CCN2 maintains alpha5beta1 integrin signaling in mouse chondrocytes

Objectives: Connective Tissue Growth Factor (CTGF, CCN2)-deficient mice died soon after birth primarily due to impaired endochondral ossification. However, the mechanism underlying impaired endochondral ossification in these mice is unclear. We investigated the mechanism at the point of interaction of integrin with CCN2 using chondrocyes isolated from Ccn2-deficient mice. Methods: To examine the involvement of CCN2 in chondrocyte function, we cultured sternal chondrocytes from Ccn2–deficient mice, and performed Northern blotting, cell proliferation assay and staining for proteoglycan. The interaction of α5 integrin with CCN2 was examined by Western blot and immunoprecipitation analysis. The levels of phospholylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK)1/2 were detected by Western blot and immunofluorescence analysis. Results: Expression of aggrecan was decreased in Ccn2 mutant chondrocytes, but there was no difference in the expression of collagen type II. Ccn2 mutant cells exhibited decreased DNA synthesis and staining of cartilage matrix compared with wild type cells. In addition, expression of α5 integrin was decreased, and adhesion to fibronectin was impaired in mutant cells. Moreover, we found that endogenous CCN2 bound directly to α5 integrin and heparan sulfate in chondrocytes. Furthermore, immunofluorescence and Western blot analysis revealed that levels of FAK and ERK1/2 phosphorylation were reduced in mutant cells spread on fibronectin for 4 hours, whereas the level of phosphorylation was maintained in wild type cells. Conclusion: These results suggest that CCN2 is an essential regulator of integrin signaling in chondrocytes, and impaired integrin signaling underlies defective endochondral ossification in Ccn2 mutants.