CCN2/CTGF interacts with fibronectin 1 and enhances cell adhesion

Objectives: CCN family member 2 / Connective tissue growth factor (CCN2/CTGF) is strongly expressed in cartilage, especially in hypertrophic chondrocyte; it modulates chondrocytes differentiation and proliferation and enhances production of extracellular matrices, cell adhesion and migration. CCN2/CTGF deficient mice show prolonged growth plates and enhanced bone fragility, suggesting that CCN2/CTGF also has an important role in endochondral ossification. Although the multiple functions of CCN2/CTGF are well known, the signal transduction pathway including its specific receptor on chondrocytes has not been identified, but there is evidence that CCN2/CTGF signaling occurs through integrin receptors and LRP1, an endocytotic receptor. Therefore we were aimed to identify the factors which modulate CCN2/CTGF function. Methods: Yeast-two hybrid screening using a cDNA library from the chondrocytic cell line HCS-2/8 and CCN2/CTGF as a bait was done. To confirm the binding, CCN2/CTGF fragment which contains one of the modules and a cDNA which picked up were cotransfected to yeast. For cell adhesion assay, fibronectin 1 was coated on the plate and HCS-2/8 cells contain recombinant CCN2/CTGF were placed and adhesive cells were counted. Results: One of the proteins interacting with CCN2/CTGF was fibronectin 1. CCN2/CTGF consists of four modules; using the yeast two hybrid assay, we showed that only the C-terminal domain (CT) of CCN2/CTGF binds to fibronectin 1. Furthermore, we demonstrated that CCN2/CTGF enhances the adhesion of HCS-2/8 cells to fibronectin 1 dose dependently. A monoclonal antibody against the CT domain partially inhibited CCN2/CTGF adhesion activity, indicating that CCN2/CTGF enhances cell adhesion activity through its CT domain. Conclusion: CCN2/CTGF may be trapped by fibronectin 1 in the extracellular matrix and enhances fibronectin 1 binding to its integrin receptor, thereby enhancing integrin activation and cell adhesion.