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Vol. 12, Issue 1, 27-36, January 2001
1 Mediates Epithelial to
Mesenchymal Transdifferentiation through a RhoA-dependent Mechanism
Vanderbilt-Ingram Cancer Center, Departments of Cancer Biology and Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232
Submitted June 29, 2000; Revised September 6, 2000; Accepted November 7, 2000  |
ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Transforming growth factor-
1 (TGF-) can be tumor suppressive,
but it can also enhance tumor progression by stimulating the complex
process of epithelial-to-mesenchymal transdifferentiaion (EMT). The
signaling pathway(s) that regulate EMT in response to TGF- are not
well understood. We demonstrate the acquisition of a fibroblastoid
morphology, increased N-cadherin expression, loss of junctional
E-cadherin localization, and increased cellular motility as markers for
TGF--induced EMT. The expression of a dominant-negative Smad3 or
the expression of Smad7 to levels that block growth inhibition and
transcriptional responses to TGF- do not inhibit mesenchymal
differentiation of mammary epithelial cells. In contrast, we show that
TGF- rapidly activates RhoA in epithelial cells, and that blocking
RhoA or its downstream target p160ROCK, by the expression
of dominant-negative mutants, inhibited TGF--mediated EMT. The data
suggest that TGF- rapidly activates RhoA-dependent signaling
pathways to induce stress fiber formation and mesenchymal characteristics.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Transforming growth factor-1 (TGF-) regulates growth,
differentiation, and epithelial transformation in the multistep
processes of tumorigenesis, wound healing, and embryogenesis. Based on
studies using cultured cells, transgenic mice, and human tumors, an
emerging model suggests the TGF- signaling pathway acts as a tumor
suppressor, but it can act as a promoter of tumor progression during
the later stages of tumorigenesis (McLeod et al., 1990
; Han
et al., 1993; Pierce et al., 1995; Cui et
al., 1996; Oft et al., 1998; Portella et
al., 1998). Although a role for TGF- as an autocrine
transforming and morphogenic factor in epithelial cells is well
established, our understanding of its intracellular signaling
mechanisms is limited. This report identifies intracellular TGF-
effectors and new mechanistic insight into TGF--mediated
fibroblastic conversion of mammary epithelial cells, a process likely
involved in tumor invasion and metastasis.
TGF- signals through an activated heteromeric complex of type I and
type II serine/threonine kinase receptors (Wrana et al., 1994). Subsequent cytoplasmic signaling involves the phosphorylation of
ligand-specific SMAD proteins, Smad2 and/or Smad3, that act as
intermediates for transcriptional regulation and cell cycle arrest in
conjunction with Smad4 in epithelial cells (reviewed in Kretzschmar and
Massague, 1998). RhoA, Rac1, and Jun N-terminal kinase (JNK) can
promote SMAD-mediated signaling, whereas negative regulators of
SMAD-mediated transcription include Smad7, RhoB, and calmodulin
(reviewed in Engel et al., 1998b). Smad7 specifically prevents the access and subsequent phosphorylation of Smad2 and Smad3
to the activated TGF- receptor complex before downstream amplification of the signaling cascade (Nakao et al., 1997).
Additionally, the activation of the H-Ras oncogene leads to suppression
of SMAD signaling (Calonge and Massague, 1999; Kretzschmar et
al., 1999). Parallel TGF--mediated transcriptional regulation
has been shown to include the mitogen-activated protein kinase
family (Atfi et al., 1997; Engel et al., 1999)
and the potentiation of phosphatidylinositol 3-kinase
(PI3-kinase) activity (Krymskaya et al., 1997; Higaki and
Shimokado, 1999). The initiation of multiple signaling pathways downstream of the activated receptor complex results in the pleotropic effects of TGF-.
Acquisition of a spindle-shaped morphology, delocalization of
E-cadherin from cell junctions, and elevated N-cadherin expression are
hallmarks of mesenchymal phenotypic conversion of mammary epithelia in
cell culture and in tumor invasion (Nieman et al., 1999). As
regulators of the actin cytoskeleton and cadherin junctions, the Rho
family of small GTPases is commonly implicated in these processes
(Bishop and Hall, 2000). Microinjection of RhoA, Rac1, or Cdc42 into
fibroblasts triggers the formation of stress fibers, lamellipodia, or
filopodia, respectively (Ridley and Hall, 1992; Ridley et
al., 1992). Rac1 and Cdc42 are involved in the establishment and
maintenance of epithelial intercellular adhesions (Braga et al., 1997; Hordijk et al., 1997; Takaishi et
al., 1997; Zhong et al., 1997); in contrast, RhoA
activation is implicated in the reversion of the epithelioid phenotype
toward a migratory, fibroblastoid morpholology of NIH3T3 cells (Sander
et al., 1999). The activated GTP-bound form of RhoA
associates specifically with multiple protein kinases. Among these,
p160 Rho-associated coiled-coil-containing protein kinase
(p160ROCK) regulates actin stress fiber formation
and integrin activation (Ishizaki et al., 1997).
Thus, data indicate an active role for small GTPases in the maintenance
and dynamic regulation of intercellular adhesion as well as having a
cytoskeleton-independent role in cell transformation, both alone and in
the context of H-Ras activation.
Because TGF- treatment of mammary epithelial cells recapitulates
this transition in culture, we explored the involvement of small
GTPases and candidate downstream effectors in TGF--induced EMT. We
show here that EMT induced by TGF- requires RhoA signaling. Importantly, we demonstrate that TGF- rapidly activates RhoA in
epithelial nontransformed mouse mammary cell line (NMuMG), mink lung
cell line (Mv1Lu), pancreatic tumor cell line (BxPc3), and primary
mouse keratinocytes, but not in fibroblastic NIH3T3 cells or TGF-
type I receptor deficient mink lung cells (R1B).
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Reagents and Constructs
TGF-1 was supplied by R&D Systems (Minneapolis, MN), LY294002
and curcumin was purchased from Sigma (St. Louis, MO). SCH51344 was a
gift from Dr. C.C. Kumar (State University of New York at Stony Brook,
NY) (Walsh et al., 1997) and LPA (1-oleoyl) was from Avanti
Polar Lipids (Alabaster, AL). The 3TP-Lux-reporter construct was
obtained from J. Massagué (Memorial Sloan-Kettering Cancer Center, New York, NY). The cDNA constructs encoding Smad3-FLAG (Dr. Rik Derynck, University of California, San Francisco, CA), Smad7-FLAG (Dr. Peter ten Dijke, Ludwig Institute for Cancer Research, Uppsala, Sweden), JNK1APF, N17Rac1 (Dr. Lynn
Cross, National Institutes of Health, Bethesda, MD), N19-RhoA (Dr. Lynn
Cross), QL-RhoA (Dr. Lynn Cross), KD-IA p160ROCK
(Dr. Shuh Narumiya, Kyoto University, Kyoto, Japan), and green fluorescent protein (GFP) (Dr. Fred H. Kant, Columbia University, New
York, NY) were subcloned into the pBabe retroviral vector.
Thymidine Incorporation and Luciferase Reporter Assay
NMuMG cells (American Type Culture Collection, Manassas, VA)
stably expressing Smad3 and Smad7 as well as the parental NMuMG cell
line were plated in 24-well plates for thymidine incorporation and
3TP-Lux reporter assays. To assay cell growth, cells were treated for
24 h with TGF- and loaded with
[3H]thymidine 2 h
before harvesting. The cells were washed and lysates measured with a
scintillation counter. Transcriptional activation was tested in cells
transfected with the 3TP-Lux luciferase (firefly) reporter construct
cDNA in conjunction with a cytomegalovirus-driven renela luciferase
plasmid (Promega, Madison, WI). Dual-luciferase assays were performed
on lysed cells as indicated by the manufacturer, Promega, and measured
on a Monolight 2010 luminometer (Analytical Luminescence Laboratory,
San Diego, CA). The ratios of firefly and renela luciferase
measurements were calculated in normalizing the reporter data in
relative luminescent units.
Retroviral Transduction and Cell Culture
Phoenix packaging cells (Kinsella and Nolan, 1996) were
transfected with retroviral constructs with Superfect (Qiagen,
Chatsworth, CA) according to manufacturer recommendations to
produce culture supernatants containing virus. We established matched
isogenic clones from the NMuMG parent cell line. Each of the lines
selected for further studies were TGF- sensitive for growth
inhibition and EMT (Bhowmick and Moses, unpublished data). NMuMG
cells were infected with virus by culturing the cells for 18 h in
1:1 Phoenix conditioned media: fresh DMEM, 10% fetal calf serum, 10 µg/ml insulin, supplemented with 4 ng/ml Polybrene (Sigma). The cells were subjected to various treatments, assaying for protein expression, or cultured in puromycin-containing media for the establishment of
stable cell lines 48 h after transduction.
Immunofluorescent Detection
Cells grown on coverslips to be stained for E- or N-cadherin (Transduction Laboratories, Lexington, KY) were fixed in ice-cold 100% methanol and subsequently permeabilized in phosphate-buffered saline containing 0.1% Triton X-100 for 10 min each step. Nonspecific sites were blocked with 3% milk; diluted primary antibody (1:1000) was incubated for 1 h, and visualized using secondary antibody conjugated to Cy2 or Cy3 (Sigma) fluorescence on a Zeiss Axovert fluorescence microscope. F-actin was stained by fixing the cells in 4% paraformaldehyde followed by incubation with Texas Red-conjugated phalloidin (Molecular Probes).
Western Blotting and p160ROCK Kinase Assay
Cells transfected using Superfect (Qiagen) or retrovirally transduced were washed in ice-cold phosphate-buffered saline, lysed (in 50 mM HEPES [pH 7.5], 150 mM NaCl, 0.2 mM vanadate, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 1% NP-40, and 0.1% SDS), briefly sonicated, and clarified by centrifugation. Protein (10 µg) was separated on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose, and immunoblotted with appropriate antibodies. Respective secondary horseradish peroxidase-conjugated antibodies (Amersham Pharmacia Biotech, Piscataway, NJ) were used, and were visualized with an enhanced chemiluminescence system (Amersham Pharmacia Biotech). Activity of p160ROCK was determined by immunoprecipitating myc-tagged p160ROCK from 200 µg of total cell lysate for incubation with 10 µg of histone, 5 mM [32P]ATP in 50 mM HEPES (pH 7.4) and 0.5 mM MgCl2 for 8 min at 30°C. The reaction was stopped by the addition of Laemmli sample buffer and separated on a 15% polyacrylamide gel for visualization by autoradiography.
GTPase Activity Assays
The biochemical activity assays were performed essentially as
described previously by Reid et al. (Ren et al.,
1999). A glutathione S-transferase (GST) fusion protein of
the Rho binding domain (RBD, a kind gift from Dr. Martin A. Schwartz,
Scripps Institute, La Jolla CA), rhotekin (Reid et al.,
1996), was used (Ren et al., 1999). For each measurement,
one 100-mm dish of cells was lysed in 1% NP-40, 50 mM Tris, pH 7.4, 10% glycerol, 100 mM NaCl, and 10 mM MgCl2. The
GST-RBD precoupled to agarose-glutathione beads (Sigma) was used to
precipitate GTP-bound RhoA from cleared lysates of cells for 30 min at
4°C for subsequent immunoblotting for RhoA, similar
to that described previously (Ren et al., 1999). A GST fusion protein of the PAK3 binding domain (PBD, a kind gift from Dr.
Gary M. Bokoch, Scripps Institute) was used to capture GTP-bound Rac1
and Cdc42 for subsequent visualization by
immunoblotting in a similar manner (Benard et
al., 1999).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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EMT in mammary epithelial cells is characterized by the
acquisition of spindle morphology and increased motility, with
changes in cadherin expression and localization. We used
pharmacological and genetic approaches to affect candidate-signaling
pathways in TGF--mediated EMT. SMAD- and RhoGTPase-dependent
pathways figured prominently given their previously demonstrated
involvement in TGF- signal transduction.
TGF--mediated morphological changes of NMuMG cells were accompanied
by a loss of E-cadherin junctional localization as reported previously
(Miettinen et al., 1994; Piek et al., 1999) as
well as our finding of the emergence of N-cadherin expression at cell margins (Figure 1). Recently, a
relationship between N-cadherin expression with elevated motility and
invasive characteristics of mammary tumor cells has been reported
(Nieman et al., 1999). Interestingly, immunoblot
analysis showed no change in E-cadherin or cadherin-associated
-catenin expression levels in response to TGF-. However,
N-cadherin expression, absent from epithelioid NMuMG cells, was
observed after 3 h of TGF- treatment and remained elevated
through the 48-h time course examined (Figure 1A). Examination of
TGF--treated cells showed disruption of cell-cell adhesions and a
change in cell morphology to a spindle shape in association with the
loss of E-cadherin junctional localization and the appearance of
N-cadherin staining at the plasma membrane (Figure 1B). Concomitant acquisition of actin stress fibers was also observed (Piek et al., 1999) (see below). However, the cells undergoing EMT remained sensitive to TGF- growth inhibition during fibroblastic conversion (Figure 2C). These data suggest that
TGF- initiates a program of EMT that correlates with changes in
cadherin expression and localization.
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We initially addressed the role of SMADs in TGF--mediated EMT by
the overexpression of antagonists of the SMAD signaling pathway. Stable
retroviral infections with GFP (control), FLAG-Smad3 (a Smad3 deletion
of the C-terminal-activating phosphorylation site that behaves in a
dominant-negative manner; Zhang et al., 1996), and
FLAG-Smad7 (an inhibitor of SMAD signaling; Nakao et al.,
1997) cDNA were expressed and confirmed by epifluorescence microscopy
and immunoblotting (Figure 2, A and B). The transduced cells displayed 
80% inhibition of TGF- transcriptional activation of the 3TP-Lux promoter, and diminished responsiveness to
TGF--mediated growth inhibition as expected from previous reports
(Zhang et al., 1996; Nakao et al., 1997; Itoh
et al., 1998) (Figure 2C). However, Smad3- or
Smad7-expressing cells treated with TGF- acquired a fibroblastic
morphology with a concomitant loss of junctional E-cadherin staining
and gain of plasma membrane N-cadherin localization similar to control
cells infected with retrovirus containing the GFP gene (Figure
3). These data suggest that
TGF--induced EMT is unaffected by decreased SMAD signaling as
reflected by SMAD-dependent growth and transcriptional responses.
Additionally, TGF--mediated stress fiber formation was not
inhibited by the down-regulation of SMAD signaling.
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To identify TGF- effectors that contribute to EMT, we next blocked
signaling pathways that have been previously attributed to changes in
cellular morphology through the retroviral transduction of
dominant-negative RhoA, Rac1, and JNK. Transient expression of
dominant-negative N17Rac1 or JNKAPF in NMuMG
cells did not alter EMT induction by TGF-; however, dominant-negative N19-RhoA blocked the mesenchymal transition (Figure
4). E-cadherin expression was maintained
at the cell junctions and N-cadherin expression was not observed in
cells transduced with N19-RhoA retrovirus after 24 h of 5-ng/ml
TGF- treatment. Assays for G-protein activation suggested the
efficacy of retroviral expression of RhoA and Rac1 mutants (Figures
5 and 6). Pharmacological inhibition of
the transcription factor activator protein-1 (downstream of JNK) with
curcumin, and specific Rac1 inhibition with 5 M SCH51344 (Walsh
et al., 1997) did not block TGF--mediated EMT; however, protein synthesis inhibition, with 50 µg/ml cycloheximide did block
TGF--mediated EMT (our unpublished data). Thus, the
recruitment of RhoA-dependent signaling pathways and nascent protein
synthesis are involved in TGF--mediated EMT.
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We (Engel et al., 1999) and others (Atfi et al.,
1997) have demonstrated the role of RhoA in TGF- transcriptional
signaling, as a positive cofactor for the JNK and SMAD signaling
pathways. However, no direct evidence for RhoA activation by TGF-
has been presented. Thus, we performed assays for the activation state of Rho-GTPases by measuring GTP-bound RhoA, as described by Ren et al. (1999), with NMuMG, Mv1Lu, R1B, primary mouse
keratinocytes, BxPc3, and NIH3T3 cells in response to treatment with
TGF- or lysophosphatic acid (LPA) as positive control (Figure 5).
The incubation of cells with LPA for 5 min resulted in a maximal 5-fold increase in RhoA-GTP over untreated cells (Figure 5A, lane 2). TGF-
treatment of the NMuMG cells showed nearly a 4-fold accumulation of
RhoA-GTP. Another control included the observation of elevated levels
of GTP-RhoA in constitutively active QL-RhoA and diminished GTP-RhoA
signal in N19-RhoA retrovirally transduced NMuMG cells (Figure 5B).
TGF- treatment of NMuMG, Mv1Lu cells, primary mouse keratinocytes,
or BxPc3 cells exhibited RhoA activation within 5 min, with a maximal
4-6-fold increase in GTP-bound RhoA was observed at 10 min. This was
followed by a rapid decrease in RhoA activity to baseline levels by 15 min of TGF- incubation. There was little change in the level of
expression of RhoA in each of the epithelial cell types that display
TGF--mediated changes in morphology. However, NIH3T3 and R1B cells
exhibited little or no change in TGF--induced RhoA activation over
basal levels (Figure 5C). TGF- treatment for 48 h showed
similar expression levels of RhoA (Bhowmick and Moses, unpublished
data). We further found that a 1 h pretreatment with
LY294002 (a specific PI3-kinase inhibitor) was largely ineffective in
blocking the TGF--mediated RhoA activation, suggesting the rapid
increase in RhoA-GTP is independent of PI3-kinase activity (Figure 5A,
lanes 6 and 7). The transient retroviral expression of a constitutively
active RhoA (QL-RhoA) in NMuMG cells did not result in spontaneous
mesenchymal transition, nor did it effect TGF--mediated EMT
(Bhowmick and Moses, unpublished data). This indicates that RhoA
activation is necessary, but not sufficient for the induction of EMT in
these cells.
The evidence of TGF--mediated PI3-kinase (Krymskaya et
al., 1997) and RhoA activity suggested the potential for the
activation of other GTPases such as Rac1 or Cdc42. We tested this
possibility by GTP-Rac1 and GTP-Cdc42 affinity precipitation with
GST-PAK3 binding domain, after immunoblotting for Rac1
or Cdc42 (Benard et al., 1999). GTPS loading of NMuMG cells
showed appreciable Rac1 activation (as a control); however, neither
GTPase exhibited detectable activation by TGF- over basal levels
(Figure 6). TGF- is observed to
activate RhoA but not Rac1 or Cdc42 in NMuMG cells.
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Thus, our findings suggest the potential involvement of RhoA-selective
downstream targets in TGF--mediated responses. We focused on the
action of p160ROCK as a potential mediator of
TGF--induced component of EMT, namely, the regulation of actin
cytoskeleton and adherens junction integrity and NMuMG cell motility.
Myc-tagged p160ROCK cDNA was transfected into
NMuMG cells and subjected to in vitro kinase assays after TGF-
stimulation by using histone as substrate. Elevated
p160ROCK activity was detected by 10 min, peaking
at a 4-fold level above control by 30 min of TGF- treatment (Figure
7A). The introduction of a
p160ROCK cDNA, encoding a kinase dead protein
unable to interact with Rho (KD-IA; Ishizaki et al., 1997),
into NMuMG cells provided further insight into the role of
p160ROCK in TGF- signaling. Cells retrovirally
transduced with KD-IA p160ROCK cDNA manifested
E-cadherin delocalization from adherens junctions, but did not acquire
stress fibers and did not adopt a mesenchymal phenotype in response to
TGF- (Figure 7B). Phalloidin staining showed notable cell-cell
junctions in the untreated cells, whereas a gap between cells was
observed in TGF--treated cells that maintained cortical actin
expression. A p160ROCK inhibitor, HA1077
[1-5-(isoquinolinesulfonyl)-homopiperazine HCl; Alexis Biochemicals],
at 1 µM concentration also inhibited stress fiber formation and the
fibroblastic morphology induced by TGF- (unpublished data).
These data suggest that regulation of actin cytoskeletal organization
and E-cadherin junction integrity diverge at RhoA, with the former
involving p160ROCK, and the latter requiring
other signaling factors.
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We next carried out motility studies to correlate the changes in cell
morphology observed in EMT with their migratory potential. Previous
studies showed that PI3-kinase and RhoA signaling could be
independently involved in the migratory phenotype of fibroblasts (Price
et al., 1999; O'Connor et al., 2000). NMuMG
cells transduced with GFP (as a control), Smad3, N19-RhoA, or KD-IA
p160ROCK retrovirus incubated with or without
TGF- or LY294002 were measured for their migration through 8 µM
pore size polycarbonate filters. A 6-fold increase in cell migration
was observed in TGF--treated over control, nontreated cells (Figure
8). The expression of Smad3 failed to
antagonize either basal or TGF--mediated NMuMG cell motility. The
TGF--induced migration was almost completely inhibited by treatment
with 10 M LY294002. And the expression of N19-RhoA antagonized the
motility of the cells, as did KD-IA p160ROCK to a
lesser extent.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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There is now considerable evidence that
autocrine/paracrine TGF- action is important in developmental
processes (Brown et al., 1996) as well as in the invasion
and metastatic spread of carcinoma cells. The NMuMG cells that we used
were isolated from normal mammary glands and do not form malignant
lesions when injected into nude mice (Hynes et al., 1985).
However, H-Ras-transformed fibroblastoid NMuMG cells are E-cadherin
negative and become fully invasive in vitro and in vivo (Van den
Broecke et al., 1996). Oft et al. (1996) reported
that fully polarized mammary epithelial cells are converted to a
fibroblastoid morphology by TGF-. Using a dominant-negative type II
TGF- receptor construct, this same group derived evidence indicating
that autocrine TGF- stimulation was necessary for invasion and
metastasis (Oft et al., 1998). In transgenic mice with
keratinocyte targeted TGF- expression, outgrowth of benign
papillomas was inhibited, consistent with TGF-'s growth inhibitory
role. However, those tumors that escaped growth inhibition by TGF-
manifested a higher rate of malignant conversion, often characterized
by an invasive, spindle cell phenotype (Cui et al., 1996).
This phenotype required TGF- receptor function (Portella et
al., 1998), suggesting separation in signal transduction pathways
downstream of the receptor complex. Clinical data from hereditary
nonpolyposis colon cancer patients support the hypothesis that TGF-
signaling is important in metastasis. Hereditary nonpolyposis colon
cancer patients frequently develop proximal colon cancers with
microsatellite instability (MSI) (Thibodeau et al., 1993). Approximately 90% of colon carcinomas with MSI have inactivating mutations of TRII (Parsons et al., 1995), and MSI is
significantly correlated with a reduced incidence of metastases and
increased patient survival (Gryfe et al., 2000; Thibodeau
et al., 1993), suggesting that complete loss of TRII in
carcinomas results in less aggressive tumors. In addition, cells from
Smad3 knockout mice have a diminished growth inhibitory response to
TGF- (Datto et al., 1999; Yang et al., 1999),
yet these mice exhibit accelerated wound healing and develop invasive,
metastasizing colorectal carcinomas (Zhu et al., 1998;
Ashcroft et al., 1999). TGF- signal transduction has been
traditionally associated with SMAD signaling for the regulation of gene
expression and growth inhibition. Our results point to an alternative
signaling pathway for TGF- via RhoA activation in the positive
regulation of EMT.
TGF- regulates at least two components of EMT progression through
RhoA-dependent pathways, the regulation of the actin cytoskeleton and
stability of adherens junctions. Both changes occur in the presence of
inhibitors of SMAD or JNK signaling (Figures 3 and 4). However,
TGF--induced SMAD- and JNK-mediated transcriptional activation are
both suggested to be dependent on RhoA activity (Atfi et
al., 1997; Engel et al., 1999). They do not, however, exclude a role for SMAD signaling in EMT, as determined by the expression pattern of E-cadherin, N-cadherin, actin, and cellular motility. Indeed, others have reported that overexpression of Smad2 and
Smad3 in the context of concomitant expression of constitutively active
TGF- type I receptor induces EMT of NMuMG cells (Piek et
al., 1999). Because dominant-negative constructs rarely abolish endogenous protein activity, it may be that EMT requires significantly lower Smad3 activity than that required for the activation of 3TP-Lux
or growth inhibition.
Our data suggest TGF- stimulation of the
RhoA/p160ROCK signaling pathway is necessary for
the acquisition of stress fibers and a fibroblastic morphology in NMuMG
and primary mouse keratinocytes. Additionally, the expression of
dominant-negative N19-RhoA in NMuMG cells blocked TGF--mediated
EMT. The observed rapid GTP loading of RhoA in Mv1Lu cells correlates
with the previously recognized loss of cell-cell adhesion and
acquisition of actin stress fibers in these cells upon TGF-
treatment (Azuma et al., 1996). Furthermore, the lack of
stimulation of RhoA activity by TGF- in R1B cells illustrates the
need for the TGF- type I receptor in RhoA signaling. Interestingly,
NIH-3T3 fibroblasts, shown to be growth stimulated by TGF- (Li
et al., 1993), exhibited no detectable GTP-RhoA accumulation
in response to TGF- treatment. This can be a result of number
potential reasons, including the differential expression of
RhoA-modifying proteins such as farnesyltransferase or
guanine-nucleotide-exchange factors (GEFs). Interestingly, BxPc3 cells,
a pancreatic metastatic tumor line not growth inhibited by TGF- with
a homologous deletion in the Smad4 gene, is TGF- responsive for RhoA
activation. This clearly indicates a bifurcation of signaling pathways
downstream of the receptor.
Recent observations show that NIH-3T3 fibroblasts can be converted to a
more epithelial morphology by elevated Rac1 activity, and their
fibroblastic morphology reverted by the restoration of RhoA activity
through the expression of constitutively active V14RhoA (Sander
et al., 1999). Interestingly, neither V14-RhoA nor N19-RhoA
affects Rac1 activity, but Rac1 activation down-regulates RhoA activity
(Sander et al., 1999). Although inhibiting Rho-GTPases by C3
microinjection is known to disrupt E-cadherin cytoskeletal links in
adherens junctions and blocks the assembly of new adherens junctions
(Hall, 1998), the expression of N19-RhoA did not produce the same
results (Figure 4). This is possibly due to redundant functions of RhoA
with other Rho proteins that N19-RhoA does not inhibit. For example,
the RhoB protein is reportedly stabilized in the cytoplasm by TGF-
(Engel et al., 1998a). The complex role of Rho proteins in
the regulation of cadherin-mediated adhesion is yet to be elucidated
(Braga et al., 1999).
We found Rac1 signaling may not be a direct signaling partner for
TGF--mediated EMT in NMuMG cells through the use of N17-Rac1 expression, SCH51344 (Rac1 inhibitor (Walsh et al., 1997),
and assaying for Rac1 activation. However, our studies do not rule out
the role of Rac1 and Cdc42 in the TGF--mediated EMT in an indirect
role in the complex process of cytoskeletal reorganization. The GEFs
are thought to mediate the replacement of GTPase-bound GDP with GTP.
The rapid response in epithelial cells would suggest a potential role
for TGF- in the direct regulation of such GEFs. Although there are
as many as 30 GEF family members identified to date (Bishop and Hall,
2000), the results show a specific TGF--mediated activation of
RhoA, but not Rac1 or Cdc42, to suggest that a RhoA-specific GEF is
potentially stimulated by the activated TGF- receptor complex.
The role of PI3-kinase in the regulation of the interactions between
the plasma membrane and cytoskeleton is a subject of current study by
many groups. The dual capacity for the activation of RhoA and
PI3-kinase (Krymskaya et al., 1997) by TGF- makes this
cytokine an important target for the study of the complex process of
EMT. We found that both RhoA GTP loading is independent of PI3-kinase
activity and inhibiting PI3-kinase can inhibit the motility of in NMuMG
cells. Unfortunately, the cooverexpression of a constitutively active
RhoA (QL-RhoA) and PI3-kinase (Myr-p110) is not feasible, because the
constitutive activation of RhoA results in the rapid initiation of
apoptosis (Subauste et al., 2000; Watanabe and Akaike,
1999). To adequately mimic TGF- signaling, a transitory activation
of RhoA is required or increased GTP exchange (see references in Van
Aelst and D'Souza-Schorey, 1997).
The elegant studies of Vasioukhin et al. (2000) have given
us insight into the mechanism of adhesion junction formation by actin
polymerization and reorganization. In epithelial cells plasma membrane
spanning E-cadherin is physically tethered to the actin cytoskeleton by
-catenin, 
-catenin, and in turn several actin-binding proteins.
Clustering of E-cadherin at the cell junctions provide the proper
conformation and/or density of -catenin to bind actin and establish
a continuous epithelial sheet (Vasioukhin et al., 2000). Our
studies indicate that TGF- signaling is capable of destabilizing
E-cadherin junctions by regulating actin organization through
RhoA/p160ROCK induction. The results from the
interference of p160ROCK activity by the
expression of KD-IA p160ROCK suggest a
dichotomous role for RhoA in TGF--mediated EMT, as a dynamic
regulator of adhesion junctions and the complex mechanism of cellular
morphology. The loss of adherens junctions in TGF--treated KD-IA-p160ROCK-expressing cells resulted in a
discontinuity in the epithelial sheet. Actin localization indicated
distinct staining of cell borders that had not formed cell-cell
contacts. Because TGF- treatment does not cause E-cadherin
expression levels to change appreciably in NMuMG cells, we can
speculate that E-cadherin relocalizes to the cytoplasm in a
p160ROCK-independent manner. Thus, the
organization of actin at the cell borders does not necessarily
presuppose the formation of adherens junctions. The role of other RhoA
effectors in TGF--mediated actin cytoskeletal organization remains
to be determined. Likely other TGF-/RhoA downstream signals such as
those involved in vesicular trafficking of E-cadherin from the plasma
membrane are involved in the disassembly of adherens junctions. The
identified interactions among RhoA, E-cadherin, and actin (Braga
et al., 1999) suggest various methods of TGF-/RhoA
regulation of cadherin junctions.
In summary we show that 1) TGF- activates RhoA and
p160ROCK, 2) N19-RhoA blocks TGF--mediated
EMT, and 3) p160ROCK inhibition blocks actin
cytoskeleton rearrangement and motility induced by TGF-. These
findings indicate a signaling cascade involving RhoA in
TGF--induced EMT. The biological activity of TGF--mediated RhoA
signaling is not limited to its capacity to mediate the actin
reorganization observed in many cell types. RhoA is currently
established as a positive regulatory factor in cell-cell contacts,
secretion, vesicular trafficking, and transformation. These studies
further suggest TGF- signaling pathways for growth inhibition and
tumor suppression may be separable from those pathways involved in EMT.
Hence, it may be possible to generate antagonists of the EMT pathways
useful for inhibiting tumor invasion and metastasis without blocking
the desirable tumor suppressive effects of TGF-.
| |
ACKNOWLEDGMENTS |
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We are grateful to Drs. William Grady, Brian Law, and Bart Lutterbach for their critical reading of the manuscript. Fluorescence and phase contrast microscopy images were acquired through the use of the Vanderbilt University Medical Center Cell Imaging Core Resource (supported by National Institutes of Health Grants CA-68485 and DK-20593). This work was supported by a National Institutes of Health training grant CA-09592 and Department of Defense, US Army Medical Research and Materiel Command Grant BC-991184 (to N.A.B.), Public Health Service Grants CA-42572 and CA-85492 (to H.L.M.), CA-62212 (to C.L.A.), Department of Defense, US Army Medical Research and Materiel Command Grant DAMD-17-98-1-8262 (to C.L.A.), and Vanderbilt-Ingram Cancer Center support Grant CA-68485.
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FOOTNOTES |
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* Corresponding author. E-mail address: hal.moses{at}mcmail.vanderbilt.edu.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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R. Vogelmann, M.-D. Nguyen-tat, K. Giehl, G. Adler, D. Wedlich, and A. Menke TGF{beta}-induced downregulation of E-cadherin-based cell-cell adhesion depends on PI3-kinase and PTEN J. Cell Sci., October 15, 2005; 118(20): 4901 - 4912. [Abstract] [Full Text] [PDF]
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L. Levy and C. S. Hill Smad4 Dependency Defines Two Classes of Transforming Growth Factor {beta} (TGF-{beta}) Target Genes and Distinguishes TGF-{beta}-Induced Epithelial-Mesenchymal Transition from Its Antiproliferative and Migratory Responses Mol. Cell. Biol., September 15, 2005; 25(18): 8108 - 8125. [Abstract] [Full Text] [PDF]
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R. A. Deaton, C. Su, T. G. Valencia, and S. R. Grant Transforming Growth Factor-{beta}1-induced Expression of Smooth Muscle Marker Genes Involves Activation of PKN and p38 MAPK J. Biol. Chem., September 2, 2005; 280(35): 31172 - 31181. [Abstract] [Full Text] [PDF]
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 T L Veveris-Lowe, M G Lawrence, R L Collard, L Bui, A C Herington, D L Nicol, and J A Clements Kallikrein 4 (hK4) and prostate-specific antigen (PSA) are associated with the loss of E-cadherin and an epithelial-mesenchymal transition (EMT)-like effect in prostate cancer cells Endocr. Relat. Cancer, September 1, 2005; 12(3): 631 - 643. [Abstract] [Full Text] [PDF]
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J. N. Kloth, G. J. Fleuren, J. Oosting, R. X. de Menezes, P. H.C. Eilers, G. G. Kenter, and A. Gorter Substantial changes in gene expression of Wnt, MAPK and TNF{alpha} pathways induced by TGF-{beta}1 in cervical cancer cell lines Carcinogenesis, September 1, 2005; 26(9): 1493 - 1502. [Abstract] [Full Text] [PDF]
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P. Kang and K.K.H. Svoboda Epithelial-Mesenchymal Transformation during Craniofacial Development Journal of Dental Research, August 1, 2005; 84(8): 678 - 690. [Abstract] [Full Text] [PDF]
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S. Yang, C. Zhong, B. Frenkel, A. H. Reddi, and P. Roy-Burman Diverse Biological Effect and Smad Signaling of Bone Morphogenetic Protein 7 in Prostate Tumor Cells Cancer Res., July 1, 2005; 65(13): 5769 - 5777. [Abstract] [Full Text] [PDF]
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S. Patel, K.-i. Takagi, J. Suzuki, A. Imaizumi, T. Kimura, R. M Mason, T. Kamimura, and Z. Zhang RhoGTPase Activation Is a Key Step in Renal Epithelial Mesenchymal Transdifferentiation J. Am. Soc. Nephrol., July 1, 2005; 16(7): 1977 - 1984. [Abstract] [Full Text] [PDF]
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N. Boyer Arnold and M. Korc Smad7 Abrogates Transforming Growth Factor-{beta}1-mediated Growth Inhibition in COLO-357 Cells through Functional Inactivation of the Retinoblastoma Protein J. Biol. Chem., June 10, 2005; 280(23): 21858 - 21866. [Abstract] [Full Text] [PDF]
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 L O'Rear, L Longobardi, M Torello, B K Law, H L Moses, F Chiarelli, and A Spagnoli Signaling cross-talk between IGF-binding protein-3 and transforming growth factor-{beta} in mesenchymal chondroprogenitor cell growth J. Mol. Endocrinol., June 1, 2005; 34(3): 723 - 737. [Abstract] [Full Text] [PDF]
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B. C. Willis, J. M. Liebler, K. Luby-Phelps, A. G. Nicholson, E. D. Crandall, R. M. du Bois, and Z. Borok Induction of Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells by Transforming Growth Factor-{beta}1: Potential Role in Idiopathic Pulmonary Fibrosis Am. J. Pathol., May 1, 2005; 166(5): 1321 - 1332. [Abstract] [Full Text] [PDF]
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W. M. Grady Transforming Growth Factor-{beta}, Smads, and Cancer Clin. Cancer Res., May 1, 2005; 11(9): 3151 - 3154. [Full Text] [PDF]
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C. Prunier and P. H. Howe Disabled-2 (Dab2) Is Required for Transforming Growth Factor {beta}-induced Epithelial to Mesenchymal Transition (EMT) J. Biol. Chem., April 29, 2005; 280(17): 17540 - 17548. [Abstract] [Full Text] [PDF]
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U. Valcourt, M. Kowanetz, H. Niimi, C.-H. Heldin, and A. Moustakas TGF-{beta} and the Smad Signaling Pathway Support Transcriptomic Reprogramming during Epithelial-Mesenchymal Cell Transition Mol. Biol. Cell, April 1, 2005; 16(4): 1987 - 2002. [Abstract] [Full Text] [PDF]
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L. Vardouli, A. Moustakas, and C. Stournaras LIM-kinase 2 and Cofilin Phosphorylation Mediate Actin Cytoskeleton Reorganization Induced by Transforming Growth Factor-{beta} J. Biol. Chem., March 25, 2005; 280(12): 11448 - 11457. [Abstract] [Full Text] [PDF]
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 R. L. Elliott and G. C. Blobe Role of Transforming Growth Factor Beta in Human Cancer J. Clin. Oncol., March 20, 2005; 23(9): 2078 - 2093. [Abstract] [Full Text] [PDF]
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 B. Ozdamar, R. Bose, M. Barrios-Rodiles, H.-R. Wang, Y. Zhang, and J. L. Wrana Regulation of the Polarity Protein Par6 by TGF{beta} Receptors Controls Epithelial Cell Plasticity Science, March 11, 2005; 307(5715): 1603 - 1609. [Abstract] [Full Text] [PDF]
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M. Maeda, K. R. Johnson, and M. J. Wheelock Cadherin switching: essential for behavioral but not morphological changes during an epithelium-to-mesenchyme transition J. Cell Sci., March 1, 2005; 118(5): 873 - 887. [Abstract] [Full Text] [PDF]
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R. T. Clements, F. L. Minnear, H. A. Singer, R. S. Keller, and P. A. Vincent RhoA and Rho-kinase dependent and independent signals mediate TGF-{beta}-induced pulmonary endothelial cytoskeletal reorganization and permeability Am J Physiol Lung Cell Mol Physiol, February 1, 2005; 288(2): L294 - L306. [Abstract] [Full Text] [PDF]
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A. K. Kamaraju and A. B. Roberts Role of Rho/ROCK and p38 MAP Kinase Pathways in Transforming Growth Factor-{beta}-mediated Smad-dependent Growth Inhibition of Human Breast Carcinoma Cells in Vivo J. Biol. Chem., January 14, 2005; 280(2): 1024 - 1036. [Abstract] [Full Text] [PDF]
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 R. M Greene and M M. Pisano Recent advances in understanding transforming growth factor {beta} regulation of orofacial development Human and Experimental Toxicology, January 1, 2005; 24(1): 1 - 12. [Abstract] [PDF]
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D. Wang, Q. Shen, X.-M. Xu, Y.-Q. Chen, and M.-H. Wang Activation of the RON receptor tyrosine kinase attenuates transforming growth factor-{beta}1-induced apoptotic death and promotes phenotypic changes in mouse intestinal epithelial cells Carcinogenesis, January 1, 2005; 26(1): 27 - 36. [Abstract] [Full Text] [PDF]
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S. Xie, M. B. Sukkar, R. Issa, U. Oltmanns, A. G. Nicholson, and K. F. Chung Regulation of TGF-{beta}1-induced connective tissue growth factor expression in airway smooth muscle cells Am J Physiol Lung Cell Mol Physiol, January 1, 2005; 288(1): L68 - L76. [Abstract] [Full Text] [PDF]
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K. J. Simpson, A. S. Dugan, and A. M. Mercurio Functional Analysis of the Contribution of RhoA and RhoC GTPases to Invasive Breast Carcinoma Cancer Res., December 1, 2004; 64(23): 8694 - 8701. [Abstract] [Full Text] [PDF]
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A. Masszi, L. Fan, L. Rosivall, C. A. McCulloch, O. D. Rotstein, I. Mucsi, and A. Kapus Integrity of Cell-Cell Contacts Is a Critical Regulator of TGF-{beta}1-Induced Epithelial-to-Myofibroblast Transition: Role for {beta}-Catenin Am. J. Pathol., December 1, 2004; 165(6): 1955 - 1967. [Abstract] [Full Text] [PDF]
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C. Bierkamp, S. Bonhoure, A. Mathieu, P. Clerc, D. Fourmy, L. Pradayrol, C. Seva, and M. Dufresne Expression of Cholecystokinin-2/Gastrin Receptor in the Murine Pancreas Modulates Cell Adhesion and Cell Differentiation in Vivo Am. J. Pathol., December 1, 2004; 165(6): 2135 - 2145. [Abstract] [Full Text] [PDF]
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A. V. Bakin, A. Safina, C. Rinehart, C. Daroqui, H. Darbary, and D. M. Helfman A Critical Role of Tropomyosins in TGF-{beta} Regulation of the Actin Cytoskeleton and Cell Motility in Epithelial Cells Mol. Biol. Cell, October 1, 2004; 15(10): 4682 - 4694. [Abstract] [Full Text] [PDF]
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O. De Wever, W. Westbroek, A. Verloes, N. Bloemen, M. Bracke, C. Gespach, E. Bruyneel, and M. Mareel Critical role of N-cadherin in myofibroblast invasion and migration in vitro stimulated by colon-cancer-cell-derived TGF-{beta} or wounding J. Cell Sci., September 15, 2004; 117(20): 4691 - 4703. [Abstract] [Full Text] [PDF]
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K. Yoshinaga, H. Inoue, T. Utsunomiya, H. Sonoda, T. Masuda, K. Mimori, Y. Tanaka, and M. Mori N-Cadherin Is Regulated by Activin A and Associated with Tumor Aggressiveness in Esophageal Carcinoma Clin. Cancer Res., September 1, 2004; 10(17): 5702 - 5707. [Abstract] [Full Text] [PDF]
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B. Hinz, P. Pittet, J. Smith-Clerc, C. Chaponnier, and J.-J. Meister Myofibroblast Development Is Characterized by Specific Cell-Cell Adherens Junctions Mol. Biol. Cell, September 1, 2004; 15(9): 4310 - 4320. [Abstract] [Full Text] [PDF]
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F. Tian, S. D. Byfield, W. T. Parks, C. H. Stuelten, D. Nemani, Y. E. Zhang, and A. B. Roberts Smad-Binding Defective Mutant of Transforming Growth Factor {beta} Type I Receptor Enhances Tumorigenesis but Suppresses Metastasis of Breast Cancer Cell Lines Cancer Res., July 1, 2004; 64(13): 4523 - 4530. [Abstract] [Full Text] [PDF]
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S. Nakajima, R. Doi, E. Toyoda, S. Tsuji, M. Wada, M. Koizumi, S. S. Tulachan, D. Ito, K. Kami, T. Mori, et al. N-Cadherin Expression and Epithelial-Mesenchymal Transition in Pancreatic Carcinoma Clin. Cancer Res., June 15, 2004; 10(12): 4125 - 4133. [Abstract] [Full Text] [PDF]
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T. Ito, J. D. Williams, D. Fraser, and A. O. Phillips Hyaluronan Attenuates Transforming Growth Factor-{beta}1-Mediated Signaling in Renal Proximal Tubular Epithelial Cells Am. J. Pathol., June 1, 2004; 164(6): 1979 - 1988. [Abstract] [Full Text] [PDF]
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S. Edlund, M. Landstrom, C.-H. Heldin, and P. Aspenstrom Smad7 is required for TGF-{beta}-induced activation of the small GTPase Cdc42 J. Cell Sci., May 1, 2004; 117(9): 1835 - 1847. [Abstract] [Full Text] [PDF]
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 J. Malmstrom, H. Lindberg, C. Lindberg, C. Bratt, E. Wieslander, E.-L. Delander, B. Sarnstrand, J. S. Burns, P. Mose-Larsen, S. Fey, et al. Transforming Growth Factor-{beta}1 Specifically Induce Proteins Involved in the Myofibroblast Contractile Apparatus Mol. Cell. Proteomics, May 1, 2004; 3(5): 466 - 477. [Abstract] [Full Text] [PDF]
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 S. C. McKarns, R. H. Schwartz, and N. E. Kaminski Smad3 Is Essential for TGF-{beta}1 to Suppress IL-2 Production and TCR-Induced Proliferation, but Not IL-2-Induced Proliferation J. Immunol., April 1, 2004; 172(7): 4275 - 4284. [Abstract] [Full Text] [PDF]
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