p38 mitogen-activated protein kinase is required for TGFß-mediated fibroblastic transdifferentiation and cell migration
Andrei V. Bakin1,
Cammie Rinehart1,
Anne K. Tomlinson1 and
Carlos L. Arteaga1,2,3,*
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
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Fig. 1. Inhibition of TGFß-mediated EMT and p38MAPK activation by H-7 kinase
inhibitor. (A) NMuMG mammary epithelial cells were grown on glass coverslips
for 24 hours and treated (bottom row) or not (top row) with 2 ng/ml TGFß1
for 24 hours. Where indicated, cells were co-incubated with 20 µM H-7.
Phase contrast images were taken at 200x magnification. (B-E) Immunoblot
analysis of whole-cell extracts from NMuMG cells treated with 2 ng/ml
TGFß1 for the indicated times. Kinase inhibitors were added 60 minutes
before TGFß treatment. (B) Immunoblot detection of phospho-Smad2 and
total Smad2. (C) Detection of phospho-p38MAPK total p38MAPK. (D) Inhibition of
TGFß-induced ATF2 phosphorylation by H-7. Immunoblots with antisera to
phospho-ATF2 and total ATF2. (E) TGFß-induced phosphorylation of MKK3/6
in cells co-treated with various concentrations of H-7 or 5 µM BIM-I, a PKC
inhibitor. (F) Luciferase activity in NMuMG transfected with Smad-dependent
reporter pSBE-Lux and pCMV-Rl vectors and treated with 1 ng/ml TGFß1 for
16 hours in the absence or presence of 20 µM H-7. Each bar represents the
mean±s.d. of three wells.
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Fig. 2. Blockade of TGFß-induced EMT by SB202190. (A) NMuMG cells grown on
glass coverslips were treated (bottom row) or not (top row) with 2 ng/ml
TGFß1 for 24 hours in the absence or presence of 10 µM SB202190. Phase
contrast images were taken at 200x magnification. (B) phospho-Smad2 and
total Smad2 immunoblot analysis of whole-cell extracts from cells treated with
2 ng/ml TGFß1 in the absence or presence of SB202190. (C) Immunoblots
with antisera to phospho-ATF2 and total ATF2. SB202190 inhibits
TGFß-induced phosphorylation of ATF2.
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Fig. 3. Activation of the p38MAPK pathway in response to TGFß. NMuMG cells
were incubated in serum-free medium for 4 hours before addition of TGFß1.
(A) Immunoblot analyses with antibodies to phospho-Smad2, phospho-MKK3/6 and
phospho-p38MAPK, and total Smad2, MKK3/6 and p38MAPK. (B) Detection of p38MAPK
kinase activity in whole-cell extracts from NMuMG cells treated with 2 ng/ml
TGFß1 using GST-ATF2 as substrate. The products were separated by
SDS-PAGE and transferred onto nitrocellulose-membrane.  -32P
incorporation into ATF2 was quantitated using PhosphorImager. The membrane was
probed with antibody to p38MAPK. (C) Immunoblot detection of TGFß1
dose-dependent effect on p38MAPK phosphorylation at 60 minutes in NMuMG cells.
(D) Induction of p38MAPK phosphorylation by 2 ng/ml TGFß1 at 60 minutes
in SiHa cells.
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Fig. 4. Effect of kinase mutant TGFß receptors on TGFß-induced EMT and
activation of the p38MAPK pathway. (A) Immunoblot analyses of whole-cell
extracts from NMuMG cells infected with retrovirus encoding TßRII-K277R
or control virus (Gabe). Cells were treated with 2 ng/ml TGFß1 for 60
minutes. Expression of HA-tagged TßRII-K277R was detected with antisera
to the HA-epitope. Dominant-negative TßRII-K277R inhibits phosphorylation
of Smad2, MKK3/6 and p38MAPK in response to TGFß. Membranes were
re-probed with antibodies to total Smad2 and p38MAPK. (B) Immunoblot analyses
of whole-cell extracts from NMuMG cells infected with retroviruses encoding
HA-tagged wild-type (WT) TßRI/Alk5, kinase-inactive Alk5-K232R, and
kinase-active Alk5-T204D. Cells were treated with 2 ng/ml TGFß1 for 60
minutes, and protein extracts were probed with antibodies to phospho-MKK3/6,
phospho-p38MAPK and total p38MAPK. Membranes were reprobed with antisera to
the HA-epitope. (C) Phase contrast images of NMuMG cells expressing wild-type
Alk5 (Alk5-WT), Alk5-K232R, and TßRII-K277R. Cells grown on glass
coverslips were untreated (top row) or treated (bottom row) with 2 ng/ml
TGFß1 for 24 hours. (D) NMuMG cells expressing Alk5-T204D were untreated
or treated with 15 µM SB202190 for 24 hours. Phase-contrast images were
recorded at 200x magnification.
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Fig. 8. Dominant-negative Rac1N17 blocks TGFß-mediated activation of p38MAPK
and EMT. NMuMG cells were infected with retrovirus encoding Rac1N17 or control
virus (BMN) and treated with 2 ng/ml TGFß1. (A) Detection of Rac1N17
expression with antisera to Rac1. Cells infected with Rac1N17 show a higher
level of Rac1 expression. (B) Immunoblot detection of MKK3/6 and p38MAPK
phosphorylation in control (BMN) and Rac1N17 expressing cells. (C) Immunoblot
with antisera to phospho-Smad2 and total Smad2. (D) Microscopic images from
NMuMG cells infected with control retrovirus (BMN) or retrovirus encoding
Rac1N17. Cells grown on glass coverslips were treated with 2 ng/ml TGFß1
for 24 hours. Cells were stained with phalloidin-Texas Red (actin). Actin and
interference-contrast images (DIC) were recorded from the same cells. Bar, 15
µm.
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Fig. 5. Effect of dominant-negative MKK3AL and p38AGF on TGFß-mediated EMT.
(A) Immunoblot analysis of p38MAPK and ATF2 phosphorylation in NMuMG cells
transfected with empty vector (BMN) or plasmid encoding HA-tagged MKK3AL.
Thirty-six hours after transfection cells were treated with 2 ng/ml TGFß1
for 60 minutes. Whole-cell extracts were probed with phospho-specific
antisera, and re-probed with antisera to total protein. Expression of MKK3AL
was detected with antisera to the HA-epitope. (B) Immunoblot detection of
Smad2 phosphorylation in MKK3AL-expressing cells. (C) Phase-contrast images of
NMuMG cells infected with control (BMN) retrovirus or retroviruses encoding
dominant-negative MKK3 (MKK3AL) or p38 (p38AGF). Cells were untreated
(top row) or treated with 2 ng/ml TGFß1 for 24 hours. Images were
recorded at 200x magnification. (D) Immunoblot detection of Flag-tagged
p38AGF in NMuMG cells infected with p38AGF encoding retrovirus compared with
control retrovirus (BMN).
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Fig. 6. Effect of p38MAPK inhibitors on TGFß-induced reorganization of the
actin cytoskeleton. (A) NMuMG cells grown on glass coverslips were incubated
with 2 ng/ml TGFß1 for 24 hours in the absence or presence of 15 µM
SB202190. Cells were fixed and stained with phalloidin-Texas Red (actin).
Actin staining and interference-contrast images (DIC) were recorded from the
same cells. Note cell elongation and actin stress fibers formation in
TGFß-treated cells in the absence of the p38MAPK inhibitor compared with
cells treated with the p38MAPK inhibitor. (B) Actin cytoskeleton in NMuMG
cells infected with MKK3AL or control (BMN) retroviruses. Cells were treated
with 2 ng/ml TGFß1 for 24 hours and stained with phalloidin-Texas Red.
Bars, 15 µm.
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Fig. 7. Rac1 is involved in TGFß-mediated activation of p38MAPK. (A)
Immunoblot analysis of p38MAPK and ATF2 phosphorylation in cells expressing
Rac1N17 and treated with 2 ng/ml TGFß1. (B) p38MAPK phosphorylation in
cells expressing RhoAN19 or RhoAQ63L. (C) NMuMG cells were treated with 2
ng/ml TGFß1 for 15 minutes. Cell lysates were clarified and used for
affinity precipitation with 8 µg of GST-PBD. Proteins bound to GST-PBD were
separated on SDS-PAGE, transferred to nitrocellulose membrane and blotted with
antibody to Rac1. The inset at the top-left shows the total signal detected
using cell lysate pre-exchanged with either GTPS or GDP as described in
Materials and Methods. (D) 293T cells were transfected with control plasmid
(BMN), kinase-inactive Alk5K232R, kinase-active Alk5T204D or dominant-negative
Rac1N17. Cells were lysed 48 hours after transfection. Cell lysates were
clarified and used for affinity precipitation with 8 µg of GST-PBD as
described above. The bottom inset shows the Rac1 signal detected in total cell
lysates. (E) Confocal images of F-actin in NMuMG cells treated with 2 ng/ml
TGFß1 for 15 minutes and stained with phalloidin-Texas Red. Arrows
indicate the spots of actin polymerization at the cell edges.
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Fig. 9. Involvement of p38MAPK in TGFß-mediated cell migration. (A,B) NMuMG or
MDA-MB-231 cells (1x105/well) were seeded in the upper
chamber of 5 µm pore transwells and 2 ng/ml TGFß1 was added to the
lower chamber. Cells were incubated for 16 hours in the absence or presence of
SB202190, a p38MAPK inhibitor. Cells migrating through pores were stained and
counted from four random fields. Experiments were performed in duplicates.
Values are the mean ±s.d. of cells per field. Migration of NMuMG cells
expressing kinase-inactive TßRII-K277R was compared with cells infected
with control Gabe virus. (B) Blockade of MDA-MB-231 cell migration with 10
µM SB202190. (C) Wound closure in monolayers of MDA-MB-231 and 4T1 cells
following 16 hours of treatment with 2 ng/ml TGFß1 in the absence or
presence of 10 µM SB202190. Phase contrast images were recorded at
100x magnification. Similar results were obtained three times.
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© The Company of Biologists Ltd 2002
