SRF is a nuclear repressor of Smad3-mediated TGF-β signaling

Lee, Ho Jae; Yun, Chohee; Lim, Seunghwan; Kim, B C; Baik, Kim G.; Kim, J M; Kim, W H; Kim, S J

doi:10.1038/sj.onc.1209774

Cited by 34 publications

(31 citation statements)

References 53 publications

(50 reference statements)

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“…TGF-␤1-induced gene transcription (29,49,50). Smad3 has been shown to interact with MKL1 whereas the MKL1/Smad3 complex activates slug expression (40).…”

Section: Discussionmentioning

confidence: 99%

MKL1 mediates TGF-β1-induced α-smooth muscle actin expression in human renal epithelial cells

Elberg¹,

Chen²,

Elberg³

et al. 2008

American Journal of Physiology-Renal Physiology

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Elberg G, Chen L, Elberg D, Chan MD, Logan CJ, Turman MA. MKL1 mediates TGF-␤1-induced ␣-smooth muscle actin expression in human renal epithelial cells. Am J Physiol Renal Physiol 294: F1116-F1128, 2008. First published March 12, 2008 doi:10.1152/ajprenal.00142.2007.-Transforming growth factor-␤1 (TGF-␤1) is known to induce epithelial-mesenchymal transition in the kidney, a process involved in tubulointerstitial fibrosis. We hypothesized that a coactivator of the serum response factor (SRF), megakaryoblastic leukemia factor-1 (MKL1), stimulates ␣-smooth muscle actin (␣-SMA) transcription in primary cultures of renal tubular epithelial cells (RTC), which convert into myofibroblasts on treatment with TGF-␤1. Herein, we study the effect of MKL1 expression on ␣-SMA in these cells. We demonstrate that TGF-␤1 stimulation of ␣-SMA transcription is mediated through CC(A/T) 6-rich GG elements known to bind to SRF. These elements also mediate the MKL1 effect that dramatically activates ␣-SMA transcription in serum-free media. MKL1 fused to green fluorescent protein localizes to the nucleus and induces ␣-SMA expression regardless of treatment with TGF-␤1. Using proteasome inhibitors, we also demonstrate that the proteolytic ubiquitin pathway regulates MKL1 expression. These data indicate that MKL1 overexpression is sufficient to induce ␣-SMA expression. Inhibition of endogenous expression of MKL1 by small interfering RNA abolishes TGF-␤1 stimulation of ␣-SMA expression. Therefore, MKL1 is also absolutely required for TGF-␤1 stimulation of ␣-SMA expression. Western blot and immunofluorescence analysis show that overexpressed and endogenous MKL1 are located in the nucleus in non-stimulated RTC. Chromatin immunoprecipitation assay demonstrates that TGF-␤1 induces binding of endogenous SRF and MKL1 to the ␣-SMA promoter in chromatin. Since MKL1 constitutes a potent factor regulating ␣-SMA expression, modulation of endogenous MKL1 expression or activity may have a profound effect on myofibroblast formation and function in the kidney.epithelial-mesenchymal transition; myocardin; ubiquitin; transcription; myofibroblast RENAL FIBROSIS IS A COMMON feature of various kidney diseases leading to end-stage renal failure (15,44). This process is characterized by the accumulation of myofibroblasts defined by the expression of ␣-smooth muscle actin (␣-SMA). These cells are major contributors to the increased extracellular matrix deposition seen in kidney fibrosis (16,69). A number of studies demonstrate that renal tubular cells (RTC) can convert to myofibroblasts on epithelial-mesenchymal transition (EMT) stimulated by transforming growth factor-␤ (TGF-␤) (9,11,24,45,69).The regulation of ␣-SMA transcription has been extensively studied in smooth muscle cells and in cells from the myocardium and skeletal muscle, which express ␣-SMA in adults and embryos, respectively (66). Studies on the ␣-SMA promoter from chickens, rats, mice, and humans highlight the importance of cell context and species differences for ␣-SMA transcriptional regulat...

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“…TGF-␤1-induced gene transcription (29,49,50). Smad3 has been shown to interact with MKL1 whereas the MKL1/Smad3 complex activates slug expression (40).…”

Section: Discussionmentioning

confidence: 99%

MKL1 mediates TGF-β1-induced α-smooth muscle actin expression in human renal epithelial cells

Elberg¹,

Chen²,

Elberg³

et al. 2008

American Journal of Physiology-Renal Physiology

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“…This raises a key issue: the overall outcome will depend upon the interplay between Smad3 and its partners, as dictated by the level of Smad3 expression together with the presence and activation state of Smad3-interacting proteins, as determined by the cell type, stimulus and developmental stage. Indeed, Smad3 is a highly promiscuous molecule, which is known to interact with many proteins involved in EMT/EMyT, including Sp1 [Subramanian et al, 2004], ␤ -catenin [Tian and Phillips, 2002], Kruppellike factors [Hu et al, 2007], MRTF [Morita et al, 2007] SRF [Lee et al, 2007] and others [Brown et al, 2008] [Hinz, 2007]. Could this be (partly) related to differences in their Smad3 levels and turnover?…”

Section: Arguments 2: the Sma Promoter Does Contain Sbes And Overexprmentioning

confidence: 99%

“…Interestingly, SRF can directly interact with Smad3 as well [Qiu et al, 2003;Lee et al, 2007]. This complex was proposed to exert enhanced transcriptional activity on CArGs (an effect that may be promyogenic) [Qiu et al, 2003] but reduced the activity on SBE (a mechanism whereby SRF can inhibit the apoptotic effect of Smad3) [Lee et al, 2007]. Another layer of complexity, namely the intrinsic role of the MRTF in EMyT and its interplay with Smad3, will be discussed in the Antithesis section.…”

Section: Thesis: Smad3 Is a Key Mediator Of Emt And Mf Generationmentioning

confidence: 99%

“…Consistent with this interpretation, our promoter studies have shown that a deletion mutant of MRTF, which has diminished capacity to bind Smad3, is much less sensitive to the MRTF-inhibiting action of Smad3. Further, since Smad3 can directly bind to SRF as well [Lee et al, 2007], it is conceivable that an SRF/Smad3 interaction might also contribute to the inhibition of SMA expression. In any case, these results depict Smad3 as an efficient break on the myogenic program.…”

Section: Antithesis: Smad3 Is a Negative Regulator Of The Myogenic Prmentioning

confidence: 99%

“…The SRF/MRTF complex targets the CC(A/T)richTT (CArG) cis-elements or CArG boxes, present in the SMA promoter and in many other cytoskeletal or muscle genes, which together constitute the 'CArGome' [Sun et al, 2006]. Interestingly, SRF can directly interact with Smad3 as well [Qiu et al, 2003;Lee et al, 2007]. This complex was proposed to exert enhanced transcriptional activity on CArGs (an effect that may be promyogenic) [Qiu et al, 2003] but reduced the activity on SBE (a mechanism whereby SRF can inhibit the apoptotic effect of Smad3) [Lee et al, 2007].…”

Section: Thesis: Smad3 Is a Key Mediator Of Emt And Mf Generationmentioning

confidence: 99%

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Smaddening Complexity: The Role of Smad3 in Epithelial-Myofibroblast Transition

Masszi

Kapùs²

2010

Cells Tissues Organs

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Epithelial-mesenchymal transition (EMT) has emerged as a major mechanism in the pathogenesis of organ fibrosis. The epithelium has been proposed to be a significant source of matrix-producing fibroblasts and of myofibroblasts (MFs), a motile and contractile cell type hallmarked by the expression of α-smooth muscle actin (SMA). Importantly, tissue accumulation of MFs shows strong correlation with the severity and progression of fibrotic diseases. The pleiotropic cytokine transforming growth factor-β₁ has been long known as the chief inducer of fibrosis, EMT and MF generation. Accordingly, receptor Smads (Smad2 and particularly Smad3), the direct targets of the activated transforming growth factor-β receptor have been implicated as critical mediators in fibrogenesis and EMT. However, evidence is accumulating that the role of Smad3 is complex and apparently controversial; in fact, Smad3 may differentially affect the various components of EMT, including the loss of epithelial markers (de-epithelialization), the production of extracellular matrix (fibrogenesis) and the expression of SMA (myogenic program). In this review, we revisit the role of Smad3 in epithelial-myofibroblast transition (EMyT). We first summarize the evidence supporting the thesis that Smad3 is a key mediator of EMT and MF generation; next, we present evidence supporting the antithesis that Smad3 is in fact a negative regulator of SMA expression and the activation of the myogenic program in the epithelium; finally, we propose a synthesis, which depicts Smad3 as a timekeeper and context-dependent modulator of EMyT. We suggest that EMyT is composed of an early, mesenchymal, Smad3-promoted phase and a late, myogenic, Smad3-inhibitable phase.

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Uncoupled investigation of scaffold modulus and mesh size on smooth muscle cell behavior

Muñoz-Pinto

Bulick

Hahn

2009

J Biomedical Materials Res

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Although scaffold material properties are known to critically impact cell behavior, it has proven difficult to correlate specific cell responses to isolated scaffold parameters, inhibiting rational design of scaffold material properties. The aim of this study was to validate a systematic approach for evaluating the influence of initial scaffold modulus and mesh size on cell extracellular matrix (ECM) deposition and phenotype. Poly(ethylene glycol) diacrylate (PEGDA) hydrogels were selected for this study because of their tunable material properties. Following screening of six distinct PEGDA hydrogels, three formulations were identified which permitted uncoupled investigation of scaffold mesh size and modulus within the target incremental modulus range of approximately 100-300 kPa. Smooth muscle cells (SMCs) were encapsulated within these three formulations, and cell ECM deposition and phenotype were evaluated following 21 days of culture. Although elastin content appeared to be correlated with scaffold mesh size and modulus to a similar degree, levels of collagen and serum response factor (SRF), a key regulator of SMC phenotype, were more strongly correlated with mesh size. To gain insight into the cell signaling underlying these observed correlations, variations in cell metabolic state and in RhoA signaling were semi-quantitatively evaluated. Both RhoA activity, which is largely modulated by scaffold mechanics in 2D, and cell metabolic activity were highly correlated with hydrogel mesh size. These results indicate that the effects of scaffold mechanics on RhoA activity in 3D may be distinct from those in 2D and underscore the need for uncoupled investigation of scaffold parameters on cell behavior. Furthermore, the present data suggest that RhoA signaling and cell metabolic regulation may be closely linked.

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SRF is a nuclear repressor of Smad3-mediated TGF-β signaling

Cited by 34 publications

References 53 publications

MKL1 mediates TGF-β1-induced α-smooth muscle actin expression in human renal epithelial cells

MKL1 mediates TGF-β1-induced α-smooth muscle actin expression in human renal epithelial cells

Smaddening Complexity: The Role of Smad3 in Epithelial-Myofibroblast Transition

Uncoupled investigation of scaffold modulus and mesh size on smooth muscle cell behavior

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