Cell contact-dependent regulation of epithelial-myofibroblast transition via the rho-rho kinase-phospho-myosin pathway

Abstract

Epithelial-mesenchymal-myofibroblast transition (EMT), a key feature in organ fibrosis, is regulated by the state of intercellular contacts. Our recent studies have shown that an initial injury of cell-cell junctions is a prerequisite for transforming growth factor-beta1 (TGF-beta1)-induced transdifferentiation of kidney tubular cells into alpha-smooth muscle actin (SMA)-expressing myofibroblasts. Here we analyzed the underlying contact-dependent mechanisms. Ca(2+) removal-induced disruption of intercellular junctions provoked Rho/Rho kinase (ROK)-mediated myosin light chain (MLC) phosphorylation and Rho/ROK-dependent SMA promoter activation. Importantly, myosin-based contractility itself played a causal role, because the myosin ATPase inhibitor blebbistatin or a nonphosphorylatable, dominant negative MLC (DN-MLC) abolished the contact disruption-triggered SMA promoter activation, eliminated the synergy between contact injury and TGF-beta1, and suppressed SMA expression. To explore the responsible mechanisms, we investigated the localization of the main SMA-inducing transcription factors, serum response factor (SRF), and its coactivator myocardin-related transcription factor (MRTF). Contact injury enhanced nuclear accumulation of SRF and MRTF. These processes were inhibited by DN-Rho or DN-MLC. TGF-beta1 strongly facilitated nuclear accumulation of MRTF in cells with reduced contacts but not in intact epithelia. DN-myocardin abrogated the Ca(2+)-removal- +/- TGF-beta1-induced promoter activation. These studies define a new mechanism whereby cell contacts regulate epithelial-myofibroblast transition via Rho-ROK-phospho-MLC-dependent nuclear accumulation of MRTF.

Figure 1.

Contact disassembly induces Rho/Rho kinase– dependent myosin phosphorylation and SMA-promoter activation. (A) Confluent LLC-PK1 cell cultures were serum-starved for 3 h and then preincubated with a Ca²⁺-containing NaCl-based medium for 10 min. Subsequently the medium was aspirated and either replaced with the same solution (control) or with a Ca²⁺-free solution containing 1 mM EGTA (no Ca) to rapidly disrupt the intercellular contacts. Five minutes later the cells were lysed, and samples of equal protein content were subjected to the Rho activity assay as described in Materials and Methods. Total Rho was determined from the same lysates. One representative blot of three separate experiments is shown. Densitometry (bars) was performed for each experiment, and Rho activation was expressed as fold increase compared with the control. (B) LLC-PK1 cells were grown on coverslips to confluence, and after various treatments were stained with anti-monophospho-MLC antibody: (a) No treatment; (b) cells were exposed to acute Ca²⁺ removal for 5 min using EGTA as in A; (c and d) for chronic Ca²⁺ removal, the normal, serum-free DMEM was replaced with a nominally Ca²⁺-free DMEM for 24 h. Thirty minutes before Ca²⁺ removal cells were preincubated with vehicle (c) or 10 μM Y-27632 (d), which remained present throughout the whole experiment. To visualize cells, nuclei were stained with DAPI; and (e and e′) cells grown to confluence were transfected with Myc-tagged DN-Rho for 24 h, exposed to nominally Ca²⁺-free conditions for an additional 24 h, and then doubly stained for monophospho-MLC (red) and for the Myc epitope (green). (C) The frequency of peripheral phospho-MLC staining was quantified in control and DN-Rho expressing cells after 24-h incubation in nominally Ca²⁺-free DMEM. Note that more than 60% of controls cells showed peripheral myosin phosphorylation, whereas this response was negligible in DN-Rho expressing cells. (n = 3, in each experiment >60 cells were counted in each cell population). (D) Confluent cells were transfected with pSMA-Luc plus pRL-TK along with either empty vector (pcDNA3.1) or with DN-Rho (see Materials and Methods). After 24 h the cells were incubated in serum-free (Cont) or serum- and Ca²⁺-free DMEM (no Ca) for an additional 24 h, followed by determination of luciferase activity (n = 3). (E) The same conditions as in D, except cells were treated for 30 min before Ca²⁺ depletion with vehicle or 10 μM Y-27632 (n = 3).

Figure 2.

Inhibition of myosin ATPase activity or myosin phosphorylation strongly suppresses the contact disruption–induced activation of the SMA promoter and its potentiation by TGF-β1. (A) Confluent monolayers were transfected with p-SMA-Luc and pRL-TK, and after 24 h were treated with vehicle or 100 μM blebbistatin for 2.5 h. Subsequently the cells were incubated in serum-free, Ca²⁺-containing or Ca²⁺-free DMEM, in the presence or absence of blebbistatin. After 4 h, 10 ng/ml TGF-β1 was added to the samples where indicated. Sixteen hours later the cells were lysed, and their luciferase activity was determined (n = 3). (B) Ca²⁺ removal does not act through increasing receptor availability for TGF-β1. Cells were transfected with the TGF-β1–responsive SBE reporter (p-SBE-Luc) and left untreated or challenged with Ca²⁺ removal, TGF-β1, or the combination of these stimuli as in A. (C) DN-MLC inhibits the Ca²⁺ removal–induced MLC phosphorylation. Cells grown on coverslips in 6-well plates were transfected with Myc-tagged DN-MLC for 24 h, incubated in serum and Ca²⁺-free DMEM for another 24 h, and then fixed and doubly stained for the Myc epitope (green) and phospho-MLC (red). Note the absence of pMLC staining in the clusters of transfected cells. (D) The effect of DN-MLC on the activation of the SMA promoter. Confluent cells were transfected with empty vector (pcDNA3) or DN-MLC, and after 24 h were subjected to Ca²⁺ removal where indicated. Four hours later, 10 ng/ml TGF-β1 was added for 20 h to the indicated samples, followed by lysis and determination of luciferase activity. (E) Cells were transfected with pGL3-SMA-Luc, an alternative vector harboring the same 765-base pairs SMA promoter region as PA3-SMA-Luc. Other conditions were identical as in D. (F) Cells were transfected with wild-type (WT) MLC. Other conditions were identical with D.

Figure 3.

The effect of myosin inhibition on F-actin organization and content. (A) LLC-PK1 cells were either left untreated or exposed to blebbistatin as in Figure 2A, and then fixed and stained with rhodamine phallodin. F-actin arrangement was visualized near the apical surface (top, microvilli) and the ventral surface (bottom, level of stress fibers). (B) Cells were transfected with Myc-tagged DN-MLC as in Figure 2C, and then doubly stained using an anti-Myc antibody (green) and rhodamine phalloidin (red). No obvious differences were observed in the organization of F-actin between control and DN-MLC–expressing cells. (C) Quantification of the cellular F-actin content. Cells were treated with blebbistatin (blebbi) as in Figure 2A or with 10 μM latrunculin B (LB) for 2 h (as a positive control), and their total F-actin content was determined with the phalloidin extraction assay as described in Materials and Methods. To assess the effect of DN-MLC, transiently transfected cells were exposed to a selection procedure using G-418, until >85% transfection efficiency was achieved. The total F-actin in these cells was then measured as above. (D) Total actin was assessed by Western blotting under the same conditions as in C.

Figure 4.

Contact disassembly facilitates the nuclear accumulation of serum response factor (SRF) in a Rho and MLC phosphorylation–dependent manner. (A) Confluent monolayers were serum-starved for 3 h and then bathed in Ca²⁺-free DMEM for the indicated times. Cells were then fixed and stained for SRF. (B) Nuclear extracts were prepared from Control or Ca²⁺-deprived (3 h) cells followed by Western blotting for SRF and H3 histones as a nuclear marker. (C) Confluent cells were transfected with constitutive active Myc-tagged Rho (CA-Rho) for 24 h, and then doubly stained for SRF (red) and Myc (green). Note the robust nuclear accumulation of the transfected cells compared with their nontransfected neighbors. (D) Cells were transfected with Myc-tagged dominant negative Rho (DN-Rho) for 24 h followed by incubation in nominally Ca²⁺-free DMEM for another 24 h. Cells were then fixed and stained for SRF (red) and Myc (green). To facilitate the identification of the same cells on the two corresponding fluorescent images, successfully transfected cells or clusters of cells are circled with dashed lines. Note the substantial reduction in the nuclear SRF staining of DN-Rho–expressing cells. (E) Conditions were as in D, except the cells were transfected with Myc-tagged DN-MLC. (F) The intracellular distribution of SRF was quantified by measuring the nucleo-cytoplasmic ratio of the fluorescence intensity. For each cell determinations were made along lines drawn across the nucleus (see dashed line in E). The ratios were calculated for control (pcDNA3) and DN-MLC–transfected cells, which were incubated either in Ca²⁺-containing or nominally Ca²⁺-free medium for a day. In each category at least 60 cells were analyzed. Ca²⁺ removal significantly enhanced the nuclear accumulation of SRF (p < 10⁻¹⁰), and this effect was significantly suppressed (p < 10⁻⁶) by DN-MLC.

Figure 5.

The impact of the Rho-F-actin pathway and cell contact injury on the localization of MRTF isoforms in epithelial cells. (A) CHO cells or LLC-PK1 cells were transfected with either FLAG-tagged MRTF-A or -B and 2 d later stained with an anti-FLAG antibody. FLAG-expressing CHO cells (145 and 167 cells for MRTF-A and -B, respectively) and LLC-PK1 cells (335 and 3606 for MRTF-A and -B, respectively) were counted for nuclear, even or cytosolic distribution. These categories were objectified as described in Materials and Methods. Typical examples of distribution are shown on the right panels. (B) Confluent layers of LLC-PK1 cells were cotransfected with FLAG-MRTF-B and GFP or GFP-CA-Rho, and 48 h later analyzed for intracellular distribution of FLAG staining. Note that CA-Rho induced large nuclear accumulation of MRTF-B. (C) Cells were either transfected with FLAG-MRTF-B alone or along with p190 RhoGAP, and after 48 h, cells were serum-deprived for 3 h. Subsequently, the medium was replaced with Ca²⁺-free DMEM where indicated, and after 2.5 h incubation the cells were fixed and stained with an anti-FLAG antibody. Data are from 3 to 9 separate experiments, and in each category 300-1800 cells were counted. Ca²⁺ removal raised the percentage of cells with fully nuclear distribution from 10.1 ± 1.8 to 26.2 ± 5.1% (p < 0.005, n = 9), and this effect was entirely prevented by Rho-GAP. (D) In a and a′ typical images showing the entirely nuclear accumulation of MRTF-B (FLAG-staining, red) in a GFP-CA-Rho–expressing cell (green); (b) jasplakinolide (Jas) treatment (0.5 μM for 12 h) induces strong nuclear accumulation of the transfected MRTF-B.

Figure 6.

Jasplakinolide or the overexpression of MRTF isoforms is sufficient to induce SMA protein synthesis in tubular cells. (A) Cells were transfected with the pSMA-Luc/pRL-TK system for 24 h and either left untreated for a day, or treated with jasplakinolide (Jas, 0.5 μM) for 24 h or for the last 3 h of this 24-h period. SMA promoter activity is expressed as fold increase compared with the untreated sample. (B) Cells were left untreated or exposed to Jas for 24 h, lysed, and subjected to Western blotting using an anti-SMA antibody. (C) Tubular cells were cotransfected with the pSMA-Luc/pRL-TK and either MRTF-A or -B. Twenty-four hours later SMA promoter activity was determined as in A. (D) Untransfected controls (none) or cells transiently transfected with MRTF-A or -B for 48 h were lysed, and subjected to SDS-PAGE followed by Western blotting using an anti-SMA antibody. Control cells do not express SMA, whereas both MRTF-A and -B were able to induce SMA expression. The response was stronger in the case of MRTF-A in agreement with the strong nuclear localization and greater SMA promoter–activating capacity of this construct. (E) Cells were transfected with MRTF-A or -B, and after 48 h, fixed and stained for SMA (red), FLAG (green) to visualize successful transfection, and the nuclear dye DAPI to visualize every cell.

Figure 7.

Contact disassembly induces Rho- and MLC phosphorylation–dependent nuclear translocation of endogenous MRTF in epithelial cells. (A) In a and b cells were serum-deprived for 3 h and then placed into either Ca²⁺-containing or Ca²⁺-free DMEM for 24 h. Cells were then fixed and stained for endogenous MRTF using a polyclonal antibody raised against BSAC, the mouse MKL1 or MRTF-A protein. (c and c′) Cells were transfected with Myc-tagged CA-Rho and 24 h later fixed and stained for endogenous MRTF (red) and Myc (green). Note the large nuclear accumulation of MRTF in the CA-Rho-expressing cells. (d–d″) Cells were transfected with Myc-tagged DN-Rho and 24 h later subjected to Ca²⁺ removal for 24 h, fixed, and stained for Myc (green), endogenous MRTF (red), and all nuclei were visualized with DAPI (blue). Note the reduced nuclear accumulation of MRTF in the DN-Rho–expressing cells compared with their untransfected neighbors. (e–e″) Similar experiments were performed as in d, except the cells were transfected with DN-MLC. Note the substantial nuclear translocation in many nontransfected cells and the preservation of nuclear exclusion in the DN-MLC–expressing cells. (B) Distribution of endogenous MRTF was quantified in each transfected group. The number of evaluated cells was as follows: control, 283; No Ca, 438; CA-Rho, 52; DN-Rho, 52; DN-MLC, 224.

Figure 8.

The effect of TGF-β1 in confluent and subconfluent layers on the intracellular distribution of endogenous MRTF and MLC phosphorylation. (A) Cells were grown to 100% confluence or approx. 30% confluence (subconfluent) and left untreated and fixed or treated with 10 ng/ml TGF-β1 for the indicated times and then fixed and stained for MRTF. The bar diagram on the right indicates the intracellular distribution of endogenous MRTF in cells at the periphery of cellular islands, under control conditions or after treatment for the indicated times with TGF-β1. (B) Confluent or subconfluent layers were left untreated or exposed to TGF-β1 for 16 h and then fixed and stained for pMLC. Nuclei were visualized by DAPI. (C) The acute effect of Ca²⁺ removal and TGF-β1 on myosin phosphorylation. Cells were treated with normal or EGTA-containing medium in the absence or present of 10 ng/ml TGF-β1 for 15 min and then processed for Western blotting with the anti-pMLC antibody as described in Materials and Methods. Tubulin was used as loading control. (D) A wound was generated in a confluent monolayer with a rubber policeman, and 6 h later the cells were fixed and stained either for MRTF or for pMLC. (E) Cells were seeded sparsely, transfected with Myc-tagged DN-MLC, and then treated daily with 10 ng/ml TGF-β1 for 3 d. Cells were then fixed and doubly stained for SMA and for the Myc epitope. Note the robust SMA expression in the control cells and the absence of SMA expression in the DN-MLC–expressing cells. To quantify the effect, three separate experiments were performed, in which 910 randomly selected control (nontransfected) cells and 311 DN-MLC–expressing cells were assessed for SMA expression (p < 2.0 × 10⁻⁵).

Figure 9.

Dominant negative MRTF inhibits the contact disassembly–induced activation of the SMA promoter and suppresses the synergism between contact disruption and TGF-β1. (A) Localization of DN myocardin (DN-MyoC). Cells transfected with FLAG-tagged DN-MyoC for 24 h were serum-starved, incubated in Ca²⁺-containing or Ca²⁺-free medium for 24 h, fixed, and stained using an anti-FLAG antibody. Note the predominantly nuclear localization of the construct irrespective of the state of the intercellular contacts. (B) Cells were cotransfected with pSMA-Luc/pRL-TK along with either empty vector (pcDNA3) or DN-MyoC for 24 h, and then exposed to Ca²⁺ removal, 10 ng/ml TGF-β1, or the combination of these treatment as in Figure 2D.

Figure 10.

The synergistic effect of cell contact injury and TGF-β1 in the complex regulation of the SMA promoter. Contact disassembly activates Rho which, in turn stimulates mDia (1; Copeland and Treisman, 2002) and ROK (2). The former process leads to increased actin polymerization, whereas the latter activates the LIMK/cofilin pathway (2a) and stimulates MLC phosphorylation (2b). The LIMK/cofilin pathway may stabilize F-actin via decreased severing (Geneste et al., 2002). Enhanced MLC phosphorylation may contribute to SMA expression by acting through various nonexclusive mechanisms: it might promote actin polymerization/stabilization (i), may directly participate in the nuclear translocation or retention of MRTF (ii), or might be required for the internalization of cell contact components (iii; Ivanov et al., 2004). Increased nuclear accumulation of MRTF acts in concert with SRF through the CArG boxes. In addition, contact injury triggers the internalization of β-catenin, which—through yet unidentified mechanisms—potentiates the activation of the SMA promoter (Masszi et al., 2004). Finally, TGF-β1 activates a multitude of signaling pathways, which through various transcription factors impact on the TCE and SBE cis elements. TGF-β1 may also contribute to Rho activation; however, this effect in itself is insufficient to provoke long-lasting MLC phosphorylation, which has a permissive effect on the activation of the SMA promoter. The underlined processes or pathways are addressed in the current study. Question marks denote potential mechanisms. The relative contribution of the primary, permissive, and potentiating mechanisms remains to be defined.