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INTRODUCTION |
Although large amounts of epidermal growth factor
(EGF)1 are found in the
synovial fluid of rheumatoid arthritic cartilage (1, 2), the role of
EGF in the pathophysiology of arthritic disease is not fully
understood. EGF is known to inhibit chondrocyte differentiation in
Meckel's cartilage (3) and chondrogenesis in chick limb bud
mesenchymal cells (4, 5). EGF is also known to inhibit accumulation of
sulfated proteoglycan in articular chondrocytes (6, 7), suggesting that
EGF not only inhibits chondrocyte differentiation but also causes loss
of differentiated chondrocyte phenotype. Articular chondrocytes are
characterized by their synthesis of cartilage-specific structural
macromolecules such as type II collagen and aggrecan, and loss of
differentiated chondrocyte phenotype or dedifferentiation causes
cessation of type II collagen expression and induction of fibroblastic
types I and III collagens (8). Although the contribution of
dedifferentiation in the pathophysiology of arthritis is poorly
understood, chondrocyte dedifferentiation is observed in arthritic
cartilage, suggesting that EGF contribute to arthritis.
EGF stimulates expression of inflammatory molecules such as
cyclooxygenase-2 (COX-2) and prostaglandin E2
(PGE2) in various cell types including cell lines
originating from chondrocytes (9). However, there are no published data
regarding EGF effects on COX-2 expression and PGE2
production in primary culture articular chondrocytes. Although the role
of EGF in PGE2 production is not clear, pro-inflammatory
cytokines such as interleukin-1
(IL-1
) cause PGE2
production, which is found in arthritic joints in large amounts.
PGE2 is a major mediator of cartilage inflammation,
cartilage and juxta-articular bone erosion, and angiogenesis (10-15).
The rate-limiting steps in PG synthesis are hydrolysis of phospholipids to produce free arachidonic acid (catalyzed by phospholipase
A2) and conversion of arachidonic acid to PGE2,
which is catalyzed by the two isoforms of COX (16, 17). COX-1 is
constitutively expressed, whereas COX-2 is rapidly induced in response
to a wide variety of cytokines such as IL-1
and growth factors
including EGF. COX-2 expression is regulated at both transcription and
post-transcription levels (18-21). Although the molecular mechanism
underlying COX-2 expression is not fully understood, several studies
indicate that its expression is regulated by mitogen-activated protein
kinase (MAPK) subtypes including extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), p38 kinase, and c-Jun N-terminal kinase, depending on the types of extracellular stimuli and cells (20-24). In
addition to regulation of COX-2 expression and/or activity, ERK and p38
MAPK are involved in the regulation of chondrocyte dedifferentiation
caused by serial subculture (25) or nitric-oxide production (26, 27),
suggesting these MAPKs regulate both dedifferentiation and inflammatory
responses such as COX expression and PGE2 production.
The current study investigated the role of EGF in the maintenance of
chondrocyte phenotype, COX-2 expression, and PGE2
production. We also examined the functional relationship between
EGF-induced dedifferentiation and COX-2 expression and characterized
signaling pathways involved in EGF action. We report that EGF
stimulates dedifferentiation, COX-2 expression, and PGE2
production in articular chondrocytes via ERK1/2 and p38 kinase
signaling and that EGF-induced dedifferentiation further
potentiates inflammatory responses in articular chondrocytes.
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EXPERIMENTAL PROCEDURES |
Isolation and Monolayer Culture of Rabbit Articular
Chondrocytes--
Articular chondrocytes were isolated from cartilage
slices of 2-week-old New Zealand White rabbits by enzymatic digestion as described previously (25). Cartilage slices were dissociated enzymatically in 0.2% collagenase type II (381 units/mg, Sigma) in Dulbecco's modified Eagle's medium (Invitrogen). Individual cells were suspended in Dulbecco's modified Eagle's medium
supplemented with 10% (v/v) bovine calf serum, 50 µg/ml
streptomycin, and 50 units/ml penicillin. Cells were plated on culture
dishes at a density of 5 × 104 cells/cm2.
The medium was replaced every 2 days, and cells were confluent after
approximately 5 days. The 2.5-day cell cultures were treated with EGF
(Invitrogen). The following pharmacological agents were added 1 h
prior to EGF: PD98059 (Calbiochem) to inhibit MEK1/2 (28); SB203580
(Calbiochem) to inhibit p38 kinase (29); and tyrphostin AG1478 (Biomol,
Plymouth Meeting, PA) to antagonize EGF receptors (30). In some
experiments, passage (P) 0 cells were subcultured to P3 by plating
cells at a density of 5 × 104 cells/cm2.
Differentiation status of articular chondrocytes was determined by
examining the accumulation of sulfated glycosaminoglycan with Alcian
blue staining or expression of type II collagen by immunoblot analysis
as described previously (25).
Three-dimentional Cultures of Chondrocytes--
Dedifferentiated
chondrocytes at P4 were three-dimensionally cultured in alginate gel
beads or as pellets as described previously (25). Cells were suspended
in 1.25% sodium alginate prepared in 20 mM HEPES (pH 7.4)
containing 0.15 M NaCl at a cell density of 2 × 106 cells/ml. The cell suspension was slowly dropped into a
gelatin solution (5 mM HEPES (pH 7.4), 102 mM
CaCl2). Cells in alginate gel beads were cultured in
complete Dulbecco's modified Eagle's medium and refed every other day
and were recovered by solubilizing alginate with 2 volumes of 50 mM EDTA and 10 mM HEPES (pH 7.4). For pellet
culture, dedifferentiated cells were resuspended in complete
Dulbecco's modified Eagle's medium at a density of 2 × 105 cells/ml and 1 ml of aliquots was pelleted by
centrifugation. Cells in alginate gel beads and pellets were incubated
for up to 8 days.
Cartilage Explant Culture and Immunohistochemistry--
Rabbit
joint cartilage explants (~125 mm3) were fixed in 4%
paraformaldehyde for 24 h at 4 °C, dehydrated with graded
ethanol, embedded in paraffin, and sectioned into 4-µm slices as
described previously (31). The sections were stained by standard
procedures using Alcian blue or antibody against type II collagen or
COX-2 and visualized by developing with a kit purchased from DAKO
(Carpinteria, CA).
Immunoblot Analysis--
Whole cell lysates were prepared by
extracting proteins using a buffer containing 50 mM
Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, and 0.1%
sodium dodecylsulfate supplemented with protease inhibitors (10 µg/ml
leupeptin, 10 µg/ml pepstatin A, 10 µg/ml aprotinin, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride) and
phosphatase inhibitors (1 mM NaF and 1 mM
Na3VO4). The proteins were size-fractionated by
SDS-polyacrylamide gel electrophoresis and transferred to a
nitrocellulose membrane. The nitrocellulose sheet was then blocked with
3% nonfat dry milk in Tris-buffered saline. COX-2 was detected using
antibody purchased from Cayman Chemical (Ann Arbor, MI), and type II
collagen was detected using antibodies purchased from Chemicon
International Inc. (Temecula, CA). The bands were visualized using
peroxidase-conjugated secondary antibodies and chemiluminescence.
MAPK Assay--
ERK1/2 activation was examined using immunoblot
analysis as described previously (25) using antibodies specific to
activated threonine 202- and tyrosine 204-phosphorylated ERK1/2 (Cell
Signaling Technology, Beverly, MA). p38 kinase activity was determined
by immune complex kinase assays as described previously (26, 27). Cells
were lysed in a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,
1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
-glycerophosphate, and inhibitors of proteases and
phosphatases as described above. Total cell lysates were precipitated with polyclonal anti-p38 kinase antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and the immune complexes were collected using protein
A-Sepharose beads. The beads were resuspended in 20 µl of kinase
reaction buffer containing 25 mM Tris-HCl (pH 7.5), 5 mM dithiothreitol, 0.1 mM sodium orthovanadate,
10 mM MgCl2, 5 µCi of
[
-32P]ATP, and 1 µg of activating transcription
factor-2 protein as a substrate (New England Biolabs, Beverly, MA). The
kinase reaction was performed for 30 min at 30 °C, and
phosphorylated activating transcription factor-2 was detected by
autoradiography following gel electrophoresis.
PGE2 Assay--
PGE2 production was
determined by measuring the levels of cellular and secreted
PGE2 using an assay kit (Amersham Biosciences). P0 cells or
dedifferentiated P4 cells were seeded in standard 96-well microtiter
plates at 2 × 104 cells/well. After the indicated
treatments, total PGE2 was quantified according to the
manufacturer's protocol. PGE2 levels were calculated against a standard curve of PGE2 and normalized against the
amount of genomic DNA.
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RESULTS |
EGF Causes Dedifferentiation of Articular Chondrocytes--
To
examine the effects of EGF on articular cartilage chondrocyte
differentiation, cartilage explant cultures were treated with 10 ng/ml
EGF for 48 h, and expression of cartilage-specific matrix
molecules were determined. EGF caused a dramatic loss of type II
collagen and sulfated proteoglycan as determined by immunohistochemical staining and Alcian blue staining, respectively (Fig.
1A). EGF also inhibited type
II collagen expression in primary culture articular chondrocytes in a
dose- and time-dependent manner, and this inhibition was
blocked when cells were pretreated with the EGF receptor antagonist
AG1478 (Fig. 1B). Similarly, EGF treatment of primary
culture cells blocked the accumulation of sulfated proteoglycan in a
dose-dependent manner (Fig. 1C), and the
inhibition was also prevented by AG1478 (Fig. 1D).
These results indicate that EGF induces dedifferentiation of articular
chondrocytes in both cartilage explants and primary culture cells.

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Fig. 1.
EGF causes dedifferentiation of articular
chondrocytes. A, cartilage explants were
untreated or treated with 10 ng/ml EGF for 72 h. Type II collagen
and proteoglycan were detected by immunohistochemical staining
and Alcian blue staining, respectively. B, primary culture
chondrocytes were treated with EGF (5 ng/ml) for various time periods
(upper panel) or with the indicated concentrations of EGF
for 48 h (middle panel). Alternatively, cells were
untreated or treated with 250 nM AG1478 (AG)
(EGF receptor antagonist) and then exposed to EGF (5 ng/ml) for 48 h (lower panel). Expression of type II collagen was detected
using immunoblot analysis. Con, control. C and
D, primary culture chondrocytes were treated with various
concentrations of EGF for 48 h (C), or the cells were
treated with or without AG1478 and then EGF (5 ng/ml) for 48 h
(D). Accumulation of sulfated glycosaminoglycan was
quantified by Alcian blue staining. The data represent results of a
typical experiment (A and B) or mean values ± S.D. (C and D) from at least four independent
experiments.
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EGF-induced COX-2 Expression and PGE2
Production--
The effect of EGF on COX-2 expression and
PGE2 production was investigated using both cartilage
explant cultures and primary culture chondrocytes. In cartilage
explants, EGF stimulated COX-2 expression as determined by
immunohistochemical staining (Fig. 2A). In primary culture
chondrocytes, EGF increased protein levels of COX-2 in a time- and
dose-dependent manner as determined by immunoblot analysis
(Fig. 2B). COX-2 was detected 1 h after EGF treatment,
and levels peaked at 12 h. The EGF effects on primary culture
chondrocytes were completely blocked by AG1478 pretreatment (Fig.
2B). Consistent with the induction of COX-2 expression, EGF
stimulated PGE2 production (Fig. 2C) that was
detectable 1 h after EGF addition (Fig. 2D) and was
blocked by AG1478 (Fig. 2E). These data indicate that EGF
not only causes dedifferentiation of articular chondrocytes but also
stimulates COX-2 expression and PGE2 production.

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Fig. 2.
EGF increases COX-2 expression and
PGE2 production in articular chondrocytes.
A, cartilage explants were untreated or treated with
5 ng/ml EGF for 72 h, and COX-2 expression was determined by
immunohistochemical staining. B, primary culture
chondrocytes were treated with 5 ng/ml EGF for the indicated periods
(upper panel) or with the indicated concentrations of EGF
for 48 h (middle panel). Chondrocytes were treated with
or without 250 nM AG1478 (AG) and then EGF (5 ng/ml) for 48 h (lower panel). COX-2 expression was
determined by immunoblot analysis. Con, control.
C and D, chondrocytes were treated with the
indicated concentrations of EGF for 24 h (C) or for the
indicated time periods with 5 ng/ml EGF (D). Alternatively,
cells were treated with or without 250 nM AG1478 and then
EGF (5 ng/ml) for 24 h (E). Levels of cellular and
secreted PGE2 were determined by kit assay. The data in
A and B represent results of a typical experiment
(n = 4), and the data in C-E represent mean
values ± S.D. (n = 4).
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MAPK Regulates EGF-induced COX-2 Expression, PGE2
Production, and Dedifferentiation--
EGF (5 ng/ml) activated both
ERK1/2 and p38 kinase in articular chondrocytes (Fig.
3A, upper panel),
and activation of both kinases was inhibited by AG1478 (Fig.
3A, lower panel). To determine whether ERK1/2
activation is associated with dedifferentiation and/or COX-2 expression
and PGE2 production, chondrocytes were treated with EGF in
the absence or presence of PD98059, which inhibits ERK1/2 activation
(Fig. 3B, upper panel). PD98059 blocked EGF-stimulated dedifferentiation as indicated by the resumption of type
II collagen expression (Fig. 3B, upper panel) and
sulfated proteoglycan accumulation (Fig. 3C). PD98059
partially blocked EGF-stimulated COX-2 expression (Fig. 3B,
upper panel) but completely blocked PGE2
production (Fig. 3D). Inhibition of p38 kinase with SB203580
(Fig. 3B, lower panel) completely blocked both
EGF-stimulated COX-2 expression (Fig. 3B, lower
panel) and PGE2 production (Fig. 3D). In
contrast to the effects of ERK inhibition, the inhibition of p38 kinase
with SB203580 did not significantly alter the EGF-induced decrease in
type II collagen expression (Fig. 3B, lower
panel) and synthesis of proteoglycans (Fig. 3C). Taken
together, these results indicate that EGF-induced dedifferentiation is
regulated by ERK1/2 activity but not by p38 kinase activity, whereas
both ERK1/2 and p38 kinase regulate COX-2 expression and
PGE2 production.

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Fig. 3.
ERK1/2 and p38 kinase regulate
dedifferentiation and COX-2 expression/PGE2
production. A, chondrocytes were treated with 5 ng/ml
EGF for the indicated periods (upper panel), or cells were
treated with or without 250 nM AG1478 (AG) for
30 min prior to EGF treatment (lower panel). ERK1/2
phosphorylation was detected using immunoblot analysis with
anti-phospho-ERK1/2 antibodies. p38 kinase activity was determined by
immune complex kinase assay using activating transcription factor-2 as
a substrate. Expression of ERK1/2 and p38 kinase was determined by
immunoblot analysis. B and C, articular
chondrocytes were treated with 5 ng/ml EGF in the absence or presence
of the indicated concentrations of PD98059 (PD) or SB203580
(SB). ERK phosphorylation, type II collagen
(Coll-II) and COX-2 were detected by immunoblot analysis.
p38 kinase activity was determined by immune complex kinase assay using
activating transcription factor-2 as a substrate (B).
Accumulation of sulfated glycosaminoglycan was quantified by Alcian
blue staining (C). ERK phosphorylation and p38 kinase
activity were measured after cells were treated with EGF for 30 min,
whereas the expression of type II collagen, COX-2, and sulfated
glycosaminoglycan were determined after cells were treated with EGF for
48 h. Con, control. D, chondrocytes were
untreated or treated with 20 µM PD98059 or SB203580 and
exposed to 5 ng/ml EGF for 24 h. PGE2 levels were
determined using an assay kit. The data are from a typical experiment
(A and B) or are mean values ± S.D.
(C and D) from more than four independent
experiments.
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Differentiation Status-dependent COX-2 Expression and
PGE2 Production--
Because EGF in articular chondrocytes
causes both dedifferentiation and COX-2 expression/PGE2
production, we next examined the functional relationship between
EGF-induced dedifferentiation and inflammatory responses.
Differentiation status-dependent COX-2 expression and
PGE2 production were examined first. Serial subculturing of
chondrocytes caused dedifferentiation as demonstrated by cessation of
type II collagen expression (Fig.
4A). This chondrocyte
phenotype loss was accompanied by significantly increased levels of
COX-2 protein (Fig. 4A) and PGE2 production
(Fig. 4B). When these dedifferentiated P3 cells were
redifferentiated into chondrocytes by three-dimensional culture in
alginate gel (Fig. 4C) or by pellet culture (Fig.
4D), COX-2 protein levels dropped to those observed in
differentiated P0 cells. Therefore, COX-2 expression and
PGE2 production were inversely related to the
differentiation status of articular chondrocytes.

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Fig. 4.
Differentiation status dependent
COX-2 expression and PGE2 production.
A, P0 chondrocytes were serially subcultured to P3,
and levels of type II collagen and COX-2 were determined by immunoblot
analysis. B, PGE2 production was compared
between P0 cells and P3 cells by using an assay kit. C and
D, dedifferentiated P3 cells were cultured
three-dimensionally in alginate beads (C) or in pellets
(D) for up to 8 days. Type II collagen and COX-2 expressions
were determined by immunoblot analysis. The data represent typical
results or mean values ± S.D. from more than four independent
experiments. M, monolayer.
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COX-2 Expression and PGE2 Production Do Not Mediate
EGF-induced Dedifferentiation--
Because PGE2 production
is known to modulate chondrocyte differentiation (32-34), we next
examined whether COX-2 expression and the resulting PGE2
production in EGF-treated cells are involved in dedifferentiation of
chondrocytes. The COX-2 inhibitors indomethacin or NS398 (35)
significantly blocked EGF-induced PGE2 production as
expected (Fig. 5A). However,
EGF-induced inhibition of sulfated proteoglycan accumulation (Fig.
5B) and type II collagen expression (Fig. 5C)
were not affected by indomethacin or NS398. In addition, treatment of
P0 cells with exogenous PGE2 did not significantly affect
proteoglycan synthesis (Fig. 5B) or type II collagen
expression (Fig. 5C). These data indicate that COX-2
expression and PGE2 production are not involved in
EGF-induced dedifferentiation of chondrocytes.

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Fig. 5.
EGF-induced COX-2 expression and/or
PGE2 production does not affect dedifferentiation.
A and B, chondrocytes was treated with
indomethacin (Indo, 25 µg/ml) or NS398 (2.5 µM) and then with EGF (5 ng/ml) for 24 h.
Alternatively, 200 ng/ml PGE2 was added to chondrocytes for
48 h. PGE2 was measured using an assay kit
(A). Accumulation of sulfated proteoglycan was quantified
using Alcian blue staining (B). C, chondrocytes
were treated with the indicated concentrations of indomethacin
(upper panel) or NS398 (middle panel) for 1 h prior to EGF (5 ng/ml) addition. Alternatively, chondrocytes were
treated with the indicated concentrations of exogenous PGE2
for 48 h. Type II collagen expression was determined by immunoblot
analysis. The data in A and B represent mean
values ± S.D., and the data in C represent a typical
experiment (n = 4).
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Dedifferentiation of Chondrocytes Potentiates COX-2 Expression and
PGE2 Production--
We examined the effect of EGF on
COX-2 expression and PGE2 production in dedifferentiated
cells. We found that EGF further increased COX-2 expression (Fig.
6A) and PGE2
production in dedifferentiated P3 cells (Fig. 6B). Although
the sensitivity of dedifferentiated chondrocytes to EGF was much less
than that of P0 cells, the levels of COX-2 and PGE2 were
much higher in dedifferentiated chondrocytes. In an attempt to reveal
the mechanisms underlying increased expression of COX-2 and
PGE2 production in dedifferentiated cells, the
differentiation status-dependent activity of MAPK subtypes
was examined. Serial subculture of chondrocytes to P3 caused sustained
activation of both ERK1/2 and p38 kinase (Fig.
7A). The inhibition of ERK
activity with PD98059 but not p38 kinase activity with SB203580
partially maintained type II collagen expression in P3 cells (Fig.
7B). Blocking ERK activity did not significantly affect
dedifferentiation-induced increase of COX-2 protein level (Fig.
7B), but it did inhibit COX-2 activity as demonstrated by
the inhibition of PGE2 production (Fig. 7C). The
inhibition of p38 kinase with SB203580 blocked both dedifferentiation
and EGF-induced potentiation of COX-2 expression (Fig. 7B)
and PGE2 production (Fig. 7C). The above results
suggest that dedifferentiation-induced activation of ERK1/2 and p38
kinase causes increased COX-2 expression and PGE2
production in articular chondrocytes.

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Fig. 6.
EGF potentiates dedifferentiation-induced
COX-2 expression and PGE2 production. A and
B, differentiated P0 chondrocytes and dedifferentiated P3
cells were treated with 5 ng/ml EGF for 48 h. Levels of type II
collagen and COX-2 were determined by immunoblot analysis
(A). PGE2 levels were determined using an assay
kit (B). The data represent results of a typical experiment
(A) or mean values ± S.D. (B) from at least
four independent experiments.
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Fig. 7.
ERK1/2 and p38 kinase mediates
dedifferentiation-induced increase of COX-2 expression and
PGE2 production. A, chondrocytes were
subcultured to P3, and ERK1/2 and p38 kinase activity were determined
by immunoblot analysis and immune complex kinase assay, respectively.
B and C, differentiated P0 chondrocytes or
dedifferentiated P3 cells were either untreated (Con) or
treated with 20 µM PD98059 (PD) or 20 µM SB203580 (SB) for 1 h and then exposed
to vehicle alone or 5 ng/ml EGF for 24 h. Type II collagen and
COX-2 were detected using immunoblot analysis (B).
PGE2 production was measured using an assay kit and
normalized by determining the amount of total genomic DNA. The data
represent results of a typical experiment or mean value ± S.D.
from at least four independent experiments.
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DISCUSSION |
Chondrocytes in normal articular cartilage are a unique cell type
in that their differentiated phenotype is reversible. Chondrocyte phenotype is regulated by a balance of anabolic and catabolic molecular
reactions that are involved in maintaining homeostasis of cartilage
tissue (8). Differentiated chondrocytes lose their phenotype and
transform into fibroblast-like cells upon exposure to soluble factors
such as IL-1
(36), retinoic acid (31, 37), and nitric oxide (26, 38)
or during serial subculture in vitro (25, 39). Such a
destruction of homeostasis is believed to be involved in the
pathophysiology of arthritis (8, 40). Because large amounts of EGF are
found in the synovial fluid of arthritic cartilage (1, 2), it is likely
that EGF contributes to the disease, although the role of EGF is poorly
understood. In this study, we demonstrated that EGF caused both
dedifferentiation and inflammatory responses such as COX-2 expression
and PGE2 production. We also demonstrated that
dedifferentiation of chondrocytes by serial subculture caused
significant increases in COX-2 expression and PGE2
production, even though COX-2 expression and PGE2
production did not affect differentiation status. Therefore, our
results indicate that EGF-induced dedifferentiation of articular
chondrocytes amplifies EGF-induced inflammatory responses and suggest
that dedifferentiation of chondrocytes may worsen arthritic cartilage inflammation.
A significant finding of this study is that EGF caused both
dedifferentiation and COX-2 expression/PGE2 production in
articular chondrocytes and that dedifferentiation itself also resulted
in these COX-2 and PGE2 responses. Previous reports (41,
42) indicate that dedifferentiation of chondrocytes caused reduction in
responsiveness to the inflammatory cytokines such as IL-1
with
respect to COX-2 expression and PGE2 production. Our
current results also indicate that dedifferentiated cells are much less sensitive to EGF-induced increases in PGE2 production. For
instance, PGE2 production increased >5-fold in
differentiated P0 cells following EGF treatment, whereas only a
~2-fold increase was observed in dedifferentiated P3 cells (Fig. 6).
The amount of PGE2 produced per well by dedifferentiated
cells either in the absence or presence of EGF was much less than that
observed in differentiated P0 cells. However, when the amount of
PGE2 produced was normalized against genomic DNA to
quantify PGE2 production per cell, significantly higher
amounts of PGE2 were produced by dedifferentiated cells (Fig. 6). Similar results were observed when chondrocytes were treated
with IL-1
(data not shown). Therefore, our observations clearly
indicate that dedifferentiated chondrocytes produce more PGE2, although sensitivity to EGF or IL-1
is much lower.
In addition to PGE2 production, the levels of COX-2 protein
were much higher in serially subcultured dedifferentiated cells compared with differentiated P0 cells (Fig. 4). We also observed that
dedifferentiation of chondrocytes by exposure to phorbol ester, nitric
oxide, or retinoic acid caused significantly elevated expression of
COX-2 (data not shown). The results of this study are different to
those reported by Thomas et al. (42) in which IL-1
-treated differentiated and dedifferentiated immortalized chondrocytes showed similar COX-2 mRNA levels. This discrepancy may
be the result of different cell origins and culture systems. Those
studies compared responses of an immortalized human articular chondrocyte cell line with responses of cells cultured as monolayers and three-dimensionally. This study used primary rabbit articular chondrocytes cultured in monolayers. Indeed, although EGF is known to
induce COX-2 expression in various cell types including cell lines
originating from chondrocytes (9), no data are available regarding the
responses of primary culture articular chondrocytes to EGF. Thus, our
current observation is the first to characterize the effect of EGF on
primary chondrocytes.
In this study, we also demonstrated that EGF-induced COX-2 expression
and PGE2 production is regulated by ERK1/2 and p38 kinase signaling. The regulation of COX-2 expression has been shown to occur
at both transcriptional and post-transcriptional levels, and structural
information on the COX-2 gene promoter revealed binding
sites for several transcription factors found in many cell types such
as NF
B and CAAT enhancer-binding protein, which are involved
in transcription regulation of COX-2 (18, 19, 22, 23). The stability of
COX-2 mRNA regulated by sequences within the 3'-untranslated region
is also an important regulatory mechanism of COX-2 expression (20, 21).
In addition, several studies linked COX-2 expression with MAPK subtypes
including ERK1/2, p38 kinase, and c-Jun N-terminal kinase (20-24).
Therefore, our observation that EGF-induced expression and activity of
COX-2 are regulated by ERK1/2 and p38 kinase signaling is consistent with the observations of others (20-24). The increased COX-2
expression and PGE2 production in dedifferentiated
chondrocytes are mediated by the
dedifferentiation-dependent activation of ERK1/2 and p38 kinase (Fig. 7). It has been shown that dedifferentiation of
chondrocytes by exposure to IL-1
or retinoic acid significantly
increased the number of cell surface EGF receptors without changing
receptor affinity (43). This finding suggests that dedifferentiating chondrocytes by serial subculture may increase EGF receptor expression and serum factors such as EGF may stimulate COX-2 expression and PGE2 production.
The effect of PGE2 on chondrocytes depends on the culture
system, microenvironment, and physiological conditions (14, 33). PGE2 exerts anabolic effects such as synthesis of
proteoglycan and type II collagen and catabolic effects such as
enhancing matrix degradation (14, 15). For example, PGE2
has been shown to promote chondrocyte differentiation in addition to
its role in inflammation by increasing type II collagen expression
(32-34). However, the expression and activation of COX-2 and resultant PGE2 production are also believed to contribute to
cartilage destruction by altering matrix degradation via matrix
metalloproteinases (15). Indeed, the administration of a COX-2
inhibitor can repress joint inflammation and cartilage destruction in
animal models of arthritis (44, 45). In our culture system, we observed
that neither the inhibition of COX-2 expression and PGE2
production nor the addition of exogenous PGE2 affected
EGF-induced modulation of chondrocyte phenotype, whereas
dedifferentiation potentiated inflammatory responses, i.e.
COX-2 expression and PGE2 production. Therefore, it is
likely that EGF-induced dedifferentiation further elevates COX-2
expression and PGE2 production and that EGF found in
arthritic cartilage synovial fluid contributes to inflammation and
cartilage destruction during arthritic disease.