Runx2 in the Perichondrial Osteoblasts Enhances Terminal Differentiation of Chondrocytes Through Nell1 Induction

Runx2 plays essential roles in osteoblast differentiation and chondrocyte maturation. Runx2 in the perichondrium has been reported to inhibit chondrocyte maturation through Fgf18 induction. To further investigate the functions of Runx2 in the perichondrium, we generated Runx2^fl/-Cre mice by crossing Runx2^fl/+, Runx2^+/-, and 2.3-kb Col1a1 Cre mice and compared them with Runx2^fl/- mice at E15.5, when the endochondral bones were cartilaginous. Skeletal preparation of the upper limbs in Runx2^fl/-Cre mice showed reduced mineralization of the humerus and scapula, and histological analysis of the femurs showed delays in the terminal differentiation of chondrocytes, as indicated by the absence of mineralization and Spp1 expression in the cartilage and osteoblast differentiation in the perichondrium, compared to those in Runx2^fl/- mice. mRNA sequence analysis showed that the expression of Nell1, which encodes a secreted protein that enhances chondrocyte maturation, in Runx2^fl/-Cre femurs was more than two-fold lower than that in Runx2^fl/- femurs. Nell1 expression was reduced in the perichondrium of Runx2^fl/-Cre femurs compared to that in Runx2^fl/- femurs. Nell1 expression was upregulated by Runx2 overexpression and downregulated by Runx2 siRNA. These findings indicate that Runx2 in perichondrial osteoblasts enhances the terminal differentiation of chondrocytes by inducing Nell1 expression.