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-

Receptor activator of nuclear factor-B ligand (RANKL), osteoprotegerin (OPG), and macrophage-colony stimulating factor play essential roles in the regulation of osteoclastogenesis. Runx2-deficient (Runx2 ؊/؊ ) mice showed a complete lack of bone formation because of maturational arrest of osteoblasts and disturbed chondrocyte maturation. Further, osteoclasts were absent in these mice, in which OPG and macrophage-colony stimulating factor were normally expressed, but RANKL expression was severely diminished. We investigated the function of Runx2 in osteoclast differentiation. A Runx2 ؊/؊ calvaria-derived cell line (CA120 -4), which expressed OPG strongly but RANKL barely, severely suppressed osteoclast differentiation from normal bone marrow cells in co-cultures. Adenoviral introduction of Runx2 into CA120 -4 cells induced RANKL expression, suppressed OPG expression, and restored osteoclast differentiation from normal bone marrow cells, whereas the addition of OPG abolished the osteoclast differentiation induced by Runx2. Addition of soluble RANKL (sRANKL) also restored osteoclast differentiation in co-cultures. Forced expression of sRANKL in Runx2 ؊/؊ livers increased the number and size of osteoclast-like cells around calcified cartilage, although vascular invasion into the cartilage was superficial because of incomplete osteoclast differentiation. These findings indicate that Runx2 promotes osteoclast differentiation by inducing RANKL and inhibiting OPG. As the introduction of sRANKL was insufficient for osteoclast differentiation in Runx2 ؊/؊ mice, however, our findings also suggest that additional factor(s) or matrix protein(s), which are induced in terminally differentiated chondrocytes or osteoblasts by Runx2, are required for osteoclastogenesis in early skeletal development.In the process of endochondral ossification, chondrocytes mature to hypertrophic chondrocytes, matrix around terminally differentiated chondrocytes (terminal hypertrophic chondrocytes) is mineralized, blood vessels invade into the calcified cartilage, and cartilage is replaced by bone (1). Osteoclasts accelerate these processes by resorption of the calcified matrix leading to bone marrow formation. Osteoclasts differentiate from hematopoietic precursor cells through direct contact with osteoblastic/stromal cells (2). Recently, osteoprotegerin (OPG) 1 / osteoclastogenesis inhibitory factor, which is an inhibitor of osteoclast differentiation (3, 4), and receptor activator of NF-B (RANK) ligand (RANKL)/tumor necrosis factor-related activation-induced cytokine/OPG ligand/osteoclast differentiation factor, which is an inducer of osteoclast differentiation (5-8), were identified. RANKL, which is expressed on the surface of osteoblastic/stromal cells or released as a soluble factor, binds to its receptor RANK (9, 10), which is expressed on the surface of osteoclast precursors and osteoclasts, and induces osteoclast differentiation and activation. OPG, which binds RANKL with higher affinity than RANK, acts as a decoy receptor for RANKL and in...

Runx2 (runt-related transcription factor 2) is an important transcription factor for chondrocyte differentiation as well as for osteoblast differentiation. To investigate the function of Runx2 in chondrocytes, we isolated chondrocytes from the rib cartilage of Runx2-deficient (Runx2–/–) mice and examined the effect of Runx2 deficiency on chondrocyte function and behavior in culture for up to 12 days. At the beginning of the culture, Runx2–/– chondrocytes actively proliferated, had a polygonal shape and expressed type II collagen; these are all characteristics of chondrocytes. However, they gradually accumulated lipid droplets that stained with oil red O and resembled adipocytes. Northern blot analysis revealed that the expression of adipocyte-related differentiation marker genes including PPARγ (peroxisome proliferator-activated receptor γ), aP2 and Glut4 increased over time in culture, whereas expression of type II collagen decreased. Furthermore, the expression of Pref-1, an important inhibitory gene of adipogenesis, was remarkably decreased. Adenoviral introduction of Runx2 or treatment with transforming growth factor-β, retinoic acid, interleukin-1β, basic fibroblast growth factor, platelet-derived growth factor or parathyroid hormone inhibited the adipogenic changes in Runx2–/– chondrocytes. Runx2 and transforming growth factor-β synergistically upregulated interleukin-11 expression, and the addition of interleukin-11 to the culture medium reduced adipogenesis in Runx2–/– chondrocytes. These findings indicate that depletion of Runx2 resulted in the loss of the differentiated phenotype in chondrocytes and induced adipogenic differentiation in vitro, and show that Runx2 plays important roles in maintaining the chondrocyte phenotype and in inhibiting adipogenesis. Our findings suggest that these Runx2-dependent functions are mediated, at least in part, by interleukin-11.