Biotechnology Co., Ltd.). The His-tagged Smad3 (pcDNA6/V5/His-Smad3) construct was generated by a PCR-based approach from pRK5/F-Smad3 into the vector pcDNA6/V5. FLAG-tagged Smads (pRK5-Smad2, pRK5-Smad3, or pRK5-Smad4), HA-tagged Smad3(3S-A), TGF-{beta}RI(T204D), and (CAGA)12-MLP-Lux constructs were kindly provided by Dr. Xiaofan Wang (Duke University). The HA-tagged ubiquitin plasmid was a gift from Dr. Ying Zhang (NCI, National Institutes of Health, Bethesda, MD). The STAT3 construct (pRC/CMV/STAT3) and its response reporter (pGL2-M67/sie) were provided by Dr. Xinyuan Fu (Yale University). The pBS/U6/CHIPi1 and 2 were constructed according to a previously published protocol (23). pEGFP-N1 was purchased from Clontech, and the pBS/U6 vector and pBS/U6/EGFPi were kindly provided by Dr. Yang Shi (Harvard University) (52).

Mammalian Cell Lines and Transfections—Mv1Lu (mink lung epithelial cells), HEK293T,COS-7, HepG2, and SkOV3 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37 °C in 5% CO2-containing atmosphere. BaF3 cells from a bone marrow-derived murine interleukin-3 dependent pro-B cell line were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 50 µM {beta}-mercaptoethanol, and 5% conditioned medium from the WEHI3 cells as a source of inteleukin-3. The U937 cell line was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and 50 µM {beta}-mercaptoethanol. Lipofectamine 2000 (Invitrogen) and the CalPhos mammalian transfection system (Clontech) were used for transient transfection for Mv1Lu, COS-7, and 293T cells. For establishment of the HA-CHIP-expressing stable Mv1Lu cells, pEFneo/HA-CHIP was transfected into Mv1Lu cells using Lipofectamine 2000 according to the manufacturer's instructions. As for BaF3/HA-CHIP cells, the pEFneo/HA-CHIP plasmid was transfected into the BaF3 cells using an electroporation procedure. Briefly, 1 x 106 cells were mixed with 15 µg of plasmid in ice-cold PBS, placed in a chamber, and subjected to 240 V (t {approx} 35–40 ms) with a BTX ECM 399 electroporation system (BTX division of Genetronics Inc.). Selection was initiated 24 h after transfection using 2 mg/ml G418 (Invitrogen), and the resultant clones were characterized by Western blot and maintained in medium containing 0.5 mg/ml G418.

Immunoprecipitation and Immunoblotting—Metabolic labeling immunoprecipitation and immunoblotting were performed as described (23). The bands were quantified with PhosphorImager software (Amersham Biosciences). Anti-FLAG (M2, Sigma), anti-HA (F-7) (mouse; Santa Cruz Biotechnology), anti-Myc (9E10) (mouse; Santa Cruz Biotechnology), anti-Smad2/3 (mouse; BD Transduction Laboratories), anti-p-Smad2/3 (rabbit; Cell Signaling), anti-JunB (C-11) (mouse; Santa Cruz Biotechnology), anti-EGFP (rabbit; Santa Cruz Biotechnology), and anti-actin (1–19) (goat; Santa Cruz Biotechnology) antibodies were used in the experiments. In some cases, transfected cells were treated with 50 µM MG132 (Calbiochem) or 7.5 ng/ml TGF-{beta}1 (R&D Systems, Inc.) as indicated.

RNA Isolation and RT-PCR Analysis—Total RNAs from 5 x 106 cells were extracted with 1 ml of Trizol (Invitrogen) plus 200 µl of chloroform. The supernatant was precipitated with an equal volume of isopropanol. The RNA pellet was re-suspended in sterile water. 1 µg of total RNA was used as template for one-step RT-PCR analysis (Takara Biotechnology Co., Ltd) according to the protocol. The primers designed to amplify the fragment in the C-terminal (U-box domain) of human and mouse CHIP were 5'-tataggatccaagaagaagcgctggaacagc-3' (forward) and 5'-tatactcgagtcagtagtcctccacccagccatt-3' (reverse). The reaction mixture was first incubated at 50 °C for 30 min for reverse transcription and then denatured at 94 °C for 2 min before PCR cycles. 26 cycles of PCR were conducted under the conditions of 94 °C for 50 s, 55 °C for 50 s, and 72 °C for 1 min. The reaction products were extended at 72 °C for 10 min before storing at 4 °C. The RT-PCR for {beta}-actin was performed with the same aliquoted samples as an internal control.

Pulse-Chase Assay—Transiently transfected COS-7 cells growing in the 12-well plate were pre-incubated in methionine/cysteine-deficient medium for 1 h and then pulsed for metabolic labeling for 10 min with 200 µCi/ml of [35S]methionine/[35S]cysteine (PerkinElmer Life Sciences) and chased at different time points. The cells were lysed and subjected to anti-FLAG (M2) immunoprecipitation and separated by SDS-PAGE. The gels were dried and 35S-labeled F-Smad3 was visualized with a PhosphorImager.

Ubiquitination Assay in Vivo—293T cells were transfected with the related constructs to express HA-ubiquitin, His-tagged Smad3, and Myc-tagged CHIP proteins as indicated in the presence of 50 µM MG132 for 5 h. 7.5 ng/ml TGF-{beta}1 was used as a final concentration for treatment of the cells. The cells were lysed, precipitated, and immunoblotted as described previously (23).

Luciferase Assays—Luciferase assays were carried out in Mv1Lu cells by co-transfecting (CAGA)12-MLP-Lux and different constructs as indicated. The vectors were used to balance the transfection. 6 h after transfection, the cells were treated with TGF-{beta}1 (7.5 ng/ml) for 24 h. Luciferase activities were measured by the Dual luciferase assay system (Promega) in Top Count (Packard). The Renilla luciferase internal control vector (pRL-TK) (Promega) was always used for calibration of the even transfection efficiency. 20 ng/ml leukemia inhibitor factor (LIF) (R&D Systems, Inc.) was used in the experiment for the STAT3 luciferase activity assay.

[3H]Thymidine Incorporation Assay—5 x 103 cells/well were seeded in 96-well plates. The cells were starved for 24 h in serum-free medium and treated with various concentrations of TGF-{beta}1 for 72 or 96 h in triplicate. [methyl-3H]thymidine (Amersham Biosciences) was added at 1 µCi/well 4 h before termination of incubation. The incorporated [3H]thymidine was counted with Top Count (Packard).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CHIP Can Significantly Reduce the Sensitivity of TGF-{beta} Signaling As Demonstrated by Smad3-dependent Transcriptional Activities—To test the role of CHIP on TGF-{beta} signal transduction, we determined whether CHIP had any effect on Smad3-dependent transcriptional activities that could be measured by the luciferase reporter (CAGA)12-MLP-Lux. We performed the experiments in TGF-{beta} response cells (Mv1Lu) and found that 7.5 ng/ml TGF-{beta} stimulated the luciferase activity 5.6-fold over control (Fig. 1A, left columns). The same dose of TGF-{beta}, on the other hand, did not stimulate the reporter activity in cells overexpressing CHIP (Fig. 1A, right columns). This result suggests that overexpression of CHIP can desensitize cells in response to TGF-{beta} signaling.



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  		FIG. 1.
CHIP inhibits Smad3 transcriptional activity. A, CHIP inhibits the luciferase activities induced by TGF-{beta}1. Mv1Lu cells were transfected with (CAGA)12-MLP-Lux, pcDNA6/HA-CHIP, or control vector pcDNA6. 6 h after transfection, the cells were treated with (filled columns) or without (open columns) TGF-{beta}1 (7.5 ng/ml) for 24 h. Luciferase activities were measured. B, CHIP has no effect on STAT3 activity. A reporter construct coding the STAT3 binding site (M67) pGL2-M67/sie was used for the demonstration of STAT3 activity. STAT3 and CHIP were co-overexpressed as indicated with (filled columns) or without (open columns) the treatment of LIF for 48 h after transfection. Luciferase activities were measured as described above. C, CHIP inhibits the luciferase activities induced by overexpression of a constitutively active TGF-{beta} receptor. Mv1Lu cells were transfected with TGF-{beta}RI(T204D) with (filled columns) or without (open columns) the overexpression of the HA-CHIP protein together with the reporter construct (CAGA)12-MLP-Lux. D, CHIP inhibits the luciferase activities induced by overexpression of Smad3 and Smad4. Mv1Lu cells were transfected with the TGF-{beta} luciferase reporter constructs (CAGA)12-MLP-Lux, pRK5/FLAG-Smad3, and pRK5/FLAG-Smad4 (indicated as Smad3 + 4), with (filled columns) or without (open columns) the overexpression of HA-CHIP. 24 h after transfection, cells were extracted and measured for luciferase activity. E, CHIP inhibits Smad2 activity. The experiment was performed according to that described for panel D, except that Smad2 and Smad4 were used. In all the above experiments the assays were carried out in triplicate, the data were normalized with internal controls, and the averages are shown with the S.D.

 
To determine whether such an effect of overexpressed CHIP is specific, we conducted a similar experiment using the STAT-responsive reporter pGL2-M67/sie, which responses to several cytokines (e.g. LIF) in the presence of STAT3 (43). Cells were co-transfected with the STAT3 reporter and STAT3 in the presence or absence of the overexpressed CHIP protein. When these cells were treated with LIF, we observed that the overexpressed CHIP failed to inhibit the LIF-induced STAT3 reporter (Fig. 1B). These results, together with what we observed as described in the above paragraph, indicate that CHIP functions specifically in the Smad-regulated pathway.

To further confirm the ability of overexpressed CHIP to reduce TGF-{beta} signaling, we tested CHIP effects in another overexpression system that involves the constitutively active TGF-{beta} type I receptor (TGF-{beta}RI(T204D)). In this system, the overexpressed TGF-{beta}RI(T204D) stimulated the (CAGA)12-MLP-Lux reporter luciferase activities to 82-fold over the control (Fig. 1C, open columns). Upon co-transfection of CHIP with TGF-{beta}RI(T204D), the luciferase activity was decreased to the background level (Fig. 1C, filled columns). In a third overexpression system, Smad3 and Smad4 were co-expressed with the luciferase reporter in the presence or absence of CHIP. Whereas Smad3 and Smad4 overexpression in Mv1Lu cells led to 153-fold increase in the luciferase activities (Fig. 1D, open columns), similar to results reported by others (44–46), CHIP co-expression dramatically decreased the Smad3 and Smad4 responses to the background level (Fig. 1D, filled columns). These results suggest that overexpressing CHIP could effectively block the transcriptional modulator activities of Smad3 and Smad4. These experiments were repeated four times in triplicate, and the results were consistent. Similar data were also obtained in COS-7 and 293T cells (data not shown). In yet another experiment, we found that overexpression of CHIP also blocked the activity of Smad2 (Fig. 1E). These data together suggest that overexpressed CHIP can negatively regulate TGF-{beta} signaling.

Knocking Down CHIP by siRNA Can Sensitize TGF-{beta} Signaling—The above experiments showed that overexpression of the CHIP protein could inhibit (or attenuate) TGF-{beta} signaling. Conversely, we wanted to examine whether knocking down the endogenous CHIP could promote or facilitate the TGF-{beta} signaling. To address this question, we created two siRNA constructs with specific sequences of CHIP driven by the U6 promoter (designated pBS/U6/CHIPi1 (23) and pBS/U6/CHIPi2). Transfection of these two constructs into 293T cells significantly blocked the expressed CHIP protein, whereas the vector (pBS/U6) and the construct for EGFP siRNA (pBS/U6/EGFPi) did not (Fig. 2A).



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  		FIG. 2.
CHIP siRNAs silence CHIP expression and facilitate TGF-{beta} signaling. A, CHIP siRNAs block expression of the CHIP protein specifically. 239T cells in 6-well plates were cotransfected with pRK1M/Myc-CHIP (indicated as Myc-CHIP), pBS/U6/CHIPi1, pBS/U6/CHIPi2, pBS/U6, and pBS/U6/EGFPi as indicated. pEGFP-N1 was co-transfected to express EGFP protein indicating the even transfection efficiency and siRNA specificity. The Myc-CHIP protein (top) was detected with an anti-Myc antibody, and EGFP (bottom) was detected with the anti-GFP antibody (pRK1M/Myc-CHIP, pEGFP-N1, and relevant siRNA constructs were used at a molecular ratio of 1:30). B, expression of the endogenous CHIP. The CHIP mRNAs from different cell lines as indicated were detected by RT-PCR, and {beta}-actin was used as a control for the even transcription and loading. C, CHIP siRNAs facilitate the transcriptional activities induced by a constitutively active TGF-{beta} receptor. 293T cells were transfected with (CAGA)12-MLP-Lux, TGF-{beta}RI(T204D), pBS/U6/CHIPi1, pBS/U6/CHIPi2, pBS/U6, and pBS/U6/EGFPi, followed by the measurement of luciferase activity as in Fig. 1. The endogenous CHIP expression levels are shown in the bottom section by RT-PCR. D, CHIP siRNAs facilitate TGF-{beta} activities. Using Mv1Lu cells, the experiment was performed as described for panel C except for the addition of TGF-{beta}1 in the cases of overexpression of TGF-{beta}RI(T204D) as indicated. The endogenous CHIP expression levels by RT-PCR are shown in the bottom section.

 
Knowing that 293T cells had a moderate level of endogenous CHIP expression (Fig. 2B), we transfected the CHIP siRNA constructs with the (CAGA)12-MLP-Lux reporter to this cell line. We observed that transfection of CHIP siRNA reduced the endogenous CHIP protein levels (Fig. 2, C and D, bottom sections). Interestingly, in the paired luciferase assays we found that the presence of CHIP siRNAs (1 and 2) enhanced TGF-{beta}RI(T204D)-stimulated luciferase activities by 2.5-fold (Fig. 2C, right two columns), whereas the vector (pBS/U6) and EGFP siRNA (pBS/U6/EGFPi) had no effect (Fig. 2B, middle two columns). Furthermore, when we treated the Mv1Lu cells with TGF-{beta} we also found that the CHIP siRNAs (1 and 2) could increase the luciferase activities (Fig. 2D). These experiments were repeated three times in triplicate. Our results indicate that CHIP siRNAs can facilitate TGF-{beta} signaling through blocking CHIP expression. This finding provides the independent evidence that CHIP is a negative regulator in TGF-{beta} signaling.

CHIP Can Desensitize TGF-{beta}-induced Cell Growth Arrest—If CHIP attenuates or inhibits TGF-{beta} signaling, then overexpression of CHIP would block the response of the cells to TGF-{beta}. We examined this possibility by a cell proliferation assay using two cell lines, Mv1Lu and BaF3, which were reported to respond to TGF-{beta} induced growth arrest (47). When increasing amounts of TGF-{beta} were added to the medium, the BaF3 mock cells exhibited growth arrest (Fig. 3B, line with squares). Cells stably expressing CHIP (Fig. 3A, second lane from left), however, remained resistant to the same doses of TGF-{beta} (Fig. 3B, line with diamonds). In Mv1Lu cells, we used three independent clones stably expressing the CHIP protein (Fig. 3C, first three lanes from the left) to repeat the proliferation experiment. Similar to what was observed in BaF3 cells, these CHIP-expressed cells lost the ability to respond to TGF-{beta}-induced growth arrest (Fig. 3D). These results suggest that cells with overexpressed CHIP are less sensitive to TGF-{beta} mediated cell cycle arrest, implying that CHIP can attenuate TGF-{beta} signaling.

CHIP Can Attenuate Native Gene Expression Induced by TGF-{beta}—The above data demonstrated that overexpressed CHIP could inhibit both the gene responses and the growth arrest mediated by TGF-{beta}. We further investigated whether CHIP had any effect on the expression of the native TGF-{beta} response genes. We examined JunB, an early TGF-{beta} response gene (4, 48, 49), in both BaF3 and Mv1Lu wild type cells (mock cells) with or without overexpressing CHIP. Whereas TGF-{beta} could stimulate junB gene expression in the mock cells (Fig. 4, A and B, second and sixth lane from the left), in cell lines stably expressing CHIP the JunB protein level was significantly decreased. Such effects were detectable at either 4 or 20 h after TGF-{beta} exposure (Fig. 4, A and B). These data clearly suggest that the increased expression of CHIP could inhibit the native gene expression induced by TGF-{beta}.



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  		FIG. 3.
Overexpression of CHIP antagonizes TGF-{beta}-induced growth inhibition in BaF3 and Mv1Lu cells. A and C, Western blot showing a series of single cell clones stably expressing HA-CHIP. BaF3 (A) and Mv1Lu (C) cells were stably transfected with pEFneo/HA-CHIP or pEFneo-vector. Anti-HA immunoblot analysis demonstrated the expression of the HA-tagged CHIP in the indicated clones. B and D, CHIP blocks TGF-{beta}-induced growth inhibition of BaF3 (B) and Mv1Lu (D) cells. 1 x 104 cells/ml were seeded in 96-well plates and starved for 24 h with serum-free medium. TGF-{beta}1 with various concentrations was added to BaF3 cells (B) for 96 h or Mv1Lu cells (D) for 72 h. The growth of the cells was quantified with the [3H]thymidine incorporation assay, and all of the assays were carried out in triplicate.

 



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  		FIG. 4.
Overexpression of CHIP affects the induction of junB gene expression. A Western blot shows that overexpression of CHIP inhibits the induction of JunB both in BaF3 (A) and Mv1Lu (B) cells. The parental BaF3 and Mv1Lu cells and their stably expressing HA-tagged CHIP cells (Mv1Lu/HA-CHIP and BaF3/HA-CHIP) were treated with 7.5 ng/ml TGF-{beta}1 for 4 or 20 h as indicated. Cells extracts were analyzed by immunoblotting with anti-JunB antibody (upper sections). The lower sections display the expression of actin, showing the even loading of the samples. Graphical presentations show the relative abundance of JunB at different time points with the treatment of TGF-{beta}1 after normalization with actin.

 
CHIP-mediated Degradation of Smad3 Can Occur Independent of TGF-{beta} Signaling—To understand the mechanisms underlying the effect of CHIP-mediated inhibition of TGF-{beta} signaling, we speculated that CHIP might mediate the degradation of Smad3 through ubiquitination based upon our previous studies (23). To test this hypothesis, we first examined the degradation of Smads induced by CHIP. As expected, overexpression of CHIP indeed decreased Smad3 protein levels in a manner sensitive to the proteasome inhibitor MG132, implying that CHIP may directly or indirectly induce the proteasomal degradation of Smad3 (Fig. 5A). This degradation was further confirmed by pulse-chase assays as shown in Fig. 5, B and C.

TGF-{beta} signaling has been shown to enhance Smad3 turnover (15). We tested whether the CHIP-mediated degradation of Smad3 is dependent on TGF-{beta} signaling. We assayed the level of exogenously expressed FLAG-tagged Smads and treated the cells with or without TGF-{beta} in the presence or absence of overexpressed CHIP. Western blot analysis showed that TGF-{beta} indeed initiated the degradation of the exogenous Smad3 (Fig. 5D, upper section, compare lanes 1 and 3). However, overexpression of CHIP did not show significant alteration of the degradation of Smad3 (Fig. 5D, upper section, lane 4). Actually, Smad3 degradation triggered by TGF-{beta} remained at the same rate in the presence or absence of CHIP overexpression (Fig. 5D, upper section, compare lanes 1 and 2 without overexpression of CHIP and lanes 2 and 4 with overexpression of CHIP). The quantified densities of Smad3 bands (Fig. 5D, lower section) further showed that the difference between lanes 1 and 2 (without TGF-{beta}) and that between lanes 2 and 4 (with TGF-{beta}) were almost the same. These data suggest that CHIP-mediated Smad3 degradation might be independent of TGF-{beta} signaling.

CHIP Can Mediate Smad3 Ubiquitination Independent of TGF-{beta} Signaling—Previously, we had observed that CHIP can mediate Smad1 ubiquitination (23). By optimizing our experimental procedures, we have now observed that CHIP can also induce ubiquitination of Smad3 (Fig. 6A, smear bands) in the presence of 50 µM MG132. To further test whether the CHIP-mediated Smad3 ubiquitination is subjected to the regulation of TGF-{beta} signaling, we performed an in vivo ubiquitination experiment by overexpressing Smad3 and CHIP in 293T cells treated with or without TGF-{beta}. We found that CHIP induced ubiquitination of Smad3 with or without TGF-{beta} treatment (Fig. 6B). The Smad3(3S-A) mutant, which could not be phosphorylated by TGF-{beta} (50), was also tested for its ubiquitination by CHIP. Results from our in vivo ubiquitination experiment indicated that the mutant Smad3(3S-A) was also ubiquitinated by CHIP (Fig. 6C). These data implied that the CHIP-induced Smad3 ubiquitination occurred independently of TGF-{beta} signal activation.



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  		FIG. 5.
CHIP mediates Smad3 degradation independent of TGF-{beta} signaling. A, overexpression of CHIP results in degradation of Smad3 in a dose-dependent manner. 293T cells were transfected with the plasmids to overexpress FLAG-tagged Smads and Myc-tagged CHIP proteins in the presence (left) or absence (right)of50 µM MG132. An immunoblot was performed with the anti-FLAG (M2) antibody (top). The middle section indicates the dose-dependent increased expression of CHIP, and the bottom section shows even loading of the samples. Graphical presentation shows quantitative densities of the relative F-Smad3 abundance in the transfection of different doses of Myc-CHIP after normalization with co-expressed EGFP. B, overexpression of CHIP decreases the metabolic stability of F-Smad3. COS-7 cells cotransfected with F-Smad3 and Myc-tagged CHIP constructs were subjected to a pulse-chase metabolic labeling with [35S]methionine/[35S]cysteine. The pulse time was 10 min, and chase times are as indicated. F-Smad3 was immunoprecipitated with an anti-FLAG (M2) antibody. C, quantitative presentation of the pulse-chase experiments. Four independent pulse-chase experiments were performed. The 35S-labeled F-Smad3 signals were quantified and valued relative to that at time 0. The data are represented as the average and standard errors. D, the degradation of Smad3 by CHIP is independent of TGF-{beta}signaling. 293T cells transfected with F-Smad3 and Myc-tagged CHIP constructs were treated with or without 7.5 ng/ml TGF-{beta}1 in the presence or absence of MG132 as indicated. Immunoblotting was performed (upper section), and the quantitative data obtained as described for panel A are shown in the lower section.

 
Because CHIP was reported as an E3 ligase containing a U-box domain, we tested the role of the U-box in CHIP-mediated Smad3 ubiquitination. We used a mutant of CHIP (H260Q), which contained a point mutation within the U-box and lacked its ubiquitin E3 ligase, to perform the ubiquitination experiment. As shown in Fig. 6B, the CHIP (H260Q) mutant exhibited significantly reduced ability to mediate Smad3 ubiquitination. In luciferase assays, the mutant CHIP (H260Q) also failed to inhibit Smad3-mediated transcriptional activation (Fig. 6D). These data indicate that CHIP functions as an E3 ligase for Smad3 and that such activity is dependent on its U-box.

CHIP Maintains Basal Level of Smad3 but Does Not Affect the Kinetics—The above experiments (Fig. 5) demonstrated that CHIP mediates degradation of the exogenously overexpressed Smad3. To investigate whether CHIP can change the basal level of the endogenous Smad proteins, we stably expressed CHIP in both Mv1Lu and BaF3 cells and tested the levels of the endogenous Smad3 in the presence or absence of TGF-{beta}. Results of our Western blot analysis showed that the endogenous Smad3 protein level in cells with stably overexpressed CHIP was greatly decreased compared with that in wild type cells (Fig. 7A, compare lanes 1 with 5, upper two sections). The presence of CHIP, however, did not prevent TGF-{beta} to enhance the accumulation of Smad3 (Fig. 7A, comparing lanes 1 to 4 with lanes 5 to 8, upper two sections), even though the basal levels under CHIP were dramatically lower.

Finally, we examined the kinetics of degradation of phosphorylated Smad3 under the treatment of TGF-{beta} with or without CHIP. We found that TGF-{beta} could mediate phosphorylation of Smad3 in the Mv1Lu cell lines regardless of whether or not CHIP was stably expressed (Fig. 7C). Apparently, the degradation rate of the phosphorylated Smad3 was not affected by the overexpressed CHIP protein (Fig. 7, D and E). These results thus suggest that CHIP mainly regulates the basal Smad3 protein level and that its effect does not seem to affect the kinetics of the TGF-{beta} signaling.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
TGF-{beta} signaling has been intensively studied during the past two decades (1–3). It is now known that TGF-{beta} signaling is mediated by Smad proteins in the cytoplasm where Smads are phosphorylated and translocated into the nucleus after activation of TGF-{beta} receptors (2, 9). It has been documented that several proteins at different levels could control the activities of Smads (R-Smads and common Smads) to regulate TGF-{beta} signaling (2, 3). One of the most advanced signs of progress in understanding the regulation of TGF-{beta} signal transduction is the study of the termination mechanism of the signaling regulated by ubiquitin-proteasome-dependent degradation (11, 51). TGF-{beta} signaling could be tightly controlled by positive and negative regulators (10) via the accumulation and subsequent degradation of the phosphorylated Smads (R-Smads) (15). The down-regulation of activated Smad proteins could be a critical mechanism for effectively turning off TGF-{beta} signaling to avoid excess stimulation. To date, several E3 ligases have been identified as participating in terminating the TGF-{beta} signaling. Among them, SCF/Roc1 and Smurf1/2 were reported as mediating the degradation of the phosphorylated Smads proteins (24, 28, 29). In this report, we proposed that CHIP, a newly recognized E3 ubiquitin ligase, could regulate the basal level of Smad3 through a ubiquitin-dependent degradation pathway and, thereby, desensitized the cells to responding to TGF-{beta} signaling. We observed that the overexpression of CHIP could significantly attenuate TGF-{beta} signaling based on luciferase reporter assays, cell growth arrest experiments, and measurements of TGF-{beta} induced gene expression. Conversely, knocking down the endogenous CHIP protein was found to significantly enhance the sensitivity of TGF-{beta} signaling. These data thus indicate that CHIP is a negative regulator for TGF-{beta} signal transduction.



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  		FIG. 6.
CHIP mediates Smad3 ubiquitination without TGF-{beta} signaling. A, overexpression of CHIP induces ubiquitination of Smad3 in vivo. 293T cells were transfected with the plasmids to overexpress His-Smad3, HA-ubiquitin, and Myc-CHIP or Myc-CHIP(H260Q) proteins as indicated in the presence (+) or absence (-) of 50 µM of MG132 for 4 h before harvest. The cell lysates were precipitated with nickel-nitrilotriacetic acid (Ni-NTA). The complex was separated and blotted with anti-HA antibody. The smear indicates the ubiquitinated Smad3 (top section, third and sixth lanes from the left). The expressions of CHIP and Smad3 are demonstrated as indicated in the middle and bottom sections. IB, immunoblot. B, the ubiquitination of Smad3 induced by CHIP is independent of TGF-{beta} signaling. 293T cells were cotransfected with His-tagged Smad3, HA-tagged ubiquitin (HA-Ub), and Myc-tagged CHIP constructs with (+) or without (-) TGF-{beta}1 treatment in the presence of MG132. The ubiquitination experiment was carried out as described for panel A (upper section). The middle and lower sections indicate the expression of Smad3 and CHIP proteins. PP, precipitation; IB, immunoblot. C, Smad3(3S-A) is ubiquitinated by CHIP. 293T cells were transfected with F-Smad3, HA-Smad3(3S-A), His-ubiquitin, and Myc-CHIP expression plasmids as indicated in the presence of MG132. The cell lysates were precipitated (PP) with nickel-nitrilotriacetic acid (Ni-NTA) and blotted (IB) with the anti-Smad2/3 antibody. Expressions of CHIP and Smad3 are demonstrated in the middle and bottom panels. D, CHIP(H260Q) mutant loses the ability to inhibit Smad3 transcriptional activity. The wild type and mutant CHIP were overexpressed as indicated in Mv1Lu cells. Luciferase activities were measured according to the protocol described under "Materials and Methods."

 
Interestingly, unlike Roc1 and Smurf1/2, in which the degradation of Smads was dependent on TGF-{beta} stimulation (24, 25, 29, 30, 32), CHIP could decrease the total Smad3 level independently of the TGF-{beta} activation. Our data demonstrated that TGF-{beta} signaling was unnecessary for CHIP to mediate Smad3 ubiquitination and degradation. It appears that the CHIP-mediated degradation of Smad3 was independent of the phosphorylation of Smad3. For example, we observed that the Smad3(3S-A) mutant, which lost the feature of phosphorylation, could be equally ubiquitinated (Fig. 6C) and degraded by CHIP (data not shown). Taken together, our results suggest that CHIP may desensitize the cell's response to TGF-{beta} by decreasing the basal Smad3 level.

Our findings have added to the general understanding that TGF-{beta} signaling could be controlled by a broad spectrum of E3 ligases. Differing from SCF/Roc1, a typical E3 ligase containing a RING finger domain, and Smurf1/2, a newly identified E3 ligase containing a HECT domain, CHIP contains a U-box domain, which was classified as a new member of the E3 ligase family. Originally, CHIP was reported as mediating the degradation of misfolded or unfolded proteins (36, 37, 40–42). This work proposes a new function for CHIP. Earlier, we had observed that CHIP could interact with Smad1, 2, and 4 and mediate their ubiquitination (23). We now have evidence that CHIP could also degrade Smad3 in a manner independent of TGF-{beta} signaling. Our findings suggest that CHIP may regulate the basal levels of multiple Smads through a proteasome degradation mechanism and, in turn, can modulate the sensitivity of TGF-{beta} signaling.



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  		FIG. 7.
Overexpression of CHIP affects endogenous Smad2/3 protein levels but does not affect the kinetics of Smad2/3. Mv1Lu and Mv1Lu/HA-CHIP cells were treated with 7.5 ng/ml TGF-{beta}1 from 0 to 5 h as indicated. A, the basal level of the endogenous Smad2/3 is decreased in the stably expressed CHIP cells. A Western blot was carried out with anti-Smad2/3. The expressions of the endogenous Smad2/3 and HA-CHIP are shown as indicated. B, graphical presentation shows the relative abundance of the endogenous Smad2/3 at different time points with the treatment of TGF-{beta}1 after normalization with actin. C, phorspho-Smad2/3 (p-Smad2/3) is not affected by overexpressing CHIP. The phospho-Smad3 levels at different time points after treatment of TGF-{beta}1 are shown by Western blot using an anti-phospho-Smad2/3 antibody. D and E, graphical presentations show the relative abundance of the phospho-Smad2/3 levels. The phospho-Smad3 level after TGF-{beta}1 treatment for 1 h was used as 100, and the relative values were plotted in different time points (E).

 

   FOOTNOTES
 
* This work was supported by the 985 Program of Tsinghua University, the Tsinghua-Yue-Yuen Medical Sciences Fund, and National Natural Science Foundation of China Grants 39970369, 30070703, 30470888, and 30030050, 973 Project Grants 2001CB510006 and 2002CB5130007, Beijing Science Research Grant H020220020310, and a special fund from Tsinghua Yuan-Xing Pharmaceuticals, Shen-Zhen, China. 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

§ These authors contributed equally to this work. Back

|| To whom correspondence should be addressed. Tel.: 86-10-62785076; Fax: 86-10-62773624; E-mail: zhijiec{at}mail.tsinghua.edu.cn.

1 The abbreviations used are: TGF-{beta}, transforming growth factor-{beta}; CHIP, carboxyl terminus of Hsc70-interacting protein; EGFP, enhanced green fluorescent protein; F-Smad3, FLAG-tagged Smad3; HA, hemagglutinin; LIF, leukemia inhibitory factor; MLP, major late promoter; PBS, phosphate-buffered saline; R-Smad, receptor-regulated Smad; RT, reverse transcription; siRNA, small interfering RNA; STAT3, signal transducer and activator of transcription 3. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Ying Zhang for HA-ubiquitin plasmids. We are grateful to Dr. Yang Shi from Harvard University for the constructs of pBS/U6 and pBS/U6/EGFPi and the protocol for RNAi design. We also thank Dr. Xinhua Feng from the Baylor College of Medicine for kindly providing related plasmids and helpful suggestions for writing the paper. We thank Dr. J. Massague for visiting Tsinghua University to offer suggestions for the project. Above all, this study would not have been possible without support and encouragement from Nanming Zhao, Xiufang Zhang, Yang Xiang-Yang, and other colleagues in Tsinghua University. We especially thank Drs. Donald C. Chang and Tongwen Wang for the helpful discussion on the manuscript writing.



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