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
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ABSTRACT |
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New
research suggests that, during tubulointerstitial fibrosis,
-smooth muscle actin (SMA)-expressing mesenchymal cells might derive
from the tubular epithelium via epithelial-mesenchymal transition
(EMT). Although transforming growth factor-
1
(TGF-
1) plays a key role in EMT, the underlying cellular
mechanisms are not well understood. Here we characterized
TGF-
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-
1 treatment caused delocalization and downregulation
of cell contact proteins (ZO-1, E-cadherin,
-catenin), cytoskeleton
reorganization (stress fiber assembly, myosin light chain
phosphorylation), and robust SMA synthesis. TGF-
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-
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-
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-1; kidney proximal tubule
cells
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INTRODUCTION |
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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
-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)-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-
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-
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-1-induced remodeling of
cell contacts in mammary epithelial cells (8). The
involvement of Rho in TGF-
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-
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-1-induced EMT and SMA expression can be
analyzed reliably. Here we show that TGF-
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-
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-
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-
1 responsiveness of the SMA
promoter in LLC-PK1 cells.
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METHODS |
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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-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-Plasmids
A 765-bp piece of the ratTransient 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-Western Blotting
Cells cultured with or without TGF-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-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, DH5Immunofluorescence Microscopy and Phalloidin Staining
Cells were cultured on 25-mm coverslips and treated with TGF-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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Characterization of TGF-1-Induced EMT in
LLC-PK1 Cells
Morphology and cell contact proteins.
To assess whether TGF-1 induces characteristic EMT in
the proximal tubule cell line LLC-PK1, 20-30%
confluent cultures were treated with 4 ng/ml TGF-
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-
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
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
-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-
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-
1 caused delocalization of E-cadherin and
-catenin from the cell periphery (Fig. 1E-H). Moreover,
increased cytosolic
-catenin staining was observed that was
frequently accompanied with enhanced perinuclear/nuclear labeling (Fig.
1H). Consistent with this, TGF-
1 increased
the amount of
-catenin in the nuclear fraction, as revealed by
Western blots (Fig. 1I). In addition to relocalization,
TGF-
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
-catenin was of smaller magnitude (Fig. 1J,
bottom), consistent with the observed cytosolic/nuclear
accumulation of this protein.
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Cytoskeletal reorganization.
Next we studied the effect of TGF-1 on cytoskeletal
structure. Control LLC-PK1 cells exhibited a strong
peripheral F-actin ring with slim central stress fibers (Fig.
2A), whereas
TGF-
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-
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-
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-
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-
1 exposure resulted in the accumulation of the
phosphorylated forms of MLC.
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SMA expression.
-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-
1 SMA protein
expression using Western blotting and immunfluorescence microscopy. No
SMA was detected in control LLC-PK1 cells, whereas
TGF-
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-
1 induced
intense labeling in
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-
1-treated cells and was present independent of the
overall magnitude of SMA expression.
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Role of Rho in TGF-1-Induced SMA Expression and
Cytoskeletal Reorganization
Effect of TGF-1 on Rho activity in
LLC-PK1 cells.
The TGF-
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-
1 induces detectable changes in the amount of active
(GTP-bound) Rho in LLC-PK1 cells. Cell lysates from control
and TGF-
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-
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-
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-
1 treatment resulted in a
later Rho stimulation. Consistent with this notion, we found that,
after 24 h of TGF-
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-
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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Involvement of Rho in the TGF-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-
1 treatment. Second, we investigated whether
interference with the activation of endogenous Rho could prevent
TGF-
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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DISCUSSION |
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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-1
exposure of LLC-PK1 cells induces an authentic EMT
characterized by tight and adherens junction disassembly, nuclear
translocation of
-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-
1 exposure and
support the intriguing concept that SMA-positive myofibroblast-like
cells can derive from the kidney epithelium itself. Accordingly,
TGF-
1 may promote renal fibrosis not only by stimulating
myofibroblasts but also by inducing their formation from the tubular
epithelium. Because myofibroblasts secrete TGF-
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-1-induced SMA expression. We considered the small
GTPase Rho as a candidate to mediate the above TGF-
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-
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-
1-stimulated prostate cancer cells. The biphasic Rho
response is consistent with and may explain the kinetics of other
TGF-
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-
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-
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-
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-1-induced stress fiber formation and MLC phosphorylation. Similarly, DN-ROK abrogated F-actin reorganization upon TGF-
1 treatment. These
results indicate that the Rho/ROK pathway is indispensable for
TGF-
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-1-induced promoter
activation. Interestingly, the dependence of the
TGF-
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-
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-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-
1
inducibility showed similar structural requirements, supporting the
concept that Rho is a key factor in mediating the effect of
TGF-
1 on SMA transcription. The fact that Rho acts through CArG boxes implicates SRF as the responsible transactivator. We
found that TGF-
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-
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-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-
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-
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-
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-1 inducible factors.
TGF-
1 stimulates a multitude of signaling pathways and
drives gene expression via several transcriptional activators
(37, 39). Interestingly, TGF-
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-
1 treatment. Interestingly, our ongoing
studies suggest that
-catenin signaling might also be necessary for
efficient SMA expression.
In summary, our work shows that Rho plays a critical role in
TGF-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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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