Role of SARA (SMAD Anchor for Receptor Activation) in Maintenance of Epithelial Cell Phenotype

By inducing epithelial-to-mesenchymal transition (EMT), transforming growth factor-β (TGF-β) promotes cancer progression and fibrosis. Here we show that expression of the TGF-β receptor-associated protein, SARA (Smad anchor for receptor activation), decreases within 72 h of exposure to TGF-β and that this decline is both required and sufficient for the induction of several markers of EMT. It has been suggested recently that expression of the TGF-β signaling mediators, Smad2 and Smad3, may have different functional effects, with Smad2 loss being more permissive for EMT progression. We find that the loss of SARA expression leads to a concomitant decrease in Smad2 expression and a disruption of Smad2-specific transcriptional activity, with no effect on Smad3 signaling or expression. Further, the effects of inducing the loss of Smad2 mimic those of the loss of SARA, enhancing expression of the EMT marker, smooth muscle α-actin. Smad2 mRNA levels are not affected by the loss of SARA. However, the ubiquitination of Smad2 is increased in SARA-deficient cells. We therefore examined the E3 ubiquitin ligase Smurf2 and found that although Smurf2 expression was unaltered in SARA-deficient cells, the interaction of Smad2 and Smurf2 was enhanced. These results describe a significant role for SARA in regulating cell phenotype and suggest that its effects are mediated through modification of the balance between Smad2 and Smad3 signaling. In part, this is achieved by enhancing the association of Smad2 with Smurf2, leading to Smad2 degradation. By inducing epithelial-to-mesenchymal transition (EMT), transforming growth factor-β (TGF-β) promotes cancer progression and fibrosis. Here we show that expression of the TGF-β receptor-associated protein, SARA (Smad anchor for receptor activation), decreases within 72 h of exposure to TGF-β and that this decline is both required and sufficient for the induction of several markers of EMT. It has been suggested recently that expression of the TGF-β signaling mediators, Smad2 and Smad3, may have different functional effects, with Smad2 loss being more permissive for EMT progression. We find that the loss of SARA expression leads to a concomitant decrease in Smad2 expression and a disruption of Smad2-specific transcriptional activity, with no effect on Smad3 signaling or expression. Further, the effects of inducing the loss of Smad2 mimic those of the loss of SARA, enhancing expression of the EMT marker, smooth muscle α-actin. Smad2 mRNA levels are not affected by the loss of SARA. However, the ubiquitination of Smad2 is increased in SARA-deficient cells. We therefore examined the E3 ubiquitin ligase Smurf2 and found that although Smurf2 expression was unaltered in SARA-deficient cells, the interaction of Smad2 and Smurf2 was enhanced. These results describe a significant role for SARA in regulating cell phenotype and suggest that its effects are mediated through modification of the balance between Smad2 and Smad3 signaling. In part, this is achieved by enhancing the association of Smad2 with Smurf2, leading to Smad2 degradation. Epithelial-to-mesenchymal transition (EMT) 3The abbreviations used are:EMTepithelial to mesenchymal transitionαSMAsmooth muscle α-actinTGFtransforming growth factorR-Smadreceptor-regulated SmadE1ubiquitin-activating enzymeE2ubiquitin carrier proteinE3ubiquitin-protein isopeptide ligasesiRNAsmall interfering RNAshRNAshort hairpin RNAHKC-CHKC-S, HKC-Sd2, HKC cells stably expressing control, SARA, or Smad2 shRNA, respectively. describes a process by which cells lose their relatively differentiated epithelial characteristics and show increased migratory or synthetic properties. This transition is evidenced by loss of proteins involved in cell-cell junctions, such as E-cadherin and occludin, and by an increase in proteins, such as vimentin and fibronectin, as well as de novo production of smooth muscle α-actin (αSMA) (1Lee J.M. Dedhar S. Kalluri R. Thompson E.W. J. Cell Biol. 2006; 172: 973-981Crossref PubMed Scopus (1741) Google Scholar). epithelial to mesenchymal transition smooth muscle α-actin transforming growth factor receptor-regulated Smad ubiquitin-activating enzyme ubiquitin carrier protein ubiquitin-protein isopeptide ligase small interfering RNA short hairpin RNA HKC-S, HKC-Sd2, HKC cells stably expressing control, SARA, or Smad2 shRNA, respectively. Phenotypic transitions are important in the differentiation of tissues during morphogenesis. Dedifferentiation plays a role in tumor progression by increasing cellular invasiveness and in tissue fibrosis through the generation of extracellular matrix-producing myofibroblasts. The importance of EMT in kidney disease has been demonstrated in experimental models where blocking EMT attenuates fibrosis (2Liu Y. J. Am. Soc. Nephrol. 2004; 15: 1-12Crossref PubMed Scopus (990) Google Scholar). Since TGF-β is a potent initiator of renal EMT, this represents one way in which aberrant TGF-β signaling stimulates fibrosis in the kidney. The TGF-β superfamily consists of TGF-β1, TGF-β2, TGF-β3, activins, and bone morphogenic proteins. These proteins are widely expressed in virtually all mammalian cell types, as are their downstream signaling mediators, the Smad proteins. TGF-β signaling is initiated when ligand-bound TGF-β type II receptor binds to and phosphorylates the TGF-β type I receptor (3Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Peptide Growth Factors and Their Receptors. Springer-Verlag, Heidelberg, Germany1990: 419-472Google Scholar, 4Piek E. Heldin C.H. Ten Dijke P. FASEB J. 1999; 13: 2105-2124Crossref PubMed Scopus (754) Google Scholar, 5Shi Y. Massagué J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4939) Google Scholar). Phosphorylation of the TGF-β type I receptor in its cytoplasmic GS region leads to its activation and its ability to activate the receptor-regulated Smads (R-Smads), Smad2 and Smad3, by C-terminal serine phosphorylation. Once phosphorylated, the R-Smads form a heteromultimeric complex with the common mediator (Co)-Smad (Smad4) and accumulate in the nucleus to regulate transcriptional responses (3Roberts A.B. Sporn M.B. Sporn M.B. Roberts A.B. Peptide Growth Factors and Their Receptors. Springer-Verlag, Heidelberg, Germany1990: 419-472Google Scholar, 4Piek E. Heldin C.H. Ten Dijke P. FASEB J. 1999; 13: 2105-2124Crossref PubMed Scopus (754) Google Scholar, 5Shi Y. Massagué J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4939) Google Scholar). Smad protein expression can be modulated through proteasomal degradation (6Izzi L. Attisano L. Oncogene. 2004; 23: 2071-2078Crossref PubMed Scopus (219) Google Scholar, 7Feng X.H. Derynck R. Annu. Rev. Cell Dev. Biol. 2005; 21: 659-693Crossref PubMed Scopus (1565) Google Scholar). This ubiquitin-mediated proteolysis occurs via a cascade of enzymatic reactions of a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3), with E3 being the defining factor for substrate selectivity. Smurf2 (Smad ubiquitination regulatory factor-2) is one such E3 ligase. Smurf2 belongs to the HECT domain ubiquitin ligase family and has recently been suggested to be specific for the degradation of Smad2 (8Lin X. Liang M. Feng X.H. J. Biol. Chem. 2000; 275: 36818-36822Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 9Tan R. He W. Lin X. Kiss L.P. Liu Y. Am. J. Physiol. Renal Physiol. 2008; 294: F1076-F1083Crossref PubMed Scopus (64) Google Scholar). In addition to ubiquitin ligases, numerous proteins have been identified that promote Smad expression, stability, activation, and assembly into transcription-regulatory complexes (10Böttinger E.P. Bitzer M. J. Am. Soc. Nephrol. 2002; 13: 2600-2610Crossref PubMed Scopus (672) Google Scholar). Several of these proteins control the localization of Smads and TβRs and the interaction of Smads with the receptor complex (11Runyan C.E. Poncelet A.C. Schnaper H.W. Cell. Signal. 2006; 18: 2077-2088Crossref PubMed Scopus (26) Google Scholar, 12Brown K.A. Pietenpol J.A. Moses H.L. J. Cell. Biochem. 2007; 101: 9-33Crossref PubMed Scopus (312) Google Scholar). One such protein, SARA (Smad Anchor for Receptor Activation), was initially described as a recruitment factor for R-Smad to the TβR (13Tsukazaki T. Chiang T.A. Davison A.F. Attisano L. Wrana J.L. Cell. 1998; 95: 779-791Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar). It contains both a Smad-binding domain, which interacts with Smad2 and Smad3 (14Wu G. Chen Y.G. Ozdamar B. Gyuricza C.A. Chong P.A. Wrana J.L. Massagué J. Shi Y. Science. 2000; 287: 92-97Crossref PubMed Scopus (238) Google Scholar), and a C-terminal, TβR complex-interacting region (13Tsukazaki T. Chiang T.A. Davison A.F. Attisano L. Wrana J.L. Cell. 1998; 95: 779-791Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar). SARA was therefore proposed to play a role in presenting R-Smads to the receptor for phosphorylation. The initial report describing this molecule suggested that SARA and Smad2 are associated basally and then dissociate upon receptor activation. In contrast, our previous studies of kidney cells demonstrated very little basal association of SARA with Smad2 but high levels of interaction upon TGF-β1 stimulation (15Runyan C.E. Schnaper H.W. Poncelet A.C. J. Biol. Chem. 2005; 280: 8300-8308Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The specific role of SARA in TGF-β1-mediated Smad signaling remains poorly understood. A previous study in COS7 cells showed that SARA was required for Smad2-dependent signaling but not for Smad3-dependent signaling (16Goto D. Nakajima H. Mori Y. Kurasawa K. Kitamura N. Iwamoto I. Biochem. Biophys. Res. Commun. 2001; 281: 1100-1105Crossref PubMed Scopus (31) Google Scholar). Additionally, our previous work showed that in human kidney mesangial cells, which express nearly equal levels of Smad2 and Smad3, SARA preferentially interacts with Smad2 in response to TGF-β1 treatment (15Runyan C.E. Schnaper H.W. Poncelet A.C. J. Biol. Chem. 2005; 280: 8300-8308Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). However, another report showed that a SARA binding-deficient mutant of Smad2 could still be activated by TGF-β receptors (17Lu Z. Murray J.T. Luo W. Li H. Wu X. Xu H. Backer J.M. Chen Y.G. J. Biol. Chem. 2002; 277: 29363-29368Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Therefore, the specific role for SARA in Smad2- or Smad3-specific signaling is not fully determined. To date, with only a small number of studies on SARA expression and function, there is little information linking it to any specific disease model. SARA expression has been shown to be increased in epithelial cells derived from patients with asthma compared with normal subjects (18Semlali A. Jacques E. Plante S. Biardel S. Milot J. Laviolette M. Boulet L.P. Chakir J. Am. J. Respir. Cell Mol. Biol. 2008; 38: 202-208Crossref PubMed Scopus (43) Google Scholar) and in synovial fibroblasts from rheumatoid arthritis patients compared with osteoarthritis patients (19Pohlers D. Beyer A. Koczan D. Wilhelm T. Thiesen H.J. Kinne R.W. Arthritis Res. Ther. 2007; 9: R59Crossref PubMed Scopus (108) Google Scholar). SARA expression may decrease as liver fibrosis develops (20Tao Y.Y. Cui H.Y. Liu C.H. Zhonghua Gan Zang Bing Za Zhi. 2006; 14: 909-913PubMed Google Scholar). There is also a study showing that SARA expression declines during the transdifferentiation of hepatic stellate cells in a model of liver fibrosis (21Liu C. Gaça M.D. Swenson E.S. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). However, in of these was it the increase or decrease in SARA expression was a mediator of the disease We therefore examined the expression of SARA plays a functional role in of human kidney epithelial cells and SARA in Smad2 or Smad3-specific to this human TGF-β1, from was as a of in and used a of was from and used a of to SARA, Smurf2, and are from E-cadherin was from was from was from and from Cell and and Smad3 from The renal epithelial cell from L. C. A. M. S. J. Full Text PDF PubMed Scopus Google Scholar), and from the in with and of fibroblasts a from J. as described W. Mori Y. A. M. J. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). in with and and between and RNA was from or HKC-S, treatment with TGF-β1 or the to the RNA with the to with the was the with the for for and for by to the of by and by the used as and and SARA, and human and and expression of the of was by with the expression of the TGF-β1 in and cells and TGF-β1 cells to cell by in and as described C.E. Schnaper H.W. Poncelet A.C. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). of and cells in a to and The of this was as the cell and the was in and to This was as the and as described C.E. Schnaper H.W. Poncelet A.C. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). cells on in with or TGF-β1 for 72 h by with blocking with cells with for h with and then with for with and a with a and to and a was The was a from of J. Biol. Chem. Full Text PDF PubMed Google Scholar). The SARA, and expression by Wrana (13Tsukazaki T. Chiang T.A. Davison A.F. Attisano L. Wrana J.L. Cell. 1998; 95: 779-791Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar). The was by The L. J.L. P. S. B. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). was with the and and as described C.E. Schnaper H.W. Poncelet A.C. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). SARA and control from SARA Smad2 and control from cells in and to and of of control or SARA TGF-β1 or control was and cells for To cell which are as in E. to for in cells and to the HKC cells with and was in a to in cell of HKC cells to cells. Once cells or used for to in the of We SARA in human kidney mesangial cells but that that cells of a mesenchymal such as mesangial cells, SARA expression cells of an epithelial such as or epithelial cells. This is demonstrated in SARA protein expression in cells of epithelial kidney HKC or with that of fibroblasts This suggested the that the transition of epithelial cells to a phenotype a in the expression of SARA. To this we a renal epithelial cell with TGF-β1 and the levels of SARA expression shown in TGF-β1 treatment for and in a in SARA to the of an increase in smooth muscle α-actin (αSMA) expression. cells and a epithelial cell also showed SARA expression in response to TGF-β1 treatment not Further, we that the TGF-β1-mediated loss of SARA the of mRNA expression This was for HKC as well as for cells. Therefore, of SARA in response to TGF-β1 is not to kidney epithelial cells. the SARA expression and the increased expression required TGF-β1 in the of the type I TGF-β receptor of EMT, the loss of E-cadherin expression, was also affected by TGF-β1 within this and of the of Smad2 that as as in of TGF-β1 treatment also the Smad2 expression. To the in SARA and expression by TGF-β1 we SARA in HKC epithelial cells that the levels high of TGF-β1 We found that when SARA levels expression was no by TGF-β1 of SARA in cells also expression not the loss of SARA expression, this response was not specific to kidney epithelial cells. Further, although TGF-β1 the of an in the of an of the for SARA this induction not when SARA was these suggest that the in SARA expression is not with an increase in but may play an important role in inducing its expression. Since high levels of SARA expression to cells to epithelial we levels of SARA expression was sufficient to associated with EMT. We the SARA expression levels in HKC shown in compared with a control, SARA SARA expression to a to the by of TGF-β1 The levels of SARA expression by associated with basal levels of expression, TGF-β1 growth a Smad-binding protein that not show expression in response to TGF-β1 was as a control A increase in expression was in cells with SARA We this by a SARA cell of HKC a to express an to SARA. shown in cells showed expression of SARA compared with cells with a control We the effect of SARA on markers of EMT in cells. with SARA cells basal expression of compared with the cells of E-cadherin expression or of in cells showed that these However, of the basal expression of showed little to no between and cells not Therefore, although the loss of SARA in expression of proteins with EMT, it may not a We examined the loss of SARA the response to TGF-β1 by to the expression of the increase in type I shown in mRNA was by TGF-β1 in control cells but in cells. This response in cells in addition to its effects, the loss of SARA also the of the cellular response to it was loss of SARA be sufficient to markers of EMT, one was that the expression of this mediator of TGF-β1 signaling could the balance of Smad or expression. We therefore examined the of Smad2 or Smad3 in or cells. shown in SARA expression in the cells, the of Smad2 was the of Smad3 Further, the of Smad2 in response to TGF-β1 treatment was in cells of the Smad2-specific showed a TGF-β1 induction in cells This response was in cells The activation of this Smad2-specific could be by SARA into the cells, that the loss of the TGF-β1 response in cells was to the loss of SARA expression In contrast, of SARA not TGF-β1 activation of the Smad3-dependent and the response was by SARA to the cells these suggest that SARA plays an important role in Smad2 and transcriptional but to be for Smad3 activation in response to In our studies of Smad2 activation in cells, we that these cells to have expression of Smad2 In as shown in Smad2 expression was by in cells compared with but Smad3 expression was not TGF-β1 leads to the loss of SARA expression, and the loss of SARA expression to to a loss of Smad2 expression, then of TGF-β1 treatment also to the cells of Therefore, the loss of Smad2 expression that we found in response to of TGF-β1 treatment is with that which be by the loss of SARA these To a loss of SARA expression levels of we examined this was a transcriptional shown in showed no in Smad2 although SARA mRNA was between and cells. This suggested that the loss of SARA expression Smad2 the protein Since Smads are to be through ubiquitin we the ubiquitination of Smad2 or Smad3 by and for shown in Smad2 was more in cells compared with control the ubiquitination of Smad3 was in control and cells. To to the loss of SARA Smad2 we examined the expression of Smurf2, which is an E3 ubiquitin ligase proposed to be specific for Smad2 Smad3 (8Lin X. Liang M. Feng X.H. J. Biol. Chem. 2000; 275: 36818-36822Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 9Tan R. He W. Lin X. Kiss L.P. Liu Y. Am. J. Physiol. Renal Physiol. 2008; 294: F1076-F1083Crossref PubMed Scopus (64) Google Scholar). cells express levels of Smurf2, be to have Smad2 expression. However, when we compared Smurf2 the of protein expression or not we found no between and cells. We therefore examined the interaction between Smad2 and Smurf2 by of Smad2 more associated Smurf2 from cells from control cells This interaction was also in of Smurf2 from or cells interaction between Smurf2 and Smad3 was in Smurf2 from control or cells not that this is a Smad2-specific the effects of SARA expression from Smad2 expression and activity, then cells of Smad2 to those of SARA. To this we used a to stably express a Smad2 in HKC cells. shown in the cells expressing Smad2 Smad2 expression compared with cells expressing the control The of Smad2 was in response to TGF-β1 treatment in the Smad3 was in these cells To cells basal expression of we with the with or Smad2 shown in cells a basal expression of which could be by of increase in basal expression could also be the protein in cells our suggest that a decline in SARA expression results in a in Smad2 expression levels through interaction as well as a in Smad2 signaling and increased expression of a mesenchymal of Smad2 the effects of loss of SARA on expression. of from cells or HKC with Smad2 show Smad2 expression in cells. cells with or TGF-β1 for show and Smad2 compared with treatment of cells. or cells with an and or Smad2 and by h for from a of showing that protein expression is in cells compared with cells. The from to control cells that are or to may is described as a in which lose cell-cell and show increased These and functional by in the in a synthetic cell type that extracellular This to as EMT, is by a number of specific in protein expression, expression of E-cadherin and increased expression of these not the cell as a are as markers of the process that leads to EMT. TGF-β is a common initiator of EMT. Therefore, our that of EMT can be by the of SARA that in the expression or of this of TGF-β1 signaling may be an important in the of the suggest that the loss of SARA leads to production in epithelial cells through the specific of Smad2 signaling and expression, which may through SARA has been demonstrated to both Smad2 and Smad3 (13Tsukazaki T. Chiang T.A. Davison A.F. Attisano L. Wrana J.L. Cell. 1998; 95: 779-791Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar). However, in our previous studies of human mesangial cells, we found although Smad2 and Smad3 expressed equal of SARA showed with a small of Smad3 (15Runyan C.E. Schnaper H.W. Poncelet A.C. J. Biol. Chem. 2005; 280: 8300-8308Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). results show that a loss of SARA expression not Smad3-dependent signaling or Smad3 expression. This is with previous studies that a SARA binding-deficient mutant of Smad3 to TGF-β1 (16Goto D. Nakajima H. Mori Y. Kurasawa K. Kitamura N. Iwamoto I. Biochem. Biophys. Res. Commun. 2001; 281: 1100-1105Crossref PubMed Scopus (31) Google Scholar). The loss of Smad2 has been suggested in studies to in expression with EMT K.A. Pietenpol J.A. Moses H.L. J. Cell. Biochem. 2007; 101: 9-33Crossref PubMed Scopus (312) Google Scholar). In kidney epithelial cells, basal expression of increased as Smad2 was via P. Biochem. J. 2006; PubMed Scopus Google Scholar), and in studies Smad2 mesenchymal cell of EMT W. A. J. D. L. R. E.P. Mol. Cell. Biol. 2006; PubMed Scopus Google Scholar). Smad2 promotes increased EMT was in a study J. G. Li A. W. M. E. J. 2008; Google Scholar). In that it was shown that with of Smad2 and progression of when compared with The poorly differentiated and EMT, and it was demonstrated that EMT mediated by a loss of Smad2 was to an increase in expression of the E-cadherin transcriptional Therefore, the that the loss of SARA leads to a concomitant loss of Smad2 expression that SARA to EMT in through effects on However, to date, we have been to in expression between and E. T. S. J. and H. W. The loss of SARA expression Smad2 signaling is not that SARA has been described by our and as an protein for interaction (13Tsukazaki T. Chiang T.A. Davison A.F. Attisano L. Wrana J.L. Cell. 1998; 95: 779-791Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar, C.E. Schnaper H.W. Poncelet A.C. J. Biol. Chem. 2005; 280: 8300-8308Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, H. N. M. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar, S. A. S. J. Cell Biol. 2002; PubMed Scopus Google Scholar). However, our that SARA also may Smad2 expression is and in the E3 ubiquitin ligase Smurf2 has been shown to and However, this interaction to the levels of and Smad3 expression (8Lin X. Liang M. Feng X.H. J. Biol. Chem. 2000; 275: 36818-36822Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar, 9Tan R. He W. Lin X. Kiss L.P. Liu Y. Am. J. Physiol. Renal Physiol. 2008; 294: F1076-F1083Crossref PubMed Scopus (64) Google Scholar). the loss of SARA increased association of Smad2 and Smurf2 is not TGF-β1 receptors have been shown to through or C. A.F. Wrana J.L. Cell Biol. 2003; PubMed Scopus Google Scholar). SARA is associated with the receptor in the and Smad2-dependent signaling is enhanced. In contrast, in the the receptor with and Smurf2, in of Smad2 signaling and receptor expression C. A.F. Wrana J.L. Cell Biol. 2003; PubMed Scopus Google Scholar). This study found more of the receptor to more of the receptor was associated with Therefore, a by which SARA more complex is that the loss of SARA more of the and receptor-associated to where it interacts with However, in the of SARA expression, the of association is the of the complex remains a of The of the SARA, the TGF-β and Smurf2 is complex and is the of studies in our on our showing that SARA expression is important in an epithelial cell to these as well as the of SARA is and TGF-β1 the of SARA may studies in disease EMT. We and for as described