Runx2 is an essential transcription factor for osteoblast differentiation. However, the functions of Runx2 in postnatal bone development remain to be clarified. Introduction of dominant-negative (dn)-Runx2 did not inhibit Col1a1 and osteocalcin expression in mature osteoblastic cells. In transgenic mice that expressed dn-Runx2 in osteoblasts, the trabecular bone had increased mineralization, increased volume, and features of compact bone, and the expression of major bone matrix protein genes was relatively maintained. After ovariectomy, neither osteolysis nor bone formation was enhanced and bone was relatively conserved. In wild-type mice, Runx2 was strongly expressed in immature osteoblasts but downregulated during osteoblast maturation. These findings indicate that the maturity and turnover rate of bone are determined by the level of functional Runx2 and Runx2 is responsible for bone loss in estrogen deficiency, but that INTRODUCTIONBone is composed of compact bone and cancellous bone. In long bones, the shaft (cortical bone) consists of compact bone, and the inside of the shaft (trabecular bone), which is a threedimensional lattice of branching bony spicules, consists of cancellous bone. Compact bone is mature bone, because it is composed of densely packed, highly organized collagen fibrils with high mineralization, and is relatively resistant to osteolysis. In contrast, cancellous bone is less mature, because it is composed of loosely organized collagen fibrils with low mineralization, and it is easily resorbed and plays an important role in calcium homeostasis (Marks and Odgren, 2002). Runt-related transcription factor 2 (Runx2) is a transcription factor that belongs to the Runx family and is involved in many aspects of skeletal development (Komori, 2005). Upon forming a heterodimer with core binding factor  (Cbf), Runx2 acquires DNA-binding activity and regulates transcriptional activity (Kundu et al., 2002;Miller et al., 2002;Yoshida et al., 2002;Kanatani et al., 2006). There are two Runx2 isoforms, type I Runx2 and type II Runx2, which have different N-termini, and type I Runx2 is more dependent on Cbfb than type II Runx2 for their functional activities (Kanatani et al., 2006). Runx2-deficient mice lack osteoblasts and show a complete lack of bone formation, demonstrating that Runx2 is essential for osteoblast differentiation (Komori et al., 1997;Otto et al., 1997). Runx2 also plays important roles in chondrocyte maturation, maintenance of the chondrocyte phenotype, and vascular invasion into cartilage (Komori, 2005;Zelzer et al., 2001). Furthermore, Runx2 regulates RANKL and OPG expression stimulating osteoclast differentiation (Enomoto et al., 2003). These findings indicate that Runx2 functions as a key molecule in skeletal development.The DNA-binding sites of Runx2 in major bone matrix protein genes including the Col1a1, osteopontin, bone sialoprotein, and osteocalcin genes, have been identified, and Runx2 induced the expression of these genes or activated their promoters (Ducy et al., 1997(Ducy et...
Functional involvement of the Notch pathway in osteoblastic differentiation has been previously investigated using the truncated intracellular domain, which mimics Notch signaling by interacting with the DNAbinding protein CBF-1. However, it is unclear whether Notch ligands Delta1 and Jagged1 also induce an identical cellular response in osteoblastic differentiation. We have shown that both Delta1 and Jagged1 were expressed concomitantly with Notch1 in maturating osteoblastic cells during bone regeneration and that overexpressed and immobilized recombinant Delta1 and Jagged1 alone did not alter the differentiated state of MC3T3-E1 and C2C12 cells. However, they augmented bone morphogenetic protein-2 (BMP2)-induced alkaline phosphatase activity and the expression of several differentiation markers, except for osteocalcin, and ultimately enhanced calcified nodule and in vivo ectopic bone formation of MC3T3-E1. In addition, both ligands transmitted signal through the CBF-1-dependent pathway and stimulated the expression of HES-1, a direct target of Notch pathway. To test the necessity of Notch signaling in BMP2-induced differentiation, Notch signaling was inhibited by the dominant negative extracellular domain of Notch1, specific inhibitor, or small interference RNA. These treatments decreased alkaline phosphatase activity as well as the expression of other differentiation markers and inhibited the promoter activity of Id-1, a target gene of the BMP pathway. These results indicate the functional redundancy between Delta1 and Jagged1 in osteoblastic differentiation whereby Delta1/Jagged1-activated Notch1 enhances BMP2-induced differentiation through the identical signaling pathway. Furthermore, our data also suggest that functional Notch signaling is essential not only for BMP2-induced osteoblast differentiation but also for BMP signaling itself.
The strength of bone depends on bone quantity and quality. Osteocalcin (Ocn) is the most abundant noncollagenous protein in bone and is produced by osteoblasts. It has been previously claimed that Ocn inhibits bone formation and also functions as a hormone to regulate insulin secretion in the pancreas, testosterone synthesis in the testes, and muscle mass. We generated Ocn-deficient (Ocn -/-) mice by deleting Bglap and Bglap2. Analysis of Ocn -/ mice revealed that Ocn is not involved in the regulation of bone quantity, glucose metabolism, testosterone synthesis, or muscle mass. The orientation degree of collagen fibrils and size of biological apatite (BAp) crystallites in the c-axis were normal in the Ocn -/bone. However, the crystallographic orientation of the BAp c-axis, which is normally parallel to collagen fibrils, was severely disrupted, resulting in reduced bone strength. These results demonstrate that Ocn is required for bone quality and strength by adjusting the alignment of BAp crystallites parallel to collagen fibrils; but it does not function as a hormone.
Reduced mechanical stress is a major cause of osteoporosis in the elderly, and the osteocyte network, which comprises a communication system through processes and canaliculi throughout bone, is thought to be a mechanosensor and mechanotransduction system; however, the functions of osteocytes are still controversial and remain to be clarified. Unexpectedly, we found that overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteoblast and osteoclast differentiation were unaffected by BCL2 transgene in vitro. However, the cortical bone mass increased due to enhanced osteoblast function and suppressed osteoclastogenesis at 4 months of age, when the frequency of TUNEL-positive lacunae reached 75%. In the unloaded condition, the trabecular bone mass decreased in both wild-type and BCL2 transgenic mice at 6 weeks of age, while it decreased due to impaired osteoblast function and enhanced osteoclastogenesis in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Rankl and Opg were highly expressed in osteocytes, but Rankl expression in osteoblasts but not in osteocytes was increased at unloading in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Sost was locally induced at unloading in wild-type mice but not in BCL2 transgenic mice, and the dissemination of Sost was severely interrupted in BCL2 transgenic mice, showing the severely impaired osteocyte network. These findings indicate that the osteocyte network is required for the upregulation of Rankl in osteoblasts and Sost in osteocytes in the unloaded condition. These findings suggest that the osteocyte network negatively regulate bone mass by inhibiting osteoblast function and activating osteoclastogenesis, and these functions are augmented in the unloaded condition at least partly through the upregulation of Rankl expression in osteoblasts and that of Sost in osteocytes, although it cannot be excluded that low BCL2 transgene expression in osteoblasts contributed to the enhanced osteoblast function.
Orthodontic tooth movement is achieved by mechanical loading; however, the biological mechanism involved in this process is not clearly understood owing to the lack of a suitable experimental model. In the present study, we established an orthodontic tooth movement model in mice using a Ni-Ti closed coil spring that was inserted between the upper incisors and the upper first molar. Histological examination demonstrated that the orthodontic force moved the first upper molar mesially without necrosis of the periodontium during tooth movement. The number of TRAP-positive osteoclasts on the pressure side significantly increased in a time-dependent manner. Quantitative real time-based reverse transcription-polymerase chain reaction analysis demonstrated increased levels of mRNA for cathepsin K. Immunohistochemical staining revealed the expression of tumor necrosis factor-alpha (TNFalpha) in periodontium on the pressure side of the first molar during orthodontic tooth movement. When this tooth movement system was applied to TNF type 1 receptor-deficient mice and TNF type 2 receptor-deficient mice, tooth movement observed in TNF type 2 receptor-deficient mice was smaller than that in the wild-type mice and TNF type 1 receptor-deficient mice. The number of TRAP-positive osteoclasts on the pressure side was significantly small in TNF type 2 receptor-deficient mice compared with that in TNF type 1 receptor-deficient mice on day 6 after application of the appliance. The present study indicates that TNFalpha signaling plays some important roles in orthodontic tooth movement.
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