Branch site mutated mice revealed distinct roles of two Runx2 isoforms in bone development

Runx2 is a key regulator of osteoblast differentiation and chondrocyte maturation. However, the distinct functions of the two functional isoforms remain to be clarified. Transcription of two isoforms, Runx2-I and Runx2-II, starts from the proximal and distal promoters upstream of exons 2 and 1, respectively. To investigate the functions of the two isoforms, we generated a novel mouse model (Runx2-br^mut/mut), in which intron 1 splicing for Runx2-II was disrupted by mutating the branch site essential for splicing. Runx2-II was severely reduced but Runx2-I was increased in Runx2-br^mut/mut mice as compared with those in Runx2-br^wt/wt mice. Although Runx2-II was about three times higher than Runx2-I in E15.5 limbs and newborn calvaria of Runx2-br^wt/wt mice, Runx2-II was extremely lower than Runx2-I in Runx2-br^mut/mut mice. Endochondral ossification was retarded in Runx2-br^mut/mut mice, but the delay was milder than in Runx2 ^+/- mice, and the primary spongiosa formation was impaired due to the reduced osteoblasts. The development of calvaria in the newborn was retarded similar to Runx2 ^+/- mice, which showed cleidocranial dysplasia, but it was much less affected than in Runx2 ^+/- mice at 8 weeks of age, and the suture mesenchymal cell proliferation increased. Furthermore, clavicle development was less affected than that in Runx2 ^+/- mice throughout their lives. The trabecular and cortical bones in the femurs of Runx2-br^mut/mut mice were lower than those of Runx2-br^wt/wt mice owing to the reduced bone formation, and the strength of the bones was also weaker. Osteoblast differentiation was impaired in Runx2-br^mut/mut mice. Overexpression of Runx2-II failed to affect endogenous Runx2 expression in vitro, and Runx2 knockdown by siRNA failed to affect the proximal promoter activity. These findings indicated that both isoforms contribute to endochondral ossification, Runx2-I can compensate for Runx2-II in endochondral ossification, Runx2-II plays important roles in osteoblast differentiation, and Runx2-I plays important roles in the development of calvaria and clavicles, at least in part, by enhancing suture mesenchymal cell proliferation. Our findings also showed that a minimal amount of Runx2-II is necessary for the efficient function of Runx2-I, indicating a basal requirement of Runx2-II in bone development, but that Runx2 expression is not autoregulated by Runx2.