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...

Summary. Runx2 is an essential transcription factor for bone and tooth development whose function in odontoblast differentiation remains to be clarified. To pursue this issue, we examined tooth development in Runx2 transgenic mice under the control of Col1a1 promoter (Tg(Col1a1-Runx2) mice). Endogenous Runx2 protein was detected in the nuclei of preodontoblasts, immature odontoblasts, mesenchymal cells in the dental sac, and osteoblasts, while transgene expression was detected in odontoblasts and osteoblasts. Odontoblasts in Tg(Col1a1-Runx2) mice lost their columnar shape and dentin was deposited around the odontoblasts, which were cuboid or flat in shape. The dentin in Tg(Col1a1-Runx2) mice was thin and possessed lacunae that contained odontoblasts and bone canaliculi-like structures, while predentin and Received May 7, 2008This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (19592120) and the President's Discretionary Fund of Nagasaki University, Japan.Address for correspondence: Prof. Toshihisa Komori, MD, PhD, Department of Cell Biology, Unit of Basic Medical Sciences, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan Tel: +81-95-819-7630, Fax: +81-95-819-7633 E-mail: komorit@nagasaki-u.ac.jp dentinal tubules were absent. We examined the expression of dentin matrix protein genes, Col1a1 and dentin sialophosphoprotein (DSPP), by in situ hybridization, and dentin matrix proteins, osteocalcin, osteopontin, and dentin matrix protein 1 (DMP1) as well as an intermediate fi lament, nestin, by immunohistochemistry to characterize odontoblasts in Tg(Col1a1-Runx2) mice. Results showed Col1a1 expression was down-regulated, DSPP expression was lost, and nestin expression was severely decreased in the odontoblasts of Tg(Col1a1-Runx2) mice. Further, the expressions of osteocalcin, osteopontin, and DMP1 were up-regulated in odontoblasts, although the upregulation of osteocalcin expression was transient. These findings indicate that Runx2 inhibits the terminal differentiation of odontoblasts, and that Runx2 induces transdifferentiation of odontoblasts into osteoblasts forming a bone structure. Thus, Runx2 expression has to be downregulated during odontoblast differentiation to acquire full odontoblast differentiation for dentinogenesis.

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