Protein Kinase C Regulates Chondrogenesis of Mesenchymes via
Mitogen-activated Protein Kinase Signaling*
Sung-Hee
Chang
,
Chun-Do
Oh
,
Myung-Soon
Yang
,
Shin-Sung
Kang
,
Young-Sup
Lee§,
Jong-Kyung
Sonn¶, and
Jang-Soo
Chun
From the Department of
Biology and
§ Biochemistry, College of Natural Sciences and
¶ Teacher's College, Kyungpook National University,
Taegu 702-701, Korea
 |
ABSTRACT |
A possible regulatory mechanism of protein kinase
C (PKC) in the chondrogenesis of chick limb bud mesenchymes has been
investigated. Inhibition or down-regulation of PKC resulted in the
activation of a mitogen-activated protein kinase subtype Erk-1 and the
inhibition of chondrogenesis. On the other hand, inhibition of Erk-1
with PD98059 enhanced chondrogenesis and relieved PKC-induced blockage of chondrogenesis. Erk-1 inhibition, however, did not affect expression and subcellular distribution of PKC isoforms expressed in mesenchymes nor cell proliferation. The results suggest that PKC regulates chondrogenesis by modulating Erk-1 activity. Inhibition or depletion of
PKC inhibited proliferation of chondrogenic competent cells, and Erk-1
inhibition did not affect PKC modulation of cell proliferation. However, PKC-induced modulation of expression of cell adhesion molecules involved in precartilage condensation was reversed by the
inhibition of Erk-1. Expression of N-cadherin was detected at the early
period of chondrogenesis. Inhibition or depletion of PKC induced
sustained expression of N-cadherin, and Erk-1 inhibition blocked the
effects of PKC modulation. The expression of integrin
5
1 and
fibronectin was found to be increased transiently during chondrogenesis. Depletion or inhibition of PKC caused a continuous increase of the expression of these molecules throughout the culture period, and Erk-1 inhibition abolished the modulating effects of PKC.
Because reduction of the examined cell adhesion molecule expression is
a prerequisite for the progression of chondrogenesis after cell
condensation, our results indicate that PKC regulates chondrogenesis by
modulating expression of these molecules via Erk-1 signaling.
 |
INTRODUCTION |
Prechondrogenic mesenchymes differentiate to chondrocytes when
they become closely packed in the limb buds of chick embryo or during
in vitro micromass culture of mesenchymes (1-3).
Chondrogenesis in vitro requires transient proliferation of
cells to increase the number of chondrogenic competent cells, and the
increased number of cells undergoes extensive cell-cell interaction
such as precartilage condensation, which is one of the earliest
morphogenetic events (1-3). Precartilage condensation is regulated by
several cell adhesion molecules such as N-cadherin (4-6) and
extracellular matrix (ECM)1
molecules including fibronectin, type I collagen, laminin, and tenascin
(7-10). Thereafter, a large amount of cartilage-specific ECM
components such as sulfated proteoglycans and type II collagen are
synthesized until individual chondrocytes are surrounded by the ECM (1,
2). Several groups of extracellular molecules have been found to
regulate chondrogenesis during the stages of cell proliferation and
precartilage condensation. For example, a fibroblast growth factor
appears to enhance chondrogenesis by increasing the number of
chondrogenic competent cells (11), whereas transforming growth factor
family members enhance chondrogenic differentiation by promoting
precartilage condensation (12, 13).
Although much progress has been made in finding a mechanism governing
chondrogenesis at levels of cell proliferation and precartilage condensation, the signal transduction pathways involved in
chondrogenesis are poorly understood. Increasing evidence indicates
that protein kinase C (PKC) might play a key role in chondrogenic
differentiation (14-17), as shown by the fact that PKC activities in
both cytosol and particulate membrane fractions of mesenchymes
increased during chondrogenesis (14). The activities of PKC seem to be
required for chondrogenic differentiation of mesenchymes because
inhibition or down-regulation of PKC blocks chondrogenesis (15) and
also causes dedifferentiation of rabbit costal and articular
chondrocytes (16, 17). PKC is a multigene family composed of 11 known
isoforms (18-20). Chick limb bud mesenchymes express
,
,
,
and
/
isoforms, and depletion of PKC
and
is sufficient to
inhibit chondrogenesis (15), suggesting that PKC
and/or
induces
chondrogenic differentiation of mesenchymes.
Although the requirement of PKC in chondrogenesis has been clearly
established, its molecular mechanism in the regulation of
chondrogenesis is not evident. To elucidate a possible mechanism involved in PKC action, our effort in this study has been focused on
whether the regulatory role of PKC in chondrogenesis is mediated by
mitogen-activated protein (MAP) kinase and also on whether PKC and/or
MAP kinase regulates chondrogenesis at stages of proliferation of
chondrogenic competent cells and precartilage condensation.
 |
EXPERIMENTAL PROCEDURES |
Micromass Culture of Mesenchymes--
Mesenchymes were derived
from the distal tips of Hamburger-Hamilton stage 23/24 embryo limb buds
(3) of fertilized white Leghorn chicken eggs as described previously
(15). The cells were suspended at a density of 2.0 × 107 cells/ml in Ham's F-12 medium containing 10% fetal
calf serum, unless otherwise indicated. Chondrogenesis was induced by
adding the cells in 15-µl drops to 35-mm or 60-mm culture dishes. The cells were incubated for 60 min at 37 °C to allow attachment and then cultured in Ham's F-12 medium containing 10% fetal calf serum, 50 µg/ml streptomycin, and 50 units/ml penicillin either in the absence or presence of various reagents as described in each
experiment. Chondrogenic differentiation of mesenchymes was determined
immunocytochemically by examining the expression of type II collagen as
described below. Alternatively, cells cultured for various time periods
were stained with Alcian blue to localize the sulfated cartilage
matrix. Alcian blue bound to sulfated glucosaminoglycans was extracted
with 4 M guanidine-HCl and quantitated by measuring the
absorbance at 600 nm (15).
Immunofluorescence Microscopy and
Immunocytochemistry--
Mesenchymes cultured for the indicated time
periods were fixed with 3% paraformaldehyde in phosphate-buffered
saline for 10 min at room temperature, and the fixed cells were washed
and incubated for 45 min with anti-collagen type II monoclonal antibody
(Developmental Studies Hybridoma Bank, University of Iowa, Iowa City,
IA) containing 5% fetal calf serum. The cells were then washed with
phosphate-buffered saline and incubated with rhodamine-conjugated goat
anti-mouse IgG antibody for another 45 min. The specimen samples were
covered with glycerol and examined with a Nikon Optiphot fluorescence microscope. Alternatively, the cells incubated with anti-collagen type
II monoclonal antibody were washed with phosphate-buffered saline and
decorated with biotinylated anti-mouse IgG for 30 min. The cells were
incubated with Vectastain ABC kit (Vector Laboratories, Inc.,
Burlingame, CA) for 30 min, and type II collagen was visualized by
developing with DAB substrate solution kit (Vector Laboratories, Inc.)
by the procedure recommended by the manufacturer.
Cell Fractionation--
Separation of cytosolic and particulate
membrane fractions was performed as described previously (21, 22).
Briefly, cells micromass cultured for the indicated time periods were
scraped in buffer A (20 mM Tris-HCl, pH 7.5, containing
0.25 M sucrose, 2 mM EGTA, 2 mM
EDTA, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride). The cells were sonicated for 6 s twice and centrifuged at 100,000 × g for 1 h. The supernatant was designated as the
cytosolic fraction. The pellet was extracted with buffer B (20 mM Tris-HCl, pH 7.5, containing 1% SDS, 150 mM
NaCl, 1 mM EGTA, 1 mM EDTA, and protease inhibitors as described above). After centrifugation at 15,000 × g for 15 min, the supernatant was saved as the particulate
membrane fraction.
Western Blot Analysis--
Total cell lysate was prepared from
cells micromass cultured for various time periods by extracting
proteins with a lysis buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, and
0.1% deoxycholate). Proteins were separated by 8% polyacrylamide gel
electrophoresis containing 0.1% SDS and transferred to nitrocellulose
membrane. The nitrocelluose sheet was blocked with 3% nonfat dry milk
in Tris-buffered saline. PKC isoforms were detected with
isoform-specific anti-PKC monoclonal antibodies for
,
,
,
,
/
, and µ (Transduction Laboratories, Lexington, KY) or with
polyclonal antibodies for
,
, and
(Santa Cruz Biotechnology,
Santa Cruz, CA) and
(Biomol, Plymouth Meeting, PA). Expression of
cell adhesion molecules was determined by using antibodies purchased
from following sources: rabbit anti-chick N-cadherin polyclonal
antibody from Sigma Chemical Co. (St. Louis, MO), rabbit anti-human
fibronectin polyclonal antibody from Upstate Biotechnology Inc. (Lake
Placid, NY), mouse anti-chick integrin
5 monoclonal antibody from
Developmental Studies Hybridoma Bank, and rabbit anti-human integrin
1 polyclonal antibody from Chemicon (Temecula, CA). The blots were
developed using a peroxidase-conjugated secondary antibody, and
proteins were visualized by the ECL system (21, 22).
MAP Kinase Assay--
Mesenchymes were micromass cultured in the
absence or presence of various reagents for the indicated time periods.
Proteins were extracted with a buffer (50 mM Tris-HCl, pH
7.4, containing 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS,
0.1% deoxycholate, 5 mM sodium fluoride, 1 mM
sodium orthovanadate, and 1 mM 4-nitrophenyl phosphate).
Expression of MAP kinase subtypes Erk-1 and -2 was determined by
Western blotting using antibodies obtained from Santa Cruz
Biotechnology. Activation of MAP kinase was examined by determining its
phosphorylation state using antibody specific to phosphorylated Erk-1
and -2 (New England Biolabs, Beverly, MA).
Cell Proliferation--
Mesenchymes were micromass cultured
(three spots/35-mm dish) in the absence or presence of various reagents
for the indicated time periods. Individual cells were suspended with
0.1% each of trypsin and collagenase, and the number of viable cells
was counted in triplicate using a hemocytometer.
 |
RESULTS |
PKC Regulates Chondrogenesis by Modulation of MAP
Kinase--
Micromass-cultured mesenchymes derived from chick limb
buds underwent spontaneous differentiation into chondrocytes,
demonstrated by the expression of type II collagen, a
cartilage-specific molecule (Fig.
1A). Immunofluorescence
microscopy revealed that the expressed type II collagen was localized
in cartilage nodules (Fig. 1B). The expression of type II
collagen was reduced when the cells were cultured in the presence of
Go6976 to inhibit PKC (23) or phorbol 12-myristate 13-acetate (PMA) to
down-regulate PKC (Fig. 1A). Similar results were obtained
when the micromass-cultured cells were stained with Alcian blue to
localize the sulfated cartilage matrix (data not shown). Quantitation
of chondrogenesis by measuring an absorbance of Alcian blue extract
indicated that inhibition or down-regulation of PKC reduced
chondrogenesis to 29 and 32% of the control, respectively (Fig.
1C). The above results indicate that PKC activity is
required for chondrogenic differentiation of mesenchymes in in
vitro micromass culture.

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Fig. 1.
PKC is required for the chondrogenic
differentiation of mesenchymes in vitro. Panel
A, type II collagen expression. Mesenchymes were micromass
cultured for 1 (a) or 4 (b) days in the presence
of vehicle alone or for 4 days in the presence of 10 nM PMA
(c) or 0.5 µM Go6976 (d).
Expression of type II collagen was determined by immunocytochemistry.
Panel B, localization of type II collagen in cartilage
nodules. Mesenchymes were cultured for 4 days, and distribution of type
II collagen was examined by immunofluorescence microscopy
(a). Phase-contrast microscopy of the micromass culture is
shown in (b). Panel C, quantitation of
chondrogenesis. The cells cultured for 4 days in the presence of
vehicle as a control, 0.5 µM Go6976, or 10 nM
PMA, were stained with Alcian blue to localize sulfated proteoglycan.
Chondrogenesis was quantitated by measuring an absorbance of
extractable Alcian blue at 600 nm (n = 4).
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Because Erk-1 and -2 subtypes of MAP kinase are downstream targets of
PKC (24-26), it was felt of importance to examine whether regulation
of chondrogenesis by PKC was mediated by MAP kinase signaling. Erk-1
was expressed at constant level during chondrogenesis (Fig.
2A); however, its
phosphorylation was high at early time periods of micromass culture,
i.e. culture day 1, and reduced as chondrogenesis proceeded
(Fig. 2B). Erk-2 was not easily detectable with the antibody
employed, probably because of its low level of expression. When a large
amount of protein was used, the expression and phosphorylation patterns
of Erk-2 were similar to those of Erk-1 (data not shown). In subsequent
experiments, therefore, we examined only Erk-1. Phosphorylation of
Erk-1 was almost completely blocked when mesenchymes were cultured in
the presence of PD98059, a specific inhibitor of MAP kinase kinase
(27). In contrast, Erk-1 phosphorylation was enhanced significantly
when mesenchymes were cultured in the presence of PMA or Go6976 (Fig.
2B), reagents that inhibit chondrogenesis (Fig. 1). As shown
in Fig. 2A, treatment of mesenchymes with PD98059, Go6976,
or PMA during micromass culture of mesenchymes did not affect the
expression level of Erk-1.

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Fig. 2.
Inhibition or down-regulation of PKC enhances
Erk-1 phosphorylation. Mesenchymes were micromass cultured for the
indicated time periods in the presence of vehicle alone as a control,
10 µM PD98059, 10 nM PMA, or 0.5 µM Go6976. Expression (A) and phosphorylation
(B) of Erk-1 were determined by Western blotting. The data
represent a typical experiment conducted more than five times
(A and B) with similar results.
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Enhancement of Erk-1 phosphorylation by inhibition or down-regulation
of PKC suggested that phosphorylation and/or activation of Erk-1 could
inhibit chondrogenesis. To explore this possibility, the effect of
inhibition of Erk-1 with PD98059 on chondrogenic mesenchyme
differentiation was determined. As shown in Fig.
3A, treatment of the cells
with PD98059 enhanced type II collagen expression. Also, Alcian blue
staining indicated that PD98059 treatment enhanced chondrogenesis up to
2-fold (Fig. 3B) at concentrations in the range of 1-20
µM. Thus, it is concluded that inactivation of Erk-1
resulted in an enhanced chondrogenesis, whereas Erk-1 activation by
inhibition or depletion of PKC was inhibitory for chondrogenesis.

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Fig. 3.
Inhibition of Erk-1 enhances
chondrogenesis. Panel A, mesenchymes were cultured for
4 days in the presence of vehicle alone (a) or 10 µM PD98059 (b), and the expression of type II
collagen was examined by immunocytochemistry. Panel B,
mesenchymes were cultured for the indicated time periods in the
presence of vehicle alone as a control, 10 µM PD98059,
0.5 µM Go6976, or 10 nM PMA. Chondrogenesis
was quantitated by staining sulfated proteoglycan with Alcian blue and
reading the absorbance of bound Alcian blue extract at 600 nm.
Panel C, mesenchymes were micromass cultured for 4 days in
the presence of various concentrations of PD98059, and chondrogenesis
was quantitated. The data in A represent results of a
typical experiment; the data in B and C represent
the average of six independent experiments with standard
deviation.
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We next explored the possibility of whether the inhibition of
chondrogenesis by down-regulation or inhibition of PKC was caused by
the activation of Erk-1. It was found that the inhibition of Erk-1 with
PD98059 blocked PMA- or Go6976-induced reduction of type II collagen
expression (Fig. 4A) or Alcian
blue staining (Fig. 4B), suggesting that Erk-1 activation
was responsible for the inhibition of chondrogenesis. PD98059
treatment, however, did not recover chondrogenesis completely when the
cells were treated with high concentrations of PMA or Go6976,
i.e. 10 nM and 0.5 µM,
respectively (Fig. 4B). As expected, more Erk-1 was phosphorylated at high concentrations of PKC modulators, and
consequently PD98059 was less effective in blocking Erk-1
phosphorylation (Fig. 4C). The ability of PD98059 to recover
chondrogenesis was proportional to the degree of inhibition of Erk-1
phosphorylation. Taken together, our results suggest that regulation of
chondrogenesis by PKC is mediated through Erk-1.

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Fig. 4.
Inhibition of Erk-1 relieves blocking of
chondrogenesis induced by the inhibition or down-regulation of
PKC. Panel A, cells were cultured in the presence of 5 nM PMA (a), 5 nM PMA and 10 µM PD98059 (b), 0.25 µM Go6976
(c), 0.25 µM Go6976 and 10 µM
PD98059 (d) for 4 days. Chondrogenic differentiation was
determined by staining type II collagen. The data represent results of
a typical experiment conducted four times. Panel B,
mesenchymes were micromass cultured in the presence of 10 µM PD98059 and various concentrations of PMA or Go6976
for 4 days, and chondrogenesis was quantitated by reading an absorbance
of Alcian blue extract. The data represent the relative value of
absorbance of Alcian blue extract against control culture
(i.e. cells cultured in the absence of PMA, Go6976, and
PD98059) and average values of four independent experiments with
standard deviation. Panel C, mesenchymes were micromass
cultured in the presence of 10 µM PD98059 and various
concentrations of PMA or Go6976 for the indicated time periods.
Phosphorylation of Erk-1 was determined by Western blotting. The data
represent the results of a typical experiment conducted four times with
similar results.
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Stimulatory Effect of Erk-1 Inhibition on Chondrogenesis Is Not the
Result of Modulation of PKC--
In addition to the inhibition of
Erk-1, a possibility of PD98059 modulation of expression and activity
of PKC isoforms in chondrogenesis was examined. We have shown
previously (15) that mesenchymes derived from chick limb buds contained
multiple PKC isoforms such as
,
,
,
, and
/
. However,
anti-PKC
antibody (Transduction Laboratories Inc.) used in previous
study cross-reacted with PKC
(according to the manufacturer). When
PKC
-specific antibodies obtained from Santa Cruz Biotechnology or
Life Technologies, Inc. were used in this study, no immunoreactive band
was detected in mesenchymes cultured up to 5 days (data not shown),
indicating that PKC
was expressed at an insignificant level in
mesenchymes during chondrogenesis. In addition, PKC
and µ, which
were not examined in the previous study (15), were also expressed at insignificant levels, with the corresponding antibodies failing to
detect these proteins (data not shown). We therefore examined the
effect of Erk-1 inhibition on the expression and subcellular distribution of PKC isoforms
,
,
, and
/
. Although
treatment of mesenchymes with PD98059 enhanced chondrogenesis up to
2-fold (Fig. 3), the amounts and expression patterns of PKC isoforms were essentially the same among the cells cultured in the presence or
absence of PD98059 (Fig. 5A).
In addition, translocation of cytosolic PKC isoforms to the particulate
membrane fractions was not affected by the inhibition of Erk-1 (Fig.
5B). Thus, it is highly unlikely that the inhibition of
Erk-1 enhances chondrogenesis by modulating PKC. Taken together, these
results strongly suggest that Erk-1 is a downstream target of PKC and
that the action of PKC on chondrogenesis is exerted through the
modulation of Erk-1.

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Fig. 5.
Inhibition of Erk-1 does not affect
expression and subcellular distribution of PKC isoforms.
Panel A, mesenchymes were cultured up to 4 days in the
absence or presence of 10 µM PD98059, and the expression
of PKC isoforms was detected by Western blotting. Panel B,
cells cultured in the absence or presence of 10 µM
PD98059 for 4 days were fractionated into cytosolic (c) and
particulate membrane (m) fractions. The distribution of PKC
isoforms was detected by Western blotting. The data represent a typical
experiment conducted four times (A and B) with
comparable results.
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Effects of PKC and MAP Kinase on the Proliferation of Chondrogenic
Competent Cells--
Because chondrogenesis of mesenchymes is cell
density-dependent and requires transient proliferation of
chondrogenic competent cells (1, 2), a question arises as to whether
PKC and Erk-1 are involved in chondrogenesis at the stage of cell
proliferation. As shown in Figs. 6,
A and B, compared with the control culture, treatment of cells with 10 nM PMA or 0.5 µM
Go6976 for 4 days reduced cell proliferation by 43 and 51%,
respectively; however, neither affected cell viability throughout the
culture periods, as determined by trypan blue exclusion experiments:
more than 97% of the cells was viable in all treatments (data not
shown). Thus, the reduction in cell number by PKC modulation was caused by reduced cell proliferation. In contrast to the inhibitory effect of
PKC modulation on cell proliferation, the inhibition of Erk-1 with
PD98059, a condition that enhanced chondrogenesis (Fig. 3), did not
affect proliferation of the cells (Fig. 6A). This indicates that the stimulatory effect of Erk-1 inhibition on chondrogenesis is
not caused by enhanced cell proliferation. Furthermore, the inhibition
of PMA- or Go6976-induced cell proliferation was not relieved by
the inhibition of Erk-1 with PD98059 (Fig. 6B), although chondrogenesis was recovered by the inhibitor (Fig. 4).

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Fig. 6.
PKC but not Erk-1 regulates cell
proliferation. Panel A, the number of cells was counted
after micromass culture of mesenchymes for the indicated time periods
in the presence of vehicle alone as a control, 10 nM PMA,
0.5 µM Go6976, or 10 µM PD98059. The data
represent the percent increase in cell number against day 1 culture.
Panel B, cells were cultured for 4 days in the presence of
the indicated concentrations of PMA or Go6976 and in the presence or
absence of 10 µM PD98059, and the number of cells was
counted. The data represent the relative number of cells against
control culture, i.e. cells cultured in the absence of PMA,
Go6976, and PD98059. The data represent the average values of five
independent experiments with standard deviation (A and
B).
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To confirm further that the stimulatory effect of PD98059 was
independent of cell proliferation, we explored a question of whether
PD98059 enhanced chondrogenesis at a low density of cells. When
micromass culture was performed at reduced cell density of 2 × 107 cells/ml to 1, 0.5, or 0.25 × 107
cells/ml, chondrogenesis was reduced to 41, 13, and 8% of the control,
as shown in Fig. 7, A and
B. This confirms the fact that chondrogenesis in culture is
cell density-dependent. PD98059 treatment of the cells at a
density of 0.25 × 107 cells/ml did not induce
chondrogenesis; a slight but significant increase at 0.5 × 107 cells/ml, however, enhanced chondrogenesis dramatically
at a density of 1 × 107 cells/ml (1.8-fold increase
against control). This suggested that there was a certain threshold
density of cells to undergo differentiation even in the presence of
PD98059. In addition to the modulation of chondrogenesis, micromass
culture of mesenchymes at a reduced cell density (1 or 0.5 × 107 cells/ml) resulted in enhancement of Erk-1
phosphorylation and less increase of PKC
and
expression compared
with those in cells cultured at a density of 2 × 107
cells/ml (data not shown). It also appeared that the stimulatory effect
of PD98059 on chondrogenesis at low cell density (i.e. 1 × 107 cells/ml) was independent of cell
proliferation because PD98059 did not affect cell numbers at a density
of 1 × 107 cells/ml as well as 2 × 107 cells/ml (Fig. 7C). These results clearly
indicated that the inhibition of Erk-1 with PD98059 stimulated
chondrogenesis without mediation of cell proliferation. The results
also led us to conclude that the recovery of chondrogenesis by PD98059
in the cells treated with PMA or Go6976 was independent of
proliferation of chondrogenic competent cells.

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Fig. 7.
Inhibition of Erk-1 enhances chondrogenesis
at low cell density. Panel A, expression of type II
collagen was determined by immunocytochemistry in mesenchymes micromass
cultured at density of 2 × 107 cells/ml
(a) or 1 × 107 cells/ml in the absence
(b) or presence (c) of 10 µM
PD98059 for 4 days. Panel B, mesenchymes were micromass
cultured at the indicated density of cells in the absence ( ) or
presence (+) of 10 µM PD98059 for 4 days. Chondrogenesis
was quantitated by reading an absorbance of Alcian blue extract.
(panel C) The number of cells was counted in cells cultured
at the indicated density for 4 days in the absence or presence of 10 µM PD98059. The data in panel A represent the
results of a typical experiment; the data in panels B and
C represent the average of four independent experiments with
standard deviation.
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Effects of PKC and MAP Kinase on the Expression of Cell Adhesion
Molecules--
The role of PKC and Erk-1 in the expression of cell
adhesion molecules and ECM components was investigated in an attempt to understand their roles in precartilage condensation. Expression of
N-cadherin, which mediates cell-cell interaction (28), was high in
1-day culture and was reduced as chondrogenesis proceeded (Fig.
8). However, down-regulation of PKC with
PMA or inhibition with Go6976 caused sustained expression of N-cadherin
throughout the culture period. In contrast, the inhibition of Erk-1
accelerated the decrease of N-cadherin expression (Fig. 8). This
indicated that activities of PKC and Erk-1 had opposing effects on
N-cadherin expression.

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Fig. 8.
Regulation of cell adhesion molecule
expression by PKC and Erk-1 during chondrogenesis. Mesenchymes
were cultured in the presence of vehicle alone as a control, 10 nM PMA, 0.5 µM Go6976, and/or 10 µM PD98059 for the indicated time periods. The expression
of N-cadherin, integrins 5 and 1, and fibronectin was determined
by Western blotting. The data represent a typical experiment conducted
more than six times with similar results.
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Because the interaction of cells with ECM in addition to cell-cell
interaction had been known to regulate chondrogenesis at the stage of
precartilage condensation (1, 2), a possibility of PKC and Erk-1
regulation of the expressions of integrin and its ligand during
chondrogenesis was examined, particularly integrin
5
1 and its
ligand fibronectin. Expressions of integrins
5 and
1 increased
transiently during chondrogenesis; their expressions were detected in
the cells cultured for 1 day, increased as chondrogenesis proceeded,
and subsequently decreased at the later stage of chondrogenic differentiation, i.e. the 5th day of culture (Fig. 8).
Down-regulation or inhibition of PKC caused a continuous increase of
integrins
5 and
1 expression throughout the culture period. In
contrast, the inhibition of Erk-1 accelerated the decrease of integrins
5 and
1 expression in the 4th and 5th day cultures. Similar to
the above, the expression of fibronectin also increased transiently during chondrogenesis (Fig. 8), increased continuously throughout the
culture period when PKC was down-regulated or inhibited, and finally
Erk-1 inhibition promoted the decrease of fibronectin expression at the
later period of micromass culture (Fig. 8). Therefore, the activity of
PKC appeared to be required for the decrease of expression of integrins
5 and
1 and fibronectin at the later period of micromass culture,
whereas Erk-1 activation resulting from PKC inhibition or depletion
increased the expression of these molecules. Because the reduction of
integrin
5
1 and its ligand fibronectin expression after cell
condensation is necessary for chondrogenesis to progress (10, 29),
regulation of the expression of these molecules by PKC and Erk-1
appears to be well correlated with the regulation of
chondrogenesis.
Finally, we investigated the question of whether the modulation of
expression of cell adhesion molecules by PKC was mediated by MAP kinase
signaling. As shown in Fig. 8, the sustained expression of N-cadherin
and increased expression of integrin
5,
1, and fibronectin by the
inhibition or down-regulation of PKC were abolished when Erk-1
activation was blocked with PD98059. The ability of PD98059 to block
PKC-mediated modulation of these expressions was dependent on the
concentration of PMA or Go6976 (data not shown). Thus, the degree of
recovery of chondrogenesis by treating cells with PD98059 appeared to
be proportional to the degree of PD98059 to block PKC. Taken together,
these results indicate that the regulatory activity of PKC in the
expression of cell adhesion molecules and ECM component is mediated by
Erk-1 signaling.
 |
DISCUSSION |
Several lines of evidence support the hypothesis that chondrogenic
differentiation of mesenchymes is regulated by PKC (14-17). PKC was
initially thought to regulate chondrogenesis in a negative way because
staurosporine, a potent inhibitor of PKC, enhanced chondrogenesis (30).
However, it was found later that the stimulatory effect of
staurosporine was caused by effects other than its ability to inhibit
PKC (31). PKC has been implicated in a variety of cellular processes
including growth and differentiation of many types of cells (18).
Regulation of cellular processes by PKC has been suggested to be
mediated by direct regulation of gene expression and/or regulation of
activities of other signaling molecules such as MAP kinase (18-20). It
is well known that direct stimulation of PKC with phorbol ester leads
to an activation of MAP kinase (32, 33) which is a common intermediate
in intracellular signaling cascades (24-26).
Studies on PKC regulation of MAP kinase indicated that stimulation of
PKC activated MAP kinase signaling (32, 33). Unexpectedly, in the
present study we found that the inhibition or down-regulation of PKC
resulted in the activation of Erk-1 and that the activated Erk-1 was
responsible for the inhibitory role of PKC in chondrogenesis. This
conclusion was derived from the observations that phosphorylation of
Erk-1 was related to the inhibition of chondrogenesis (Fig. 2) and that
the inhibition of Erk-1 phosphorylation relieved the inhibition of
chondrogenesis induced by PKC (Fig. 4). The signaling pathway leading
to the activation of Erk-1 by the inhibition or down-regulation of PKC
is not currently known. Because stimulation of PKC is known to activate
Erk-1 and -2 by the stimulation of upstream signaling molecules such as
Raf (32, 33), it is not likely that activation of Erk-1 is induced by
the stimulation of Raf as a consequence of PKC inhibition or depletion.
Rather, it is quite possible that inhibition or depletion of PKC
inactivates MAP kinase phosphatase, which enhances MAP kinase
activation (24-26).
Subtypes of MAP kinase, Erk-1 and -2, are thought to play a key role in
the signaling process of many types of cellular differentiation, acting
as an inhibitor or stimulator for cellular differentiation, depending
on the types of differentiation. For instance, activation of both Erk-1
and -2 is required for the differentiation of fibroblasts to adipocytes
(34), and maturation of immature T cells also requires MAP kinase
activity because expression of catalytically inactive MAP kinase kinase
inhibits T cell maturation (35). In contrast to the above, it has been
suggested that inactivation of Erk-2 is required for C2C12 myoblasts to
initiate myogenesis (36). Inactivation of MAP kinase is also observed
during enterocyte differentiation (37). We demonstrated in this study
that inactivation of Erk-1 is required for the initiation of
chondrogenesis.
Extracellular molecules known to regulate chondrogenesis exert their
effects at the level of cell proliferation and/or precartilage condensation (11-13). Therefore, we investigated the role of PKC and
Erk-1 in the regulation of proliferation of chondrogenic competent cells and the expression of cell adhesion molecules known to be involved in precartilage condensation. Because inhibition or
down-regulation of PKC reduces cell proliferation (Fig. 6), PKC
activity appeared to be required for the proliferation of chondrogenic
competent cells. However, the inhibition of cell proliferation by PKC
was eliminated not by inhibiting Erk-1 (Fig. 6), whereas chondrogenesis was recovered significantly (Fig. 4). In addition, the inhibition of
Erk-1 in the absence of PKC modulation, a condition that enhanced chondrogenesis, did not affect proliferation of chondrogenic competent cells, and PD98059 enhanced chondrogenesis at low cell density without
affecting cell proliferation (Fig. 7). Therefore, we conclude that the
stimulation and recovery of chondrogenesis by Erk-1 inhibition were not
the result of enhancement of cell proliferation.
Precartilage condensation is a process that reduces intercellular
spaces and formation of extensive cell-cell contacts between prechondrogenic mesenchymes (1, 2, 29). The biochemical events leading
to cell condensation are not yet understood clearly. However, both
cell-cell and cell-ECM interactions have been considered to play a role
in precartilage condensation. Therefore, the possibility of whether PKC
and MAP kinase regulate chondrogenesis by modulating the expression of
cell adhesion molecules and ECM components such as N-cadherin, integrin
5
1, and fibronectin was investigated.
N-Cadherin has been known to be expressed in prechondrogenic
mesenchymes and during cell condensation (4, 5), and blockage of its
function inhibited precartilage condensation (6). However, it was not
expressed in chondrocytes that were completely surrounded by
cartilage-specific ECM molecules (4). Our results (Fig. 8) also
indicated that N-cadherin was expressed at the early period of
micromass culture, and its expression decreased as chondrogenesis proceeded. Inhibition or down-regulation of PKC resulted in sustained expression of N-cadherin, whereas inhibition of Erk-1 enhanced the
decrease of N-cadherin expression at the later period of micromass culture. In addition, the effect of PKC on N-cadherin expression was
blocked by the inhibition of Erk-1, suggesting that PKC action was
mediated by Erk-1. A question of whether the modulation of N-cadherin
expression by PKC and Erk-1 is a determinant of chondrogenesis or
whether the modulation of chondrogenesis results in the altered expression of N-cadherin remains to be clarified. However, our data
clearly indicate that PKC and Erk-1 signaling is closely coupled with
the regulation of N-cadherin expression during chondrogenesis.
In this study, we presented evidence that the expression of fibronectin
and its receptor integrin
5
1 is regulated by PKC through Erk-1
signaling pathway, especially at the later stage of micromass culture,
i.e. in cells cultured for 4-5 days (Fig. 8). Thus, the
increased expression of integrin
5
1 and fibronectin at the later
stage of micromass culture upon inhibition or depletion of PKC is
closely correlated with the inhibition of chondrogenesis. On the other
hand, the decreased expression of these molecules upon Erk-1 inhibition
is correlated with an enhancement of chondrogenesis. The above results
are consistent with the facts that an interaction of cells with
fibronectin via integrin
5
1 is necessary for cell condensation to
occur and that a reduction of fibronectin and its integrin
5
1
receptor after cell aggregation is necessary for the progression of
cartilage differentiation (10, 29, 38). Indeed, it has been known that
enhanced expression of fibronectin exerts negative effects on
chondrogenesis (39-41). We demonstrated in this study that PKC
activity that inactivates Erk-1 is required to reduce the expression of
integrin
5
1 and fibronectin and thereby to initiate the
progression of chondrogenic differentiation. Because the reduction of
fibronectin and integrin
5
1 occurs after the cell aggregation
(29), our results suggest also that PKC and Erk-1 are not directly
related to cell condensation. It is not yet known how the reduction of
integrin
5
1 and fibronectin expression causes progression of
chondrogenesis and how Erk-1 regulates expression of cell adhesion
molecules and ECM components during chondrogenesis. We postulate that
Erk-1 modulates synthesis and/or activation of transcription factors
that regulate the expression of cell adhesion molecules and ECM
components. Further work is needed to define downstream events that
lead to the modulation of cell adhesion molecule expression.
 |
ACKNOWLEDGEMENTS |
We are grateful to Drs. Woon Ki Paik for
critical reading of the manuscript and Mahn Joon Ha (Ajou University)
for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by grants from the Korea Ministry of
Education (BSRI-97-4402), Korea Science and Engineering Foundation (971-0503-014-2), and Hallym Academy of Science, Hallym
University.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.
To whom correspondence should be addressed: Dept. of Biology,
College of Natural Sciences, Kyungpook National University, Pookgu,
Taegu 702-701, Korea. Tel.: 82-53-950-5353; Fax: 82-53-953-3066; E-mail: jschun{at}bh.kyungpook.ac.kr.
1
The abbreviations used are: ECM, extracellular
matrix; PKC, protein kinase C; MAP kinase, mitogen-activated protein
kinase; PMA, phorbol 12-myristate 13-acetate.
 |
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