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Requirement of transforming growth factor activated kinase 1 for transforming growth factor induced ╨Ю┬▒-smooth muscle actin expression and extracellular matrix contraction in fibroblasts.

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ARTHRITIS & RHEUMATISM
Vol. 60, No. 1, January 2009, pp 234–241
DOI 10.1002/art.24223
© 2009, American College of Rheumatology
Requirement of Transforming Growth Factor ␤–Activated
Kinase 1 for Transforming Growth Factor ␤–Induced
␣-Smooth Muscle Actin Expression and
Extracellular Matrix Contraction in Fibroblasts
Xu Shi-wen,1 Sunil K. Parapuram,2 Daphne Pala,2 Yunliang Chen,3 David E. Carter,4
Mark Eastwood,3 Christopher P. Denton,1 David J. Abraham,1 and Andrew Leask2
Objective. Fibrosis is believed to occur through
normal tissue remodeling failing to terminate. Tissue
repair intimately involves the ability of fibroblasts to
contract extracellular matrix (ECM), and enhanced
ECM contraction is a hallmark of fibrotic cells in
various conditions, including scleroderma. Some fibrogenic transcriptional responses to transforming growth
factor ␤ (TGF␤), including ␣-smooth muscle actin
(␣-SMA) expression and ECM contraction, require
focal adhesion kinase/Src (FAK/Src). The present study
was undertaken to assess whether TGF␤-activated ki-
nase 1 (TAK1) acts downstream of FAK/Src to mediate
fibrogenic responses in fibroblasts.
Methods. We used microarray, real-time polymerase chain reaction, Western blot, and collagen gel
contraction assays to assess the ability of wild-type and
TAK1-knockout fibroblasts to respond to TGF␤1.
Results. The ability of TGF to induce TAK1 was
blocked by the FAK/Src inhibitor PP2. JNK phosphorylation in response to TGF␤1 was impaired in the
absence of TAK1. TGF␤ could not induce matrix contraction or expression of a group of fibrotic genes,
including ␣-SMA, in the absence of TAK1.
Conclusion. These results suggest that TAK1 operates downstream of FAK/Src in mediating fibrogenic
responses and that targeting of TAK1 may be a viable
antifibrotic strategy in the treatment of certain disorders, including scleroderma.
Supported by the Canadian Institutes of Health Research, the
Canadian Foundation for Innovation, the Arthritis Research Campaign, the Raynaud’s and Scleroderma Association, and the Scleroderma Society. Dr. Parapuram is recipient of a postdoctoral fellowship
from the Canadian Institutes of Health Research, Canadian Scleroderma Research Group. Ms Pala was recipient of a Network of Oral
Research Training and Health scholarship for undergraduate dental
research and is recipient of an Ontario Graduate Scholarship in
Science and Technology at the University of Western Ontario. Dr.
Leask is a New Investigator of the Arthritis Society (Scleroderma
Society of Ontario) and recipient of an Early Researcher Award from
the Ontario Ministry of Research and Innovation.
1
Xu Shi-wen, PhD, Christopher P. Denton, PhD, FRCP,
David J. Abraham, PhD: University College London (Royal Free
Campus), London, UK; 2Sunil K. Parapuram, PhD, Daphne Pala,
MSc, Andrew Leask, PhD: University of Western Ontario, London,
Ontario, Canada; 3Yunliang Chen, PhD, Mark Eastwood, PhD: University of Westminster, London, UK; 4David E. Carter, MSc: London
Regional Genomics Centre, London, Ontario, Canada.
Dr. Denton has received consulting fees, speaking fees,
and/or honoraria from Actelion and Encysive (less than $10,000 each).
Dr. Abraham has received consulting fees, speaking fees, and/or
honoraria from Actelion and Encysive (less than $10,000 each).
Address correspondence and reprint requests to Andrew
Leask, PhD, CIHR Group in Skeletal Development and Remodeling,
Department of Physiology and Pharmacology, Schulich School of
Medicine and Dentistry, Dental Sciences Building, University of
Western Ontario, London, Ontario N6A 5C1, Canada. E-mail:
Andrew.Leask@schulich.uwo.ca.
Submitted for publication April 30, 2008; accepted in revised
form October 3, 2008.
Normal tissue repair involves migration of fibroblasts into the wound. The fibroblasts then synthesize
and remodel extracellular matrix (ECM), resulting in
wound closure. These events involve the ability of fibroblasts to attach to ECM through specialized cell surface
structures called focal adhesions (1). Persistently activated adhesion and adhesive signaling is a hallmark of
fibrotic cells, including those cultured from lesional
areas in patients with the fibrotic autoimmune disease
systemic sclerosis (SSc; scleroderma) (2,3). Adhesion to
ECM involves integrins, whose signals are transmitted
by focal adhesion kinase (FAK), which is present at focal
adhesions and is phosphorylated after integrin-mediated
cell attachment (4). FAK has been classically considered
to mediate fibroblast attachment to ECM; however, a
priori it is also possible that it may be involved in
transducing signals from growth factors (5). Indeed, we
have recently found that the ability of transforming
234
NECESSITY OF TAK1 FOR FIBROBLAST RESPONSES TO TGF␤
235
TAK1 to investigate the contribution of TAK1 to mediation of transcriptional responses to TGF␤ in fibroblasts. Our results provide new insights into the contribution of TAK1 to tissue repair and remodeling
responses in fibroblasts and suggest that targeting of
TAK1 may be a viable antifibrotic treatment approach.
MATERIALS AND METHODS
Figure 1. Requirement for focal adhesion kinase in transforming
growth factor ␤ (TGF␤) induction of TGF␤-activated kinase 1 (TAK1)
phosphorylation in fibroblasts. A, Fibroblasts from lesional areas of
skin from patients with systemic sclerosis (SSc) and age-, sex-, and
site-matched controls were serum starved for 24 hours and subjected
to Western blot analysis with anti-TAK1 and anti–phospho-TAK1
antibodies. Cells from 4 normal subjects and 4 SSc patients were used.
B, Fibroblasts from tak⫹/⫹ mice were serum starved and left untreated
or treated with TGF␤1 (4 ng/ml) for 30 minutes. Cells were pretreated
with PP2 (10 ␮M) for 45 minutes prior to addition of TGF␤ or were
not pretreated. In parallel experiments, fibroblasts from fak⫹/⫹ and
fak⫺/⫺ mice were similarly tested. Cell extracts were prepared and
subjected to Western blot analysis with anti–phospho-TAK1 and
anti-TAK1 antibodies as indicated. A total of 4 independent experiments were performed, and representative blots are shown.
growth factor ␤ (TGF␤) to induce expression of messenger RNA (mRNA) for profibrotic genes involves
FAK (6). FAK was required in order for TGF␤ to
induce JNK, and the FAK/MEKK1/JNK pathway was
required for TGF␤ to induce myofibroblast formation,
as identified by ␣-smooth muscle actin (␣-SMA) expression and the ability of fibroblasts to contract ECM (6).
TGF␤-activated kinase 1 (TAK1), a member of
the MAPKKK family, is thought to be a key modulator
of the inducible transcription factors NF-␬B and activator protein 1, and therefore plays a crucial role in
regulating the genes that mediate inflammation. In
fibroblasts, TAK1 deletion does not impair the ability of
TGF␤ to activate the Smad pathway (7). However,
Smad-independent pathways are known to mediate the
action of TGF␤; thus, the effect of loss of TAK1 on
transcriptional responses in fibroblasts is unclear. We
used wild-type fibroblasts and fibroblasts deficient in
Cell culture and Western blot analysis. Fibroblasts
derived from E9.75 wild-type (tak⫹/⫹) and homozygous mutant (tak⫺/⫺) mouse embryos were immortalized by stably
transfecting linearized SV40 large T antigen plasmid (7). For
some experiments, fibroblasts obtained from E8.5 FAK wildtype and FAK-knockout mouse embryos (American Type
Culture Collection, Rockville, MD) were used. In addition,
dermal fibroblasts from affected areas in patients with SSc and
from age-, sex- and site-matched controls were used. Cells
were grown in Dulbecco’s modified Eagle’s medium containing
5% fetal calf serum (FCS), 2 mM L-glutamine, antibiotics (100
units/ml penicillin and 100 ␮g/ml streptomycin), and 1 mM
sodium pyruvate (Invitrogen, Burlington, Ontario, Canada).
Cells were serum starved for 24 hours and then left untreated
or treated with TGF␤1 (4 ng/ml; R & D Systems, Minneapolis,
MN). Whole cell protein extracts were produced and subjected
to Western blot analysis using anti-TAK, anti–phospho-TAK,
anti-JNK, anti–phospho-JNK1 (all from Cell Signaling Technology, Vancouver, British Columbia, Canada), anti-CCN2
(Abcam, Cambridge, UK), anti–␣-SMA (Sigma, St. Louis,
MO), or anti-GAPDH (Abcam). Intensity of signals relative to
GAPDH controls was calculated using densitometry, and
averages and standard deviations of data obtained from 3
independent experiments were calculated.
RNA quality assessment, probe preparation, and GeneChip hybridization and analysis. Microarray analysis was
performed as previously described (6,8,9). GeneChips were
processed at the London Regional Genomics Centre (Robarts
Research Institute, London, Ontario, Canada [http://
Figure 2. Impairment of transforming growth factor ␤ (TGF␤)–
induced JNK phosphorylation in the absence of TGF␤-activated
kinase 1. Fibroblasts from tak⫹/⫹ mice (wild-type [WT]) or tak⫺/⫺
mice (knockout [KO]) were serum starved and left untreated or
treated with TGF␤1 (4 ng/ml) for 1 hour. Cell extracts were prepared
and subjected to Western blot analysis with anti-JNK1 or anti–
phospho-JNK1 antibodies. A total of 4 independent experiments were
performed, and representative blots are shown.
236
SHI-WEN ET AL
Figure 3. Necessity of TGF␤-activated kinase 1 for TGF␤ induction of ␣-smooth muscle actin (␣-SMA) protein and mRNA in fibroblasts. A,
Fibroblasts from tak⫹/⫹ and tak⫺/⫺ mice were serum starved and left untreated or treated with TGF␤1 (4 ng/ml) for 24 hours. Proteins were
subjected to Western blot analysis with anti–␣-SMA, anti-CCN2 (anti–connective tissue growth factor [anti-CTGF]), or anti-GAPDH antibodies. In
tak⫺/⫺ mouse cells, ␣-SMA protein was not induced, whereas CCN2 induction was observed. Densitometry of the blots resulting from 4 separate
experiments was performed, and quantitative results for ␣-SMA (upper graph) and CCN2 (lower graph) are shown. Bars from left to right show the
mean and SD results in untreated tak⫹/⫹ mouse fibroblasts, untreated tak⫺/⫺ mouse fibroblasts, TGF␤-treated tak⫹/⫹ mouse fibroblasts, and
TGF␤-treated tak⫺/⫺ mouse fibroblasts, respectively. ⴱ ⫽ P ⬍ 0.05 versus TGF␤-treated tak⫺/⫺ mouse fibroblasts (upper graph) or versus untreated
tak⫹/⫹ and tak⫺/⫺ mouse fibroblasts (lower graph). B, Fibroblasts from tak⫹/⫹ and tak⫺/⫺ mice were serum starved and left untreated or treated
with TGF␤1 (4 ng/ml) for 6 hours. Real-time polymerase chain reaction analysis was performed with primers detecting ␣-SMA, CCN2, and
ribosomal RNA (rRNA); samples were standardized to rRNA. Values are the mean ⫾ SD fold increase in response to TGF␤ (in relation to basal
expression in WT and KO cells [set at 1]); SDs within groups were ⬍10%. Induction of mRNA for ␣-SMA, but not CCN2, was impaired in tak⫺/⫺
cells. ⴱ ⫽ P ⬍ 0.05 versus TGF␤-treated tak⫹/⫹ cells. See Figure 2 for other definitions.
www.lrgc.ca]). Briefly, RNA was harvested using TRIzol (Invitrogen, Carlsbad, CA), quantified, and quality was assessed
with a Degradometer (www.dnaarrays.org) (Agilent, Palo Alto,
CA). Biotinylated complementary RNA (cRNA) was prepared
from 10 ␮g of total RNA, according to instructions in the
Affymetrix GeneChip Technical Analysis Manual (Affymetrix,
Santa Clara, CA). Double-stranded cDNA was synthesized
using Superscript II (Invitrogen) and oligo(dT) 24 primers.
Biotin-labeled cRNA was prepared by in vitro transcription
using the Bizarre High-Yield RNA Transcript Labeling kit
(Enzo Brioche, New York, NY) with biotinylated UTP and
CTP. Fifteen micrograms of labeled cRNA was hybridized to
Mouse Genome 430 2.0 GeneChips for 16 hours at 45°C as
described (Affymetrix). GeneChips were stained with
streptavidin–phycoerythrin, an antibody solution, and a second
streptavidin–phycoerythrin solution. Liquid handling was performed using a GeneChip Fluidics Station 450. GeneChips
were scanned with a GeneChip Scanner 3000 (Affymetrix).
Signal intensities of genes were determined with
GCOS1.2 (Affymetrix), using default values for the statistical
NECESSITY OF TAK1 FOR FIBROBLAST RESPONSES TO TGF␤
237
Figure 4. Necessity of TGF␤-activated kinase 1 (TAK1) for TGF␤ induction of collagen gel contraction in fibroblasts. A, Floating gel contraction
assay. Fibroblasts from tak⫹/⫹ and tak⫺/⫺ mice were placed in collagen gel lattices. After polymerization, lattices were detached from tissue culture
plates and left untreated or treated with TGF␤1 (4 ng/ml) for 24 hours. Contraction was monitored by measuring gel weight. Values are the mean
and SD from 3 independent experiments. ⴱ ⫽ P ⬍ 0.05 versus untreated control WT cells. B, Fibroblast-populated collagen lattice assay. Fibroblasts
from tak⫹/⫹ and tak⫺/⫺ mice were placed in collagen gel lattices. After polymerization, lattices were left untreated or treated with TGF␤1 (4 ng/ml)
for 24 hours. Contractile forces were monitored over 24 hours as previously described (13). Experiments were performed 3 times; results of a
representative experiment are shown. 3-D ⫽ 3-dimensional (see Figure 2 for other definitions).
expression algorithm parameters, a target signal of 150 for all
probe sets, and a normalization value of 1. Normalization was
performed with GeneSpring 7.2 (Agilent). An RMA preprocessor was used to import data from the .cel files. Data were
first transformed (measurements ⬍0.01 set to 0.01) and then
normalized per chip to the fiftieth percentile, and per gene to
wild-type control samples. Experiments were performed twice,
and fold changes were identified using the GeneSpring filter.
The change between treated and untreated samples had to be
at least 2-fold for a transcript to be identified as being altered.
These criteria had to be met in both sets of experiments; the
variation between the data points in each set was ⬍25%. Probe
sets that were up-regulated by ⱖ2 fold, as well as 52 probe sets
that were down-regulated (0.5-fold and below), were further
analyzed using the above-mentioned software packages and
DAVID (http://david.abcc.ncifcrf.gov/home.jsp) (1,2). The analysis was carried out at high stringency and generated 12
functional clusters.
Real-time polymerase chain reaction (PCR). Cells
were serum starved for 24 hours and treated with 4 ng TGF␤
for 6 hours. Total RNA was isolated using TRIzol, and the
integrity of the RNA was verified by gel electrophoresis or with
an Agilent bioanalyzer. For initial time course analysis, total
RNA (25 ng) was reverse transcribed, and amplified using
TaqMan Assays-on-Demand (Applied Biosystems, Foster City,
CA) in a 15-␮l reaction volume containing 2 unlabeled primers and
6-carboxyfluorescein–labeled TaqMan MGB probe. Samples were
combined with TaqMan One-step Master Mix (Applied Biosystems). Amplified sequences were detected using the ABI Prism 7900
HT sequence detector according to the instructions of the manufac-
turer (PerkinElmer Cetus, Vaudreuil, Quebec, Canada). Triplicate
samples were run, and transcripts and expression values were standardized to values obtained with control 28S RNA primers using the
⌬⌬Ct method as previously described (9–11). Variation within
samples was ⬍10%.
Floating collagen gel cultures and quantitation of gel
contraction. Collagen gel experiments were performed essentially as previously described (12). Briefly, 24-well tissue culture plates were precoated with bovine serum albumin.
Trypsinized fibroblasts were suspended in MCDB medium and
mixed with collagen solution (1 part 0.2M HEPES [pH 8.0], 4
parts collagen [Vitrogen-100, 3 mg/ml] [Cohesion Technologies, Palo Alto, CA], and 5 parts MCDB X2), yielding a final
concentration of 80,000 cells per ml and 1.2 mg/ml collagen.
Collagen/cell suspension (1 ml) was added to each well. After
polymerization, gels were detached from wells by adding 1 ml
MCDB medium. Contraction of the gel was quantified based
on loss of gel weight and decrease in gel diameter over a
24-hour period. For inhibition experiments, cells were preincubated in the presence of inhibitor for 30 minutes prior to
initiation of the assay.
Fibroblast-populated collagen lattices (FPCLs). Measurement of contractile force generated within a
3-dimensional, tethered floating fibroblast–populated collagen
lattice was performed as described previously (13,14). Using
1 ⫻ 106 cells/ml of collagen gel (First Link, Birmingham, UK),
we determined the force generated across the collagen lattice,
with a culture force monitor. This instrument is capable of
measuring the minute forces exerted by cells within a collagen
lattice (13,14) over 24 hours as fibroblasts attach, spread,
238
SHI-WEN ET AL
Table 1. Genes whose expression was increased ⬎2-fold in wild-type
(WT) but not in tak-knockout (KO) mouse embryonic fibroblasts*
Fold increase
Affymetrix ID
WT
KO
Cell motility
genes
1428650_at
1426955_at
1452587_at
2.1
2.4
2.2
1.7
0.9
0.8
1416157_at
1417122_at
1450413_at
2.2
2.3
2.9
0.8
0.9
1.8
2.4
2.0
1.2
1.5
2.4
2.1
2.2
3.0
2.2
0.9
0.8
1.0
1.9
1.8
1448416_at
1423606_at
1417455_at
2.4
2.8
2.1
1.0
1.3
1.5
1448228_at
1448123_s_at
1416121_at
1419089_at
3.9
3.8
3.6
3.3
0.7
0.5
0.5
1.0
1449335_at
2.9
1.0
1452445_at
1452595_at
2.2
2.2
1.7
1.1
1416786_at
1421198_at
Extracellular matrix
genes
1426955_at
1418599_at
1416741_at
1460302_at
1451527_at
Gene name
Tensin 1
Procollagen type XVIII ␣1
Actin-related protein 2
homolog
Vinculin
Vav 3
Platelet-derived growth
factor, B
Activin A receptor type 1
␣V integrin
Procollagen type XVIII ␣1
Procollagen type XI ␣1
Procollagen type V ␣1
Thrombospondin 1
Procollagen c-endopeptidase
enhancer 2
Matrix Gla protein
Periostin
Transforming growth factor
␤3
Lysyl oxidase
Beta_IG
Lysyl oxidase
Tissue inhibitor of
metalloproteinases 3
Tissue inhibitor of
metalloproteinases 3
ADAM-28
ADAMTS-4
* Values are the mean from 2 independent experiments.
migrate, and differentiate into myofibroblasts. Briefly, a rectangular fibroblast-seeded collagen gel was cast and floated in
medium with 2% FCS in the presence or absence of TGF␤1 (4
ng/ml, 24 hours), tethered to 2 flotation bars on either side of
the long edges, which were in turn attached to a ground point
at one end and a force transducer at the other. Cell-generated
tensional forces in the collagen gel were detected by the force
transducer and the data logged into a personal computer.
Graphic readings were produced every 15 seconds, providing a
continuous output of force (dynes; 1 ⫻ 10⫺5N) generated
(13,14).
Statistical analysis. Student’s paired and unpaired
t-tests were used to assess the significance of differences
between treatment groups. P values less than 0.05 were considered significant.
RESULTS
Necessity of FAK/Src for TGF␤ induction of TAK
phosphorylation in fibroblasts. We previously showed
that the profibrotic protein endothelin 1 (ET-1) induces
myofibroblast formation through a JNK-dependent
mechanism, and that constitutive ET-1–dependent JNK
activation is a key feature of fibrotic fibroblasts in SSc
(15). TGF␤ can further induce JNK via a FAKdependent mechanism, leading to myofibroblast formation (6). Moreover, we found that TAK was constitutively phosphorylated in SSc fibroblasts (Figure 1A). To
investigate the control mechanisms through which
TGF␤ can induce a fibrotic phenotype in fibroblasts, we
evaluated the role of TAK1 in this pathway.
We first used fibroblasts cultured from wild-type
mice to verify, using Western blot analysis with anti–
phospho-TAK1 and anti-TAK1 antibodies, that TGF␤
could induce TAK in fibroblasts (Figure 1); enhanced
adhesive signaling is a feature of fibrotic cells (3,16). To
assess whether FAK was required in order for TGF␤ to
induce JNK, fibroblasts were pretreated with the FAK/
Src inhibitor PP2 prior to addition of TGF␤. The
resultant protein extracts were subjected to Western blot
analysis with anti–phospho-JNK and anti-JNK antibodies. TGF␤ induced TAK1 phosphorylation in fibroblasts,
and this was significantly impaired by PP2 (Figure 1B).
Moreover, TAK phosphorylation was reduced in response to TGF␤ in FAK-knockout mouse fibroblasts
(Figure 1B). These results suggested that TGF␤ induces
TAK1 in fibroblasts, in a FAK-dependent manner.
Impairment of TGF␤-induced JNK phosphorylation in the absence of TAK1. The ability of TGF␤ to
induce the Smad pathway does not involve TAK1 (7).
Since FAK/Src is required for JNK activation in fibroblasts (6), we assessed whether the ability of TGF␤ to
induce JNK phosphorylation was impaired in the absence of TAK1. Cells were serum starved for 24 hours
and then treated with TGF␤ for 1 hour. Protein was then
harvested from the cell layers and subjected to Western
blot analysis with anti-JNK1 and anti–phospho-JNK1
antibodies. The results demonstrated that the ability of
TGF␤ to induce JNK1 phosphorylation was impaired in
TAK1-deficient mouse fibroblasts (Figure 2).
Impairment of TGF␤-induced ␣-SMA expression and ECM contraction in the absence of TAK1. We
then wished to examine the contribution of FAK/Srcdependent TAK1 activation to the ability of TGF␤ to
induce a tissue remodeling phenotype in fibroblasts. To
address this, we first assessed whether TGF␤-induced
myofibroblast formation was impaired in TAK1deficient fibroblasts. Wild-type mouse fibroblasts were
left untreated or treated with TGF␤ for 24 hours, and
cells were then subjected to Western blot analysis with
an anti–␣-SMA antibody. TGF␤ potently increased
anti–␣-SMA protein expression in fibroblasts from
NECESSITY OF TAK1 FOR FIBROBLAST RESPONSES TO TGF␤
239
Figure 5. Inability of TGF␤ to induce expression of mRNA for various tissue
remodeling molecules in tak⫺/⫺ fibroblasts. Fibroblasts from tak⫹/⫹ and tak⫺/⫺ mice
were serum starved and left untreated or treated with TGF␤1 (4 ng/ml) for 6 hours.
Real-time polymerase chain reaction analysis was performed with primers detecting
vinculin, tissue inhibitor of metalloproteinases 3 (TIMP-3), thrombospondin 1
(TSP-1), and lysyl oxidase (LOX). Samples were standardized to ribosomal RNA.
Values are the mean ⫾ SD fold increase in response to TGF␤ (in relation to basal
expression in WT and KO cells [set at 1]) in 1 representative experiment (of 3
performed); SDs within groups were ⬍10%. ⴱ ⫽ P ⬍ 0.05 versus TGF␤-treated
tak⫹/⫹ cells. See Figure 2 for other definitions.
tak⫹/⫹ mice, but not in fibroblasts from tak⫺/⫺ mice
(Figure 3A). Results were confirmed by real-time PCR
analysis of mRNA harvested from cells that were untreated or were treated with TGF␤ for 6 hours (Figure
3B). Intriguingly, the ability of TGF␤ to induce CCN2
protein and mRNA, an action not involving JNK (17),
was not impaired in tak⫺/⫺ mouse embryonic fibroblasts.
Moreover, TGF␤ was able to induce tak⫹/⫹
mouse fibroblasts, but not tak⫺/⫺ mouse fibroblasts, to
contract in a floating collagen gel matrix (Figure 4A).
Similar results were obtained using an FPCL assay, in
which force generation across a floating collagen gel
matrix fixed at one end was examined (Figure 4B).
Collectively, these results are consistent with the notion
that FAK/TAK-dependent JNK activation is essential to
the ability of TGF␤ to signal in fibroblasts and, in
particular, in TGF␤-induced ␣-SMA expression and
matrix contraction.
Impairment of TGF␤-induced expression of fibrogenic genes in takⴚ/ⴚ mouse fibroblasts. To evaluate
the contribution of TAK1 to the ability of TGF␤ to
induce gene expression in fibroblasts, we cultured tak⫹/⫹
and tak⫺/⫺ mouse fibroblasts to 80% confluence and
serum starved the cells for 24 hours. Cells were then left
untreated or treated with TGF␤ (4 ng/ml) for an additional 6 hours. Total RNA was prepared from these
cells, reverse transcribed, and applied to Affymetrix
MOE430 arrays. Experiments were performed twice,
and average induction values were obtained.
Analysis of the data by GeneSpring revealed 265
transcripts that were increased ⬎2-fold by TGF␤ in
tak⫹/⫹ mouse fibroblasts. Of these, 194 were not induced ⬎2-fold in tak⫺/⫺ mouse fibroblasts. Cluster
analysis using DAVID revealed that expression of profibrotic transcripts involved in cell motility and ECM
was reduced in response to TGF␤ in TAK1-deficient
cells. These transcripts included genes for thrombospondin 1, tissue inhibitor of metalloproteinases 3,
vinculin, lysyl oxidase, and several collagens (Table 1).
Results obtained using microarray analysis for these
transcripts were verified by real-time PCR analysis of
RNA isolated from tak⫹/⫹ and tak⫺/⫺ mouse embryonic
fibroblasts that were left untreated or treated with
TGF␤ for 6 hours (Figure 5). Collectively, these findings
240
SHI-WEN ET AL
suggest that TAK1 is required for a subset of TGF␤
responses in fibroblasts, including the induction of certain profibrotic genes.
DISCUSSION
In the present study, we examined the signaling
events downstream of TGF␤ and FAK/Src that lead to
induction of a tissue remodeling phenotype. TGF␤
promotes ECM production and contraction in fibroblasts, leading to deposition of granulated tissue (18).
Although most studies have focused on the general
TGF␤ signaling pathway, including TGF␤ receptors and
Smads, in controlling ECM production and remodeling,
it is now understood that additional pathways mediate
the action of TGF␤ (18). In the potential development
of therapies that target these pathways, it is hoped that
wound healing and scarring responses could be affected
while other, perhaps beneficial, effects of TGF␤ are left
intact (18).
MAP kinase cascades play key roles in driving
tissue repair and fibrogenesis in response to TGF␤; for
example, both the Ras/MEK/ERK and FAK/JNK cascades control the expression of TGF␤ target genes in
fibroblasts, in a gene-specific manner (6,17,19). Both of
these pathways appear to work independent of the Smad
pathway (6,17,19). Previously, we have shown that JNK
mediates TGF␤ induction of ET-1 and that constitutive
JNK activation, mediated by endogenous ET-1 production, contributes at least partially to the persistent
fibrotic phenotype of scleroderma lung fibroblasts (15).
Moreover, we have demonstrated that JNK activation in
response to TGF␤ in normal fibroblasts depends on
FAK/Src (6). In this study, we showed that TGF␤induced JNK phosphorylation is impaired in the absence
of TAK1. Moreover, deletion of TAK1 resulted in a
failure of fibroblasts to support not only JNK activation
in response to TGF␤, but also the expression of mRNA
for ␣-SMA and a group of profibrotic TGF␤-induced
genes, and ECM contraction. All of these responses
required JNK and FAK in wild-type mouse fibroblasts
(6).
It was of interest that, in the FPCL assay, TAK1deficient cells contracted less well than did wild-type
cells. This suggests that TAK1 is required for basal
fibroblast function. Indeed, microarray analysis demonstrated that TAK1-deficient fibroblasts have reduced
gene expression even in the absence of added TGF␤
(results not shown). It should also be noted that the
FPCL assay measures myofibroblast induction in response to mechanical tension as well as to factors such as
cell migration (13). Thus, it is indeed possible that TAK1
deficiency may also affect basal fibroblast function.
Exploring this possibility was beyond the scope of the
current study. Since TAK1-deficient mice died early in
development, our studies with TAK1-knockout mouse
cells required use of a transformed line derived from
multiple embryos, in order to obtain sufficient cells for
analysis (7). It is therefore theoretically possible that
biases in clonal selection may have contributed to the
present results. Overall, however, our findings suggest
that, in fibroblasts, TAK is required downstream of
FAK/Src to mediate a subset of profibrotic responses to
TGF␤.
TAK1 was constitutively phosphorylated in fibroblasts isolated from 4 SSc patients, relative to healthy
control fibroblasts. This raises the intriguing possibility
that TAK1 inhibitors (although we are aware of none
that are readily commercially available) might be used to
combat the fibrosis observed in SSc. It is important to
note that since TAK1-deficient mice die in utero (7), it
is possible that TAK1 is responsible for functions other
than those described herein. Further evaluation of the
suitability of TAK1 as a therapeutic target for fibrosis
must await the development of conditionally deleted
mouse strains.
In conclusion, the findings reported herein provide evidence that the ability of TGF␤ to induce ␣-SMA
expression and myofibroblast formation in fibroblasts
depends on TAK1. Compared with general blockade of
TGF␤ signaling by antagonizing TGF␤ receptors or
Smads (18,20), it is likely that modifying TAK1 activity
may be more suitable for control of tissue remodeling
and development of selective antifibrotic therapies for
disorders such as scleroderma.
AUTHOR CONTRIBUTIONS
Dr. Leask had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Shi-wen, Pala, Denton, Abraham, Leask.
Acquisition of data. Shi-wen, Parapuram, Pala, Chen, Carter, Eastwood.
Analysis and interpretation of data. Shi-wen, Pala, Carter, Eastwood,
Denton, Abraham, Leask.
Manuscript preparation. Shi-wen, Denton, Abraham, Leask.
Statistical analysis. Shi-wen.
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expressions, matrix, muscle, contractile, induced, growth, transforming, factors, extracellular, smooth, activ, requirements, kinases, activated, fibroblasts
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