The differentiation of mesenchymal cells into chondrocytes and chondrocyte proliferation and maturation are fundamental steps in skeletal development. Runx2 is essential for osteoblast differentiation and is involved in chondrocyte maturation. Although chondrocyte maturation is delayed inVertebrate skeletons are constructed through the formation of bone structures, a process that is achieved by intramembranous or endochondral ossification. Intramembranous bones, which are directly formed by osteoblasts, are restricted to the cranial vault, some facial bones, and parts of the mandible and clavicle, whereas the rest of the skeleton is composed of endochondral bones that are formed as a cartilaginous template which is then replaced by bone. In early skeletal development, mesenchymal cells condense and acquire the phenotypes of chondrocytes including the ability to produce Col2a1 and proteoglycan. In the process of endochondral ossification, immature chondrocytes proliferate, and chondrocytes at the center of the cartilaginous skeleton begin to mature to become prehypertrophic chondrocytes, which express parathyroid hormone/parathyroid hormone-related peptide (Pthlh) receptor (Pthr1) and Indian hedgehog (Ihh). The prehypertrophic chondrocytes further mature to hypertrophic chondrocytes, which express Col10a1. Upon the terminal differentiation of chondrocytes, the terminal hypertrophic chondrocytes express osteopontin, the matrix is mineralized, vascular vessels invade the calcified cartilage, and finally the cartilage is replaced by bone. Chondrocyte proliferation and differentiation occur in an organized manner and result in the formation of a growth plate that is composed of layers of chondrocytes at different stages of differentiation, in-

Targeted disruption of core binding factor α1 (Cbfa1) showed that Cbfa1 is an essential transcription factor in osteoblast differentiation and bone formation. Furthermore, both in vitro and in vivo studies showed that Cbfa1 plays important roles in matrix production and mineralization. However, it remains to be clarified how Cbfa1 controls osteoblast differentiation, bone formation, and bone remodelling. To understand fully the physiological functions of Cbfa1, we generated transgenic mice that overexpressed Cbfa1 in osteoblasts using type I collagen promoter. Unexpectedly, Cbfa1 transgenic mice showed osteopenia with multiple fractures. Cortical bone, which was thin, porous, and enriched with osteopontin, was invaded by osteoclasts, despite the absence of acceleration of osteoclastogenesis. Although the number of neonatal osteoblasts was increased, their function was impaired in matrix production and mineralization. Furthermore, terminally differentiated osteoblasts, which strongly express osteocalcin, and osteocytes were diminished greatly, whereas less mature osteoblasts expressing osteopontin accumulated in adult bone. These data indicate that immature organization of cortical bone, which was caused by the maturational blockage of osteoblasts, led to osteopenia and fragility in transgenic mice, demonstrating that Cbfa1 inhibits osteoblast differentiation at a late stage.

Runx2 and phosphatidylinositol 3-kinase (PI3K)–Akt signaling play important roles in osteoblast and chondrocyte differentiation. We investigated the relationship between Runx2 and PI3K-Akt signaling. Forced expression of Runx2 enhanced osteoblastic differentiation of C3H10T1/2 and MC3T3-E1 cells and enhanced chondrogenic differentiation of ATDC5 cells, whereas these effects were blocked by treatment with IGF-I antibody or LY294002 or adenoviral introduction of dominant-negative (dn)–Akt. Forced expression of Runx2 or dn-Runx2 enhanced or inhibited cell migration, respectively, whereas the enhancement by Runx2 was abolished by treatment with LY294002 or adenoviral introduction of dn-Akt. Runx2 up-regulated PI3K subunits (p85 and p110β) and Akt, and their expression patterns were similar to that of Runx2 in growth plates. Treatment with LY294002 or introduction of dn-Akt severely diminished DNA binding of Runx2 and Runx2-dependent transcription, whereas forced expression of myrAkt enhanced them. These findings demonstrate that Runx2 and PI3K-Akt signaling are mutually dependent on each other in the regulation of osteoblast and chondrocyte differentiation and their migration.

During skeletogenesis, cartilage develops to either permanent cartilage that persists through life or transient cartilage that is eventually replaced by bone. However, the mechanism by which cartilage phenotype is specified remains unclarified. Core binding factor α1 (Cbfa1) is an essential transcription factor for osteoblast differentiation and bone formation and has the ability to stimulate chondrocyte maturation in vitro. To understand the roles of Cbfa1 in chondrocytes during skeletal development, we generated transgenic mice that overexpress Cbfa1 or a dominant negative (DN)-Cbfa1 in chondrocytes under the control of a type II collagen promoter/enhancer. Both types of transgenic mice displayed dwarfism and skeletal malformations, which, however, resulted from opposite cellular phenotypes. Cbfa1 overexpression caused acceleration of endochondral ossification due to precocious chondrocyte maturation, whereas overexpression of DN-Cbfa1 suppressed maturation and delayed endochondral ossification. In addition, Cbfa1 transgenic mice failed to form most of their joints and permanent cartilage entered the endochondral pathway, whereas most chondrocytes in DN-Cbfa1 transgenic mice retained a marker for permanent cartilage. These data show that temporally and spatially regulated expression of Cbfa1 in chondrocytes is required for skeletogenesis, including formation of joints, permanent cartilages, and endochondral bones.