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Am J Physiol Renal Physiol 284: F911-F924, 2003. First published December 27, 2002; doi:10.1152/ajprenal.00183.2002
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Vol. 284, Issue 5, F911-F924, May 2003

Central role for Rho in TGF-beta 1-induced alpha -smooth muscle actin expression during epithelial-mesenchymal transition

András Masszi1,2, Caterina Di Ciano1, Gábor Sirokmány1, William T. Arthur3, Ori D. Rotstein1, Jiaxu Wang4, Christopher A. G. McCulloch4, László Rosivall2, István Mucsi2,5,6, and András Kapus1

1 Department of Surgery, The Toronto General Hospital and University Health Network, Toronto, Ontario M5G 1L7; 4 Canadian Institutes of Health Research Group in Matrix Dynamics, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada M5S 3E2; 2 Institute of Pathophysiology, Hungarian Academy of Sciences and Semmelweis University Nephrology Research Group, Budapest H-1089; 5 First Department of Internal Medicine, Faculty of Medicine and 6 Faculty of Medicine, Department of Behavioural Sciences, Semmelweis University, Budapest, Hungary H-1083; and 3 Department of Cell and Developmental Biology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

New research suggests that, during tubulointerstitial fibrosis, alpha -smooth muscle actin (SMA)-expressing mesenchymal cells might derive from the tubular epithelium via epithelial-mesenchymal transition (EMT). Although transforming growth factor-beta 1 (TGF-beta 1) plays a key role in EMT, the underlying cellular mechanisms are not well understood. Here we characterized TGF-beta 1-induced EMT in LLC-PK1 cells and examined the role of the small GTPase Rho and its effector, Rho kinase, (ROK) in the ensuing cytoskeletal remodeling and SMA expression. TGF-beta 1 treatment caused delocalization and downregulation of cell contact proteins (ZO-1, E-cadherin, beta -catenin), cytoskeleton reorganization (stress fiber assembly, myosin light chain phosphorylation), and robust SMA synthesis. TGF-beta 1 induced a biphasic Rho activation. Stress fiber assembly was prevented by the Rho-inhibiting C3 transferase and by dominant negative (DN) ROK. The SMA promoter was activated strongly by constitutively active Rho but not ROK. Accordingly, TGF-beta 1-induced SMA promoter activation was potently abrogated by two Rho-inhibiting constructs, C3 transferase and p190RhoGAP, but not by DN-ROK. Truncation analysis showed that the first CC(A/T)richGG (CArG B) serum response factor-binding cis element is essential for the Rho responsiveness of the SMA promoter. Thus Rho plays a dual role in TGF-beta 1-induced EMT of renal epithelial cells. It is indispensable both for cytoskeleton remodeling and for the activation of the SMA promoter. The cytoskeletal effects are mediated via the Rho/ROK pathway, whereas the transcriptional effects are partially ROK independent.

Rho kinase; epithelial-mesenchymal transdifferentiation; transforming growth factor-beta 1; kidney proximal tubule cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TUBULOINTERSTITIAL FIBROSIS is the common pathomechanism whereby a variety of chronic kidney diseases progress to end-stage renal failure (14, 48). The process is characterized by excessive deposition of extracellular matrix (ECM) components and the consequent destruction of normal tissue architecture. A key factor in the underlying pathology is the progressive accumulation of myofibroblasts, the main producers of the ECM (61). Indeed, both clinical studies and animal models indicate a strong positive correlation between the loss of kidney function and the number of myofibroblasts or the expression of alpha -smooth muscle actin (SMA; see Refs. 11, 44, and 68), a hallmark of the myofibroblast phenotype (18, 53). Despite the recognition of their central importance in disease progression, the origin of myofibroblasts has not been elucidated completely. Although in different organs myofibroblasts may derive from smooth muscle cells (49) and resident fibroblasts (23, 45), intriguing new studies suggest that they may also arise from transdifferentiation of the tubular epithelium (19, 66). Recent studies provide evidence that epithelial-mesenchymal transition (EMT) contributes to the generation of kidney fibroblasts during experimental renal fibrosis (30, 42, 58) and that tubular epithelial cells have the ability to transdifferentiate into SMA-positive mesenchymal cells termed as myofibroblasts (19, 40, 66). During EMT, epithelial cells lose their polygonal morphology and adhesive cell contacts and acquire fibroblast-like characteristics, including elongated shape, expression of mesenchymal markers, and increased motility (7, 19, 58). Further transdifferentiation toward the myofibroblast phenotype may ensue, as indicated by the expression of SMA (40, 66). Importantly, similar processes appear to participate in the clinical pathogenesis of kidney fibrosis, as evidenced by the presence of cell populations that stain positively for both epithelial and mesenchymal markers and express SMA (31, 47).

The multifunctional cytokine transforming growth factor (TGF)-beta 1 is a potent inducer of EMT in several tissues (10) and has been shown to provoke SMA production in various cell types (25, 40, 42). Moreover, TGF-beta 1 is both a product and an activator of myofibroblasts (46) and has been identified as a major mediator of kidney fibrosis (9). However, the mechanism whereby TGF-beta 1 treatment of epithelial cells triggers SMA expression remains to be clarified.

A recent study has implicated the small GTPase Rho and its downstream effector Rho kinase (ROK) in TGF-beta 1-induced remodeling of cell contacts in mammary epithelial cells (8). The involvement of Rho in TGF-beta 1 signaling is of particular interest since this small G protein is not only a major organizer of the cytoskeleton (24) but has been shown to regulate gene expression (27, 34, 36, 50). Specifically, Rho has been found to be necessary for the constitutive expression of SMA in smooth muscle cells (34). These observations led us to hypothesize that the Rho/ROK pathway might play an important role in the TGF-beta 1-induced SMA expression in kidney epithelial cells.

The regulation of the SMA promoter is complex (35) and shows substantial tissue specificity (25, 56). Importantly, its basal activity and inducibility markedly differ between smooth muscle cells, fibroblasts, and endothelial cells (25, 56). However, despite the increasingly recognized pathological significance of SMA expression by epithelial cells, the regulation of the promoter has not been hitherto investigated in this cellular context.

To address these issues, we established a renal cell culture model in which TGF-beta 1-induced EMT and SMA expression can be analyzed reliably. Here we show that TGF-beta 1-treated LLC-PK1 proximal tubule cells undergo EMT that manifests in loss of cell contacts, cytoskeleton remodeling, myosin light chain (MLC) phosphorylation, and SMA expression. TGF-beta 1 triggers a biphasic activation of Rho in LLC-PK1 cells. Using various Rho- and ROK-interfering constructs, we provide evidence that Rho, but not ROK, activation is indispensable for the TGF-beta 1-induced SMA promoter activation. Our results show that the first serum response factor (SRF)-binding cis-element (CArG B box) is essential for both Rho inducibility and TGF-beta 1 responsiveness of the SMA promoter in LLC-PK1 cells.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials and Antibodies

DMEM (1,000 mg/l glucose), Hank's balanced salt solution, PBS, FBS, penicillin/streptomycin, and trypsin were from GIBCO-BRL (Burlington, ON). We purchased human recombinant TGF-beta 1 from Sigma (St Louis, MO). The ROK inhibitor compound Y-27632 was from Calbiochem (San Diego, CA). Anti-beta -catenin, anti-MLC, anti-phospho-MLC (specific for the diphosphorylated form of MLC), anti-Myc (9E10), anti-Rho, and anti-SRF antibodies were purchased from Santa Cruz Biotechnology (San Francisco, CA). Anti-E-cadherin was from BD Transduction Laboratories (Mississauga, ON); anti-cortactin and anti-ZO-1 were from Upstate Biotechnology (Lake Placid, NY); anti-alpha -SMA (1E4) was from Sigma; and mouse anti-Histone antibody was from Chemicon. Rhodamine and Alexa (488)-labeled phalloidin were from Molecular Probes (Eugene, OR). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG antibodies were purchased from Amersham Biosciences (Uppsala, Sweden), and anti-goat antibody was from Santa Cruz. FITC and Cy3-labeled anti-goat, anti-rabbit, and anti-mouse secondary antibodies were from Jackson Immunoresearch Laboratories (West Grove, PA). Enhanced chemiluminescence reagent was from Amersham Biosciences.

Cells

LLC-PK1 is a well-characterized and widely used proximal tubular epithelial cell line from the pig (28). LLC-PK1 cells (Cl4) stably expressing the rabbit AT1 receptor were a kind gift from Dr. R. Harris (12). Cells were kept in DMEM containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified incubator at 37°C under 5% CO2. Cells were treated with 4 ng/ml TGF-beta 1 from 30% confluence for the indicated times. Morphological changes were detected by phase-contrast microscopy.

Plasmids

A 765-bp piece of the rat alpha -SMA promoter ligated in a promoterless luciferase vector (PA3-Luc) designated as p765-SMA-Luc was a kind gift from Dr. R. A. Nemenoff (21). The constructs p155/LacZ, p92/LacZ, and p56/LacZ (later designated as p155, p92, and p56) contain the first 155, 92, and 56 bp of the rat alpha -SMA promoter, respectively, and were inserted in a beta -galactosidase vector (pUC19/AUG). The original construct, p547/LacZ was obtained from Dr. G. K. Owens. The thymidine kinase-driven Renilla luciferase vector (pRL-TK; Promega) was used as an internal control. The plasmid encoding the Myc-tagged constitutively active (Q63L) form of RhoA was provided by Dr. G. Downey. Expression vectors encoding the Myc-tagged constitutively active catalytic domain of p160 Rho-associated kinase I (ROK-CAT) and the dominant-negative form of the kinase [ROK-RB/PH (TT) designated here as DN-ROK] were a kind gift of Dr. Kozo Kaibuchi (43). Vectors encoding C3 transferase (4) and p190RhoGAP (62) were described previously. To generate p190RhoGAP-green fluorescence protein (GFP) expression plasmid, the p190RhoGAP insert was excised from pKH3-p190RhoGAP with BamHI and EcoRI and then ligated in pEGFP-C1 that had been digested with BglII and EcoRI (BamHI and BglII are compatible). pEGFP vector was from Clontech Laboratories (Palo Alto, CA), and pcDNA3 was from Invitrogen (Burlington, ON).

Transient Transfection and Reporter Enzyme Assays

Cells were plated on six-well plates 1 day before transfection. At 30% of confluence, cells were transfected with 1 µg of the corresponding DNA using 2.5 µl FuGENE6 (Roche Molecular Biochemicals, Indianapolis, IN). For the SMA-luciferase construct (p765-SMA-Luc), cells were transfected with 0.5 µg promoter plasmid, 0.1 µg pRL-TK, and either 2 µg of the specific construct or 2 µg of the empty expression plasmid (pcDNA3) per well. After a 16-h transfection period, cells were washed with Hank's balanced salt solution and incubated in serum-free DMEM for 3 h. This was followed by treatment with either vehicle or 10 ng/ml TGF-beta 1 (dissolved in 4 mM HCl and 0.1% albumin) for 24 h. Subsequently, the cells were lysed in 250 µl of passive lysate buffer (Promega) and exposed to one cycle of freezing/thawing (-80°C/37°C), and the samples were clarified by centrifugation (13,000 rpm at 4°C, 5 min). Firefly and Renilla luciferase enzyme activities were determined in an aliquot of the supernatant using the Dual-Luciferase Reporter Assay Kit (Promega) and a Berthold Lumat LB 9507 luminometer according to the manufacturer's instructions. Results are expressed as a normalized ratio obtained by dividing the firefly luciferase activity by the Renilla luciferase activity of the same sample. Each transfection was done in duplicate, and determinations for each group were repeated at least three times. For the beta -galactosidase-coupled SMA promoters, cells were transfected with 1 µg promoter construct, 0.1 µg pRL-TK, and 2 µg of either the empty vector or the RhoAQ63L construct per well. We followed a similar time schedule as for the luciferase constructs, but the cells were scraped in 250 µl of 100 mM potassium phosphate buffer (pH 7.8) supplemented with 1 mM dithiothreitol. After three freeze-thaw cycles, the lysates were cleared by centrifugation, and beta -galactosidase activity was determined by the luminescent beta -galactosidase kit (Clontech Laboratories). Renilla luciferase activity was measured from an aliquot of the same sample by the renilla luciferase reporter assay system kit (Promega) following the manufacturer's protocol. The endogenous beta -galactosidase activity of nontransfected cells was determined and subtracted from the total values. The transfection-dependent beta -galactosidase activity was then normalized to the Renilla luciferase activity of the same sample. Cotransfection efficiency was assessed by immunofluorescence. Cells were transfected with GFP and the Myc-labeled construct of interest (RhoAQ63L, ROK-CAT, or DN-ROK) using 0.5 and 2 µg DNA, respectively, and the percentage of Myc-expressing cells in the GFP-expressing population was determined by immunostaining the epitope tag. In agreement with our previous data (59), >= 90% of green cells were Myc positive. In the case of C3 transferase, which was not labeled with an epitope, we used a functional assay. After cotransfection of C3 transferase with GFP, we stained the cells with rhodamine phalloidin and compared the F-actin structure in GFP-positive and -negative cells. Abnormal F-actin structure (stress fiber disruption and major reduction in staining) was observed in 97% of the green cells. Transfection with GFP alone had no effect.

Western Blotting

Cells cultured with or without TGF-beta 1 on 10-cm dishes were scraped in 800 µl ice-cold Triton buffer [30 mM HEPES (pH 7.4), 100 mM NaCl, 1 mM EGTA, 20 mM NaF, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 µl/ml protease inhibitory cocktail (Pharmingen BD Biosciences), and 1 mM Na3VO4]. The samples were clarified by centrifugation at 13,000 rpm for 5 min at 4°C. We added 2× Laemmli buffer [375 mM Tris (pH 6.8), 10% SDS, 20% glycerol, 0.005% bromphenol blue, and 2% beta -mercaptoethanol] to the supernatants and boiled for 5 min. Protein concentrations were determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were separated on 10% SDS-polyacrylamide gels with a Bio-Rad Protean II apparatus and transferred to nitrocellulose membranes (Bio-Rad). Blots were blocked in Tris-buffered saline and 0.1% Tween 20 (TBS-T) containing 5% albumin for 1 h. The membranes were incubated with primary antibody (diluted in TBS-T containing 1% albumin) for 1 h, washed extensively, and incubated with the appropriate peroxidase-conjugated secondary antibody for another hour. After the final washes, immunoreactive bands were visualized by enhanced chemiluminescence reaction. The bands were quantified using a Bio-Rad GS-690 Imaging Densitometer and the Molecular Analyst software (Bio-Rad), as in Ref. 32. Data are presented as representative blots of at least three similar experiments.

MLC Phosphorylation

To detect changes in MLC phosphorylation, the nonphosphorylated, mono- and diphosphorylated forms of MLC were separated using nondenaturating urea-glycerol PAGE. Cells plated on 10-cm dishes and grown until confluence were serum starved for 3 h, treated with vehicle or 10 ng/ml TGF-beta 1 for 4 h, and then lysed in 1.5 ml of acetone containing 10% trichloracetate and 10 mM dithiothreitol. The lysates were spun down, and the resulting pellet was washed in 1 ml pure acetone. The solvent was aspirated, and the pellet was air-dried at room temperature for 1 h. The samples were then dissolved in 200 µl sample buffer [20 mM Tris (pH 8.6), 23 mM glycine, 8 M urea, 234 mM sucrose, 10 mM dithiothreitol, and 0.01% bromphenol blue] with periodic agitation and subjected to urea-glycerol PAGE using a 12% gel containing 40% glycerol, 20 mM Tris (pH 8.6), and 23 mM glycine, as described previously (59). Separated proteins were transferred to nitrocellulose membranes, and Western blotting was performed using an anti-MLC antibody.

Preparation of Glutathione-S-Transferase-Rho-Binding Domain Beads and Measurement of RhoA Activity

This method is a pulldown affinity assay based on the ability of the Rho-binding domain (RBD) of Rhotekin (amino acids 7-89) to selectively bind the GTP-loaded form of Rho. The recombinant glutathione-S-transferase (GST)-RBD protein was prepared from Escherichia coli, as described previously (5) with slight modification. Briefly, DH5alpha E. coli culture transformed with pGEX plasmid encoding GST-RBD was pelleted and dissolved in 10 ml STE buffer [10 mM Tris (pH 8.0), 150 mM NaCl, and 1 mM EDTA] supplemented with 5 mM dithiothreitol, 100 µg/ml lysozyme, 20 µl/ml protease inhibitory cocktail, and 1 mM PMSF. For completing bacterial lysis, we applied two cycles of French Press (900 psi) and added 1% sarcosyl for 10 min. The supernatant was purified by centrifugation, supplemented with 1% Triton X-100, and incubated with 1 ml gluthatione-Sepharose 4B beads (Amersham Pharmacia Biotech) for 1 h at 4°C by constant agitation. The beads were washed three times with 10 ml of STE buffer containing 1% Triton X-100 and then three times with STE buffer alone and stored at 4°C. To determine the short-term effects of TGF-beta 1 on Rho activity, confluent cells were serum starved for 3 h and then exposed to 10 ng/ml TGF-beta 1 for the indicated time periods. To determine the long-term effect of TGF-beta 1, the cells were grown to 60% confluence and then exposed to TGF-beta 1 for 1-3 days. After treatment, the cells were washed with ice-cold PBS, lysed, and scraped in 800 µl lysis buffer [100 mM NaCl, 50 mM Tris (pH 7.6), 20 mM NaF, 10 mM MgCl2, and 1% Triton X-100, supplemented with 0.1% SDS, 0.5% sodium deoxycholate, 20 µl/ml protease inhibitor cocktail, 1 mM Na3VO4, and 1 mM PMSF]. The detergent-insoluble fraction was removed by centrifugation, and the lysates were incubated with 10-15 µg of GST-RBD for 45 min at 4°C. The beads were washed three times with 1 ml lysis buffer supplemented only with 1 mM Na3VO4 and boiled in 25 µl of 2× Laemmli buffer for 5 min. The bead-associated proteins were resolved by 15% SDS-PAGE, and the captured Rho protein was detected by Western blotting using an anti-Rho antibody.

Immunofluorescence Microscopy and Phalloidin Staining

Cells were cultured on 25-mm coverslips and treated with TGF-beta 1 or vehicle for the indicated periods. After the coverslips were washed with PBS, cells were fixed in 4% paraformaldehyde (PFA) for 30 min. PFA was quenched with PBS containing 100 mM glycine, and the coverslips were washed thoroughly with PBS. Cells were permeabilized for 20 min in PBS containing 0.1% Triton X-100. For phalloidin staining, cells were incubated for 1 h with rhodamine-labeled phalloidin in 1:100 dilution. For E-cadherin staining, cells were fixed and permeabilized in ice-cold methanol for 5 min. Nonspecific binding was blocked with 5% albumin in PBS for 1 h. Subsequently, the coverslips were incubated with the primary antibodies for 1 h. After being washed six to eight times with PBS, samples were incubated with the fluorescently labeled secondary antibodies for 1 h. The coverslips were washed and mounted on slides using Fluorescence Mounting Medium (Dako Diagnostics Canada, Mississauga, ON). Samples were viewed using a Nikon Eclipse TE200 microscope (100× objective) coupled to a Hamamatsu cooled charge-coupled device camera (C4742-95) controlled by the Simple PCI software.

Preparation of Nuclear Extracts and EMSAs

Nuclear extracts were prepared according to a modified Digman's method, as described previously (63). The double-stranded CArG B oligonucleotide (5'-GAGGTCCCTATAGGTTTGTG-3') was synthesized and purified commercially (GIBCO-BRL). The EMSA probe was generated by end labeling single-stranded oligonucleotide (20 µM) with 150 µCi [32P]ATP (3,000 Ci/mmol; Mandel) using T4 polynucleotide kinase. The labeled single-stranded oligonucleotide was annealed and purified from unincorporated nucleotide using ProbeQuant TM G-50 Micro columns (Pharmacia Biotech). For EMSAs, the samples were incubated for 30 min at room temperature in 20 µl of a reaction mixture containing 1× binding buffer [10 mM Tris · HCl (pH 7.5), 100 mM KCl, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 5% glycerol], ~50 pg (50,000 cpm) labeled probe, 10 µg nuclear extract, and 0.25 µg poly(dA-dT). Samples were separated by electrophoresis on 4.5% polyacrylamide gels at 150 volts in 45 mM Tris borate and 1 mM EDTA. For supershift assays, 10 µg nuclear extracts were preincubated for 15 min with 2 µl SRF antibody.

Statistical Analysis

Data are presented as representative blots from three similar experiments or as means ± SE for the number of experiments (n) indicated. Statistical significance was determined by Student's t-test or ANOVA (one-way ANOVA; Prism, GraphPad Software).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of TGF-beta 1-Induced EMT in LLC-PK1 Cells

Morphology and cell contact proteins. To assess whether TGF-beta 1 induces characteristic EMT in the proximal tubule cell line LLC-PK1, 20-30% confluent cultures were treated with 4 ng/ml TGF-beta 1, and the subsequent changes were followed by phase-contrast and immunofluorescence microscopy. Vehicle-treated control cells formed islands within which individual cells showed typical polygonal appearance and were tightly attached to each other (Fig. 1A). In contrast, cells treated with TGF-beta assumed an elongated shape, and many cells lost contact with their neighbors (Fig. 1B). These characteristics developed gradually; the effect was discernible after 24 h, whereas after 3 days approx 80% of the cells exhibited fibroblast-like shape. To visualize the reorganization of tight junctions and adherent junctions, cells were immunostained for ZO-1 and for E-cadherin and beta -catenin. In control cells, ZO-1 accumulated at the cell boundary, forming a sharp narrow line, and showed a faint punctate labeling in the cytosol (Fig. lC). On TGF-beta 1 treatment, the peripheral staining became discontinuous, and ZO-1 accumulated in rod-like structures that were perpendicular to the cell membrane (Fig. 1D). TGF-beta 1 caused delocalization of E-cadherin and beta -catenin from the cell periphery (Fig. 1E-H). Moreover, increased cytosolic beta -catenin staining was observed that was frequently accompanied with enhanced perinuclear/nuclear labeling (Fig. 1H). Consistent with this, TGF-beta 1 increased the amount of beta -catenin in the nuclear fraction, as revealed by Western blots (Fig. 1I). In addition to relocalization, TGF-beta 1 induced a marked reduction in the overall expression of both adheren junction proteins (Fig. 1J). However, although a 3-day treatment resulted in a dramatic loss (>80%) of E-cadherin (Fig. 1J, top), the decrease in beta -catenin was of smaller magnitude (Fig. 1J, bottom), consistent with the observed cytosolic/nuclear accumulation of this protein.


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  		Fig. 1.   Transforming growth factor (TGF)-beta 1 induces disassembly of cell-cell contacts and downregulation of junctional proteins in LLC-PK1 cells. Cells grown on tissue culture dishes (A and B) or glass coverslips (C-H) were either treated with vehicle alone (Control; A, C, E, and G) or exposed to 4 ng/ml TGF-beta 1 (B, D, F, and H) for 3 days. Morphological changes were examined by phase-contrast microscopy (A and B). Distribution of the tight junction component ZO-1 (C and D) and the adherens junction proteins E-cadherin (E and F) and beta -catenin (G and H) were visualized by immunofluorescent microscopy, as described in METHODS. For A and B ×10 and for C-H ×100 objectives were used. I: cells were treated with vehicle (-) or 4 ng/ml TGF-beta 1 (+) for 3 days, and nuclear lysates were prepared and then probed for beta -catenin (top). To test for loading of nuclear proteins, the same blot was stripped and reprobed with an anti-Histone antibody (bottom). The effect of TGF-beta 1 on the total amount of E-cadherin and beta -catenin was analyzed by Western blotting (J). Lysates from cells treated as in I and containing an equal amount of protein were subjected to SDS-PAGE, blotted on nitrocellulose, and probed with the corresponding primary and secondary antibodies.

Cytoskeletal reorganization. Next we studied the effect of TGF-beta 1 on cytoskeletal structure. Control LLC-PK1 cells exhibited a strong peripheral F-actin ring with slim central stress fibers (Fig. 2A), whereas TGF-beta 1-treated cells showed a decrease in marginal F-actin but contained much thicker central stress fibers that were mostly oriented parallel to the long axis of the cells (Fig. 2B). TGF-beta 1 has been reported to increase MLC phosphorylation in endothelial cells (29), a process associated with cell contact remodeling (22, 33). Therefore, we analyzed whether a similar phenomenon occurs in LLC-PK1 cells. Staining for the diphosphorylated (active) form of MLC gave only background and nuclear labeling in controls (Fig. 2C) but visualized distinct cytosolic filaments in TGF-beta 1-treated cells (Fig. 2D). This finding was substantiated by biochemical means; with the use of urea-glycerol PAGE, we separated the nonphosphorylated and mono- and diphosphorylated forms of MLC in lysates obtained from control and TGF-beta 1-challenged cells. The various forms were visualized by Western blotting using an anti-MLC antibody that reacts independently of phosphorylation status. Figure 2G shows that TGF-beta 1 exposure resulted in the accumulation of the phosphorylated forms of MLC.


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  		Fig. 2.   TGF-beta 1 induces cytoskeleton reorganization and myosin light chain (MLC) phosphorylation in LLC-PK1 cells. LLC-PK1 cells were treated with vehicle alone (Control; A, C, and E) or 4 ng/ml TGF-beta 1 (B, D, and F) for 3 days and then fixed and permeabilized. A and B: F-actin was visualized by rhodamine-phalloidin staining. C and D: distribution of the diphosphorylated form of MLC (ppMLC) was detected by immunoflourescence microscopy after staining with an antibody specific for ppMLC. This antibody gave nuclear labeling that was not different in control and TGF-beta 1-treated cells. Note the presence of a large number of ppMLC-positive cytosolic fibers that were seen exclusively in the TGF-beta 1-treated cells. (Bar: 10 µm). G: to separate differentially phosphorylated forms of MLC, serum-depleted cells were left untreated (-) or exposed to TGF-beta 1 for 4 h (+) and lysed. Lysates were subjected to nondenaturing urea-glycerol PAGE, followed by Western blotting with a nonphospho-specific anti-MLC antibody, as described in METHODS. Under our conditions, each form of MLC (non-, mono-, and diphosphorylated) ran as a doublet. This was verified by using a monophospho-specific antibody and by inducing maximal MLC phosphorylation with the phosphatase inhibitor calyculin A (data not shown). E and F: cortactin was visualized using a specific monoclonal anti-cortactin antibody. Arrow in F indicates cortactin accumulation in a large lamellopodium.

Mesenchymal transdifferentiation results in a motile phenotype, characterized by leading edge formation (51). To address whether TGF-beta 1 can elicit such an effect in LLC-PK1 cells, we stained the cells for cortactin, a sensitive marker of cortical cytoskeleton dynamics and actin-based motility (64). In untreated cells, cortactin was dispersed evenly in the cytosol with a faint accumulation along the entire cell periphery (Fig. 2E). In TGF-beta 1-treated cells, cortactin distribution became highly polarized, visualizing large lamellipodia that developed in many cells (Fig. 2F). This morphology is suggestive of increased migratory potential of the TGF-beta 1-treated LLC-PK1 cells.

SMA expression. alpha -SMA expression is a marker of myofibroblasts, a cell type that represents an advanced phase of EMT (11, 48, 68). We therefore investigated the effect of TGF-beta 1 SMA protein expression using Western blotting and immunfluorescence microscopy. No SMA was detected in control LLC-PK1 cells, whereas TGF-beta 1 exposure induced strong SMA expression by 3 days, which slightly increased further by 6 days (Fig. 3A). Accordingly, immunofluorescence images showed only weak background staining in control cells, whereas a 3-day exposure to TGF-beta 1 induced intense labeling in approx 60% of the cells (see below). Importantly, the newly synthesized SMA assembled in thick fibers (Fig. 3B). This filamentous pattern was the most robust morphological marker of the effect, since it was observed exclusively in TGF-beta 1-treated cells and was present independent of the overall magnitude of SMA expression.


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  		Fig. 3.   TGF-beta 1 induces alpha -smooth muscle actin (SMA) expression and stimulates SMA promoter activity in LLC-PK1 cells. A: cells were treated with vehicle (-) or 4 ng/ml TGF-beta 1 for the indicated times and lysed. Equal amounts of protein from these lysates were subjected to Western blotting using an anti-SMA antibody. B: control or TGF-beta 1-treated (3 days) cells were fixed and stained for SMA. C: cells grown on 6-well plates were cotransfected with a 765-bp sequence of the rat SMA promoter coupled to the firefly luciferase gene (p765-SMA-Luc, 0.5 µg/well) and with Renilla luciferase (pRL-TK, 0.1 µg/well), as described in METHODS. Later (1 day), the cells were treated with vehicle (Control) or with 10 ng/ml TGF-beta 1 for 24 h. At the end of this period, the cells were lysed, and luciferase activities of the samples were measured by luminometry. TGF-beta 1 caused a 3.52 ± 0.26-fold (n = 12) increase in the normalized p765-SMA-Luc activity (P < 0.05).

To investigate the effect of TGF-beta 1 on SMA gene transcription in a kidney epithelial cell setting, LLC-PK1 cells were cotransfected with a construct encoding a 765-bp sequence of the SMA promoter fused to the firefly luciferase gene (21), along with Renilla luciferase. This cotransfection method provides a highly reliable way to correct for potential differences in transfection efficiency. Figure 3C shows that a 24-h exposure to TGF-beta 1 induced a 3.52 ± 0.26-fold (n = 12) increase in SMA promoter activity, indicating that it rapidly and efficiently stimulated the promoter in LLC-PK1 cells. Taken together, our data show that TGF-beta 1 induced the transformation of LLC-PK1 proximal tubule cells from an epithelial to a mesenchymal/myofibroblast-like phenotype. This EMT manifested in characteristic shape changes, downregulation of tight and adherens junction components, cytosolic and nuclear beta -catenin accumulation, F-actin reorganization, MLC phosphorylation, leading edge formation, and robust de novo SMA synthesis, presumably through transcriptional activation. In contrast to MCT cells (42), epidermal growth factor failed to induce any of the above changes in LLC-PK1 cells (data not shown). This observation suggests that, in terms of EMT, LLC-PK1 cells are selectively responsive to TGF-beta 1.

Role of Rho in TGF-beta 1-Induced SMA Expression and Cytoskeletal Reorganization

Effect of TGF-beta 1 on Rho activity in LLC-PK1 cells. The TGF-beta 1-induced changes in cytoskeletal organization and SMA expression raised the possibility that the small GTPase Rho may be a central mediator of these effects, since Rho is known to increase stress fiber formation and MLC phosphorylation (24) and has been shown to stimulate SMA expression in muscle cells (34). To test this hypothesis, first we measured whether TGF-beta 1 induces detectable changes in the amount of active (GTP-bound) Rho in LLC-PK1 cells. Cell lysates from control and TGF-beta 1-treated cells were incubated with a GST fusion protein containing the RBD of Rhotekin, which selectively captures the active form of Rho (5). TGF-beta 1 exposure caused a rapid and transient increase in the amount of GTP-Rho. The effect was visible after 1 min, peaked around 5 min, and decayed thereafter (Fig. 4A). The cytoskeletal changes (i.e., stress fiber assembly and enhanced MLC phosphorylation) observed after a 3-day TGF-beta 1 treatment raised the possibility that the basal Rho activity might be chronically elevated in the transformed cells. We tested whether, in addition to immediate Rho stimulation, TGF-beta 1 treatment resulted in a later Rho stimulation. Consistent with this notion, we found that, after 24 h of TGF-beta 1 treatment, an elevation in Rho activity was noticeable again compared with the control. This late-onset Rho activation further increased and persisted throughout the whole course (3 days) of the experiment (Fig. 4B). Thus TGF-beta 1 induced a biphasic change in Rho activity in LLC-PK1 cells; a rapid and transient response was followed by a chronic elevation.


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  		Fig. 4.   Rho is activated by TGF-beta 1 in a biphasic manner in LLC-PK1 cells. A: cells grown on 10-cm tissue culture dishes were serum starved for 3 h and then treated either with vehicle (-) or with 10 ng/ml TGF-beta 1 for the indicated times and lysed. To capture active Rho (i.e., GTP bound), the lysates were incubated with glutathione beads covered with a glutathione-S-transferase (GST) fusion protein containing the Rho-binding domain of Rhotekin, as described in METHODS. Beads were then separated by centrifugation, and the captured Rho was detected by Western blotting (Active). To verify that equal amounts of Rho were subjected to the assay, an aliquot of the cell lysates was taken from each sample before incubating the rest with the fusion protein, and the total amount of Rho was determined by Western blotting (Total). In five separate experiments, a 5-min treatment with TGF-beta 1 caused a 1.7 ± 0.3-fold (P < 0.05) increase in the amount of active Rho. B: cells were cultured the presence of 10 ng/ml TGF-beta 1 for the indicated times, after which the samples were processed as in A. Note that, after 24 h of TGF-beta 1 treatment, Rho activation became noticeable again and increased further by days 2 and 3 despite the lower total Rho content of the transformed cells.

Involvement of Rho in the TGF-beta 1-induced responses. Next we addressed whether there is a causal relationship between the observed Rho activation and the subsequent changes in SMA expression and cytoskeleton organization. We applied two approaches. First, we tested whether constitutively active Rho elicits similar cytoskeletal responses in LLC-PK1 cells as observed after TGF-beta 1 treatment. Second, we investigated whether interference with the activation of endogenous Rho could prevent TGF-beta 1-induced cytoskeletal and transcriptional effects. Cells were transfected with a construct encoding a GTPase-defective (and thereby constitutively active) Myc-tagged Rho mutant (RhoAQ63L). Later (2 days), the cells were doubly stained using anti-Myc antibody and either Alexa(488)-phalloidin or anti-diphospho-MLC antibody. RhoAQ63L-expressing cells showed abundant thick stress fibers (Fig. 5A) and substantially increased labeling for diphospho-MLC (Fig. 5B). Having confirmed the efficiency of RhoAQ63L in exerting strong cytoskeletal effects, we tested whether it can drive the SMA promoter by cotransfecting RhoAQ63L with the SMA reporter system. RhoAQ63L provoked a 4.72 ± 0.52-fold (n = 14) increase in SMA promoter activity, indicating that Rho is a potent activator of this construct in LLC-PK1 cells (Fig. 5C).


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  		Fig. 5.   Constitutively active RhoA induces cytoskeletal responses and stimulates the SMA promoter in LLC-PK1 cells. A and B: cells grown on coverslips were transfected with a constitutively active mutant of Myc-tagged RhoA (RhoAQ63L). F-actin structure was visualized by Alexa(488)-phalloidin staining (A), whereas MLC phosphorylation was detected using anti-ppMLC goat primary and FITC-labeled anti-goat secondary antibody (B). To identify successfully transfected cells, the samples were also stained with an anti-Myc mouse primary and a Cy3-labeled anti-mouse secondary antibody (Myc). Arrows on corresponding images (top and bottom) indicate identical cells. Note that RhoAQ63L caused robust stress fiber assembly and led to the formation of ppMLC-positive fibers and increased peripheral ppMLC staining. C: cells grown on 6-well plates were cotransfected with p765-SMA-Luc (0.5 µg/well), pRL-TK (0.1 µg/well), and either 2 µg of the empty vector (Control) or the active Rho mutant (RhoAQ63L), as described in METHODS. SMA promoter activity was detected 48 h later by luminometry. Active Rho induced a 4.72 ± 0.52-fold (n = 14) increase in SMA promoter activity (P < 0.05).

To interfere with endogenous Rho activity before TGF-beta 1 challenge, we used two constructs that inhibit Rho by distinct mechanisms. We expressed either Clostridium botulinum C3 transferase (C3), which selectively ADP-ribosylates and thereby inactivates Rho (27, 65), or the GFP-tagged version of p190RhoGAP that enhances the endogenous GTPase activity of Rho, thereby terminating its action (4). C3 or p190RhoGAP was cotransfected with the SMA reporter system, and 24 h later the cells were exposed to TGF-beta 1 for 1 day. Expression of C3 or p190RhoGAP did not significantly change the basal luciferase expression but strongly inhibited the TGF-beta 1-induced rise in SMA promoter activity (Fig. 6). These findings show that Rho activity is indispensable for the TGF-beta 1-induced upregulation of the SMA promoter.


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  		Fig. 6.   Inhibition of Rho abrogates the TGF-beta 1-induced stimulation of the SMA promoter. Cells were cotransfected with p765-SMA-Luc (0.5 µg), pRL-TK (0.1 µg), and either 2 µg of empty vector (Control) or C3 transferase (C3) or p190Rho GTPase-activating protein (GAP)-green fluorescence protein (GFP; p190RhoGAP). A day after transfection, the cells were serum starved for 3 h and then exposed to vehicle (veh) or 10 ng/ml TGF-beta 1 for 24 h, as indicated. Subsequently, p765-SMA-Luc activity was determined. Values were normalized to the basal activity measured in vehicle-treated control cells. The TGF-beta 1-induced degree of activation (TGF-beta 1/veh) in each group was as follows: control: 3.45 ± 0.38; C3: 1.72 ± 0.18; p190RhoGAP: 1.69 ± 0.25 (for control vs. C3 and control vs. p190RhoGAP P < 0.05).

To discern whether the TGF-beta 1-induced cytoskeletal reorganization and in situ SMA protein expression are also Rho dependent, we compared the effects of TGF-beta 1 in the absence and presence of Rho inhibition. Successful transfection with the C3 construct was verified by expression of the cotransfected GFP (see METHODS), whereas the expression of p190RhoGAP-GFP could be visualized directly. Expression of GFP alone did not interfere with the F-actin structure under control or stimulated conditions (Fig. 7A, left). In contrast, C3 caused a complete loss of stress fibers in untreated cells and prevented the TGF-beta 1-induced remodeling of the F-actin cytoskeleton (Fig. 7A, right). Furthermore, although GFP alone had no effect on the diphospho-MLC staining (Fig. 7B, left), inhibition of Rho prevented the TGF-beta 1-induced accumulation of diphospho-MLC (Fig. 7B, right). Importantly, TGF-beta 1 failed to induce normal SMA upregulation in C3-expressing cells, although it had a strong effect in nontransfected or only GFP-expressing cells (Fig. 8A). To quantify these effects, we determined the percentage of SMA-expressing cells after TGF-beta 1 treatment in nontransfected, GFP-transfected, and GFP plus C3-transfected cells (Fig. 8B). A 3-day exposure to TGF-beta 1 induced SMA expression in 61 ± 9% of control cells. Expression of GFP alone resulted in a modest reduction in the percentage of SMA-positive cells (to 41 ± 6%), whereas cotransfection with C3 abolished SMA expression. These findings clearly indicate that C3 strongly inhibited SMA expression, and the absence of SMA in individual C3-expressing cells cannot be accounted for by the less than complete transformation observed in the control cells. Furthermore, overexpression of p190RhoGAP also abrogated TGF-beta 1-induced SMA protein expression (Fig. 8C). These findings confirmed that Rho is required for endogenous SMA expression in LLC-PK1 cells.


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  		Fig. 7.   Inhibition of Rho prevents the TGF-beta 1-induced stress fiber assembly and MLC phosphorylation in LLC-PK1 cells. Cells grown on coverslips were either cotransfected with C3 transferase (2 µg) and GFP (0.5 µg; to identify the C3-expressing cells) or transfected with GFP (2 µg) alone, as indicated at top. Cells were then incubated without (control) or with 4 ng/ml TGF-beta 1 for 3 days and fixed. Samples were processed to visualize F-actin using rhodamine-phalloidin (A) or anti-ppMLC and a Cy3-labeled anti-goat secondary antibody (B). Arrows indicate identical areas on the corresponding images obtained by the red and green filters. Note that TGF-beta 1 induced robust stress fiber formation and MLC phosphorylation in nontransfected or only GFP-expressing cells, but it failed to do so in C3-expressing cells.



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  		Fig. 8.   In situ SMA expression is abolished by C3 transferase and p190RhoGAP. A: cells were transfected with GFP alone or cotransfected with C3 and GFP, exactly as described in Fig. 7. Cells were then left untreated or exposed to 4 ng/ml TGF-beta 1 for 3 days, fixed, and stained for SMA using monoclonal anti-SMA and a Cy3-labeled anti-mouse secondary antibody. Arrows point to successfully transfected cells and indicate identical areas of the corresponding images. B: quantification of the inhibition of SMA expression by C3. The percentage of SMA expressors was determined in nontransfected, GFP-transfected, or GFP + C3-transfected cells after 3 days of TGF-beta 1 treatment. The results are from 3 independent experiments. In each experiment, 50-100 cells were counted in each category. C: cells were transfected with the p190RhoGAP-GFP construct (2 µg), the expression of which can be directly visualized. Treatments and staining for SMA were performed as in A. Arrows point to successfully transfected cells and indicate identical areas of the corresponding images.

ROK is a downstream effector of Rho that has been implicated as a central mediator of Rho's cytoskeletal effects (3). However, the involvement of ROK in Rho-dependent gene transcription is controversial and remains to be elucidated (13, 34, 38, 50). We therefore aimed to assess the role of ROK in SMA promoter activity. In the first set of experiments, we expressed the Myc-tagged catalytic domain of ROK (ROK-CAT) that acts in a constitutively active manner (43, 59). To verify that ROK-CAT expression was sufficient to exert functional effects, we compared the actin cytoskeleton organization in control and ROK-CAT-expressing (Myc-positive) cells. In ROK-CAT expressors, the actin skeleton showed significant alterations as follows: thick F-actin fibers were formed that radiated from central F-actin foci or patches (Fig. 9A). In contrast to the marked cytoskeletal effects, the same construct caused only marginal changes in SMA promoter activity, inducing a <1.5-fold increase in SMA-luciferase expression (Fig. 9B). Although ROK-CAT in itself was insufficient to mimic the effect of Rho on the SMA promoter, it was conceivable that it might contribute to the TGF-beta 1-induced effect. To address this, we transfected cells with a Myc-tagged kinase-deficient ROK (DN-ROK) that has been shown to act as a dominant-negative mutant (43, 59). DN-ROK caused almost complete stress fiber disassembly and strongly inhibited the cytoskeletal reorganizing effects of TGF-beta 1 (Fig. 9C). In contrast, DN-ROK did not reduce the magnitude of the TGF-beta 1-induced SMA promoter activation and caused only a slight inhibition (approx 25%) when the TGF-beta 1-induced increases were compared between mock-transfected and DN-ROK-transfected cells. Consistent with this finding, long-term pretreatment of the cells with the ROK inhibitor Y-27632 failed to significantly change the SMA promoter activity in TGF-beta 1-stimulated cells and caused only partial inhibition in the TGF-beta 1-induced fold-increase in SMA promoter activation (Fig. 9D). Collectively, these observations suggest that ROK is neither sufficient nor absolutely required for SMA expression, and a substantial part of the TGF-beta 1 effect on SMA transcription appears to be mediated by a Rho-dependent but ROK-independent mechanism.


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  		Fig. 9.   Differential involvement of Rho kinase (ROK) in the TGF-beta 1-induced F-actin reorganization and SMA promoter activation. A: cells were transfected with the Myc-tagged constitutive active catalytic domain of Rho kinase (ROK-CAT, 1 µg). Later (3 days), the cells were fixed and doubly stained for F-actin using Alexa(488)-phalloidin (top) and with an anti-Myc antibody (bottom) to identify ROK-CAT expressors. B: cells were cotransfected with p765-SMA-Luc (0.5 µg), pRL-TK (0.1 µg), and either 2 µg of empty vector (control) or ROK-CAT. p765-SMA-Luc activity was determined 72 h later [control: 1 ± 0.03 vs. ROK-CAT: 1.49 ± 0.17 (n = 6); P < 0.05]. C: cells were transfected with a vector encoding the Myc-tagged dominant-negative form of ROK (DN-ROK, 2 µg), and either left untreated (control, top) or exposed to 4 ng/ml TGF-beta 1 (bottom) for 3 days. After being fixed, the cells were doubly stained as in A. To clearly visualize the differences in the F-actin structure of DN-ROK-expressing cells and their nonexpressing neighbors, the staining for ROK (red) and F-actin (green) is shown separately and also as merged images. Identically oriented arrows point to the same transfected cell. D: cells were cotransfected with p765-SMA-Luc (0.5 µg), pRL-TK (0.1 µg), and either empty vector (control) or the DN-ROK (2 µg) construct. The cells were then treated with vehicle or 4 ng/ml TGF-beta 1 in the absence or presence of 20 µM Y-27632, as indicated. After 24 h, luciferase activity was determined as above. Values were normalized to the basal activity measured in vehicle-treated control cells. The TGF-beta 1-induced degree of activation (TGF-beta 1/veh) in each group was as follows: control: 2.95 ± 0.19; DN-ROK: 2.59 ± 0.12; Y-27632: 1.89 ± 0.05; control vs. DN-ROK is not significant; control vs. Y-27632, P < 0.05.

The regulation of SMA expression is complex, since the promoter contains a variety of positive and negative regulatory sequences, the relative importance of which depends on the cellular context (25, 56). The following experiments were performed to identify the Rho-dependent region of the SMA promoter in LLC-PK1 cells and to investigate the relationship between Rho-inducible and TGF-beta 1-responsive cis elements. We used a beta -galactosidase reporter system containing the first 155 bp of the SMA promoter (p155) and two truncations (p92 and p56) of this sequence. p155 has been shown to confer TGF-beta 1 responsiveness and provide maximal transcriptional activity in various cell types (25, 26, 56). It contains two CArG elements (B and A in 5' right-arrow 3' direction, respectively) that are binding sites for the SRF, a TGF-beta 1 control element (TCE) whose trans factor has not yet been fully identified (1), and a TATA box. The various constructs and the results obtained after their transfection are shown in Fig. 10A. In LLC-PK1 cells, TGF-beta 1 induced a 2.4 ± 0.07-fold (n = 6) increase in the reporter activity of the p155 construct. Importantly, cotransfection with RhoAQ63L resulted in a 5.16 ± 0.07-fold (n = 6) activation of the promoter. This level of stimulation is similar to that obtained using the 765-bp construct, suggesting that Rho responsiveness is confined to the first 155-bp promoter region. The basal activity of p92, which lacks the CArG B box, decreased by approx 50% compared with p155. More importantly, the inducibility of this construct dramatically differed from p155; TGF-beta 1 caused only a slight rise in promoter activity (1.39 ± 0.05-fold, n = 6), whereas the effect of RhoAQ63L was essentially abolished (1.25 ± 0.09-fold increase). A further truncation involving the CArG A box (p56) resulted in complete loss of both basal activity and stimulation either by TGF-beta 1 or RhoAQ63L. These results unambiguously show that the CArG B box is required for the Rho inducibility of the SMA promoter. Furthermore, in agreement with earlier findings obtained with other cell types, the CArG box is also essential for the TGF-beta 1 responsiveness of the SMA promoter (25, 26).


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  		Fig. 10.   CC(A/T)richGG (CArG) B box is essential for the Rho inducibility of the SMA promoter in LLC-PK1 cells. Cells were transfected with 1 µg of the indicated SMA promoter construct (p155, p92, or p56) along with RhoAQ63L (2 µg) or empty vector plus pRL-TK (0.1 µg). Later (1 day), cells were exposed to vehicle or 10 ng/ml TGF-beta 1 for 24 h, as indicated. At the end of this period, the beta -galactosidase and renilla luciferase activities of the samples were determined as described in METHODS. Values were normalized to the basal activity of p155 measured in vehicle-treated cells. The basal activity of p92 was 54.11 ± 3.87% of p155, whereas it dropped to approx 0% in the case of p56 (P < 0.05 for both). The degrees of stimulation caused by TGF-beta 1 treatment or RhoAQ63L expression in each group compared with its own untreated control (veh) were as follows: p155, for TGF-beta 1 2.4 ± 0.07, for RhoAQ63L 5.16 ± 0.07; P < 0.05 for both; p92, for TGF-beta 1 1.42 ± 0.09; P < 0.05; for RhoAQ63L: 1.26 ± 0.09; P = 0.04; p56, nondetectable. B: cells exposed to vehicle or 4 ng/ml TGF-beta 1 for 72 h were lysed, and the lysates were probed by Western blotting using an anti-SRF antibody. C: EMSAs were performed on nuclear extracts from vehicle- and TGF-beta 1-treated (72 h) cells using the CArG B box-specific probe, as described in METHODS. Where indicated, the extracts were incubated with anti-SRF-antibodies. Arrows on bottom show the TGF-beta 1-induced binding of the probe; arrow on top points to the supershifted band. In the absence of anti-SRF antibody, the lower mobility band was not observed, even at longer exposure times.

CArG boxes are targets of SRF, a member of the MAD box transcription factor family that is known to regulate SMA expression (35, 54). To assess whether changes in SRF content might contribute to TGF-beta 1-induced, Rho-dependent SMA expression in LLC-PK1 cells, we analyzed lysates from control and TGF-beta 1-treated cells by Western blotting using an anti-SRF antibody. As shown in Fig. 10B, TGF-beta 1 exposure (72 h) caused a marked increase in SRF content of the cells. The robust increase in cellular SRF may contribute to the in situ activation of the SMA promoter. In support of this notion, TGF-beta 1 treatment resulted in enhanced binding of the CArG B probe to nuclear extracts, and the addition of an anti-SRF antibody induced the appearance of a band with further reduced mobility.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To explore the signaling mechanisms involved in transdifferentiation of renal epithelial cells, we set up a cell culture model using LLC-PK1 cells, a well-known and stable porcine proximal tubule cell line. We found that TGF-beta 1 exposure of LLC-PK1 cells induces an authentic EMT characterized by tight and adherens junction disassembly, nuclear translocation of beta -catenin, increased stress fiber formation, MLC phosphorylation, cortactin redistribution, and the expression of the myofibroblast marker SMA. These findings confirm and extend recent studies that described various aspects of EMT in rat and human proximal tubule cells (19, 66, 67). Together these observations imply that epithelial-mesenchymal transformation is a genuine response of proximal tubule cells on chronic TGF-beta 1 exposure and support the intriguing concept that SMA-positive myofibroblast-like cells can derive from the kidney epithelium itself. Accordingly, TGF-beta 1 may promote renal fibrosis not only by stimulating myofibroblasts but also by inducing their formation from the tubular epithelium. Because myofibroblasts secrete TGF-beta 1 (46), this mechanism could create a positive feedback loop that may contribute to the progressive nature of the fibrotic disease.

Having established the EMT model, we intended to identify key signaling events underlying the cytoskeletal changes and particularly the TGF-beta 1-induced SMA expression. We considered the small GTPase Rho as a candidate to mediate the above TGF-beta 1 effects, since Rho 1) has a central role in stress fiber formation (24), 2) induces MLC phosphorylation via both ROK-mediated direct phosphorylation and inhibition of MLC phosphatase (20), and 3) has recently been implicated in constitutive SMA expression by smooth muscle cells (34). Consistent with this hypothesis, we found that TGF-beta 1 causes a biphasic Rho stimulation in LLC-PK1 cells: a rapid and transient peak, similar to that observed in mammary epithelial cells (8), followed by a late-onset (>12 h) elevation. While our manuscript was prepared for submission, a paper was published by Edlund et al. (15) reporting similar biphasic Rho activation in TGF-beta 1-stimulated prostate cancer cells. The biphasic Rho response is consistent with and may explain the kinetics of other TGF-beta 1-induced signaling events, e.g., the bimodal activation of c-Jun kinase, a process dependent on or potentiated by Rho (6, 17). The mechanism(s) whereby TGF-beta 1 stimulates Rho remains to be clarified. The process may involve rapid activation of guanine nucleotide exchange factors (GEFs) and/or the inhibition of GTPase-activating proteins (GAPs) or GDP dissociation inhibitors (60). Regarding the late phase, several additional mechanisms may be evoked. For example, TGF-beta 1 was found to stimulate the synthesis of NET1, a RhoA-specific GEF (55), and to stabilize RhoB by inhibiting its degradation (16). Importantly, cadherin disengagement has been shown to potently stimulate Rho activity (41). In our case, this may be a crucial factor, since in LLC-PK1 cells TGF-beta 1 induced not only the delocalization of E-cadherin (as in mammary epithelial cells; see Ref. 8) but also a dramatic decrease in the level of this protein.

The functional significance of Rho activation is evidenced by the fact that inhibition of Rho prevented TGF-beta 1-induced stress fiber formation and MLC phosphorylation. Similarly, DN-ROK abrogated F-actin reorganization upon TGF-beta 1 treatment. These results indicate that the Rho/ROK pathway is indispensable for TGF-beta 1-induced cytoskeleton remodeling. Presumably, an increase in contractility is one of the major mechanisms whereby Rho promotes EMT, since elevated cell tension itself has been shown to contribute to contact remodeling (33) and fibroblastic transformation of epithelial cells (69).

The other crucial mechanism appears to be a Rho-dependent change in gene transcription. We found that active Rho strongly stimulated the SMA promoter in LLC-PK1 cells, whereas two Rho inhibitory constructs prevented the TGF-beta 1-induced promoter activation. Interestingly, the dependence of the TGF-beta 1-provoked SMA promoter activation on Rho and ROK markedly differed since ROK caused only marginal promoter activation and DN-ROK exerted only a slight inhibitory effect. In accordance with this, pharmacological inhibition of ROK resulted in only partial reduction in the SMA response. Thus the TGF-beta 1-induced SMA activation is mediated by Rho-dependent but partially ROK-independent mechanisms.

Previous studies have shown that the first 125 bp of the SMA promoter (p125) drive maximal reporter expression in smooth muscle cells and provide moderate basal activity in fibroblasts and endothelial cells (25, 26, 56). This region contains two CC(A/T)richGG (CArG B and A) cis-acting elements and a recently described TCE upstream of a TATA box. The CArG motifs are binding sites for the transcriptional activator SRF, whereas the TCE likely interacts with Kruppel factor-like transactivators (1, 35). Interestingly, intact CArG boxes are essential for the high constitutive expression of SMA in smooth muscle cells (26, 56), but their role is not critical for the basal expression in fibroblasts and endothelial cells (25). In contrast, each functional domain of p125 (or p155) was found to be required for TGF-beta 1 responsiveness (25, 26). Further upstream regions are responsible for suppressing expression in nonmuscle cells and harbor muscle-type specific regulatory sequences. We found that, in epithelial cells, active Rho potently stimulated both the longer (765-bp) and the minimal (p155) promoter constructs. Moreover, the level of activation was similar, suggesting that Rho targets the p155 sequence and acts primarily by driving the minimal promoter rather than removing a constraint acting on the upstream sequences. Consistent with this notion, deletion of CArG B, which reduced but did not eliminate basal transcription, entirely abolished Rho responsiveness. Importantly, Rho and TGF-beta 1 inducibility showed similar structural requirements, supporting the concept that Rho is a key factor in mediating the effect of TGF-beta 1 on SMA transcription. The fact that Rho acts through CArG boxes implicates SRF as the responsible transactivator. We found that TGF-beta 1 markedly increases SRF protein in LLC-PK1 cells, and this effect is likely to contribute to the dramatic rise in SMA synthesis. Our EMSAs support the involvement of SRF in the TGF-beta 1-induced SMA response; however, the participation of additional cis-elements is also possible.

The mechanism whereby Rho activates or induces the expression of SRF is not well understood. Rho has been suggested to stimulate the serum response element of the c-fos promoter and the constitutive expression of the SMA promoter through changes in actin organization (34, 57). It has been proposed that actin monomers inhibit the action of SRF, and Rho relieves this block by promoting actin polymerization. This mechanism may certainly contribute to the TGF-beta 1-induced upregulation of the SMA promoter in LLC-PK1 cells. However, additional factors are likely to participate, since inhibition of Rho or ROK had similar effects on TGF-beta 1-induced cytoskeletal reorganization, but the Rho-inhibitory constructs exerted a stronger inhibition on the SMA promoter than DN-ROK. Consistent with the notion of partial dissociation between cytoskeletal and transcriptional effects, various Rho effector loop mutants that fail to affect the cytoskeleton have been shown to induce strong activation of SRF-dependent transcription (50). Moreover, we found that RhoN19, a dominant-negative mutant that disrupted stress fibers in LLC-PK1 cells, did not prevent the TGF-beta 1-induced SMA response (data not shown). It must be emphasized that the mechanism of action of RhoN19 is different from C3 transferase or p190RhoGAP, since the latter proteins inactivate endogenous Rho itself, whereas RhoN19 competes for activating factors and/or downstream effectors. Therefore, RhoN19 may have a differential capability to interfere with distinct Rho partners and/or Rho isoforms. This notion is substantiated by the finding that RhoN19 was unable to counteract SRF activation induced by certain Rho mutants (50) and had only a moderate inhibitory effect on the phenylephrine-induced c-Fos serum response element activation in myocytes (38). In contrast, RhoN19 potently inhibited the TGF-beta 1-induced dissociation of E-cadherin in mammary epithelial cells (8). Considered together, these observations suggest that partially overlapping but distinct downstream pathways are involved in the Rho-dependent cytoskeletal remodeling and SMA expression and that the cytoskeletal effects may be essential for cell contact remodeling but not for SMA induction.

Although Rho activation is a prerequisite for SMA synthesis, it may not be sufficient in itself, since transfection of active Rho alone was unable to induce significant increases in SMA immunoreactive protein in LLC-PK1 cells (data not shown). Analogous observations were made in fibroblasts, where injection of active Rho induced expression of extrachromosomal SRF reporter genes but not chromosomal templates (2). The most plausible explanation for this phenomenon is the requirement for additional TGF-beta 1 inducible factors. TGF-beta 1 stimulates a multitude of signaling pathways and drives gene expression via several transcriptional activators (37, 39). Interestingly, TGF-beta 1-induced expression of extra domain-A fibronectin was found to precede and be required for SMA expression by myofibroblasts (52). Such requirement for additional factors may also explain why SMA protein expression is seen only after 2-3 days of TGF-beta 1 treatment. Interestingly, our ongoing studies suggest that beta -catenin signaling might also be necessary for efficient SMA expression.

In summary, our work shows that Rho plays a critical role in TGF-beta 1-induced cytoskeleton remodeling and SMA synthesis during epithelial-mesenchymal/myofibroblast transdifferentiation. Future studies should define whether pharmacological interference with the Rho pathway might signify a therapeutically relevant approach to lessen organ fibrosis.


    ACKNOWLEDGEMENTS

We are indebted to Drs. K. Burridge, G. P. Downey, K. Kaibuchi, R. A. Nemenoff, and G. K. Owens for providing various constructs used in this study. We thank Dr. K. Szászi for valuable discussions.


    FOOTNOTES

This work was supported by grants from the Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council of Canada (to A. Kapus), the Heart and Stroke Foundation of Canada (to C. McCulloch), and National Scientific Research Funds (OTKA T026223, T 034409, T029260, ETT 232, and FKFP 0316; to I. Mucsi and L. Rosivall). A. Kapus is a CIHR scholar. A. Masszi was a recipient of the Eötvös Hungarian State Fellowship. I. Mucsi is a Békésy Postdoctoral Fellow of the Hungarian Ministry of Education. W. T. Arthur was supported by General Medical Sciences Grant GM-29860.

Address for reprint requests and other correspondence: A. Kapus, Toronto Hospital, Dept. of Surgery, Transplantation Research, Rm. CCRW 2-850, 101 College St., Toronto, Ontario, Canada M5G 1L7 (E-mail: akapus{at}transplantunit.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published December 27, 2002;10.1152/ajprenal.00183.2002

Received 9 May 2002; accepted in final form 14 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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