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Published ahead of print on September 7, 2006
J Am Soc Nephrol 17: 2781-2791, 2006
© 2006 American Society of Nephrology
doi: 10.1681/ASN.2005101055
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Pathophysiology of Renal Disease and Progression

Downregulation of SnoN Expression in Obstructive Nephropathy Is Mediated by an Enhanced Ubiquitin-Dependent Degradation

Ruoyun Tan*,{dagger}, Jinglan Zhang*, Xiaoyue Tan*, Xianghong Zhang*, Junwei Yang{dagger} and Youhua Liu*

* Division of Cellular and Molecular Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and {dagger} Renal Division, Department of Medicine, The First Affiliated Hospital, Nanjiang Medical University, Nanjing, China

Address correspondence to: Dr. Youhua Liu, Department of Pathology, University of Pittsburgh, S-405 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15261. Phone: 412-648-8253; Fax: 412-648-1916; E-mail: liuy{at}upmc.edu

Received for publication October 12, 2005. Accepted for publication July 19, 2006.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Smad transcriptional co-repressor SnoN acts as an antagonist that tightly controls the trans-activation of TGF-beta/Smad target genes. SnoN protein is reduced progressively in the fibrotic kidney after obstructive injury, suggesting that the loss of Smad antagonist is a critical event that leads to an uncontrolled fibrogenic signaling. However, the mechanism underlying SnoN downregulation remains unknown. This study investigated the regulation and mechanism of renal SnoN expression in vivo. Whereas SnoN protein was markedly diminished, its mRNA levels remained relatively constant in the obstructed kidney after ureteral ligation. An increased ubiquitination and proteasome-dependent degradation of SnoN was found in obstructed kidney, compared with sham controls. Smad ubiquitination regulatory factor-2, an E3 ubiquitin ligase, was induced and formed a complex with SnoN in vivo. In vitro, TGF-beta1 promoted SnoN protein degradation, which was mediated by ubiquitination and a proteasome-dependent mechanism. SnoN constitutively interacted with another Smad co-repressor, Ski, and they formed ternary complex with Smad2/3 upon TGF-beta1 stimulation. However, ectopic expression of Ski did not alter the degradation rate of SnoN. Blockage of SnoN degradation by proteasome inhibitor abolished TGF-beta1–mediated {alpha}-smooth muscle actin and fibronectin induction, suggesting that SnoN degradation could be necessary for TGF-beta1 to exert its fibrogenic action. Furthermore, knockdown of Smad ubiquitination regulatory factor-2 expression by small interfering RNA strategy led to an increase in SnoN abundance and inhibited the TGF-beta1–mediated gene transcription. These results indicate that downregulation of SnoN expression in the obstructed kidney is mediated by an enhanced ubiquitin-dependent degradation. Preservation of SnoN by inhibiting its degradation may be a novel strategy for targeting hyperactive Smad signaling in renal fibrotic diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TGF-beta is widely recognized as a key fibrogenic cytokine that plays a critical role in the pathogenesis of renal fibrosis (1–4). Extensive investigations over the past decade have demonstrated unambiguously the importance of TGF-beta in the initiation and progression of chronic kidney disease (CKD) after various injuries. Induction of TGF-beta expression is found in virtually every type of CKD (1,2,5). In vitro, TGF-beta1 stimulates myofibroblastic activation from glomerular mesangial cells and interstitial fibroblasts (6,7) and promotes tubular epithelial to mesenchymal transition (8,9). Overexpression of active TGF-beta1 in transgenic mice induces renal glomerular and interstitial fibrotic lesions (10). Conversely, inhibition of TGF-beta via various strategies, including antisense inhibition of TGF-beta expression and blockade of TGF-beta action by neutralizing antibody or soluble receptor, attenuates renal fibrosis and ameliorates progressive loss of kidney function in animal models (11–14).

TGF-beta signaling is transduced from cell surface to the nucleus through transmembrane type I and type II serine/threonine kinase receptors and their downstream mediators known as Smads (2,15). On stimulation of TGF-beta, Smad2 and Smad3 undergo phosphorylation, which triggers their interaction with Smad4. The Smad complex then is translocated into the nucleus and binds to the specific cis-acting element in the regulatory region of TGF-beta target genes and directs their transactivation. Given the vital importance of TGF-beta in such diverse biologic processes as matrix homeostasis, cell growth and differentiation, and immune modulation, TGF-beta/Smad signaling is tightly constrained by a negative controlling mechanism in normal physiologic conditions. Several proteins, including Ski (Sloan-Kettering Institute proto-oncogene), SnoN (Ski-related novel gene, non Alu-containing), and TGIF (TG-interacting factor) have been identified as Smad transcriptional co-repressors (16–19). Through various mechanisms, they antagonize Smad action and block Smad-mediated gene transcription. Therefore, the abundance and activity of Smad transcriptional co-repressors in a particular cellular context may determine the ultimate response of the cells to TGF-beta stimulation.

Earlier studies from our laboratory demonstrated that SnoN protein is downregulated progressively in the fibrotic kidney after obstructive injury (20). Loss of Smad transcriptional co-repressor seems to have a profound impact on Smad signaling, because knockdown of SnoN via small interfering RNA (siRNA) inhibition dramatically sensitizes renal tubular epithelial cells to TGF-beta1 stimulation (20). Consistently, increased SnoN expression, either via transfection of SnoN expression vector (20) or through induction by hepatocyte growth factor (21), suppresses TGF-beta/Smad-mediated gene transcription. These observations accentuate that the loss of Smad antagonist is a critical, pathogenic event that may lead to an uncontrolled Smad signaling. However, the mechanism underlying SnoN reduction in the diseased kidney remains unknown.

In this study, we investigated the regulation and mechanism of SnoN expression in the fibrotic kidney after obstructive injury. Our results demonstrate that downregulation of SnoN in vivo is mediated by an enhanced ubiquitin-dependent degradation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal Model
Male CD-1 mice that weighed 18 to 22 g were obtained from Harlan Sprague-Dawley (Indianapolis, IN). They were housed in the animal facilities of the University of Pittsburgh Medical Center, with free access to food and water. Animals were treated humanely by use of the protocols that were approved by the Institutional Animal Use and Care Committee at the University of Pittsburgh. Unilateral ureteral obstruction (UUO) was performed using an established procedure (22). Groups of mice (n = 4) were killed at days 1, 3, 7, and 14 after UUO, respectively. One group of sham-operated mice (n = 4) was killed at day 7 after surgery. One part of the kidneys was fixed in 10% phosphate-buffered formalin, followed by paraffin embedding for immunohistochemical studies. The remaining kidneys were snap-frozen in liquid nitrogen and stored at –80°C for RNA and protein extractions.

RNA Isolation and Northern Blot Analysis
Total RNA was extracted from the kidney tissue using an Ultraspec RNA isolation system according to the instructions specified by the manufacturer (Biotecx Laboratories, Houston, TX). Northern blot analysis for SnoN expression was carried out by the procedures described previously (21,23). SnoN cDNA probe was provided by R. Weinberg (MIT, Cambridge, MA). After autoradiography, membranes were stripped and rehybridized with rat glyceraldehyde-3-phosphate dehydrogenase probe to ensure equal loading of each lane.

Reverse Transcriptase–PCR
Reverse transcriptase–PCR was performed as described elsewhere (24). Briefly, the first-strand cDNA synthesis was carried out by using a Reverse Transcription System kit according to the instructions of the manufacturer (Promega, Madison, WI). PCR amplification was performed on HotStar TaqMaster Mix Kit (Qiagen, Valencia, CA). The primer sequences were as follows: SnoN 5'-GATACACCATCGGGAATGGA-3' (sense) and 5'-AGATGTGAGCGACACATTCG-3' (antisense); {alpha}-smooth muscle actin ({alpha}-SMA) 5'-CCCAGCCAAGCACTGTCA-3' (sense) and 5'-TCCAGAGTCCAGCACGATG-3' (antisense); fibronectin 5'-CCACCCCCATAAGGCATAGG-3' (sense) and 5'-GTAGGGGTCAAAGCACGAGTCATC-3' (antisense); and beta-actin 5'-TCAAGATCATTGCTCCTCCTGAGC-3' (sense) and 5'-TGCTGTCACCTTCACCGTTCCAGT-3' (antisense). Expression levels of mRNA were calculated after normalizing with housekeeping gene beta-actin.

Endogenous SnoN Degradation Assay
The endogenous SnoN degradation was assessed by an in vitro assay in tissue homogenates that were prepared from sham and UUO kidneys at 7 d after surgery, according to the method described previously (25,26). Briefly, 100 µg of the kidney homogenates from a pool of four mice per group were incubated in 100 µl of ubiquitination mixture (50 mM Tris-HCl [pH 8.3], 5 mM MgCl2, 2 mM dithiothreitol, 5 mM ATP, and 2 mg/ml ubiquitin) for 0, 4, 8, and 16 h at 37°C, respectively. After incubation, the mixture were heated at 100°C for 5 min in SDS sample buffer, followed by immunoblotting with anti-SnoN antibody. The degradation assay also was performed in the presence of 1x proteasome inhibitor mix (0.25 mM MG132 and 0.25 mM MG115; EMD Biosciences, San Diego, CA).

In Vitro SnoN Ubiquitination Assay
SnoN ubiquitination activity in kidney tissues was detected by an in vitro assay using exogenous hemagglutinin (HA)-tagged SnoN protein as substrates, as described elsewhere (25,26). Human kidney proximal tubular epithelial cells (HKC) were transfected with HA-tagged SnoN expression vector (27). HA-tagged SnoN was immunoprecipitated by anti-HA antibody and used for in vitro ubiquitination assay. Kidney tissues from sham and UUO mice at 3, 7, and 14 d after surgery (pool from four mice per group) were lysed in Triton X-100 lysis buffer and centrifuged at 30,000 x g overnight at 4°C. The supernatants (100 µg) were incubated with 6 µl of immunopurified HA-tagged SnoN protein and 100 µl of ubiquitination mixture (50 mM Tris-HCl [pH 8.3], 5 mM MgCl2, 2 mM dithiothreitol, 5 mM ATP, 2 mg/ml ubiquitin, 50 µg/ml ubiquitin aldehyde, 10 mM phosphocreatinine, 0.28 units/ml phosphocreatinine kinase, 1x protease inhibitor mix, 1x proteasome inhibitor mix, 100 nM E1, and 1 µM UbcH5b; BostonBiochem, Cambridge, MA) for 1 h at 37°C. After incubation, the reactions were boiled for 5 min in SDS sample buffer, followed by immunoblotting with anti-ubiquitin antibody.

Western Blot Analysis
Detection of protein expression by Western blot was carried out according to the established protocols described previously (22). The primary antibodies used were as follows: Anti-SnoN (sc-9595), anti–c-Ski (ss-9140), anti–Smad ubiquitination regulatory factor-2 (Smurf2; sc-25511), anti-ubiquitin (sc-8017), and anti-actin (sc-1616; Santa Cruz Biotechnology, Santa Cruz, CA); anti–{alpha}-SMA (clone 1A4) and anti–{alpha}-tubulin (T-9026; Sigma, St. Louis, MO); anti–glyceraldehyde-3-phosphate dehydrogenase (Ambion, Austin, TX); and anti-fibronectin (clone 10; BD Transduction Laboratories, Lexington, KY). Quantification was performed by measurement of the intensity of the signals with the use of National Institutes of Health Image analysis software.

Immunohistochemical Staining
Immunohistochemical staining was performed using established procedures as described previously (3). Briefly, paraffin-embedded kidney sections from the UUO and sham-operated mice were prepared by a routine procedure and stained with the specific primary antibody against Smurf2. As a negative control, the primary antibody was replaced with nonimmune normal goat IgG, and no staining occurred.

Cell Culture, Treatment, and Transient Transfection
HKC were cultured in DMEM and Ham’s F-12 medium (1:1) supplemented with 5% FBS (Invitrogen, Carlsbad, CA), as described previously (22). Cells typically were seeded at approximately 70% confluence in complete medium that contained 5% FBS for 24 h, and then serum-starved for 16 h. For SnoN degradation assay, HKC were transiently transfected with pHA-SnoN expression vector (27) and then treated with cycloheximide (Sigma) at the concentrations of 10 µg/ml in the absence or presence of recombinant human TGF-beta1 (2 ng/ml; R&D Systems, Minneapolis, MN) for various periods of time as indicated. In some experiments, cells were pretreated with MG132 at the concentrations as indicated for 3 h, followed by various treatments. Transient transfection of HKC was carried out by using Lipofectamine 2000 according to the instructions specified by the manufacturer (Invitrogen). The empty pcDNA3 vector was used as a negative control.

For siRNA inhibition studies, HKC were transfected with control siRNA (sc-37007) or Smurf2 siRNA (sc-41675; Santa Cruz Biotechnology) at the final concentration of 120 nM by using oligofectamine reagent according to the instructions specified by the manufacturer (Invitrogen). Whole-cell lysates were collected for assessment of the expression of Smurf2 and SnoN by Western blot analyses.

Immunoprecipitation
Immunoprecipitation experiments were performed using similar methods as described previously (21). Briefly, cell or kidney tissue lysates were centrifuged at 12,000 x g for 10 min at 4°C. The resulting supernatants were collected for immunoprecipitation. After preclearing with normal host IgG, the lysates were immunoprecipitated overnight at 4°C with 2 µg of various primary antibodies, respectively, followed by precipitation with 30 µl of protein A/G Plus-Agarose (Santa Cruz Biotechnology) for 3 h at 4°C. The primary antibodies used were as follows: Anti-SnoN, anti-Smurf2, and anti-Smad2/3 (Santa Cruz Biotechnology). The precipitated complexes were boiled for 5 min in SDS sample buffer, followed by immunoblotting with various antibodies as indicated.

Reporter Constructs and Luciferase Assay
The reporter construct p3TP-Lux was provided by Dr. J. Massague (Memorial Sloan-Kettering Cancer Center, New York, NY). HKC were co-transfected by using Lipofectamine 2000 reagent with p3TP-Lux (0.5 µg), control siRNA, or Smurf2 siRNA (100 nM). A fixed amount (0.05 µg) of internal control reporter Renilla reniformis luciferase driven under thymidine kinase (TK) promoter (pRL-TK; Promega) also was co-transfected for normalizing the transfection efficiency. The transfected cells were incubated in serum-free medium for 16 h and then treated without or with TGF-beta1 (2 ng/ml) for an additional 48 h. Luciferase assay was performed using the Dual Luciferase Assay System kit according to the manufacturer’s protocols (Promega). Relative luciferase activity (arbitrary unit) was reported as fold induction over the controls after normalization for transfection efficiency.

Immunofluorescence Staining
Indirect immunofluorescence staining was performed using an established procedure (22). Cells were incubated with the anti-fibronectin antibody, followed by staining with FITC-conjugated secondary antibody. Cells also were stained with 4',6-diamidino-2-phenylindole, HCl to visualize the nuclei. Slides were viewed with a Nikon Eclipse E600 Epi-fluorescence microscope equipped with a digital camera (Melville, NY). In each experimental setting, immunofluorescence images were captured with identical light exposure times.

Statistical Analyses
Statistical analysis was performed using SigmaStat software (Jandel Scientific Software, San Rafael, CA). Comparisons between groups were made using one-way ANOVA, followed by the t test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SnoN Is Downregulated in Obstructive Nephropathy by a Mechanism Independent of Gene Transcription and mRNA Stability
Figure 1A shows that SnoN was markedly downregulated in the obstructed kidney at 7 d after UUO, consistent with a previous report (20). To explore the potential mechanism by which mediates SnoN reduction, we examined the steady-state level of SnoN mRNA in the obstructed kidney at various time points after UUO. Surprisingly, SnoN mRNA abundance remained relatively constant throughout the entire experimental period, ranging from day 1 to 14 after UUO, as shown by Northern blot analysis (Figure 1B). For further confirmation of this observation, a semiquantitative reverse transcriptase–PCR approach was used to assess SnoN mRNA level. As shown in Figure 1, C and D, there was no significant alteration in the abundance of SnoN mRNA in the sham and obstructed kidneys at various time points after UUO. Therefore, it seems that the downregulation of SnoN expression in the fibrotic kidney is not caused primarily by an alteration in its gene transcription or mRNA stability; rather, it probably results from an enhanced protein degradation.


Figure 1
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  		Figure 1. SnoN protein but not its mRNA is downregulated in the obstructed kidney after unilateral ureteral obstruction (UUO). (A) Western blot analysis demonstrates the downregulation of SnoN protein in obstructive nephropathy. Whole-tissue homogenates were prepared from the sham and obstructed kidneys at day 7 after UUO, respectively. Numbers 1, 2, 3, and 4 indicate each individual mouse in a given group. (B) Northern blot analysis shows the SnoN mRNA levels in the obstructed kidney at various times after UUO. Total RNA were isolated from different groups as indicated and hybridized with cDNA probes of SnoN and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. Representative Northern blot shows the results from two mice per group. (C and D) Semiquantitative reverse transcriptase–PCR (RT-PCR) analyses display little change in SnoN mRNA levels in the obstructed kidneys after UUO. (C) Representative RT-PCR result. (D) Graphic representation of the relative abundance of SnoN mRNA levels in different groups as indicated after normalization with beta-actin. Data are presented as mean ± SEM of four mice per group. No statistical difference in SnoN mRNA levels was found among the groups.

 
Increased Ubiquitination and Degradation of SnoN in the Obstructed Kidney
We then investigated the degradation of endogenous SnoN protein in the obstructed kidney after UUO. By using an in vitro degradation assay, we found an increased degradation of SnoN protein in the obstructed kidney, compared with the sham controls (Figure 2A). SnoN protein in the homogenates that were prepared from the obstructed kidneys at 7 d after UUO degraded rapidly. After 16 h of incubation, >80% of SnoN protein in the homogenates vanished (Figure 2B). Under the same conditions, only 20% of SnoN protein in the homogenates that were prepared from the sham kidney disappeared (Figure 2). Moreover, treatment with proteasome inhibitors largely prevented the degradation of SnoN in the obstructed kidney (Figure 2C). These observations suggest an increased proteasome-mediated degradation of SnoN protein in the obstructed kidney.


Figure 2
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  		Figure 2. An enhanced ubiquitination and degradation of SnoN protein in the obstructed kidney. (A) Western blot analysis shows an enhanced degradation of SnoN protein in obstructive nephropathy. Whole-tissue homogenates were prepared from the sham and obstructed kidneys at 7 d after UUO. A pool of the homogenates from four mice per group was incubated with ubiquitination reaction mixture for various periods of time as indicated, followed by immunoblotting with antibodies against SnoN and GAPDH, respectively. A longer exposure time was used to detect SnoN protein in the samples from the obstructed kidney (right). (B) Quantitative determination of SnoN protein levels at various time points in different groups as indicated. Relative SnoN abundances are presented after comparison with the controls (100) in each group. (C) Blockade of SnoN protein degradation by the proteasome inhibitors. Whole-tissue homogenates were incubated with ubiquitination reaction mixture in the absence or presence of the proteasome inhibitors (0.25 mM MG132 and 0.25 mM MG115) at 37°C for 8 h. The reaction mixtures were immunoblotted with antibodies against SnoN and GAPDH, respectively. (D and E) Ubiquitination activity for exogenous SnoN is enhanced in the obstructed kidney. Exogenous SnoN protein was prepared by transfection of human kidney tubular epithelial cells (HKC) with hemagglutinin (HA)-tagged SnoN expression plasmid. (D) The immunoprecipitated HA-SnoN protein was incubated at 37°C for 1 h with kidney homogenates from different groups as indicated. The reaction mixtures were immunoblotted with anti-ubiquitin antibody. (E) Quantitative determination of the poly-ubiquitinated SnoN abundance shown in D.

 
To provide further evidence for the potential involvement of the ubiquitin-proteasome pathway in SnoN degradation, we examined the ubiquitination activity for exogenous SnoN protein in tissue homogenates from the sham and obstructed kidneys. To this end, HA-tagged exogenous SnoN protein was prepared by transient transfection of tubular epithelial HKC with pHA-SnoN expression vector, followed by immunoprecipitation with anti-HA antibody. The immunoprecipitated HA-SnoN protein was incubated with the kidney homogenates in an in vitro ubiquitination assay. As shown in Figure 2D, anti-ubiquitin antibody detected significant smeared bands of the poly-ubiquitinated SnoN protein in the reactions using the homogenates from the obstructed kidney (Figure 2D, lanes 2 through 4). However, much less smeared bands was observed in the samples from sham controls (Figure 2D, lane 1). Quantitative determination revealed a more than eight-fold induction of the polyubiquitinated SnoN abundance when the homogenates from the obstructed kidney at 7 d after UUO were used, compared with the sham controls (Figure 2E). These data suggest that ubiquitination activity for SnoN protein is enhanced in the obstructed kidney.

Smurf2 Is Induced and Physically Interacts with SnoN In Vivo
We found that Smurf2, an E3 ubiquitin ligase, was significantly induced in the obstructed kidney in a time-dependent manner (Figure 3A). Western blot analysis showed that Smurf2 induction took place in the obstructed kidneys at day 3, peaked at day 7, and was sustained at least to day 14 after UUO. Quantitative determination on renal Smurf2 abundance at various time points after UUO are presented in Figure 3B. Figure 3, C and D, exhibited the localization of Smurf2 protein in sham and obstructed kidneys at 7 d after UUO, respectively. Smurf2 staining was weak in normal kidney (Figure 3C). However, the staining for Smurf2 protein was intensified in the obstructed kidney at 7 d after UUO. Smurf2 was localized predominantly in the nuclei of renal tubular epithelial cells in the obstructed kidney. This pattern of cellular localization for Smurf2 is in harmony with the nuclear staining of SnoN (20). Of note, Smurf1 level was low in the kidney, and it did not change significantly after UUO (data not shown).


Figure 3
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  		Figure 3. Smad ubiquitination regulatory factors 2 (Smurf2) is induced in the obstructed kidney. (A) Western blot analysis shows the induction of Smurf2 expression in the obstructed kidneys after UUO in a time-dependent manner. Whole-tissue homogenates from various groups as indicated were probed with antibodies against Smurf2 and GAPDH, respectively. Numbers 1, 2, 3, and 4 indicate each individual mouse in a given group. (B) Graphic representation of the relative abundance (fold induction over sham controls) of Smurf2 in different groups as indicated after normalization with GAPDH. Data are presented as mean ± SEM of four mice per group. *P < 0.001 versus sham controls. (C and D) Representative micrographs show the expression and localization of Smurf2 in the sham (C) and obstructed (D) kidneys. Smurf2 was localized mainly in the nuclei of renal tubular epithelial cells in the obstructed kidney at 7 d after UUO. Bar = 20 µm. (E) Smurf2 physically interacts with SnoN in the obstructed kidney. Whole-tissue homogenates were immunoprecipitated with specific antibody against Smurf2, followed by immunoblotting with anti-SnoN and anti-Smurf2 antibodies, respectively.

 
We next investigated the potential interaction between Smurf2 and SnoN in the obstructed kidney. Physical association between Smurf2 and SnoN clearly was evident, as SnoN was readily detectable in the immunocomplexes that were precipitated by anti-Smurf2 antibody (Figure 3E). Whereas Smurf2 was increased in the obstructed kidney in a time-dependent manner, SnoN protein precipitated by Smurf2 remained relatively unchanged (Figure 3E). This suggests that SnoN protein perhaps is unstable and subjected to degradation upon binding to Smurf2.

TGF-beta1 Promotes SnoN Degradation In Vitro
To investigate further the mechanism underlying SnoN degradation, we used renal tubular epithelial cells as an in vitro system. As demonstrated in Figure 4, in the absence of new protein synthesis after treatment with cycloheximide, SnoN abundance gradually reduced in a time-dependent manner. However, TGF-beta1 markedly and rapidly promoted SnoN protein degradation in HKC. At 1 h after TGF-beta1 treatment, SnoN level was reduced by >90%, compared with the controls. Under the identical conditions, approximately 60% of SnoN protein still was intact when the cells were incubated in the absence of TGF-beta1.


Figure 4
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  		Figure 4. TGF-beta1 promotes SnoN protein degradation in renal tubular epithelial cells in vitro. HKC were incubated with cycloheximide (CHX; 10 µg/ml) in the absence or presence of TGF-beta1 (2 ng/ml) for various periods of time as indicated. (A) Whole-cell lysates were immunoblotted with anti-SnoN and anti-actin antibodies, respectively. (B) Graphic representation of the relative abundance of SnoN protein shown in A. SnoN abundance was expressed as the percentage of the value in controls (100).

 
To explore whether TGF-beta1 promotes SnoN degradation via a ubiquitination- and proteasome-dependent pathway, we pretreated the tubular epithelial cells with proteasome inhibitor. As shown in Figure 5A, proteasome inhibitor (MG132) was able to abolish the TGF-beta1–mediated SnoN downregulation. Furthermore, TGF-beta1 clearly induced the ubiquitination of SnoN protein. When the immunocomplexes that were precipitated with anti-SnoN antibody were probed with anti-ubiquitin, smeared bands of the poly-ubiquitinated SnoN were markedly increased in the presence of TGF-beta1 (Figure 5B). These results suggest that TGF-beta1 may be responsible for triggering or promoting SnoN degradation in kidney epithelial cells.


Figure 5
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  		Figure 5. TGF-beta1 promotes SnoN protein degradation by a ubiquitination- and proteasome-dependent pathway. (A) Western blot analysis shows that proteasome inhibitor (MG132) abolished the TGF-beta1–mediated SnoN degradation. HKC were transiently transfected with pHA-SnoN expression vector to increase SnoN abundance. The cells then were preincubated without or with 15 µM MG132 for 3 h, followed by treatment with CHX (10 µg/ml) and TGF-beta1 (2 ng/ml) for various periods of time as indicated. (B) TGF-beta1 increases the ubiquitination of SnoN protein. HKC were preincubated without or with MG132 for 3 h, followed by treatment with TGF-beta1 (2 ng/ml) for various periods of time as indicated. Whole-cell lysates were immunoprecipitated with anti-SnoN antibody, followed by immunoblotting with anti-ubiquitin.

 
SnoN Forms Complex with Ski, but Its Degradation Is Independent of Ski Abundance
Because another Smad transcriptional co-repressor, Ski, also is downregulated in the fibrotic kidney (20), we sought to determine whether Ski interacts with SnoN in tubular epithelial cells and, if so, whether Ski abundance affects the stability of SnoN. Figure 6 shows that SnoN constitutively formed a complex with Ski at basal conditions, as Ski was detectable in the immunocomplexes that were precipitated by anti-SnoN antibody (Figure 6A, lane 7). Furthermore, the SnoN/Ski formed a ternary complex with activated Smad2/3 upon TGF-beta1 stimulation (Figure 6A, lane 8). Either SnoN or Ski alone also could form complex with activated Smad2/3 (Figure 6A, lanes 4 and 6).


Figure 6
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  		Figure 6. SnoN forms ternary complex with Ski and Smad2/3, and ectopic expression of Ski fails to stabilize SnoN. (A) Complex formation among SnoN, Ski, and Smad2/3 in HKC. HKC were transiently transfected with empty vector pcDNA3, pHA-Ski, pHA-SnoN, or pHA-Ski plus pHA-SnoN expression plasmids for 24 h, followed by incubation with or without TGF-beta1 (2 ng/ml) for 1 h. Whole-cell lysates were immunoprecipitated with anti-SnoN or anti-Smad2/3 antibodies, respectively. The immune complexes were probed with specific antibodies against Ski or HA, respectively. (B) Ectopic expression of Ski fails to stabilize SnoN. Whole-cell lysates from the various groups were immunoblotted with anti-SnoN or anti-GAPDH antibodies, respectively. (C and D) Ectopic expression of Ski does not affect the degradation rate of SnoN in HKC. HKC were transiently transfected with empty vector pcDNA3 or pHA-Ski expression plasmids, followed by incubation with CHX (10 µg/ml) and TGF-beta1 (2 ng/ml) for various periods of time as indicated. (C) Western blot analysis shows the expression of SnoN, Ski, and actin, respectively. (D) Densitometric analysis shows no significant difference in SnoN protein degradation after overexpression of Ski.

 
Ectopic expression of Ski was found to have little effect on the stability of SnoN protein (Figure 6B). TGF-beta1 was able to induce SnoN downregulation, regardless of the abundance of cellular SnoN protein (Figure 6B, lane 2 versus 4 and lane 6 versus 8). The degradation rate of SnoN essentially was identical in the HKC that were transfected with either Ski expression vector (pHA-Ski) or empty vector (pcDNA3; Figure 6, C and D). Therefore, loss of Ski is not the cause of decreased SnoN.

SnoN Degradation Is Required for TGF-beta1 to Exert Its Fibrogenic Action
To understand the impact of SnoN degradation on TGF-beta1 function, we examined the effects of blockade of SnoN degradation on TGF-beta1–mediated fibrogenic response in renal epithelial cells. As shown in Figure 7, A and B, treatment with proteasome inhibitor MG132 completely abolished the {alpha}-SMA and fibronectin mRNA expression that was induced by TGF-beta1 in HKC. Likewise, MG132 abrogated the TGF-beta1–mediated fibronectin protein expression and its extracellular assembly in tubular epithelial cells (Figure 7, C through H).


Figure 7
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  		Figure 7. Inhibition of SnoN degradation by proteasome inhibitor abolishes TGF-beta1–mediated {alpha}-smooth muscle actin ({alpha}-SMA) and fibronectin expression in renal epithelial cells. HKC were pretreated with or without 10 µM MG132 for 1 h and then treated with TGF-beta1 (2 ng/ml) for 24 h. (A and B) RT-PCR demonstrates the relative mRNA abundance after various treatments as indicated. (A) Representative RT-PCR results. (B) Quantitative determination of relative mRNA levels (fold induction over controls). Data are presented as mean ± SEM of three experiments. *P < 0.01 versus normal control; {dagger}P < 0.01 versus TGF-beta1 group; **P < 0.001 versus normal control; {dagger}{dagger}P < 0.001 versus TGF-beta1 group. (C) Whole-cell lysates were immunoblotted with antibodies against fibronectin and {alpha}-tubulin, respectively. (D) Graphic representation of the relative abundance of fibronectin after normalization with {alpha}-tubulin. Data are presented as mean ± SEM of three experiments. *P < 0.05 versus normal control (lane 1); {dagger}P < 0.05 versus TGF-beta1 group (lane 2). (E through H) Immunofluorescence staining shows the expression and deposition of fibronectin after various treatments: Control (E), TGF-beta1 alone (F), MG132 alone (G), and TGF-beta1 plus MG132 (H). Bar = 10 µm.

 
To establish further the relevance of Smurf2 and SnoN regulation to TGF-beta1 function, we knocked down Smurf2 expression in HKC by an siRNA strategy. As shown in Figure 8, A and B, knockdown of Smurf2 expression increased SnoN protein abundance in tubular epithelial cells. It is interesting that downregulation of Smurf2 significantly suppressed the TGF-beta1–mediated gene transcription in a functional promoter (p3TP-Lux) reporter assay (Figure 8C). Similarly, knockdown of Smurf2 abolished the fibronectin induction in response to TGF-beta1 stimulation in HKC (Figure 8, D and E). As reported previously (20), knockdown of SnoN enhanced the TGF-beta1–mediated gene transcription and fibronectin induction. Together, it seems that SnoN degradation could be necessary for TGF-beta1 to exert its fibrogenic action.


Figure 8
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  		Figure 8. Knockdown of Smurf2 expression inhibits TGF-beta1–mediated gene transcription and fibronectin expression in tubular epithelial cells. (A and B) Knockdown of Smurf2 expression stabilizes the expression of SnoN. HKC were transiently transfected with control small interfering RNA (siRNA) or Smurf2 siRNA (120 nM). (A) Representative Western blots showed the expression levels of Smurf2, SnoN, and {alpha}-tubulin at 72 h after transfection. (B) Graphic representation of the relative abundance of Smurf2 and SnoN protein levels after normalization with {alpha}-tubulin. Data relative to the controls (1.0) are presented as mean ± SEM of three experiments. *P < 0.01, **P < 0.001 versus control siRNA group. (C) Knockdown of Smurf2 expression inhibits the TGF-beta1–mediated gene transcription. HKC were transiently co-transfected with p3TP-Lux luciferase reporter construct and control siRNA or Smurf2 siRNA, followed by incubation without or with 2 ng/ml TGF-beta1 for 48 h. Relative luciferase activities (arbitrary unit) were calculated after normalization of transfection efficiency and are presented as mean ± SEM of six experiments. **P < 0.001 versus control siRNA group in the absence of TGF-beta1; {dagger}{dagger}P = 0.001 versus control siRNA group in the presence of TGF-beta1. (D) Inhibition of Smurf2 expression blocks TGF-beta1–mediated fibronectin expression in tubular epithelial cells. HKC were transiently transfected 120 nM control siRNA or Smurf2 siRNA for 24 h and then treated with TGF-beta1 for an additional 48 h. Whole-cell lysates were immunoblotted with antibodies against fibronectin and GAPDH, respectively. (E) Immunofluorescence staining shows the fibronectin expression and deposition after various treatments as indicated. Bar = 10 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Hyperactive TGF-beta/Smad signaling is widely considered as a major pathogenic mediator in the genesis and progression of chronic renal fibrosis after various injuries. Whereas extensive studies are focused on the induction and activation of the positive components of this signal pathway in the diseased kidney, we recently demonstrated a progressive downregulation of Smad transcriptional co-repressors in the fibrotic kidney after UUO. This suggests that the loss of the negative controlling mechanism may be equally important in amplifying the fibrogenic Smad signaling. This study was carried out to address specifically the mechanism underlying SnoN suppression under pathologic conditions. Our results indicate that the downregulation of SnoN is mediated primarily by an enhanced ubiquitination/proteasome-dependent degradation, rather than by a reduced gene transcription. Functionally, SnoN degradation may be a prerequisite for TGF-beta to exert its fibrogenic action, because preservation of SnoN by proteasome inhibitor blocks TGF-beta1–mediated {alpha}-SMA and fibronectin expression. Furthermore, stabilization of SnoN by knockdown of Smurf2 leads to an inhibition of the TGF-beta1–mediated gene transcription. Therefore, these findings establish that dysregulation of ubiquitination and proteasomal degradation pathway may be critical in rendering an uncontrolled TGF-beta/Smad signaling, thereby playing an imperative role in the pathogenesis of chronic renal fibrosis.

SnoN protein is expressed robustly in normal kidney, which makes the Smad signaling tightly constrained under physiologic conditions. Evidence suggests that SnoN can effectively block TGF-beta/Smad signaling through diverse mechanisms (28). SnoN has been shown to interact directly with Smad proteins in the nuclei and block the ability of the Smads to trans-activate gene expression (28,29). Recent studies indicated that SnoN also sequesters Smads in the cytoplasm (30). Furthermore, SnoN may play a role in recruiting other transcriptional co-repressors, such as the nuclear hormone receptor co-repressor and mammalian Sin3 ortholog, and prevents Smads from binding to the transcriptional co-activator p300/CBP (28). In this context, downregulation of SnoN protein in the fibrotic kidney could sensitize renal cells to TGF-beta stimulation and amplifies the fibrogenic signal (20). In this study, we demonstrated that SnoN reduction in the obstructed kidney is mediated primarily through an enhanced ubiquitination and subsequent proteasomal degradation pathway. This conclusion is supported by several lines of evidence. First, SnoN reduction is not caused primarily by a decrease in its mRNA expression, suggesting that it likely is independent of gene transcription and mRNA stability. Second, an increased ubiquitin-proteasomal degradation activity against SnoN is found in the obstructed kidney. Third, Smurf2, a specific E3 ubiquitin ligase that is implicated in the degradation of Smad signaling components (31,32), is induced in the obstructed kidney. Finally, TGF-beta1, a fibrogenic cytokine that is markedly upregulated in the fibrotic kidney (1,2), triggers and/or promotes SnoN degradation in tubular epithelial cells, and SnoN degradation is a prerequisite for TGF-beta1–mediated fibrogenic action. Nevertheless, at this stage, we cannot completely rule out the possibility that a decreased SnoN protein translation also may play a role in mediating the SnoN protein reduction in the obstructed kidney.

Protein degradation is a tightly regulated event that is essential for controlling many cellular processes. Among several proteolytic systems documented, the ubiquitin-proteasome pathway is a complex one that requires energy and consists of a highly organized cascade of enzymatic reactions that select, tag, and execute the orderly destruction of a wide variety of physiologically important proteins (33–35). Not surprising, defects in the ubiquitin-proteasomal proteolytic pathway that render protein substrates more or less susceptible to degradation are linked to many devastating disorders, ranging from cancer to neurodegenerative diseases and kidney failure (33–35). It has been shown that components of the Smad signaling pathway including SnoN are subjected to ubiquitin-proteasome–mediated degradation under both physiologic and pathologic circumstances (18,32,36,37). The finding that an increased ubiquitination and proteasomal degradation is directed against SnoN in the obstructed kidney suggests that SnoN is targeted specifically for ubiquitin-dependent destruction in vivo in the fibrotic state. These observations, together with previous reports on Smad2 and Smad7 degradation (25,26), suggest a general theme that alteration in the ubiquitin-proteasomal degradation of key signaling regulators may result in disturbance of vital cellular signaling, thereby leading to kidney cell malfunction and contributing to the pathogenesis of CKD.

Covalent attachment of ubiquitin to target proteins that are destined for degradation is initiated by several sequential steps that are controlled by the activity of E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and a specific E3 ubiquitin ligase. It is commonly believed that the E3 ubiquitin ligase plays a decisive role in defining the substrate selectivity and the subsequent degradation by the 26S proteasomes (33,34). In this regard, the observation that Smurf2, an E3 ubiquitin ligase, is induced in the obstructed kidney provides significant insights into the mechanism of SnoN degradation. Smurf2 belongs to a family of E3 ligases that contain the so-called HECT domain that is homologous to the E6-AP COOH terminus (31,33). Unlike its functional cousin, Smurf1, that primarily targets bone morphogenetic protein signaling such as Smad1, Smad5, and Smad8, Smurf2 selectively targets Smad2 but not Smad3 for degradation (25,31,36,38). Smurf2 also interacts with Smad7 in the nucleus and induces the nuclear export of Smad7 (32,37), which in turn targets TGF-beta type I receptor and enhances its degradation. In this study, we have shown that Smurf2 is localized in the nucleus and physically associate with SnoN, strongly suggesting that Smurf2 is a ubiquitin E3 ligase that targets nuclear SnoN for proteasome-dependent degradation. In accordance with this, the upregulation of Smurf2 in the nucleus of kidney cells is closely correlated with reduction of SnoN after UUO (20). However, it remains to be determined whether other E3 ubiquitin ligases, such as the anaphase-promoting complex (39,40), also may be implicated in the ubiquitination and subsequent proteasome-dependent degradation of SnoN in the obstructed kidney.

Studies from an in vitro model system suggest that TGF-beta1 can trigger and/or promote SnoN degradation in tubular epithelial cells (Figures 4 and 5). This is consistent with earlier reports indicating that activated Smad3 plays a crucial role in SnoN degradation in other types of cells (28,39). TGF-beta1 also is shown to induce Smurf2 gene expression (41). Because TGF-beta1 is induced markedly and progressively in virtually every type of CKD (1,2,8), it is plausible that TGF-beta1 also plays a critical role in SnoN degradation in vivo. This raises an interesting possibility that TGF-beta1 may modulate Smad signaling via dual mechanisms, with activating positive Smad signaling on the one hand and targeting Smad antagonist SnoN for ubiquitin-dependent degradation on the other hand.

The results presented herein may suggest new strategies in designing future therapeutics for CKD, in which hyperactive Smad signaling is a causative factor for the evolution of tissue scarring. It seems conceivable that preservation of SnoN by inhibiting its degradation may be a novel approach for targeting hyperactive Smad signaling in renal fibrosis. This view evidently is supported by the observation that blockade of SnoN degradation either by proteasome inhibitor or by knockdown of Smurf2 effectively prevents TGF-beta1–mediated fibrogenic response. Further studies clearly are warranted to evaluate the applicability and the efficacy of this strategy in experimental animal models for the prevention and treatment of chronic renal insufficiency.


   Acknowledgments
 
This work was supported by National Institutes of Health grants DK054922, DK061408, DK064005, and DK071040.


   Footnotes
 
Published online ahead of print. Publication date available at www.jasn.org.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Border WA, Noble NA: TGF-beta in kidney fibrosis: A target for gene therapy. Kidney Int 51 : 1388 –1396, 1997[Medline]
  2. Bottinger EP, Bitzer M: TGF-beta signaling in renal disease. J Am Soc Nephrol 13 : 2600 –2610, 2002[Abstract/Free Full Text]
  3. Schnaper HW, Hayashida T, Hubchak SC, Poncelet AC: TGF-beta signal transduction and mesangial cell fibrogenesis. Am J Physiol Renal Physiol 284 : F243 –F252, 2003[Abstract/Free Full Text]
  4. Eddy AA: Molecular basis of renal fibrosis. Pediatr Nephrol 15 : 290 –301, 2000[CrossRef][Medline]
  5. Liu Y: Renal fibrosis: New insights into the pathogenesis and therapeutics. Kidney Int 69 : 213 –217, 2006[CrossRef][Medline]
  6. Dai C, Liu Y: Hepatocyte growth factor antagonizes the profibrotic action of TGF-beta1 in mesangial cells by stabilizing Smad transcriptional corepressor TGIF. J Am Soc Nephrol 15 : 1402 –1412, 2004[Abstract/Free Full Text]
  7. Yang J, Dai C, Liu Y: Hepatocyte growth factor suppresses renal interstitial myofibroblast activation and intercepts Smad signal transduction. Am J Pathol 163 : 621 –632, 2003[Abstract/Free Full Text]
  8. Yang J, Liu Y: Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis. Am J Pathol 159 : 1465 –1475, 2001[Abstract/Free Full Text]
  9. Liu Y: Epithelial to mesenchymal transition in renal fibrogenesis: Pathologic significance, molecular mechanism, and therapeutic intervention. J Am Soc Nephrol 15 : 1 –12, 2004[Abstract/Free Full Text]
  10. Mozes MM, Bottinger EP, Jacot TA, Kopp JB: Renal expression of fibrotic matrix proteins and of transforming growth factor-beta (TGF-beta) isoforms in TGF-beta transgenic mice. J Am Soc Nephrol 10 : 271 –280, 1999[Abstract/Free Full Text]
  11. Ma LJ, Jha S, Ling H, Pozzi A, Ledbetter S, Fogo AB: Divergent effects of low versus high dose anti-TGF-beta antibody in puromycin aminonucleoside nephropathy in rats. Kidney Int 65 : 106 –115, 2004[CrossRef][Medline]
  12. Ziyadeh FN, Hoffman BB, Han DC, Iglesias-De La Cruz MC, Hong SW, Isono M, Chen S, McGowan TA, Sharma K: Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-beta antibody in db/db diabetic mice. Proc Natl Acad Sci U S A 97 : 8015 –8020, 2000[Abstract/Free Full Text]
  13. Isaka Y, Akagi Y, Ando Y, Tsujie M, Sudo T, Ohno N, Border WA, Noble NA, Kaneda Y, Hori M, Imai E: Gene therapy by transforming growth factor-beta receptor-IgG Fc chimera suppressed extracellular matrix accumulation in experimental glomerulonephritis. Kidney Int 55 : 465 –475, 1999[CrossRef][Medline]
  14. Isaka Y, Tsujie M, Ando Y, Nakamura H, Kaneda Y, Imai E, Hori M: Transforming growth factor-beta 1 antisense oligodeoxynucleotides block interstitial fibrosis in unilateral ureteral obstruction. Kidney Int 58 : 1885 –1892, 2000[CrossRef][Medline]
  15. Massague J: How cells read TGF-beta signals. Nat Rev Mol Cell Biol 1 : 169 –178, 2000[CrossRef][Medline]
  16. Prunier C, Pessah M, Ferrand N, Seo SR, Howe P, Atfi A: The oncoprotein Ski acts as an antagonist of transforming growth factor-beta signaling by suppressing Smad2 phosphorylation. J Biol Chem 278 : 26249 –26257, 2003[Abstract/Free Full Text]
  17. Wotton D, Massague J: Smad transcriptional corepressors in TGF beta family signaling. Curr Top Microbiol Immunol 254 : 145 –164, 2001[Medline]
  18. Stroschein SL, Wang W, Zhou S, Zhou Q, Luo K: Negative feedback regulation of TGF-beta signaling by the SnoN oncoprotein. Science 286 : 771 –774, 1999[Abstract/Free Full Text]
  19. Wotton D, Lo RS, Swaby LA, Massague J: Multiple modes of repression by the Smad transcriptional corepressor TGIF. J Biol Chem 274 : 37105 –37110, 1999[Abstract/Free Full Text]
  20. Yang J, Zhang X, Li Y, Liu Y: Downregulation of Smad transcriptional corepressors SnoN and Ski in the fibrotic kidney: an amplification mechanism for TGF-beta1 signaling. J Am Soc Nephrol 14 : 3167 –3177, 2003[Abstract/Free Full Text]
  21. Yang J, Dai C, Liu Y: A novel mechanism by which hepatocyte growth factor blocks tubular epithelial to mesenchymal transition. J Am Soc Nephrol 16 : 68 –78, 2005[Abstract/Free Full Text]
  22. Yang J, Liu Y: Blockage of tubular epithelial to myofibroblast transition by hepatocyte growth factor prevents renal interstitial fibrosis. J Am Soc Nephrol 13 : 96 –107, 2002[Abstract/Free Full Text]
  23. Yang J, Shultz RW, Mars WM, Wegner RE, Li Y, Dai C, Nejak K, Liu Y: Disruption of tissue-type plasminogen activator gene in mice reduces renal interstitial fibrosis in obstructive nephropathy. J Clin Invest 110 : 1525 –1538, 2002[CrossRef][Medline]
  24. Li Y, Spataro BC, Yang J, Dai C, Liu Y: 1,25-dihydroxyvitamin D inhibits renal interstitial myofibroblast activation by inducing hepatocyte growth factor expression. Kidney Int 68 : 1500 –1510, 2005[CrossRef][Medline]
  25. Togawa A, Yamamoto T, Suzuki H, Fukasawa H, Ohashi N, Fujigaki Y, Kitagawa K, Hattori T, Kitagawa M, Hishida A: Ubiquitin-dependent degradation of Smad2 is increased in the glomeruli of rats with anti-thymocyte serum nephritis. Am J Pathol 163 : 1645 –1652, 2003[Abstract/Free Full Text]
  26. Fukasawa H, Yamamoto T, Togawa A, Ohashi N, Fujigaki Y, Oda T, Uchida C, Kitagawa K, Hattori T, Suzuki S, Kitagawa M, Hishida A: Down-regulation of Smad7 expression by ubiquitin-dependent degradation contributes to renal fibrosis in obstructive nephropathy in mice. Proc Natl Acad Sci U S A 101 : 8687 –8692, 2004[Abstract/Free Full Text]
  27. Sun Y, Liu X, Ng-Eaton E, Lodish HF, Weinberg RA: SnoN and Ski protooncoproteins are rapidly degraded in response to transforming growth factor beta signaling. Proc Natl Acad Sci U S A 96 : 12442 –12447, 1999[Abstract/Free Full Text]
  28. Luo K: Ski and SnoN: Negative regulators of TGF-beta signaling. Curr Opin Genet Dev 14 : 65 –70, 2004[CrossRef][Medline]
  29. Liu X, Sun Y, Weinberg RA, Lodish HF: Ski/Sno and TGF-beta signaling. Cytokine Growth Factor Rev 12 : 1 –8, 2001[CrossRef][Medline]
  30. Krakowski AR, Laboureau J, Mauviel A, Bissell MJ, Luo K: Cytoplasmic SnoN in normal tissues and nonmalignant cells antagonizes TGF-beta signaling by sequestration of the Smad proteins. Proc Natl Acad Sci U S A 102 : 12437 –12442, 2005[Abstract/Free Full Text]
  31. Lin X, Liang M, Feng XH: Smurf2 is a ubiquitin E3 ligase mediating proteasome-dependent degradation of Smad2 in transforming growth factor-beta signaling. J Biol Chem 275 : 36818 –36822, 2000[Abstract/Free Full Text]
  32. Kavsak P, Rasmussen RK, Causing CG, Bonni S, Zhu H, Thomsen GH, Wrana JL: Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation. Mol Cell 6 : 1365 –1375, 2000[CrossRef][Medline]
  33. Debigare R, Price SR: Proteolysis, the ubiquitin-proteasome system, and renal diseases. Am J Physiol Renal Physiol 285 : F1 –F8, 2003[Abstract/Free Full Text]
  34. Mani A, Gelmann EP: The ubiquitin-proteasome pathway and its role in cancer. J Clin Oncol 23 : 4776 –4789, 2005[Abstract/Free Full Text]
  35. Gao M, Karin M: Regulating the regulators: Control of protein ubiquitination and ubiquitin-like modifications by extracellular stimuli. Mol Cell 19 : 581 –593, 2005[CrossRef][Medline]
  36. Zhang Y, Chang C, Gehling DJ, Hemmati-Brivanlou A, Derynck R: Regulation of Smad degradation and activity by Smurf2, an E3 ubiquitin ligase. Proc Natl Acad Sci U S A 98 : 974 –979, 2001[Abstract/Free Full Text]
  37. Asano Y, Ihn H, Yamane K, Kubo M, Tamaki K: Impaired Smad7-Smurf-mediated negative regulation of TGF-beta signaling in scleroderma fibroblasts. J Clin Invest 113 : 253 –264, 2004[CrossRef][Medline]
  38. Fukuchi M, Fukai Y, Masuda N, Miyazaki T, Nakajima M, Sohda M, Manda R, Tsukada K, Kato H, Kuwano H: High-level expression of the Smad ubiquitin ligase Smurf2 correlates with poor prognosis in patients with esophageal squamous cell carcinoma. Cancer Res 62 : 7162 –7165, 2002[Abstract/Free Full Text]
  39. Stroschein SL, Bonni S, Wrana JL, Luo K: Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN. Genes Dev 15 : 2822 –2836, 2001[Abstract/Free Full Text]
  40. Wan Y, Liu X, Kirschner MW: The anaphase-promoting complex mediates TGF-beta signaling by targeting SnoN for destruction. Mol Cell 8 : 1027 –1039, 2001[CrossRef][Medline]
  41. Ohashi N, Yamamoto T, Uchida C, Togawa A, Fukasawa H, Fujigaki Y, Suzuki S, Kitagawa K, Hattori T, Oda T, Hayashi H, Hishida A, Kitagawa M: Transcriptional induction of Smurf2 ubiquitin ligase by TGF-beta. FEBS Lett 579 : 2557 –2563, 2005[CrossRef][Medline]



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