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INTRODUCTION |
During limb development, a cartilaginous template is formed from
mesenchymal condensations that perform the skeletal elements (1).
Subsequent longitudinal bone growth is dependent upon the process of
endochondral ossification whereby chondrocytes sequentially proliferate
and differentiate. Chondrocyte differentiation is marked by profound
physical and biochemical changes, including a 5-10-fold increase in
volume and expression of alkaline phosphatase and type X collagen (2,
3). The process of chondrocyte differentiation culminates in
calcification of the matrix and cellular apoptosis (4-6). The
calcified matrix subsequently serves as the template for primary bone formation.
Endochondral ossification is regulated by local growth factors
including PTHrP1, which has been defined as a critical
regulator of the rate of endochondral ossification. Mice null for
either PTHrP (7) or its receptor (8) display accelerated chondrocyte
differentiation and therefore abnormal endochondral bone formation. In
contrast, animals that overexpress PTHrP exhibit delay in chondrocyte
terminal differentiation (9). Humans with an activating mutation in the
PTH/PTHrP receptor have Jansen's metaphyseal chondrodysplasia, characterized by disorganization of the growth plate and delayed chondrocyte terminal differentiation (10). Thus, PTHrP signaling is
critical for normal growth plate morphology and function.
PTHrP receptor activation stimulates both protein kinase A
(PKA) and protein kinase C (PKC)
signaling. Activation of these signaling pathways mediates important
effects and in chondrocytes PKA signaling is associated with
proliferation, stimulation of proteoglycan synthesis, and inhibition of
alkaline phosphatase activity (11). PTHrP also stimulates phospholipase
C activity with metabolism of membrane phospholipids and production of
diacyl glycerol and inositol phosphate (12, 13). Inositol phosphate stimulates the release of intracellular calcium stores while the regeneration of diacylglycerol leads to activation of protein kinase C. While much is known about the upstream signaling events immediately
following receptor ligation, there is little information on the
downstream transcription factors involved in mediating PTHrP effects in
chondrocytes and their relationship with PKA and PKC signaling.
One of the potential downstream signaling targets of PKA is the cyclic
AMP response element-binding protein (CREB) (14). This transcription
factor is a CREB/ATF family member that is constitutively present in
the nucleus. CREB binds to a DNA consensus cAMP response element (CRE)
primarily as a homodimer, via a leucine zipper domain. CREB is
activated by PKA-mediated phosphorylation within its P-box domain (15),
which permits its interaction with p300/CBP and other coactivators,
leading to gene transcription. Thymocytes and T cells from transgenic
mice expressing a dominant-negative form of CREB specifically in these
cells display a profound proliferative defect and G1
cell-cycle arrest in response to a number of different activation
signals (16). ATF-2 deficiency, another member of the CREB/ATF family,
has been shown to induce chondrodysplasia and neurological
abnormalities in mutant mice (17). Although the role of CREB in
cartilage development or bone formation has not been investigated, the
finding that CREB KO mouse has a dwarf phenotype (18) combined with the
established important role of CREB in PKA mediated events in other
cells, suggests that this transcription factor may be involved in
regulating some of the critical events in chondrocyte differentiation.
PTHrP also activates the transcription factor AP-1, a complex formed
through interactions between Fos and Jun family members (19, 20). These
interactions are also mediated by leucine zipper domains to form
Fos/Jun heterodimers or Jun/Jun homodimers (21). The protein complex
binds to the phorbol 12-myristate 13-acetate response element
(TRE), a specific cis-acting DNA consensus sequence in the
promoter region of target genes. AP-1-regulated genes appear to have an
important role in skeletal physiology. Transgenic mice overexpressing
c-Fos develop bone and cartilaginous tumors (22), while Fos-Jun double
transgenic mice develop osteosarcomas at a higher frequency (23). PTHrP
stimulates c-Fos mRNA and protein synthesis in osteoblasts (24),
and in chondrocytes PTHrP was found to increase c-Jun and JunD mRNA
and protein levels (25). AP-1 activation has been shown to be mediated
by several different signaling pathways including PKA and PKC (26,
27).
In this work we have characterized CREB and c-Fos as two important
regulators of cartilage development and have investigated their
activation by PKA and PKC signaling. We have shown that their
activation occurs rapidly in response to PTHrP stimulation and that
interference with their function results in alteration of cartilage
phenotype produced by PTHrP. This represents the first report
documenting a critical role for CREB in cartilage development.
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MATERIALS AND METHODS |
Chondrocyte Cell Culture--
Embryonic cephalic sternal
chondrocytes (day 13) were prepared and cultured as described (28).
After isolation and primary culture for 5-7 days, floating cells were
plated in secondary cultures at 2.5 × 105
cells/cm2 in Dulbecco's modified Eagle's medium
containing 10% NuSerum IV (Collaborative Biomedical, Bedford MA), 4 units/ml hyaluronidase (Sigma), and 2 mM
L-glutamate (Sigma). After 6 days, upper sternal chondrocytes (USC) were harvested and plated in 6-well plates for the
transient transfection assay and alkaline phosphatase or 60-mm dishes
for Northern analysis. PTHrP (10
7 M) was
added to the cultures in some experiments.
Western Blotting--
Cell were washed with cold
phosphate-buffered saline and lysed on ice in Golden lysis buffer (29)
supplemented with protease inhibitor mixture tablets (Roche Molecular
Biochemicals), 1 mM sodium orthovanadate, 1 mM
EGTA, 1 mM NaF, 1 µM microcysteine. Insoluble
material was removed by centrifugation at 12,000 × g. The protein concentration of the soluble material was estimated using
Coomassie Plus Protein Assay kit (Pierce, Rockford, IL). 25 µg of
extracts was assayed by SDS-polyacrylamide gel electrophoresis. After
transfer to a nitrocellulose membrane (Schleicher and Schuell), the
blots were probed with the following antibodies: anti-CREB or
anti-phospho-CREB antibody (Upstate Biotechnology, Lake Placid, NY) at
a dilution 1:500, anti-c-Fos, anti-ATF-2, anti-CREM, and anti-ATF-1
(Santa Cruz) at a dilution 1:1000. In one case, the membrane was
striped and reprobed with anti-
-actin (Sigma) in a dilution
1:8000 to serve as a loading control. Horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse polyclonal antibodies (Bio-Rad) were used as secondary antibody. The immune complexes were detected using ECL (Amersham Pharmacia Biotech).
Gel-shift and Supershift Assay--
Nuclear protein extracts
were prepared as previously described (30) were analyzed by
electrophoretic mobility shift assay. 5 µg of this nuclear extracts
from upper sternal chondrocytes treated with PTHrP for 30 min, 1 h, 2 h, or 4 h were incubated with 1 µg of poly(dI-dC)
(Amersham Pharmacia Biotech) and 2 ng of 32P-end-labeled
CRE or TRE consensus oligos (Santa Cruz) and run on a 5%
polyacrylamide gel. Supershifts were performed with antibodies specific
for CREB/ATF and Fos/Jun family members. The incubation with antibodies
was 20 min prior to the addition of oligonucleotides.
Transfections and Luciferase Assay--
For this set of
experiments we have used Stratagene's Path detect in vivo
signal transduction pathway reporting systems, which are a series of
inducible reporter vectors that contain the firefly luciferase reporter
gene driven by a basic promoter (TATA box) plus a defined inducible
enhancer element: four repeats of CRE (CRE-Luc) or seven repeats of the
TRE (TRE-Luc). The transfection efficiency was controlled for by
co-transfecting pRL vector from Promega and determining the
Renilla uniformis luciferase activity. The upper
sternal chondrocytes were transiently transfected in serum-free media
(supplemented with insulin and triiodothyronine) (31) with these
plasmids using the transfection reagent Fugene-6 (Roche Molecular
Biochemicals). Twelve hours after transfection, a PTHrP
(10
7 M) treatment was given to the cells for
24 h. Two plasmids expressing the PKA catalytic subunit and
360-672 amino acids from MEKK were used as positive controls for the
CRE/TRE-Luc reporting systems, respectively. The following inhibitors
purchased from Calbiochem were used: H-89, 10 µM for PKA
inhibition, GO6976, 12 µM for PKC inhibition, 1 µM KN-93 for CaM kinase II, and 50 µM
PD98059 for MEKK. We have also used a dominant negative CREB and c-Fos
in the pRC-CMV vector, as previously described (32). In the
co-transfection experiments 1 µg of reporter and 1 µg of dominant
negative (or empty vector for control) were co-transfected in a ratio
1:3 with the transfection reagent. Other reporters used were c-Fos
promoter and several cyclin D1 promoter constructs from Dr. R. Pestell (Albert Einstein College of Medicine). Constructs employed were the
following: 1745-bp full-length cyclin D1 promoter, 66 bp of the
promoter encompassing the CRE site situated at 52 bp and 1745 mut and
66 mut that have the CRE site mutated as previously described (33).
Luciferase activity in cell lysate was measured using Luciferase assay
system (Promega) and an Optocomp luminometer (MGM Instruments).
Expression of ACREB and AFOS using Retroviral Systems--
Chick
embryonic fibroblasts grown in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum, 0.2% fetal chick serum and
penicillin/streptomycin were transfected with the replication competent
avian sarcoma virus RCASBP(A) alone and RCASBP(A) containing dominant
negative c-Fos and CREB inserts. Cells were passaged three times to
allow the spreading of the virus. At confluence, growth media was
changed to low serum (Dulbecco's modified Eagle's medium with 10%
NuSerum) for collection of virus. Viral supernatants were collected at
24-h intervals for 3 days. At the time of secondary plating,
chondrocytes were incubated with fresh viral supernatant for 48 h
followed by addition of PTHrP in some of the samples.
Northern Blot Analysis--
Total RNA was extracted from
cultures using the RNAeasy kit (Qiagen, Valencia, CA). 5 µg of the
total RNA was run on a 1.2% agarose gel containing 17.5% formaldehyde
and transferred to a GeneScreen Plus membrane (PerkinElmer Life
Sciences). The RNA was UV cross-linked to the membrane. Northern
analysis was performed using denaturing formaldehyde/agarose gels as
described (34). A synthetic type X oligonucleotide was end labeled as
previously described (34). Prehybridization was performed in QuickHyb
solution (Stratagene, La Jolla, CA) for 20 min at 68 °C.
Hybridization was done at 73 °C for 1 h. The blot was washed
twice for 15 min with 2 × SSC and 0.1% SDS, followed by a 30-min
wash with 0.1 × SSC and 0.1% SDS. The blot was exposed to X-Omat
AR film (Kodak, Rochester, NY) for autoradiography.
Alkaline Phosphatase Activity--
Alkaline phosphatase activity
was measured as previously described (35). Culture medium was aspirated
from chondrocytes cultured in 6-well plates, which then were rinsed
with 150 mM NaCl. One ml of reaction buffer containing 0.25 M 2-methyl-2-aminopropanol, 1 mM
MgCl2, and 2.5 mg/ml p-nitrophenyl phosphate
(Sigma) at pH 10.3 was added to the wells at 37 °C. The reaction was
stopped after 30 min by the addition of 0.5 ml of 0.3 M
Na3PO4 (pH 12.3). The alkaline phosphatase
activity was determined spectrophotometrically at 410 nm by comparison
with standard solutions of p-nitrophenol and an appropriate blank.
Fluorescence-activated Cell Sorter Analysis--
Flow cytometry
was performed using a fluorescence-activated cell sorter Calibur
cytometer and the Cell Quest (Becton-Dickinson, Franklin Lakes, NJ)
plotting program. 2-3 × 106 cells were resuspended
in 70% EtOH at 4 °C for at least 12 h. The cells were then
resuspended in 1 × phosphate-buffered saline with 1 mg/ml RNase
and incubated for 30 min at room temperature. Finally cells were
stained with propidium iodide (10 µg/ml in phosphate-buffered saline)
and run through the flow cytometer.
[3H]Thymidine Incorporation--
Chondrocytes
plated at 200,000 cells/well in a 6-well plate were treated with PTHrP
for 24 h and incubated with 2 µCi/ml [3H]thymidine
(PerkinElmer Life Sciences) in the last 4 h. Cells were washed
with cold 5% trichloroacetic acid two times and then the cells were
left in 0.5 ml of 1 N NaOH, 0.1% Triton X-100 overnight at
4 °C. 100-µl aliquots were added to 4 ml of Ecoscint fluid and
assayed for 3H incorporation.
Statistics--
Statistical comparisons were made between
the groups using a way analysis of variance (ANOVA). Significance was
considered present when the p value was less than 0.05 and
is denoted in each of the figures.
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RESULTS |
Activation of Signaling Pathways by PTHrP--
The effects of
PTHrP stimulation on CREB/ATF activation were investigated in sternal
chondrocytes at various times following treatment over a 2-h time
course experiment. The level of CREB protein remained constant as
determined by Western blotting (Fig. 1A). However, phosphorylated
CREB (pCREB) levels were markedly increased following this treatment.
The increase in pCREB was detectable within 5 min, maximal by 30 min,
and returned to baseline levels 2 h after PTHrP stimulation (Fig.
1A). Following PTHrP stimulation, there was also an increase
in c-Fos protein levels that was observed as early as 30 min, was
maximal by 1 h, and returned to basal levels by 4 h (Fig.
1B). To determine the effect of PTHrP stimulation on CREB
DNA binding activity in chondrocytes, gel mobility shift assays were
performed. These experiments revealed minimal differences over a 4-h
time course following PTHrP stimulation (Fig.
2A). To characterize this
protein-DNA complex, supershift experiments using specific antibodies
to members of the CREB/ATF family were performed. In this experiment,
only CREB binding to this consensus sequence was detected (Fig.
2B), although ATF-2 and CREM proteins could be detected in
these extracts by Western blot (Fig. 2C). These findings
suggest that CREB protein levels and DNA binding activity remain
constant following PTHrP treatment. However, PTHrP may modulate gene
expression in sternal chondrocytes by inducing CREB phosphorylation and
therefore increasing its transcriptional activity.

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Fig. 1.
PTHrP induces phosphorylation of CREB and
stimulates c-Fos protein production. Phosphorylation of CREB was
measured at different time points following PTHrP (10 7
M) treatment. Protein samples (25 µg/lane) were subjected
to Western blot analysis using two different polyclonal rabbit IgG
antibodies: one specific to the phosphorylated form of CREB
( -pCREB), the other that recognizes both phosphorylated and
un-phosphorylated forms of CREB ( -CREB) (A). PTHrP
stimulation of c-Fos protein was assayed by Western blot using an
antibody directed against c-Fos protein (B). A control for
loading equal amounts of protein was performed by stripping the blot
and reprobing it with an anti- -actin antibody.
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Fig. 2.
PTHrP induces AP-1, but not CREB DNA binding
activity. Nuclear extracts (5 µg/lane) obtained from USC
stimulated with PTHrP for various time points, were incubated with or
without anti-c-Fos antibody, followed by addition of a radioactive
labeled consensus AP-1 probe (A). Nuclear extracts (5 µg/lane) from USC were also incubated with radioactively labeled CRE
oligonucleotides and then gel shift assay was performed (A).
Nuclear extracts (5 µg/lane) from USC were preincubated with
antibodies against different CREB/ATF or Fos family members, before
addition of radioactive labeled CRE or AP-1 oligonucleotides and then
gel shift assay was performed (B). Western blots with
antibodies against different CREB/ATF family members were done with
whole cell lysates from USC (C).
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To assess AP-1 activation following PTHrP stimulation, gel mobility
shift assays were performed using a probe containing the TRE consensus
sequence. PTHrP stimulated a marked increase in DNA binding (Fig.
2A). The increase was detectable at 30 min, peaked at 1 h, and remained elevated 4 h following PTHrP treatment. Addition
of an antibody directed against c-Fos protein inhibited DNA binding
(Fig. 2A), showing the presence of c-Fos protein in the gel
mobility shift complex, consistent with the increase in c-Fos protein
expression observed on Western blot. In contrast, incubation with
antibodies directed against Fra1, ATF-2, and CREM did not affect DNA
binding (Fig. 2B).
To determine whether this apparent stimulation of CREB and AP-1
signaling is associated with an increase in gene transcription, sternal
chondrocytes were transfected with CRE and TRE luciferase reporter
constructs and treated with PTHrP (Fig.
3). PTHrP (10
7
M) treatment resulted in transcriptional activation of both
the TRE (23-fold) and CRE (103-fold) constructs. As a positive control we co-transfected the reporter constructs with either activated MAPK
kinase kinase (MEKK) or PKA constructs. In these experiments the MEKK
construct was a more potent stimulator of the TRE reporter, while the
PKA construct caused about a 40-fold stimulation of both the CRE and
the TRE reporters (Fig. 3A). These latter findings indicate
the presence of cross-talk between these signaling pathways in
chondrocytes following PTHrP stimulation. Furthermore, PKA signaling in
these cells results in activation of both CRE and TRE mediated
transcription.

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Fig. 3.
Pharmacological inhibition of PTHrP-induced
CREB and AP-1-mediated transcription. USC were transfected in
serum-free media with 1 µg of luciferase reporter: CRE-Luc or
TRE-Luc, respectively, and stimulated with PTHrP (10 7
M) for 24 h or co-transfected with 1 µg of reporter
and 2 ng of the MEKK or PKA catalytic subunit expression constructs for
comparison (A). The cells were then assayed for luciferase
activity (* denotes statistical significance at p 0.005, when compared with untreated control). USC were transfected in
serum-free media with 1 µg of CRE-Luc (B) or TRE-Luc
(C) and pretreated with pharmacological inhibitors as
indicated, for 45 min at 37 °C, followed by PTHrP treatment
(10 7 M) for 24 h in the presence of
inhibitors. The relative luciferase activities (mean ± S.E.;
n = 3) are presented. (* denotes statistical
significance at p 0.005 and # denotes statistical
significance at p 0.01 when compared with untreated
control; ** denotes statistical significance at p 0.005 when compared with the PTHrP-treated sample.)
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To determine the relative effects of the PKA, PKC, mitogen-activated
protein kinase, and calmodulin kinase signaling pathways on CRE
activation, the CRE reporter was transfected into sternal chondrocytes
and the cultures were treated with PTHrP in the presence and absence of
pharmacological inhibitors of these various pathways (Fig.
3B). Inhibition of PKA signaling with H89 resulted in a 95%
inhibition of CRE activation, consistent with the role of PKA as a
potent inducer of CREB phosphorylation. Inhibitors of PKC (GO6976, 45%
inhibition), mitogen-activated protein kinase (PD98059, 27%
inhibition), and calmodulin kinase (KN93, 17% inhibition) all had less
potent effects. PKA inhibition also had the greatest effect on the TRE
reporter (80% inhibition), while inhibition of MAP kinase signaling
resulted in a 37.7% decrease in luciferase activity. In contrast,
inhibition of PKC and calmodulin kinase did not affect PTHrP-mediated
activation of the TRE reporter (Fig. 3C).
To confirm our findings regarding CREB and AP-1 transcriptional
activation following PTHrP treatment, constructs expressing dominant
negative c-Fos (AFOS) and CREB (ACREB) were each co-transfected into
sternal chondrocytes with reporter constructs for the CRE and TRE (Fig.
4). The ACREB construct completely
inhibited CRE activation by PTHrP, while the AFOS construct did not
inhibit PTHrP activation of the CRE (Fig. 4A). Moreover, the
AFOS construct induced an increase in the PTHrP stimulation of CRE
reporter. A possible explanation for this finding is that AP-1
inhibition may result in greater availability of the transcriptional
coactivator, CBP (CREB-binding protein), for CREB-mediated signaling.
In contrast, both ACREB and AFOS constructs inhibited activation of the
TRE reporter following stimulation by PTHrP (Fig. 4B).
Co-transfection of both constructs reduced the detectable luciferase
activities to below basal levels. These findings further confirm PTHrP
activation of PKA and phosphorylation of CREB, leading to CRE dependent
transcription.

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Fig. 4.
AFOS and ACREB inhibition of PTHrP activation
of CRE-Luc, TRE-Luc, and the c-Fos promoter. USC were transfected
in serum-free media with 1 µg of luciferase reporter: CRE-Luc
(A), TRE-Luc (B), or c-Fos promoter
(C); and 1 µg of empty vector, AFOS, ACREB, or a
combination of both (0.5 + 0.5 µg). Twelve hours after transfection,
cells were washed, followed by 24 h treatment with or without
PTHrP (10 7 M). The relative luciferase
activities (mean ± S.E.; n = 3) are presented. (*
denotes statistical significance at p 0.005 when
compared with untreated control, ** denotes statistical significance at
p 0.005 when compared with PTHrP treated
sample.)
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In contrast, activation of AP-1 by CREB is likely indirect and may be
mediated by stimulation of c-Fos transcription. To test this, a c-Fos
promoter-reporter construct was transfected into sternal chondrocytes.
PTHrP treatment resulted in a 91-fold stimulation in luciferase
activity (Fig. 4C). Co-transfection of these cultures with
the ACREB construct blocked this PTHrP stimulation of the cFos
promoter, while the AFOS construct had no effect, further supporting a
model of PTHrP activation of AP-1 through CREB-dependent transcription of c-Fos.
PTHrP Effects on Maturation Mediated by CREB and
AP-1--
PTHrP regulates the rate of chondrocyte terminal
differentiation. To see if PTHrP inhibits terminal differentiation
through CREB or AP-1 signaling, we investigated the expression of
markers of chondrocyte maturation, type X collagen (colX)
mRNA expression, and alkaline phosphatase activity, in cell
cultures following PTHrP treatment. Short-term experiments were
performed using transiently transfected chondrocytes (2 days), while
longer term experiments (7 days) were performed with chondrocytes
infected with a replication competent retrovirus that permitted
sustained expression in a larger percentage of cells. In the absence of
PTHrP, ACREB, and AFOS had minimal effects in both the
transiently transfected and retrovirally infected cultures (Fig.
5, A and B).
However, in the presence of PTHrP, transient transfection of ACREB and
AFOS constructs stimulated type X collagen (colX) mRNA
expression, 1.6- and 1.3-fold, respectively (Fig. 5A),
compared with vector-transfected control chondrocytes. These signaling
pathways appeared to be synergistic, since co-transfection of both
ACREB and AFOS resulted in even greater colX mRNA
expression (5.2-fold). Effects of the dominant negative signaling
molecules were much greater when the constructs were introduced by
retroviral infection. Alone, both ACREB and AFOS infection resulted in
a larger induction of colX in PTHrP-treated cultures
compared with minimal increases in retroviral infected control samples
(Fig. 5B).

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Fig. 5.
PTHrP inhibits collagen type X collagen
(col X) expression that is relieved by AFOS and ACREB
expression. For the transient transfection experiments
(A), USC were transfected in growth media with 2.5 µg of
empty vector, AFOS, ACREB, or both, in a ratio 1:4 with Superfect
transfection reagent (Qiagen). Twelve hours after transfection, cells
were washed and treated with PTHrP (10 7 M) or
left untreated for 48 h. Changes in colX were measured
by Northern blot of mRNA. The ethidium bromide-stained 18 S rRNA
was used as a loading control. For the long-term experiments
(B), USC were incubated for 2 days with fresh viral
supernatant of: empty RCAS, or RCAS carrying either AFOS or ACREB.
Chondrocytes were cultured for 7 days in the presence or absence of
PTHrP.
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In PTHrP-treated cultures, transient transfection with both ACREB
and AFOS resulted in only small increases in alkaline phosphatase activity (data not shown). Much larger effects on alkaline phosphatase activity were observed when the cultures were infected with retroviral constructs, consistent with the findings observed with the other maturation marker, type X collagen. In the PTHrP-treated cultures, ACREB doubled and AFOS tripled alkaline phosphatase activity (Fig. 6) compared with smaller increases in
untreated samples (1.3- and 1.7-fold increases). Collectively, these
findings demonstrate that PTHrP effects on maturation are mediated by
both CREB and AP-1 signaling. Furthermore, while the findings suggest
basal AP-1 and CREB signaling, these transcription factors are much more relevant in the presence of PTHrP.

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Fig. 6.
PTHrP inhibition of alkaline phosphatase
activity is relieved by AFOS and ACREB expression. USC were
incubated for 2 days with fresh viral supernatant of respective viral
constructs: empty RCAS, or RCAS carrying either AFOS or ACREB.
Chondrocytes were cultured for 7 days in the presence or absence of
PTHrP (10 7 M). Each treatment group
represents the mean alkaline phosphatase activity of triplicate samples
normalized for protein concentration. (* denotes statistical
significance at p 0.025 and denotes
statistical significance at p 0.0001 when compared
with control.)
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CREB and AP-1 Effects on Chondrocyte Proliferation--
To
evaluate the effects of PTHrP on proliferation, sternal chondrocytes
were treated with PTHrP and analyzed by fluorescence-activated cell
sorter 24 h latter (Table I). PTHrP
treatment resulted in more than a 2-fold increase in the number of
cells entering the cell cycle and undergoing DNA synthesis (S + G2 + M). Since cyclin D1 is a required for the transition
from the G1 to S phase, we next investigated the effect of
PTHrP and downstream signals on the cyclin D1 promoter (Fig.
7). PTHrP treatment caused a 5.6-fold increase in luciferase activity by the full-length (1745 bp) cyclin D1
promoter (Fig. 7A). A point mutation at the CRE site
(position 52 bp) results in a 30% reduction in PTHrP mediated
stimulation. Similar experiments with a truncated 66-bp promoter
construct that includes the CRE-binding site, results in a 2.1-fold
stimulation following PTHrP treatment. This induction was lost in a
mutant construct containing a point mutation that disrupts the CRE
consensus sequence. Co-transfection of the cultures with the AFOS or
ACREB constructs, either alone or in combination, further confirmed a
role for the CRE site in PTHrP-mediated cyclin D1 promoter activation. Transfection of the dominant negative ACREB construct completely inhibited the PTHrP induction of both the full-length and truncated 66-bp cyclin D1 promoter. In contrast, transfection of the AFOS construct did not significantly alter the induction of the full-length construct and caused only a partial reduction in the truncated promoter
(Fig. 7B). These findings confirm the effects of PTHrP on
proliferation and suggest that the effects on cyclin D1 are primarily
related to CREB signaling.
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Table I
PTHrP stimulation increases the number of chondrocytes entering the
cell cycle
USC were stimulated with PTHrP for the indicated time points and cell
cycle analysis was performed by fluorescence-activated cell sorter as
described under "Materials and Methods."
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Fig. 7.
PTHrP activation of the cyclin D1 promoter is
diminished by perturbation of CREB signaling. In A, USC
were transfected in serum-free media with 1 µg of the following
luciferase reporters: 1745 bp, 1745-bp mut, 66 bp, 66-bp mut. After
transfection, cells were washed, and incubated with or without PTHrP
(10 7 M) for 24 h. The relative
luciferase activities (mean ± S.E.; n = 3) are
presented. (* denotes statistical significance at p 0.025 when compared with the PTHrP-treated sample for the 1745-bp
reporter, ** denotes statistical significance at p 0.005 when compared with PTHrP-treated sample for the 66-bp reporter.)
In B, USC were transfected in serum-free media with 1 µg
of the luciferase reporters: 1745 or 66 bp and 1 µg of the empty
vector, AFOS, ACREB, or both. After transfection, cells were washed,
and incubated with or without PTHrP (10 7 M)
for 24 h. The relative luciferase activities (mean ± S.E.;
n = 3) are presented. (* denotes statistical
significance at p 0.005 when compared with the
untreated control, ** denotes statistical significance at
p 0.005 when compared with PTHrP-treated
sample.)
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To further define the role of AP-1 and CREB on PTHrP mediated
proliferative effects, chondrocytes were treated with PTHrP in cultures
transfected with AFOS or ACREB alone or in combination (Fig.
8). ACREB resulted in a 20% reduction in
thymidine incorporation and 25% AFOS reduction. A statistically
significant 30% reduction in thymidine incorporation
(p < 0.05) was observed when cultures were
co-transfected with both constructs. Thus, similar to the effects
observed on differentiation, the regulation of chondrocyte proliferation by PTHrP is mediated by both AP-1 and CREB signaling, with interactive and synergistic effects observed between these pathways.

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Fig. 8.
PTHrP stimulation of chondrocyte
proliferation is inhibited by AFOS and ACREB. USC were transfected
in growth media with 1 µg of the empty vector, AFOS, ACREB, or
together in a ratio 1:3. Twelve hours after transfection, cells were
washed and treated with PTHrP (10 7 M) for
24 h. [3H]Thymidine labeling was performed as
described under "Experimental Procedures." (* denotes statistical
significance at p 0.005 when compared with the
untreated control, ** denotes statistical significance at
p 0.05 when compared with PTHrP-treated
sample.)
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DISCUSSION |
While PTHrP is known to be a critical regulator of
chondrocyte proliferation and differentiation, the signaling pathways
through which this factor acts remain to be elucidated. The current
article demonstrates that both CREB and AP-1 activation are critical to PTHrP signaling in chondrocytes. In these chondrocytes, CREB DNA binding activity is constitutive. However, PTHrP treatment leads to
CREB phosphorylation within 5 min. In contrast AP-1 DNA binding activity is induced by PTHrP, with effects initially observed 1 h
following treatment. Consistent with these findings PTHrP activated CRE
and TRE reporter constructs, and both signaling pathways were found to
be important mediators of PTHrP effects on chondrocyte phenotype.
Alone, PTHrP suppressed maturation and stimulated proliferation of the
chondrocyte cultures. However, in the presence of dominant negative
inhibitors of CREB and c-Fos, these PTHrP effects were suppressed and
chondrocyte maturation was accelerated. Moreover, in combination, the
effects of dominant negative cFos and CREB were synergistic, suggesting
interaction between these signaling pathways during chondrocyte differentiation.
Prior studies have demonstrated that CREB binds constitutively to
the CRE consensus sequence and is activated following phosphorylation by upstream kinases (36, 37). The cAMP-activated protein kinase A (PKA)
dependent pathway is the classic activator of CREB and acts through
phosphorylation of a critical serine residue located at position
133 (15, 38). However, other signals, including the calcium/calmodulin
signaling (CaM kinases II and IV) (39), PKC signaling (40), and Ras
signaling mediated by the serine/threonine kinase RSK2 also regulate
CREB through phosphorylation at Ser133 or other sites (36,
41). In our pharmacological studies with inhibitors of these kinases,
we found that the PKA pathway is the one through which the most potent
signal is transduced.
Similarly, AP-1 activation is also stimulated by numerous
pathways, but here we found that following PTHrP stimulation, PKA signaling is most critical for its transcriptional activity. Inhibition of PKA signaling with the drug H-89, or co-transfection of ACREB, resulted in nearly complete inhibition of AP-1 transcriptional activation, as measured by reporter assay. Maximal transcriptional activation of AP-1 regulated genes is mediated by c-Fos/c-Jun heterodimers, and is highly dependent upon c-Fos protein expression. Previously, it has been shown that c-Fos transcription is induced by
CREB activation (42-44). Here we find evidence that this mechanism is
also operative in chondrocytes following PTHrP stimulation. Our
transient transfection experiments here demonstrated the induction of a
c-Fos promoter by CREB as previously described (43, 45, 46). Consistent
with these findings was the increase in c-Fos protein and the delay in
AP-1 DNA binding activity, which was maximally increased at 1 h
following PTHrP stimulation, while an increase in CREB phosphorylation
could be detected within 5 min. Thus, our data support a mechanism
whereby PTHrP rapidly activates CREB through PKA signaling, and results
in the subsequent transcriptional activation of
PKA/CREB-dependent genes. One of these genes is likely to
be c-Fos, which following its de novo synthesis, enhances
AP-1 signaling (43, 45, 46). These events result in a delayed
enhancement of AP-1 signaling through c-Fos transcription, and are
consistent with PTHrP effects reported in other cell systems (43,
44).
The primary role for cyclic AMP/CREB signals in the regulation of
chondrocyte differentiation is supported by genetic experiments. CREB
knockout mice has a dwarf phenotype and die in the neonatal period of
respiratory distress, similar to PTHrP knockout animals. Recently, a
mutant PTH receptor with normal phospholipase C signaling, but
deficient G
s signaling has been expressed in chimeric
mice (47). Cells with deficient G
s signaling underwent
premature maturation in the growth plate, while wild type cells had a
normal rate of differentiation. In contrast, mice that express a mutant PTHrP receptor with normal G
s signaling, but deficient
phospholipase C signaling, have normal size, are fertile, and do not
have reduced rates of chondrocyte differentiation (48). These genetic
experiments support our findings in that cyclic AMP/PKA signal
transduction regulates the activation of the two important
transcription factors, CREB and AP-1, that mediate PTHrP effects in
chondrocytes. In contrast, PKC signaling appears less relevant for
these events both in vivo and in vitro.
To establish whether either CREB or AP-1 transcriptional
regulation is important for the effect of PTHrP on chondrocyte
phenotype, experiments were performed using ACREB and AFOS. In the
absence of PTHrP, the effects of these signaling inhibitors was
minimal, suggesting relatively low basal activation of these signaling pathways. However, large effects were observed in the presence of
PTHrP, consistent with the activation of CREB and induction of c-Fos by
PTHrP. Although the magnitude of the individual signaling effects was
greater in retroviral infected cultures, the transient transfection
experiments demonstrated interactive effects between CREB and AP-1
signaling in chondrocyte maturation.
Chondrocyte maturation was determined by the expression of type X
collagen and alkaline phosphatase, both of which are markedly elevated
during chondrocyte differentiation and are important for the normal
process of endochondral ossification (49, 50). As a suppresser of
chondrocyte differentiation, PTHrP inhibited both type X collagen and
alkaline phosphatase activity. Here we used two strategies to examine
the role of CREB and AP-1 in this process: use of transient
transfection and long-term retroviral expression vectors. Although
transient transfection allows for modest expression for a shorter
period of time, it also permits use of both dominant-negative
constructs in combination. Thus, the effects we observed in these
experiments were smaller but we were able to define synergistic effects
and show interdependence between AP-1 and CREB mediated effects. In
contrast, the effects observed in the retrovirally infected cultures
were greater and further confirmed the importance of these
transcription factors in PTHrP mediated signaling events. The phenotype
that we obtained by inhibiting CREB and AP-1 signaling is similar to
the phenotype of the PTHrP knockout mouse; accelerated chondrocyte
differentiation of chondrocytes, resulting in premature endochondral
ossification at many sites (7, 51).
Although, PTHrP has an accepted role as an inhibitor of maturation, its
role in proliferation is controversial. While Lee et al.
(52), observed similar expression of H4-histone mRNA (marker for
S-phase of the cell cycle) in wild-type and PTHrP-deficient animals,
others have shown diminished [3H]thymidine incorporation
and a marked reduction in the number of proliferating chondrocytes in
the knockout animals (51, 53). In vitro studies have
supported an important role for PTHrP in chondrocyte proliferation
(54-57). In the current experiments, PTHrP doubled the number of cells
entering mitosis, and stimulated [3H]thymidine
incorporation and cyclin D1 promoter activity in chondrocytes. This
increase was diminished by inhibiting AP-1 and CREB signaling. One
crucial step for G1 progression through the cell cycle
appears to be induction of cyclin D1 expression during growth factor
stimulated cell cycle re-entry (33). Consistent with reports in the
literature (58), our results point out a role of CREB/ATF and AP-1
family members in the activation of cyclin D1 following PTHrP stimulation.
Chondrocytes integrate a complex array of signals during the process of
differentiation. PTHrP is one of the major regulators but other factors
such as Indian hedgehog, bone morphogenetic proteins, and transforming
growth factor-
also affect the differentiation through overlapping
or complementary pathways (54, 59, 60). Our experiments show that both
CREB and AP-1 transcription factors are important regulators of the
rate of chondrocyte differentiation. Additional studies will be
required to define how other growth factors interact with CREB and AP-1
signaling and thereby modulate PTHrP effects during chondrocyte differentiation.