Roles of heterotypic CCN2/CTGF-CCN3/NOV and homotypic CCN2-CCN2 interactions in expression of the differentiated phenotype of chondrocytes.
To identify proteins that regulate CCN2 activity, we carried out GAL4-based yeast two-hybrid screening with a cDNA library derived from a chondrocytic cell line, HCS-2/8. CCN2/CTGF and CCN3/NOV polypeptides were picked up as CCN2-binding proteins, and CCN2–CCN2 and CCN2–CCN3 binding domains were identified. Direct binding between CCN2 and CCN3 was confirmed by coimmunoprecipitation in vitro and in vivo and surface plasmon resonance, and the calculated dissociation constants (K(d)) were 1.17 × 10(-9) m for CCN2 and CCN2, and 1.95 × 10(-9) m for CCN2 and CCN3. Ectopically overexpressed green fluorescent protein–CCN2 and Halo–CCN3 in COS7 cells colocalized, as determined by direct fluorescence analysis. We present evidence that CCN2–CCN3 interactions modulated CCN2 activity such as enhancement of ACAN and col2a1 expression. Curiously, CCN2 enhanced, whereas CCN3 inhibited, the expression of aggrecan and col2a1 mRNA in HCS-2/8 cells, and combined treatment with CCN2 and CCN3 abolished the inhibitory effect of CCN3. These effects were neutralized with an antibody against the von Willebrand factor type C domain of CCN2 (11H3). This antibody diminished the binding between CCN2 and CCN2, but enhanced that between CCN3 and CCN2. Our results suggest that CCN2 could form homotypic and heterotypic dimers with CCN2 and CCN3, respectively. Strengthening the binding between CCN2 and CCN3 with the 11H3 antibody had an enhancing effect on aggrecan expression in chondrocytes, suggesting that CCN2 had an antagonizing effect by binding to CCN3.
 Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan Biodental Research Center, Okayama University Dental School, Japan.
Mitsuhiro
M
Hoshijima
 Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan Biodental Research Center, Okayama University Dental School, Japan.
Takako
T
Hattori
 Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan Biodental Research Center, Okayama University Dental School, Japan.
Eriko
E
Aoyama
 Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan Biodental Research Center, Okayama University Dental School, Japan.
Takashi
T
Nishida
 Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan Biodental Research Center, Okayama University Dental School, Japan.
Takashi
T
Yamashiro
 Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan Biodental Research Center, Okayama University Dental School, Japan.
Masaharu
M
Takigawa
D053674:Aggrecans / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002454:Cell Differentiation; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism*; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D047468:Immunoprecipitation; D055514:Nephroblastoma Overexpressed Protein / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D055503:Protein Multimerization; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020349:Surface Plasmon Resonance; D020798:Two-Hybrid System Techniques
D053674:Aggrecans; C526317:CCN2 protein, human; C526319:CCN3 protein, human; C499177:COL2A1 protein, human; D024043:Collagen Type II; D055514:Nephroblastoma Overexpressed Protein; D012333:RNA, Messenger; D055513:Connective Tissue Growth Factor