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.
Jiang et al. (Fri,) studied this question.