From the Musculoskeletal Research Laboratory,
Department of Orthopaedics and Rehabilitation and the
Department
of Cellular and Molecular Physiology, Pennsylvania State University
College of Medicine, Hershey, Pennsylvania 17033
Received for publication, June 8, 2000, and in revised form, November 6, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Parathyroid hormone (PTH) and its related peptide
regulate endochondral ossification by inhibiting chondrocyte
differentiation toward hypertrophy. However, the intracellular pathway
for transducing PTH/PTH-related peptide signals in chondrocytes
remains unclear. Here, we show that this pathway is mediated by
mitogen-activated protein kinase (MAPK) p38. Incubation of hypertrophic
chondrocytes with PTH (1-34) induces an inhibition of p38 kinase
activity in a time- and dose-dependent manner. Inhibition
of protein kinase C prevents PTH-induced p38 MAPK inhibition, whereas
inhibition of protein kinase A has no effect. Thus, protein kinase C,
but not protein kinase A, is required for the inhibition of p38 MAPK by
PTH. Treatment of hypertrophic chondrocytes by PTH or by p38 MAPK
inhibitor SB203580 up-regulates Bcl-2, suggesting that Bcl-2 lies
downstream of p38 MAPK in the PTH signaling pathway. Inhibition of p38
MAPK in hypertrophic chondrocytes by either PTH, SB303580, or both
together leads to a decrease of hypertrophic marker type X collagen
mRNA and an increase of the expression of prehypertrophic marker
cartilage matrix protein. Therefore, inhibition of p38 converts a
hypertrophic cell phenotype to a prehypertrophic one, thereby
preventing precocious chondrocyte hypertrophy. Taken together, these
data suggest a major role for p38 MAPK in transmitting PTH signals to
regulate chondrocyte differentiation.
During endochondral ossification, chondrocytes undergo a
differentiation process including proliferation, maturation,
hypertrophy, and apoptosis (1). Cells from these different stages of
differentiation synthesize specific molecules, for example, type X
collagen is synthesized specifically by hypertrophic chondrocytes (2,
3), whereas cartilage matrix protein
(CMP)1/matrilin-1, is a
marker of prehypertrophic chondrocytes (1). Parathyroid hormone (PTH)
and parathyroid hormone-related peptide (PTHrP) are major regulators of
the chondrocyte differentiation process. PTH and PTHrP both bind to PTH
receptors, which belong to G protein-coupled receptors (4). In a growth
plate, PTH receptors are expressed at specific stages during
chondrocyte differentiation, with the highest level at the
prehypertrophic to hypertrophic transition (5). This suggests that PTH
receptors may be important for the regulation of chondrocyte
differentiation from the prehypertrophic state to the hypertrophic
state. Indeed, overexpression of PTHrP or its constitutively active
receptor in growth plates of transgenic mice delays endochondral bone
formation (6), whereas mice with ablation of the PTH/PTHrP receptor
gene develop skeletal dysplasia because of accelerated chondrocyte hypertrophy (7-9). Thus, the PTH/PTHrP receptor plays a fundamental role in the control of endochondral bone formation by transducing signals inhibiting chondrocyte hypertrophy. However, the intracellular signaling mechanism for PTH/PTHrP to regulate chondrocyte
differentiation remains unknown.
The PTH/PTHrP receptor may exert its biological action via at least two
signaling pathways, i.e. elevating intracellular cAMP, thereby activating cAMP-dependent protein kinase A (PKA) or
activating phospholipase C that results in an increase in intracellular
free calcium and activation of protein kinase C (PKC) (10). It has also
been shown that Bcl-2, an anti-apoptosis molecule, is also involved in
the PTH/PTHrP signaling pathway in chondrocytes (11). In this study, we
will determine the involvement of PKA and PKC pathways in transducing
PTH/PTHrP signals to regulate chondrocyte differentiation. Furthermore,
the signaling molecules downstream of PKA or PKC that mediate PTH/PTHrP
regulation of chondrocyte differentiation will be defined. We will
present the evidence that a stress-induced mitogen-activated protein
kinase (MAPK) p38 is a central link to connect PTH signaling to the
regulation of chondrocyte hypertrophy.
Recent studies indicate that stress-induced MAPK superfamily, which
includes p38 MAPK and c-Jun NH2-terminal kinase (JNK), is
important for regulating cell differentiation and apoptosis by
transmitting extracellular signals to the nucleus (12). However, the
roles of JNK and p38 MAPK in the regulation of chondrocyte terminal
differentiation-hypertrophy are not known. p38 MAPK and JNK are serine
and threonine protein kinases that are activated by osmotic pressure,
stress, and cytokines (13). In addition, several growth factors and G
protein-coupled receptors may also activate p38 MAPK and JNK (14). The
functional difference between p38 MAPK and JNK can be distinguished
using a selective kinase inhibitor SB203580 that inhibits only p38 but
not JNK activities (15). The aim of this study is to test whether
stress-activated MAPKs are involved in the regulation of chondrocyte
hypertrophy by PTH. If so, which specific MAPK pathway regulates
chondrocyte differentiation? We show here that in primary chick
hypertrophic chondrocytes PTH inhibits p38 MAPK activity in a dose- and
time-dependent manner but has little effect on JNK
activity. The inhibition of p38 MAPK activity by PTH appears to be
mediated by PKC. Furthermore, we demonstrate that inhibition of p38
MAPK activity is a signaling mechanism responsible for regulation of
chondrocyte terminal differentiation by PTH/PTHrP.
Materials--
PTH (1-34), myelin basic protein (MBP),
calphostin C, and thyroxin were purchased from Sigma. Dibutyryl-cAMP
and H89 were obtained from Calbiochem (La Jolla, CA). SB203580 was from
BIOMOL (Plymouth Meeting, PA). Electrophoresis reagents were obtained from Bio-Rad. Agarose-conjugated anti-phosphotyrosine antibody (clone
4G10) and c-Jun (169) GST were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-p38 MAPK, anti-JNK1, anti-BCL-2, and
protein A/G were purchased from Santa Cruz Biotechnology Inc (Santa
Cruz, CA). Monoclonal antibodies including anti-CMP and anti-link
protein were prepared as described previously (16). Horseradish
peroxidase-linked anti-rabbit/mouse secondary antibodies were obtained
from Pierce. [ Cell Culture and Preparation of Cell Lysates--
Primary
chondrocyte cultures were established as described previously (1).
Briefly, hypertrophic chondrocytes were obtained from the cephalic part
of sterna cartilage from 17-day-old embryonic chickens, and
prehypertrophic chondrocytes were obtained from the caudal part of the
cartilage. We chose 17-day-old embryonic chickens because the cephalic
part of sterna cartilage contains early hypertrophic chondrocytes that
just started to synthesize type X collagen (3). Cartilage pieces were
incubated with 0.1% testicular hyaluronidase, 0.3% collagenase for 30 min at 37 °C. The cells were replaced with fresh medium containing
enzymes, and incubation was continued for additional 1 h. Cells
were collected by centrifugation and maintained in plating medium
supplemented with 10% fetal bovine serum in a humidified atmosphere of
5% CO2 and 95% O2 at 37 °C. At about
80-90% confluence, cells were incubated with 0.5% fetal bovine serum
medium overnight before experiments. After cells were treated with PTH
or other agents for designated time, reactions were stopped by
aspirating the medium and washing twice with cold phosphate-buffered
saline. For immunoblot and protein phosphorylation analysis, cells were
lysed in buffer A (50 mM Tris, pH 7.4, 150 mM
NaCl, 0.25% sodium deoxycholate, 3 mM
Na3VO4, 1 mM EGTA, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride). For the kinase activity assay, cells were lysed in buffer B (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 20 mM In Vitro Immune Complex Kinase Assay--
After an aliquot (200 µg of proteins) of cell lysate was incubated with 1 µg of anti-p38
MAPK or 2 µg of anti-JNK1 overnight at 4 °C, 15 µl of protein
A/G Plus (Santa Cruz, CA) was then added to the tubes for additional
2 h of incubation. Immune complex was washed three times with
buffer B and twice with kinase assay buffer (20 mM Tris, pH
7.5, 2 mM EGTA, 20 mM MgCl2, 12.5 mM Immunoblotting and Tyrosine Phosphorylation Assay--
To
determine tyrosine phosphorylation levels of p38 MAPK, 0.5 mg of
supernatant protein was incubated overnight at 4 °C with 10 µl of
agarose-conjugated anti-phosphotyrosine monoclonal antibody (4G10).
Immunoprecipitates were collected and washed three times with buffer A. The immune complex was then resuspended in 40 µl of sample buffer and
separated by 12% SDS gel electrophoresis. After transferred to
nitrocellulose membranes, the blots were probed with anti-p38 MAPK
antibody. The signals were visualized with Supersignal Western blot
Detection System (Pierce). For determining protein expression of Bcl-2,
CMP, and link protein, 20 µg of protein was loaded in each lane of
10% SDS-polyacrylamide gels. After electrophoresis, the gels were
transferred to nitrocellulose membranes and probed with the
corresponding antibody. The signals were then visualized as described above.
Real Time Quantitative RT-PCR--
After cells were treated with
PTH or other reagents, total cellular RNA was isolated from
chondrocytes with RNeasy isolation kit (Qiagen). The relative content
of mRNA was quantified by real time quantitative PCR using
respective probe and primer for CMP or type X collagen. For chick CMP
mRNA, the forward and reverse primers were
5'-AGGGCTTCACGCTGAACAAT-3' and 5'-AGGGCAGATCCTGACCCAC-3', respectively.
The internal probe was 5'-(FAM)-TGGGAAGACCTGCAGTGCTTGCAGT-(TAMRA)-3'. They were designed according to GenBankTM accession numbers
12346-12354 (19). The forward and reverse primers for Data Analysis--
Data are expressed as the means ± S.E.
and analyzed by analysis of variance followed by Newman-Keuls test.
Statistical significance is considered at p < 0.05. In
some cases, Student's t test was also used when indicated.
PTH Inhibits p38 MAPK Activity and Tyrosine
Phosphorylation--
To determine whether p38 MAPK activity correlates
with chondrocyte hypertrophy, we examined the basal level of p38 MAPK
activity in both prehypertrophic and hypertrophic chondrocytes. As
shown in Fig. 1A, the basal
p38 MAPK activity in hypertrophic chondrocytes was at least 10-fold
higher than that of prehypertrophic cells (prehypertrophic, 1 ± 0.11; hypertrophic, 11.2 ± 0.37, n = 3, p < 0.01, t test). Therefore, p38 MAPK
activity was elevated in hypertrophic chondrocytes, thus correlating
with the differentiation state of chondrocytes. The difference of p38
MAPK activity between prehypertrophic and hypertrophic cells was not
due to the difference of the expression levels of p38 MAPK protein,
because there was no significant difference between the p38 protein
levels from these two populations of cells, as assessed by Western blot
analysis of p38 MAPK (Fig. 1A).
To determine whether PTH, which suppressed hypertrophy of chondrocytes
(9), had an effect on the kinase activity of p38 MAPK, hypertrophic
chondrocytes were treated with PTH (1-34) for various time periods or
with various dosages (Fig. 1B). An inhibition of p38 MAPK by
100 nM PTH was observed at 10 min of treatment, and the
inhibition lasted until 60 min when the kinase recovered to the basal
level (Fig. 1, B and C). Dose response study of
PTH indicated that both 1 and 100 nM PTH treatment of
chondrocytes for 10 min inhibited p38 MAPK significantly, but 0.01 nM PTH did not (Fig. 1, B and C). To
further confirm the inhibitory effect of PTH on p38 MAPK activity,
tyrosine phosphorylation of p38 MAPK was determined during the time
course of PTH treatment. Because p38 MAPK activation requires
phosphorylation of both threonine and tyrorine residues in the
molecule, the level of tyrosine phosphorylation of p38 MAPK reflects
the extent of kinase activation (17). As shown in Fig. 1D,
tyrosine phosphorylation of p38 MAPK was decreased after 10 min of PTH
treatment and returned to the basal level after 60 min. This was in
agreement with the time course of the PTH inhibition of p38 MAPK
activity in hypertrophic chondrocytes. Thus, PTH inhibited p38 MAPK
activity in hypertrophic chondrocytes in a dose- and
time-dependent manner. In contrast, treatment of hypertrophic chondrocytes with PTH had no significant effect on the
activity of JNK, another major stress-activated MAPK (Fig. 2).
PKC but Not PKA Is Involved in Inhibition of p38 MAPK by
PTH--
To determine whether inhibition of p38 MAPK by PTH was
mediated by the PKC or PKA pathway, hypertrophic chondrocytes were incubated with specific inhibitors of PKC or PKA pathways. As shown in
Fig. 3, inhibition of PKC by calphostin
C, a selective PKC inhibitor, did not alter basal p38 activity (Fig.
3A, compare Cal.C with Control).
However, calphostin C completely abolished PTH-mediated inhibition of
p38 MAPK activity (Fig. 3A, compare Cal.C+PTH
with PTH). This suggested that PKC was required for PTH-induced inhibition of p38 MAPK activity. In contrast, treatment of
cells with either H-89, a selective PKA inhibitor (Fig. 3B, compare H-89+PTH with PTH), or cell permeable
cAMP, an activator of PKA (Fig. 3A, compare
cAMP+PTH with PTH), had no effect on the
inhibition of p38 MAPK activity by PTH. This indicated that the PKA
pathway was not involved in the inhibition of p38 MAPK activity by
PTH.
Inhibition of p38 MAPK Is Associated with Up-regulation of
Bcl-2--
To test whether the inhibition of p38 MAPK activity by PTH
involved Bcl-2, a recently identified component of the PTH
pathway regulating chondrocyte differentiation (11), the protein
levels of Bcl-2 in hypertrophic chondrocytes were determined by Western blot analysis. PTH treatment at 100 nM stimulated Bcl-2
protein expression, whereas treatment with 100 ng/ml thyroxine, a
chondrocyte differentiation inducer (21), did not alter the Bcl-2
protein level (Fig. 4A). Thus,
PTH specifically stimulated the Bcl-2 protein level in
hypertrophic chondrocytes.
To determine whether the inhibition of p38 MAPK activity by PTH led to
the up-regulation of Bcl-2, hypertrophic chondrocytes were treated with
100 nM PTH; 10 µM SB203580, a specific
inhibitor of p38 kinase activity; or a combination of both PTH and
SB203580. The up-regulation of Bcl-2 by PTH (Fig. 4B,
PTH) can be mimicked by the inhibition of p38 MAPK by
SB203580 (Fig. 4B, SB203580). In addition, a
similar extent of the increase of Bcl-2 level was observed when cells
were treated with both SB203580 and PTH (Fig. 4B,
SB+PTH). These data suggested that inhibition of p38 MAPK was involved in PTH-regulated Bcl-2 expression.
Specific Inhibition of p38 MAPK Activity by SB 203580 in
Hypertrophic Chondrocytes--
Although it is known that SB203580 at
10 µM, the concentration used in this study, inhibits
activity of p38 MAPK but not that of JNK or ERK (22), it has been
reported recently that SB203580 at the 5-10 µM range
could inhibit interleukin-2-induced PKB (Akt) activation in CT6 cells
(18). To determine whether SB203580 also inhibited PKB activation in
hypertrophic chondrocytes, hypertrophic chondrocytes were incubated
with SB203580 at various concentrations, and kinase activities of both
p38 MAPK and PKB were determined. As shown in Fig.
5, SB203580 inhibited p38 MAPK in a
dose-dependent manner. In contrast, no significant
inhibition of PKB activity was observed in these SB203580-treated
cells. This result was verified by an in vitro assay, in
which lysates of hypertrophic chondrocytes were incubated with SB203580
for 5 min, followed by determination of PKB activity. No significant
inhibitory effects of SB203580 on PKB activity was observed (Control,
3207 ± 72; 1 µM SB203580, 2984 ± 124; 10 µM SB203580, 3023 ± 97; arbitrary units,
n = 3). Thus, the optimal concentration of SB 203580 for specific inhibition of p38 MAPK may depend on cell types. In
hypertrophic chondrocytes, 10 µM SB 203580 inhibited p38
MAPK activity but not PKB activity.
PTH Differentially Regulates CMP and Inhibition of p38 MAPK Is Associated with Down-regulation of Type X
Collagen by PTH--
To determine whether p38 MAPK is involved in
PTH-regulated chondrocyte differentiation by differentially altering
the levels of type X collagen and CMP, we incubated hypertrophic
chondrocytes with 100 nM PTH in the absence or presence of
10 µM SB203580 for 1 day. Real-time quantitative RT-PCR
analysis showed that p38 MAPK inhibitor SB203580 significantly
inhibited the level of Inhibition of p38 MAPK Is Associated with Up-regulation of CMP by
PTH--
Inhibition of p38 MAPK activity by SB203580 stimulated the
CMP mRNA level to a similar extent as treatment with both PTH and SB together (Fig. 8A).
Treatment with PTH alone also stimulated the CMP mRNA level but to
a lesser degree (Fig. 8A). This suggested that inhibition of
p38 MAPK could be achieved by PTH and some unknown elements, which led
to up-regulation of the CMP mRNA level. In agreement with the
mRNA data, a parallel increase of CMP protein level was observed by
the same treatment (Fig. 8B, CMP). In contrast, although the level of link protein, a protein expressed throughout chondrocyte differentiation process, was up-regulated by PTH, this
stimulatory effect was not achieved by either SB treatment alone or SB
and PTH treatment together (Fig. 8B, LP). This
indicated that other signaling mechanism that did not involve p38 MAPK
might contribute to the PTH stimulation of LP expression. Furthermore, the up-regulation of CMP by PTH, SB, or PTH and SB treatment together (Fig. 8, A and B, CMP), as well as the
down-regulation of type X collagen by these treatment (Fig. 7),
corresponded to the inhibition of p38 MAPK activity during these
treatment (Fig. 8C). This further supported the conclusion
that the inhibition of p38 MAPK activity was responsible for both
up-regulation of CMP and down-regulation of type X collagen by PTH.
The endochondral ossification process, which consists of
chondrocyte proliferation, maturation, hypertrophy, and apoptosis, requires precise regulation of these differentiation events.
It has become clear in recent years that a major regulator of
chondrocyte hypertrophy is the PTH/PTHrP signaling pathway (7-9).
Surprisingly, very little is known about the intracellular molecules
that transduce PTH/PTHrP signals to regulate chondrocyte hypertrophy.
In this study, we present the first evidence that inhibition of
chondrocyte hypertrophy by PTH is mediated by an inhibition of p38 MAPK
activity. We propose a model in which p38 MAPK is a central link in the PTH/PTHrP signaling pathway to regulate chondrocyte hypertrophy by connecting PKC in the upstream and Bcl-2 in the downstream (Fig.
9). The PTH-p38 MAPK pathway prevents
precocious chondrocyte hypertrophy by simultaneously inhibiting type X
collagen, a marker of hypertrophic chondrocytes, and stimulating
CMP, a marker of prehypertrophic chondrocytes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was purchased from
PerkinElmer Life Sciences. Ham's F-12 medium was from Life
Technologies, Inc.
-glycerophosphate 1 mM EGTA, 20 mM NaF, 3 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin and
leupeptin, and 1% Nonidet P-40). Lysates were centrifuged 12,000 × g for 15 min at 4 °C to precipitate debris. The
protein content of supernatant was determined. Aliquots of supernatant
were used for immunoprecipitation or immunoblotting (see below).
-glycerophosphate, 1 mM dithiothreitol,
0.2 mM Na3VO4). Kinase activity
assays were performed in kinase assay buffer in the presence of 50 µM [
-32P]ATP (5 µCi), 0.2 mg/ml MBP
(for p38 MAPK), or 2 µg of c-Jun GST (for JNK), as described
previously (17). After incubation at 30 °C for 20 min, the reaction
was terminated by addition of 2× sample buffer. The products were
resolved by 12% SDS-polyacrylamide gel electrophoresis. The gels were
stained with Coomassie Blue to confirm loading of equal amounts of
substrate. The phosphorylation of MBP was visualized by autoradiography
and quantified by PhosphorImager. To determine PKB activity, cell
lysate was immunoprecipitated with anti-AKT1/2 antibody (Santa Cruz,
CA), and kinase activity assay was performed using cross-tide peptide
(Upstate Biotechnology, Inc.) as substrate, as previously described
(18).
1 type X
collagen were 5'-AGTGCTGTCATTGATCTCATGGA-3' and
5'-TCAGAGGAATAGAGACCATTGGATT-3', respectively. The internal probe of
Col X cDNA was 5'-(FAM)- TCAAGTGTGGCTCCAGATGCCAAA-(TAMRA)-3'. They
were designed according to GenBankTM accession number
M13496 (20). Quantification of mRNAs of CMP and type X collagen was
performed by real time quantitative RT-PCR with a PerkinElmer ABI PRISM
7700 Sequence Detection System, as described previously (28). RT-PCR
reaction was performed with AmpliTAQ Gold polymerase (PerkinElmer) with
20 ng of total RNA for each reaction. The 18 S RNA was amplified at the
same time and used as an internal control. The cycle threshold (Ct) values for 18 S RNA and that of samples were measured and calculated by
computer software (PerkinElmer). Relative transcript levels were
calculated as x = 2
Ct, in which
Ct =
E
C, and
E =Ctexp
Ct18 s;
C = Ctctl
Ct18
s.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (33K):
[in a new window]
Fig. 1.
A, the basal level of p38 MAPK activity
corresponds to the differentiation stage of chondrocytes.
Phospho-MBP, hypertrophic chondrocytes exhibited higher
levels of p38 MAPK activity, in comparison with those of
prehypertrophic cells. A representative autoradiograph is shown. Equal
amounts of lysates from those cells were incubated with an anti-p38
MAPK antibody. Immune complex was collected, and p38 MAPK kinase
activity assay was performed using MBP as substrate in the presence of
[ -32P]ATP. p38 MAPK, the protein levels of
p38 MAPK remained the same between prehypertrophic and hypertrophic
cells. A representative Western blot is shown. Equal amount of lysates
was loaded in each lane. Western blot analysis was performed with an
antibody against p38 MAPK. n = 3. B and
C, PTH inhibits p38 MAPK activity in a dose- and
time-dependent manner. Hypertrophic chondrocytes were
incubated with either 100 nM PTH for indicated time periods
or with various dosages of PTH for 10 min. Equal amount of lysates from
those cells was incubated with an anti-p38 MAPK antibody. Immune
complex was collected, and p38 MAPK kinase activity assay was performed
in the presence of [
-32P]ATP using MBP as substrate.
*, p < 0.05, significant difference compared with
control (zero time and dose). D, PTH inhibits p38 MAPK
tyrosine phosphorylation. Hypertrophic chondrocytes were treated with
100 nM PTH for indicated time periods. Equal amount of cell
lysates was immunoprecipitated (IP) with an anti-tyrosine
antibody 4G10. The immunoprecipitates were separated by
SDS-polyacrylamide gel electrophoresis and blotted with an anti-p38
MAPK antibody. A representative immunoblot is shown. Similar results
were obtained from three experiments.
View larger version (12K):
[in a new window]
Fig. 2.
PTH does not affect JNK activity.
Hypertrophic chondrocytes were treated with 100 nM PTH for
the indicated time periods. Cell lysate was incubated with an anti-JNK1
antibody, and the immune complex was collected. The JNK activity assay
was performed in the presence of [ -32P]ATP with
GST-c-Jun as substrate. The kinase activity was quantified using a
PhosphorImager. The graph is the summary of three independent
experiments.
View larger version (24K):
[in a new window]
Fig. 3.
PKC is involved in the inhibition of p38 MAPK
by PTH. A, hypertrophic chondrocytes were treated with
100 nM PTH for 10 min (PTH), 1 µM
PKC inhibitor calphostin C for 2 h (Calphostin C), 0.5 mM PKA activator cAMP for 10 min (cAMP), cAMP
for 10 min plus PTH for additional 10 min (PTH + cAMP), or
calphostin C for 2 h plus PTH for additional 10 min (Cal.C + PTH). p38 MAPK activity was determined by a kinase assay with MBP
as phosphorylation substrate. B, cells were treated with 1 µM H-89 for 1 h (H-89), 100 nM PTH for 10 min (PTH), or H-89 for 1 h
plus PTH for additional 10 min (H-89+PTH). A representative
autoradiograph is shown for each set of experiments. Bar
graphs show the averages of four independent experiments. *,
p < 0.01, significant difference compared with control
(untreated).
View larger version (25K):
[in a new window]
Fig. 4.
Inhibition of p38 MAPK is associated with
up-regulation of Bcl-2 by PTH. A, hypertrophic cells
were treated with 100 nM PTH (PTH), 100 ng/ml
thyroxine (TH), or untreated (Control) for 3 days. Cell lysates were then prepared for Western blot analysis with an
anti-Bcl-2 antibody. B, to determine the possible role of
p38 MAPK in PTH-mediated Bcl-2 expression, chondrocytes were
preincubated with 10 µM SB203580 (SB), a
selective p38 MAPK inhibitor, for 2 h before100 nM PTH
was added for additional 3 days. Equal amount of cell lysate was loaded
to a SDS-polyacrylamide gel and blotted with an anti-Bcl-2 antibody.
The immunoreactive proteins were detected by the ECL system for Western
blot. A representative blot is shown for each set of experiments.
Bar graphs show the averages of quantified data from three
independent experiments. *, p < 0.01, significant
difference compared with control.
View larger version (13K):
[in a new window]
Fig. 5.
SB203580 does not inhibit PKB (Akt) activity
in hypertrophic chondrocytes. Cells were incubated with SB203580
at various concentrations for 2 h before collection for
preparation of cell lysates. Kinase assay of immune complex was then
performed for p38 MAPK and PKB using different substrates. *,
p < 0.01, significant difference compared with 0 µM SB203580, n = 3.
1 type X mRNA
Expression--
To determine the downstream effects of PTH regulation
of chondrocyte hypertrophy, we quantified the levels of mRNA of
type X collagen, a marker of hypertrophic chondrocytes (2, 3), and that
of CMP/matrilin-1, a marker of prehypertrophic chondrocytes (1). Real
time quantitative RT-PCR indicated that
1 type X mRNA was
significantly decreased after 1 day of treatment of hypertrophic chondrocytes with 100 nM PTH, with maximal inhibition
achieved after 3 days (Fig. 6). Thus, PTH
inhibited chondrocyte hypertrophy. In contrast, PTH induced a
significant increase of CMP mRNA after 1 day of treatment (2.6 ± 0.21-fold), and after 2 days of treatment (1.7 ± 0.1-fold)
(Fig. 6). This suggested that PTH converted hypertrophic chondrocytes
to the prehypertrophic phenotype, of which CMP was the marker. This
change of CMP mRNA level, however, was transient. After 3 days of
treatment with PTH, the CMP mRNA level returned to the basal level
(Fig. 6).
View larger version (14K):
[in a new window]
Fig. 6.
PTH differentially regulates CMP and collagen
X mRNA expression. Hypertrophic chondrocytes were treated with
100 nM PTH for the indicated number of days. Total RNA was
isolated from these cells using RNAeasy Mini Kit (Qiagen). The mRNA
of CMP and collagen X (COL X) was quantified by real time
quantitative RT-PCR. *, p < 0.01, significant
difference compared with no treatment, n = 4.
1 type X mRNA (Fig.
7), mimicking the effect of PTH
treatment. In addition, treatment with both PTH and SB203580 showed a
similar extent of inhibition of
1 type X mRNA as that of
treatment with SB203580 alone (Fig. 7). These data suggested that
inhibition of p38 MAPK activity was involved in the inhibition of
1
type X mRNA by PTH.
View larger version (14K):
[in a new window]
Fig. 7.
Inhibition of p38 MAPK is associated with
down-regulation of type X collagen mRNA by PTH. Hypertrophic
chondrocytes were incubated 24 h with 100 nM PTH
(PTH), 0.1% Me2SO, a vehicle for SB (Con.), 10 µM SB203580 (SB), or with SB for 2 h
before 100 nM PTH was added for additional 22 h
(SB+PTH). The mRNA for type X collagen was then
quantified by real time quantitative PCR. *, p < 0.01, significant difference compared with control, n = 3.
View larger version (20K):
[in a new window]
Fig. 8.
Inhibition of p38 MAPK is associated with
up-regulation of CMP by PTH. A, CMP mRNA.
Hypertrophic chondrocytes were incubated for 24 h with 100 nM PTH (PTH), 0.1% Me2SO, a vehicle
for SB203580 (Con.), 10 µM SB203580
(SB), or with SB203580 for 2 h before 100 nM PTH was added for additional 22 h
(SB+PTH). The mRNA for CMP was then quantified by real
time quantitative PCR. *, p < 0.01, significant
difference compared with control. #, p < 0.01, significant difference compared with PTH, n = 4. B, CMP protein. After treatment with reagents as described
above, equal amounts of cell lysate were electrophoresed and blotted
with an anti-CMP antibody (CMP) or an anti-link protein
antibody (LP). The experiments were repeated three
times with similar results. Representative blots are shown.
C, p38 MAPK activity. After 15 min of treatment with
reagents as indicated, cell lysate from those cells was prepared and
incubated with an anti-p38 MAPK antibody. Immune complex was collected,
and p38 MAPK kinase activity assay was performed using MBP as substrate
in the presence of [ -32P]ATP. The data shown are the
averages of three independent experiments. *, p < 0.01, significant difference compared with control. #,
p < 0.05, significant difference compared with
PTH.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
[in a new window]
Fig. 9.
Summary of signaling cascades in PTH-mediated
chondrocyte differentiation. E, extracellular;
M, cell membrane; C, cytoplasm; N,
nucleus.
It has been shown previously that PTH and PTHrP, which bind to a common PTH/PTHrP receptor (4), employ at least two pathways for transducing signals, i.e. activation of PKA via Gs-coupled mechanism, or activation of PKC via a Gq-coupled mechanism (10). In addition, both PKA and PKC have been reported to regulate the p38 MAPK pathway, and the specificity of regulation seems to vary depending on cell types (17, 23, 24). In this study, we have shown that it is the PKC pathway that is responsible for transducing PTH signals to inhibit p38 MAPK activity in hypertrophic chondrocytes. PKC has also been implicated in regulating chondrogenesis of mesenchymal cells (25). Our data provide evidence that p38 MAPK maybe a downstream signaling molecule in PKC-regulated chondrocyte differentiation.
We have also shown that inhibition of p38 MAPK activity is responsible
for transducing PTH signals to prevent chondrocyte hypertrophy. The
activity of p38 is elevated more than 10-fold in hypertrophic
chondrocytes. Treatment with PTH inhibits such elevation, thereby
preventing hypertrophy. This inhibition is specific, because PTH has no
effect on the activities of JNK, another major stress-activated MAPK.
Both p38 and JNK exist in chondrocytes, and they are activated by
interleukin-1 and TNF-, which may lead to chondrocyte apoptosis
and cartilage degradation (26). We have shown here that inhibition of
p38 MAPK alone is sufficient to prevent chondrocyte terminal
differentiation to hypertrophy. Inhibition of p38 MAPK, even in the
absence of PTH signaling, causes downstream effects including
up-regulation of Bcl-2 and CMP and down-regulation of type X collagen.
This suggests that p38 may play a role not only in transmitting the PTH
signals but also in the general control of chondrocyte hypertrophy.
Our study also connects the PTH-p38 MAPK pathway to the regulation of Bcl-2 level in hypertrophic chondrocytes. Previous transgenic experiments have shown that Bcl-2 is a component of the PTH signaling pathway (11). We show here that the Bcl-2 level is regulated by the p38 MAPK activity. Bcl-2 is an anti-apoptosis molecule that maintains cellular calcium homeostasis (27, 28). Furthermore, it has been shown that chondrocyte calcium metabolism regulates its hypertrophic phenotype (29, 30) and that chondrocyte hypertrophy is closely associated with apoptosis (31). However, it was not known how chondrocyte hypertrophy is related to apoptosis and calcium metabolism. Our finding that p38 MAPK regulates both Bcl-2 and chondrocyte hypertrophy through PTH signaling may reconcile all of these findings.
It is interesting to note that the PTH-p38 MAPK pathway not only inhibits the hypertrophic marker type X collagen but also stimulates the prehypertrophic marker CMP. This indicates that PTH prevents chondrocyte terminal differentiation not only by inhibiting hypertrophy but also by actively converting a hypertrophic phenotype to a prehypertrophic one. This is consistent with the finding that hypertrophic chondrocytes can be converted into prehypertrophic chondrocytes (1, 32) in a "retro-differentiation" process (1). However, this reversion is transient, as evidenced by the temporal stimulation of CMP in hypertrophic cells by PTH. A possible explanation for this phenomenon may be the desensitization of PTH/PTHrP receptors, which is common for G protein-coupled receptors upon prolonged exposure to ligands (33, 34). Receptor desensitization could be due to phosphorylation of the receptors induced by agonist and mediated by PKA, PKC, or other G protein-coupled receptor kinases (34, 35). For example, it has been demonstrated that phosphorylation of PTH/PTHrP receptors by prolonged PTH incubation desensitizes the receptors, probably via a G protein receptor kinase (34). Indeed, in PTHrp overexpression transgenic mice, endochondral ossisfication is delayed but eventually occurred (6). Our data suggest that one way to overcome such transient nature of retro-differentiation is to consistently inhibit the downstream p38 MAPK activity.
In summary, our study presents an important intracellular pathway for
PTH signaling in hypertrophic chondrocytes. This pathway links
extracellular PTH/PTHrP signals to intracellular regulatory molecules,
including PKC, p38 MAPK, and Bcl-2, and ultimately to the regulation of
chondrocyte differentiation and synthesis of extracellular matrix
molecules such as type X collagen and CMP. The elucidation of this
pathway may have strong implications for our understandings of skeletal
development, which is potently regulated by PTH/PTHrP.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Deb Grove for assistance in performing real time RT-PCR.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants AG14399, AG00811, and AG17021 from the National Institutes of Health (to Q. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dept. of Pharmacology and Physiology, MCP Hahnemann University, Philadelphia, PA 19121.
¶ Supported by the Arthritis Foundation.
** To whom correspondence should be addressed. Tel.: 717-531-4835; Fax: 717-531-7583; E-mail: qchen@psu.edu.
Published, JBC Papers in Press, November 29, 2000, DOI 10.1074/jbc.M004990200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CMP, cartilage matrix protein; PTH, parathyroid hormone; PTHrP, PTH-related peptide; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; PKC, protein kinase C; PKA, protein kinase A; PKB, protein kinase B; RT, reverse transcription; PCR, polymerase chain reaction; MBP, myelin basic protein; GST, glutathione S-transferase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Chen, Q., Johnson, D. M., Haudenschild, D. R., and Goetinck, P. F. (1995) Dev. Biol. 172, 293-306[CrossRef][Medline] [Order article via Infotrieve] |
2. |
Schmid, T. M.,
and Linsenmayer, T. F.
(1983)
J. Biol. Chem.
258,
9504-9509 |
3. | Gibson, G. J., and Flint, M. H. (1985) J. Cell Biol. 101, 277-284[Abstract] |
4. | Abou-Samra, A. B., Juppner, H., Force, T., Freeman, M. W., Kong, X. F., Schipani, E., Urena, P., Richards, J., Bonventre, J. V., Potts, J. T., Jr., Kronenberg, H., and Segre, G. V. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2732-2736[Abstract] |
5. |
Iwamoto, M.,
Jikko, A.,
Murakami, H.,
Shimazu, A.,
Nakashima, K.,
Iwamoto, M.,
Takigawa, M.,
Baba, H.,
Suzuki, F.,
and Kato, Y.
(1994)
J. Biol. Chem.
269,
17245-17251 |
6. |
Schipani, E.,
Lanske, B.,
Hunzelman, J.,
Luz, A.,
Kovacs, C. S.,
Lee, K.,
Pirro, A.,
Kronenberg, H. M.,
and Juppner, H.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
13689-13694 |
7. | Karaplis, A. C., Luz, A., Glowacki, J., Bronson, R. T., Tybulewicz, V. L. J., Kronenberg, H. M., and Mulligan, R. C. (1984) Genes Dev. 8, 277-289[Abstract] |
8. | Amizuka, N., Warshawsky, H., Henderson, J. E., Goltzman, D., and Karaplis, A. C. (1994) J. Cell Biol. 126, 1611-1623[Abstract] |
9. |
Chung, U. I.,
Lanske, B.,
Lee, K. C.,
Li, E.,
and Kronenberg, H.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
13030-13035 |
10. | Abou-Samra, A. B., Jueppner, H., Westerberg, D., Potts, J. T., Jr., and Segre, G. V. (1989) Endocrinology 124, 1107-1113[Abstract] |
11. |
Amling, M.,
Neff, L.,
Tanaka, S.,
Inoue, D.,
Kuida, K.,
Weir, E.,
Philbrick, W. M.,
Broadus, A. E.,
and Baron, R.
(1997)
J. Cell Biol.
136,
205-213 |
12. |
Cobb, M. H.,
and Goldsmith, E. J.
(1995)
J. Biol. Chem.
270,
14843-14846 |
13. | Paul, A., Wilson, S., Belham, C. M., Robinson, C. J., Scott, P. H., Gould, G. W., and Plevin, R. (1997) Cell. Signalling 9, 403-410[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Morooka, T.,
and Nishida, E.
(1998)
J. Biol. Chem.
273,
24285-24288 |
15. |
Young, P. R.,
McLaughlin, M. M.,
Kumar, S.,
Kassis, S.,
Doyle, M. L.,
McNulty, D.,
Gallagher, T. F.,
Fisher, S.,
McDonnell, P. C.,
Carr, S. A.,
Huddleston, M. J.,
Seibel, G.,
Porter, T. G.,
Livi, G. P.,
Adams, J. L.,
and Lee, J. C.
(1997)
J. Biol. Chem.
272,
12116-12121 |
16. | Chen, Q., Johnson, D. M., Haudenschild, D. R., Tondravi, M. M., and Goetinck, P. F. (1995) Mol. Biol. Cell 6, 1743-1753[Abstract] |
17. |
Zhen, X.,
Uryu, K.,
Wang, H. Y.,
and Friedman, E.
(1998)
Mol. Pharmacol.
54,
453-458 |
18. |
Lali, F. V.,
Hunt, A. E.,
Turner, S. J.,
and Foxwell, B. M.
(2000)
J. Biol. Chem.
275,
7395-7402 |
19. |
Kiss, I.,
Deak, F.,
Holloway, R. G., Jr.,
Delius, H.,
Mebust, K. A.,
Frimberger, E.,
Argraves, W. S.,
Tsonis, P. A.,
Winterbottom, N.,
and Goetinck, P. F.
(1989)
J. Biol. Chem.
264,
8126-8134 |
20. |
LuValle, P.,
Ninomiya, Y.,
Rosenblum, N. D.,
and Olsen, B. R.
(1988)
J. Biol. Chem.
263,
18378-18385 |
21. | Ballock, R. T., and Reddi, A. H. (1994) J. Cell Biol. 126, 1311-1318[Abstract] |
22. | Cuenda, A., Rouse, J., Doza, Y. N., Meier, R., Cohen, P., Gallagher, T. F., Young, P. R., and Lee, J. C. (1995) FEBS Lett. 364, 229-233[CrossRef][Medline] [Order article via Infotrieve] |
23. | Berra, E., Municio, M. M., Sanz, L., Frutos, S., Diaz-Meco, M. T., and Moscat, J. (1997) Mol. Cell. Biol. 17, 4346-4354[Abstract] |
24. | Dohlman, H. G., Thorner, J., Caron, M. G., and Lefkowitz, R. J. (1991) Annu. Rev. Biochem. 60, 653-688[CrossRef][Medline] [Order article via Infotrieve] |
25. | Yang, M. S., Chang, S. H., Sonn, J. K., Lee, Y. S., Kang, S. S., Park, T. K., and Chun, J. S. (1998) Mol. Cells 8, 266-271[Medline] [Order article via Infotrieve] |
26. |
Geng, Y.,
Valbracht, J.,
and Lotz, M.
(1996)
J. Clin. Invest.
98,
2425-2430 |
27. |
He, H.,
Lam, M.,
McCormick, T. S.,
and Distelhorst, C. W.
(1997)
J. Cell Biol.
138,
1219-1228 |
28. | Rajpurohit, R., Mansfield, K., Ohyama, K., Ewert, D., and Shapiro, I. M. (1999) J. Cell. Physiol. 179, 287-296[CrossRef][Medline] [Order article via Infotrieve] |
29. | Bonen, D. K., and Schmid, T. M. (1991) J. Cell Biol. 115, 1171-1178[Abstract] |
30. | Wu, Q., and Chen, Q. (2000) Exp. Cell Res. 256, 383-391[CrossRef][Medline] [Order article via Infotrieve] |
31. | Gibson, G., Lin, D. L., and Roque, M. (1997) Exp. Cell Res. 233, 372-382[CrossRef][Medline] [Order article via Infotrieve] |
32. | Zerega, B., Cermelli, S., Bianco, P., Cancedda, R., and Cancedda, F. D. (1999) J. Bone Miner. Res. 14, 1281-1289[Medline] [Order article via Infotrieve] |
33. | Hausdorff, W. P., Caron, M. G., and Lefkowitz, R. J. (1990) FASEB J. 4, 2881-2889[Abstract] |
34. | Mitchell, J., and Goltzman, D. (1990) Endocrinology 126, 2650-2660[Abstract] |
35. | Blind, E., Bambino, T., and Nissenson, R. A. (1995) Endocrinology 136, 4271-4277[Abstract] |