JBC Anatrace, Inc.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M701991200 on March 30, 2007

J. Biol. Chem., Vol. 282, Issue 21, 15534-15540, May 25, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/21/15534    most recent
M701991200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Poncelet, A.-C.
Right arrow Articles by Runyan, C. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Poncelet, A.-C.
Right arrow Articles by Runyan, C. E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Cell Phenotype-specific Down-regulation of Smad3 Involves Decreased Gene Activation as Well as Protein Degradation*

Anne-Christine Poncelet{ddagger}§1, H. William Schnaper§, Ruoyun Tan, Youhua Liu, and Constance E. Runyan§

From the {ddagger}Department of Medicine, University of Washington School of Medicine, Seattle, Washington 98109, §Department of Pediatrics, Northwestern University, Feinberg School of Medicine, Chicago, Illinois 60611, and Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Received for publication, March 7, 2007 , and in revised form, March 27, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Signaling by transforming growth factor-beta (TGF-beta), a regulator of several biological processes, including renal fibrosis, is mediated, in part, by the Smad proteins. Tight control of Smad level and activity is critical for proper TGF-beta biological functions. Here, we have investigated the mechanisms involved in regulating Smad3 expression. In human glomerular mesangial cells, Smad3 protein levels were specifically reduced by 24 h of TGF-beta1 treatment, whereas Smad2 and Smad4 levels were not. TGF-beta1 increased endogenous Smad3 ubiquitination, and proteasome inhibitor treatment blocked TGF-beta1-mediated Smad3 down-regulation resulting in accumulation of ubiquitinated Smad3. These data support the concept that Smad3 down-regulation occurs via degradation by the ubiquitin/proteasome machinery. However, changes in Smad3 protein levels were also paralleled by changes in Smad3 mRNA expression. TGF-beta1 did not decrease Smad3 mRNA stability, but it significantly inhibited Smad3 promoter activity. In renal tubular epithelial cells, decreased Smad3 levels were observed only after exposure to TGF-beta1 for longer time periods (5–7 days) that paralleled epithelial-to-mesenchymal transition, as determined by increased expression of smooth muscle {alpha}-actin and decreased expression of E-cadherin. Decline in Smad3 expression also occurred in kidneys after unilateral ureteral obstruction, a model of tubulointerstitial fibrosis associated with TGF-beta up-regulation and epithelial-to-mesenchymal transition. Our data show for the first time that TGF-beta1 modulates the expression of a receptor-activated Smad at both the protein and transcriptional level. Smad3 down-regulation could represent a feedback loop controlling TGF-beta signaling in a cell phenotype-specific manner.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Glomerulosclerosis and tubulointerstitial fibrosis are the consequences of excessive extracellular matrix (ECM)2 accumulation and lead to end-stage renal failure (1). Transforming growth factor-beta (TGF-beta) is a pleiotropic cytokine that regulates a variety of physiological and pathological processes in a cell type-specific manner and plays a central role in the development of fibrosis. In mesenchymal cells, such as fibroblasts, activated hepatic stellate cells, and glomerular mesangial cells, TGF-beta leads to ECM accumulation (2–4). TGF-beta contributes to embryogenesis, fibrosis, and carcinogenesis, in part, by stimulating epithelial-to-mesenchymal transition (EMT) (5, 6). TGF-beta inhibits proliferation in non-transformed epithelial, hematopoietic, and endothelial cells. It also stimulates differentiation of leukocytes and apoptosis of cells such as podocytes, hepatocytes, and renal tubular and prostatic epithelial cells (3, 4, 7–9).

TGF-beta family receptors transduce signals to the nucleus through the Smads. These proteins are divided into receptor-regulated or R-Smads Smad1, -2, -3, -5, and -8; common-partner or co-Smad Smad4; and inhibitory Smads or I-Smads Smad6 and -7 (10–13). A critical role for Smad3 as a mediator of fibrosis has been established using in vitro and in vivo models examining various tissues and organs (14). Using TGF-beta1-treated human mesangial cells as our model for kidney fibrosis, we have shown that Smad3 is necessary for TGF-beta1-stimulated {alpha}2(I) collagen (COL1A2) promoter activity (15, 16). However, 24 h of TGF-beta1 treatment decreases Smad3 levels in these cells (15).

Recent studies have shown that several members of the Smad protein family can be degraded by the ubiquitin-proteasome pathway (17). However, only a few reports have examined Smad3 turnover. In addition, these studies were performed mainly with overexpressed proteins, with Smad3 ubiquitination observed in cells transfected with Smad3 and ubiquitin, in the presence of a proteasome inhibitor and/or overexpression of an E3 ubiquitin ligase (18–20).

Here, we have investigated the regulation of the expression of endogenous Smad3, a central participant in the mechanisms by which TGF-beta1 triggers events that contribute to fibrotic diseases. Our results indicate that both increased degradation and decreased promoter activity contribute to the TGF-beta1-mediated decrease in Smad3 expression.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Reagents were purchased from the following vendors. Active human recombinant TGF-beta1 was from R & D Systems (Minneapolis, MN); rabbit anti-phospho-Smad3 (Ser-433/435) and mouse anti-ubiquitin from Cell Signaling Technology (Beverly, MA); mouse anti-Smad4 (B-8), mouse anti-Smad1/2/3 (H-2), rabbit anti-Dab2, anti-mouse IgG-horseradish peroxidase, and anti-goat IgG-horseradish peroxidase from Santa Cruz Biotechnology (Santa Cruz, CA); anti-rabbit IgG-horseradish peroxidase, luciferase, and beta-galactosidase assay systems from Promega (Madison, WI); rabbit anti-Smad3, rabbit anti-Smad2, and protein G-Sepharose from Invitrogen and Zymed Laboratories Inc.; mouse anti-E-cadherin from BD Transduction Laboratories; actinomycin D, cycloheximide, proteasome inhibitor I, lactacystin, and Z-Leu-Leu-al (MG132) from Sigma. The Alk5/TbetaRI inhibitor SB-431542 (21) was a generous from gift Dr. N. Laping (GlaxoSmithKline). Stock solutions were made as follows: 4 µg/ml TGF-beta1 in 4 mM HCl containing 1 mg/ml bovine serum albumin; 10 mM proteasome inhibitor I, lactacystin, and MG132 in Me2SO; 1 mg/ml actinomycin D in Me2SO; 1 mg/ml cycloheximide in water.

Cell Culture—Human glomerular mesangial cells were isolated and grown in Dulbecco's modified Eagle's medium/F-12 with 20% newborn calf serum, penicillin/streptomycin, Hepes buffer, sodium pyruvate, 8 µg/ml insulin, and glutamine as described previously (15). The renal tubular epithelial cell line HKC, obtained from Dr. L. Racusen (22), was cultured in Dulbecco's modified Eagle's medium/F-12 supplemented with 10% fetal bovine serum, penicillin/streptomycin, Hepes buffer, and glutamine.

Animals—Male CD-1 mice that weighed ~18–20 g were obtained from Harlan Sprague-Dawley (Indianapolis, IN). Unilateral ureteral obstruction (UUO) was performed as described previously (23). Sham-operated mice had their ureters exposed and manipulated but not ligated. Mice were killed 3, 7, and 14 days after surgery. Kidneys were snap-frozen in liquid nitrogen and stored at -80 °C before protein extractions.

Immunoprecipitation and Western Blot Analysis—Cells were switched to medium containing 1% newborn calf serum (for mesangial cells) or 0.5% fetal bovine serum (for HKC) and treated with 1 ng/ml TGF-beta1 for various time periods, leading to simultaneous harvest. In some experiments, the cells were incubated with inhibitors for 1 h prior to TGF-beta1 treatment. The cells were lysed as described previously (16). Kidney tissues were homogenized, and proteins were extracted as described previously (23). For immunoprecipitation experiments, 500 µg of lysates were immunoprecipitated with 1.25 µg of anti-Smad3 antibody and 50 µl of protein G-Sepharose. Immunoprecipitates and whole cell lysates were analyzed by immunoblotting. Densitometric analysis was performed as described previously (24).

RNA Isolation and Northern Blot—Total RNA was harvested using TRIzol (Invitrogen) according to the manufacturer's protocol and analyzed by Northern blot with cDNA probes for human Smad3 (25) and bovine 28 S ribosomal RNA (provided by Dr. H. Sage, University of Washington, Seattle, WA) as described previously (15).

Transient Transfection and Luciferase Assay—Transfection was performed with the FuGENE 6 transfection reagent (Roche Applied Science), and luciferase and beta-galactosidase activities were measured as previously described (15, 26). Briefly, 8–10 x 104 cells were seeded in 6-well plates. Eighteen hours later, the cells were switched to 1% newborn calf serum-containing medium and transfected with the Smad3-LUC constructs along with cytomegalovirus-beta-galactosidase (Invitrogen) as a control of transfection efficiency. One hour later, 1 ng/ml TGF-beta1 or control vehicle was added, and the cells were harvested after 18–24 h. Luciferase assay results were normalized for beta-galactosidase activity. Experimental points were performed in triplicates in several independent experiments.

Plasmid Constructs—The -290/+157 Smad3-LUC construct containing Smad3 promoter sequences was generated as follows. The Smad3 promoter sequences from -1733/+157 or -1034/+157 obtained by PCR amplification using the Advantage GC genomic polymerase mix (Clontech laboratories) and human genomic DNA (Roche Applied Science) as a template were subcloned into the pGL3basic vector, which carries the luciferase reporter gene without a promoter (Promega). The resulting clones were used to generate the 5' deletion mutant -290/+157 Smad3-LUC by digestion with SmaI and religation with the T4 DNA ligase (Invitrogen). Two clones were verified by sequencing.

Statistical Analysis—Statistical differences between experimental groups were determined by analysis of variance using the StatView, version 4.02, software program for Macintosh. Values of p < 0.05 by Fisher's protected least significant difference were considered significant. Differences between two comparative groups were further analyzed by unpaired Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Smad3 Protein Down-regulation—In studying activation of the Smad pathway in human glomerular mesangial cells, we observed that Smad3 protein levels were decreased in response to TGF-beta1 (15). However, we also have shown that Smad3 is required for TGF-beta1-stimulated COL1A2 gene expression, as overexpression of a Smad3 dominant-negative mutant or mutation of the Smad binding site in the COL1A2 promoter blocks its induction by TGF-beta1 (15, 16). Here we sought to study the mechanism by which TGF-beta1 regulates Smad expression. Smad3 (but not Smad2 or Smad4) was significantly decreased by 24 h of TGF-beta1 treatment in mesangial cells (Fig. 1A). In these cells, Smad3 levels remained low for up to 72 h of incubation with TGF-beta1 (data not shown). It should be noted that Smad2 protein expression was increased after 6 h of treatment with TGF-beta1 but returned to control levels at 24 h (Fig. 1A). We next examined whether TGF-beta1-induced Smad3 down-regulation is dependent on an active TGF-beta receptor complex. Fig. 1B shows that the TGF-beta type I receptor (Alk5/TbetaRI) kinase inhibitor SB-431542 blocked TGF-beta1-induced Smad3 phosphorylation and down-regulation, suggesting that decreased Smad3 expression requires an active Alk5/TbetaRI.

Role of the Ubiquitin-Proteasome Pathway—Because polyubiquitination is generally considered to be a marker of protein degradation by the proteasome pathway (27), we examined the extent of Smad3 ubiquitination in response to TGF-beta1. Fig. 2 shows that endogenous Smad3 was polyubiquitinated after 24 h of TGF-beta1 stimulation in mesangial cells. Treatment with proteasome inhibitors prevented the decrease in Smad3 protein levels (Fig. 3) and led to accumulation of polyubiquitinated Smad3 (data not shown). These data suggest that the TGF-beta1-induced Smad3 down-regulation occurs via the ubiquitin-proteasome pathway.


Figure 1
View larger version (30K):
[in this window]
[in a new window]

  		FIGURE 1.
Smad3 protein is down-regulated in human renal glomerular mesangial cells. A, serum-deprived mesangial cells (MC) were treated with 1 ng/ml TGF-beta1 for different time periods leading to simultaneous harvest. Cell lysates were analyzed by immunoblotting with anti-Smad1/2/3, anti-Smad3, anti-Smad4 or anti-phospho-Smad3. Top, representative blots. Bottom, densitometric analysis of several independent experiments performed with mesangial cells. Values are expressed as the ratio between treated and control cells for each Smad protein. *, p < 0.002; n ≥ 7. B, Smad3 down-regulation requires an active Alk5/TbetaRI. Mesangial cells were pretreated for 1 h with 5 µM SB-431542 or Me2SO as vehicle control before incubation with TGF-beta1 for 24 h. Lysates were examined by immunoblotting. ns, nonspecific band.

 


Figure 2
View larger version (56K):
[in this window]
[in a new window]

  		FIGURE 2.
Smad3 ubiquitination after TGF-beta1 treatment. Lysates from mesangial cells treated with TGF-beta1 for the indicated time periods were immunoprecipitated with anti-Smad3 (Smad3 IP). Smad3 IP complexes were analyzed by Western blotting with anti-ubiquitin or anti-Smad1/2/3. Whole cell lysates (WCL) were analyzed in parallel to show the effect of TGF-beta1 on the Smad expression in these samples. Lb, IP with lysis buffer alone; * indicates the position of Smad3.

 
Smad3 Gene Expression—Previously, we showed that, in addition to affecting Smad3 protein expression, TGF-beta1 modulates Smad3 mRNA levels in mesangial cells, with Smad3 mRNA expression significantly decreased following 4 h of incubation with TGF-beta1, whereas Smad2 and Smad4 mRNA levels remained unchanged (15). To determine whether the effect of TGF-beta1 on Smad3 down-regulation was direct or not, we examined whether cycloheximide affected Smad3 mRNA expression. Fig. 4A shows that cycloheximide alone increased Smad3 mRNA levels. However, it impaired TGF-beta1-induced inhibition, suggesting that de novo protein synthesis may be required for Smad3 mRNA down-regulation in response to TGF-beta1. Next, we investigated whether TGF-beta1 affects Smad3 mRNA stability. An analysis of mRNA half-life using actinomycin D to inhibit transcription (Fig. 4B) showed that TGF-beta1 did not increase the rate of turnover for Smad3 mRNA, suggesting that the TGF-beta1-induced decline in Smad3 mRNA level is not due to decreased mRNA stability.

Regulation of Smad3 Promoter Activity—Our results in Fig. 4B suggest that TGF-beta1 inhibits mesangial cell Smad3 expression by a transcriptional mechanism. To evaluate this, we generated a Smad3 promoter luciferase reporter construct and assessed the effect of TGF-beta1 on Smad3 promoter activity in transient transfection experiments. The Smad3-LUC construct showed increased activity of 137-fold over the empty vector (Fig. 5). Moreover, TGF-beta1 treatment significantly inhibited Smad3 promoter activity, whereas it had no effect on the control empty reporter construct pGL3basic. These data suggest that, in addition to decreasing Smad3 protein stability via the ubiquitin-proteasome pathway (Fig. 2), TGF-beta1 also inhibits Smad3 gene transcription.

Smad3 Down-regulation in Cells with a Myofibroblastic Phenotype—Next, we sought to compare the effect of TGF-beta1 on Smad3 expression in various cell types to determine whether the TGF-beta1-induced Smad3 down-regulation is a general cell response. In contrast to what we observed in mesangial cells, treatment with TGF-beta1 for 24 h did not affect the expression of any of the three Smad proteins examined in renal tubular epithelial HKC cells, although the Smad pathway was activated by TGF-beta1 as determined by increased Smad3 phosphorylation (Fig. 6A). Similarly, we did not observe any Smad3 decline within this time frame in other epithelial cells, including the mouse mammary gland epithelial cell line NMuMG and mink lung epithelial cell line Mv1Lu (data not shown). Increasing evidence suggests that renal myofibroblasts, whose activation is thought to play a critical role in the progression of renal fibrosis by contributing to ECM overproduction, may derive at least in part from tubular epithelial cells via EMT (1, 28, 29). Because TGF-beta has been shown to induce renal tubular cell EMT (30), we examined whether prolonged TGF-beta1 treatment affects Smad3 expression following myofibroblastic phenotypic changes in HKC. Time course experiments showed a significant decrease in Smad3 levels after 7 days of exposure to TGF-beta1 (Fig. 6B). In contrast, Smad2 and Smad4 levels remained unchanged. The timing of the decline in Smad3 expression coincided with a change to a myofibroblastic phenotype, as demonstrated by increased expression of smooth muscle {alpha}-actin ({alpha}-SMA), a myofibroblast marker, and decreased expression of E-cadherin, an epithelial cell marker (Fig. 6B), and enhanced collagen I accumulation (data not shown).


Figure 3
View larger version (33K):
[in this window]
[in a new window]

  		FIGURE 3.
Proteasome inhibitors prevent TGF-beta1-mediated Smad3 down-regulation. Mesangial cells were pretreated for 1 h with 10 µM proteasome inhibitor I or Me2SO as vehicle control before the addition of TGF-beta1 for 24 h. Lysates were analyzed by immunoblotting with anti-Smad1/2/3 or anti-Smad4. Top, representative blots. Bottom, densitometric analysis of at least four independent experiments in which the proteasome pathway was inhibited with proteasome inhibitor I, lactacystin, or MG132. *, p = 0.0005; NS, not significant compared with levels in control cells for each Smad examined.

 


Figure 4
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 4.
Inhibition of Smad3 mRNA expression by TGF-beta1. A, requirement for de novo protein synthesis. Mesangial cells were pretreated with 10 µg/ml cycloheximide (CHX) or vehicle control (control) for 1 h before incubating with or without TGF-beta1 for 24 h. Total RNA was analyzed by Northern blot using Smad3 and 28 S cDNA probes. Left, a representative experiment is shown. Right, graphic representation of two independent experiments showing Smad3/28 S ratio in TGF-beta1-treated cells versus untreated cells, either in the presence (CHX) or absence (control) of cycloheximide. B, TGF-beta1 does not decrease Smad3 mRNA stability. Cells were treated with 250 ng/ml actinomycin D (ActD) or Me2SO as vehicle control in the presence or absence of TGF-beta1 for 2, 4, 7, or 24 h. Total RNA was analyzed by Northern blot using Smad3 and 28 S cDNA probes. The graph shows Smad3/28S ratio at each time point examined for at least two independent experiments, with the ratio for control cells being arbitrarily set at 1.

 


Figure 5
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 5.
Inhibition of Smad3 promoter activity by TGF-beta1. Mesangial cells were transfected with the Smad3-LUC construct (containing Smad3 promoter sequences from -290 to +157 cloned in front of the luciferase reporter gene) or the empty reporter construct pGL3basic, along with a cytomegalovirus-beta-galactosidase construct as a control for transfection efficiency. After treatment with TGF-beta1 or vehicle control for 18–24 h, lysates were harvested, and luciferase and beta-galactosidase activities were measured. *, p = 0.0004; n ≥ 4.

 
Next, we examined whether Smad3 down-regulation occurs in HKC via a mechanism similar to what we observed in human mesangial cells. Fig. 7A shows that Smad3 was polyubiquitinated in HKC after 3–7 days of TGF-beta1 treatment, suggesting that Smad3 protein is targeted for degradation by the proteasome pathway in these cells, although in delayed fashion, consistent with the occurrence of EMT. In addition, incubation with TGF-beta1 led to a progressive decrease in Smad3 mRNA expression (Fig. 7B). These results suggest that, similar to what we demonstrated in human mesangial cells, Smad3 down-regulation in HKC following long term treatment with TGF-beta1 involves both increased protein degradation and inhibition of transcription.

Unilateral ureteral obstruction (UUO) is a model of tubulointerstitial fibrosis associated with TGF-beta up-regulation that causes EMT of renal tubules and ECM accumulation (23, 31, 32). Thus, we examined Smad3 expression after UUO. Smad3 levels were progressively decreased in the affected kidney following UUO in mice (Fig. 8). The decline was concomitant with increased {alpha}-SMA expression after 3 days, up to 14 days of obstruction. These changes correlate with increased TGF-beta and TbetaRI expression in renal tubular epithelium of the obstructed kidney (31) as well as increased TGF-beta1 mRNA (data not shown). Together, our findings suggest that Smad3 down-regulation is a cell type-specific event, occurring in response to TGF-beta1 in cells with a fibroblastoid phenotype but not in epithelial cells.


Figure 6
View larger version (30K):
[in this window]
[in a new window]

  		FIGURE 6.
Smad expression in tubular epithelial cell. A, treatment with TGF-beta1 for 24 h does not affect renal tubular epithelial cell Smad3 levels. HKC cells were treated for the indicated time periods with TGF-beta1 leading to simultaneous harvest. Lysates were analyzed with the indicated antibodies. B, Smad3 down-regulation occurs in epithelial cells following EMT. HKC cells were treated for 3, 5, or 7 days with TGF-beta1, and lysates were analyzed by immunoblotting. Top, representative blots. Bottom, densitometric analysis of several independent experiments. *, p < 0.002; n ≥ 2.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Because Smad3 plays a critical role in wound healing and the pathogenesis of fibrosis, determining how Smad3 is regulated is essential to understanding the mechanisms of these processes. Here we show that TGF-beta1 down-regulates Smad3 levels in a cell phenotype-specific manner. This down-regulation is due to both increased degradation via the proteasome pathway and decreased gene transcription.

Only a few studies have examined Smad3 turnover (18–20). Potential E3 ubiquitin ligases reported to target Smad3 for degradation are the RING finger ubiquitin ligase ROC1 and the carboxyl terminus of Hsc70-interacting protein CHIP. The role of ROC1 is still controversial. Fukuchi et al. (18) have suggested that overexpressed ROC1 interacts with activated Smad3 and triggers its degradation in COS7 cells, whereas Inoue et al. (19) were unable to detect interaction between Smad3 and ROC1 in HEK293T cells even after TGF-beta stimulation. Overexpression of CHIP in several cell lines decreases basal Smad3 levels but independently of TGF-beta signaling (20). It is important to note that, in these studies, overexpressed Smad3 and ubiquitin were used and required overexpression of the E3 ligase complex ROC1-SCFFbw1a or CHIP and/or the presence of proteasome inhibitor to detect Smad3 ubiquitination. In contrast, we show a decline in endogenous Smad3 in mesangial cells within 24 h in response to TGF-beta1 and that TGF-beta1 treatment increases ubiquitination of endogenous Smad3 targeting it for degradation by the proteasome machinery. Whether ROC1, CHIP, or other E3 ligases play a role in endogenous Smad3 down-regulation remains to be examined. Interestingly, protein and mRNA expression of the closely related Smad2 are not affected by 24 h of treatment with TGF-beta1 in human mesangial cells (present report and Ref. 15). Because Smad2 and Smad3 have distinct roles that may also be cell type-specific (33–35), differential regulation of Smad2 and Smad3 are likely to affect how cells respond to TGF-beta.


Figure 7
View larger version (38K):
[in this window]
[in a new window]

  		FIGURE 7.
Increased ubiquitination of Smad3 protein and decreased Smad3 mRNA expression in HKC cells. A, Lysates from HKC cells treated with TGF-beta1 for the indicated time periods were immunoprecipitated with anti-Smad3 (Smad3 IP) before immunoblotting with anti-ubiquitin or anti-Smad3. B, Total RNA from HKC cells treated with TGF-beta1 for various times was analyzed by Northern blot using Smad3 and 28S cDNA probes. Top, Results from a representative experiment are shown. Bottom, Densitometric analysis of 3 independent experiments showing Smad3 mRNA levels following TGF-beta1 treatment in HKC.

 


Figure 8
View larger version (52K):
[in this window]
[in a new window]

  		FIGURE 8.
Decline in Smad3 levels correlates with induction of {alpha}-SMA expression in the obstructed kidneys after UUO. Kidney lysates from sham-operated animals at 7 days or UUO at 3, 7, or 14 days were analyzed by immunoblotting with anti-Smad3, anti-{alpha}-SMA as a myofibroblastic marker, or anti-Dab2 as a control for loading. Top, results from two independent sets of animals are shown. Bottom, densitometric analysis of Smad3 levels. Data are presented as mean ± S.E. of four animals per group. *, p < 0.0001 001 versus sham control group.

 
TGF-beta induces ECM accumulation in fibroblasts and mesenchymal cells such as glomerular mesangial cells, whereas it inhibits proliferation and/or induces apoptosis and EMT in several epithelial cells, including renal tubular epithelial cells (2, 5–7). The interactions of the Smad cascade with other signaling pathways contribute to the specificity of the response (36, 37). Here we have identified a cell response that is both phenotype- and Smad type-specific. This specific Smad3 down-regulation may play a role in modulating TGF-beta signals.

In addition to decreasing Smad3 protein levels, our data show that TGF-beta1 also modulates Smad3 mRNA expression. Although Smad3 mRNA down-regulation has been observed in two immortalized human lung epithelial cell lines, BEASB2B and HPL1A (38), and in normal skin and lung fibroblasts (39, 40), the mechanism by which this occurs has not been described. Here we show that the TGF-beta1-induced decrease in Smad3 mRNA expression is not due to an alteration in mRNA stability but rather to decreased promoter activity. In addition, our experiment with cycloheximide suggests that Smad3 down-regulation by TGF-beta1 requires de novo synthesis of proteins, such as transcription factors and/or cytokines, that might act as autocrine or paracrine mediators. Of note, cycloheximide alone increases expression of Smad3 mRNA. This may result from increased stability of mRNA because of the inhibition of the synthesis of proteins involved in RNA degradation or relief of transcriptional repression.

Although TGF-beta-enhanced, ubiquitin-mediated Smad3 degradation has been reported previously, the data suggest that Smad3 expression is compensated by concomitant increased Smad3 synthesis in the time frame examined (18–20). Thus, the result of this response appears to be that turnover is increased, but protein levels are stable. Our data suggest that a critical additional event in mesenchymal cells, inhibition of promoter activity, plays an important role in decreasing Smad3 expression. This is the first report showing regulation of an R-Smad promoter activity by TGF-beta1. Because the decline in Smad3 is phenotype-specific, we hypothesize that inhibition of Smad3 gene expression could be part of a feedback loop controlling TGF-beta signaling. Increased expression of the inhibitor Smad7 has been associated with inhibition of TGF-beta signaling (41). Smad7 may regulate TGF-beta signaling by inhibiting R-Smad activation by TbetaRI (41, 42) or by enhancing TbetaRI degradation (43, 44). PIASy (protein inhibitor of activated STAT) and TLP (TRAP-1-like protein) may also negatively regulate TGF-beta/Smad3-mediated signaling (45, 46). Here, we suggest an additional mechanism for controlling TGF-beta responses that involves a decrease in Smad3 expression by inhibition of gene transcription as well as increased protein degradation.

Our data show that, although tubular epithelial cell Smad3 levels are unaffected by 24 h of treatment with TGF-beta1, prolonged exposure to TGF-beta1 leads to a significant decline in Smad3 expression. These changes parallel decreased E-cadherin and increased {alpha}-SMA expression, suggesting that decreased Smad3 expression occurs with EMT. Similar results were obtained in kidney following UUO, which is associated with EMT of the tubules. Because Smad3 has been shown to be essential for the initiation of EMT after UUO and in cultured tubular epithelial cells (32), the decline in Smad3 levels following EMT suggest that Smad3 down-regulation could represent a feedback loop controlling TGF-beta1 responses. Interestingly, in a model of lung fibrosis induced by bleomycin, Smad3 expression decreased during the reparative phase of lung injury at days 8 and 12 (40). In addition, Kelley et al. found reduced Smad3 protein levels in a model of cystic fibrosis compared with those in control mice (47). It is intriguing that rat and mouse UUO kidneys show increased nuclear localization of phospho-Smad2/3 in some glomerular and tubulointerstitial cells (48, 49). Because we detected a decrease in total Smad3 expression in mouse UUO kidneys, this raises questions regarding whether the active or inactive form of Smad3 is targeted for degradation following UUO and whether and how the residual active Smad3 contributes to renal fibrosis.

In summary, we have shown that TGF-beta1 down-regulates Smad3 in cells with a myofibroblastic phenotype. The down-regulation involves protein degradation via the ubiquitin-proteasome pathway and inhibition of gene transcription. Because Smad3 is a key mediator of fibrosis, understanding the causes and effects of changes in Smad3 expression may provide a model to suggest specific means to block fibrotic diseases.


   FOOTNOTES
 
* This work was supported by NIDDK, National Institutes of Health Grants K01 DK64074 (to A.-C. P.), R01 DK061408 (to Y. L.), and R01 DK49362 (to H. W. S.) and by the Children's Memorial Research Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Medicine, UW Medicine South Lake Union, University of Washington School of Medicine, 815 Mercer St., Box 358050, Rm. 254, Seattle, WA 98109. Tel.: 206-897-1967; Fax: 206-897-1300; E-mail: annechr{at}u.washington.edu.

2 The abbreviations used are: ECM, extracellular matrix; EMT, epithelial-to-mesenchymal transition; TGF-beta, transforming growth factor-beta; E3, ubiquitin-protein isopeptide ligase; UUO, unilateral ureteral obstruction; {alpha}-SMA, smooth muscle {alpha}-actin. Back


   ACKNOWLEDGMENTS
 
The Alk5/TbetaRI inhibitor SB-431542 was a generous gift from Dr. N. Laping (GlaxoSmithKline Pharmaceuticals). We express our gratitude to Dr. Christine Abrass for helpful comments.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Liu, Y. (2006) Kidney Int. 69, 213-217[CrossRef][Medline] [Order article via Infotrieve]
  2. Border, W. A., and Noble, N. A. (1997) Kidney Int. 51, 1388-1396[Medline] [Order article via Infotrieve]
  3. Blobe, G. C., Schiemann, W. P., and Lodish, H. F. (2000) N. Engl. J. Med. 342, 1350-1358[Free Full Text]
  4. Gressner, A. M., Weiskirchen, R., Breitkopf, K., and Dooley, S. (2002) Front. Biosci. 7, 793-807[CrossRef]
  5. Zavadil, J., and Bottinger, E. P. (2005) Oncogene 24, 5764-5774[CrossRef][Medline] [Order article via Infotrieve]
  6. Roberts, A. B., Tian, F., Byfield, S. D., Stuelten, C., Ooshima, A., Saika, S., and Flanders, K. C. (2006) Cytokine Growth Factor Rev. 17, 19-27[CrossRef][Medline] [Order article via Infotrieve]
  7. Bottinger, E. P., and Bitzer, M. (2002) J. Am. Soc. Nephrol. 13, 2600-2610[Abstract/Free Full Text]
  8. Schuster, N., and Krieglstein, K. (2002) Cell Tissue Res. 307, 1-14[CrossRef][Medline] [Order article via Infotrieve]
  9. ten Dijke, P., Goumans, M. J., Itoh, F., and Itoh, S. (2002) J. Cell. Physiol. 191, 1-16[CrossRef][Medline] [Order article via Infotrieve]
  10. Piek, E., Heldin, C.-H., and ten Dijke, P. (1999) FASEB J. 13, 2105-2124[Abstract/Free Full Text]
  11. Attisano, L., and Wrana, J. L. (2000) Curr. Opin. Cell Biol. 12, 235-243[CrossRef][Medline] [Order article via Infotrieve]
  12. Feng, X. H., and Derynck, R. (2005) Annu. Rev. Cell Dev. Biol. 21, 659-693[CrossRef][Medline] [Order article via Infotrieve]
  13. Massagué, J., Seoane, J., and Wotton, D. (2005) Genes Dev. 19, 2783-2810[Abstract/Free Full Text]
  14. Flanders, K. C. (2004) Int. J. Exp. Pathol. 85, 47-64[CrossRef][Medline] [Order article via Infotrieve]
  15. Poncelet, A.-C., de Caestecker, M. P., and Schnaper, H. W. (1999) Kidney Int. 56, 1354-1365[CrossRef][Medline] [Order article via Infotrieve]
  16. Poncelet, A.-C., and Schnaper, H. W. (2001) J. Biol. Chem. 276, 6983-6992[Abstract/Free Full Text]
  17. Izzi, L., and Attisano, L. (2004) Oncogene 23, 2071-2078[CrossRef][Medline] [Order article via Infotrieve]
  18. Fukuchi, M., Imamura, T., Chiba, T., Ebisawa, T., Kawabata, M., Tanaka, K., and Miyazono, K. (2001) Mol. Biol. Cell 12, 1431-1443[Abstract/Free Full Text]
  19. Inoue, Y., Kitagawa, M., Onozaki, K., and Hayashi, H. (2004) J. Interferon Cytokine Res. 24, 43-54[CrossRef][Medline] [Order article via Infotrieve]
  20. Xin, H., Xu, X., Li, L., Ning, H., Rong, Y., Shang, Y., Wang, Y., Fu, X. Y., and Chang, Z. (2005) J. Biol. Chem. 280, 20842-20850[Abstract/Free Full Text]
  21. Laping, N. J., Grygielko, E., Mathur, A., Butter, S., Bomberger, J., Tweed, C., Martin, W., Fornwald, J., Lehr, R., Harling, J., Gaster, L., Callahan, J. F., and Olson, B. A. (2002) Mol. Pharmacol. 62, 58-64[Abstract/Free Full Text]
  22. Racusen, L. C., Monteil, C., Sgrignoli, A., Lucskay, M., Marouillat, S., Rhim, J. G., and Morin, J. P. (1997) J. Lab. Clin. Med. 129, 318-329[CrossRef][Medline] [Order article via Infotrieve]
  23. Yang, J., and Liu, Y. (2002) J. Am. Soc. Nephrol. 13, 96-107[Abstract/Free Full Text]
  24. Runyan, C. E., Schnaper, H. W., and Poncelet, A.-C. (2003) Am. J. Physiol. 285, F413-F422
  25. Zhang, Y., Feng, X., We, R., and Derynck, R. (1996) Nature 383, 168-172[CrossRef][Medline] [Order article via Infotrieve]
  26. Runyan, C. E., Schnaper, H. W., and Poncelet, A. C. (2004) J. Biol. Chem. 279, 2632-2639[Abstract/Free Full Text]
  27. Ciechanover, A. (2005) Nat. Rev. Mol. Cell Biol. 6, 79-87[CrossRef][Medline] [Order article via Infotrieve]
  28. Kalluri, R., and Neilson, E. G. (2003) J. Clin. Investig. 112, 1776-1784[CrossRef][Medline] [Order article via Infotrieve]
  29. Liu, Y. (2004) J. Am. Soc. Nephrol. 15, 1-12[Abstract/Free Full Text]
  30. Fan, J. M., Ng, Y. Y., Hill, P. A., Nikolic-Paterson, D. J., Mu, W., Atkins, R. C., and Lan, H. Y. (1999) Kidney Int. 56, 1455-1467[CrossRef][Medline] [Order article via Infotrieve]
  31. Yang, J., and Liu, Y. (2001) Am. J. Pathol. 159, 1465-1475[Abstract/Free Full Text]
  32. Sato, M., Muragaki, Y., Saika, S., Roberts, A. B., and Ooshima, A. (2003) J. Clin. Investig. 112, 1486-1494[CrossRef][Medline] [Order article via Infotrieve]
  33. Piek, E., Ju, W. J., Heyer, J., Escalante-Alcalde, D., Stewart, C. L., Weinstein, M., Deng, C., Kucherlapati, R., Bottinger, E. P., and Roberts, A. B. (2001) J. Biol. Chem. 276, 19945-19953[Abstract/Free Full Text]
  34. Kretschmer, A., Moepert, K., Dames, S., Sternberger, M., Kaufmann, J., and Klippel, A. (2003) Oncogene 22, 6748-6763[CrossRef][Medline] [Order article via Infotrieve]
  35. Roberts, A. B., Russo, A., Felici, A., and Flanders, K. C. (2003) Ann. N. Y. Acad. Sci. 995, 1-10[Medline] [Order article via Infotrieve]
  36. Javelaud, D., and Mauviel, A. (2005) Oncogene 24, 5742-5750[CrossRef][Medline] [Order article via Infotrieve]
  37. Moustakas, A., and Heldin, C. H. (2005) J. Cell Sci. 118, 3573-3584[Abstract/Free Full Text]
  38. Yanagisawa, K., Osada, H., Masuda, A., Kondo, M., Saito, T., Yatabe, Y., Takagi, K., Takahashi, T., and Takahashi, T. (1998) Oncogene 17, 1743-1747[CrossRef][Medline] [Order article via Infotrieve]
  39. Mori, Y., Chen, S. J., and Varga, J. (2000) Exp. Cell Res. 258, 374-383[CrossRef][Medline] [Order article via Infotrieve]
  40. Zhao, Y., and Geverd, D. A. (2002) Biochem. Biophys. Res. Commun. 294, 319-323[CrossRef][Medline] [Order article via Infotrieve]
  41. Nakao, A., Afrakthe, M., Morén, A., Nakayama, T., Christian, J. L., Heuchel, R., Itoh, S., Kawabata, M., Heldin, N.-E., Heldin, C.-H., and ten Dijke, P. (1997) Nature 389, 631-635[CrossRef][Medline] [Order article via Infotrieve]
  42. Hayashi, H., Abdollah, S., Qiu, Y., Cai, J., Xu, Y.-Y., Grinnell, B. W., Richardson, M. A., Topper, J. N., Gimbrone, M. A., Jr., Wrana, J. L., and Falb, D. (1997) Cell 89, 1165-1173[CrossRef][Medline] [Order article via Infotrieve]
  43. Kavsak, P., Rasmussen, R. K., Causing, C. G., Bonni, S., Zhu, H., Thomsen, G. H., and Wrana, J. L. (2000) Mol. Cell 6, 1365-1375[CrossRef][Medline] [Order article via Infotrieve]
  44. Ebisawa, T., Fukuchi, M., Murakami, G., Chiba, T., Tanaka, K., Imamura, T., and Miyazono, K. (2001) J. Biol. Chem. 276, 12477-12480[Abstract/Free Full Text]
  45. Imoto, S., Sugiyama, K., Muromoto, R., Sato, N., Yamamoto, T., and Matsuda, T. (2003) J. Biol. Chem. 278, 34253-34258[Abstract/Free Full Text]
  46. Felici, A., Wurthner, J. U., Parks, W. T., Giam, L. R., Reiss, M., Karpova, T. S., McNally, J. G., and Roberts, A. B. (2003) EMBO J. 22, 4465-4477[CrossRef][Medline] [Order article via Infotrieve]
  47. Kelley, T. J., Elmer, H. L., and Corey, D. A. (2001) Am. J. Respir. Cell Mol. Biol. 25, 732-738[Abstract/Free Full Text]
  48. Lan, H. Y., Mu, W., Tomita, N., Huang, X. R., Li, J. H., Zhu, H. J., Morishita, R., and Johnson, R. J. (2003) J. Am. Soc. Nephrol. 14, 1535-1548[Abstract/Free Full Text]
  49. Inazaki, K., Kanamaru, Y., Kojima, Y., Sueyoshi, N., Okumura, K., Kaneko, K., Yamashiro, Y., Ogawa, H., and Nakao, A. (2004) Kidney Int. 66, 597-604[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Renal Physiol.Home page
R. Tan, W. He, X. Lin, L. P. Kiss, and Y. Liu
Smad ubiquitination regulatory factor-2 in the fibrotic kidney: regulation, target specificity, and functional implication
Am J Physiol Renal Physiol, May 1, 2008; 294(5): F1076 - F1083.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
T. Hayashida, M.-H. Wu, A. Pierce, A.-C. Poncelet, J. Varga, and H. W. Schnaper
MAP-kinase activity necessary for TGFbeta1-stimulated mesangial cell type I collagen expression requires adhesion-dependent phosphorylation of FAK tyrosine 397
J. Cell Sci., December 1, 2007; 120(23): 4230 - 4240.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/21/15534    most recent
M701991200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Poncelet, A.-C.
Right arrow Articles by Runyan, C. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Poncelet, A.-C.
Right arrow Articles by Runyan, C. E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?