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