BMP-2-enhanced chondrogenesis involves p38 MAPK-mediated down-regulation of Wnt-7a pathway.

Abstract

The bone morphogenetic protein (BMP) family has been implicated in control of cartilage development. Here, we demonstrate that BMP-2 promotes chondrogenesis by activating p38 mitogen-activated protein kinase (MAPK), which in turn downregulates Wnt-7a/b-catenin signaling responsible for proteasomal degradation of Sox9. Exposure of mesenchymal cells to BMP-2 resulted in upregulation of Sox9 protein and a concomitant decrease in the level of b-catenin protein and Wnt-7a signaling. In agreement with this, the interaction of Sox9 with b-catenin was inhibited in the presence of BMP-2. Inhibition of the p38 MAPK pathway using a dominant negative mutant led to sustained Wnt-7a signaling and decreased Sox9 expression, with consequent inhibition of precartilage condensation and chondrogenic differentiation. Moreover, overexpression of b-catenin caused degradation of Sox9 via the ubiquitin/26S proteasome pathway. Our results collectively indicate that the increase in Sox9 protein resulting from downregulation of b-catenin/Wnt-7a signaling is mediated by p38 MAPK during BMP-2 induced chondrogenesis in chick wing bud mesenchymal cells.

Full text

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Mol. Cells, Vol. 22, No. 3, pp. 353-359






BMP-2-Enhanced Chondrogenesis Involves p38 MAPK-mediated
Down-Regulation of Wnt-7a Pathway

Eun-Jung Jin1, Sun-Young Lee1, Young-Ae Choi1, Jae-Chang Jung1, Ok-Sun Bang3, and Shin-Sung Kang1, 2,*
1 Department of Biology, College of Natural Sciences, Kyungpook National University, Daegu 702-701, Korea;
2 Daegu Center, Korea Basic Science Institute, Daegu 702-701, Korea;
3 School of Biological Sciences, Seoul National University, Seoul 151-742, Korea.

(Received September 20, 2006; Accepted November 13, 2006)

The bone morphogenetic protein (BMP) family has
been implicated in control of cartilage development.
Here, we demonstrate that BMP-2 promotes chondro-
genesis by activating p38 mitogen-activated protein
kinase (MAPK), which in turn downregulates Wnt-
7a/β-catenin signaling responsible for proteasomal deg-
radation of Sox9. Exposure of mesenchymal cells to
BMP-2 resulted in upregulation of Sox9 protein and a
concomitant decrease in the level of β-catenin protein
and Wnt-7a signaling. In agreement with this, the in-
teraction of Sox9 with β-catenin was inhibited in the
presence of BMP-2. Inhibition of the p38 MAPK path-
way using a dominant negative mutant led to sustained
Wnt-7a signaling and decreased Sox9 expression, with
consequent inhibition of precartilage condensation and
chondrogenic differentiation. Moreover, overexpression
of β-catenin caused degradation of Sox9 via the ubiq-
uitin/26S proteasome pathway. Our results collectively
indicate that the increase in Sox9 protein resulting
from downregulation of β-catenin/Wnt-7a signaling is
mediated by p38 MAPK during BMP-2 induced chon-
drogenesis in chick wing bud mesenchymal cells.

Keywords: β-Catenin; Chondrogenesis; p38 MAPK; Sox9;
Wnt-7a.


Introduction

Chondrogenic differentiation of mesenchymal cells, a
prerequisite for cartilage formation in developing limbs,
requires cell proliferation followed by precartilage con-
densation (DeLise et al., 2000; Knudson and Knudson

* To whom correspondence should be addressed.
Tel: 82-53-950-5349; Fax: 82-53-953-3066
E-mail: kangss@knu.ac.kr
2001; Sandell and Adler, 1999). A number of growth fac-
tors, including fibroblast growth factor (Grotewold and
Ruther, 2002; Solchaga et al., 2005), insulin-like growth
factors (Longobardi et al., 2003; Phornphutkul et al.,
2004), transforming growth factor (TGF)-βs (Blaney
Davison et al., 2006; Goessler et al., 2005; Jin et al.,
2006), and bone morphogenetic proteins (Denker et
al.,1999; Fisher et al., 2006; Zhang and Stott, 2004) have
been implicated in this differentiation process.
Bone morphogenetic proteins (BMPs) are multifunctional
regulators of cell growth and differentiation (Bi et al.,
1999; Yi et al., 2000; Zhao et al., 1997), and BMP-2 is
one of the signals that control the recruitment of cells into
developing cartilaginous condensations. In the murine
mesenchymal stem cell line C3H10T1/2, BMP-2 stimu-
lates chondrogenic differentiation and upregulates Type II
collagen, aggrecan, and chondrocyte/cartilage-specific
factors (Fischer et al., 2002; Gomes et al., 2006; Izzo et
al., 2002). BMP-2 stimulation of chondrogenesis in
C3H10T1/2 cells has been shown to result in repression
of Wnt-7a, pointing to antagonism between Wnt-7a and
BMP-2 during mesenchymal condensation (Fischer et al.,
2002; Stott et al., 1999). BMPs induce the expression of
specific markers of chondrogenesis by regulating Sox and
homeobox proteins (Baur et al., 2000; Boulet and Capec-
chi, 2004; Shea et al., 2003; Tsumaki et al., 2002; Zhang
et al., 2003). Sox proteins, high mobility DNA-binding
transcription factors, are important downstream mediators
of the BMP-2 signaling pathway (Zehentner et al., 1999).
Members of this family bind and activate chondrocyte-
specific enhancers in genes encoding various collagens
(Bi et al., 1999; Lefebvre et al., 1997; Zhou et al., 1998).
In particular, Sox9 stimulates the expression of colla-
gen II and collagen α2 genes in embryonic stem cells

Abbreviations: BMP, bone morphogenetic protein; MAPK, mito-
gen-activated protein kinase; PNA, peanut agglutinin.
Molecules
and
Cells
©KSMCB 2006

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354 BMP-2-Induced Up-Regulation of Sox9

(Bi et al., 1999) and immortalized cell lines (Zehentner et
al., 1999). Moreover, genetic studies on Sox9 heterozygous
mutant mice and floxed alleles of Sox9 demonstrate that
the protein is required for several sequential steps of chon-
drogenesis and cartilage development (Akiyama et al.,
2002; Bi et al., 1999). However, the mechanism by which
BMP-2 regulates Sox9 remains to be clarified. In the pre-
sent study, we show that BMP-2-induced downregulation
of Wnt-7a/β-catenin signaling via p38 MAPK results in
enhanced levels of Sox9 protein. We also explore the inter-
actions between Sox9 and β-catenin that induce Sox9
ubiquitination.


Materials and Methods

Cell culture and treatment Micromass cultures of mesenchy-
mal cells derived from the distal tips of Leghorn chick embryos
at Hamburger-Hamilton (HH) stage 22/23 were established, as
described previously (Bang et al., 2000). Cells were suspended
at a density of 2 × 107 cells/ml in Ham’s F-12 medium contain-
ing 10% fetal bovine serum, 100 IU/ml penicillin, and 50 μg/ml
streptomycin (Gibco Invitrogen, USA). Three 15 μl drops of the
suspensions were plated in 35 mm Corning culture dishes, and
incubated for 1 h at 37°C under 5% CO2 to allow attachment.
Cultures were maintained for the indicated times, or 24 h, in the
absence or presence of 100 ng/ml BMP-2 (R&D Systems, Min-
neapolis, MN) or 30 µM MG132 (Calbiochem, USA).

Analysis of differentiation and cellular condensation Chon-
drogenesis was measured by Alcian blue staining of sulfated
cartilage glycosaminoglycans. Alcian blue-bound sulfated gly-
cosaminoglycans were extracted with 6 M guanidine-HCl, and
quantified by measuring the absorbances of the extracts at 600
nm. Binding of peanut agglutinin (PNA) was used as a specific
marker for precartilage condensation (Maleski and Knudson,
1996). Briefly, cultures were rinsed twice with 0.02 M PBS, pH
7.2, fixed in methanol: acetone (1:1) for 1 min, air-dried, and
incubated with 100 μg/ml biotinylated PNA (Sigma, USA) for 1
h. PNA binding was visualized with VECTASTAIN ABC and a
DAB substrate solution kit (Vector Laboratories Inc., USA).

Preparation of conditioned medium for Wnt-7a Wnt-7a- or
control-conditioned medium (CM) was prepared according to a
previous report (Lyu and Joo, 2005). Briefly, mouse fibroblast
L929 cells were transfected with the Wnt-7a expression vector
or empty vector, using Lipofectamine reagent (Gibco Invitro-
gen), to generate stable cell lines. Expression of Wnt-7a was
confirmed by RT-PCR. After growing to 90% confluence, con-
trol and Wnt-7a-expressing L929 cells were washed and main-
tained in serum-free Dulbecco’s modified Eagle’s medium for
36 h. Conditioned media were clarified by centrifugation at
10,000 × g for 5 min, followed by filtration (0.2 µm pore size),
and concentrated 20 times by ultrafiltration in Amicon stirred
cells (Millipore, USA) using a YM membrane with a 10 kDa
molecular mass cut-off.

Transient transfection Five μg of a dominant-negative mutant
of p38 MAPK (pCMV5-Flag-p38AGF, a kind gift from Dr. Roger
Davis, Howard Hughes Medical Institute, USA), with the phos-
phorylation motif Thr-Gly-Tyr of p38MAPK replaced with the
phosphorylation-defective motif Ala-Gly-Phe to block its phos-
phorylation by MKK6, or constitutively active β-catenin, was
electroporated into isolated mesenchymal cells, using a square
wave generator (BTX-830; Gentronics, USA) with 20 msec 200
square pulses. A myc-tag was added to the C terminus of β-
catenin by the polymerase chain reaction. The β-catenin/Myc
construct was cloned into the BamHI site of pcDNA3 vector
(Gibco Invitrogen, USA). Mutagenesis of serine 37 was per-
formed using a Quick-Change site-directed mutagenesis kit
(Stratagene, USA). Electroporated chondroblasts were micro-
mass-cultured at a density of 2 × 107 cells/ml.

Immunoprecipitation and Western blotting Cultures were
lysed in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM
Tris, pH 8.0) containing protease and phosphatase inhibitors.
Lysates were centrifuged at 13,000 × g for 10 min at 4°C to
remove cell debris, and incubated with 1.5 μg of antibody
against Sox9. Immunocomplexes were precipitated by incuba-
tion with protein A-Sepharose for 1 h at 4°C, and analyzed by
SDS-PAGE and Western blotting. Twenty μg of protein per lane
was electrophoresed on a 10% polyacrylamide gel containing
0.1% SDS, and transferred to a nitrocellulose membrane
(Schleicher and Schuell, Germany). Membranes were incubated
for 1 h at room temperature in Tris-buffered saline (20 mM Tris-
HCl, pH 8.0, 137 mM NaCl) containing 0.1% Tween 20 and
10% non-fat dry milk (blocking buffer), and probed with anti-
bodies (1:1,000 dilution) against ubiquitin (Sigma), N-cadherin,
β-catenin (BD Transduction Lab., USA), type II collagen, Sox9,
Erk, and p38 MAPK (Santa Cruz Biotech, USA), as well as
phospho-specific antibodies against Erk, and pATF-2 or phos-
pho-p38 MAPK (Signal Transduction Laboratory, USA). Anti-
HSP70 antibody was from StressGen Biotechnologies Group
(Victoria, Canada). Blots were developed using peroxidase-
conjugated secondary antibody (1:5,000 dilution), and reactive
proteins were visualized with the ECL system (Amersham, UK).

RT-PCR Total RNA was isolated using TRIzol (Gibco Invitro-
gen, USA) on days 1, 3, and 5 of culture. Next, cDNA was syn-
thesized by reverse transcription of 2 μg RNA in 20 μl of Mas-
ter mix containing 200 U/μl Superscript III (Gibco Invitrogen,
USA), 5 mM MgCl2, PCR Buffer II, 1 mM dNTP, 1 U/μl RNase
inhibitor, and 2.5 mM oligo dT on a Perkin-Elmer GeneAmp
PCR system 9600 (Wellesley, USA) at 42°C for 55 min and
99°C for 5 min. Synthesized cDNA samples were subjected to
30 cycles of amplification at 94°C (denaturing) for 40 s, 65°C
(annealing) for 40 s, and 72°C (extension) for 40 s. Subse-
quently, the PCR products were electrophoresed on a 2.0% aga-
rose gel. The following primers were employed: 5′-CCCC-
AACGCCATCTTCAA-3′ (antisense), 5′-CTGCTGAT GCCGT-

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Eun-Jung Jin et al. 355

AGGTA-3′ (sense) for Sox9, 5′-CTGAAAGTGGGTAGCAG-
AGAAGCA-3′, 5′-CTCGAGTACTGGTGCGTGTTGTAA-3′ for
Wnt-7a, and 5′-GATGGGTGTCAA CCATGAGAAA-3′, 5′-
ATCAAAGGTGG AAGAATGGCTG-3′ for GAPDH.


Results and Discussion

Chondrogenesis comprises three main steps: chondrogenic
lineage commitment, mesenchymal cell condensation, and
differentiation into cartilage. The condensation phase in-
volves synthesis of specific adhesion molecules that alter
adhesive properties and cell-cell interactions (Ide et al.,
1994; Tavella et al., 1994). After mesenchymal condensa-
tion is complete, prechondrogenic cells differentiate fur-
ther to produce specific cartilage matrices containing type
II collagen and aggrecan. Several recent studies show that
BMP signaling is required for precartilagenous condensa-
tion and differentiation of precursors into chondrocytes in
C3H10T1/2 and ADTC5 mesenchymal cell lines (Fischer
et al., 2002; Izzo et al., 2002; Yoon and Lyons, 2004;
Yoon et al., 2005). However, there is limited information
on the molecular mechanism of BMP-2 action in chon-
drogenesis of primary mesenchymal cells during embry-
onic development.
In the present study, BMP-2 significantly enhanced cel-
lular condensation and chondrogenesis of chick wing bud
mesenchymal cells, as determined by PNA and Alcian
blue staining, respectively (Fig. 1A). In addition, the lev-
els of chondrogenic proteins, type II collagen and Sox9,
were markedly increased (Fig. 1B). There are reports that
BMP-2 stimulates the production of transcripts of Sox9,
which is one of the earliest markers expressed in cells
undergoing condensation and is required for type II colla-
gen expression (Ng et al., 1997; Park et al., 2005). Based
on these data we propose that Sox9 plays a critical role in
BMP-2-induced chondrogenic differentiation of chick
limb bud mesenchymal cells.
Chondrogenesis is initiated by precartilage condensa-
tion via cell-cell adhesion (DeLise et al., 2000). N-
cadherin, a key Ca2+-dependent cell adhesion molecule,
mediates cell-cell adhesion in association with β-catenin
(Conacci-Sorrell et al., 2002). Perturbation of N-cadherin
function using antibodies (Oberlender and Tuan, 1994a)
or dominant-negative cadherin molecules (DeLise and
Tuan, 2002a) results in developmental delays and de-
formities (Oberlender and Tuan, 1994b). It has also been
shown that sustained expression of N-cadherin contributes
to inhibition of chondrogenesis (DeLise and Tuan, 2002b;
Tufan and Tuan, 2001). These findings suggest that strict
temporal regulation of N-cadherin and its association with
β-catenin is required for chondrogenesis. Therefore, we
analyzed the expression of N-cadherin and β-catenin after
exposure to BMP-2. The levels of N-cadherin, a cell-cell
adhesion protein, increased in the early stages of conden-
A







B





Fig. 1. BMP-2 stimulates chondrogenic differentiation of wing
bud mesenchymal cells. Mesenchymal cells were cultured at a
density of 2 × 107 cells/ml with or without 100 ng/ml of BMP-2
for 24 h. A. Cells were stained with PNA on day 3 and Alcian
blue on day 5 of culture. B. Changes in the levels of type II col-
lagen and Sox9 in control and BMP-2-treated cultures were
analyzed by Western blotting on the indicated days. HSP70
served as a loading control. Data are typical results from at least
four independent experiments.


sation in the presence of BMP-2, and decreased in the
later stages (Fig. 2A), consistent with previous in vitro
findings on the requirement for N-cadherin in BMP-2-
induced chondrogenesis (Denker et al., 1999; Haas and
Tuan, 1999). Furthermore, BMP-2 treatment also led to
an early increase in β-catenin protein (Fig. 2A), a known
N-cadherin-associated Wnt signal transducer in mouse
C3H10T1/2 cells, a multipotent mesenchymal cell line
(Fischer et al., 2002), implying functional cross-talk be-
tween BMP-2 and the Wnt signaling pathways. RT-PCR
data also revealed a significant decrease in Wnt-7a mRNA
in the presence of BMP-2 (Fig. 2B). Our results indicate
that stimulation of cell condensation by BMP-2 involving
the time-dependant regulation of N-cadherin and β-
catenin proteins and downregulation of Wnt-7a signaling,
which in turn decreases β-catenin protein, is responsible
for the chondro-stimulatory effect of BMP-2. Wnt-7a and
BMP-2 may have antagonistic effects on cartilage differ-
entiation, and a gradient of the two proteins is possibly
involved in defining the boundaries of initial precartilage
condensation (Stott et al., 1999).
At least two distinct pathways mediate BMP signaling;
specifically, the canonical Smad pathway and a mitogen-
activated protein kinase (MAPK) pathway (Hatakeyama
et al., 2003; Jadlowiec, 2006; Ju et al., 2000; Seto et al.,
2004). The p38 MAPK pathway contributes to the initia-
tion of chondrogenic cell condensation in ATDC5 cells
(Nakamura et al., 1999), and ERK1/2 activation cross-
interacts with BMP-2-induced signaling to regulate chon-
drogenesis in a positive manner (Seghatoleslami et al.,

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356 BMP-2-Induced Up-Regulation of Sox9

A





B




Fig. 2. BMP-2 inhibits Wnt-7a/β-catenin signaling during chon-
drogenic differentiation. A. Changes in the levels of N-cadherin
and β-catenin in control and BMP-2-treated cultures were as-
sessed by Western blotting. HSP70 served as a loading control.
B. Alterations in the mRNA level of Wnt-7a were determined by
RT-PCR on the indicated days. GAPDH was used as a loading
control. Data are typical results from at least four independent
experiments.


2003). Previously, we showed that ERK1/2 and p38 MAPK
act as negative and positive regulators of chondrogenesis,
respectively (Oh et al., 2000; Yoon et al., 2000). However,
the properties of BMP signaling in chondrocytes mediated
by p38 MAPKs are currently unclear. Accordingly, we in-
vestigated the possible involvement of MAPK signaling in
BMP-2-induced chondrogenesis. Phosphorylation of p38
MAPK, but not ERK, was increased in response to BMP-2
during chondrogenesis of wing bud mesenchymal cells (Fig.
3A). To determine the role of p38 MAPK during chondro-
genesis, a dominant-negative construct (dn-p38 MAPK) was
electroporated into mesenchymal cells, and its effects on
chondrogenesis and the Wnt signaling pathway were ana-
lyzed. Transient expression of the dominant-negative con-
struct inhibited phosphorylation of p38 MAPK in the pres-
ence of BMP-2 (Fig. 3B, upper panel), and this downregula-
tion of p38 MAPK activity prevented the suppression of
Wnt-7a mRNA production by BMP-2 (Fig. 3B, lower panel).
This suggests that p38 MAPK is responsible for the down-
regulation of expression of Wnt-7a by BMP-2 signaling.
To further establish the function of the Wnt-7a and p38
MAPK pathways in BMP-2- induced chondrogenesis, we
added conditioned medium (CM) from L929 cells that
secrete Wnt-7a protein, or electroporated BMP-2-treated
cultures with the dn-p38 MAPK construct. Addition of
Wnt-7a-containing CM or inactivation of p38 MAPK us-
ing dn-p38 MAPK blocked BMP-2-stimulated precarti-
lage condensation, as well as chondrogenic differentiation,
as determined by PNA and Alcian blue staining, respec-
tively (Fig. 3C). Moreover, addition of Wnt-7a or electro-
poration of dn-p38 MAPK antagonized the effect of
BMP-2 by increasing β-catenin and decreasing Sox9 lev-
els (Fig. 3D). Our findings indicate that the BMP-2-
induced increase in Sox9 protein and decrease in Wnt-
A C





B







a=







Fig. 3. p38 MAPK signaling is involved in BMP-2-induced
downregulation of the Wnt-7a/β-catenin signaling pathway. A.
Mesenchymal cells were treated with 100 ng/ml of BMP-2 for 24
h or left untreated. Changes in Erk and p38 MAPK phosphoryla-
tion levels were determined by Western blotting using pERK and
pATF-2 antibodies, respectively, on the indicated days. B. Mesen-
chymal cells were electroporated with pcDNA3.1 vector as a con-
trol (-) or with a dominant-negative construct of p38 MAPK (dn-
p38 MAPK, +), and then Wnt-7a mRNA was measured by RT-
PCR after the indicated days in the presence of BMP-2 (lower
panel). The transfection efficiency was confirmed by Western
blotting using a phospho-p38 MAPK antibody (upper panel). C.
Wing mesenchymal cells were treated with Wnt-7a-conditioned
medium or electroporated with dn-p38 MAPK, and cultured in the
presence of BMP-2. Cells were stained with PNA on day 3 and
Alcian blue on day 5 (upper panel). Chondrogenesis was assessed
by measuring the absorbance of bound Alcian blue at 600 nm
(lower panel). D. Changes in the levels of β-catenin and Sox9 on
day 3 of culture were determined by Western blotting. HSP70
served as a loading control. Data are representative of at least four
independent experiments.


7a/β-catenin signaling are mediated by p38 MAPK, and
that these alterations in the steady-state levels of Sox9 or
β-catenin in mesenchymal cells trigger BMP-2-induced
chondrogenic differentiation. Based on these observations,
we investigated the functional relationship between β-
catenin and Sox9 protein during BMP-2-induced chondro-
genic differentiation.
β-catenin, an effector of the Wnt signaling pathway, is
necessary and sufficient to suppress the differentiation of
mesenchymal cells into Sox9-positive skeletal precursors

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Eun-Jung Jin et al. 357

A











B





C






Fig. 4. β-catenin interacts with Sox9 and induces the ubiquitin-
dependent degradation of Sox9. Wing bud mesenchymal cells
were transfected with a Myc-tagged constitutively active β-
catenin expression vector. A. The transfection efficiency was
confirmed by Western blotting using an anti-myc antibody (up-
per panel). Cells were stained with PNA on day 3 and Alcian
blue on day 5 of culture (lower panel). B. Mesenchymal cells
expressing mock or constitutively active β-catenin were cultured
in the presence of BMP-2, and treated with 30 μM MG132 for 5
h on day 3 of culture, or left untreated. Changes in Sox9 level
were determined by Western blotting. C. Mesenchymal cells
were harvested 36 h after electroporation with the β-catenin
expression vector, and immunoprecipitated with anti-Sox9 anti-
body. Immunoprecipitates were subjected to Western blot analy-
sis with anti-Sox9 and -ubiquitin antibodies.


(Hill et al., 2005). To determine the role of Wnt/β-catenin
signaling as modulated by BMP-2, cells were electropo-
rated with a Myc-tagged constitutively active β-catenin
expression vector. Overexpression of β-catenin after tran-
sient transfection was confirmed by Western blotting with
anti-Myc antibody (Fig. 4A, upper panel), and its effect on
precartilage condensation and chondrogenesis was ana-
lyzed by PNA and Alcian blue staining, respectively. Over-
expression of β-catenin in the primary mesenchymal cells
caused a marked inhibition of cellular condensation and
chondrogenesis (Fig. 4A, lower panel).
The transcription factor, Sox9, is degraded by the ubiq-
uitin-proteasome system and stabilized by a K398A muta-
tion in the ubiquitin target site (Akiyama et al., 2004). Re-
cent reports show that interactions between Sox9 and β-
catenin promote the mutual degradation of both proteins
via the ubiquitin-proteasome system, and maintain their
relative levels to control chondrocyte differentiation (Aki-
yama et al., 2004). Accordingly, we examined whether
overexpression of β-catenin affects the level of Sox9 pro-
tein in mesenchymal cells. Cells were electroporated with
the β-catenin expression vector, cultured in the presence of
BMP-2, and exposed to MG132, a proteasome inhibitor, for
5 h before cell harvest. The induction of Sox9 protein by
BMP-2 was suppressed by overexpression of β-catenin and
restored by treatment with the proteasome inhibitor (Fig
4B). These data strongly suggest that Sox9 is degraded by
the ubiquitin-proteasome system. To confirm that interac-
tions between Sox9 and β-catenin modulate the ubiquitina-
tion of Sox9, we measured ubiquitinated Sox9 proteins
prior to proteasomal degradation by immunoprecipitation.
After electroporation of β-catenin, extracts were immuno-
precipitated, separated by SDS-PAGE, and probed with
anti-Sox9 or anti-ubiquitin antibody. The presence of
polyubiquitinated Sox9 was confirmed by Western blotting
with an anti-ubiquitin antibody. As shown in Fig. 4C,
polyubiquitinated Sox9 accumulated only during β-catenin
overexpression, indicating that β-catenin binding to Sox9
stimulates the attachment of ubiquitin. Our results demon-
strate that β-catenin interacts with Sox9 in mesenchymal
cells, and triggers its destruction via a ubiquitination-
dependent proteasome pathway during BMP-2-induced
chondrogenesis.
Inactivation of Sox9 in mouse limb buds using the
Cre/loxP recombination system before cellular condensa-
tion results in complete blocking of mesenchymal conden-
sation and subsequent cartilage formation, implying that the
protein is required for mesenchymal condensation (Aki-
yama et al., 2002). In mouse chondrocytes, Sox9 and β-
catenin have opposing roles in chondrogenesis: increased
expression of Sox9 resembles the phenotype produced by
loss of function of β-catenin, and conversely, loss of Sox9
expression resembles the phenotype produced by constitu-
tively active β-catenin (Akiyama et al., 2004). Moreover,
several studies point to opposite effects of BMP-2 on Sox9
and β-catenin. BMP-2 upregulates Sox9 mRNA in bovine
synovium-derived progenitor cells, but downregulates β-
catenin protein in C3H10T1/2 cells (Fischer et al., 2002;
Park et al., 2005; Uusitals et al., 2001).
In conclusion, BMP-2 signals downregulate the Wnt-
7a/β-catenin pathway by activating p38 MAPK. This re-
sults in inhibition of proteasomal degradation of Sox9 and
promotes cellular condensation and chondrogenesis in wing
bud mesenchyme.


Acknowledgment This work was supported by the Korea Re-
search Foundation (KRF-2003-070-C00033).