A transcription factor, Cbfa1, which belongs to the runt-domain gene family, is expressed restrictively in fetal development. To elucidate the function of Cbfa1, we generated mice with a mutated Cbfa1 locus. Mice with a homozygous mutation in Cbfa1 died just after birth without breathing. Examination of their skeletal systems showed a complete lack of ossification. Although immature osteoblasts, which expressed alkaline phophatase weakly but not Osteopontin and Osteocalcin, and a few immature osteoclasts appeared at the perichondrial region, neither vascular nor mesenchymal cell invasion was observed in the cartilage. Therefore, our data suggest that both intramembranous and endochondral ossification were completely blocked, owing to the maturational arrest of osteoblasts in the mutant mice, and demonstrate that Cbfa1 plays an essential role in osteogenesis.

Abstract. The implantation of bone morphogenetic protein (BMP) into muscular tissues induces ectopic bone formation at the site of implantation. To investigate the mechanism underlying this process, we examined whether recombinant bone morphogenetic protein-2 (BMP-2) converts the differentiation pathway of the clonal myoblastic cell line, C2C12, into that of osteoblast lineage. Incubating the cells with 300 ng/ml of BMP-2 for 6 d almost completely inhibited the formation of the multinucleated myotubes expressing troponin T and myosin heavy chain, and induced the appearance of numerous alkaline phosphatase (ALP)-positive cells. BMP-2 dose dependently induced ALP activity, parathyroid hormone (PTH)-dependent 3',5'-cAMP production, and osteocalcin production at concentrations above 100 ng/ml. The concentration of BMP-2 required to induce these osteoblastic phenotypes was the same as that required to almost completely inhibit myotube formation. Incubating primary muscle cells with 300 ng/ml of BMP-2 for 6 d also inhibited myotube formation, whereas induced ALP activity and osteocalcin production. Incubation with 300 ng/ml of BMP-2 suppressed the expression of mRNA for muscle creatine kinase within 6 h, whereas it induced mRNA expression for ALP, PTH/PTH-related protein (PTHrP) receptors, and osteocalcin within 24--48 h. BMP-2 completely inhibited the expression of myogenin mRNA by day 3. By day 3, BMP-2 also inhibited the expression of MyoD mRNA, but it was transiently stimulated 12 h after exposure to BMP-2. Expression of Id-1 mRNA was greatly stimulated by BMP-2. When C2C12 cells pretreated with BMP-2 for 6 d were transferred to a colony assay system in the absence of BMP-2, more than 84 % of the colonies generated became troponin T-positive and ALP activity disappeared. TGF-/31 also inhibited myotube formation in C2C12 cells, and suppressed the expression of myogenin and MyoD mRNAs without inducing that of Id-1 mRNA. However, no osteoblastic phenotype was induced by TGF-/31 in C2C12 cells. TGF-/31 potentiated the inhibitory effect of BMP-2 on myotube formation, whereas TGF-/31 reduced ALP activity and osteocalcin production induced by BMP-2 in C2C12 cells. These results indicate that BMP-2 specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells, but that the conversion is not heritable.S EVERAL lines of evidence indicate that osteoblasts, chondrocytes, myocytes, and adipocytes are all derived from a common progenitor cell called undifferentiated mesenchymal cells (Taylor and Jones, 1979; Grigoriadis et al., 1988Grigoriadis et al., , 1990 Yamaguchi and Kahn, 1991). During the process of their differentiation, progenitor cells acquire specific phenotypes depending upon the differentiated cell types under the control of respective regulatory factors (for reviews see Rodan and Rodan, 1984;Owen, 1988; Wlodar-Address all correspondence to Dr. Tatsuo Suda, Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Tokyo 142, ski, 1990). The di...

In autoimmune arthritis, traditionally classified as a T helper (Th) type 1 disease, the activation of T cells results in bone destruction mediated by osteoclasts, but how T cells enhance osteoclastogenesis despite the anti-osteoclastogenic effect of interferon (IFN)-γ remains to be elucidated. Here, we examine the effect of various Th cell subsets on osteoclastogenesis and identify Th17, a specialized inflammatory subset, as an osteoclastogenic Th cell subset that links T cell activation and bone resorption. The interleukin (IL)-23–IL-17 axis, rather than the IL-12–IFN-γ axis, is critical not only for the onset phase, but also for the bone destruction phase of autoimmune arthritis. Thus, Th17 is a powerful therapeutic target for the bone destruction associated with T cell activation.

We developed a co-culture system with mouse spleen cells and osteoblastic cells to examine the role of osteoblasts in osteoclast formation. When mouse spleen cells and osteoblastic cells isolated from fetal mouse calvariae were co-cultured in the presence of 10 nM 1 alpha, 25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], numerous tartrate-resistant acid phosphate (TRACP)-positive mononuclear and multinucleated cells were formed within 8 days. Neither the same co-cultures without the vitamin nor separate cultures of either spleen cells or osteoblastic cells with the vitamin produced TRACP-positive cells. Salmon calcitonin (CT) markedly increased cAMP production in the co-cultures treated with 1 alpha,25(OH)2D3. Autoradiographic studies clearly demonstrated that [125I]-CT specifically bound to the TRACP-positive cells formed in the co-cultures with the vitamin. When spleen cells and osteoblastic cells were co-cultured on dentine slices in the presence of 1 alpha,25(OH)2D3, numerous resorption lacunae were formed on the slices. Neither co-cultures of alveolar macrophages and osteoblastic cells nor those of spleen cells and mouse skin-derived fibroblasts induced TRACP-positive cells even in the presence of 1 alpha,25(OH)2D3. When spleen cells and osteoblastic cells were cultured separately from each other by a membrane filter (0.45 micron), no TRACP-positive cells were formed. These results indicate that osteoblastic cells are required for the differentiation of osteoclast progenitors in splenic tissues into multinucleated osteoclasts.

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.

Osterix/Sp7, a member of the Sp1 transcription factor family, plays an essential role in bone formation and osteoblastogenesis. Although Osterix has been shown to be induced by BMP2 in a mesenchymal cell line, the molecular basis of the regulation, expression and function of Osterix during osteoblast differentiation, is not fully understood. Thus we examined the role of BMP2 signaling in the regulation of Osterix using the mesenchymal cell lines C3H10T1/2 and C2C12. Osterix overexpression induced alkaline phosphatase activity and osteocalcin expression in C2C12 cells and stimulated calcification of murine primary osteoblasts. Considering that Runx2 overexpression induces Osterix, these results suggest that Osterix functions as downstream of Runx2. Surprisingly, BMP2 treatment induced Osterix expression and alkaline phosphatase activity in mesenchymal cells derived from Runx2-deficient mice. Furthermore, overexpression of Smad1 and Smad4 upregulated Osterix expression, and an inhibitory Smad, Smad6, markedly suppressed BMP2-induced Osterix expression in the Runx2-deficient cells. Moreover, overexpression of a homeobox gene, Msx2, which is up-regulated by BMP2 and promotes osteoblastic differentiation, induced Osterix expression in the Runx2-deficient cells. Knockdown of Msx2 clearly inhibited induction of Osterix by BMP2 in the Runx2-deficient mesenchymal cells. Interestingly, microarray analyses using the Runx2-deficient cells revealed that the role of Osterix was distinct from that of Runx2. These findings suggest that Osterix is regulated via both Runx2-dependent and -independent mechanisms, and that Osterix controls osteoblast differentiation, at least in part, by regulating the expression of genes not controlled by Runx2.Osteoblasts are differentiated from multipotent mesenchymal cells (1). This differentiation process is regulated by several cytokines, including bone morphogenetic proteins, transforming growth factor ␤, Wnt, and hedgehog (2-5). Among them, BMP2 (bone morphogenetic protein 2) is one of the most powerful cytokines that promote differentiation of mesenchymal cells into osteoblasts in vitro and induce bone formation in vivo (2). BMP2 exhibits this osteogenic action by activating Smad signaling and by regulating transcription of osteogenic genes such as ALP, type I collagen, osteocalcin, and bone sialoprotein (Bsp) 2 (6). Runt-related gene 2 (Runx2)/Core-binding factor 1 (Cbfa1), an essential transcription factor for osteoblast differentiation and bone formation (7) and responsible gene for cleidocranial dysplasia (8), directly regulates the expression of osteocalcin and Bsp (9). BMP2 is known to control the expression and functions of Runx2 through Smad signaling (10 -12). These findings have established the importance of the BMP2-SmadRunx2 axis in osteoblastogenesis.Osterix, an Sp1 transcription family member, is up-regulated by BMP2 during osteoblastic differentiation (13). Osterix has also been reported to inhibit chondrogenesis (14). Mice deficient in the Osterix gene show no bone formation...

Abstract. The in vitro effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on osteogenic and myogenic differentiation was examined in two clonal cell lines of rat osteoblast-like cells at different differentiation stages, ROB-C26 (C26) and ROB-C20 (C20). The C26 is a potential osteoblast precursor cell line that is also capable of differentiating into muscle ceils and adipocytes; the C20 is a more differentiated osteoblastic cell line. Proliferation was stimulated by rhBMP-2 in C26 cells, but inhibited in C20 cells, rhBMP-2 greatly increased alkaline phosphate (ALP) activity in C26 cells, but not in C20 cells. The steady-state level of ALP mRNA was also increased by rhBMP-2 in C26 cells, but not in C20 cells. Production of 3',5'-cAMP in response to parathyroid hormone (PTH) was dose-dependently enhanced by adding rhBMP-2 in both C26 and C20 cells, though the stimulatory effect was much greater in the former. There was neither basal expression of osteocalcin mRNA nor its protein synthesis in C26 cells, but they were strikingly induced by rhBMP-2 in the presence of lot,25-dihydroxyvitamin D3. rhBMP-2 induced no appreciable changes in procollagen mRNA levels of type I and type UI in the two cell lines. Differentiation of C26 cells into myotubes was greatly inhibited by adding rhBMP-2. The inhibitory effect of rhBMP-2 on myogenic differentiation was also observed in clonal rat skeletal myoblasts (L6). Like BMP-2, TGF-fll inhibited myogenic differentiation. However, unlike BMP-2, TGF-fll decreased ALP activity in both C26 and C20 cells. TGF-/$1 induced neither PTH responsiveness nor osteocalcin production in C26 cells, but it increased PTH responsiveness in C20 cells. These results clearly indicate that rhBMP-2 is involved, at least in vitro, not only in inducing differentiation of osteoblast precursor cells into more mature osteoblast-like cells, but also in inhibiting myogenic differentiation.CTOPIC bone formation is elicited at intramuscular sites by implantation of bone inducing factors contained in demineralized bone matrix (28,35,36). This indicates that cells of the osteoblast lineage have a close relationship with those of the muscular lineage in their ontogeny, and the development of the two cell lineages may be mutually regulated by some factor(s) stored in bone matrix. The components and the action mechanism of these bone inducing factors had long remained obscure. This is mainly due to the difficulties of purification of these factors and the lack of suitable in vitro bioassay systems.Recently, a number of laboratories have isolated bioactive proteins which induce cartilage and/or bone formation at the sites implanted (1,13,30,39,40). Human cDNAs for seven different bone morphogenetic proteins (BMPs), t BMP-1, BMP-5, BMP-6 (Vgr-1 [141), and BMP-7 (OP-1125, 30]) have been cloned (41,42). The sequences deduced from these cDNAs have indicated that BMP-2 through BMP-7 are members of transforming growth factorfl (TGF-fl) superfamily (14,25,41,42). Furthermore, active recombinant human bone mor...

Osteoblasts arise from common progenitors with chondrocytes, muscle and adipocytes, and various hormones and local factors regulate their differentiation. We review here regulation of osteoblast differentiation mediated by the local factors such as bone morphogenetic proteins (BMPs) and hedgehogs and the transcription factor, core-binding factor ␣-1 (Cbfa1). BMPs are the most potent regulators of osteoblast differentiation among the local factors. Sonic and Indian hedgehogs are involved in osteoblast differentiation by interacting with BMPs. Cbfa1, a member of the runt domain gene family, plays a major role in the processes of a determination of osteoblast cell lineage and maturation of osteoblasts. Cbfa1 is an essential transcription factor for osteoblast differentiation and bone formation, because Cbfa1-deficient mice completely lacked bone formation due to maturation arrest of osteoblasts. Although the regulatory mechanism of Cbfa1 expression has not been fully clarified, BMPs are an important local factor that up-regulates Cbfa1 expression. Thus, the intimate interaction between local factors such as BMPs and hedgehogs and the transcription factor, Cbfa1, is important to osteoblast differentiation and bone formation. (Endocrine Reviews 21: [393][394][395][396][397][398][399][400][401][402][403][404][405][406][407][408][409][410][411] 2000)