You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


Abbreviations used in this paper: α-SMA, α--smooth muscle actin; CA, constitutive active; ChIP, chromatin immunoprecipitation; DN, dominant negative; EMT, epithelial--mesenchymal transition; HMGA2, high mobility group A2; LMB, leptomycin B; MRTF, myocardin-related transcription factor; SBE, Smad-binding element; SRF, serum response factor.

Introduction
============

Epithelial--mesenchymal transition (EMT) is a critical process occurring during embryonic development and in fibrosis and tumor progression ([@bib17]; [@bib37]). TGF-β is a major inducer of EMT, which triggers dissociation of cell--cell contacts and remodeling of the actin cytoskeleton, permitting adherent epithelial cells to scatter and migrate directionally through the extracellular matrix ([@bib49]). Binding of TGF-β to its receptor leads to phosphorylation of its downstream targets Smad2 and 3, and then the phosphorylated Smad2 and 3 form complexes with cytoplasmic Smad4 ([@bib44]). The Smad complexes translocate into the nucleus where they regulate transcription of target genes through binding to specific cis-elements within their promoter regions ([@bib52]; [@bib15]). Recent studies suggest that Smads play a critical role in TGF-β--induced EMT by regulating transcription of their target genes ([@bib51]; [@bib40]). TGF-β is also reported to activate several signaling cascades, such as the extracellular signal--related mitogen-activated protein kinase ([@bib50]), p38 mitogen-activated protein kinase ([@bib47]), phosphatidylinositol 3 kinase ([@bib16]), and Rho pathways ([@bib2]), which also contribute to TGF-β--induced EMT, respectively.

Many kinds of transcription factors, such as zinc finger transcriptional factors Snail ([@bib1]; [@bib7]), Slug ([@bib31]), and basic helix-loop-helix transcription factor Twist ([@bib45]), have been shown as regulators of EMT. They directly repress the transcription of E-cadherin, leading to dissociation of cell--cell contacts. Although TGF-β up-regulates the expression of these EMT regulators in some epithelial cell lines, the molecular mechanism underling their expressions is not fully understood. In this connection, two transcriptional regulators, the hairy/enhancer of split-related transcriptional repressor (Hey1) and high mobility group A2 (HMGA2), have been identified as more upstream regulators of EMT ([@bib51]; [@bib38]). TGF-β1 stimulation rapidly and transiently induces the expression of *hey1* and *hmga2* genes via activation of Smad2/3 signaling, resulting in up-regulation of *snail* and *slug* expressions. It is, however, unclear how Hey1 and HMGA2 are involved in the expression of these EMT regulators.

Myocardin-related transcription factor (MRTF) family members MRTF-A and -B (also known as MAL and MKL1/2) have been reported to be coactivators of serum response factor (SRF)--dependent transcription ([@bib43]; [@bib23]). Myocardin is restrictedly expressed in smooth and cardiac muscles and regulates the differentiation of these muscle types via transactivation of the appropriate differentiation marker genes ([@bib42]). In contrast, MRTFs are broadly distributed in tissues and cells ([@bib43]). Although MRTFs are also involved in muscle differentiation ([@bib32]; [@bib19]), their functions in nonmuscle cells are largely unclear, except for regulation of mammary myoepithelial differentiation ([@bib20]; [@bib35]). The activity of MRTFs is regulated via their nuclear translocation, which is triggered by activation of the Rho signaling pathway ([@bib23]), and TGF-β also affects the subcellular localization of MRTF-A ([@bib11]; [@bib14]). Here, we investigated the involvement of MRTFs in TGF-β1--induced EMT and demonstrated that MRTFs induce the *slug* expression in response to TGF-β stimulation coupling with the Smad pathway. Additionally, MRTFs also regulate reorganization of the actin cytoskeleton mediated through transcriptional activation of actin cytoskeletal genes. Thus, MRTFs are critical mediators for TGF-β1--induced EMT with their dual functions.

Results
=======

Rho-dependent activation of MRTFs by TGF-β1 induces EMT in MDCK cells
---------------------------------------------------------------------

We first examined the location of MRTFs in the *Canis familiaris* kidney epithelial (MDCK) cells, and found both MRTF-A and -B mostly in the cytosol. In response to TGF-β1 stimulation, MRTF-A, but not -B, translocated into the nucleus after 24 h of the stimulation ([Fig. 1 A](#fig1){ref-type="fig"}). The nuclear translocation of MRTFs is reportedly regulated by Rho activity ([@bib23]). In MDCK cells, the Rho inhibitor exoenzyme C3 completely suppressed the nuclear translocation of MRTF-A ([Fig. 1 A](#fig1){ref-type="fig"}). We also monitored endogenous MRTF activity with an SRF-dependent luciferase reporter assay, using a construct containing the SRF-binding cis-elements (3xCArG). TGF-β1 enhanced the reporter activity after 12 to 24 h of stimulation, and Rho kinase inhibitor Y27632 or Rho inhibitor C3 suppressed this activation ([Fig. 1 B](#fig1){ref-type="fig"}). To clarify the MRTF dependency of this promoter activation, MDCK cells were transfected with dominant-negative (DN) MRTF-A lacking its N-terminal RPEL motifs and C-terminal transactivation domain. The DN--MRTF-A protein forms nonproductive heterodimers with endogenous MRTF-A and -B, and therefore inhibits the function of endogenous MRTFs ([@bib8]; [@bib32]). DN--MRTF-A suppressed the TGF-β1--induced activation of the 3xCArG-Luc construct ([Fig. 1 B](#fig1){ref-type="fig"}), suggesting that TGF-β1 enhances the MRTF-A\'s function by promoting its Rho-mediated nuclear translocation.

![**Involvement of MRTFs in TGF-β1--induced EMT.** (A) MDCK cells were cultured with or without TGF-β1 for 24 h. In experiments using Rho inhibitor TAT-C3, 50 μg/ml TAT-C3 was added to the culture medium before TGF-β1 stimulation. The cells were fixed and stained with anti--MRTF-A or -B antibodies (green). The nuclei were stained with Hoechst 33342 (blue). Bars, 20 μm. (B) MDCK cells were transfected with 3xCArG-Luc, with or without pcDNA3.1--DN--MRTF-A or pcDNA3.1-C3. 18 h after the transfection, the cells were treated with TGF-β1 for the indicated number of hours and the luciferase activities were measured. In experiments using Rho kinase inhibitor Y27632, 20 μM Y27632 was added to the culture medium along with TGF-β1. Error bars represent SD from three independent experiments. (C) CA--MRTF-A cells (clone C2), DN--MRTF-A cells (clone D2), and parental MDCK cells (WT) were cultured with or without TGF-β1 for 24 h, and then stained with anti--E-cadherin antibody (green) and Alexa 568--conjugated phalloidin (red). Bars, 20 μm. (D) Phase-contrast images of MDCK and CA--MRTF-A cells (clone C2), which were cultured in DME-FCS without TGF-β1. Bars, 50 μm. (E) MDCK cells were transfected with siRNAs against MRTF-A (MRTF-A siRNA1 or 2) and -B (MRTF-B siRNA1 or 2). Scrambled siRNA was used as the control. After the transfection, the cells were cultured with or without TGF-β1 for 24 h, and then stained with anti--E-cadherin antibody (green) and Alexa 568--conjugated phalloidin (red). Bar, 20 μm. (F) DN--MRTF-A cells (clones D2, D10, and D12) and parental MDCK cells were cultured with or without TGF-β1 (left). CA--MRTF-A (clones C2, C3, and C12) and parental MDCK cells were cultured without TGF-β1 (right). The expression levels of epithelial and mesenchymal marker proteins were compared by immunoblotting. (G) MDCK cells were transfected with MRTF-A and -B siRNAs or control siRNA, and then cultured with or without TGF-β1. The expression levels of MRTF-A and -B and epithelial and mesenchymal marker proteins were compared by immunoblotting.](jcb1791027f01){#fig1}

To analyze MRTFs\' function in EMT, we isolated stable MDCK cell lines expressing constitutive-active (CA) MRTF-A and DN--MRTF-A, respectively. The CA--MRTF-A protein, which lacks its N-terminal RPEL motifs, localizes in the nucleus without TGF-β1 stimulation. The parental MDCK cells grew as monolayers maintained by cell--cell junctions. CA--MRTF-A clones, however, showed a mesenchymal morphology without TGF-β1 stimulation. That is, they grew as scattered, individual cells that formed stress fibers ([Fig. 1, C and D](#fig1){ref-type="fig"}; and Fig. S1, A and B, available at <http://www.jcb.org.cgi/content/full/jcb.200708174/DC1>). In contrast, DN--MRTF-A clones did not respond to TGF-β1 by entering EMT ([Figs. 1 C](#fig1){ref-type="fig"} and S1 C). To confirm the effect of the loss of MRTFs\' function, we designed two siRNAs (siRNA1 and siRNA2) for each MRTF whose targeting sequences are specific to MRTF-A and -B. Consistent with our DN experiments, knockdown of MRTF-A and -B by the siRNAs suppressed the TGF-β1--induced EMT ([Fig. 1 E](#fig1){ref-type="fig"}).

In MDCK cells, treatment with TGF-β1 led to decrease in expression levels of epithelial markers E-cadherin and β-catenin and increase in expression of mesenchymal markers N-cadherin and vimentin ([Fig. 1 F](#fig1){ref-type="fig"}). In contrast, almost no such changes were seen in DN--MRTF-A clones in response to TGF-β1, and CA--MRTF-A clones expressed mesenchymal markers at high levels instead of epithelial markers, even in the absence of TGF-β1 ([Fig. 1 F](#fig1){ref-type="fig"}). Depletion of MRTF-A and/or -B by siRNAs also suppressed the TGF-β1--induced expression changes in epithelial and mesenchymal markers ([Fig. 1 G](#fig1){ref-type="fig"}). Double depletion of MRTFs yielded a more severe phenotype than depletion of either MRTF alone, suggesting that MRTF-A and -B are both important in EMT. As shown in [Fig. 1 A](#fig1){ref-type="fig"}, however, MRTF-B seemed not to translocate into the nucleus upon TGF-β1 stimulation. In various cell types, MRTFs continuously shuttle between the cytosol and the nucleus, and therefore they are mostly observed in the cytosol under steady-state conditions ([@bib48]; [@bib11]; [@bib41]). To clarify the translocation of MRTFs induced by TGF-β1, MDCK cells were treated with the nuclear export inhibitor leptomycin B (LMB). Although both MRTFs remained in the cytosol in the absence of TGF-β1, they translocated to the nucleus when TGF-β1 was added along with LMB ([Fig. 2](#fig2){ref-type="fig"}). Collectively, these data indicate that TGF-β1 enhances the Rho-dependent nuclear translocation of both MRTF-A and -B, resulting in induction of EMT in MDCK cells.

![**Subcellular localization of MRTF-A and -B under the LMB-treated conditions.** MDCK cells were cultured with or without TGF-β1 for 24 h, and then treated with LMB for 1 h. The cells were fixed and stained with anti--MRTF-A and -B antibodies (green), Hoechst 33342 (blue), and Alexa 568--conjugated phalloidin (red). Bar, 20 μm.](jcb1791027f02){#fig2}

MRTFs mediate TGF-β1--induced expression of the slug gene
---------------------------------------------------------

Several transcriptional regulators, including Snail, Slug, and Twist, have been reported to induce EMT through the transcriptional repression of E-cadherin ([@bib31]; [@bib1]; [@bib7]; [@bib45]). In MDCK cells, TGF-β1 markedly induced the expression of *slug*, but not of *snail* or *twist*, within 24 h ([Fig. 3 A](#fig3){ref-type="fig"}). Meanwhile, [@bib27] previously reported TGF-β1--induced *snail* expression in MDCK II cells. We confirmed that TGF-β1 weakly and transiently induced *snail* expression in MDCK II cells, but not in MDCK cells, whereas *slug* expression was highly and continually induced for 3 d in both cell lines ([Fig. 3 B](#fig3){ref-type="fig"}). In our CA--MRTF-A clones, *slug* expression was strikingly higher than that in the parental cells, and the TGF-β1--induced up-regulation of *slug* expression was suppressed in DN--MRTF-A clones and MRTF-A/B--depleted cells ([Fig. 3, C--E](#fig3){ref-type="fig"}). [@bib5] reported that the ectopic expression of *slug* was sufficient to down-regulate epithelial marker expression and up-regulate mesenchymal markers in MDCK cells. Collectively, these results imply that MRTFs activated by TGF-β1 predominantly induce *slug* expression, leading to EMT in MDCK cells. Although TGF-β1 did not increase *twist* expression in MDCK cells, CA--MRTF-A clones exhibited high *twist* expression ([Fig. 3 C](#fig3){ref-type="fig"}). In DN--MRTF-A clones and MRTF-A/B--depleted cells, the basal levels of *twist* expression were lower than those in wild-type cells ([Fig. 3, D and E](#fig3){ref-type="fig"}). Thus, *twist* expression is TGF-β1 independent but MRTF dependent.

![**Expression profiles of the *slug*, *snail*, and *twist* genes.** (A) MDCK cells were cultured with TGF-β1 for the indicated number of hours, and the expression time of EMT-inducible genes (*slug*, *snail*, and *twist*) was analyzed by RT-PCR. (B) MDCK or MDCK II cells were cultured with TGF-β1 for the indicated number of days, and the expression time of *slug*, *snail*, and *twist* genes was analyzed by RT-PCR. (C) The expression levels of *slug*, *snail*, and *twist* genes in CA--MRTF-A (clones C2, C3, and C12) and their parental MDCK cells were compared by RT-PCR. (D) DN--MRTF-A (clones D2, D10, and D12) and their parental MDCK cells were cultured with or without TGF-β1 for 24 h. The expression levels of *slug*, *snail*, and *twist* genes were compared by RT-PCR. (E) MDCK cells were transfected with MRTF-A or -B siRNAs or control siRNA, and the cells were cultured with or without TGF-β1 for 24 h. The expression levels of *slug*, *snail*, and *twist* genes were compared by RT-PCR.](jcb1791027f03){#fig3}

MRTFs and Smad3 cooperatively regulate the *C. familiaris slug* promoter activity via a newly identified cis-element
--------------------------------------------------------------------------------------------------------------------

To determine the regulatory mechanism underlying the transcription of *slug* gene by MRTFs, we analyzed the promoter region of the *C. familiaris slug* gene. Within the ∼2.5-kb fragment upstream of the first exon, we found several candidate cis-elements for SRF, Smad, and SP1 ([Fig. 4 A](#fig4){ref-type="fig"} and Fig. S2, available at <http://www.jcb.org/cgi/content/full/jcb.200708174/DC1>). To identify the MRTF-response elements, a series of 5′ promoter deletion mutants of the *slug* gene (F1--F8) were created. The F1--F6 reporter constructs were equally activated by MRTF-A, but this response was completely eliminated in F7 and F8, indicating that the MRTF-response elements lie between −218 and −293. This region has two types of putative binding sequences: two for SP1 (SP1near and SP1far) and one for Smad (GCCG box--like motif; [Fig. 4 B](#fig4){ref-type="fig"}). The mutational analysis revealed that the GCCG-like and SP1near motifs were important for the basal-level and/or MRTF-dependent activity, and therefore the doubly mutated construct (F6-mutGCCG/SP1near) lost its responsiveness to MRTF-A, and showed the same activity as F7 ([Fig. 4, C and D](#fig4){ref-type="fig"}). The F1 reporter construct was also activated by TGF-β1 stimulation, and depletion of MRTF-A/B by their siRNAs led to decrease in the TGF-β1--induced activation ([Fig. 4 E](#fig4){ref-type="fig"}). Further, the GCCG-like and SP1near motifs were necessary for TGF-β1--induced activation of the *C. familiaris slug* promoter ([Fig. 4 F](#fig4){ref-type="fig"}). Thus, these results indicate that TGF-β1--MRTF signaling activates the *slug* promoter-reporter construct via the GCCG-like/SP1near motifs.

![**Promoter analysis of the *C. familiaris slug* gene.** (A) Luciferase reporter assays using a series of 5′ promoter deletion mutants of the *slug* gene (F1--F8). The ∼2.5-kb fragment upstream of the first exon contains the putative CArG box--like sequences, SBE, and SP1-binding elements. MDCK cells were transfected with these reporter constructs with or without pcDNA3.1--CA--MRTF-A, and the luciferase activities were measured 24 h after the transfection. Error bars represent SD from three independent experiments. (B) Sequence for promoter construct F6, which bears seven SP1-binding elements and the GCCG-like motifs. The core region of the MRTF-A response elements is underlined. (C) Luciferase reporter assay using a series of mutated F6 constructs, in which sites in SP1far, SP1near, and/or the GCCG-like motifs were mutated as indicated in D. Error bars represent SD from three independent experiments. (E) MDCK cells were transfected with MRTF-A and -B siRNAs or control siRNA. After the transfection, the F1 reporter construct was introduced into the cells. The cells were cultured with or without TGF-β1 for 24 h, and then the luciferase activities were measured. Error bars represent SD from three independent experiments. (F) MDCK cells were transfected with the indicated reporter constructs. The cells were then cultured with or without TGF-β1 for 24 h and the luciferase activities were measured. Error bars represent SD from three independent experiments.](jcb1791027f04){#fig4}

To determine whether the GCCG-like and SP1near motifs are sufficient for the TGF-β1 and MRTF responses, nine tandem copies of the sequence between −219 and −244 (core region) were inserted upstream of a reporter gene (9xcore region--Luc; [Fig. 5 A](#fig5){ref-type="fig"}). This reporter construct was significantly activated by TGF-β1, dependent on MRTFs\' expression ([Fig. 5 B](#fig5){ref-type="fig"}). The GCCG motif has been identified as the *Drosophila melanogaster* Smad protein (Mad/Medea)--binding cis-element ([@bib15]). The reporter assay demonstrated that Smad1, 2, or 3 were not activators of 9xcore region--Luc, whereas MRTF-A markedly activated this reporter construct ([Fig. 5, C and D](#fig5){ref-type="fig"}). The coexpression of Smads with MRTF-A, however, slightly increased the activity of this construct in MDCK cells compared with MRTF-A alone ([Fig. 5 C](#fig5){ref-type="fig"}). The synergistic effect between MRTF-A and Smad3 on the activation of 9xcore region--Luc was definitely observed in HepG2 cells ([Fig. 5 D](#fig5){ref-type="fig"}). HepG2 cells showed robust activations of 9xcore region--Luc compared with MDCK cells, which may be because of differences in their cellular contexts, such as endogenous Smad3 and MRTF-A activities and transfection efficacies. Furthermore, depletion of endogenous Smad3 by its siRNAs reduced the responsiveness of 9xcore region--Luc to exogenous expression of MRTF-A ([Fig. 5, E and F](#fig5){ref-type="fig"}). Thus, these results suggest that MRTFs and Smad3 coordinately regulate the transcription of *slug* gene via the core region.

![**Promoter analysis of the 9xcore region--Luc construct.** (A) Schematic representation of the 9xcore region--Luc construct. (B) MDCK cells were transfected with MRTF-A or -B siRNAs or control siRNA. After the transfection, the 9xcore region--Luc construct was introduced into the cells. The cells were cultured with or without TGF-β1 for 24 h, and then the luciferase activities were measured. Error bars represent SD from three independent experiments. (C and D) The 9xcore region--Luc construct was introduced into MDCK (C) or HepG2 (D) cells together with pcDNA3.1-Smad1(3E), pcDNA3.1-Smad2(2E), pcDNA3.1-Smad3(3E), and/or pcDNA3.1--CA--MRTF-A, and the luciferase activities were measured 24 h after the transfection. Error bars represent SD from three independent experiments. (E) HepG2 cells were transfected with two Smad3 siRNAs (Smad3 siRNA1 or 2) or control siRNA, respectively. 24 h after the transfection, the expression levels of Smad3 protein were analyzed by immunoblotting. (F) HepG2 cells were transfected with Smad3 siRNAs or control siRNA. After the transfection, the 9xcore region--Luc construct was introduced into the cells with various amounts of pcDNA3.1--CA--MRTF-A (0 μg \[−\], 0.3 μg \[+\], 0.6 μg \[++\], and 0.9 μg \[+++\]), and the luciferase activities were measured. Error bars represent SD from three independent experiments.](jcb1791027f05){#fig5}

MRTFs bind to the C terminus of Smad3 via their basic domains
-------------------------------------------------------------

Two research groups recently reported that myocardin binds to Smad1 and 3 and activates cardiac and smooth muscle-- specific transcription ([@bib6]; [@bib30]). In MDCK cells, endogenous MRTF-A and -B interacted with Smad3, and TGF-β1 treatment significantly increased their interactions ([Fig. 6 A](#fig6){ref-type="fig"}), suggesting that TGF-β1 not only induces nuclear translocation of MRTFs but also enhances their interactions. To determine the interacting domains of MRTFs and Smad3, deletion series of MRTF and Smad3 proteins were synthesized in vitro and their interactions were analyzed by coimmunoprecipitation. Smad3-C bound more tightly to MRTF-A--full than did Smad3-full, whereas the interaction between Smad3-NL and MRTF-A--full was very weak, indicating that the C-terminal domain of Smad3, including the MH2 domain, is largely responsible for the interaction with MRTF-A ([Fig. 6, B and C](#fig6){ref-type="fig"}). In contrast, Smad3-C bound to MRTF-A--full, MRTF-A N-term, and MRTF-A N-term ΔQ, but not to MRTF-A cent, MRTF-A C-term, or MRTF-A N-term ΔBQ ([Fig. 6, D and E](#fig6){ref-type="fig"}), indicating that the basic domain of MRTF-A is necessary for Smad3 binding. We confirmed that like MRTF-A, MRTF-B also bound to Smad3, and both MRTF-A ΔB and -B ΔB lost Smad3-binding activities ([Fig. 6 F](#fig6){ref-type="fig"}). These results indicate that the basic domain of MRTFs and the MH2 domain of Smad3 interact directly with each other.

![**Interactions between MRTF-A or -B and Smad3.** (A) Coimmunoprecipitation of the MRTFs and Smad3 expressed in MDCK cells. MDCK cells were cultured with or without TGF-β1 for 24 h. Coimmunoprecipitations were performed using nonimmune rabbit IgG (control IgG), anti-Smad3, or anti--MRTF-A or -B antibodies. The proteins in the precipitants were detected by immunoblotting with the indicated antibodies. (B) Schematic representation of the truncated mutants of Smad3. The MH1 domain, linker region, and MH2 domain are shown. The indicated numbers present amino acid positions. (C) HA-Smad3 full, HA-Smad NL, HA-Smad C, and full-length FLAG--MRTF-A proteins were synthesized in vitro, and their interactions were analyzed by coimmunoprecipitation using anti-HA antibody. (D) Schematic representation of the truncation series of MRTF-A and -B proteins. The RPEL motif, basic domain (B), Q-rich domain (Q), SAP domain, coiled-coil domain, and transactivation domain (TAD) are shown. (E) Truncation series of FLAG--MRTF-A proteins and HA-Smad3 C proteins were synthesized in vitro, and their interactions were analyzed by coimmunoprecipitation using anti-FLAG antibody. (F) FLAG--MRTF-A full, -B full, -A ΔB, and -B ΔB and HA-Smad3 full proteins were synthesized in vitro, and their interactions were analyzed by coimmunoprecipitation using anti-HA antibody.](jcb1791027f06){#fig6}

MRTF--Smad3 complex directly binds to the core region of *C. familiaris slug* promoter
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To determine whether the MRTF--Smad3 complexes bind to the DNA sequence within the *slug* core region, DNA--protein binding assay was performed using in vitro*--*translated HA-Smad3, FLAG--MRTF-A, and a biotinylated DNA probe containing the core region sequence. [@bib15] reported that Smad1 and 4 directly bind to the GCCG motif (GCCGnCGC), but Smad3 does not. Our present result, however, showed a significant interaction between Smad3 and the core region probe, which contained the GCCG-like motif (GCCGtCCC; [Fig. 7 A](#fig7){ref-type="fig"}). Expressed alone, MRTF-A did not bind to the DNA probe, although it bound significantly in the presence of Smad3 ([Fig. 7 A](#fig7){ref-type="fig"}). When a nonbiotinylated probe was added as a competitor, this interaction was markedly suppressed. Mutation of the GCCG-like motif in the competitor abrogated this interference, indicating that the MRTF--Smad3 complex binds to the core region of *slug* promoter via a Smad3--GCCG-like motif interaction. Furthermore, MRTF-A enhanced the affinity of Smad3 with the probe in a dose-dependent manner ([Fig. 7 B](#fig7){ref-type="fig"}). We also confirmed the interactions between MRTFs, Smads, and the core region sequence in vivo with chromatin immunoprecipitation (ChIP) assay and clearly showed that MRTF-A/B and Smad3, but not Smad1 or 2, bind to the core region of *slug* promoter in response to TGF-β1 stimulation ([Fig. 7 C](#fig7){ref-type="fig"}).

![**Binding of MRTF/Smad3 to the core region sequence of *C. familiaris slug* promoter.** (A) DNA--protein binding assay was performed using a biotinylated core region DNA probe and in vitro*--*synthesized HA-Smad3 full and FLAG--MRTF-A full. The proteins that bound to the DNA probe were detected by immunoblotting with the indicated antibodies. (B) Synergistic effect of MRTF-A on the binding of Smad3 to the DNA probe containing the core region in a dose-dependent manner. (C) ChIP assay for MRTF-A and -B and Smad1, 2, and 3 binding to the endogenous core region sequence of the *slug* gene. MDCK cells were cultured with or without TGF-β1 for 24 h, and the ChIP assay was performed using nonimmune rabbit IgG (control IgG), anti--MRTF-A and -B, and anti-Smad1, 2, and 3 antibodies. PCR was performed to determine the *slug* promoter sequence surrounding the core region.](jcb1791027f07){#fig7}

MRTFs also regulate reorganization of the actin cytoskeleton in TGF-β1--induced EMT
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During EMT, TGF-β1 induces stress fiber formation instead of disappearance of cortical actin bundles. We demonstrated that TGF-β1--stimulated MDCK cells and CA--MRTF-A clones exhibit marked stress fiber formation with the loss of cortical actin bundles ([Fig. 1 C](#fig1){ref-type="fig"} and [Fig. 8 A](#fig8){ref-type="fig"}). The actin remodeling in TGF-β1--induced EMT appears to be regulated through the Rho pathway, which modulates reorganization of the actin cytoskeleton by actin polymerization and depolymerization ([@bib2]). Unexpectedly, the TGF-β1--induced actin remodeling was completely suppressed by the protein synthesis inhibitor cycloheximide ([Fig. 8 A](#fig8){ref-type="fig"}), suggesting de novo protein synthesis is required. The expression of α--smooth muscle actin (α-SMA), a well-known mesenchymal marker, increased during EMT ([Fig. 8 B](#fig8){ref-type="fig"}). Moreover, we observed the up-regulation of caldesmon and tropomyosin ([Fig. 8 B](#fig8){ref-type="fig"}), which enhance stress fiber formation in various cell lines ([@bib33]; [@bib21]). β-Actin protein slightly increased, but the expression levels of focal adhesion proteins vinculin and talin were unchanged ([Fig. 8 B](#fig8){ref-type="fig"}). Recently, we reported that MRTF-A and -B regulate reorganization of the actin cytoskeleton in fibroblast NIH 3T3 cells through transcriptional regulation of actin cytoskeletal genes, including *caldesmon* and *tropomyosin 1* ([@bib24]). In CA--MRTF-A clones, expression levels of the actin cytoskeletal proteins were markedly increased ([Fig. 8 C](#fig8){ref-type="fig"}). In contrast, the TGF-β1--induced up-regulation of these proteins was severely repressed in DN--MRTF-A clones ([Fig. 8 C](#fig8){ref-type="fig"}). The depletion of MRTF-A/B also repressed the TGF-β1 induction of cytoskeletal proteins ([Fig. 8 D](#fig8){ref-type="fig"}), suggesting that MRTFs activate the transcription of these genes in response to TGF-β1 stimulation. Importantly, the promoter regions of *caldesmon* ([@bib46]), *tropomyosin* ([@bib25]), α-*SMA* ([@bib4]), and β*-actin* ([@bib22]) all contain a CArG box or CArG box--like motif (Fig. S3, available at <http://www.jcb.org/cgi/content/full/jcb.200708174/DC1>) and their transcription is regulated by SRF. In our reporter assay, TGF-β1 enhanced the activities of these promoters in a CArG box--dependent manner ([Fig. 8 E](#fig8){ref-type="fig"}) and these activations were suppressed by DN--MRTF-A ([Fig. 8 F](#fig8){ref-type="fig"}). These results strongly suggest that once activated by TGF-β1, MRTFs also enhance the SRF/CArG-mediated transcription of actin cytoskeletal genes, which leads to remodeling of the actin cytoskeleton.

![**MRTFs regulate actin remodeling induced by TGF-β1 in MDCK cells.** (A) MDCK cells were cultured with or without TGF-β1 and/or cycloheximide (CHX) for 24 h. Cells were then fixed and stained with Alexa 568--conjugated phalloidin. Bar, 20 μm. (B) MDCK cells were cultured with or without TGF-β1 for 24 h. The expression levels of the indicated cytoskeletal proteins were compared by immunoblotting. (C) DN--MRTF-A (clones D2, D10, and D12) and parental MDCK cells were cultured with or without TGF-β1 (left). CA--MRTF-A (clones C2, C3, and C12) and parental MDCK cells were cultured without TGF-β1 (right). The expression levels of the actin cytoskeletal proteins were compared by immunoblotting. (D) MDCK cells were transfected with MRTF-A or -B siRNAs or control siRNA and cultured with or without TGF-β1. The expression levels of MRTF-A and -B and cytoskeletal proteins were compared by immunoblotting. (E and F) Luciferase reporter assays were performed using promoter constructs for the *caldemon*, *tropomyosin 1*, α-*SMA*, and β*-actin* genes that contain a native (WT CArG) or mutated (mut CArG) CArG box (Fig. S3, available at <http://www.jcb.org/cgi/content/full/jcb.200708174/DC1>). MDCK cells were transfected with these cytoskeletal promoter constructs. 18 h after the transfection, the cells were cultured with or without TGF-β1 for 24 h, and the luciferase activities were measured. For F, the pcDNA3.1--DN--MRTF-A vector was transfected along with the cytoskeletal promoter constructs. Error bars represent SD from three independent experiments.](jcb1791027f08){#fig8}

MRTFs regulate TGF-β1--induced EMT in HK-2 and NMuMG cells
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To confirm whether our current model, demonstrated in MDCK cells, is applicable to other cell types, we isolated CA--MRTF-A-- and DN--MRTF-A--expressing stable cell lines of mouse mammary epithelial NMuMG cells and human renal proximal tubular epithelial HK-2 cells. Like MDCK cells, MRTFs expressed in NMuMG were translocated into the nucleus by TGF-β1 stimulation in the presence of LMB, whereas HK-2 cells showed the TGF-β1--induced nuclear translocation of MRTFs even without LMB (Fig. S4, available at <http://www.jcb.org/cgi/content/full/jcb.200708174>). In NMuMG and HK-2 clones expressing CA--MRTF-A, EMT events, namely actin remodeling associated with up-regulation of actin cytoskeletal proteins, dissociation of cell--cell adhesion, and expressional changes from epithelial to mesenchymal markers were observed without TGF-β1 stimulation ([Fig. 9, A--E](#fig9){ref-type="fig"}). In contrast, EMT events induced by TGF-β1 were suppressed in NMuMG and HK-2 clones expressing DN--MRTF-A ([Fig. 9, A--E](#fig9){ref-type="fig"}).

![**MRTF-A and -B induce EMT in mouse NMuMG cells and human HK-2 cells.** (A) NMuMG CA--MRTF-A (clones C4 and C9), DN--MRTF-A (clones D34 and D41), and parental NMuMG cells were cultured with or without TGF-β1 for 24 h and stained with Alexa 568--conjugated phalloidin. Bars, 20 μm. (B) HK-2 CA--MRTF-A (clones C1 and C3), DN--MRTF-A (clones D1 and D3), and parental HK-2 cells were cultured with or without TGF-β1 for 48 h and stained with Alexa 568--conjugated phalloidin. Bars, 20 μm. (C) Phase-contrast images of NMuMG CA--MRTF-A (clones C4 and C9) and HK-2 CA--MRTF-A (clones C1 and C3) cells cultured without TGF-β1. Bars, 50 μm. (D and E) NMuMG (WT), NMuMG CA--MRTF-A (clones C4 and C9), HK-2 (WT), and HK-2 CA--MRTF-A (clones C1 and C3) cells were cultured without TGF-β1 (left). NMuMG (WT), NMuMG DN--MRTF-A (clones D34 and D42), HK-2 (WT), and DN--MRTF-A (clones D1 and D3) cells were cultured with or without TGF-β1 for 24 h (NMuMG cell lines) or 48 h (HK-2 cell lines; right). The expression levels of the indicated proteins were compared by immunoblotting. (F) NMuMG cells were cultured with TGF-β1 for the indicated number of days, and the expression time of *slug*, *snail*, and *twist* genes was analyzed by RT-PCR. (G) NMuMG and NMuMG CA--MRTF-A (clones C4 and C9) cells were cultured without TGF-β1 (left). NMuMG and NMuMG DN--MRTF-A (clones D34 and D42) cells were cultured with or without TGF-β1 for 24 h (right). The expression levels of *slug*, *snail*, and *twist* genes were analyzed by RT-PCR. (H) HK-2 cells were cultured with TGF-β1 for the indicated number of days, and the expression time course of *slug*, *snail*, and *twist* genes was analyzed by RT-PCR. (I) HK-2 and HK-2 CA--MRTF-A (clones C1 and C3) cells were cultured without TGF-β1 (left). HK-2 and DN--MRTF-A (clones D1 and D3) cells were cultured with or without TGF-β1 for 24 h (right). The expression levels of *slug*, *snail*, and *twist* genes were analyzed by RT-PCR.](jcb1791027f09){#fig9}

In NMuMG cells, TGF-β1 up-regulated the expression of *slug*, *snail*, and *twist*, and these up-regulations were maintained for at least 3 d after TGF-β1 stimulation ([Fig. 9 F](#fig9){ref-type="fig"}). Ectopic CA--MRTF-A markedly induced the expression of *slug*, *snail*, and *twist*, whereas ectopic DN--MRTF-A suppressed the TGF-β1--induced expression of these genes ([Fig. 9 G](#fig9){ref-type="fig"}), indicating that TGF-β1--MRTF signaling regulates the induction of all these genes in NMuMG cells. In contrast, HK-2 cells exhibited TGF-β1 induction of *slug* expression but not of *twist* expression ([Fig. 9, H and I](#fig9){ref-type="fig"}). TGF-β1 also increased *snail* expression in HK-2 cells, but this induction was transient, just as observed in MDCK II cells ([Fig. 9 H](#fig9){ref-type="fig"}). In HK-2 cells, ectopic CA--MRTF-A markedly induced *slug* expression, and ectopic DN--MRTF-A suppressed the TGF-β1--induced *slug* expression ([Fig. 9 I](#fig9){ref-type="fig"}). Both CA--MRTF-A and DN--MRTF-A, however, did not affect *snail* and *twist* expressions ([Fig. 5, H and I](#fig5){ref-type="fig"}), suggesting that MRTFs don\'t directly link to their expressions in HK-2 cells. Thus, *slug* expression was closely regulated by the TGF-β1--MRTF signaling in NMuMG and HK-2 cells, just as observed in MDCK cells, whereas *snail* and *twist* expressions were variously regulated in these cell lines.

MRTF--Smad3 complex regulates the human slug promoter activity via GCCG-like motif
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We further analyzed the molecular mechanism of human *slug* transcription by MRTFs and Smad3. Within the ∼1.1-kb fragment upstream of the first *slug* exon, there are several candidate cis-elements, including the GCCG-like motif (GCCGgCCC; [Fig. 10, A and B](#fig10){ref-type="fig"}; and Fig. S5, available at <http://www.jcb.org/cgi/content/full/jcb.200708174/DC1>). Reporter assays using a series of human *slug* promoter constructs bearing point mutations showed that TGF-β1 and MRTF/Smad3 activated the human *slug* promoter mainly via the GCCG-like motif, similar to the *C. familiaris slug* promoter ([Fig. 10, C and D](#fig10){ref-type="fig"}). DNA--protein binding assay revealed that the MRTF--Smad3 complex bound to a DNA probe containing the GCCG-like motif of human *slug* gene, but not to a mutated one ([Fig. 10 E](#fig10){ref-type="fig"}). ChIP assay further showed the binding of MRTF/Smad3 to the GCCG-like motif of human *slug* promoter in a TGF-β1--dependent manner ([Fig. 10 F](#fig10){ref-type="fig"}). In the reporter assay, a Smad-binding element (SBE) appeared to be slightly involved in the activation of *slug* promoter construct by TGF-β1 and MRTF/Smad3 ([Fig. 10, C and D](#fig10){ref-type="fig"}). The results of DNA--protein binding and ChIP assays, however, indicate that the binding of MRTFs/Smad3 to the SBE was faint compared with that to the GCCG-like motif ([Fig. 10, G--I](#fig10){ref-type="fig"}). Thus, the contribution of the SBE to *slug* promoter activity is rather weak. In addition to the GCCG-like motif and SBE, two CArG box--like sequences also contribute to the responsibility of the *slug* promoter construct to exogenous MRTF ([Fig. 10 D](#fig10){ref-type="fig"}), raising a possibility that, in addition to the GCCG-like motif, the CArG box--like sequences may be synergistically involved in the TGF-β1--mediated induction of human *slug* gene.

![**MRTF/Smad3 also regulates the human *slug* promoter activity.** (A) Schematic representation of human *slug* promoter construct and its site-mutated series. Within the ∼1.1-kb fragment upstream of the first exon, a potential SBE element, CArG box--like sequences, and GCCG-like motif exist. (B) Sequence alignment of GCCG-like motifs within the *C. familiaris slug* and human *slug* promoter regions. The indicated numbers present upstream positions of nucleic acid from start codon. (C) HepG2 cells were transfected with the site-mutated reporter constructs. X-axis numbers indicate the numbers of reporter construct presented in A. The cells were cultured with or without TGF-β1 for 24 h, and then luciferase activities were measured. Error bars represent SD from three independent experiments. (D) HepG2 cells were transfected with the site-mutated reporter constructs along with pcDNA3.1--CA--MRTF-A and/or pcDNA3.1-Smad3(3E). X-axis numbers indicate the numbers of reporter construct presented in A. The luciferase activities were measured 24 h after the transfection. Error bars represent SD from three independent experiments. (E) DNA--protein binding assay was performed using a biotinylated DNA probe containing the human *slug* GCCG-like motif and in vitro*--*synthesized FLAG--MRTF-A and HA-Smad3. (F) HK-2 cells were cultured with or without TGF-β1 for 24 h, and ChIP assay was performed using nonimmune rabbit IgG (control IgG), anti-Smad1, 2, and 3, and anti--MRTF-A and -B antibodies. PCR was performed to determine the human *slug* promoter sequence surrounding the GCCG-like motif. (G) DNA--protein binding assay was performed using a biotinylated DNA probe containing the human *slug* SBE and in vitro*--*synthesized FLAG--MRTF-A and HA-Smad3. (H) The amounts of HA-Smad3 protein bound to the DNA probes of human *slug* GCCG-like motif or SBE were compared by DNA--protein binding assay. (I) ChIP assay for MRTF-A and -B and Smad1, 2, and 3 bound to the endogenous SBE motif within the human *slug* gene. PCR was performed to determine the human *slug* promoter sequence surrounding the SBE. (J) Scheme of dual functions of MRTFs in TGF-β1--induced EMT. TGF-β1 stimulation induces the phosphorylation and thereby the nuclear translocation of Smad3. TGF-β1 also activates Rho, which results in translocation of MRTFs into the nucleus. The MRTF--Smad3 complex binds to the GCCG-like motif of *slug* gene and enhances *slug* transcription, resulting in the cells becoming scattered in the absence of cell--cell contacts. In addition, the MRTF--SRF complex enhances the transcription of various actin cytoskeletal genes via the CArG box in their promoter regions, leading to actin remodeling.](jcb1791027f10){#fig10}

Discussion
==========

The studies presented here clearly demonstrated that TGF-β1 triggers the nuclear translocation of MRTFs, which activates the two parallel pathways during EMT ([Fig. 10 J](#fig10){ref-type="fig"}). One pathway up-regulates the expression of EMT-regulating genes, such as *slug*, via MRTFs, Smad3, and GCCG-like motifs, leading to dissociation of cell--cell contacts. The other up-regulates the expression of actin cytoskeletal genes via MRTFs, SRF, and CArG box, resulting in remodeling of the actin cytoskeleton.

Recent studies demonstrate that MRTFs associate with monomeric G-actin through their RPEL motifs, which anchor MRTFs in the cytoplasm ([@bib23]; [@bib28]). Activated Rho reduces the cytoplasmic G-actin pool by enhancing actin polymerization, and then triggers the dissociation of MRTFs from G-actin, resulting in the nuclear translocation of MRTFs. TGF-β1 stimulation enhances the Rho activity in many kinds of epithelial cell lines ([@bib2], [@bib3]; [@bib39]). We demonstrated that TGF-β1 induces the nuclear translocation of MRTF via activation of the Rho pathway. However, MRTF-A began to accumulate in the nucleus 12--24 h after TGF-β1 stimulation, and the CArG promoter construct was also activated by TGF-β1 with a similar time course ([Fig. 1, A and B](#fig1){ref-type="fig"}). These responses are rather slow for a direct regulation by activated Rho, raising the possibility that an additional factor is required for the nuclear accumulation of MRTFs. [@bib41] recently demonstrated that the nuclear/cytosolic localization of MRTF-A and -B is determined by the balance between nuclear import and export. LMB treatment alone did not lead to accumulation of MRTF-A and -B in the nucleus. In TGF-β1--treated MDCK cells, nuclear import of MRTF-A would surpass its nuclear export, and thereby MRTF-A was accumulated in the nucleus ([Fig. 1 A](#fig1){ref-type="fig"}). In our and other research group\'s studies, MRTF-B seems to have a lower ability in nuclear import or a higher ability in nuclear export ([@bib11]; [@bib24]). In fact, nuclear accumulation of MRTF-B was observed in TGF-β1--treated MDCK cells only under the LMB-treated conditions ([Fig. 2](#fig2){ref-type="fig"}). These differences may be caused by different affinities of MRTF-A and -B with nuclear import or export machineries. TGF-β1 is also known to trigger the nuclear translocation of Smad complexes mediated through their phosphorylation ([@bib44]). This raises a possibility that the Smad complexes may take MRTFs into the nucleus through their direct interactions after TGF-β1 stimulation. However, it is generally accepted that the nuclear translocation of Smad complexes is promptly occurring after TGF-β1 stimulation in a Rho- independent manner, suggesting that the nuclear translocation of Smad and MRTF are regulated by different mechanisms.

Recent studies have demonstrated that several transcriptional regulators, such as Slug, Snail, and Twist, are involved in EMT ([@bib31]; [@bib1]; [@bib7]; [@bib45]). Because it is difficult to obtain suitable Slug antibody for immunoblotting, we analyzed the expression level of *slug* mRNA. In MDCK cells, the expression of *slug*, but not of *snail* or *twist*, was induced by TGF-β1 ([Fig. 3, A and B](#fig3){ref-type="fig"}). In MDCK II and HK-2 cells, *slug* and *snail* expressions were induced by TGF-β1, but the *snail* induction was only transient and greatly diminished 2 d after TGF-β1 stimulation ([Figs. 3 B](#fig3){ref-type="fig"} and [9 H](#fig9){ref-type="fig"}). NMuMG cells showed significant induction of all these genes ([Fig. 9 F](#fig9){ref-type="fig"}). Thus, the transcriptional regulation of *snail* and *twist* was different in these cell lines, whereas *slug* expression was induced in common by the TGF-β1--MRTF signaling pathway. In *C. familiaris* and human cells, this *slug* induction was regulated by the same transcriptional mechanism via binding of MRTF/Smad3 to the GCCG-like motif within the *slug* promoter ([Figs. 7](#fig7){ref-type="fig"} and [10](#fig10){ref-type="fig"}). In this study, we also suggested the involvement of MRTF in *snail* and *twist* expression. In MDCK cells, *twist* expression was TGF-β1 independent but MRTF dependent ([Fig. 3](#fig3){ref-type="fig"}). The basal level of *twist* expression was altered in the MRTF loss- or gain-of-function MDCK clones, which may influence their phenotypes ([Fig. 3, C--E](#fig3){ref-type="fig"}). In NMuMG cells, all *slug*, *snail*, and *twist* genes were induced by the TGF-β1--MRTF signaling pathway, even though basal levels of *snail* and *twist* expression were also strongly suppressed by DN--MRTF-A independently of TGF-β1 ([Fig. 9 G](#fig9){ref-type="fig"}). In HK-2 cells, transient induction of *snail* by TGF-β1 was MRTF independent. Because the regulatory modes of *snail* and *twist* expressions are complicated, as demonstrated here, further studies are needed to clarify the involvement of MRTFs in their regulations.

All of the myocardin family members (myocardin and MRTF-A and -B) play important roles in muscle differentiation ([@bib42]; [@bib10]; [@bib32]; [@bib19]). Myocardin is specifically expressed in cardiac and smooth muscles ([@bib42]), and myocardin-null mice die at embryonic day 10.5 with no evidence of vascular smooth muscle differentiation in the dorsal aorta ([@bib18]). Compared with myocardin, MRTF-A and -B are more broadly expressed in various tissues and cells, including nonmuscle cells ([@bib43]). However, their roles, except for the regulation of muscle differentiation, remain largely unknown. Recently, [@bib11] have reported that contact injury or TGF-β1 stimulation induces the nuclear accumulation of SRF and MRTF-A in Lewis lung carcinoma--PK1 (CL4)--proximal tubular cells. They, however, did not discuss MRTF-A\'s role in EMT, except for induction of α-SMA expression. We performed the extensive analyses of MRTFs\' functions in EMT and revealed that MRTFs play dual roles in EMT: direct regulation of *slug* transcription and reorganization of the actin cytoskeleton. These findings provide novel insights into MRTF\'s functions in nonmuscle cells. Recently, *MRTF-A* and *-B* knockout mice were reported on ([@bib26]; [@bib20]; [@bib35]). Although disruption of the *MRTF-B* gene leads to a lethal defect in pharyngeal arch remodeling, *MRTF-A* knockout mice exhibit a deficit only in the differentiation of mammary myoepithelial cells. In this connection, our present results ([Figs. 1 G](#fig1){ref-type="fig"} and [8 D](#fig8){ref-type="fig"}) raise the possibility that MRTF-A and -B in these mice may compensate for one another in the EMT process. In *D. melanogaster*, whose genome contains only a single member of the myocardin family (MAL-D), MAL-D loss-of-function induces defects in an event similar to EMT: the actin rearrangement in and migration of border cells ([@bib34]). This evidence supports the importance of MRTFs in the EMT events.

Smad2 and 3 are major downstream targets of the TGF-β1 pathway. TGF-β1 stimulation immediately induces a nuclear translocation of Smad2/3 via their phosphorylation in a variety of cells ([@bib44]). Several DNA-binding motifs for Smads have been identified. The GCCG motif (GCCGnCGC) has been identified as a consensus binding sequence for *D. melanogaster* Smad homologue Mad/Medea and for bone morphogenetic protein--regulated Smad1 in mammals, but not for TGF-β--regulated Smad3 ([@bib15]). In our present study, we identified the GCCG-like motif (GCCGnCCC) within the *C. familiaris* and human *slug* promoter region as a TGF-β1--MRTF-responsive element to which the MRTF--Smad3 complex can bind ([Figs. 7](#fig7){ref-type="fig"} and [10](#fig10){ref-type="fig"}). Recent studies using Smad3 knockout mice provide accumulating evidences that Smad3 is required for TGF-β1--induced EMT ([@bib51]). In this connection, Hey1 and HMGA2 are identified as direct transcriptional targets of Smad2/3 in TGF-β1--induced EMT ([@bib51]; [@bib38]). Their expressions are immediately and transiently induced 1--8 h after TGF-β1 stimulation, and Hey1 regulates the immediate--early phase of TGF-β1--mediated *snail*, *slug*, and *sip1* inductions in a transient fashion. In our experiments, however, the induction level of *slug* expression was dramatically increased 12--24 h after TGF-β1 stimulation, when the activity of endogenous MRTFs was also increased, and sustained for at least 3 d ([Fig. 1 B](#fig1){ref-type="fig"}; and [Fig. 3, A and B](#fig3){ref-type="fig"}), suggesting that MRTFs and Hey1/HMGA2 would differently regulate TGF-β1--induced EMT.

This is the first paper to identify MRTFs as EMT regulators with dual functions and to uncover the transcriptional mechanism of the *slug* gene by TGF-β1. Although further studies are needed to clarify the roles of MRTFs in EMT during embryonic development and in fibrosis and tumor progression, our present results provide new insights into the molecular mechanism underlying EMT.

Materials and methods
=====================

Materials
---------

TGF-β1 (R&D Systems), protein synthesis inhibitor cycloheximide (EMD), Rho kinase inhibitor Y27632 (EMD), and nuclear export inhibitor LMB (EMD) were purchased. Cell-permeable exonuclease C3, TAT-C3, was generated in *Escherichia coli* BL21 using pGEX-KG TAT-C3 (a gift from S. Narumiya, Kyoto University, Kyoto, Japan), as previously described ([@bib24]). Antitropomyosin (TM311; Sigma-Aldrich), anti--α-SMA (1A4; Sigma-Aldrich), anti--β-actin (AC-15; Sigma-Aldrich), anti-SRF (G-20; Santa Cruz Biotechnology, Inc.), antivinculin (HVIN-1; Sigma-Aldrich), anti-talin (8d4; Sigma-Aldrich), anti--E-cadherin (BD Biosciences), anti--N-cadherin (BD Biosciences), antivimentin (Transformation Research), anti--β-catenin (BD Biosciences), anti--α-tubulin (DM1A; Sigma-Aldrich), anti-Smad3 (mouse monoclonal; M05 \[Abnova\] or rabbit polyclonal \[Invitrogen\]), anti-HA (3F10; Roche), and anti-FLAG (Sigma-Aldrich) antibodies were purchased. Anti--MRTF-A and -B antibodies were produced in New Zealand rabbits using aa 776--901 of human MRTF-A and aa 913--1061 of human MRTF-B as respective antigens and purified by affinity chromatography using their antigens. Anti-caldesmon antibody was generated as previously described ([@bib36]).

Cell culture
------------

MDCK, MDCK II, NMuMG, and HepG2 cells were cultured in DME supplemented with 10% FCS. HK-2 cells were cultured in DME/F-12 supplemented with 10% FCS. In experiments with TGF-β1, cells were cultured in DME containing 2% horse serum with/without 10 ng/ml of TGF-β1 for 24--48 h.

Expression vectors and transfection
-----------------------------------

The coding regions for mouse *MRTF-A* and *-B* and human *Smad1*, *2*, and *3* were amplified by PCR and cloned into the pcDNA3.1(+) expression vector (Invitrogen). CA--MRTF-A ([@bib24]), CA-Smad1 (Smad1(3E); [@bib29]), CA-Smad2 (Smad2(2E); [@bib12]), CA-Smad3 (Smad3(3E); [@bib9]), and DN--MRTF-A ([@bib24]) were constructed as previously described. The coding region of the C3 exoenzyme was amplified by PCR using pGEX-KG-TAT-C3 as a template, and then cloned into pcDNA3.1(+). Cells were transfected with these expression vectors using Lipofectamine 2000 (Invitrogen), TransIT-LT1 (Mirus), or nucleofector (Amaxa Biosystems). To establish stable cell lines expressing CA-- or DN--MRTF-A, MDCK and NMuMG cells were transfected with pcDNA3.1--CA--MRTF-A or pcDNA3.1--DN--MRTF-A and cultured with 100 μg/ml Geneticin (Invitrogen) to isolate drug-resistant clones. Because HK-2 cells are resistant to Geneticin, HK-2 cell lines expressing CA-- or DN--MRTF-A were isolated using Linear Hygromycin Marker (Clontech Laboratories, Inc.).

Luciferase reporter assay
-------------------------

The promoter regions of human *caldesmon* ([@bib46]), *tropomyosin 1* ([@bib25]), α−*SMA* ([@bib4]), and β*-actin* ([@bib22]) genes were amplified by PCR and cloned into pGL3-basic (Promega; Fig. S3). The promoter regions of the *C. familiaris slug* (available from GenBank/EMBL/DDBJ under accession no. [AB300658](AB300658)) and human *slug* (available from GenBank/EMBL/DDBJ under accession no. [AB300659](AB300659)) genes were amplified by PCR and cloned into pGL3-basic. To construct the 9xcore region--Luc vector, the core region sequence (5′-tcgcggccgtcccgggggcggggctg-3′) was synthesized in vitro, and nine copies of the core region fragments were tandemly inserted into pGL3-basic. The 3xCArG-Luc vector was constructed with reference to a previous paper ([@bib13]). In brief, three tandem repeats of the *c-fos* CArG box sequence (5′-attggatgtccatattaggacatct-3′) were synthesized in vitro as a single unit. The TATA box sequence of the *Xenopus* γ*-actin* gene was amplified by PCR and inserted into pGL3-basic together with the synthesized 3xCArG oligonucleotide. These constructs were introduced into MDCK or HepG2 cells along with pSV-βGal (Promega), which was used to normalize the transfection efficiency. 24 h after the transfection, the cells were lysed with Passive lysis buffer (Promega), and the luciferase and β-galactosidase activities were measured using the luciferase assay system (Promega) and Luminescent β-galactosidase detection kit II (Clontech Laboratories, Inc.), respectively.

Immunocytochemistry and microscopy
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Cells grown on coverslips were fixed using 4% paraformaldehyde in PBS for 30 min at room temperature, and then permeabilized with 0.1% Triton X-100 in PBS. The fixed cells were incubated with the primary antibody, followed by the appropriate Alexa 488--conjugated secondary antibodies (Invitrogen). To visualize actin filaments or the nuclei, Alexa 568--conjugated phalloidin (Invitrogen) or Hoechst 33342 (Invitrogen) was added with the secondary antibody solution. The stained cells were mounted in GEL/MOUNT mounting medium (Biomeda) and observed using a microscope (IX70; Olympus) with a 60×/1.25 lens (UPlanFLN; Oil Iris; Olympus) at room temperature. Fluorescent images were acquired with a camera (CoolSNAP HQ CCD; Photometrics) using MetaMorph software (MDS Analytical Technologies) and processed with Photoshop CS software (Adobe).

RT-PCR
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Total RNA was extracted from cells using TRIzol reagent (Invitrogen) and reverse transcribed using PrimeScript Reverse Transcriptase (Takara Bio Inc.). The cDNA was amplified using the following PCR primers: *slug*, 5′-atgccgcgctccttcctgg-3′/5′-gtgtgagttctaatgtgtccttgaag-3′; *snail*, 5′-accttccagcagccctacgac-3′/5′-gcagcgtgtggcttcggatg-3′; *twist*, 5′-cctcggayaagctgagcaagat-3′/5′-ctagtgggacgcggacatgga-3′; and *gapdh*, 5′-tcttcaccaccatggagaagg-3′/5′-gaaggccatgccagtgag-3′.

Coimmunoprecipitation
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Cells were lysed with lysis buffer (1 mM EDTA, 0.1% Triton X-100, 50 mM NaF, 1 mM Na~3~VO~4~, 1 mM β-glycerophosphate, and protease inhibitor cocktail tablets \[Roche\] in PBS, pH 7.4, \[Nakarai Tesque Inc.\]) and incubated on ice for 15 min. After a brief sonication, the lysates were spun at 10,000 *g* for 30 min to remove cell debris. The resulting supernatants were incubated overnight (16--24 h) with the indicated antibodies. After this incubation, protein G--Sepharose (GE Healthcare) was added to the mixtures, which were further incubated for 4 h with gentle agitation. To elute the immunocomplexes, Sepharose beads were washed four times with the lysis buffer, and then SDS sample buffer was added. To examine the interaction between MRTFs and Smad3, MRTF-A and -B, Smad3, and the products of their corresponding deletion constructs were synthesized in vitro using TNT Quick Coupled Transcription/Translocation Systems (Promega). The synthesized proteins were mixed in the lysis buffer in various combinations and their interactions were evaluated by coimmunoprecipitation.

RNA interference
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Cells were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen) and cultured for 2 d before analysis. The siRNA sequences were as follows: *C. familiaris* MRTF-A siRNA1, 5′-caggtgaattatccgaaagta-3′; *C. familiaris* MRTF-A siRNA2, 5′-ctgcgtgcatatcaagaacaa-3′; *C. familiaris* MRTF-B siRNA1, 5′-cccaagaatcctaatgacaaa-3′; *C. familiaris* MRTF-B siRNA2, 5′-accaatgaaattgttccacta-3′; human MRTF-A siRNA1, 5′-gacacctcggaattgcacttt-3′; human MRTF-A siRNA2, 5′-cccgagtcagcgaaccactgc-3′; human MRTF-B siRNA1, 5′-ggacgagagaacaactagtgg-3′; human MRTF-B siRNA2, 5′-gtatgctggaccattcacact-3′; human Smad3 siRNA1, 5′-atcaagggatttcctatggaa-3′; and human Smad3 siRNA2, 5′-aagagattcgaatgacggtaa-3′. In control experiments, scrambled siRNA (Santa Cruz Biotechnology, Inc.) was used.

DNA--protein binding assay
--------------------------

In vitro*--*synthesized FLAG--MRTF-A and/or HA-Smad3 proteins were diluted with 500 μl of DNA affinity precipitation buffer (20 mM Hepes-KOH, pH 7.9, 80 mM KCl, 1 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 10% wt/vol glycerol, and 0.1% Triton X-100) and incubated with 1 μg of biotinylated double-stranded DNA probe and 15 μg of herring sperm DNA for 1.5 h at 4°C. In competition experiments, 50 μg of nonbiotinylated DNA probe or nonbiotinylated mutated DNA probe was also added. Dynabeads M-280 Streptavidin (Invitrogen) was then added to the DNA--protein mixture, and the sample was gently mixed by rotation for 1.5 h at room temperature. The beads were washed with DNA affinity precipitation buffer, and the bound proteins were eluted with SDS sample buffer. The sequences of DNA probes were as follows: core region of *C. familiaris slug* promoter, 5′- gttcgcggccgtcccgggggcgggggctgaaa-3′; mutated core region of *C. familiaris slug* promoter, 5′-gttcgcgaaagtcccgggggcgggggctgaaa-3′; GCCG-like motif of human *slug* promoter, 5′-tcagaggcgccggcccgtccgtct-3′; mutated GCCG-like motif of human *slug* promoter, 5′-tcagaggcaaaggcccgtccgtct-3′; SBE of human *slug* promoter, 5′-aaataattgtctctaaagacccatacaa-3′; and mutated SBE of human *slug* promoter, 5′-aaataattatatctaaaaaaccatacaa-3′.

ChIP assay
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MDCK or HK-2 cells cultured in DME containing 2% horse serum with or without TGF-β1 for 24 h were used in a ChIP assay performed with a ChIP assay kit (Millipore) and anti--MRTF-A and -B and anti-Smad1, 2, and 3 antibodies. The sequences of primers used for the PCR amplification were as follows: *C. familiaris slug* core region, 5′-ccgtcctgcgccgcagcgg-3′ and 5′-ctcctgcgccgactgagcg-3′; human *slug* GCCG-like motif, 5′-cctctcagctgtgattggatc-3′ and 5′-acggcggtccctacagcatc-3′; and human SBE, 5′-gaaactggtagatactgagatgg-3′ and 5′-gaaccaccggacattctctc-3′.

Online supplemental material
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Fig. S1 shows a phenotype of MDCK cell lines expressing CA--MRTF-A (clones C3 and C12) or DN--MRTF-A (clones C10 and C12). Fig. S2 shows the full sequence of the *C. familiaris slug* promoter region used in this paper. Fig. S3 shows full sequences of promoter regions of the actin cytoskeletal genes used in this paper. Fig. S4 shows the nuclear translocation of MRTF-A and -B in response to TGF-β1 stimulation in NMuMG and HK-2 cells. Fig. S5 shows the full sequence of the human slug promoter region used in this paper. Online supplemental material is available at <http://www.jcb.org/cgi/content/full/jcb.200708174/DC1>.

This work was supported by a Grant-in-Aid for Scientific Research (15GS0312) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (K. Sobue).

[^1]: Correspondence to Kenji Sobue: <<PRESIDIO_ANONYMIZED_EMAIL_ADDRESS>>