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...

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.

Runx2 and Sp7 are essential transcription factors for osteoblast differentiation. However, the molecular mechanisms responsible for the proliferation of osteoblast progenitors remain unclear. The early onset of Runx2 expression caused limb defects through the Fgfr1–3 regulation by Runx2. To investigate the physiological role of Runx2 in the regulation of Fgfr1–3, we compared osteoblast progenitors in Sp7−/− and Runx2−/− mice. Osteoblast progenitors accumulated and actively proliferated in calvariae and mandibles of Sp7−/− but not of Runx2−/− mice, and the number of osteoblast progenitors and their proliferation were dependent on the gene dosage of Runx2 in Sp7−/− background. The expression of Fgfr2 and Fgfr3, which were responsible for the proliferation of osteoblast progenitors, was severely reduced in Runx2−/− but not in Sp7−/− calvariae. Runx2 directly regulated Fgfr2 and Fgfr3, increased the proliferation of osteoblast progenitors, and augmented the FGF2-induced proliferation. The proliferation of Sp7−/− osteoblast progenitors was enhanced and strongly augmented by FGF2, and Runx2 knockdown reduced the FGF2-induced proliferation. Fgfr inhibitor AZD4547 abrogated all of the enhanced proliferation. These results indicate that Runx2 is required for the proliferation of osteoblast progenitors and induces proliferation, at least partly, by regulating Fgfr2 and Fgfr3 expression.

Bcl2 subfamily proteins, including Bcl2 and Bcl-XL, inhibit apoptosis. As osteoblast apoptosis is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging, bone loss might be inhibited by the upregulation of Bcl2; however, the effects of Bcl2 overexpression on osteoblast differentiation and bone development and maintenance have not been fully investigated. To investigate these issues, we established two lines of osteoblast-specific BCL2 transgenic mice. In BCL2 transgenic mice, bone volume was increased at 6 weeks of age but not at 10 weeks of age compared with wild-type mice. The numbers of osteoblasts and osteocytes increased, but osteoid thickness and the bone formation rate were reduced in BCL2 transgenic mice with high expression at 10 weeks of age. The number of BrdU-positive cells was increased but that of TUNEL-positive cells was unaltered at 2 and 6 weeks of age. Osteoblast differentiation was inhibited, as shown by reduced Col1a1 and osteocalcin expression. Osteoblast differentiation of calvarial cells from BCL2 transgenic mice also fell in vitro. Overexpression of BCL2 in primary osteoblasts had no effect on osteoclastogenesis in co-culture with bone marrow cells. Unexpectedly, overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteocytes, which had a reduced number of processes, gradually died with apoptotic structural alterations and the expression of apoptosis-related molecules, and dead osteocytes accumulated in cortical bone. These findings indicate that overexpression of BCL2 in osteoblasts inhibits osteoblast differentiation, reduces osteocyte processes, and causes osteocyte apoptosis.