1 Department of Life Science, Kwangju Institute of Science and Technology,
Gwangju 500-712, Korea
2 Department of Orthopaedic Surgery, Wonkwang University School of Medicine,
Iksan 570-711, Korea
3 Laboratory of Developmental Biology and Genomics, College of Veterinary
Medicine, Seoul National University, Seoul, Korea
4 TG Biotech, Kyungpook National University, Daegu 702-701, Korea
Author for correspondence (e-mail: jschun{at}kjist.ac.kr)
Accepted 14 August 2002
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SUMMARY |
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Key words: Cartilage, Chondrocytes, Differentiation, De-differentiation, ß-Catenin, Chick
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INTRODUCTION |
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Although the maintenance of the differentiated chondrocyte phenotype is
important for cartilage homeostasis, the detailed mechanisms of the
maintenance and loss of differentiated chondrocyte phenotypes remain largely
unknown. Cell-to-cell interaction mediated by N-cadherin is an important
regulator of both precartilage condensation and its progression to cartilage
nodule during chondrocyte differentiation
(Sandell and Adler, 1999;
DeLise et al., 2000
). Based on
the importance of the strictly regulated expression of N-cadherin, it is
believed that expression of N-cadherin-related cytoskeletal components such as
- and ß-catenin play a role in chondrocyte differentiation. In
addition to the stabilization of cell-cell adhesion by interacting with
cadherin, ß-catenin is also engaged in the regulation of gene expression
by acting as a transcriptional co-activator in the regulation of several
biological functions (Ben-Ze'ev and Geiger,
1998
; Willert and Nusse,
1998
). In the presence of Wnt signal, ß-catenin escapes from
ubiquitin-dependent proteolytic degradation via the 26S proteasome, and the
accumulated ß-catenin translocates into the nucleus in association with
members of the T cell-factor (TCF)/lymphoid-enhancer-factor (LEF) family of
transcription factors to stimulate transcription of target genes.
The role of ß-catenin in the regulation of chondrogenesis or
phenotypic loss of chondrocytes during cartilage destruction remains largely
unknown. To address this issue, we investigated the function of ß-catenin
in the regulation of phenotypic changes of chondrocytes (i.e.,
differentiation, de-differentiation, and redifferentiation). In addition to in
vivo examination, we employed micromass culture of embryonic mesenchymal cells
as a model system to study chondrogenesis, a serial monolayer culture or
treatment of articular chondrocytes with RA or IL1ß to study
de-differentiation, and three-dimensional culture of de-differentiated cells
in alginate gel beads to study redifferentiation. We focused our effort on RA
and IL1ß, which are known to modulate chondrocyte phenotypes. RA is a
well-characterized soluble mediator that inhibits chondrogenesis and induces
de-differentiation of chondrocytes (Cash et
al., 1997; Hering,
1999
; Weston et al.,
2000
). IL1ß plays a major role in joint cartilage destruction
in arthritis and induces de-differentiation of chondrocytes by inhibiting
expression of cartilage-specific type II collagen and proteoglycan
(Goldring et al., 1994
;
Demoor-Fossard et al.,
1998
).
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MATERIALS AND METHODS |
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Immunohistochemistry and immunofluorescence microscopy
Wing buds of chicken embryos and spots of micromass culture were fixed in
4% paraformaldehyde for 24 hours at 4°C, dehydrated with graded ethanol,
embedded in paraffin wax and sectioned at 4 µm thickness. The sections were
stained by standard procedures using Alcian Blue or antibodies against type II
collagen (Chemicon, Temecula, CA) and ß-catenin and Jun (BD Transduction
Laboratories, Lexington, KY), and visualized by developing with a kit
purchased from DAKO (Carprinteria, CA). Immunofluorescence microscopy was also
used to determine expression and distribution of type II collagen and
ß-catenin (Yoon et al.,
2002). Briefly, chondrocytes were fixed with 3.5% paraformaldehyde
in phosphate-buffered saline (PBS) for 10 minutes at room temperature. The
cells were permeabilized and blocked with 0.1% Triton X-100 and 5% fetal calf
serum in PBS for 30 minutes. The fixed cells were washed and incubated for 1
hour with antibody (10 µg/ml) against ß-catenin or type II collagen.
The cells were washed, incubated with rhodamine- or fluorescein-conjugated
secondary antibodies for 30 minutes, and observed under a fluorescence
microscope.
Northern and western blot assay
Total RNA was isolated by a single-step guanidinium
thiocyanatephenol-chloroform method, using RNA STAT-60 (Tel-Test B,
Friendswood, TX) according to the manufacturer's protocol. Total RNA (3 µg)
was fractionated on formaldehyde/agarose gel. Rabbit type II collagen
transcript was detected with a 370-bp partial cDNA probe as previously
described (Kim et al., 2002a;
Yoon et al., 2002
). The probe
(542 bp) for ß-catenin transcript was generated by RT-PCR using a sense
primer corresponding to nucleotides -18 to +10 and an antisense primer
corresponding to nucleotides +501 to +524 of ß-catenin. For western
blotting, whole cell lysates prepared as previously described
(Kim et al., 2002a
) were
fractionated by SDS-polyacrylamide gel electrophoresis and transferred to a
nitrocellulose membrane. Proteins were detected using antibodies purchased
from the following sources: type II collagens from Chemicon, rabbit anti-chick
N-cadherin polyclonal antibody from Sigma-Aldrich, rabbit anti-human
-catenin polyclonal antibody from Santa Cruz (Santa Cruz, CA), and
mouse ß-catenin or Jun monoclonal antibodies from BD Transduction
Laboratories.
Transfection and reporter gene assays
Retroviral vector (5 µg) containing cDNA for S37A ß-catenin was
transfected to articular chondrocytes using LipofectaminePLUS (Gibco-BRL,
Gaithersburg, MD) or infected with viral supernatant for 90 minutes. The
transfected cells, which were cultured in complete medium for 48 hours, were
used for further analysis as indicated in each experiment. To investigate
ß-catenin-TCF/LEF signaling, cells were transiently transfected with 1
µg of the TCF/LEF reporters, TOPFlash (optimal LEF-binding site) or
FOPFlash (mutated LEF-binding site) (van
de Wetering et al., 1997) (Upstate Biotechnology Inc., Lake
Placid, NY), and 1 µg of pCMV-ß-galactosidase. After incubation with
IL1ß or RA for 72 hours, luciferase activity was measured and normalized
for transfection efficiency using ß-galactosidase activity.
Immunoprecipitation
Chondrocytes were lysed in Nonidet P-40 lysis buffer (1% NP-40, 150 mM
NaCl, 50 mM Tris, pH 8.0) containing inhibitors of proteases [10 µg/ml
leupeptin, 10 µg/ml pepstatin A, 10 µg/ml aprotinin and 1 mM of
4-(2-aminoethyl) benzenesulfonyl fluoride] and phosphatases (1 mM NaF and 1 mM
Na3VO4). After preclearing with protein A sepharose for
1 hour, proteins (500 µg) were incubated with antibodies against
ß-catenin or N-cadherin. The immune complex was then precipitated by the
incubation with protein A sepharose for 1 hour at 4°C. After washing with
lysis buffer, the immuncomplex was analyzed by SDS-polyacrylamide gel
electrophoresis and western blotting (Kim
et al., 2002a).
Preparation of Triton X-100 insoluble and nuclear fractions
Triton X-100 insoluble cytoskeletal fraction was prepared as described by
Stolz et al. (Stolz et al.,
1992). For the preparation of nuclear proteins, chondrocytes were
washed with PBS and homogenized in a buffer A (10 mM HEPES, pH 7.9, 10 mM KCl,
0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol containing inhibitors of
proteases and phosphatases). Following addition of 0.6% (v/v) Nonidet P-40,
the cells were incubated for 15 minutes on ice and then centrifuged at 13,000
g for 30 seconds at 4°C. The pellet was suspended in
buffer B (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA and 1 mM
dithiothreitol) containing inhibitors of proteases and phosphatases and
centrifuged at 13,000 g for 5 minutes at 4°C. The
resulting supernatant (nuclear fraction) was stored at -70°C until further
analysis.
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RESULTS |
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We employed micromass culture of chick embryonic mesenchymal cells to
determine the role of ß-catenin in chondrogenesis. Immunohistochemical
staining of type II collagen and ß-catenin
(Fig. 2A) in cross-sections of
micromass culture spot (upper panel) revealed that ß-catenin is absent in
cartilage nodules where type II collagen expressing chondrocytes are
localized. The expressed ß-catenin in chondrifying mesenchymal cells is
localized in cell-cell contacts (lower panel). Expression of ß-catenin
and N-cadherin was high at the condensation period and decreased thereafter, a
pattern opposite to that of type II collagen
(Fig. 2B, upper panel).
Treatment of chondrifying mesenchymal cells with 10 nM phorbol 12-myristate
13-acetate (PMA) to downregulate protein kinase C, 10 µM SB203580 to
inhibit p38 kinase, or 10 ng/ml epidermal growth factor, conditions that
inhibit chondrogenesis (Chang et al.,
1998; Oh et al.,
2000
; Yoon et al.,
2000
) (Fig. 2B,
middle and lower panels), blocked the decrease of ß-catenin and
N-cadherin expression. By contrast, inhibition of extracellular
signal-regulated kinase with 10 µM PD98059, which enhances chondrogenesis
(Oh et al., 2000
), potentiated
the decrease of ß-catenin and N-cadherin expression
(Fig. 2B, middle and lower
panels). Treatment with 5 mM LiCl, which causes accumulation of ß-catenin
by the inhibition of glycogen synthase kinase (GSK)-3ß via its
phosphorylation (Stambolic et al.,
1996
), resulted in inhibition of type II collagen expression
(Fig. 2C, upper and middle
panels) and proteoglycan synthesis (Fig.
2C, lower panel) and sustained expression of N-cadherin
(Fig. 2C, middle panel), which
clearly indicating a negative role of ß-catenin in chondrocyte
differentiation.
|
Accumulation and transcriptional activity of ß-catenin causes
phenotype loss of differentiated chondrocytes
We next investigated whether ß-catenin is also associated with the
maintenance of differentiated chondrocyte phenotypes using rabbit articular
chondrocytes. The ß-catenin level was low in differentiated chondrocytes
and significantly increased as cells underwent de-differentiation by a serial
monolayer culture (Fig. 3A,
upper panel), 1 µM RA treatment (Fig.
3A, middle panel), or 5 ng/ml IL1ß treatment for 72 hours
(Fig. 3A, lower panel). All of
the culture conditions caused reduction of type II collagen expression
(Fig. 3A,B lower panel) and
proteoglycan synthesis (Fig.
3B, upper panel). The elevated ß-catenin protein level in
de-differentiated cells was decreased when de-differentiated cells
redifferentiate by three-dimensional culture in alginate gel
(Fig. 3C). Therefore, the
expression level of ß-catenin is differentially regulated during de- and
redifferentiation with an inverse relationship to the degree of chondrocyte
differentiation status.
|
Northern blot analysis indicated that de-differentiation did not accompany any changes in ß-catenin transcript levels (Fig. 4A, upper panel). Phosphorylation of GSK-3ß was significantly increased in de-differentiating cells, indicating the inhibition of GSK-3ß activity (Fig. 4A, lower panel). Because GSK-3ß activity is primarily responsible for the degradation of ß-catenin via ubiquitin-proteasome system, the inhibition of GSK-3ß indicated that post-translational accumulation of ß-catenin contributes to the increased levels of ß-catenin in cells treated with RA or IL1ß. This was further supported by the observation that treatment of chondrocytes with LiCl, which inhibits GSK-3ß, resulted in increased phosphorylation of GSK-3ß, accumulation of ß-catenin and reduction of type II collagen expression (Fig. 4B). In addition, block of ß-catenin degradation by the inhibition of 26S proteasome by MG132 also resulted in increased levels of ß-catenin and cessation of type II collagen expression (Fig. 4C).
|
To determine molecular mechanisms of ß-catenin regulation of the
chondrocyte phenotype, we examined a role of ß-catenin functions as a
cytoskeletal component by determining its participation in N-cadherin-mediated
cell-to-cell adhesion and as a nuclear signaling molecule in activating the
TCF/LEF transcription factor. During chondrocyte de-differentiation caused by
1 µM RA treatment for 72 hours, expression of cell adhesion machinery
components such as N-cadherin and -catenin as well as ß-catenin
was significantly increased, whereas expression of these molecules did not
change during IL1ß (5 ng/ml for 72 hours)-mediated de-differentiation
(Fig. 5A, upper panel).
Treatment of cells with RA, but not IL1ß, also increased association of
ß-catenin with N-cadherin as determined by immunoprecipitation
experiments (Fig. 5A, lower
panel). Thus, RA-induced de-differentiation accompanied not only increased
expression of ß-catenin but also enhanced association with N-cadherin,
while IL1ß-induced de-differentiation did not accompany these changes.
This was further demonstrated by examining localization of N-cadherin and
ß-catenin in Triton-X 100 insoluble cytoskeletal fractions. Western blot
analysis (Fig. 5B) and indirect
immunofluorescence microscopy (Fig.
5C) clearly indicated that RA, but not IL1ß, increased
localization of N-cadherin and ß-catenin in the cytoskeletal fraction. In
addition, changes of cell morphology including stress fiber formation were
seen only in cells treated with RA (Fig.
5D).
|
We next assessed the possibility that ß-catenin acts as a nuclear
signaling molecule during de-differentiation through its function as a
co-activator of TCF/LEF family of transcription factors. Most of the expressed
ß-catenin is localized in cell-to-cell contacts in chondrocytes, and RA
(1 µM, 72 hours) or IL1ß (5 ng/ml, 72 hours) dramatically increased
nuclear localization of ß-catenin
(Fig. 6A). Western blot
analysis also showed significantly increased levels of ß-catenin in the
nuclear fraction (Fig. 6B, lower panel). Transcriptional activation by ß-catenin was examined by
TCF/LEF reporter gene assay using TOPFlash (optimal TCF/LEF-binding site) and
FOPFlash (mutated TCF/LEF-binding site). These reporter gene assays indicated
a transcriptionally active role for ß-catenin
(Fig. 6B, upper panel).
Consistent with the increased ß-catenin-TCF/LEF activity, expression of
known ß-catenin target genes such as Jun
(Mann et al., 1999), but not
connexin 43 (van der Heyden et al.,
1998
), was increased (Fig.
6B, lower panel). Therefore, accumulation of ß-catenin in
de-differentiating chondrocytes appears to alter the gene expression profile
of the cell by activating the TCF/LEF family of transcription factors.
|
To access the function of ß-catenin more directly in the regulation of
the chondrocyte phenotype, S37A ß-catenin, a stable non-ubiquitinatable
form of ß-catenin (Easwaran et al.,
1999), was ectopically expressed in chondrocytes. Transfection of
ß-catenin caused a dramatic increase of TCF/LEF activity
(Fig. 7A, middle panel) and
reduced accumulation of proteoglycan (Fig.
7A, right panel). Ectopic expression of ß-catenin also caused
a significant reduction of type II collagen expression and enhanced expression
of the ß-catenin target gene Jun (Fig.
7A, left panel). Double staining of type II collagen and
ß-catenin in differentiated chondrocytes transfected with S37A
ß-catenin indicated that cells highly expressing ß-catenin are
negative for type II collagen staining
(Fig. 7B), indicating that
ß-catenin expression was sufficient to cause de-differentiation of
chondrocytes.
|
Regulation of Jun expression by ß-catenin
Because the above results indicate that accumulation of ß-catenin
during de-differentiation causes increased expression of Jun, we next examined
the role of ß-catenin on Jun expression during chondrogenic
differentiation of mesenchymal cells. Distribution pattern of Jun is
essentially same as that of ß-catenin: it was highly expressed in
prechondrogenic mesenchymal cells in day 5 embryos but absent in
differentiated chondrocytes in day 6 and thereafter
(Fig. 8A). Immunohistochemical
staining of type II collagen and Jun in cross-sections of micromass culture
spot showed that Jun staining in cartilage nodules, where type II collagen
expressing chondrocytes are localized, is dramatically reduced
(Fig. 8B). Thus, the expression
and distribution pattern of ß-catenin
(Fig. 1) and Jun
(Fig. 8) is essentially same
during chondrogenesis both in vivo and in vitro. Expression level of Jun, as
determined by western blot analysis, was high at the condensation period and
decreased thereafter, a pattern similar to ß-catenin and opposite to type
II collagen (Fig. 8C).
Treatment of chondrifying mesenchymal cells with 10 nM phorbol 12-myristate
13-acetate (PMA) or 1 µM Go6976 to downregulate and inhibit protein kinase
C, respectively, blocked the decrease of Jun and ß-catenin levels with
the inhibition of chondrogenesis. By contrast, inhibition of extracellular
signal-regulated kinase with 10 µM PD98059 potentiated the decrease of Jun
and ß-catenin levels with the enhancement of chondrogenesis
(Fig. 8D). Therefore, the
expression of Jun appears to be regulated by ß-catenin during
differentiation, as well as de-differentiation of chondrocytes.
|
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DISCUSSION |
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Cell-to-cell adhesion is an essential regulatory step in chondrogenesis by
coordinating precartilage condensation and cartilage nodule formation
(Oberlender and Tuan, 1994;
Sandell and Adler, 1999
;
Woodward and Tuan, 1999
;
DeLise et al., 2000
).
N-cadherin is highly expressed and localized to the prechondroblastic cells
during mesenchymal cell condensation, and perturbation of N-cadherin function
inhibits cellular condensation and chondrogenesis
(Oberlender and Tuan, 1994
).
The expression of N-cadherin is downregulated in the later stage of
chondrogenesis, which appears to be required for the progression of
precartilage condensation to cartilage nodules
(Oberlender and Tuan, 1994
;
Chang et al., 1998
;
Oh et al., 2000
;
Yoon et al., 2000
). The
involvement of ß-catenin in the regulation of chondrogenesis has been
suggested from the observation that ectopic expression of members of Wnt genes
such as Wnt1, Wnt7a and Wnt14 (Rudnicki
and Brown, 1997
; Hartmann and
Tabin, 2001
; Tufan and Tuan,
2001
; Tufan et al.,
2002a
) or frizzled receptor for Wnt
(Tufan et al., 2002b
) inhibits
chondrogenesis. In addition, it has been suggested that stabilization of
N-cadherin-mediated cell adhesion is responsible for the Wnt inhibition of
chondrogenesis (Tufan and Tuan,
2001
; Tufan et al.,
2002a
; Tufan et al.,
2002b
). Our results indicated that most of the expressed
ß-catenin in chondrifying mesenchymal cells during condensation period or
inter-nodular area was distributed at cell-cell contacts without any obvious
nuclear localization (Fig. 2A).
Furthermore, accumulation of ß-catenin by the inhibition of GSK-3ß
blocked down regulation of N-cadherin (Fig.
2C) and inhibition of chondrogenesis accompanied elevated
expression of N-cadherin and ß-catenin
(Fig. 2B). Therefore, we
postulate that the failure to downregulate ß-catenin and N-cadherin
blocks chondrogenesis by stabilizing cell-to-cell adhesion, rather than
altering gene expression profiles by acting as a transcriptional
co-activator.
Because the transforming group of Wnt family, such as Wnt1 and Wnt7a, but
not nontransforming Wnts exerts their effects by accumulating cytosolic
ß-catenin (Shimizu et al.,
1997), our current observation that accumulation of ß-catenin
inhibits chondrogenesis is in agreement with the inhibition of chondrogenesis
by the transforming Wnts (Rudnicki and
Brown, 1997
; Stott et al.,
1999
; Tufan and Tuan,
2001
). Although no direct evidence for the role of ß-catenin
in chondrogenesis is yet available, Hartmann and Tabin
(Hartmann and Tabin, 2000
)
reported that misexpression of ß-catenin in developing chicken wing buds
accelerates chondrocyte maturation. Similar to our results, they observed
lower levels of ß-catenin mRNA in chondrocytes of the articular region of
day 7.5 chick embryo limb buds, whereas significantly high levels of
ß-catenin were observed in the cells of the perichondrium and
hypertrophic chondrocytes. Misexpression of ß-catenin showed shortening
of the cartilage elements with the slightly increased expression of markers
for hypertophic chondrocytes, including type X collagen leading to their
conclusion that ß-catenin promotes progression of differentiated
chondrocytes to a hypertrophic state. Therefore, it may be possible that the
inhibition of type II collagen expression by the accumulation of
ß-catenin during differentiation and de-differentiation of chondrocytes
is due to maturation of differentiated chondrocytes into hypertrophic
chondrocytes. However, we could not detect any increase of alkaline
phosphatase activity in LiCl-treated micromass culture of mesenchymal cells
and in articular chondrocytes treated with RA or IL1ß or transfected with
S37A ß-catenin (data not shown), indicating that the loss of type II
collagen expression is due to inhibition of chondrogenesis of mesenchymal
cells and de-differentiation of articular chondrocytes. Our results are in
good agreement with the observations by others, which indicate expression of
hypertrophic chondrocyte markers (type X collagen and alkaline phosphatase)
during micromass culture of mesenchymal cells needs much longer culture period
(1-3 weeks) (Mello and Tuan,
1999
; Boskey et al.,
2002
), and that treatment with RA
(Cash et al., 1997
;
Hering, 1999
;
Weston et al., 2000
),
IL1ß (Goldring et al.,
1994
; Demoor-Fossard et al.,
1998
) or a serial subculture
(Lefebvre et al., 1990
;
Yoon et al., 2002
) causes
de-differentiation of articular chondrocytes. On the bases of the experiments
by Hartmann and Tabin (Hartmann and Tabin,
2000
) that suggest ß-catenin misexpression promotes
chondrocyte maturation and on our in vitro experiment that indicates
inhibition of chondrogenesis by the accumulation of ß-catenin in
chondrifying mesenchymal cells, it is possible that ß-catenin inhibits
initial chondrogenic differentiation of mesenchymal cells and also promotes
maturation of the differentiated chondrocytes that is caused by escaping from
ß-catenin inhibition of chondrogenesis. Our current observation of the
de-differentiation of articular chondrocytes, rather than maturation by the
accumulation of ß-catenin in articular chondrocytes, is different from
the suggestions made by Hartmann and Tabin
(Hartmann and Tabin, 2000
).
Therefore, further investigation is necessary to reconcile these observations.
It is of interest to determine the effects of ß-catenin accumulation on
chondrocyte phenotype in three-dimensionally cultured chondrocytes that mimics
in vivo condition of chondrocytes.
In contrast to chondrocyte differentiation, phenotype loss or
de-differentiation of chondrocytes is caused by the action of ß-catenin
as a transcriptional co-activator. This was clearly demonstrated by the
observation that de-differentiation of chondrocytes caused by IL1ß
accompanied transcriptional activation by ß-catenin
(Fig. 6) without any modulation
of cell-to-cell adhesion (Fig.
5). In addition, forced expression of S37A ß-catenin, which
dramatically increased ß-catenin-TCF/LEF activity without modulation of
N-cadherin expression, caused de-differentiation of chondrocytes
(Fig. 7), indicating that the
function of ß-catenin as a nuclear signaling molecule is sufficient to
cause phenotypic loss of chondrocytes. Because changes in cell morphology and
actin cytoskeleton such as stress fiber formation were observed in cells
treated with RA but not IL1ß (Fig.
5), RA-induced ß-catenin association with N-cadherin appears
to be involved in morphological changes of chondrocytes, rather than cessation
of the expression of chondrocyte markers. Consistent with our current
observation of RA effects on chondrocytes, RA treatment caused increased
expression of N-cadherin, increased cell-to-cell adhesion, and the recruitment
of cytoplasmic ß-catenin to the membrane in epithelial and breast cancer
cells (Vermeulen et al., 1995;
Sanchez et al., 1996
).
Although increased cadherin expression can modulate ß-catenin signaling
by depleting the cytoplasmic pool of ß-catenin, our results indicate that
this is not the case in chondrocytes as RA increased ß-catenin protein in
both the cytoskeletal and nuclear fractions.
Because loss of differentiated phenotype of chondrocytes is associated with
cartilage destruction during arthritis
(Sandell and Aigner, 2001),
accumulation of ß-catenin appears to contribute to arthritic disease.
Indeed, we observed that levels of ß-catenin were significantly increased
in osteoarthritis-affected cartilage that is obtained from individuals
undergoing total knee arthroplasty with loss of type II collagen and
proteoglycan. The increase in ß-catenin protein levels was also evident
in experimental rheumatoid arthritic cartilage caused by type II collagen
injection in DBA/1 mice (data not shown). In addition, we recently showed that
ectopic expression of transcriptionally competent ß-catenin stimulated
expression of cyclooxygenase 2 in articular chondrocytes
(Kim et al., 2002b
).
Therefore, our results suggest that accumulation of ß-catenin may play a
role in the inflammatory responses and destruction of cartilage during
arthritic disease.
Although it is clear that ß-catenin causes loss of chondrocyte
phenotype by activating transcription of genes, the mechanisms of chondrocyte
phenotype loss by ß-catenin need to be further characterized.
ß-Catenin may cause cessation of type II collagen expression and
proteoglycan synthesis either directly or indirectly. The known type II
collagen promoter/enhancer sequence in human, mouse and rat does not contain
the canonical TCF/LEF-binding motif CCTTTGA/TA/TC
(van de Wetering et al.,
1997). Thus, we postulate that ß-catenin-TCF/LEF indirectly
regulates type II collagen expression by modulating an unknown
ß-catenin-LEF/TCF target gene that may inhibit type II collagen
expression and accumulation of sulfated proteoglycan. Therefore, it is of
interest to identify ß-catenin target genes in chondrocytes to define
ß-catenin regulation of chondrocyte phenotype. In this study we
identified that Jun is a target gene of ß-catenin in articular
chondrocytes. Expression pattern of Jun is essentially same as that of
ß-catenin both in vivo and in vitro, and ectopic expression of
ß-catenin caused induction of Jun expression, indicating that Jun
expression is regulated by ß-catenin during phenotype modulation of
chondrocytes. Indeed, it has been shown that Jun and Fra1 are direct target
genes of ß-catenin in colorectal carcinoma cells
(Mann et al., 1999
). Recent
study by Tufan et al. (Tufan et al.,
2002a
) also indicated that AP-1 transcription factor is a target
of Wnt-7a signal during chondrogenesis. Therefore, it is likely that AP-1
transcription factor is associated with the ß-catenin regulation of
chondrocyte differentiation and de-differentiation.
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ACKNOWLEDGMENTS |
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