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Department of Biochemistry and Molecular Dentistry (T.N., T.N., T.S., M.T.) and Biodental Research Center (T.N., T.N., M.T.), Okayama University Dental School, Okayama 700-8525; Departments of Orthopedic Surgery (T.K.) and Microbiology (K.K.), Kyoto Prefectural University of Medicine, Kyoto 602-8566; and Pharmaceutical Frontier Research Laboratories, Japan Tobacco, Inc. (T.T., K.T.), Yokohama 236-0004, Japan
Address all correspondence and requests for reprints to: Masaharu Takigawa, D.D.S., Ph.D., Department of Biochemistry and Molecular Dentistry, Okayama University Dental School, 2–5-1 Shikata-cho, Okayama 700-8525, Japan. E-mail: takigawa{at}dent.okayama-u.ac.jp
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To isolate regulatory molecules involved in many stages in the process of endochondral ossification, we recently isolated, by differential display PCR (25), several sequence tags that were more highly expressed in an immortal human chondrocytic cell line, HCS-2/8 (26, 27, 28, 29, 30), than in osteosarcoma and osteoblastic cell lines (31, 32). In these tags, no. 24 showed high homology with the sequence of connective tissue growth factor (CTGF) (33, 34, 35) complementary DNA (cDNA), and the gene, named hcs24, that included the tag no. 24 encoded CTGF protein (36). It was expressed selectively in the hypertrophic region of costal cartilage and vertebrate columns of embryonic cartilage tissues, and its expression was induced by TGFß and BMP-2 (36).
CTGF is a cysteine-rich polypeptide isolated from angioendothelial cells as a growth factor structurally and functionally related to platelet-derived growth factor (PDGF) (33). It was recognized by anti-PDGF antibodies and has PDGF-like mitogenic and chemotactic activities (33). It belongs to a special gene family, named CCN, that contains immediate early genes (cyr61 or cef10) (34). It was also found in skin fibroblasts as a repairing growth factor (35). However, detailed analysis of neither its function nor distribution in various tissues, including cartilage, has been performed.
In this study we investigated the role of CTGF/Hcs24 in the proliferation and differentiation of chondrocytes using the chondrocytic cell line HCS-2/8 and RGC cells in culture. Firstly, we transfected recombinant adenoviruses that expressed sense RNA [messenger RNA (mRNA)] of CTGF/Hcs24 continuously in HCS-2/8 cells and tested the effect of CTGF on the proliferation and differentiation of the cells. Next, we obtained recombinant CTGF/Hcs24 protein (rCTGF/Hcs24), which was produced by HeLa cells transfected with expression vectors, and tested its effect on the proliferation and differentiation of HCS-2/8 cells and RGC cells in culture.
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MEM (ICN Biomedicals, Inc.,
Costa Mesa, CA) containing 10% FBS.
Adenovirus-mediated gene transfection
We used adenovirus type 5-based recombinant virus vector, which
lacks E1A, E1B, and E3. AxCALacZ (37), a recombinant virus harboring
the Escherichia coli-galactosidase (ß-gal) gene
(lacZ) under the control of the CAG promoter, which is a
potent promoter consisting of cytomegalovirus (CMV; i.e.
enhancer), chicken ß-actin promoter, and rabbit ß-globulin
polyadenylation signal (37), and pAx1w, a control vector harboring no
foreign gene expression unit, were gifts from Dr. I. Saito (University
of Tokyo, Tokyo, Japan). The recombinant virus that expresses
CTGF/Hcs24 gene was constructed as follows. For construction of sense
RNA (mRNA)-producing vector, the latent coding sequence of CTGF/Hcs24,
which contained the whole coding region of CTGF/Hcs24 cDNA, was cloned
into the EcoRI site of pCAGGS and excised from the
SalI-HindIII site that contained CMV enhancer and
CAG promoter in the up-stream of CTGF/Hcs24 cDNA. Then, the fragments
were cloned into SwaI site of pAx1w. The recombinant
adenoviruses were propagated and titrated as previously described (38),
and adenovirus-mediated gene transfection was performed as described
previously (39). X-gal staining was performed as described previously
(39).
RT-PCR
For quantitation of mRNA expression, the sets of primers
described below were used. The numbers in parentheses are
the expected sizes (base pairs) of PCR products: CTGF/Hcs24 (primers in
the coding region of CTGF/Hcs24), 5'-GACGGCTGCGGCTGCTGC-3' and
5'-CACACCCACTCCTCGCAGCA-3' (344); aggrecan core protein,
5'-CGCGAGACCTGGGTGGATGC-3' and 5'-GAAGGGG/CAGG/CTGGATATTGC-3'
(310); type II collagen,
5'-ATGACAATCTGGCTCCCAACACTGC-3' and
5'-GACCGGCCCTATGTCCACACCGAAT-3' (364); human type X collagen,
5'-AGCCAGGGTTGCCAGGACCA-3' and 5'-TTTTCCCACTCCAGGAGGGC-3' (387); rabbit
type X collagen, 5'-GCCCAAGAGGTGCCCCTGGAATCA-3' and
5'-CCTGAGAA-AGAGGAGTGGACATAC-3' (703); and glyceraldehyde-3-phosphate
dehydrogenase (G3PDH), 5'-ACCACAGTCCATGCCATCAC-3' and
5'-TCCACCACCCTGTTGCTGTA-3' (450). Total RNA (deoxyribonuclease I
treated) was reverse transcribed to cDNA using
oligo(deoxythymidine)16 with AMV-derived reverse
transcriptase (Takara Shuzo Co. Ltd., Tokyo, Japan) for 30 min at 42 C,
and then the cDNA was amplified by the primers with Taq
polymerase (Takara Shuzo Co. Ltd.) using an unsaturated cycle number.
The amplification conditions were as follows: 95 C for 1 min, 57 C for
1 min, and 72 C for 2 min for 30 cycles for G3PDH or for 35 cycles for
others. PCR products were analyzed by agarose gel electrophoresis.
Preparation of recombinant CTGF/Hcs24
For construction of CTGF/Hcs24 expression vector, a DNA fragment
of CTGF/Hcs24 that contained the whole coding region of CTGF/Hcs24 cDNA
was cloned into pcDNA3.1(-) vector, and CTGF/Hcs24 was expressed under
the control of the CMV promoter. This expression vector was introduced
into HeLa cells by electroporation using a Gene Pulser (Bio-Rad Laboratories, Inc., Hercules, CA), and stable transformants that
were resistant to G418 were selected and maintained in ASF104
serum-free medium (Ajinomoto, Tokyo, Japan). rCTGF/Hcs24 was purified
from 35 liters of the conditioned medium of stable transformants
containing 1–3 µg/ml rCTGF by heparin affinity chromatography
(HiTrap Heparin column, Pharmacia Biotech, Uppsala,
Sweden). After loading of samples, the column was washed with PBS
containing 0.2 M NaCl, and bound proteins were eluted with
PBS containing 0.5 M NaCl. Partially purified rCTGF/Hcs24
eluted from heparin affinity chromatography was further purified by
affinity chromatography using an anti-CTGF antibody that had been
prepared by immunizing rabbits with a synthetic peptide, described
below. rCTGF/Hcs24 eluted with 0.1 M glycine buffer (pH
2.5) was neutralized with 0.1 vol 0.75 M Tris and dialyzed
against PBS. The purity of rCTGF/Hcs24 was analyzed by SDS-PAGE, silver
staining, and Western blotting.
Preparation of antibody
Anti-CTGF antibody was raised in rabbits by immunization with
synthetic peptides of CTGF composed of 20 amino acids, including the
lysine cluster in the C-terminal of CTGF. The amino acid sequence of
the peptide was the same as that of Fisp12 (mouse CTGF homolog), but
different from that of Cyr61. The IgG fraction of the anti-CTGF
antibody purified with Mab Trap G II (Pharmacia Biotech)
was used for Western blotting and neutralizing experiments. The
specificity of the antibody was confirmed by Western blotting using
synthetic peptides of CTGF or Cyr61 composed of 20 amino acids,
described above as competitors (40).
Western blotting
Proteins were separated by SDS-PAGE using the method of Laemmli
(41). Immunoblotting was carried out by the method of Towbin et
al. (42). with minor modifications. Briefly, the proteins were
transferred to a polyvinylidene difluoride membrane. The membrane was
first incubated with 500-fold diluted antirabbit CTGF IgG for 8 h
at 25 C and next with 2000-fold diluted ALPase-conjugated goat
antirabbit IgG for 90 min at 37 C. The membrane was colored with nitro
blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.
Determination of DNA synthesis
HCS-2/8 cells were inoculated at a density of 2 x
104 cells/well of 96-well plates (Sumitomo
Bakelite Co. Ltd.) in 100 µl DMEM containing 10% FBS. When they
reached subconfluence, the concentration of FBS was reduced to 0.5%,
and the cells were preincubated for 24 h. In the case of RGC
cells, the cells were inoculated at a density of 1 x
104 cells/well in 100 µl MEM containing 10%
FBS. When they reached confluence, the concentration of FBS was reduced
to 0.5%, and the cells were preincubated for 48 h. Then,
rCTGF/Hcs24 protein and/or anti-CTGF antibody dissolved in a small
volume of PBS was added to the cultures. After 22 h,
[3H]thymidine [925 terabecquerels (TBq)/mmol;
Amersham Pharmacia Biotech (Aylesbury, UK)] dissolved in
DMEM was added to the cultures at a final concentration of 740 KBq/ml,
and the cells were incubated for another 4 h. After labeling, the
cell layers were washed three times with PBS and treated successively
with 5% trichloroacetic acid and ethanol-ethyl ether (3:1, vol/vol).
Radioactivity in the residual materials was measured using a Micro
ß-PLUS (Pharmacia Biotech) as described previously
(40).
Determination of proteoglycan synthesis
Proteoglycan synthesis was assayed as described previously with
slight modification (2, 4, 18). HCS-2/8 cells and RGC cells were grown
to confluence in 48-well microplates in DMEM or MEM, respectively,
containing 10% FBS. They were then preincubated in DMEM containing
0.5% FBS for 24 h and further incubated in the same medium with
rCTGF/Hcs24 (10–100 ng/ml) and/or anti-CTGF antibody for 5 h.
Then [35S]sulfate (37 MBq/ml) dissolved in PBS
was added to the cultures at a final concentration of 370 kBq/ml, and
incubation was continued for another 17 h. After labeling, the
cultures were digested with 1 mg/ml actinase E (Kaken Pharmaceuticals,
Tokyo, Japan), and the radioactivity of the material precipitated with
cetylpyridinium chloride was measured in a scintillation counter.
Measurement of ALPase activity
RGC cells were grown in 48-well microplates at a density of
2 x 104 cells/well with MEM containing
10% FBS for 6 days. Then, the medium was replaced with MEM
containing 0.5% FBS, and the cells were incubated with rCTGF/Hcs24 for
72 h. After the incubation, the cells were rinsed three times with
ice-cold PBS, homogenized in 0.5 M Tris (pH 9.0) containing
0.9% NaCl and 1% Triton X-100 on ice, and then centrifuged at
12,000 x g for 15 min. ALPase activity in the
resultant supernatants was determined by the method of Majeska and
Rodan (43) with some modifications. The enzyme reaction was initiated
with 0.5 mM p-nitrophenyl phosphate in
0.5 M Tris-HCl (pH 9.0) containing 0.5
mM MgCl2 at 37 C and
terminated by the addition of a quarter volume of 1
M NaOH. The concentration of
p-nitrophenol generated was determined by spectrophotometry
at 410 nm. The enzyme activity is expressed as nanomoles of
p-nitrophenyl phosphate cleaved per min/well.
Statistical analysis
Unless otherwise specified, all experiments were repeated at
least twice, and similar results were obtained in the repeated
experiments. Statistical analysis was performed by Student’s
t test if necessary. Data are expressed as the mean ±
SD. P < 0.05 was considered
significant.
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) and transfected the viruses to HCS-2/8
cells. When recombinant adenoviruses that express ß-galactosidase
were transfected to HCS-2/8 cells at a multiplicity of infection of
4 x 107 plaque-forming units/ml, almost all
cells were positive for X-gal staining (Fig. 1D, X), indicating
that the cells were successfully transfected with this type of
adenoviruses and expressed ß-gal. Under the same conditions,
adenoviruses containing sense DNA of CTGF/Hcs24 were transfected to
HCS-2/8 cells. To confirm that the transfection with sense adenoviruses
resulted in successful production of CTGF/Hcs24 protein, Western
blotting of cell extracts transfected with sense adenoviruses or
control adenoviruses (Ax1w) was performed using anti-CTGF antibodies
(Fig. 1B). In the cell extracts transfected with sense adenoviruses, a
large amount of CTGF/Hcs24 protein with an apparent molecular mass of
36–38 kDa was produced. The two major bands seems to represent the
difference of glycosylation. Because the sensitivity of Western
blotting was adjusted to suitable detection of overexpressed CTGF/Hcs24
in HCS-2/8 cells transfected with sense adenoviruses (Fig. 1B, S), no
band was observed in control cultures (Fig. 1, B, C and CV), but
CTGF/Hcs24 protein was detected in the control HCS-2/8 cells at a
higher sensitivity (data not shown), consistent with the previous
finding about mRNA levels in the cells (36). Under these conditions,
cell number was calculated after 5 days of transfection when the
fastest growing cultures reached confluence. As shown in Fig. 1, C and
D, HCS-2/8 cells transfected with adenoviruses expressing CTGF/Hcs24
sense RNA (mRNA; S) grew faster than the cells transfected with control
adenoviruses (CV) and reached confluence after 5 days. The cell number
was 140% that of the control cultures.
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). The expression of G3PDH was also
estimated as a control. Figure 2A (CTGF/Hcs24) shows the products of
RT-PCR in agarose gels, and Fig. 2B (CTGF/Hcs24) shows the
densitometric analysis of the PCR products. Transfection with
CTGF/Hcs24 sense adenoviruses effectively increased the expression of
total CTGF/Hcs24 mRNA. The effect was observable after 3 days, reached
a maximum after 7 days, and then declined.
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1(X)], which is a well known marker
of hypertrophic chondrocytes, were estimated by RT-PCR (Fig. 2
). The
expression of mRNA of aggrecan core protein in HCS-2/8 cells
transfected with control adenoviruses started to increase 3 days after
transfection, reached a maximum after 7 days, and then decreased. The
expression of type X collagen started to increase after 7 days, and the
highest expression was observed after 10 days. When HCS-2/8 cells were
transfected with CTGF/Hcs24 sense adenoviruses, aggrecan expression was
up regulated after 3–10 days. The degree of increase was 2.8-fold 3
days after transfection, and 1.8-fold after 7 days. In addition,
expression of type X collagen mRNA was increased. The maximum level was
observed after 7 days and was 2.2 times the control value.
Stimulation of proliferation of HCS-2/8 cells and RGC cells by
rCTGF/Hcs24 protein
To investigate whether CTGF/Hcs24 is a direct growth-promoting
factor for HCS-2/8 cells, we next prepared rCTGF/Hcs24 and tested its
effect on the proliferation of HCS-2/8 cells. As shown in Fig. 3, rCTGF/Hcs24 was stably produced by
transfection of HeLa cells with pcDNA3.1(-) expression vector
containing the whole coding region of human CTGF/Hcs24 cDNA under the
control of CMV promoter and purified by heparin affinity chromatography
and anti-CTGF antibody affinity chromatography. About 6.2 mg protein
were purified from 35 liters conditioned medium. Figure 3B shows the
patterns of SDS-PAGE (silver staining; S) and Western blotting (W) of
the purified rCTGF/Hcs24. Like native CTGF, reported previously (33),
rCTGF/Hcs24 was observed as 36- to 38-kDa protein. This purified
preparation was added to the culture of HCS-2/8 cells. As shown in Fig. 4A, rCTGF/Hcs24 stimulated the DNA synthesis
of HCS-2/8 cells. The stimulatory effect was dose dependent. A
concentration of 50 ng/ml CTGF/Hcs24 caused the most stimulation, 1.4
times the control level (Fig. 4A). Addition of 10 µg/ml anti-CTGF
antibody effectively neutralized the stimulatory action of recombinant
protein, whereas nonimmune control serum had no effect (Fig. 5A). rCTGF/Hcs24 also stimulated the
proliferation of HCS-2/8 cells. When the cells were cultured in the
presence of 50 ng/ml rCTGF/Hcs24 for 5 days, cell number in the treated
group was 132% that in nontreated controls (Fig. 4B). rCTGF/Hcs24 also
stimulated the DNA synthesis of RGC cells (Fig. 6A). The stimulatory effect was dose
dependent, reaching a plateau at a concentration of 50 ng/ml. A
concentration of 80 ng/ml CTGF/Hcs24 caused the most stimulation, 1.8
times the control level. In addition, 10 µg/ml anti-CTGF antibody
effectively neutralized the stimulatory action of rCTGF/Hcs24, whereas
nonimmune control serum showed no such effect, indicating that the
stimulatory effect of the rCTGF/Hcs24 preparation was indeed due to
CTGF/Hcs24 itself (data not shown).
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). Addition
of 30 µg/ml anti-CTGF antibody effectively neutralized the
stimulatory effect of recombinant protein, whereas nonimmune control
serum had no effect (Fig. 5B). When rCTGF/Hcs24 was added to confluent
culture of RGC cells, it also increased proteoglycan synthesis in the
cells in a dose-dependent manner (Fig. 6B). A concentration of 50 ng/ml
rCTGF/Hcs24 had the greatest stimulatory effect on proteoglycan
synthesis in the cells. The maximal stimulation by rCTGF/Hcs24 was
comparable to those of PTH (4, 44), IGFs (12, 20), and TGFß (18, 20).
Increase in ALPase activity in RGC cells by rCTGF/Hcs24 protein
ALPase activity has been shown to be a good marker of hypertrophic
chondrocytes in calcifying cartilage (1, 10). When RGC cells were
cultured with MEM containing 10% FBS for 6 days, they reached
overconfluence. In this phase, the cells started to show the phenotype
of hypertrophic chondrocytes. When these cells were treated with
rCTGF/Hcs24, ALPase activity in the cells was increased dose
dependently, reaching a plateau at a concentration of 80 ng/ml (Fig. 6C).
Stimulation of mRNA expression of aggrecan core protein, type II
collagen, and type X collagen in RGC cells by rCTGF/Hcs24 protein
To further clarify the role of CTGF/Hcs24 in the differentiation
of chondrocytes, we next investigated the effect of rCTGF/Hcs24 on the
mRNA expression of aggrecan core protein, type II collagen, and type X
collagen in cultured RGC cells by RT-PCR (Fig. 7). When RGC cells were cultured in the
presence of 30 and 50 ng/ml rCTGF/Hcs24 for 7 days from day 2 of
culture, the mRNA levels of aggrecan core protein, type II collagen,
and type X collagen were significantly increased. Especially, mRNAs of
aggrecan core protein and type X collagen were markedly increased. The
expression of aggrecan core protein mRNA increased to about 4 times
that of the PBS control at both concentrations. The expression of type
X collagen mRNA also increased to 3–4 times that of the PBS
control.
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In the transfection experiments we at first clarified the role of
CTGF/Hcs24 in the proliferation of HCS-2/8 cells. Continuous production
of sense RNA (mRNA) of CTGF/Hcs24 in the transfected cells produced a
large amount of CTGF protein and stimulated the proliferation of
HCS-2/8 cells, resulting in an increase in cell number compared with
the controls. These findings clearly show that high level expression of
CTGF/Hcs24 is important for the proliferation of HCS-2/8 cells.
Moreover, rCTGF/Hcs24 stimulated DNA synthesis and proliferation of not
only HCS-2/8 cells, but also RGC cells in primary culture (Figs. 4 and 6), indicating that CTGF/Hcs24 is a growth-stimulating factor for
chondrocytes.
Control HCS-2/8 cells that were transfected with control adenoviruses became confluent on day 8 of culture (corresponding to 7 days after transfection). At this time, expression of mRNA of aggrecan core protein was the greatest. In addition, gene expression of type X collagen started to increase on day 8 and continued to increase until day 11 of culture (corresponding to 10 days after transfection). These findings indicate that in these conditions, HCS-2/8 cells proliferate sparsely, mature as they become confluent, and then express hypertrophic phenotype as they become overconfluent. The weak expression of type X collagen on day 1 of culture was due to contamination of cells with hypertrophic phenotype from parent cultures at the time of subculture.
When HCS-2/8 cells were transfected with adenoviruses expressing CTGF/Hcs24 sense RNA (mRNA), expression of aggrecan core protein gene, a maker of chondrocyte maturation, was increased earlier, and the maximal level was higher than the control values, suggesting that expression of CTGF/Hcs24 is important for chondrocyte maturation. Similarly, expression of type X collagen gene, a maker of chondrocyte hypertrophy, was also increased earlier, and the maximal expression level was higher than the control values, suggesting that expression of CTGF/Hcs24 is also important for chondrocyte hypertrophy. The findings that rCTGF/Hcs24 stimulated proteoglycan synthesis, a marker of chondrocyte maturation, in HCS-2/8 and RGC cells and increased ALPase activity, a marker of chondrocyte hypertrophy, in RGC cells provide direct evidence for stimulatory roles of CTGF/Hcs24 in chondrocyte maturation and hypertrophy. In addition, RT-PCR revealed that rCTGF/Hcs24 increased mRNA levels of aggrecan and collagen types II and X in RGC cells, suggesting that CTGF/Hcs24 stimulates the whole process of chondrocyte maturation and hypertrophy.
We showed previously that TGFß and BMP promote the expression of CTGF/Hcs24 mRNA in HCS-2/8 cells (36), and anti-CTGF antibodies inhibited the TGFß-induced proliferation of HCS-2/8 cells (our unpublished data). Because TGFß has been shown to be highly expressed in the hypertrophic region of cartilage tissue (49), it is feasible that TGFß stimulates the expression of CTGF/Hcs24 mRNA in the hypertrophic chondrocytes, and the CTGF/Hcs24 protein produced in the cells promotes both the proliferation of proliferating chondrocytes and the differentiation of maturing chondrocytes in a paracrine manner in vivo. It is also feasible that CTGF/Hcs24 mediates the action of BMP on chondrocytes in the early stage of chondrocyte differentiation. Further investigations are needed to clarify the regulation of CTGF/Hcs24 by other growth factors.
It is unknown how CTGF/Hcs24 exhibit multiple modes of action on
chondrocytes, but there are several possibilities. First, we previously
reported that the chondrocytic cell line HCS-2/8 has specific receptors
for CTGF/Hcs24, and the putative molecular mass of the receptors is 240
kDa (50). Because the receptors were phosphorylated by the stimulation
of rCTGF/Hcs24 (our unpublished data), they may mediate some (at least
one) of the effects of CTGF/Hcs24 on chondrocytes. Our preliminary data
revealed that the number of the receptors decreased during
differentiation of chondrocytes, but the effective concentrations of
CTGF/Hcs24 for the proliferation and expression of differentiated
functions shown in this study were almost the same. Therefore, there
might be some intracellular regulation, such as switching putative
multiple signal transduction pathways downstream of CTGF
receptors during proliferation and differentiation of chondrocytes.
Secondly, we have shown that CTGF/Hcs24 has angiogenic activity (40, 51). Lau and colleagues reported that Fisp12, the mouse ortholog of
CTGF, and another CCN family member, Cyr61, mediate endothelial cell
adhesion through integrin
vß3 and induce
angiogenesis (52, 53, 54). Therefore, it is possible that integrin
vß3 also mediates
some, but not all, effects of CTGF/Hcs24 on chondrocytes. Thirdly,
CTGF/Hcs24 has an IGF-binding protein-like domain and weakly binds to
IGFs (55). Because IGFs stimulate the proliferation and proteoglycan
synthesis (11, 12, 13), and IGF-binding proteins are known to modulate
IGF’s actions (56, 57), it is feasible that CTGF/Hcs24 modulate the
stimulatory effects of IGF on DNA and proteoglycan syntheses in
chondrocytes. Lastly, CTGF has been suggested to exhibit
growth-promoting and cell adhesion activities through its C-terminal
module, and the 10-kDa fragment (C-terminal portion of CTGF) has been
suggested to be its active form with respect to fibroblast
proliferation (48). However, a recent report about CTGF-like protein,
which lacks C-terminal module, indicates that other domains have also
some rather antagonistic activities (58). Because CTGF/Hcs24 is
produced by hypertrophic chondrocytes, and its target cells are
proliferating and maturing chondrocytes and endothelial cells,
CTGF/Hcs24 produced by hypertrophic chondrocytes may be released
directly or after processing into small fragments by an unknown
protease(s) as a paracrine factor. These CTGF/Hcs24 and its derivatives
might exhibit different activities through different pathways in
vivo. Further investigation is needed to clarify the mechanism of
the multiple modes of CTGF/Hcs24 action, but the present study of two
strategies clearly shows that CTGF/Hcs24 is a novel factor that
stimulates the proliferation and differentiation of chondrocytes in
many stages. Moreover, the finding that CTGF/Hcs24 is a novel potent
angiogenesis factor that stimulates the proliferation, migration, and
tube formation of vascular endothelial cells (51) suggests that
CTGF/Hcs24 produced by hypertrophic chondrocytes may also act on
vascular endothelial cells in bone, which is close to the hypertrophic
zone of cartilage, as a paracrine angiogenesis factor, resulting in the
replacement of cartilage by bone.
Recently, it was found that the cyr61 gene, which is a member of the CCN gene family and is expressed in mouse 3T3 fibroblasts, was also expressed in developing mouse cartilaginous elements and placental tissues (59). In addition, its encoded protein promotes chondrogenesis in mouse limb bud mesenchymal cells (60). Previously, we showed that CTGF/Hcs24 was highly expressed in the hypertrophic region of the mouse embryo (E17), and we also revealed its function on chondrocytes in this report. It is noteworthy that there are some differences in the expression patterns of cyr61 and CTGF/Hcs24. The expression of cyr61 was observed in newly formed cartilaginous elements and was diminished during the development of the vertebral column of 12.5- to 15.5-day-old embryos (59). Conversely, expression in the placenta was increased during the development of embryos (59). In the case of CTGF/Hcs24, in situ hybridization revealed a high level of expression in hypertrophic chondrocytes, but not in resting or proliferating chondrocytes of 17-day-old embryos (36). In addition, Northern blot analysis showed that the expression of CTGF/Hcs24 mRNA in the adult placenta was very weak (36). Therefore, although both cyr61 and CTGF/Hcs24 encode novel growth factors that are involved in chondrogenesis and placental formation, they might act in a complementary fashion in differential stages of development.
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Acknowledgments |
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Footnotes |
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2 On leave of absence from the Department of Oral and Maxillofacial
Surgery II, Okayama University Dental School, Okayama 700, Japan.
Received April 27, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxycholecalciferol
and 24R,25-dihydroxycholecalciferol on the proliferation and the
differentiated phenotype of rabbit costal chondrocytes in culture.
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Vß3.
J Biol Chem 273:3090–3096Vß3, promotes endothelial cell survival,
and induces angiogenesis in vivo. Mol Cell Biol 19:2958–2966This article has been cited by other articles:
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 M. Oka, S. Kubota, S. Kondo, T. Eguchi, C. Kuroda, K. Kawata, S. Minagi, and M. Takigawa Gene Expression and Distribution of Connective Tissue Growth Factor (CCN2/CTGF) During Secondary Ossification Center Formation J. Histochem. Cytochem., December 1, 2007; 55(12): 1245 - 1255. [Abstract] [Full Text] [PDF]
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 A. Leask and D. J. Abraham All in the CCN family: essential matricellular signaling modulators emerge from the bunker J. Cell Sci., December 1, 2006; 119(23): 4803 - 4810. [Abstract] [Full Text] [PDF]
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 C.-C. Chang, M.-T. Lin, B.-R. Lin, Y.-M. Jeng, S.-T. Chen, C.-Y. Chu, R. J. Chen, K.-J. Chang, P.-C. Yang, and M.-L. Kuo Effect of connective tissue growth factor on hypoxia-inducible factor 1alpha degradation and tumor angiogenesis. J Natl Cancer Inst, July 19, 2006; 98(14): 984 - 995. [Abstract] [Full Text] [PDF]
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Y. Mukudai, S. Kubota, T. Eguchi, S. Kondo, K. Nakao, and M. Takigawa Regulation of Chicken ccn2 Gene by Interaction between RNA cis-Element and Putative trans-Factor during Differentiation of Chondrocytes J. Biol. Chem., February 4, 2005; 280(5): 3166 - 3177. [Abstract] [Full Text] [PDF]
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M. Hermansson, Y. Sawaji, M. Bolton, S. Alexander, A. Wallace, S. Begum, R. Wait, and J. Saklatvala Proteomic Analysis of Articular Cartilage Shows Increased Type II Collagen Synthesis in Osteoarthritis and Expression of Inhibin {beta}A (Activin A), a Regulatory Molecule for Chondrocytes J. Biol. Chem., October 15, 2004; 279(42): 43514 - 43521. [Abstract] [Full Text] [PDF]
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S. Kubota, K. Kawata, T. Yanagita, H. Doi, T. Kitoh, and M. Takigawa Abundant Retention and Release of Connective Tissue Growth Factor (CTGF/CCN2) by Platelets J. Biochem., September 1, 2004; 136(3): 279 - 282. [Abstract] [Full Text] [PDF]
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C.-C. Chang, J.-Y. Shih, Y.-M. Jeng, J.-L. Su, B.-Z. Lin, S.-T. Chen, Y.-P. Chau, P.-C. Yang, and M.-L. Kuo Connective Tissue Growth Factor and Its Role in Lung Adenocarcinoma Invasion and Metastasis J Natl Cancer Inst, March 3, 2004; 96(5): 364 - 375. [Abstract] [Full Text] [PDF]
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 G. R. GROTENDORST, H. RAHMANIE, and M. R. DUNCAN Combinatorial signaling pathways determine fibroblast proliferation and myofibroblast differentiation FASEB J, March 1, 2004; 18(3): 469 - 479. [Abstract] [Full Text] [PDF]
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 S. Croci, L. Landuzzi, A. Astolfi, G. Nicoletti, A. Rosolen, F. Sartori, M. Y. Follo, N. Oliver, C. De Giovanni, P. Nanni, et al. Inhibition of Connective Tissue Growth Factor (CTGF/CCN2) Expression Decreases the Survival and Myogenic Differentiation of Human Rhabdomyosarcoma Cells Cancer Res., March 1, 2004; 64(5): 1730 - 1736. [Abstract] [Full Text] [PDF]
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M. Minato, S. Kubota, H. Kawaki, T. Nishida, A. Miyauchi, H. Hanagata, T. Nakanishi, T. Takano-Yamamoto, and M. Takigawa Module-Specific Antibodies against Human Connective Tissue Growth Factor: Utility for Structural and Functional Analysis of the Factor as Related to Chondrocytes J. Biochem., March 1, 2004; 135(3): 347 - 354. [Abstract] [Full Text] [PDF]
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 H. R. Mason, A. C. Lake, J. E. Wubben, R. A. Nowak, and J. J. Castellot Jr The growth arrest-specific gene CCN5 is deficient in human leiomyomas and inhibits the proliferation and motility of cultured human uterine smooth muscle cells Mol. Hum. Reprod., March 1, 2004; 10(3): 181 - 187. [Abstract] [Full Text] [PDF]
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 S. B. Connolly, D. Sadlier, N. E. Kieran, P. Doran, and H. R. Brady Transcriptome Profiling and the Pathogenesis of Diabetic Complications J. Am. Soc. Nephrol., August 1, 2003; 14(90003): S279 - 283. [Abstract] [Full Text] [PDF]
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 S. Ivkovic, B. S. Yoon, S. N. Popoff, F. F. Safadi, D. E. Libuda, R. C. Stephenson, A. Daluiski, and K. M. Lyons Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development Development, June 15, 2003; 130(12): 2779 - 2791. [Abstract] [Full Text] [PDF]
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 H. Enomoto, I. Inoki, K. Komiya, T. Shiomi, E. Ikeda, K.-i. Obata, H. Matsumoto, Y. Toyama, and Y. Okada Vascular Endothelial Growth Factor Isoforms and Their Receptors Are Expressed in Human Osteoarthritic Cartilage Am. J. Pathol., January 1, 2003; 162(1): 171 - 181. [Abstract] [Full Text] [PDF]
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J. K. G. Crean, D. Finlay, M. Murphy, C. Moss, C. Godson, F. Martin, and H. R. Brady The Role of p42/44 MAPK and Protein Kinase B in Connective Tissue Growth Factor Induced Extracellular Matrix Protein Production, Cell Migration, and Actin Cytoskeletal Rearrangement in Human Mesangial Cells J. Biol. Chem., November 8, 2002; 277(46): 44187 - 44194. [Abstract] [Full Text] [PDF]
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G. Hashimoto, I. Inoki, Y. Fujii, T. Aoki, E. Ikeda, and Y. Okada Matrix Metalloproteinases Cleave Connective Tissue Growth Factor and Reactivate Angiogenic Activity of Vascular Endothelial Growth Factor 165 J. Biol. Chem., September 20, 2002; 277(39): 36288 - 36295. [Abstract] [Full Text] [PDF]
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 S. Kondo, S. Kubota, T. Shimo, T. Nishida, G. Yosimichi, T. Eguchi, T. Sugahara, and M. Takigawa Connective tissue growth factor increased by hypoxia may initiate angiogenesis in collaboration with matrix metalloproteinases Carcinogenesis, May 1, 2002; 23(5): 769 - 776. [Abstract] [Full Text] [PDF]
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 M A E Rageh, E E-D A Moussad, A K Wilson, and D R Brigstock Steroidal regulation of connective tissue growth factor (CCN2; CTGF) synthesis in the mouse uterus Mol. Pathol., October 1, 2001; 54(5): 338 - 346. [Abstract] [Full Text] [PDF]
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I. HARTLAPP, R. ABE, R. W. SAEED, T. PENG, W. VOELTER, R. BUCALA, and C. N. METZ Fibrocytes induce an angiogenic phenotype in cultured endothelial cells and promote angiogenesis in vivo FASEB J, October 1, 2001; 15(12): 2215 - 2224. [Abstract] [Full Text] [PDF]
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B Perbal NOV (nephroblastoma overexpressed) and the CCN family of genes: structural and functional issues Mol. Pathol., April 1, 2001; 54(2): 57 - 79. [Abstract] [Full Text]
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T. Yamashiro, T. Fukunaga, N. Kobashi, H. Kamioka, T. Nakanishi, M. Takigawa, and T. Takano-Yamamoto Mechanical Stimulation Induces CTGF Expression in Rat Osteocytes Journal of Dental Research, February 1, 2001; 80(2): 461 - 465. [Abstract] [PDF]
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M. Uzumcu, M.F.A. Homsi, D.K. Ball, S. Coskun, K. Jaroudi, J.M.G. Hollanders, and D.R. Brigstock Localization of connective tissue growth factor in human uterine tissues Mol. Hum. Reprod., December 1, 2000; 6(12): 1093 - 1098. [Abstract] [Full Text] [PDF]
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C.-C. Chen, N. Chen, and L. F. Lau The Angiogenic Factors Cyr61 and Connective Tissue Growth Factor Induce Adhesive Signaling in Primary Human Skin Fibroblasts J. Biol. Chem., March 23, 2001; 276(13): 10443 - 10452. [Abstract] [Full Text] [PDF]
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T. M. Grzeszkiewicz, D. J. Kirschling, N. Chen, and L. F. Lau CYR61 Stimulates Human Skin Fibroblast Migration through Integrin alpha vbeta 5 and Enhances Mitogenesis through Integrin alpha vbeta 3, Independent of Its Carboxyl-terminal Domain J. Biol. Chem., June 8, 2001; 276(24): 21943 - 21950. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
, Control viruses; 
, sense
adenoviruses. The PCR products amplified from total RNA of HCS-2/8
cells prepared after 3, 7, and 10 days of transfection were analyzed.
The amount of PCR products in controls on day 0 were taken as 1.0, and
the relative ratio of the products is indicated in the
ordinate. Each value was corrected using the amount of
PCR products of G3PDH as a standard. Points and
bars are the averages and SDs for values
from five determinations. *, P < 0.05,
significantly different from the control virus-transfected cells.
