From the Craniofacial Developmental Biology and
Regeneration Branch, NIDCR and ¶ Laboratory of Cell Regulation
and Carcinogenesis, NCI, National Institutes of Health,
Bethesda, Maryland 20892
Received for publication, June 29, 2000, and in revised form, January 25, 2001
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ABSTRACT |
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In chondrogenesis, members of the transforming
growth factor- During development, cartilage serves as a template for most bones.
Cartilage formation is initiated with condensation of mesenchymal cells, followed by progression of chondrocyte differentiation toward
proliferation, prehypertrophy, and hypertrophy (1, 2). A number of
growth factors, such as fibroblast growth factors (3, 4), insulin-like
growth factor (5-7), transforming growth factor Among the ECM molecules, aggrecan is a major proteoglycan of cartilage
(13), and its deposition corresponds well with Alcian blue staining,
which is commonly used for identification of cartilage. Although low
levels are detected in the heart (14) and brain (15), aggrecan is
largely restricted to cartilage, and it contributes to water retention,
resistance to deformation, and the gel-like property of the cartilage.
Aggrecan-null mice develop perinatal lethal dwarfism with little ECM in
cartilage and defective chondrocyte differentiation, indicating
critical roles of aggrecan in cartilage development (16). Therefore,
aggrecan serves as a good marker for differentiated chondrocytes.
A number of in vitro studies have been performed using
primary chondrocytes and chondrogenic cell lines, such as CFK 2 (17), ATDC5 (12, 18, 19), C1 (20), C3H10T1/2 (11), and RCJ3.1C5 cells (21,
22), to elucidate the mechanisms of chondrocyte differentiation. Among
them, ATDC5 cells are a well characterized chondrogenic cell line
derived from mouse teratocarcinoma. In culture, these cells mimic the
multistep process of chondrocyte differentiation. After plating, they
proliferate until confluency, at which point they undergo growth arrest
following contact inhibition. After 4 days, these cells undergo a
phenotypic change characterized by the secretion of a variety of ECM
molecules, such as type II collagen, aggrecan, and link protein, and by
the formation of cartilaginous nodules on the culture plate (12, 18,
19). Recent studies have demonstrated that TGF- Receptor complexes of the TGF- Although the Smad pathway is widely represented in most of the cell
types and tissues studied, additional pathways may be activated
following treatment with TGF- The relative contribution of these different pathways in chondrocytic
responses to TGF- Cell Culture--
ATDC5 cells (18) were grown in Dulbecco's
modified Eagle's medium/F-12 (Life Technologies, Inc.) containing 5%
fetal bovine serum (HyClone) and 10 µg/ml insulin, 10 µg/ml
transferrin, and 10 µg selenium (ITS) (Biofluids, Inc) at
37 °C under 5% CO2. Cells were maintained at 20-80%
confluency, replacing media every other day. For inhibition of protein
synthesis, a concentration of 5 µg/ml cycloheximide (CHX, Sigma),
which blocks 95% of protein synthesis without cell toxicity, was added
to culture media at the indicated times.
Transfection and Luciferase Assay--
Cells at 50-60%
confluency were transfected with DNA constructs using
FuGeneTM 6 (Roche Molecular Biochemicals). Transfection
efficiency was monitored using a constitutively expressed GFP
expression vector under the control of the EF1 promoter, pCEFL-GFP (a
gift from J. S. Gutkind), confirming that ~65% of the
transfected cells expressed GFP under fluorescence microscopy. For
dominant inhibitory experiments of the Smad pathway, cells were
cotransfected with the following: 0.25 µg of p3TP-Lux, which is a
TGF- Immunoblot Analysis for Phosphorylation and Activation of Smad
and MAPK--
Phosphorylation of ERK1/2, JNK, and p38 MAPK was
determined with corresponding PhosphoPlus AntibodyTM kits
(New England Biolabs), respectively. Briefly, a pair of immunoblot
analyses was performed with phosphospecific antibodies or antibodies to
detect the proteins themselves. For Smad2 phosphorylation, anti-phospho-Smad2 (Upstate Biotechnology, Inc.), which detects the
C-terminal phosphorylated form of Smad2, and anti-total Smad2 (Zymed Laboratories Inc.) were used. For ERK1/2 and
Smad2 phosphorylation, cells were starved for 16 h to decrease
basal phosphorylation levels. After treatment with TGF- Nuclear Translocation Analysis--
Confluent cells were
serum-starved for 16 h, pretreated with U0126 (10 µM) or SB203580 (20 µM) for 1 h, and
treated with TGF- Quantitative RT-PC--
For analysis of Agc
transcription, cells were cultured for 12 h in a culture medium
containing 0.2% fetal bovine serum and were treated with TGF- Expression of Aggrecan Gene (Agc) Is Rapidly Induced by TGF-
Next, we used CHX to examine whether protein synthesis was required for
Agc induction. Pretreatment with CHX 30 min before TGF- The Smad Pathway Mediates Induction of Agc Expression--
To
determine the nature of this signaling response, we examined a number
of pathways previously shown to be activated by TGF- Both ERK1/2 and p38 MAPK Pathways Are Required for TGF-
Next, we used a number of known specific inhibitors of the MAPK
pathways to determine the functional significance of
TGF- Cross-talk of the Smad Pathway with p38 MAPK and ERK1/2
Pathways--
We demonstrated that both the Smad and the p38 MAPK and
ERK1/2 pathways are activated by TGF-
To determine whether these MAPK pathways are affecting the
transcriptional activity of the Smad complex, we used a heterologous transcription assay with Gal4-Smad fusion proteins. This system defines
a specific Smad-dependent transcriptional response that bypasses confounding factors associated with different response elements in a variety of other TGF- Agc Expression No Longer Requires TGF-
Next, we examined phosphorylation patterns of Smad2, ERK1/2, and p38
MAPK, mediated by TGF- In these studies, we have demonstrated that both Smad2/4 and the
p38 MAPK and ERK1/2 pathways are rapidly and transiently activated
following treatment with TGF- Contrasting with these findings in undifferentiated cells, we now show
that differentiated ATDC5 cells demonstrate a distinct pattern of
responses following stimulation with TGF- In differentiated chondrocytes, other members of the TGF- We have also shown that TGF- Finally, our studies using cycloheximide to inhibit de novo
protein synthesis also help to define the levels of transcriptional intermediates regulated by Smad2/4, p38 MAPK, and ERK1/2 pathways in
defining Agc expression. We showed that concomitant
treatment of the confluent ATDC5 cells with cycloheximide inhibited
TGF- Taken together, these data indicate that there are distinct
transcriptional cross-talk mechanisms between p38 MAPK, ERK1/2, and
Smad signaling pathways that are involved in regulating Agc expression at different stages of chondrocyte differentiation, and
these data provide new insights into the regulatory mechanisms defining
chondrocytic phenotypes. Further work will be necessary to define the
precise nature of the cartilage-specific transcriptional intermediates
regulating Agc expression.
(TGF-
) superfamily play critical roles by inducing
gene expression of cartilage-specific molecules. By using a
chondrogenic cell line, ATDC5, we investigated the TGF-
-mediated
signaling pathways involved in expression of the aggrecan gene
(Agc). At confluency, TGF-
induced Agc
expression within 3 h, and cycloheximide blocked this induction,
indicating that de novo protein synthesis is essential for
this response. At this stage, TGF-
induced rapid, transient phosphorylation of Smad2, extracellular signal-activated kinase 1/2
(ERK1/2), and p38 mitogen-activated protein kinase (MAPK). Inhibition
of the Smad pathways by transfection with a dominant negative Smad4
construct significantly reduced TGF-
-induced Agc expression, indicating that Smad signaling is essential for this response. Furthermore, an inhibitor of the ERK1/2 pathway, U0126, or
inhibitors of the p38 MAPK pathway, SB203580 and SKF86002, repressed
TGF-
-induced Agc expression in a
dose-dependent manner, indicating that ERK1/2 or p38 MAPK
activation is also required for TGF-
-induced Agc
expression in confluent ATDC5 cells. In differentiated ATDC5 cells,
persistently high basal levels of ERK1/2 and p38 MAPK phosphorylation
correlated with elevated basal Agc expression, which was
inhibited by incubation with inhibitors of these pathways. Whereas
Smad2 was rapidly phosphorylated by TGF-
and involved in the initial
activation of Agc expression in confluent cells, Smad2
activation was not required for maintaining the high level of
Agc expression. Taken together, these results suggest an
important role for transcriptional cross-talk between Smad and MAPK
pathways in expression of early chondrocytic phenotypes and identify
important changes in the regulation of Agc expression following chondrocyte differentiation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
s
(TGF-
s)1 (8-10), and bone
morphogenetic proteins (BMPs) (4, 11, 12), have been implicated in this
differentiation process. During differentiation, chondrocytes secrete
extracellular matrix (ECM) molecules characteristic of cartilage, such
as type II collagen, aggrecan, and link protein, providing an
environment that maintains the chondrocyte phenotype. Thus,
chondrocytes are defined both by their morphology and capacity to
synthesize these characteristic ECM molecules.
, BMP-2 (12), and growth/differentiation factor-5 (GDF-5, also known as cartilage-derived morphogenetic protein-1, CDMP-1) (23) rapidly induce type II collagen
expression in confluent ATDC5 cells, suggesting critical roles of
signaling by the TGF-
superfamily for chondrocyte-specific gene expression.
superfamily consist of a
ligand-binding type II receptor serine-threonine kinase that, following ligand binding, binds to and transphosphorylates the signal transducing type I serine-threonine kinase (24). These receptors, in turn, activate, through a phosphorylation event, members of a family of
downstream signaling intermediates, the Smads (25). Within this family
of proteins, the receptor-activated Smads, R-Smads, are the direct
substrates for the activated type I serine-threonine kinase and are
phosphorylated on a conserved C-terminal -SSXS motif. Smad1,
Smad5, and Smad8 are phosphorylated following activation of type I bone
morphogenetic protein (BMP) receptors, whereas Smad2 and Smad3 are
activated by the type I TGF-
and activin receptors (25). The
phosphorylated R-Smads then form heteromeric complexes with the common
Smad mediator, Smad4, which is then translocated to the nucleus. Once
in the nucleus, these Smad complexes function as transcriptional
activators, binding to specific cis-acting elements in
Smad-dependent promoters (26-29), interacting with and
recruiting a number of other DNA binding (30-36) and non-DNA binding
transcriptional activators (37), inhibitors (38-41), and bridging
coactivator proteins CBP and p300 (28, 42). Smad4 is an
essential component in many of the Smad-dependent responses (43), serving both to stabilize the Smad-transcription factor complex
(44) and to form functional interactions with critical transcriptional
adapter proteins including CBP and p300 (45).
in specific contexts. For example,
activation of Ras, extracellular signal-regulated kinase 1/2 (ERK1/2),
and c-Jun N-terminal kinase (JNK) by TGF-
signaling has been
reported in primary intestinal epithelial cells and some breast cancer
cell lines (46, 47), whereas activation of protein kinase A contributes
to TGF-
-signaling responses in murine mesangial cells (48). In
addition, TGF-
-activated kinase-1 (TAK1), a member of the MEKK
family and activator of JNK and p38 MAPK pathways (14, 49), is rapidly
activated by TGF-
in certain cell systems, notably in murine C2C12
myocytes (50).
is poorly understood. The aim of our study is to
define the contribution of specific TGF-
-dependent signaling pathways involved in the regulation of chondrocyte
differentiation. In particular, we have focused on the regulation of
aggrecan gene (Agc) expression as a unique marker gene that
is characteristic of the chondrocyte phenotype. In this paper, we
demonstrate that TGF-
rapidly induces Agc expression in
ATDC5 cells and that this response requires TGF-
-induced activation
of both R-Smad2/4 complexes as well as p38 MAPK and ERK1/2 pathways. In
contrast, following differentiation, high levels of Agc
expression are maintained without activation of R-Smad2 but are
associated with, and require, persistently high basal activation of p38
MAPK and ERK1/2 pathways. These findings suggest that there are
distinct transcriptional cross-talk mechanisms regulating
Agc expression in differentiating chondrocytes, and they
provide new insights into the regulatory mechanisms defining
chondrocytic phenotypes.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-responsive reporter containing three
12-O-tetradecanoylphorbol-13-acetate-responsive elements and
a small TGF-
-responsive element from the plasminogen activator inhibitor 1 promoter (51); 1.7 µg of a dominant negative (DN
) Smad4 construct, Smad4-(
275-322) (45); or empty vector pcDNA3 (Invitrogen); and 0.1 µg of pRLSV40 (Promega), a
Renilla luciferase expression vector driven by SV40
promoter, was used as an internal control vector. Twenty four hours
after transfection, when cells reached confluency, the culture medium
was replaced with Dulbecco's modified Eagle's medium/F-12 containing
ITS and 0.2% fetal bovine serum. After 12 h, cells were treated
with 10 ng/ml TGF-
2 (R & D Systems) for 16 h and
were lysed. Luciferase activity of cell lysate was assayed with the
Dual-Luciferase reporter assay system (Promega) using a microtiter
plate luminometer (Dynex). For inhibition studies, U0126 (Promega),
SB203580, and SKF86002 (Calbiochem) were used at the concentrations
indicated and were added 1 h before TGF-
(10 ng/ml) treatment.
U0126 is a specific inhibitor of an upstream molecule, MEK1/2, that
activates ERK1/2 (52). In preliminary experiments, a concentration of
10 µM showed optimal inhibition of ERK1/2
phosphorylation. SB203580 (53) inhibits p38 MAPK activity but not its
phosphorylation. SKF86002 (54, 55) is an inhibitor of MAPK-kinase 6 (MKK6), an activator of p38 MAPK. Concentrations of 20-50
µM showed optimal inhibition of p38 MAPK activity without
cytotoxicity. Because of the relatively short half-life of SB203580 and
SKF86002, these compounds were added to the media every 4 h during
the course of the assay. Gal4-Smad experiments were performed as
reported elsewhere (45). Briefly, cells were similarly transfected with
1 µg of p147-Gal4-Smad2 or -Smad4 (266), coupled with 1 µg of
Gal4-Luc (56) and 0.1 µg of pRLSV40 (Promega). Gal4-VP-16 was also
used as an independent transactivator for a control. After 24 h,
inhibitors were added under serum-starved conditions. After 1 h,
cells were treated with TGF-
. Cells were collected after 16 h,
and cell lysate was similarly subjected to the Dual-Luciferase reporter
assay system (Promega).
for the time
indicated, cells were washed with ice-cold phosphate-buffered saline
and collected, and cell lysate was subjected to a SDS-polyacrylamide
gel electrophoresis under a reducing condition, followed by
immunoblotting. For the inhibition studies, optimized concentrations of
U0126, SB203580, and SKF86002 were added 1 h prior to TGF-
treatment. An inhibitor of protein kinase A, H-89, was used at
concentrations of 10 µM as a negative control. ERK1/2
assay was carried out using p44/42 MAP kinase assay kit (New England
Biolabs) according to the manufacturer's protocol. For p38 MAPK
activity, p38 MAP kinase assay kit (New England Biolabs) was used
according to the manufacturer's protocol.
(10 ng/ml) for 1 h. Then cells were fixed
with 4% paraformaldehyde, permeabilized in 0.2% Triton X-100, and
incubated with an anti-Smad2/3 antibody (Transduction Laboratories)
overnight at 4 °C, followed by incubation with Alexa
FluorTM 488 (Molecular Probes) secondary antibody. Cells
were then visualized with immunofluorescence microscopy (Nikon). The
percentage of cells with Smad2/3 staining in the nucleus was determined
by observing 100 cells.
at a
concentration of 10 ng/ml for 12 h, unless otherwise indicated.
Poly(A) RNA was prepared from cells using the Micro-Fast
TrackTM kit (Invitrogen). This mRNA (200-600 ng) was
reverse-transcribed to generate cDNA using the Superscript
PreamplificationTM System (Life Technologies, Inc.). For
semiquantitative analysis of Agc expression, PCR was
performed using a set of primers, 5'-TGGAGCATGCTAGAACCCTCG-3' and
5'-GCGACAAGAAGACACCATGTG-3'. After confirmation of the Agc transcript, real time quantitative PCR was performed using the TaqManTM 7700 (PE Applied Biosystems). Sequences for a
probe and a set of primers were chosen by the Primer
ExpressTM program as follows: 5'-CCCTGGGCAGCGTGATCCTCAC-3'
for a probe, 5'-CTGCCCTTGCCCCGTA-3' for a forward primer, and
5'-GACAGGTCAAAGATGGGCTTTG-3' for a reverse primer. The probe was
labeled with fluorescent reporter dyes 6-carboxyfluorescein and
6-carboxy-N,N,N',N'-tetramethylrhodamine at 5' and 3' ends,
respectively. As an internal control, a set of primers and a probe of
rodent GAPDH labeled with VICTM (PE Applied Biosystems)
were used according to the manufacturer's protocol.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
in
ATDC5 Cells--
In confluent ATDC5 cells, RT-PCR analysis revealed
induction of Agc transcription as early as 3 h after
the treatment with TGF-
(Fig.
1A). These data were confirmed
by direct measurement of the transcript levels of Agc by a
real time quantitative RT-PCR assay. By using this assay, the fold
induction of Agc mRNA by TGF-
was 4.1 ± 0.38, 10.22 ± 2.75, 22.3 ± 9.1, and 59.6 ± 0.5 for 3, 6, 12, and 24 h, respectively, where 0 h was 1.0 ± 0.03 (mean ± S.D., n = 3, Fig. 1B). Without
TGF-
, Agc expression after 24 h was 5.35 ± 0.41 that of 0 h.
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Fig. 1.
Induction of aggrecan gene
(Agc) expression by
TGF- . A, RT-PCR analysis for
Agc expression. Electrophoresis gel of RT-PCR products
showing Agc transcript and GAPDH as control. B,
quantitation of Agc transcript using a real time RT-PCR.
TGF-
-induced Agc expression (closed circle) is
observed for 3 h. Agc expression without
treatment for 24 h is also shown (open circle). The
fold induction of Agc mRNA by TGF-
is 4.1 ± 0.38, 10.22 ± 2.75, 22.3 ± 9.1, and 59.6 ± 0.5 for 3, 6, 12, and 24 h, respectively, and 0 h is 1.0 ± 0.03. Data represent mean ± S.D. (n = 3). C,
electrophoresis gel showing inhibition of Agc expression by
cycloheximide (CHX) treatment, coupled with GAPDH expression
as control. RT-PCR products of Agc after TGF-
treatment
for 6 h are shown. TGF-
-induced Agc expression is
blocked by CHX treatment. Addition of CHX 120 min after TGF-
treatment does not inhibit Agc expression. Two independent
experiments showed the same results.
addition completely blocked transcription of Agc. When CHX was added 2 h after TGF-
treatment, Agc
transcription was observed (Fig. 1C). These results indicate
that protein synthesis initiated within 2 h of treatment with
TGF-
is required for Agc expression and suggest that this
expression requires induction of an immediate early response gene (IEG)
to a TGF-
-dependent signal.
in different
cell systems. First, we examined if Smads are involved in
Agc expression. As shown in Fig.
2A, using
phospho-Smad2-specific antibodies, Smad2 was rapidly phosphorylated
within 5 min of treatment with TGF-
and persisted for 4 h. We
next examined the functional significance of this Smad pathway
activated by TGF-
in regulating Agc expression by using a
dominant negative (DN
) Smad4 construct, Smad4-(
275-322), which
has previously been shown to inhibit TGF-
-dependent transcriptional responses in a number of different cell types and
reporter systems (45).2 When
the cells reached confluency after 24 h of transfection, cells
were starved for 12 h and treated with TGF-
for an additional 12 h. The expression of p3TP-lux reporter gene was inhibited in a
dose-dependent manner, with a maximal inhibition of 65%,
confirming down-regulation of the Smad pathway by DN-Smad4 construct
(Fig. 2B). Under the same conditions, this dominant negative
construct inhibited TGF-
-mediated Agc expression as
determined by real time RT-PCR, by ~65% (Fig. 2C). In
parallel experiments, transfection efficiency of 65 ± 10%
(mean ± S.D.) was obtained using the GFP expression construct,
suggesting that the DN-Smad4 construct strongly inhibits TGF-
signaling in the transfected cells. These results indicate involvement
of the Smad pathway in Agc expression.
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Fig. 2.
Decreased Agc expression by
inhibition of the Smad pathway with a dominant negative DN-Smad4
construct. A, immunoblot showing phosphorylation of
Smad2. Cells were treated with TGF- for the time indicated. Phospho-
and total Smad2 are shown in upper and lower
panels, respectively. B, inhibition of p3TP-Lux Smad
reporter gene expression by a dominant negative DN-Smad4 construct.
Cells were transfected with 1 µg of a dominant negative Smad4
construct, Smad4-(
275-322), 1 µg of a reporter p3TP-Lux, and 0.1 µg pRLSV40 as an internal control. After 24 h, cells were
treated with TGF-
(10 ng/ml) and were subjected to luciferase assay
as described under "Materials and Methods." C,
inhibition of Agc expression. Under the same conditions as
in B, Agc expression was measured by real time
quantitative RT-PCR analysis as indicated under "Materials and
Methods." Note similar inhibition patterns in both p3TP-Lux and
Agc expression by transfection of the DN-Smad4 construct.
Two additional independent experiments showed similar results.
-induced
Agc Gene Expression--
We next examined the effects of TGF-
on
activation of ERK1/2, SAPK/JNK, and p38 MAPK in confluent ATDC5 cells.
TGF-
increased phosphorylation of ERK1/2 5-10 times that of the
basal level. The increased phosphorylation appeared as early as 5 min,
reached the peak at 15-30 min, and was sustained until 1 h after
TGF-
treatment (Fig. 3A).
Correlation between phosphorylation and activation of ERK1/2 was
confirmed by an ERK1/2 assay system using Elk1 as a substrate (Fig.
3A, lower panel, at 15 min). Immunoblot analysis using anti-phospho-p38 showed negligible basal levels of phospho-p38 MAPK, and TGF-
dramatically phosphorylated p38 MAPK, although compared with ERK1/2 phosphorylation, phosphorylation of p38 MAPK was
delayed (Fig. 3B). JNK was not phosphorylated or activated by TGF-
, indicating that this pathway is not involved in TGF-
signaling in these cells (data not shown).
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Fig. 3.
Phosphorylation and activation of ERK1/2
(A) and p38 MAPK (B). Confluent
ATDC5 cells were treated with TGF- (10 ng/ml). At the indicated
times, cells were collected, and phosphorylation of either ERK1/2 or
p38 MAPK was analyzed. A, phosphorylation and activation of
ERK1/2 by TGF-
treatment. The 1st and the 2nd
panels show immunoblot using anti-phospho-ERK1/2 and anti-ERK1/2
molecules themselves, respectively. The 3rd and 4th
panels show activity of ERK1/2 using Elk-1 as substrate and
control immunoblot using anti-total ERK1/2 of the cell lysate,
respectively. Note the correlation of phosphorylation and activation of
ERK1/2. B, phosphorylation of p38 MAPK by TGF-
treatment.
Immunoblot analysis was performed using specific antibodies for
phospho-p38 and total p38. Upper and lower panels
indicate patterns of phospho- and total p38 MAPK in transfected cells,
detected by their specific antibodies, respectively.
-dependent p38 MAPK and ERK1/2 activation on
Agc expression. To inhibit the ERK1/2 pathway, we used
U0126, a specific inhibitor of MEK1/2, which is an upstream molecule
that activates ERK1/2 (52). For the p38 MAPK pathway, SB203580 (53) and
SKF86002 (54, 55) were used. These inhibit activity of p38 for its
substrate and that of MAPK-kinase-6 (MKK6) for p38, respectively. U0126
(1.0 and 10 µM) inhibited phosphorylation of ERK1/2 (Fig.
4A, lower panels) in a
dose-dependent manner. Both SB203580 (2.0 and 20 µM) and SKF86002 (2.0 and 20 µM) inhibited
p38 activity (Fig. 4, B and C, lower panels).
Whereas TGF-
induced Agc expression to 12.4 ± 3.4-fold, U0126 at 1.0 and 10 µM attenuated the fold induction to 6.13 ± 0.38- and 1.41 ± 0.06-fold,
respectively (Fig. 4A). Similarly, both SB203580 and
SKF86002 attenuated TGF-
-induced Agc expression in a
dose-dependent manner. Fold induction by TGF-
was
5.1 ± 0.03 and 1.31 ± 0.10 following treatment with
SB203580 at 2.0 and 20 µM and 6.55 ± 0.54 and
2.93 ± 0.13 following treatment with SKF86002 at 2.0 and 20 µM, respectively (Fig. 4, B and C). To ensure that not all protein kinase inhibitors inhibit
TGF-
-mediated induction of Agc expression, an inhibitor
of protein kinase A, H-89, was used as a negative control. H-89 did not
show inhibitory activity for TGF-
-induced Agc expression
(data not shown). These results clearly demonstrate that both ERK1/2
and p38 pathways are required for Agc expression.
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Fig. 4.
Inhibition of Agc expression
by specific inhibitors of ERK1/2 and p38 MAPK pathways. Confluent
ATDC5 cells were starved for ~16 h. Then U0126, SB203580
(SB), or SKF86002 (SKF) was added at a
concentration indicated. After 1 h, cells were treated with 10 ng/ml TGF- for 12 h; mRNA was obtained; and real time
quantitative RT-PCR analysis was performed. To confirm inhibitory
effects of each pathway, immunoblot analysis for ERK1/2 phosphorylation
(lower panel) and p38 MAPK assay using ATF-2 as a substrate
(B and C, lower panels) were performed
as described under "Materials and Methods." Agc
transcription in TGF-
-treated (solid bar) is indicated as
fold induction that nontreated (open bar), where 1.0 equals
basal transcription. Whereas TGF-
induces Agc expression
to 12.4 ± 3.4-fold, U0126 at 1.0 and 10 µM inhibits
the fold induction to 6.13 ± 0.38- and 1.41 ± 0.06-fold,
respectively (A). Both SB and SKF inhibit TGF-
-induced
Agc expression: at 2.0 and 20 µM, SB inhibits
to 5.1 ± 0.03 and 1.31 ± 0.10 and SFK to 6.55 ± 0.54 and 2.93 ± 0.13, respectively (B and C).
The data represent mean ± S.D. (n = 3). Similar
results were obtained in an additional independent experiment.
in confluent ATDC5 cells and that each of these pathways is essential for
TGF-
-dependent induction of Agc expression.
Next, we asked whether these pathways act independently or involve some
level of intracellular cross-talk. Activation of MAPK pathways has been
shown both to activate and induce nuclear translocation of Smad2 (57,
58) or, in other contexts, to inhibit TGF-
-dependent
nuclear translocation of Smad2 and -3 (59). We therefore tested whether
specific inhibitors of ERK1/2 and p38 MAPK pathways inhibit
TGF-
-dependent nuclear translocation of the Smad
proteins in ATDC5 cells. Cells were treated with the specific
inhibitors and, after 1 h of incubation with TGF-
, the number
of Smad2 positive nuclei was counted under fluorescence microscopy.
Neither U0126 (10 µM) nor SB203580 (20 µM)
inhibited TGF-
-dependent nuclear translocation of Smad
2/3 (Fig. 5A), indicating that
these MAPK pathways are not inducing nuclear translocation of Smad2/3.
Furthermore, immunoblotting with anti-phospho-Smad2 antibody, which
recognizes specific C-terminal phosphoserine residues of Smad2,
demonstrated that TGF-
increased C-terminal phosphorylation of
Smad2, but these phosphorylation events were not inhibited by U0126 or
SB203580 (Fig. 5B). These findings suggest that
TGF-
-dependent activation of p38 and ERK1/2 MAPK
pathways does not influence activation of R-Smads by TGF-
and
indicate that under the experimental conditions of these studies
neither U0126 nor SB203580 affects the ability of TGF-
receptors to
phosphorylate downstream R-Smad substrates.
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Fig. 5.
Nuclear translocation and phosphorylation of
Smad2. A, nuclear translocation of Smad2. Upper
panel shows nuclear localization of Smad2 in TGF- -treated ATDC5
cells. Smad2 is observed predominantly in nuclei at 30 min after
TGF-
treatment (right), whereas cells without treatment
contain Smad2 primarily in cytoplasm (left). Bottom
panel shows percentage of nuclei positive for Smad2 after
treatment with U126 (U) or SB203580 (SB) and
TGF-
(mean ± S.D., n = 3). No significant
effects of the inhibitors on nuclear translocation were observed.
B, phosphorylation of Smad2 after treatment with U126
(U) or SB203580 (SB) followed by TGF-
. TGF-
treatment of ATDC5 cells increased Smad2 phosphorylation. The
inhibitors did not significantly inhibit Smad2 phosphorylation. Similar
results were obtained in two additional independent experiments.
-responsive reporters. For these
experiments, we cotransfected cells with Smad2 or a transcriptionally active N-terminal truncation of Smad4, Smad4-(266-552), fused to the DNA-binding domain of Gal4 (45), together with pGal4-Luc, a
luciferase reporter gene under the control of six Gal4-responsive elements and a minimal TATA-containing promoter. This reporter construct minimized effects of inhibitors on the basal luciferase activity. As shown in Fig. 6, increasing
concentrations of U0126, SB203580, and SKF86002 inhibited
TGF-
-induced transcriptional activity of both Gal4-Smad2 and
Gal4-Smad4 systems. Whereas U0126 (10 µM) and SB203580
(20 µM) inhibited basal Gal4-Smad2 activation by 30 and
25%, TGF-
-induced activation was inhibited by 65 and 70%,
respectively. Furthermore, SKF86002 had little effect on the basal
levels, indicating that the suppression of ligand-induced reporter
activity by these inhibitors is specific. We used Gal4-VP16 as an
independent transactivator to evaluate the specific effect of these
inhibitors. Transfection of Gal4-VP16 induced activity of the reporter
luciferase gene about 95-fold. Addition of U0126 or SB203580 resulted
in no significant reduction of transactivation by Gal4-VP16 (data not
shown). These results indicate that these inhibitors specifically block
the p38 MAPK and ERK1/2 cascades. Taken together, these results
indicate that TGF-
-induced activation of p38 MAPK or ERK1/2 is
essential for transcriptional activation of Smad2 and Smad4 and that
this interaction is necessary for maximal activation of a specific
Smad-dependent transcriptional response in ATDC5 cells.
View larger version (24K):
[in a new window]
Fig. 6.
Interaction of Smad2/4 proteins with
molecules downstream of ERK1/2 and p38 MAPK pathways. Cells were
cotransfected with Gal4-Luc reporter construct, Gal4-Smad2 or
Gal4-Smad4-(266-552) expression construct, and pRLSV40 as a control.
After 24 h, inhibitors were added under a serum-starved condition.
After 1 h, cells were treated with TGF- . Cells were collected
after 16 h, and cell lysate was similarly subjected to the
Dual-Luciferase reporter assay system. Luciferase activity is expressed
as mean ± S.D. (n = 3).
following
Differentiation--
To determine whether this pattern of the Smad and
MAPK pathways was restricted to confluent ATDC5 cells or was a common
feature at all stages in chondrocyte differentiation, we performed a
time course analysis on their response to TGF-
at various stages of differentiation. First, we examined levels of Agc
expression, using real time quantitative RT-PCR. As shown in Table
I, basal Agc expression of
nontreated ATDC5 cells increased during differentiation. Agc
transcript levels increased to 87.5 ± 6.5 and 114.0 ± 1.9, at day 7 and 14 after confluency, respectively, where the level at confluency is 1.0 ± 0.02, confirming chondrocyte
differentiation in this time course experiment. Although TGF-
up-regulated Agc expression during differentiation, the fold
induction by TGF-
became attenuated to 1.5-2.4-fold at day
14, as compared with ~12-fold induction in confluent cells.
Levels of Agc transcripts in ATDC5 cells
following differentiation in ATDC5 cells.
TGF-
dramatically phosphorylated Smad2 at confluency, day 7, and day 14, indicating that receptors for TGF-
and their phosphorylation of Smad2 remain unaffected through differentiation (Fig. 7). Furthermore, low levels of
Smad2 phosphorylation in the absence of TGF-
indicate that there is
no significant autocrine TGF-
activity in these cells. In contrast,
the response of p38 MAPK and ERK1/2 to TGF-
was reversed during
differentiation. At day 7, the phosphorylation level of ERK1/2 was
unaffected by TGF-
, whereas by day 14, TGF-
decreased the high
basal phosphorylation of ERK1/2 and p38 MAPK. Phosphorylation levels of
both ERK1/2 and p38 MAPK correlate with elevated basal Agc
expression in differentiated ATDC5 cells, suggesting that activation of
both ERK1/2 and p38 MAPK may be required for continuous Agc
expression in differentiated cells. Therefore, we examined the effect
of inhibitors on Agc expression in differentiated ATDC5
cells. As shown in Fig. 8, both U0126 (10 µM) and SB203580 (20 µM) down-regulated
Agc expression in differentiated cells. These data
demonstrate the role of activated ERK1/2 and p38 MAPK for
Agc expression and together indicate that in differentiated,
as opposed to confluent, ATDC5 cells, Agc expression is
maintained without activation of the Smad pathway.
View larger version (42K):
[in a new window]
Fig. 7.
TGF- -induced
phosphorylation patterns of Smad2, ERK1/2, and p38 MAPK at different
stages (confluency (confl.), day 7, and day 14 after
confluency, as indicated in the figure) of ATDC5 cells.
Phosphorylation patterns after TGF-
treatment of 15 min for Smad2
and ERK1/2 and that of 1 h for p38 MAPK are shown.
View larger version (14K):
[in a new window]
Fig. 8.
Inhibition of Agc expression
in differentiated ATDC5 cells (day 14 after confluency) by specific
inhibitors of ERK1/2 and p38 MAPK pathways. Differentiated cells
were serum-starved for 16 h and then U0126 or SB203580
(SB) was added to the culture for 1 h. The cells were
then treated with TGF- for 12 h. mRNA was prepared, and
real time quantitative PCR analysis was performed. Both U0126 (10 µM) and SB203580 (SB, 20 µM)
inhibit Agc transcript levels by ~50%. Similar results
were obtained in two independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
in a mouse chondrogenic cell line,
ATDC5, and that the activation of all three of these pathways is
required for the transcriptional activation of Agc expression. Furthermore, we show that the TGF-
-induced
transcriptional activation of a specific Smad2/4-dependent
response element is dependent on the cooperative activation of p38 MAPK
and ERK1/2 pathways in these cells. This suggests that there may be an
important level of transcriptional cross-talk involving an interaction
between these Smad proteins and components downstream of p38 MAPK and ERK1/2 in these cells. Previous studies have shown evidence that TGF-
treatment of ATDC5 cells enhances the expression of chondrocyte differentiation markers, including type II collagen (19), and is
associated with activation of p38 MAPK and ERK1/2 pathways (23).
Furthermore, it has recently been shown that overexpression of the
inhibitory Smads 6 and 7 can block spontaneous differentiation of ATDC5
cells (60), indicating that Smad signaling is also required for this
process of cellular differentiation. Our data are consistent with these
observations, but they also now define the mechanism of cross-talk
between the MAPK and Smad signaling pathways. Furthermore, the fact
that p38 MAPK and ERK1/2 activation shows overlapping via
distinct kinetics following treatment with TGF-
and that both
pathways are required for maximal TGF-
-induced activation of
Agc expression suggests that there may be some cooperative interaction or overlap of substrates downstream of these pathways that
is involved in regulating Agc expression. It is still
unclear what components downstream of these MAPK pathways are involved in regulating this interaction.
. Although there is still
rapid induction of Smad2 phosphorylation following treatment with
TGF-
in these cells, indicating that there is an intact TGF-
receptor/Smad signaling pathway, the persistently high basal
phosphorylation of p38 MAPK and ERK1/2 is actually inhibited following
treatment with TGF-
. Furthermore, unlike the confluent ATDC5 cells,
the differentiated cells have a high basal level of Agc
expression which requires p38 MAPK and ERK1/2 activation without
activation of the Smad2, indicating that Agc expression has
escaped regulation by TGF-
-activated Smads. The role of Smad2/4
proteins in this response is less clear, as these do not seem to be
essential to maintain the high basal levels of Agc
expression seen in differentiated ATDC5 cells. Although TGF-
treatment does augment basal Agc expression, albeit to a lesser extent even in these cells, the functional requirement for the
Smad2/4 protein complex appears to be critically dependent on the flux
of p38 MAPK and ERK1/2 activation. In the presence of persistently high
levels of p38 MAPK and ERK1/2 activation in the differentiated cells,
activation of Smad2/4 is redundant, whereas at low transient levels of
p38 MAPK and ERK1/2 activation in the confluent cells, the Smad2/4
pathway interaction is essential.
superfamily may also participate in high levels of Agc
expression. GDF5 and BMP-2 are induced in differentiating ATDC5
cells.3 GDF5 has been shown
to activate p38 MAPK and ERK1/2 in ATDC5 cells, and treatment with
either of GDF5 or BMP-2 promotes chondrocyte differentiation (23).
Constitutive up-regulation of these proteins could therefore account
for the enhanced basal MAPK activation and subsequent Agc
expression following differentiation in ATDC5 cells.
-induced transcriptional activation of
Smad2/4 in confluent ATDC5 cells is absolutely dependent on the
activation of ERK1/2 and p38 MAPK pathways by TGF-
in heterologous
transcriptional response assays. Although this is an artificial system
that may not reflect the true interaction between these pathways in the
context of a physiological TGF-
-dependent response, taken
together with our observations on Agc expression, it does
suggest that additional levels of cross-talk between these pathways may
be involved in regulating these responses. One explanation for this
cross-talk is that components of these pathways interact directly in
the transcriptional complex. For example, although we do not know which
specific intermediates are substrates of the p38 MAPK and ERK1/2
pathways in confluent ATDC5 cells, we do know that Smad4 is able to
interact with a variety of different transcription factors, including
ATF2, which is a downstream substrate of p38 MAPK (50). An alternative,
although not exclusive, explanation for the cross-talk between Smad2/4
and MAPK pathways is that the activated MAPKs are themselves directly
activating the Smad2/4 complex in these cells. This may result from the
phosphorylation of specific MAPK sites in Smad2 if this phosphorylation
event is required for its transcriptional activity and/or its ability to form heteromeric complexes with Smad4. It is notable that although we did not detect phospho-Smad2 in differentiated cells in the presence
of a high basal levels of MAPK activity, it is possible that the
phospho-Smad2-specific antibody, raised against a C-terminal phosphoserine peptide, could not recognize these phosphorylated residues in other domains of the molecule. This interpretation is
compatible with our earlier findings that hepatocyte growth factor- and
epidermal growth factor-induced activation of the ERK1/2 pathway
phosphorylates Smad2 outside of the C-terminal phosphoreceptor sites
and yet enhances the transcriptional activity of Smad2/4 (57).
Furthermore, other groups studying the role of JNK/SAPK (58, 61) have
shown that transient activation of these MAPK pathways can include
phosphorylation and transcriptional activation of Smad2 and Smad3.
These findings, along with our own, contrast with the observation that
Ras transformation, which is associated with persistently high levels
of MAPK activation, inhibits TGF-
-dependent activation
of Smad2 and Smad3 (59). This suggests that the overall effects of
these MAPK pathways on the transcriptional activation of these Smad
proteins may be fundamentally dependent on both the level and
persistence of MAPK pathway activation in a particular cell type.
-induced Agc expression, indicating that this
response requires de novo protein synthesis and is not a
direct signaling event. However, a subsequent time course of treatment
with cycloheximide showed that the induction of Agc
expression by TGF-
was not blocked if cycloheximide treatment was
delayed for 2 h after treatment with TGF-
. The relatively short
time course of this effect suggests that TGF-
signaling directly
induces the transcription of an unidentified, and possibly
cartilage-specific, immediate early response gene, which may act
directly on the Agc promoter to activate gene expression.
Sox9 and scleraxis are implicated for Agc expression in
cartilage-derived cell line TC6 (62) and in osteoblastic osteosarcoma
cell line ROS17/2.8 (63), respectively. However, little expression of
Sox9 is observed in both undifferentiated and differentiated
ATDC5 cells. TGF-
also does not induce Sox9 expression in ATDC5
cells.4 Scleraxis is
expressed in undifferentiated ATDC5 cells, and its expression level is
not changed with TGF-
treatment.3 Thus, Sox9 and
Scleraxis do not appear to be involved in TGF-
-mediated induction of
aggrecan expression in ATDC5 cells.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank J. Silvio Gutkind for expression constructs and valuable comments; Anita Roberts, Ricardo Sanchez, Shige Fukuhara, Mario Chiariello, Maria J. Marinissen, Makoto Haino, Yuji Hiraki, Chisa Shukunami, Koji Kimata, Roumen Pankov, and Harry Grant for valuable comments; and Joan Massague for materials.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Institute for Molecular Science of Medicine, Aichi Medical University, Yazako, Nagakute, Aichi 480-11, Japan.
To whom correspondence should be addressed: Bldg. 30, Rm. 405, NICDR, National Institutes of Health, 30 Convent Dr. MSC 4370, Bethesda, MD 20892-4370. Tel.: 301-496-2111; Fax: 301-402-0897; E-mail:
yoshi.yamada@nih.gov.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M005724200
2 M. P. de Caestecker, unpublished data.
3 H. Watanabe, unpublished data.
4 H. Watanabe, M. P. de Caestecker, and Y. Yamada, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TGF-, transforming
growth factor-
;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
MAPK, mitogen-activated protein kinase;
ERK, extracellular
signal-regulated kinase;
BMPs, bone morphogenetic proteins;
ECMs, extracellular matrix;
JNK, c-Jun N-terminal kinase;
DN, dominant
negative;
PCR, polymerase chain reaction;
RT-PCR, reverse
transcriptase-PCR;
CHX, cycloheximide;
GFP, green fluorescent
protein.
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