1 Department of Medicine, Vanderbilt University School of Medicine, 777 Preston
Research Building, Nashville, TN 37232, USA
2 Department of Cancer Biology, Vanderbilt University School of Medicine, 777
Preston Research Building, Nashville, TN 37232, USA
3 Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, 777
Preston Research Building, Nashville, TN 37232, USA
* Author for correspondence (e-mail: carlos.arteaga{at}mcmail.vanderbilt.edu )
Accepted 23 April 2002
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Summary |
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Key words: p38MAPK, TGFß, Epithelial-mesenchymal transition, Cell migration, Rac1
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Introduction |
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Smad-dependent signaling has been shown to be required for the
antiproliferative activity of TGFß, and components of this pathway are
frequently mutated or silenced in several human cancers
(de Caestecker et al., 2000).
Tumors, however, frequently express high levels of TGFß and inhibition of
TGFß signaling has been shown to reduce tumor invasiveness and metastasis
(Akhurst and Balmain, 1999
;
Barrack, 1997
;
Cui et al., 1996
;
Hojo et al., 1999
). A number
of studies provide evidence that TGFß contributes to tumor cell invasion
and metastasis by inducing mesenchymal transdifferentiation in epithelial
cells (EMT) and stimulating cell migration
(Akhurst and Balmain, 1999
;
Barrack, 1997
;
Oft et al., 1998
). This
TGFß-mediated fibroblastic transdifferentiation is a complex process
associated with alterations in epithelial cell junctions, changes in cell
morphology, reorganization of the cell cytoskeleton, expression of
fibroblastic markers (fibronectin, vimentin), and enhancement of cell
migration (Bakin et al., 2000
;
Miettinen et al., 1994
;
Piek et al., 1999b
).
The molecular mechanisms of TGFß-mediated EMT and cell migration are
not entirely understood. Studies with TGFß receptors have shown that a
truncated TGFß/bone morphogenic protein (BMP) type I receptor, Alk2,
blocks EMT in mouse NMuMG cells (Miettinen
et al., 1994). Adenoviral expression of constitutively active
human TßRI/Alk5 together with Smad2/3 can induce EMT in these cells
(Piek et al., 1999b
).
Expression of a dominant-negative truncated form of TßRII decreases the
formation of invasive spindle tumours
(Portella et al., 1998
).
Adenoviral expression of Smad2/3 induced EMT only in the context of expression
of constitutively active Alk5 (Piek et
al., 1999b
). Overexpression of Smad7, an inhibitor of
Smad-dependent signaling, or dominant-negative Smad3 did not affect the
transdifferentiation, arguing against involvement of Smads in EMT
(Bhowmick et al., 2001a
).
Inhibition of JNK with curcumin (Bakin et
al., 2000
) or by expression of dominant-negative JNK mutant
(Bhowmick et al., 2001a
) did
not affect EMT. TGFß did not activate the Ras-Raf-ERK1/2 cascade and MEK
inhibitors (PD098059 and U0126) did not block EMT in NMuMG cells
(Bakin et al., 2000
;
Piek et al., 1999b
). We have
recently shown that the phosphatidylinositide 3-kinase (PI3K)-Akt pathway
contributes to EMT at the step of tight junction disruption
(Bakin et al., 2000
). The role
of p38MAPK in TGFß-mediated EMT has not been studied.
The p38MAPK pathway has been implicated in various biological responses to
members of the TGFß superfamily including TGFß-stimulated migration
of smooth muscle cells (Hedges et al.,
1999), neuronal differentiation of PC12 cells induced by bone
morphogenic protein 2 (BMP-2) (Iwasaki et
al., 1999
), growth/differentiation factor-5-induced chondrogenesis
of ATDC-5 cells (Nakamura et al.,
1999
), and BMP-mediated cardiomyocyte differentaition
(Monzen et al., 1999
). Studies
in Drosophila have shown that p38MAPKs are required for wing morphogenesis
downstream of decapentaplegic (Dpp), a homologue of TGFß
(Adachi-Yamada et al., 1999
).
The p38MAPK pathway has also been implicated in TGFß transcriptional
responses (Hanafusa et al.,
1999
; Kucich et al.,
2000
; Sano et al.,
1999
).
The molecular mechanism(s) of TGFß-induced activation of p38MAPK
signaling are not defined. Mammalian p38MAPKs are activated by distinct
upstream dual specificity MAPK kinases (MKK), MKK3 and MKK6
(Tibbles and Woodgett, 1999).
TGFß-activated kinase 1 (Tak1) phosphorylates MKK3/6 in TGFß and BMP
signaling (Shibuya et al.,
1998
; Yamaguchi et al.,
1995
). In addition, other MKK kinases including p21-activating
kinase (PAK1) and mixed-lineage kinase (MLK) have been shown to phosphorylate
MAPK kinases (MKK3/6) and induce p38MAPKs
(Tibbles et al., 1996
;
Zhang et al., 1995
). p38MAPK
downstream targets include MAPK-activated protein kinase-2, mitogen- and
stress-activated protein kinase-1 (MSK1), and transcription factors ATF2,
CHOP, CREB and MEF2C (Tibbles and
Woodgett, 1999
). Recent studies have found that p38MAPKs are
involved in the control of cell cytoskeleton and cell migration via
phosphorylation of paxillin and heat shock protein 27 (HSP27)
(Hedges et al., 1999
).
In these studies we found that H-7, a protein kinase inhibitor, blocks
TGFß-induced EMT and activation of the p38MAPK pathway in NMuMG mouse
mammary epithelial cells. The specific p38MAPK inhibitors, SB203580 and
SB202190, impaired TGFß-mediated changes in cell shape, the actin
cytoskeleton, and cell migration. H-7 and the p38MAPK inhibitors blocked
phosphorylation of ATF2, but did not inhibit TGFß-mediated
phosphorylation of Smad2. Expression of dominant-negative mutants (DN) of MKK3
or p38 inhibited TGFß-mediated EMT. We also showed that TGFß
activates the MKK3/6-p38MAPK-ATF2 cascade within 15 minutes and expression of
DN-MKK3 blocked TGFß-mediated activation of p38MAPK and EMT.
Kinase-inactive TGFß type II and type I (Alk5) receptors blocked EMT and
the activation of p38MAPK. Forced expression of kinase-active Alk5-T204D
induced both EMT and phosphorylation of p38MAPK in NMuMG cells.
Alk5-T204D-induced EMT was blocked by a p38MAPK inhibitor. Finally, we
demonstrated that forced expression of dominant-negative Rac1N17 blocked
TGFß-induced activation of the p38MAPK-ATF2 cascade and EMT.
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Materials and Methods |
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Cell culture
NMuMG mouse mammary epithelial cells, SiHa human cervical carcinoma cells,
MDA-MB-231 human breast cancer cells and HEK293T human kidney cells were
purchased from American Tissue Culture Collection (ATCC). Cells were cultured
as recommended by ATCC. 4T1 tumor cells were provided by F. Miller (Karmanos
Cancer Center, Detroit, MI) and cultured in 10% FBS-DMEM.
Plasmids and retroviral constructs
The retroviral vectors pGabe and pGabe-TßRII-K277R were provided by
Martin Oft (UCSF, San Francisco, CA) and have been described previously
(Oft et al., 1998). The
TßRII-K277R construct contains an HA-tag at the N-terminus. Human
wild-type Alk5, dominant-negative Alk5-K232R, and constitutively active
Alk5-T204D constructs were provided by Masahiro Kawabata (The Cancer
Institute, Tokyo, Japan). To generate pBMN-Alk5 constructs, the
EcoRI/SalI fragments of Alk5 and Alk5-K232R including the
C-terminal HA-tag were cloned in the retroviral pBMN-IRES-EGFP vector provided
by Garry Nolan (Stanford University). The pBMN-Rac1N17 was engineered by
cloning a BamHI-XhoI fragment encoding Rac1N17 from
pCDNA3-Rac1N17 (a gift of Richard Cerione, Cornell University, Ithaca, NY) at
the BamHI-SalI site of the retroviral pBMN-IRES-GFP vector.
RhoAN19 and RhoAQ63L were previously described
(Bakin et al., 2000
). The
pBMN-MKK3AL and pBMN-MKK6AL plasmids were generated by cloning
SalI-NotI fragment of MKK6AL or XhoI-NotI
fragment of MKK3AL from pCDNA3 vector into the retroviral pBMN-IRES-GFP
vector. pCDNA3-MKK3AL and pCDNA3-MKK6AL plasmids were a gift of James Woodgett
(The Ontario Cancer Institute, Toronto, Ontario). pBMN-p38AGF encoding a
dominant-negative mutant of p38
and containing N-terminal Flag epithope
was generated by cloning a HindIII-XbaI fragment of p38AGF
from pcDNA3-p38AGF at the XhoI site of pBMN-IRES-GFP. pcDNA3-p38AGF
was a gift of Roger Davies (University of Massachusetts, Worcester, MA).
Plasmids phCMV-VSVG, encoding vesicular stomatitis virus glycoprotein (VSV-G),
and pCMV gag-pol, containing the Moloney murine leukemia virus (MoMLV) gag and
pol genes, were provided by Jane Burns (University of California at San
Diego).
Retroviral infection of cells
Retroviruses were prepared by transfection of HEK293T cells with 15 µg
DNA/100 mm dish of three plasmids encoding gag/pol, VSV-G, and the target
construct, ratio 4:3:8. Supernatants from cells were collected for 3 days and
combined, filtered through 0.4 µm filters, and stored in aliquots at
-80°C. NMuMG cells were infected with supernatant containing retroviruses
in the presence of 6 µg/ml Polybrene (Sigma) as described previously
(Yee et al., 1994). Three days
later, GFP-positive cells were selected by flow cytometry. Under these
conditions more than 95% of selected cells expressed GFP at the time of
experiments.
Immunoblot analysis
Cells were incubated in serum-free medium for 4 hours prior to treatment
with TGFß1. Cells were lysed in buffer containing 20 mM Tris, pH 7.4, 137
mM NaCl, 1% NP-40, 10% glycerol, 20 mM NaF, 1 mM Na orthovanadate, 1 mM PMSF,
2 µg/ml aprotinin, and 2 µg/ml leupeptin. Protein concentrations in cell
lysates were determined by the Bradford method. Protein extracts (50
µg/lane) were separated by 12.5% SDS-PAGE and transferred to nitrocellulose
membranes (100 mA, 2.5 hours). Membranes were blocked with 5% milk in TBST for
1 hour at room temperature (RT) and then incubated with primary antibodies in
TBST plus 1% milk for 16 hours at 4°C, followed by incubation with
secondary antibodies for 1 hour at RT. Membranes were washed three times in
TBST and immunoreactive bands visualized by ECL (Pierce).
p38MAPK in vitro kinase assay
p38MAPK was precipitated from protein extracts (200 µg) with a p38MAPK
monoclonal antibody (Santa Cruz Biotechnology) for 2 hours at 4°C. An in
vitro kinase reaction was performed in a 40-µl volume by adding to the
immune complexes 1 µg GST-ATF2 and 10 µCi [-32P]ATP
(specific activity 3000 Ci/mmol, New England Nuclear) for 20 minutes at
30°C in the presence of 10 µM PKA peptide inhibitor (Calbiochem).
Reactions were terminated by the addition of Laemmli loading buffer and
heating, followed by 15% SDS-PAGE and transfer to nitrocellulose (NC)
membranes. Quantitative analysis of [
-32P]-labeled bands was
performed using a PhosphorImager (Molecular Dynamics). The same NC-membranes
were probed with a monoclonal antibody to p38MAPK.
Immunofluorescence microscopy
NMuMG cells (105cells/well) were grown in DMEM containing 5% FBS
on glass coverslips (22x22 mm) for 24 hours before treatment with 2
ng/ml TGFß1. Cells were fixed with 4% paraformaldehyde in
phosphate-buffered saline (PBS) for 10 minutes at RT and then permeabilized
with 0.05% Triton X-100 for 10 minutes. Cells were washed three times in PBS
after each treatment. Cells were blocked with 3% milk in PBS for 30 minutes at
RT, incubated for 60 minutes with primary antibodies diluted in 1% milk/PBS
(1/300 for ZO-1, 1/500 for Smad2, 1/250 for fibronectin), and then with
fluorescent secondary antibodies (1/500) for 45 minutes at RT. Microtubules
were stained for 30 minutes at RT with ß-tubulin-Cy3 diluted 1/250 in 1%
milk/PBS. Actin was stained with phalloidin-FITC (4 units/ml) or
phalloidin-Texas Red (2 units/ml). Cell nuclei were stained with 1 µg/ml
Hoechst for 10 minutes at RT. Coverslips were mounted on 25x75 mm
microslides (VWR Scientific) using AquaPolyMount (Polysciences). Fluorescent
images were captured using a Princeton Instruments cooled CCD digital camera
from a Zeiss Axiophot upright microscope.
Transcriptional assays
NMuMG cells (3x104) were seeded in 24-well plates and
transfected with 0.16 µg/ml pSBE-Lux containing 12 repeats of Smad binding
sequence (provided by J.-M. Gauthier, Laboratoire Glaxo Wellcome, Les Ulis
Cedex, France) with 0.002 µg/ml pCMV-Rl (Promega, Madison, WI) using
FuGENE6 reagent (Roche Molecular Biochemicals) according to the manufacturer's
protocol. Cells were incubated for 8 hours in 0.5% FBS-DMEM prior to treatment
with 1 ng/ml TGFß1 for 16 hours. Firefly luciferase (Luc) and Renilla
reniformis luciferase (RlLuc) activities in cell lysates were determined
using the Dual Luciferase Reporter Assay System (Promega) according to the
manufacturer's protocol in a Monolight 2010 luminometer (Analytical
Luminescence Laboratory, San Diego, CA). Luc activity was normalized to RlLuc
activity and presented as Relative Luciferase Units. All assays were done in
triplicate wells and each experiment was repeated at least twice.
Affinity precipitation of Rac using GST-PBD
A fusion protein containing the GTPase-binding domain from human PAK1 (PBD)
and glutathione S-transferase (GST) was expressed in Escherichia coli
using pGEX-4T3-GST-PBD as described (Benard
et al., 1999). pGEX-4T3-GST-PBD was kindly provided by Gary Bokoch
(Scripps Research Institute). NMuMG cells (2x107/assay) were
treated with 2 ng/ml TGFß1 for 15 minutes followed by cell lysis in 20 mM
Tris, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% NP-40, 5% glycerol, 20 mM
NaF, 1 mM sodium orthovanadate, 1 mM PMSF, 2 µg/ml aprotinin, and 2
µg/ml leupeptin in the presence of 8 µg GT-PBD. Cell lysates were
clarified by low speed centrifugation at 4°C. HEK293T cells transfected
with Rac1N17 or Alk5 mutants were lysed in the same buffer. After
clarification, cell lysates (350 µg/assay) were incubated with 8 µg
GST-PBD. To prepare cytosolic Rac1 loaded with GDP or GTP
S, cell
lysates (equivalent of 2x106 cells) were incubated for 15
minutes at 30°C in the presence of 10 mM EDTA and 100 mM GTP
S or 1
mM GDP to facilitate nucleotide exchange
(Benard et al., 1999
). The
loading reaction was terminated by addition of 60 mM MgCl2.
Affinity precipitation was performed using 15 µl of glutathione-Sepharose
4B beads (Pharmacia) for 1 hour at 4°C. The bead pellets were washed three
times with 20 mM Tris, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1%
NP-40 and 2 times in PBS. The bead pellet was finally suspended in 40 µl of
Laemmli sample buffer. Proteins were separated on 15% SDS-PAGE, transferred to
nitrocellulose membrane and immunoblotted with an antibody to Rac1
(Transduction Laboratories).
Migration assays
NMuMG or MDA-MB-231 cells (1x105/well) were plated in
DMEM/0.5%FBS in the upper chamber of 5 µm pore (24-well) transwells
(Costar, High Wycombe, UK) and incubated alone or with 2 ng/ml TGFß1 in
the absence or presence of SB202190. After 16 hours, cells were fixed in 100%
methanol and cells remaining at the top of the polycarbonate membrane were
removed with cotton swabs. The cells that had migrated through pores to the
lower surface were stained with Diff-quick stain (VWR Scientific). Membranes
were mounted on 25x75 mm microslides. Four random images were recorded
at 200x magnification and cells were counted. Experiments were performed
in duplicate.
Wound closure assay
MDA-MB-231 and 4T1 cells (1-2x105/well) were seeded in
12-well plates. Cells were incubated in serum-free medium for 32 hours prior
to wounding. The wounds were made by scraping with plastic tip across the cell
monolayer. Cells were treated with kinase inhibitors 60 minutes before
wounding. The wounded cells were treated or untreated with 2 ng/ml TGFß1.
Phase contrast images were recorded at the time of wounding (0 hours) and 16
hours thereafter. The wound closure was estimated as the ratio of the
remaining wound area relative to the initial wounded area. Experiments were
repeated at least three times.
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Results |
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|
Next, we checked whether H-7 inhibits activation of MKK3/6. We found that
TGFß-induced phosphorylation of MKK3/6 was inhibited by H-7 in a
dose-dependent manner (Fig.
1E), suggesting that H-7 inhibits a kinase upstream of MKK3/6.
This kinase is downstream of TGFß receptors as incubation with 5-40 µM
H-7 did not block phosphorylation of Smad2
(Fig. 1B). Consistent with this
result, H-7 did not block TGFß-mediated activity of Smad-dependent
luciferase reporter (Fig. 1F).
Since H-7 can inhibit PKC, we examined activation of p38MAPK in the presence
of another PKC inhibitor, bisindolylmaleimide-I (BIM-I)
(Davies et al., 2000).
Treatment of cells with doses of BIM-I (1-5 µM) that block typical PKCs
(Davies et al., 2000
) did not
affect phosphorylation of MKK3/6 in response to TGFß
(Fig. 1D). These results
suggest that H-7 impairs TGFß signaling by inhibiting activation of the
p38MAPK pathway downstream of TGFß receptors, and not through its effect
on PKCs.
p38MAPK is involved in TGFß-mediated EMT
To test whether p38MAPK is involved in EMT, we used specific inhibitors of
p38MAPK, SB202190 and SB203580 that do not affect JNK, MEK1/2 and ERK1/2
(Davies et al., 2000).
Microscopic examination showed that cell elongation induced by TGFß in
NMuMG cells was blocked by co-treatment with 10 µM SB202190
(Fig. 2A). Similarly, the
p38MAPK inhibitor blocked TGFß-induced cell elongation in cervical cancer
epithelial SiHa cells (Fig.
2A). Previous studies have shown that these p38MAPK inhibitors may
affect the kinase activity of TGFß receptors
(Eyers et al., 1998
).
Therefore, we examined their effect on TGFß-receptor-dependent
phosphorylation of Smad2. Treatment of cells with TGFß in the presence of
SB202190 did not significantly affect the expression and TGFß-induced
phosphorylation of Smad2 (Fig.
2B), whereas it reduced phosphorylation of ATF2
(Fig. 2C). Similar results were
obtained with SB203580 (data not shown).
|
TGFß activates the p38MAPK pathway in NMuMG and SiHa cells
We next examined activation of the p38MAPK pathway in response to
TGFß. Protein extracts were prepared from cells starved in serum-free
medium for 4 hours and treated with TGFß1. Phosphorylation of MKK3/6 was
detected after 15 minutes of TGFß treatment reaching a maximum at 60
minutes, whereas an increase in p38MAPK phosphorylation at Thr180/Tyr182 was
observed at 30 minutes and reached a plateau at 60 minutes
(Fig. 3A). To confirm the
immunoblot data, we tested p38MAPK-specific activity using an in vitro kinase
assay with GST-ATF2 fusion protein as substrate. Treatment with TGFß
increased -32P incorporation into GST-ATF2 in a
time-dependent fashion, sixfold at 15 minutes and reaching a maximal
stimulation of 24-fold above control by 60 minutes
(Fig. 3B). This increase in
p38MAPK kinase activity at 15 minutes may reflect a higher sensitivity of the
in vitro kinase assay compared with detection of phosphorylated p38MAPK by
immunoblot. TGFß-induced activation of p38MAPK was dose-dependent with
0.1 ng/ml being sufficient to induce phosphorylation of p38MAPK with a maximal
effect observed between 0.5 and 2 ng/ml
(Fig. 3C). Treatment of SiHa
human cervical carcinoma cells with TGFß1 for 60 minutes resulted in
phosphorylation of p38MAPK (Fig.
3D), suggesting activation of p38MAPK signaling in response to
TGFß1 in these cells.
|
Kinase activities of TGFß receptors are required for
TGFß-induced p38MAPK activation
To confirm the role of TGFß receptors in activation of p38MAPK, we
engineered cells expressing TßRII-K277R, a kinase-inactive mutant of
TGFß type II receptor (Wrana et al.,
1994). NMuMG cells were infected with retrovirus encoding
TßRII-K277R and enhanced green fluorescent protein (EGFP) or with control
retrovirus encoding EGFP only (Gabe). Fluorescent cells were selected by flow
cytometry and expression of the HA-tagged mutant receptor was confirmed by
immunoblot analysis (Fig. 4A).
TGFß-mediated phosphorylation of Smad2, MKK3/6, and p38MAPK was inhibited
in TßRII-K277R cells compared with control Gabe cells
(Fig. 4B). TßRII-K277R
also blocked EMT (Fig. 4D) and
cell migration (Fig. 8A),
indicating that TßRII kinase activity is required for these TGFß
responses.
|
|
To determine whether the activation of p38MAPK was TGFß-specific, we
expressed wild-type TßRI/Alk5 (Alk5-WT), kinase-inactive Alk5-K232R, or
kinase active Alk5-T204D (Kawabata et al.,
1995) in NMuMG cells. Alk5 mutants were expressed using a
bi-cistronic retroviral vector encoding EGFP. GFP-positive cells were selected
by flow cytometry and expression of mutants was confirmed by immunoblot
analysis (Fig. 4B).
Kinase-inactive Alk5-K232R significantly reduced TGFß-induced
phosphorylation of MKK3/6 and p38MAPK, whereas kinase active Alk5-T204D
induced phosphorylation of MKK3/6 and p38MAPK in the absence of added ligand
(Fig. 4B). Microscopic studies
showed that TGFß-induced EMT was impaired in cells expressing
kinase-inactive Alk5-K232R. Cells expressing Alk5-T204D exhibited a
fibroblastic morphology similar to Alk5-WT cells treated with TGFß for 24
hours (Fig. 4C). Treatment of
cells expressing Alk5-T204D with the p38MAPK inhibitor SB202190 reversed these
morphological changes.
MKK3/6 kinases mediate activation of p38MAPK and EMT in response to
TGFß
Dual-specificity MKK3 and MKK6 kinases have been implicated in activation
of p38MAPK (Raingeaud et al.,
1996). Phosphorylation of both kinases is induced by TGFß or
by expression of active Alk5-T204D in NMuMG cells
(Fig. 4B). Therefore, we tested
the effect of dominant-negative MKK3AL
(Huang et al., 1997
;
Zanke et al., 1996
) on
TGFß-mediated activation of p38MAPK and EMT in NMuMG cells. Expression of
HA-tagged MKK3AL reduced phosphorylation of endogenous p38MAPK and ATF2
(Fig. 5A), whereas expression
and phosphorylation of Smad2 were not affected
(Fig. 5B). Similar results were
obtained with dominant-negative MKK6AL (data not shown). Next, we examined the
effect of MKK3AL on EMT. TGFß induced EMT in NMuMG cells infected with
control retrovirus encoding EGFP only (BMN), whereas EMT was inhibited in
MKK3AL-expressing cells (Fig.
5C). SB202190, a p38MAPK inhibitor, blocks activity of p38
and p38ß but does not inhibit p38
and p38
(Davies et al., 2000
). Since,
SB202190 blocked EMT (Fig. 2A),
we tested the effect of p38AGF, a dominant-negative mutant of p38
, on
TGFß-mediated EMT. TGFß-induced morphological transformation in
NMuMG infected with retroviruses encoding p38AGF was impaired compared with
cells infected with control BMN virus (Fig.
5C). These findings suggest that MKK3/6 kinases mediate
TGFß-induced activation of p38MAPK and EMT in NMuMG cells.
|
p38MAPK is involved in TGFß-induced reorganization of the actin
cytoskeleton
We characterized the effect of p38MAPK inhibitors on reorganization of the
actin cytoskeleton in response to TGFß. Microscopic examination of
F-actin by staining with phalloidin-fluorescein showed a cortical arrangement
of actin at the cell-cell junctions without significant stress fibers
(Fig. 6A). Treatment with
TGFß1 for 24 hours induced formation of actin stress fibers arranged
along the largest cell axis. SB202190 did not significantly change the actin
organization in TGFß-untreated cells, but impaired TGFß-induced
formation of actin stress fibers (Fig.
6A). Similar blockade of stress fiber formation was observed in
cells pretreated with H-7 (data not shown). Examination of the actin
cytoskeleton in MKK3AL cells showed that MKK3AL did not affect the cortical
arrangement of actin in untreated cells, but inhibited TGFß-induced actin
stress fiber formation (Fig.
6B). These data suggest that p38MAPK contributes to the
reorganization of the actin cytoskeleton induced by TGFß during EMT.
|
Rac GTP-binding protein is involved in TGFß-induced activation
of p38MAPK and EMT
There is evidence that small GTP-binding proteins are involved in TGFß
signaling (Atfi et al., 1997;
Bakin et al., 2000
;
Bhowmick et al., 2001a
;
Engel et al., 1999
;
Mucsi et al., 1996
). Rac1 and
CDC42 have been implicated in the activation of the MKK3/6-p38MAPK cascade in
several systems (Coghlan et al.,
2000
; Tibbles et al.,
1996
; Uddin et al.,
2000
; Zhang et al.,
1995
). To test whether Rac1 or RhoA are involved in p38MAPK
activation in response to TGFß, we transfected dominant-negative RhoAN19
or Rac1N17 in NMuMG cells. Rac1N17 inhibited TGFß1-induced
phosphorylation of p38MAPK and its downstream substrate ATF2, whereas neither
dominant-negative RhoAN19 nor constitutively active RhoAQ63L did not affect
p38MAPK phosphorylation (Fig.
7A,B). These data suggest that Rac1 mediates p38MAPK activation in
response to TGFß.
|
To examine whether Rac1 activity is induced by TGFß, we performed
affinity precipitation assays using a fusion protein of the GTPase-binding
domain (amino acids 67-152) from human PAK1 (PBD) and GST. The GST-PBD fusion
protein has been shown to specifically bind active Rac1 loaded with GTP
(Benard et al., 1999).
Treatment of NMuMG cells for 15 minutes with TGFß resulted in the
increase in Rac1 binding to purified GST-PBD
(Fig. 7C). GST-PBD effectively
interacted with the active GTP
S-bound form of Rac1 but did not bind to
the inactive GDP-bound form of Rac1 (Fig.
7C, left inset). To confirm that TGFß receptors can mediate
activation of Rac1, we expressed mutants of Alk5-TßRI in HEK293T cells.
Kinase-inactive Alk5K232R reduced the level of active Rac1, whereas
kinase-active Alk5T204D increased the amount of Rac1 bound to GST-PBD.
Expression of dominant-negative Rac1N17 reduced the amount of Rac1 recovered
from GST-PBD beads (Fig. 7D).
Since active Rac1 mediates actin ruffling and lamellipodia formation
(Hall, 1998
), we examined
F-actin in NMuMG and MDA-MB-231 cells treated with 2 ng/ml of TGFß1 for
15 minutes. Confocal microscopy of cells stained with phalloidin-Texas Red
showed that TGFß induced actin ruffles, a phenotype associated with
active Rac (Fig. 7E).
In order to examine the role of Rac1 in EMT, NMuMG cells were infected with a retrovirus encoding dominant-negative Rac1N17 and Green Fluorescent Protein (GFP). Immunoblot analysis showed at least twofold higher levels of Rac1 in cells infected with Rac1N17 retrovirus compared with cells infected with control BMN virus encoding GFP only (Fig. 8A). TGFß induced phosphorylation of MKK3/6 and p38MAPK in cells infected with control retrovirus whereas, in Rac1N17 cells, this induction was significantly reduced (Fig. 8B). Rac1N17 did not significantly affect TGFß-dependent phosphorylation of Smad2 (Fig. 8C). Microscopic examination showed that TGFß1 induced cell elongation and the formation of actin stress fibers in control BMN cells, whereas these effects were impaired in cells expressing Rac1N17 (Fig. 8D). These findings suggest that Rac1 is involved in TGFß-induced EMT and activation of p38MAPK.
p38MAPK inhibitors block TGFß-mediated cell motility TGFß
stimulates chemotaxis and migration of tumor and nontumor cells
(Ashcroft et al., 1999;
Postlethwaite et al., 1987
).
Recent studies implicated p38MAPK in TGFß-induced chemotaxis of human
neutrophils (Hannigan et al.,
1998
). We next tested the effect of p38MAPK inhibitors on
TGFß-mediated migration of NMuMG (nontumor) and MDA-MB-231 (tumor) cells.
TGFß stimulated approximately threefold the chemotactic migration of
NMuMG cells through polycarbonate filters
(Fig. 9A). Migration of NMuMG
cells was significantly inhibited by SB202190, as were NMuMG cells infected
with kinase-inactive TGFß type II receptor (TßRII-K277R) compared
with those infected with control Gabe retrovirus
(Fig. 9A). TGFß stimulated
approximately sixfold migration of breast cancer MDA-MB-231 cells. This was
also blocked by SB202190 (Fig.
9B).
|
To investigate further the role of p38MAPK in TGFß-mediated cell migration, wounds were made in confluent cultures of MDA-MB-231 and 4T1 breast cancer cells. These cells are not growth inhibited by TGFß1. Addition of TGFß1 to serum-free medium accelerated the wound closure in both cell lines, whereas in the presence of the p38MAPK inhibitor the wounds stayed opened (Fig. 9D). These data suggest that p38MAPK is involved in TGFß-induced cell migration.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The H-7 studies suggested a critical role for the p38MAPK pathway in EMT.
This hypothesis was further tested using the p38MAPK specific inhibitors,
SB202190 and SB203580, which do not inhibit JNK, MEK1/2 and ERK1/2 kinases
(Davies et al., 2000).
SB202190 and SB203580 blocked TGFß-induced cell morphological changes in
NMuMG mouse mammary epithelial cells and SiHa human cervical carcinoma cells.
The p38MAPK inhibitors blocked TGFß-induced phosphorylation of ATF2, a
p38MAPK substrate, without effect on Smad2 phosphorylation, implying that
under these experimental conditions the blockade of p38MAPK did not affect
TGFß receptor kinase activity.
To test whether activation of p38MAPK by TGFß is a direct event, we
investigated the kinetics of activating phosphorylation of MKK3/6 and p38MAPK.
TGFß induced phosphorylation of Smad2 and MKK3/6 kinases with similar
kinetics (15 minutes). Phosphorylation of p38MAPK was delayed (30 minutes)
suggesting that this event requires activation of MKK3/6. We further showed
that dominant-negative mutants of MKK3 and MKK6 interfering with p38MAPK
activation (Raingeaud et al.,
1996) impaired TGFß-induced phosphorylation of p38MAPK and
ATF2, indicating that the MKK3/6-p38MAPK module mediates TGFß signaling
in NMuMG cells. The dose-dependent increase in p38MAPK activity was confirmed
by in vitro kinase assay and by phosphorylation of ATF2.
To confirm the specificity of TGFß signaling to p38MAPK we performed studies with TGFß receptor mutants. Kinase-inactive type II receptor blocked EMT and phosphorylation of Smad2 as well as MKK3/6 and p38MAPK, indicating that kinase function of TßRII is required for activation of p38MAPK and EMT. Kinase-inactive TßRI/Alk5-K232R also blocked TGFß-induced activation of the p38MAPK pathway, whereas expression of kinase active Alk5-T204D resulted in phosphorylation of MKK3/6 and p38MAPK and EMT in the absence of added TGFß1. Thus, kinase activities of both TGFß receptors are required for TGFß-induced activation of the p38MAPK pathway, and Alk5-T204D can signal to p38MAPK in the absence of added ligand. Alk5-T204D-induced EMT was inhibited by SB202190, a p38MAPK inhibitor, suggesting that p38MAPK mediates EMT induced by Alk5-T204D.
Activation of p38MAPK is mediated by Rac1/CDC42 GTP-binding proteins
(Coghlan et al., 2000;
Tibbles et al., 1996
;
Uddin et al., 2000
;
Zhang et al., 1995
). Small
GTP-binding proteins are also involved in TGFß responses
(Atfi et al., 1997
;
Bakin et al., 2000
;
Bhowmick et al., 2001a
;
Engel et al., 1999
;
Mucsi et al., 1996
). We found
that dominant-negative Rac1N17 impaired activation of the p38MAPK pathway in
NMuMG cells, whereas RhoAN19 did not block this event. Expression of Rac1N17
did not affect phosphorylation of Smad2. These data suggest that Rac1 mediates
TGFß-induced p38MAPK activation independently of Smad activation. The
mechanism of downstream signaling events is unclear. Previous studies showed
that PAK1 mediates p38MAPK activation downstream of Rac1 and CDC42
(Zhang et al., 1995
).
Furthermore, TGFß-activated kinase 1 (TAK1), has been implicated in
p38MAPK activation in response to BMP and TGFß in several cell systems
(Yamaguchi et al., 1995
).
Expression of dominant-negative Rac1N17 in NMuMG cells inhibited
TGFß1-induced changes in cell shape and the actin cytoskeleton suggesting
involvement of Rac1 in TGFß-induced EMT. This result is consistent with
other reports. For example, both D-Rac and D-p38 have been reported to
contribute to Dpp signaling during wing morphogenesis in Drosophila
(Adachi-Yamada et al., 1999;
Eaton et al., 1995
). There is
also evidence that Rac1 is required for EMT induced by hepatocyte growth
factor (HGF) in MDCK cells (Ridley et al.,
1995
; Royal et al.,
2000
). Dominant-negative Rac/CDC42 mutants inhibit oncogenic
Ras-induced cell transformation (Qiu et
al., 1997
; Qiu et al.,
1995
), and Ras has been shown to cooperate with TGFß in the
induction of EMT (Oft et al.,
1996
). In addition, Rho/Rac/CDC42 proteins are involved in
morphogenesis by regulating the actin cytoskeleton
(Hall, 1998
). Therefore, Rac1
may contribute to TGFß-induced EMT via its effects on the cell
cytoskeleton and/or via activation of the p38MAPK pathway. In NMuMG cells,
TGFß1 induced actin ruffles and activation of Rac1 within 15 minutes
(Fig. 7C,E). Expression of
kinase-inactive Alk5K232R reduced, whereas constitutively active Alk5-T204D
increased, Rac1 loading with GTP (Fig.
7D) and induced the formation of strong actin ruffles (data not
shown). These results suggest that Rac activation and actin ruffling induced
by TGFß may precede the formation of actin stress fibers, which does not
occur until 4 hours after addition of TGFß1
(Bhowmick et al., 2001a
).
Inhibitors of p38MAPK and dominant-negative MKK3AL impaired
TGFß-induced changes in cell morphology and reorganization of the actin
cytoskeleton. Expression of the dominant-negative mutant of p38 also
blocked TGFß-mediated EMT. Together, these results suggest that the
p38MAPK pathway contributes to TGFß-induced alterations in the actin
cytoskeleton and the cell shape during EMT. Consistent with this hypothesis,
p38MAPK has been shown to mediate regulation of the actin cytoskeleton in
smooth muscle myocytes in response to TGFß
(Hedges et al., 1999
), and in
H2O2-induced rapid reorganization of the actin
cytoskeleton in endothelial and mesenchymal cells
(Huot et al., 1998
). A recent
study reported involvement of p38MAPK in TGFß-mediated EMT
(Bhowmick et al., 2001b
). In
this report, adenoviral transduction of dominant-negative p38ß inhibited
TGFß-mediated EMT at the step of disruption of junctional complexes but
did not alter actin reorganization. We found that p38MAPK inhibitors and
dominant-negative MKK3AL affected actin stress fiber formation
(Fig. 6). TGFß and
Alk5T204D activated both MKK3 and MKK6 in NMuMG cells (Figs
3,
4). This suggests that
TGFß may activate multiple p38MAPK isoforms in NMuMG cells as MKK3
preferentially activates p38
and p38
, while MKK6 activates
p38MAPKs
, ß and
(Enslen et al., 1998
). Recent
studies showed that p38
and p38ß may have different functions
(Wang et al., 1998
) and
different subcellular localization (Lee et
al., 2000
). p38MAPK inhibitors block activity of both p38
and p38ß (Enslen et al.,
1998
) and MKK3AL impaired phosophorylation of p38MAPK in NMuMG
cells as measured with an antibody that recognizes both
and ß
isoforms. Therefore, multiple p38MAPKs may be involved in TGFß-induced
EMT and mediate different aspects of EMT, potentially explaining the
discrepancies with previous studies
(Bhowmick et al., 2001b
).
EMT is a complex process involving restructuring of the cell cytoskeleton,
cell membrane and cell-cell junctions. Previous studies implicated several
molecules in different aspects of EMT. Smad transcription factors have been
shown to synergize with Alk5 in induction of EMT but no specific function has
been associated with these factors (Piek
et al., 1999a). PI3K/Akt may contribute to dissolution of tight
junctions and to TGFß transcriptional responses
(Bakin et al., 2000
). RhoA/Rock
signaling has been implicated in the actin stress fiber formation
(Bhowmick et al., 2001a
). What
aspect of EMT can be mediated by p38MAPK? p38MAPK can regulate the actin
organization via HSP27 (Hedges et al.,
1999
; Huot et al.,
1998
). Therefore, p38MAPK may function in TGFß-induced
reorganization of the actin cytoskeleton in parallel or upstream of the
RhoA/Rock pathway since dn-RhoA and Y27632, a Rock kinase inhibitor, did not
affect activation of p38MAPK by TGFß (data not shown). In addition,
p38MAPK may contribute to the expression of TGFß target genes that are
casually involved in EMT because p38MAPK has been implicated in
TGFß-transcriptional responses by activating ATF2 and Sp1
(Park et al., 2000
;
Raingeaud et al., 1996
;
Sano et al., 1999
).
Finally, we investigated the role of p38MAPK in TGFß-induced migration
of mouse and human mammary epithelial cells. The p38MAPK inhibitors blocked
TGFß-stimulated migration of NMuMG, MDA-MB-231 and 4T1 cells. These
results are consistent with the proposed role of p38MAPK in TGFß-mediated
chemotaxis of human neutrophils (Hannigan
et al., 1998) and smooth muscle cells
(Hedges et al., 1999
).
Interestingly, Smad3-deficient keratinocytes and monocytes are impaired in the
chemotactic response to TGFß (Ashcroft
et al., 1999
), whereas p38MAPK inhibitors did not affect Smad2
phosphorylation (Fig. 2). These
data suggest that the p38MAPK pathway may act in parallel or in cooperation
with a Smad-dependent pathway in chemotactic responses to TGFß.
The data presented suggest that p38MAPK signaling plays a critical role in TGFß-induced EMT and cell migration. This pathway may be considered as a potential target of therapeutic interventions in neoplastic and inflammatory disorders associated with TGFß-mediated EMT.
![]() |
Acknowledgments |
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