Annexin-mediated Ca2+ Influx Regulates Growth Plate
Chondrocyte Maturation and Apoptosis*
Wei
Wang,
Jinping
Xu, and
Thorsten
Kirsch
From the Department of Orthopaedics, University of Maryland School
of Medicine, Baltimore, Maryland 21201
Received for publication, August 29, 2002, and in revised form, November 18, 2002
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ABSTRACT |
Maturation of epiphyseal growth plate
chondrocytes plays an important role in endochondral bone formation.
Previously, we demonstrated that retinoic acid (RA) treatment
stimulated annexin-mediated Ca2+ influx into growth
plate chondrocytes leading to a significant increase in cytosolic
Ca2+, whereas K-201, a specific annexin Ca2+
channel blocker, inhibited this increase markedly. The present study
addressed the hypothesis that annexin-mediated Ca2+ influx
into growth plate chondrocytes is a major regulator of terminal
differentiation, mineralization, and apoptosis of these cells. We found
that K-201 significantly reduced up-regulation of expression of
terminal differentiation marker genes, such as cbfa1,
alkaline phosphatase (APase), osteocalcin, and type I collagen in
RA-treated cultures. Furthermore, K-201 inhibited up-regulation of
annexin II, V, and VI gene expression in these cells. RA-treated chondrocytes released mineralization-competent matrix vesicles, which
contained significantly higher amounts of annexins II, V, and VI as
well as APase activity than vesicles isolated from untreated or
RA/K-201-treated cultures. Consistently, only RA-treated cultures showed significant mineralization. RA treatment stimulated the whole
sequence of terminal differentiation events, including apoptosis as the
final event. After a 6-day treatment gene expression of bcl-2, an anti-apoptotic protein, was down-regulated,
whereas caspase-3 activity and the percentage of TUNEL-positive cells were significantly increased in RA-treated cultures compared with untreated cultures. Interestingly, the cytosolic calcium chelator BAPTA-AM and K-201 protected RA-treated chondrocytes from undergoing apoptotic changes, as indicated by higher bcl-2 gene
expression, reduced caspase-3 activity, and the percentage of
TUNEL-positive cells. In conclusion, annexin-mediated Ca2+
influx into growth plate chondrocytes is a positive regulator of
terminal differentiation, mineralization, and apoptosis events in
growth plate chondrocytes.
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INTRODUCTION |
Maturation of epiphyseal growth plate chondrocytes, which plays an
important role during endochondral ossification, is accompanied by
major changes of chondrocyte morphology, biosynthetic activities, and
energy metabolism. These processes involve an ordered progression of
various cell differentiation stages, including proliferation, hypertrophic differentiation, terminal differentiation, and ultimately programmed cell death (apoptosis) (1, 2). During normal development
these sequential events are under the strict control of local and
systematic factors such as hormones and growth factors. If these
processes, however, occur during pathological conditions, they can
result in serious cartilage or bone defects. Evidence of endochondral
ossification is also seen during osteophyte formation in osteoarthritic
cartilage (3, 4). Terminal differentiation of growth plate chondrocytes
is an essential process, which primes the cartilage skeleton for its
subsequent invasion by osteoblasts and its replacement by a bone
matrix. Despite the obvious importance of these terminal
differentiation events still little is known about mechanisms
regulating these processes.
cbfa1, a member of the runt domain family of transcription
factors, was originally discovered as a key transcription factor, which
controls osteoblast differentiation. In cbfa1-null mice no
endochondral and intramembranous bone formation occurs due to an arrest
in osteoblast differentiation (5-8). Recent studies have indicated
that cbfa1 also plays an important regulative role in
terminal chondrocyte maturation. Transgenic mice, which overexpress cbfa1 in non-hypertrophic chondrocytes, display an
acceleration of endochondral ossification. Overexpression of
cbfa1 in chondrocytes of cbfa1-null mice
partially rescued the abnormalities of cbfa1-null mutant
mice. In particular, it rescued hypertrophic chondrocyte differentiation in the humerus and femur (9). Thus, cbfa1
seems to play dual functions in endochondral bone formation; it plays a
key role in osteoblast differentiation from mesenchymal precursor cells, and it has the ability to stimulate hypertrophic and terminal chondrocyte differentiation.
Chondrocyte hypertrophy and terminal differentiation are accompanied by
an increase in cytosolic calcium, [Ca2+]i
(10-12). Calcium is recognized as an important regulator of many
cellular processes, and it controls a diverse range of cell functions,
including adhesion, motility, gene expression, cell differentiation,
and proliferation. For example, the amplitude and duration of calcium
signals control differential activation of different transcription
factors in B lymphocytes (13). Calcium has been shown to play several
roles in vesiculation and the formation of vesicles. For example,
Iannotti et al. (14) have shown a correlation between
increasing [Ca2+]i and the release of matrix
vesicles (14). Matrix vesicles are small membrane-enclosed particles,
which are released from the plasma membrane of growth plate
chondrocytes and which initiate the mineralization process (15). We
have previously shown that
RA,1 which stimulates
terminal differentiation and mineralization of hypertrophic
chondrocytes, induces Ca2+ influx into growth plate
chondrocytes causing the release of mineralization-competent matrix
vesicles (16).
Annexins II, V, and VI, which are highly expressed in hypertrophic and
mineralizing growth plate cartilage, are major components of matrix
vesicles (17-19). Annexins II, V, and VI belong to a family of
Ca2+- and phospholipid-binding proteins. In addition,
annexins II, V, and VI have been shown to form Ca2+
channels in phospholipid bilayers or in liposomes (20). They also form
Ca2+ channels in matrix vesicles enabling Ca2+
influx into these particles as a possible initial step for the formation of the first mineral phase within the vesicle lumen (21).
Careful studies have provided evidence that apoptosis is the final fate
of terminally differentiated growth plate chondrocytes. Chondrocyte
apoptosis in the growth plate is centered at the site of the transition
of cartilage to bone (2, 22). These apoptotic chondrocytes show
characteristic hallmarks of apoptosis, including condensed nuclei,
DNA fragmentation, activation of caspase cascade, and
phosphatidylserine externalization. Previous studies have indicated
that elevation of [Ca2+]i is involved in the
induction of apoptosis. For example, apoptosis of cultured human
endothelial cells was inhibited by chelating extracellular calcium with
EGTA or by inhibiting the calcium influx by calcium channel blockers.
It has been suggested that elevated [Ca2+]i leads
to activation of proteases, lipases, and nucleases. All these actions
can contribute to cell death (23-25).
We have provided evidence that RA promotes annexin channel formation in
growth plate chondrocytes and that annexin-mediated Ca2+
influx into these cells controls Ca2+ homeostasis (16). To
determine the role of annexin-mediated alteration of Ca2+
homeostasis in terminal differentiation, mineralization, and apoptosis
of growth plate chondrocytes, we cotreated growth plate chondrocytes
isolated from the hypertrophic zone of day 19 embryonic chicken growth
plate cartilage with RA and K-201, a specific annexin channel blocker,
or RA and BAPTA-AM, a cytosolic Ca2+ chelator, and analyzed
the rate of terminal differentiation, mineralization, and apoptosis in
these cells.
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EXPERIMENTAL PROCEDURES |
Chondrocyte Culture--
Chondrocytes were isolated from the
hypertrophic zone of day 19 embryonic chick tibia growth plate
cartilage as described previously (19). Briefly, sliced growth plate
cartilage was digested with 0.25% trypsin and 0.05% collagenase for
5 h at 37 °C. Cells were plated at a density of 3 × 106 in 10-cm tissue culture dishes and grown in monolayer
cultures in Dulbecco's modified Eagle's medium (Invitrogen)
containing 5% fetal calf serum (Hyclone, Logan, UT), 2 mM
L-glutamine (Invitrogen), and 50 units/ml penicillin and
streptomycin (complete medium). After cultures reached confluency,
chondrocytes were cultured in the presence of 1.5 mM
phosphate and in the absence or presence of (a) 35 nM RA (Sigma-Aldrich), (b) 35 nM RA
and 2 µM 1,4-benzothiazepine derivative K-201 (JTV519)
(provided by Drs. Noboro Kareko, Dokkyo University, Tochigo, Japan and
Toshizo Tanaka, Japan Tobacco Inc., Osaka, Japan), and (c)
35 nM RA and 10 µM BAPTA-AM (Molecular Probes
Inc., Eugene, OR).
Isolation of Total RNA and Real Time PCR--
Total RNA was
isolated from untreated, RA-treated, RA/K-201-treated, and
RA/BAPTA-treated chondrocyte cultures after 1-, 3-, 5-day treatments
using RNeasy Mini Kit (Qiagen, Stanford, CA). 1 µg of RNA was
reverse-transcribed using Ominiscript RT Kit (Qiagen). A 1:100 dilution
of the resulting cDNA was used as the template to quantify the
relative content of mRNA by real time PCR (ABI PRISM 7700 sequence
detection system) using respective primers and SYBR Green. The
following primers for real time PCR were designed using Primer Express
software. Annexin II: forward primer,
5'-CATGCCTATCTGCTCTTCGTT-3'; reverse primer,
5'-AGCCACCACACCGTCCATAA-3'; annexin V: forward primer,
5'-AGAGACATCAGGCCATTTTCAGA-3'; reverse primer,
5'-CTGCCATCAGGATCTCTATTTGC-3'; annexin VI: forward primer,
5'-GCGGCTGATTGTAAGCTTGAT-3'; reverse primer, 5'-GTCGGTGGTCCAGCACTTA-3';
type I collagen (
1(I)): forward primer, 5'-CAGCCGCTTCACCTACAGC-3';
reverse primer, 5'-TTTTGTATTCAATCACTGTCTTGCC-3'; type II collagen:
forward primer, 5'-GGCAATAGCAGGTTCACGTAC-3'; reverse primer,
5'-CGATAACAGTCTTGCCCCACTT-3'; type X collagen: forward primer,
5'-AGTGCTGTCATTGATCTCATGGA-3'; reverse primer, 5'-TCAGAGGAATAGAGACCATTGGATT-3'; cbfa1: forward primer,
5'-CGCGGAGCTGCGAAAT-3'; reverse primer, 5'-ACGAATCGCAGGTCATTGAAT-3';
APase: forward primer, 5'-CCCTGACATCGAGGTGATCCT-3'; reverse primer,
5'-GGTACTCCACATCGCTGGTGTT-3'; osteocalcin: forward primer,
5'-TCGCGGCGCTGCTCACATTCA-3'; reverse primer,
5'-TGGCGGTGGGAGATGAAGGCTTTA-3'; bcl-2: forward primer, 5'-GGTGACCCGAAGCATCAAA-3'; reverse primer, 5'-AGCGACACGAAAACCCAAAC-3'. PCR reactions were performed with the TaqMan PCR master mix kit (Applied Biosystems) using 1 cycle at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and
60 °C for 1 min. The 18 S RNA was amplified at the same time and
used as an internal control. The cycle threshold values for 18 S RNA and that of the samples were measured and calculated by computer software. Relative transcript levels were calculated as
x = 2

Ct, in which

Ct =
E
C and
E = Ctexp
Ct18 S;
C = Ctctl
Ct18 S.
Isolation of Matrix Vesicles--
Matrix vesicles were isolated
from chondrocyte cultures after a 6-day treatment by enzymatic
digestion and ultracentrifugation as described previously (19).
Measurement of APase Activity and Protein Content--
APase
activity was measured using p-nitrophenyl phosphate
(Sigma-Aldrich) as a substrate as described previously (19). Protein content was analyzed by the BCA protein assay from Pierce.
SDS-PAGE and Immunoblotting--
To determine the amounts of
annexin II, V, and VI in matrix vesicles, vesicle fractions (total
protein of 30 µg) were subjected to SDS-PAGE and immunoblotted with
primary antibodies specific for annexins II, V, and VI. Samples were
dissolved in 4× NuPAGE SDS sample buffer (Invitrogen). Prior to
electrophoresis, the reducing reagent was added to the sample solution,
denatured at 70 °C for 10 min, and analyzed by electrophoresis in
10% Bis-Tris gels following the NuPAGE electrophoresis protocols.
Samples were electroblotted onto nitrocellulose filters after
electrophoresis. After blocking with a solution of low fat milk
protein, blotted proteins were immunostained with primary antibodies
followed by peroxidase-conjugated secondary antibody, and the signal
was detected by enhanced chemiluminescene (Pierce).
Alizarin Red S Staining--
To determine the degree of
mineralization chondrocyte cultures were stained with alizarin red S
after a 6-day treatment as described previously (16). Briefly,
chondrocyte cultures were fixed with 70% ethanol and then stained with
0.5% alizarin red S solution, pH 4.0, for 5 min at room temperature.
To quantify the intensity of alizarin red S staining, alizarin red
S-stained cultures were incubated with 100 mM
cetylpyridinium chloride for 1 h to solubilize and release
calcium-bound alizarin red S into solution (26). The absorbance of the
released alizarin red S staining was measured at 570 nm using a
spectrophotometer. Data were expressed as units of alizarin red S
released per mg of protein in each culture.
Caspase-3 Activity Assay--
Caspase-3 activity was determined
using the ApoAlert caspase fluorescent assay kit
(Clontech) following the manufacturer's protocol.
Briefly, after a 6-day treatment chondrocyte cultures were washed twice
with ice-cold phosphate-buffered saline (PBS), scraped into tubes, and
centrifuged at 1500 rpm for 10 min. Cell pellets were washed one more
time with ice-cold PBS and centrifuged again. Then air-dried cell
pellets were resuspended in 60 µl of chilled cell lysis buffer and
incubated on ice for 10 min. Cellular debris was removed by
centrifugation, and 50 µl of 2 × reaction buffer/dithiothreitol
mixture and 5 µl of 1 mM caspase-3 substrate (DEVD-7-amino-4-trifluoromethylcoumarin) were added to 50 µl
of each sample and incubated for 1 h at 37 °C. Caspase-3
activity was measured in a fluorimeter (Photon Technology Instruments) using the excitation wavelength of 400 nm and the emission wavelength of 505 nm. Caspase-3 activity was quantitated using
7-amino-4-trifluoromethylcoumarin standard and normalized to the
protein content in each culture.
In Situ Detection of Apoptotic Chondrocytes by TUNEL
Labeling--
Apoptotic chondrocytes in day 6 chondrocyte cultures
were detected using ApopTag in situ apoptosis detection kit
to label apoptotic cells by modifying genomic DNA utilizing terminal
deoxynucleotidyltransferase (TdT) (Intergen Co., Purchase, NY).
Briefly, chondrocytes were washed twice with PBS and fixed with 1%
paraformaldehyde/PBS solution (pH 7.4) for 10 min. Then fixed
chondrocytes were incubated with 1% Triton/PBS solution, followed by
incubation with a proteinase K solution (20 µg/ml) for 10 min at room
temperature. Samples were then incubated in 3% hydrogen peroxide/PBS
for 5 min at room temperature to quench endogenous peroxidases,
followed by rinsing with PBS and incubation with equilibration buffer.
Samples were incubated with a reaction mixture containing terminal
deoxynucleotidyltransferase enzyme and digoxigenin-labeled dNTPs at
37 °C in a humidified chamber. After 1 h, the reaction was
stopped, and digoxigenin-labeled nucleotides were detected by
peroxidase-conjugated anti-digoxigenin antibodies in a humidified
chamber for 30 min at room temperature. The signal was detected using
3,3'-diaminobenzidine as a color substrate. Sections were
counterstained with methylene green, mounted, and viewed under an
Olympus microscope. To gain insights into the extent of apoptosis in
the various chondrocyte cultures, the percentage of stained cells was
determined. 500 chondrocytes were counted in 10 randomly chosen areas
of three different cultures. Data were expressed as the mean ± S.D. of the percentage of total cells that show TUNEL staining.
Statistical Analysis--
Numerical data are presented as
mean ± S.D. (n > 4), and statistical
significance between groups was identified using the two-tailed
Student's t test (p values are reported in the
figure legends).
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RESULTS |
Treatment of hypertrophic growth plate chondrocytes with RA
induced terminal differentiation of these cells, as indicated by
up-regulation of terminal differentiation marker genes, including cbfa1 (Fig. 1A),
APase (Fig. 1B), and osteocalcin (Fig. 1C),
compared with the expression levels in untreated cells. An
approximately 9-fold increase in cbfa1 gene expression was
detected after a 3-day treatment, whereas APase gene expression was
up-regulated by ~16-fold after a 5-day treatment, and osteocalcin
gene expression increased ~16-fold after a 3-day treatment and
~14-fold after a 5-day treatment. Furthermore, alizarin red S
staining revealed that RA-treated cultures were heavily mineralized
after a 6-day treatment, whereas untreated cultures showed only little
signs of mineralization (Fig. 2).
Previously, we have shown that RA treatment led to a 3-fold increase in
[Ca2+]i of growth plate chondrocytes compared
with the concentration of untreated cells. In addition, we provided
evidence that most of this increase was mediated by Ca2+
influx through annexin channels (16). Thus, it is possible that
annexin-mediated Ca2+ influx into growth plate chondrocytes
regulates terminal differentiation events of these cells. To test this
hypothesis, we cotreated cells with RA and the annexin-specific
Ca2+ channel blocker K-201 or antibodies specific for
annexin V. Blocking annexin channel activities with K-201 led to a
significant reduction of cbfa1, APase, and osteocalcin gene
expression (Fig. 1), and mineralization of RA-treated chondrocyte
cultures (Fig. 2). We have previously shown that antibodies specific
for annexin V blocked its Ca2+ channel activities (26).
Cotreatment of RA-treated cultures with antibodies specific for annexin
V also significantly reduced the rate of mineralization compared with
the degree of mineralization in RA-treated cultures (Fig. 2).

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Fig. 1.
Quantitative real time PCR analysis of
cbfa1 (A), alkaline phosphatase
(APase) (B), and osteocalcin (C) gene
expression in untreated, RA-, and RA/K-201-treated growth plate
chondrocytes. Total RNA was isolated from day 1, 3, and 5 untreated, RA-, and RA/K-201-treated chondrocytes. Gene expressions of
cbfa1, APase, and osteocalcin were detected by quantitative
real time PCR. Data were obtained from triplicated PCR reactions of
three different cultures, and values are mean ± S.D. (*,
p 0.01; **, p 0.05; RA
versus RA/K-201 treatment).
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Fig. 2.
Extent of matrix mineralization in
chondrocyte cultures treated with RA, RA and K-201, or RA and
antibodies specific for annexin V. Hypertrophic growth plate
chondrocytes were treated with RA, RA/K-201, or RA/anti-annexin V IgGs
for 6 days. A, alizarin red S staining of untreated,
RA-treated, RA/K-201-treated, and RA/anti-annexin V IgG-treated
cultures. Note the intense staining in RA-treated cultures, while
untreated, RA/K-201-, and RA/anti-annexin V IgG-treated cultures showed
little staining. B, to quantitate alizarin red S staining,
alizarin red S-stained cultures were incubated with 100 mM
cetylpyridium chloride for 1 h. The alizarin red staining released
into the solution was collected, diluted when necessary, and read as
units of alizarin red released (1 unit is equivalent to 1 unit of
absorbance density at 570 nm) per mg of protein. Data were obtained
from four different experiments, and values are mean ± S.D. (*,
p 0.01; RA-treated versus
RA/K-201-treated cultures).
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RA treatment also up-regulated annexin II, V, and VI gene expression.
K-201 significantly reduced the up-regulation of annexin II, V, and VI
gene expression in RA-treated cultures to levels similar to untreated
cultures (Fig. 3, A,
B, and C). Type I collagen gene expression was
up-regulated in RA-treated cultures (Fig. 4A), whereas type II collagen
gene expression was down-regulated in these cultures (Fig.
4B). Cultures cotreated with RA and K-201 showed levels of
type I and II collagen gene expression similar to levels in untreated
cultures (Fig. 4, A and B). Type X collagen gene
expression was not affected by RA or RA/K-201 treatment (Fig. 4C).

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Fig. 3.
Quantitative real time PCR analysis of
annexin II (A), V (B), and VI
(C) gene expression in untreated, RA-, and
RA/K-201-treated growth plate chondrocytes. Total RNA was isolated
from day 1, 3, and 5 untreated, RA-, and RA/K-201-treated chondrocytes.
Gene expressions of annexin II, V, and VI were detected by quantitative
real time PCR. Data were obtained from triplicated PCR reactions of
three different cultures, and values are mean ± S.D. (*,
p 0.01; RA versus RA/K-201
treatment).
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Fig. 4.
Quantitative real time PCR analysis of type I
(A), II (B), and X
(C) collagen gene expression in untreated, RA-treated,
and RA/K-201-treated growth plate chondrocytes. Total RNA was
isolated from day 1, 3, and 5 untreated, RA-, and RA/K-201-treated
chondrocytes. Gene expressions of type I collagen (A), type
II collagen (B), and type X collagen (C) were
detected by quantitative real time PCR. Data were obtained from
triplicated PCR reactions of three different cultures, and values are
mean ± S.D. (*, p 0.01; **, p 0.05; RA versus untreated or RA/K-201 treatment).
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We have demonstrated that RA treatment led to the release of
mineralization-competent APase and annexin II-, V-, and VI-containing matrix vesicles, whereas untreated chondrocytes released vesicles that
contain no or little APase activity and annexins II, V, and VI (16; see
also Fig. 5). Cotreatment of chondrocytes
with RA and K-201 significantly reduced the amounts of APase activity (Fig. 5A) and annexins II, V, and VI (Fig. 5B) in
matrix vesicle fractions. Thus, annexin-mediated Ca2+
influx into growth plate chondrocytes regulates expression of terminal
differentiation marker genes, the release of APase- and annexin II-,
V-, and VI-containing matrix vesicles, and subsequent mineralization.

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Fig. 5.
APase activity (A) and
amount of annexins II, V, and VI (B) in matrix
vesicles isolated from untreated, RA-, and RA/K-201-treated growth
plate chondrocytes. After a 3-day treatment, matrix vesicles were
isolated from the cell layer of untreated, RA-, and RA/K-201-treated
chondrocytes using enzymatic digestion and serial ultracentrifugation.
A, APase activity was significantly increased in matrix
vesicles isolated from RA-treated cultures compared with the activity
in vesicles isolated from untreated and RA/K-201-treated cultures. Data
were obtained from four different experiments, and values are mean ± S.D. (*, p 0.01; RA versus RA/K-201
treatment). B, matrix vesicle fractions (30 µg of total
protein) isolated from untreated, RA-, and RA/K-201-treated cultures
were subjected to SDS-PAGE and immunoblotting using antibodies specific
for annexin II, V, or VI. The optical densities of annexin bands were
quantitated by densitometry. The optical densities of annexin bands in
matrix vesicle fractions isolated from untreated cultures were set as
1. Data were obtained from four different experiments, and values are
mean ± S.D. (*, p 0.01; RA versus
RA/K-201 treatment).
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It is now well established that the final fate of terminally
differentiated chondrocytes is apoptosis (2). Therefore, we addressed
the question of whether RA triggers the whole cascade of terminal
differentiation events including apoptosis and whether annexin-mediated
Ca2+ influx into chondrocytes is also involved in the
regulation of apoptotic changes. To determine the degree of apoptosis
in the various treated chondrocyte cultures we measured
bcl-2 gene expression and caspase-3 activity and performed
TUNEL labeling. A 5-day treatment with RA led to a significant decrease
in gene expression of bcl-2, an anti-apoptotic protein (27)
(Fig. 6). In contrast, caspase-3 activity, an active cell death protease involved in the execution phase
of apoptosis (28), was more than 5-fold elevated in cultures treated
for 6 days with RA compared with untreated cells (Fig. 7). Cotreatment of cultures with RA and
the cytosolic Ca2+ chelator BAPTA-AM abolished the decrease
in bcl-2 gene expression (Fig. 6) and the increase in
caspase-3 activity (Fig. 7), suggesting that cytosolic calcium is
directly involved in the regulation of apoptotic events. Interestingly,
bcl-2 gene expression was also higher in RA/K-201-treated
cells than in RA-treated cells (Fig. 6), whereas caspase-3 activity was
lower (Fig. 7). In addition, TUNEL labeling revealed that in RA-treated
cultures more than 10% of cells were TUNEL-positive, whereas only
~2% were TUNEL-positive in untreated and RA/BAPTA-treated cells, and
~4% of cells were TUNEL-positive in RA/K-201-treated cultures (Fig.
8).

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Fig. 6.
Quantitative real time PCR analysis of
bcl-2 gene expression in untreated, RA-, RA/K-201-,
and RA/BAPTA-treated growth plate chondrocytes. Total RNA was
isolated from day 1, 3, and 5 untreated, RA-, RA/K-201-, and
RA/BAPTA-treated chondrocytes. Gene expression of bcl-2 was
detected by quantitative real time PCR. Data were obtained from
triplicated PCR reactions of three different cultures, and values are
mean ± S.D. (*, p 0.01; RA versus
RA/K-201 treatment, RA versus RA/BAPTA treatment).
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Fig. 7.
Caspase-3 activity in untreated, RA-,
RA/K-201-, and RA/BAPTA-treated growth plate chondrocytes. After a
6-day treatment, caspase-3 activities in untreated, RA-, RA/K-201-, and
RA/BAPTA-treated chondrocytes were determined using the ApoAlert
caspase fluorescent assay kit as described under "Experimental
Procedures." Caspase-3 activity was calibrated with AFC calibration
curve and normalized to the protein content in each culture. Data were
obtained from four different experiments, and values are mean ± S.D. (*, p 0.01; RA versus RA/K-201
treatment, RA versus RA/BAPTA treatment).
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Fig. 8.
In situ detection of apoptotic
chondrocytes in untreated, RA-, RA/ K-201-, and RA/BAPTA-treated
growth plate chondrocyte cultures. A, after a 6-day
treatment apoptotic chondrocytes in untreated, RA-, RA/K-201-, and
RA/BAPTA-treated chondrocyte cultures were detected using TUNEL
labeling as described under "Experimental Procedures." Note the
more positively stained chondrocytes in RA-treated culture compared
with untreated, RA/K-201-, and RA/BAPTA-treated cultures. B,
500 cells in 10 randomly chosen areas were counted in untreated, RA-,
RA/K-201-, and RA/BAPTA-treated chondrocyte cultures. Data were
obtained from four different experiments and are expressed as the
means ± S.D. of the percentage of total cells that show TUNEL
labeling (*, p 0.01; RA versus RA/K-201
treatment, RA versus RA/BAPTA treatment).
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 |
DISCUSSION |
In this study we show that RA triggers a whole series of terminal
differentiation events, including up-regulation of terminal differentiation marker genes (APase, cbfa1, osteocalcin),
release of mineralization-competent matrix vesicles, subsequent
mineralization, and finally apoptosis. RA also induces annexin-mediated
Ca2+ influx into growth plate chondrocytes. Blocking
annexin Ca2+ channel activities inhibited the whole series
of terminal differentiation events, including up-regulation of terminal
differentiation marker gene expression, release of
mineralization-competent matrix vesicles, extracellular matrix
mineralization, and apoptosis. These findings clearly establish the
prominent regulatory function of annexin-mediated Ca2+
influx into growth plate chondrocytes in terminal differentiation and
apoptosis of these cells.
RA is known to regulate transcription after binding to the retinoic
acid receptor complex. Three RA receptors have been identified, retinoic acid receptors
,
, and
. RA binds to one of these receptors, and this receptor complex then dimerizes with another receptor, retinoid X receptor (RXR). These receptor complexes then
directly activate gene expression of transcription factors and other
genes (29). Our study, however, indicates that RA also uses other
mechanisms to regulate cell differentiation. As shown in this and
previous studies, RA induces annexin channel formation in the plasma
membrane of growth plate chondrocytes leading to an increased cytosolic
calcium concentration (16). This increased cytosolic calcium
concentration leads to further up-regulation of annexin and other
terminal differentiation marker gene expression and causes the release
of mineralization-competent matrix vesicles and the induction of
apoptotic events.
It is not clear how RA induces annexin channel formation. However,
several possibilities can be envisioned. RA may bind to membrane
receptors yet to be discovered. This binding activates an initial
increase in cytosolic calcium concentration. Annexins require a certain
Ca2+ concentration to bind to phospholipids (20). The
initial increase in cytosolic calcium may then lead to a relocation of
annexins from the cytoplasma to the plasma membrane and channel
formation causing a further boost in cytosolic calcium. Alternatively,
RA might have similar effects on the membrane as vitamin D, which has
been shown to increase plasma membrane fluidity (30). Increased membrane fluidity might favor annexin channel formation. A third possibility is that annexins bind directly to RA, and this binding favors annexin channel formation. This possibility is supported by
recent findings showing that annexin II binds directly to vitamin D and
acts as an alternative vitamin D receptor (31).
RA up-regulates gene expression of cbfa1, APase, and
osteocalcin. These genes are considered terminal differentiation
markers and are expressed by late hypertrophic chondrocytes in the
growth plate. cbfa1, a member of the runt-domain family of
transcription factors, is expressed in osteoblasts and hypertrophic
chondrocytes (6-9). In cbfa1-deficient mice no endochondral
and intramembranous ossification occurs because of an arrest of
osteoblast differentiation (7, 8). A disturbance of chondrocyte
differentiation, especially terminal differentiation, was also observed
in these mice (8). Continuous expression of cbfa1 in
non-hypertrophic chondrocytes induced hypertrophic differentiation and
endochondral ossification (9). Furthermore, overexpression of
cbfa1 in chick immature chondrocytes induced type X collagen
and MMP-13 expression, APase activity, and extensive matrix
mineralization (32). These findings indicate that cbfa1 is
an important regulatory factor in chondrocyte terminal differentiation.
Our results show that annexin-mediated Ca2+ influx into
hypertrophic chondrocytes sequentially activates gene expression of
cbfa1, APase, and osteocalcin. Interestingly, cbfa1 directly regulates gene expression of osteocalcin and
other genes (33). Thus, it is possible that annexin-mediated alteration of Ca2+ homeostasis might control terminal differentiation
and apoptotic events through the regulation of cbfa1 gene expression.
Terminal differentiation is also accompanied by extracellular matrix
remodeling and alteration of collagen gene expression. When
chondrocytes undergo hypertrophic changes they turn on type X collagen
gene expression and down-regulate type II collagen synthesis (34).
Furthermore, previous studies in vivo and in vitro have demonstrated that terminal differentiated mineralizing chicken growth plate chondrocytes express type I collagen and other
bone-related proteins, including osteocalcin and osteopontin (35, 36).
Our study showing that RA treatment leads to down-regulation of type II
collagen and up-regulation of type I collagen gene expression confirms
these previous findings and indicates that alteration of
Ca2+ homeostasis is involved in regulating collagen gene
expression during growth plate development. In addition, our findings
reveal that RA or RA/K-201 treatment did not affect type X collagen
gene expression, indicating that type X collagen expression in growth plate chondrocytes used in this study was already at a high level. These results are consistent with previous findings demonstrating that
type X collagen synthesis was greatly up-regulated in immature growth
plate chondrocytes after RA treatment but remained unchanged in
RA-treated mature hypertrophic growth plate chondrocytes (37, 38).
Annexin-mediated alteration of Ca2+ homeostasis
up-regulates annexin II, V, and VI and APase gene expression. The
up-regulation of annexin and APase gene expression might be required
for the release of mineralization-competent matrix vesicles. These
vesicles contain large amounts of annexins II, V, and VI and APase
activity. Annexins II, V, and VI also form Ca2+ channels in
matrix vesicles (19, 21). Thus, annexin channel formation seems to play
multiple functions in terminal differentiation of growth plate
chondrocytes. Firstly, channel formation alters Ca2+
homeostasis, which controls terminal differentiation events, and
secondly annexin channel formation in matrix vesicles allows Ca2+ influx into these particles as a possible initial step
of mineral formation.
RA does not only induce mineralization of growth plate chondrocytes,
but it triggers the whole cascade of terminal differentiation events,
including apoptosis. Apoptosis, or programmed cell death, has been
shown to be the final event of chondrocyte differentiation (2, 22).
Induction of apoptosis by RA has also been demonstrated in other cell
types, including leukemia cells, thymocytes, neuroblastoma cell lines,
and articular chondrocytes (39-42). We show that RA down-regulates
bcl-2 gene expression and stimulates caspase-3 activity. In
addition, the percentage of TUNEL-positive cells was significantly
higher in RA-treated cells compared with the number of apoptotic cells
in untreated cultures. bcl-2 belongs to a rapidly expanding
family of genes implicated in the control of apoptosis. Up-regulation
of bcl-2 by PTHrP delays maturation of growth plate
chondrocytes toward hypertrophy and subsequent apoptosis (27). In
contrast, caspase-3 is an active cell death protease involved in the
execution phase of apoptosis (28). Interestingly, apoptotic changes
were significantly reduced in cultures cotreated with RA and BAPTA or
RA and K-201, suggesting that annexin-mediated Ca2+ influx
into growth plate chondrocytes is involved in the regulation of the
complete terminal differentiation program, including apoptosis. Thus,
RA and RA-mediated annexin Ca2+ channel formation appear to
stimulate a similar sequence of events as observed in growth plate
cartilage. A recent study has demonstrated high amounts of retinoids in
the perichondrium and that implantation of beads containing RA
antagonist near the humeral anlagen drastically decreased chondrocyte
hypertrophy and terminal differentiation. In addition, retinoic acid
receptor
is expressed in hypertrophic and terminally differentiated
chondrocytes (43). Thus, it is likely that RA and the resulting annexin
Ca2+ channel formation play important regulatory roles in
the regulation of terminal differentiation events in the growth plate
during endochondral ossification.
Annexin V has also been shown to mediate Ca2+ influx
induced by hydrogen peroxide into B-lymphocytes (44). Thus, annexins not only form Ca2+ channels in chondrocytes but also other
cell types. In addition, B-lymphocytes lacking annexin V are resistant
to apoptosis (45). Ca2+ is known to be required for
apoptosis, and it is known that Ca2+ can cause apoptosis by
itself under conditions of Ca2+ overload. Our study
demonstrates that the cytosolic calcium chelator BAPTA-AM or the
annexin channel blocker K-201 significantly inhibits apoptosis of
growth plate chondrocytes. These findings suggest that annexin-mediated
Ca2+ influx or related annexin functions in various cell
types can lead to a Ca2+ overload and apoptosis resulting
from this overload.
Hypertrophic and terminally differentiated growth plate chondrocytes
express three annexins (annexins II, V, and VI). Previous studies have
shown that all three annexins can form Ca2+ channels and
that K-201 inhibits Ca2+ channel activities of all three
annexins (21). However, it is not clear, whether all three annexins
form Ca2+ channels in growth plate chondrocytes
independently and mediate Ca2+ influx into these cells. Our
previous findings show that each antibody fraction specific for annexin
II, V, or VI partially inhibited increases in
[Ca2+]i in growth plate chondrocytes, suggesting
that all three annexins contributed to Ca2+ influx into
these cells (16). However, antibodies specific for annexin V inhibited
mineralization of growth plate chondrocytes to a degree similar to
K-201 (see Fig. 2). Other studies have shown that peroxide-mediated
Ca2+ influx was altered only in B cells lacking annexin V
but not in cells lacking annexin II (44). In addition, only B cells lacking annexin V but not cells lacking annexin II were resistant to
apoptosis (45). Future studies have to establish whether only annexin V
modulates Ca2+ homeostasis of growth plate chondrocytes and
is involved in the regulation of chondrocyte terminal differentiation,
whether all three annexins form Ca2+ channels independently
and regulate Ca2+ influx into growth plate chondrocytes, or
whether the interactions between the three annexins are required for
optimal annexin V channel activities in growth plate chondrocytes.
Previous studies from our and other laboratories have shown the
expression of hypertrophic and terminal differentiation markers, including annexins II, V, and VI, APase, osteopontin, osteocalcin, and
type X collagen in osteoarthritic cartilage (17, 18, 46-49). In
addition, mineralization and apoptosis were detected in osteoarthritic cartilage (17, 50-52). Thus, it is possible that up-regulation of
annexin gene expression in osteoarthritic cartilage might lead to
annexin-mediated Ca2+ influx into articular chondrocytes
and subsequent stimulation of terminal differentiation events in these
cells. Terminal differentiation events are required for endochondral
bone formation during normal development; however, if these events
occur under pathological conditions, such as osteoarthritis, they will
lead to cartilage destruction. If these annexins are as essential for
terminal differentiation in osteoarthritic cartilage as they are in
growth plate cartilage during endochondral ossification, they could be
promising targets for therapies.
 |
FOOTNOTES |
*
This work was supported by NIAMS, National Institutes of
Health Grants AR 43732 and AR46245 (to T. K.).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 Orthopaedics,
University of Maryland School of Medicine, 22 South Greene St.,
Baltimore, MD 21201. Tel.: 410-706-2417; Fax: 410-706-0028; E-mail:
tkirsch@umoa.umm.edu.
Published, JBC Papers in Press, November 22, 2002, DOI 10.1074/jbc.M208868200
 |
ABBREVIATIONS |
The abbreviations used are:
RA, retinoic
acid;
APase, alkaline phosphatase activity;
RAR, retinoic acid
receptor;
PBS, phosphate-buffered saline;
BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid acetoxymethyl ester;
TUNEL, terminal
deoxynucleotidyltransferase-mediated dUTP nick end labeling.
 |
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