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Endocrinology Vol. 141, No. 1 264-273
Copyright © 2000 by The Endocrine Society

ARTICLES

Effects of CTGF/Hcs24, a Product of a Hypertrophic Chondrocyte-Specific Gene, on the Proliferation and Differentiation of Chondrocytes in Culture1

Tohru Nakanishi, Takashi Nishida, Tsuyoshi Shimo2, Kappei Kobayashi, Toshikazu Kubo, Takuya Tamatani, Katsunari Tezuka and Masaharu Takigawa

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recently, we cloned a messenger RNA (mRNA) predominantly expressed in chondrocytes from a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, by differential display PCR and found that its gene, named hcs24, was identical with that of connective tissue growth factor (CTGF). Here we investigated CTGF/Hcs24 function in the chondrocytic cell line HCS-2/8 and rabbit growth cartilage (RGC) cells. HCS-2/8 cells transfected with recombinant adenoviruses that generate CTGF/Hcs24 sense RNA (mRNA) proliferated more rapidly than HCS-2/8 cells transfected with control adenoviruses. HCS-2/8 cells transfected with recombinant adenoviruses that generate CTGF/Hcs24 sense RNA expressed more mRNA of aggrecan and type X collagen than the control cells. To elucidate the direct action of CTGF/Hcs24 on the cells, we transfected HeLa cells with CTGF/Hcs24 expression vectors, obtained stable transfectants, and purified recombinant CTGF/Hcs24 protein from conditioned medium of the transfectants. The recombinant CTGF/Hcs24 effectively promoted the proliferation of HCS-2/8 cells and RGC cells in a dose-dependent manner and also dose dependently increased proteoglycan synthesis in these cells. In addition, these stimulatory effects of CTGF/Hcs24 were neutralized by the addition of anti-CTGF antibodies. Furthermore, the recombinant CTGF/Hcs24 effectively increased alkaline phosphatase activity in RGC cells in culture. Moreover, RT-PCR analysis revealed that the recombinant CTGF/Hcs24 stimulated gene expression of aggrecan and collagen types II and X in RGC cells in culture. These results indicate that CTGF/Hcs24 directly promotes the proliferation and differentiation of chondrocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE PROCESS of endochondral ossification, growth cartilage cells first proliferate and become mature chondrocytes, which produce much extracellular matrix, mainly composed of aggrecan and type II collagen (1, 2, 3). Then, the cells become hypertrophic chondrocytes, which produce type X collagen and alkaline phosphatase (ALPase), and the matrix is mineralized and invaded by capillary sprouts; finally, cartilage is replaced by bone (1, 2, 3). Many kinds of hormones, such as PTH and the active form of vitamin D3 (4, 5, 6); vitamins, such as ascorbic acid and retinoic acid (7, 8, 9, 10); and growth factors, such as insulin-like growth factors (IGFs) (11, 12, 13), basic fibroblast growth factor (14, 15), transforming growth factor-ß (TGFß) (16, 17, 18), and bone morphogenetic proteins (BMPs) (19), have been shown to be involved in the process. However, these factors stimulate the proliferation and differentiation of chondrocytes at a specific stage and inhibit them at other stages. For example, IGF-I stimulates the proliferation and proteoglycan synthesis but inhibits chondrocyte hypertrophy (12, 13), whereas basic fibroblast growth factor stimulates the proliferation of rabbit growth cartilage (RGC) cells but inhibits their proteoglycan synthesis and terminal differentiation (20, 21). On the other hand, TGFß stimulates both proliferation and proteoglycan synthesis of RGC cells (18) but inhibits further differentiation of the cells (22). BMPs also stimulate the proliferation and differentiation of chondrocytes, especially in the early stage of chondrocyte differentiation (23, 24) but have not been shown to induce angiogenesis, which is involved in the final step of endochondral ossification.

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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The human chondrosarcoma-derived chondrocytic cell line HCS-2/8 (26, 27, 28, 29, 30) was inoculated at a density of 5 x 104 cells/cm2 in 10-cm diameter dishes or 2 x 104 cells/cm2 in 96-well plates (Sumitomo Bakelite Co. Ltd., Tokyo, Japan) and cultured in DMEM (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) containing 10% FBS (Upstate Biotechnology, Inc., Lake Placid, NY). RGC cells were isolated from growth cartilage of ribs of young rabbits as described previously (2, 4). The isolated cells were inoculated at a density of 2 x 104 cells/cm2 in 10-cm diameter dishes, 24-well plates, or 96-well plates (Sumitomo Bakelite Co. Ltd.) and cultured in {alpha}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 {alpha}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 {alpha}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 {alpha}MEM containing 10% FBS for 6 days. Then, the medium was replaced with {alpha}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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Proliferation of HCS-2/8 cells transfected with recombinant adenoviruses generating sense RNA of CTGF/Hcs24
We constructed recombinant adenoviruses that express sense RNA (mRNA) of CTGF/Hcs24 under the control of CAG promoter (Fig. 1AGo) 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. 1DGo, 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. 1BGo). 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. 1BGo, S), no band was observed in control cultures (Fig. 1Go, 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. 1Go, 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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  		Figure 1. Effect of transfection with adenoviruses that express sense RNA of CTGF/Hcs24 on the proliferation of HCS-2/8 cells. A, Construction of adenoviruses that express sense RNA of CTGF/Hcs24. B, Western blotting of the cell lysates prepared from control or sense adenovirus transfected cells using anti-CTGF antibodies. HCS-2/8 cells were inoculated at a density of 1 x 105 cells/35-mm dish. Next day, the medium was removed, and a 100-µl solution of adenoviruses (final concentration, 4 x 107 plaque-forming units), which express CTGF/Hcs24 sense RNA (mRNA), was added. After 1-h incubation at 37 C, the cells were fed with 1 ml serum-containing medium. Cell lysates were prepared 5 days after the infection. About 20 µg of each cell lysate were subjected to SDS-PAGE, and Western blotting was performed as described in Materials and Methods. C, Untreated; CV, transfected with control virus with no insert; S, transfected with sense (mRNA) virus. The arrow indicates the major bands corresponding to intact CTGF/Hcs24 protein. Because the sensitivity of Western blotting was adjusted for suitable detection of overexpressed CTGF/Hcs24 (B, S), no band was observed in control cultures (B, C and CV), but at a higher sensitivity CTGF/Hcs24 was observed in control cultures (data not shown). C, Growth rate of the recombinant adenovirus-transfected HCS-2/8 cells. HCS-2/8 cells were inoculated, and adenoviruses were transfected as described in B. Cell number was estimated 5 days after the infection when the cells were in logarithmic growth phase. S, Transfected with CTGF/Hcs24 sense virus; X, transfected with ß-gal-producing virus; CV, transfected with control virus with no insert. The ordinate indicates the cell number in 35-mm dishes. Cell numbers were calculated at eight points in three culture dishes and are given as the mean ± SD. *, P < 0.01, significantly different from the control virus-insfected cells. D, Phase contrast microphotographs of HCS-2/8 cells transfected with adenoviruses that express CTGF/Hcs24 sense RNA (mRNA; S), ß-gal (X), or control vector (CV), which contains no insert DNA. The transfection was performed under the same conditions as in B. Photomicrographs were taken 3 days after the transfection. Magnification, x33.

 
Expression of mRNA of CTGF/Hcs24, aggrecan core protein, and type X collagen in HCS-2/8 transfected with recombinant adenoviruses generating sense RNA of CTGF/Hcs24
To confirm successful transfection of the adenoviruses at the mRNA level, RT-PCR with a primer set to amplify CTGF/Hcs24 mRNA was performed using the RNA prepared from the transfected HCS-2/8 cells after 3, 7, and 10 days of transfection (Fig. 2Go). The expression of G3PDH was also estimated as a control. Figure 2AGo (CTGF/Hcs24) shows the products of RT-PCR in agarose gels, and Fig. 2BGo (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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  		Figure 2. Effect of transfection with adenoviruses that express sense RNA of CTGF/Hcs24 on the differentiation of HCS-2/8 cells. A, Adenoviruses that express CTGF/Hcs24 sense RNA (mRNA; lanes 3, 5, and 7) were transfected to HCS-2/8 cells as described in Fig. 1BGo. Control viruses were also transfected to HCS-2/8 cells (lanes 2, 4, and 6). Total RNA was prepared from the cells when adenoviruses were transfected (lane 1) and 3 days (lanes 2 and 3), 7 days (lanes 4 and 5), and 10 days (lanes 6 and 7) after the transfection. RT-PCR was performed using the primers described in Materials and Methods. CTGF/Hcs24 primers in the coding region of CTGF/Hcs24 were used: aggrecan core protein (AGR), collagen type X [{alpha}1(X)], and G3PDH as a typical control. B, Densitmetric analysis of the results of RT-PCR. {blacksquare}, Control viruses; {circ}, 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.

 
Under the same conditions, expression of aggrecan core protein (AGR), which is a maker of differentiated chondrocytes, and that of type X collagen [{alpha}1(X)], which is a well known marker of hypertrophic chondrocytes, were estimated by RT-PCR (Fig. 2Go). 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. 3Go, 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 3BGo 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. 4AGo, 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. 4AGo). Addition of 10 µg/ml anti-CTGF antibody effectively neutralized the stimulatory action of recombinant protein, whereas nonimmune control serum had no effect (Fig. 5AGo). 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. 4BGo). rCTGF/Hcs24 also stimulated the DNA synthesis of RGC cells (Fig. 6AGo). 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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  		Figure 3. Production of rCTGF/Hcs24 protein from HeLa cells. A, Construction of CTGF/Hcs24 expression vector under the control of CMV promoter. B, Silver staining (lane S) and Western blotting (lane W) of rCTGF/Hcs24 (0.5 and 0.1 µg, respectively) purified from the conditioned medium of HeLa cells transformed with CTGF/Hcs24 expression vector.

 


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  		Figure 4. Effects of rCTGF/Hcs24 protein on DNA and proteoglycan syntheses of HCS-2/8 cells in culture. A, For estimation of DNA synthesis, HCS-2/8 cells were inoculated at a density of 2 x 104 cells/well of 96-well plates in 100 µl DMEM containing 10% FBS. In the subconfluent stage, the concentration of FBS was reduced to 0.5%, and the culture was preincubated for 24 h. Then, purified rCTGF/Hcs24 was added to the cultures at the concentrations indicated. DNA synthesis was measured by the incorporation of [3H]thymidine after 22 h as described in Materials and Methods. Points and bars are the averages and SDs for triplicate cultures. The ordinate indicates the percentage of the PBS-treated control value. *, P < 0.05; **, P < 0.01 (significantly different from the control cultures). For estimation of proteoglycan synthesis, confluent cultures of HCS-2/8 cells were preincubated in DMEM containing 0.5% FBS for 24 h and then incubated in the same medium with or without rCTGF/Hcs24 (10–100 ng/ml) for 5 h. Then, [35S]sulfate (37 MBq/ml) dissolved in PBS was added to the cultures (370 kBq/ml final), and incubation was continued for another 17 h. Points and bars are the averages and SDS for triplicate cultures. *, P < 0.05 (significantly different from the control cultures). B, For estimation of cell proliferation, HCS-2/8 cells were inoculated at a density of 50,000 cells/well in 24-well microplates in 1 ml DMEM containing 10% FBS and 50 ng/ml rCTGF/Hcs24. Cell numbers were calculated at eight points in three culture dishes on days 3 and 6 and are given as the means ± SDs. *, P < 0.05 (significantly different from the control cultures).

 


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  		Figure 5. Effect of anti-CTGF antibody on CTGF/Hcs24-induced DNA and proteoglycan syntheses in HCS-2/8 cells. A, For estimation of DNA synthesis, HCS-2/8 cells were grown and preincubated as described in Fig. 4AGo. B, For estimation of proteoglycan synthesis, confluent cultures of HCS-2/8 cells were preincubated as described in Fig. 4BGo. The cultures were then treated with 50 ng/ml rCTGF/Hcs24 (lane 2), 50 ng/ml rCTGF/Hcs24 plus 10 µg/ml (A) or 30 µg/ml (B) anti-CTGF antibody (lane 3), 50 ng/ml rCTGF/Hcs24 plus preimmune serum (lane 4), or PBS (lane 1). DNA and proteoglycan syntheses were determined as described in Materials and Methods. Columns and bars are the means and SDs from triplicate cultures. *, P < 0.05, significantly different from the control cultures. **, P < 0.05, significantly different from the preimmune serum-treated cultures.

 


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  		Figure 6. Effects of rCTGF/Hcs24 protein on the proliferation, proteoglycan synthesis, and ALPase activity RGC cells in culture. A, For estimation of DNA synthesis, RGC cells were inoculated at a density of 1 x 104 cells/well of 96-well plates in 100 µl {alpha}MEM containing 10% FBS. When they reached subconfluence, the cells were incubated in 100 µl {alpha}MEM containing 0.5% FBS for 48 h, and then rCTGF/Hcs24 was added to the cultures at the concentrations indicated. PBS was added to the control culture. DNA synthesis was measured 22 h later as described in Materials and Methods. Points and bars are the averages and SDs for triplicate cultures. *, P < 0.05; **, P < 0.01 (significantly different from the control cultures). B, For estimation of proteoglycan synthesis, confluent cultures of RGC cells were preincubated in DMEM containing 0.5% FBS for 24 h and then incubated in the same medium with rCTGF/Hcs24 (10–100 ng/ml) for 5 h. PBS was added to the control culture. Then, [35S]sulfate (37 MBq/ml) dissolved in PBS was added to the cultures (final concentration, 370 kBq/ml), and incubation was continued for another 17 h. Points and bars are the averages and SDs for triplicate cultures. **, P < 0.01 (significantly different from the control cultures). C, For estimation of ALPase activity, 2 x 104 cells/well of RGC cells were grown in 48-well microplates with {alpha}MEM containing 10% FBS until they became slightly overconfluent. Then, the medium was replaced with {alpha}MEM containing 0.5% FBS, and rCTGF/Hcs24 was added at the concentrations indicated. PBS was added to the control culture. ALPase activity was determined 72 h after the addition. The enzyme activity was indicated as nanomoles of p-nitrophenol (PNP) per µg protein. Points and bars are the averages and SDs for triplicate cultures. *, P < 0.05; **, P < 0.01 (significantly different from the 0-time control cultures).

 
Stimulation of proteoglycan synthesis in HCS-2/8 cells and RGC by rCTGF/Hcs24 protein
Proteoglycan synthesis is a good marker of differentiated chondrocytes (3, 4). When rCTGF/Hcs24 was added to the culture of HCS-2/8 cells, proteoglycan synthesis increased dose dependently, reaching a maximum at a concentration of 50 ng/ml (Fig. 4AGo). Addition of 30 µg/ml anti-CTGF antibody effectively neutralized the stimulatory effect of recombinant protein, whereas nonimmune control serum had no effect (Fig. 5BGo). 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. 6BGo). 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 {alpha}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. 6CGo).

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. 7Go). 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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  		Figure 7. The effect of rCTGF/Hcs24 on the mRNA expression of aggrecan core protein, type II collagen, and type X collagen in RGC cells. RGC cells were inoculated at a density of 8 x 104 cells/35-mm dish in {alpha}MEM containing 10% FBS on day 0, and rCTGF/Hcs24 (30 or 50 ng/ml) was added to the culture on day 2. The medium was changed on days 2, 4, and 7. On day 9, total RNA was isolated from the cells for RT-PCR analysis. The resulting PCR products were analyzed on agarose gel electrophoresis, and the density of each band was quantitated. Lane 1, Control; lane 2, 30 ng/ml rCTGF/Hcs24; lane 3, 50 ng/ml rCTGF/Hcs24. The amounts of the products are indicated on the ordinate as the expression ratio based on the amounts of PCR products of 18S ribosome RNA as a standard. Points and bars are the averages and SDs for for values from five determinations. *, P < 0.05; **, P < 0.01 (significantly different from the control cultures).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we used two strategies to investigate the roles of CTGF/Hcs24 in the proliferation and differentiation of chondrocytes: gene transfection with adenoviruses expressing CTGF/Hcs24 sense RNA (mRNA) and preparation and usage of rCTGF/Hcs24 for chondrocytes. The adenovirus system is a powerful tool for introducing foreign DNA units into cells with high frequency (45, 46, 47). The chondrosarcoma-derived chondrocytic cell line HCS-2/8 was previously shown to be efficiently transfected with adenoviruses carrying ß-galactosidase, TGFß, or heat shock protein genes (39). These genes were continuously expressed in the cells for at least 21 days. Therefore, experiments for gene transfection were carried out using the HCS-2/8 cells to investigate the role of CTGF/Hcs24. To further investigate the role of CTGF/Hcs24, we applied rCTGF/Hcs24 on HCS-2/8 cells and RGC cells in primary culture. For production of rCTGF/Hcs24, we used stable transformants of mammalian cells (HeLa cells). The reason for this is that rCTGF/Hcs24 produced by E. coli had been predicted to be biologically inactive because of the high content of cysteine residues in CTGF protein. As a result, three bands of rCTGF/Hcs24 with molecular masses of 36–38 kDa were obtained, and these differences are probably due to differential glycosylation because CTGF protein has two glycosylation sites (33). The low molecular mass form of 20 kDa detected by Western blotting should be a processed form of CTGF/Hcs24, because Brigstock et al. (48) identified 10-, 16-, and 20-kDa CTGF-immunoreactive proteins in uterine secretory fluids and also showed that the 10-kDa protein was active. Therefore, although the preparation of rCTGF/Hcs24 used in this study was composed of several bands, they all are CTGF/Hcs24 or CTGF/Hcs24-related protein.

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. 4Go and 6Go), 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 {alpha}vß3 and induce angiogenesis (52, 53, 54). Therefore, it is possible that integrin {alpha}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.


   Acknowledgments
 
We thank Dr. Kojiro Takahashi and Miss Takako Hattori for useful discussions, and Mrs. Etsuko Fujisawa for secretarial assistance.


   Footnotes
 
1 This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (to T.N. and M.T.), grants from the the Naito Foundation (to M.T.), the Nakatomi Health Science Foundation (to M.T.), and the Foundation for Growth Science in Japan (to M.T.), and Research for the Future Programme of The Japan Society for the Promotion of Science (JSPS) (Project: Biological Tissue Engineering, JSPS-RFTF98100201). Back

2 On leave of absence from the Department of Oral and Maxillofacial Surgery II, Okayama University Dental School, Okayama 700, Japan. Back

Received April 27, 1999.


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 Results
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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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 J. Cell Sci.Home page
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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 JNCI J Natl Cancer InstHome page
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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 J. Biol. Chem.Home page
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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 J. Biol. Chem.Home page
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.