Toshihisa Komori

Targeted Disruption of Cbfa1 Results in a Complete Lack of Bone Formation owing to Maturational Arrest of Osteoblasts

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.

Runx2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12.

ABSTRACT When C2C12 pluripotent mesenchymal precursor cells are treated with transforming growth factor β1 (TGF-β1), terminal differentiation into myotubes is blocked. Treatment with bone morphogenetic protein 2 (BMP-2) not only blocks myogenic differentiation of C2C12 cells but also induces osteoblast differentiation. The molecular mechanisms governing the ability of TGF-β1 and BMP-2 to both induce ligand-specific responses and inhibit myogenic differentiation are not known. We identified Runx2/PEBP2αA/Cbfa1, a global regulator of osteogenesis, as a major TGF-β1-responsive element binding protein induced by TGF-β1 and BMP-2 in C2C12 cells. Consistent with the observation that Runx2 can be induced by either TGF-β1 or BMP-2, the exogenous expression of Runx2 mediated some of the effects of TGF-β1 and BMP-2 but not osteoblast-specific gene expression. Runx2 mimicked common effects of TGF-β1 and BMP-2 by inducing expression of matrix gene products (for example, collagen and fibronectin), suppressing MyoD expression, and inhibiting myotube formation of C2C12 cells. For osteoblast differentiation, an additional effector, BMP-specific Smad protein, was required. Our results indicate that Runx2 is a major target gene shared by TGF-β and BMP signaling pathways and that the coordinated action of Runx2 and BMP-activated Smads leads to the induction of osteoblast-specific gene expression in C2C12 cells.

Differential Requirements for Runx Proteins in CD4 Repression and Epigenetic Silencing during T Lymphocyte Development

T lymphocytes differentiate in discrete stages within the thymus. Immature thymocytes lacking CD4 and CD8 coreceptors differentiate into double-positive cells (CD4(+)CD8(+)), which are selected to become either CD4(+)CD8(-)helper cells or CD4(-)CD8(+) cytotoxic cells. A stage-specific transcriptional silencer regulates expression of CD4 in both immature and CD4(-)CD8(+) thymocytes. We show here that binding sites for Runt domain transcription factors are essential for CD4 silencer function at both stages, and that different Runx family members are required to fulfill unique functions at each stage. Runx1 is required for active repression in CD4(-)CD8(-) thymocytes whereas Runx3 is required for establishing epigenetic silencing in cytotoxic lineage thymocytes. Runx3-deficient cytotoxic T cells, but not helper cells, have defective responses to antigen, suggesting that Runx proteins have critical functions in lineage specification and homeostasis of CD8-lineage T lymphocytes.

Regulation of osteoblast differentiation by transcription factors

Runx2, osterix, and β‐catenin are essential for osteoblast differentiation. Runx2 directs multipotent mesenchymal cells to an osteoblastic lineage, and inhibits them from differentiating into the adipocytic and chondrocytic lineages. After differentiating to preosteoblasts, β‐catenin, osterix, and Runx2 direct them to immature osteoblasts, which produce bone matrix proteins, blocking their potential to differentiate into the chondrocytic lineage. Runx2 inhibits osteoblast maturation and the transition into osteocytes, keeping osteoblasts in an immature stage. Other transcription factors including Msx1, Msx2, Dlx5, Dlx6, Twist, AP1(Fos/Jun), Knox‐20, Sp3, and ATF4 are also involved in osteoblast differentiation. To gain an understanding of bone development, it is important to position these transcription factors to the right places in the processes of osteoblast differentiation. J. Cell. Biochem. 99: 1233–1239, 2006.

Maturational disturbance of chondrocytes in Cbfa1-deficient mice.

Cbfa1, a transcription factor that belongs to the runt‐domain gene family, plays an essential role in osteogenesis. Cbfa1‐deficient mice completely lacked both intramembranous and endochondral ossification, owing to the maturational arrest of osteoblasts, indicating that Cbfa1 has a fundamental role in osteoblast differentiation. However, Cbfa1 was also expressed in chondrocytes, and its expression was increased according to the maturation of chondrocytes. Terminal hypertrophic chondrocytes expressed Cbfa1 extensively. The significant expression of Cbfa1 in hypertrophic chondrocytes was first detected at embryonic day 13.5 (E13.5), and its expression in hypertrophic chondrocytes was most prominent at E14.5–16.5. In Cbfa1‐deficient mice, whose entire skeleton was composed of cartilage, the chondrocyte differentiation was disturbed. Calcification of cartilage occurred in the restricted parts of skeletons, including tibia, fibula, radius, and ulna. Type X collagen, BMP6, and Indian hedgehog were expressed in their hypertrophic chondrocytes. However, osteopontin, bone sialoprotein, and collagenase 3 were not expressed at all, indicating that they are directly regulated by Cbfa1 in the terminal hypertrophic chondrocytes. Chondrocyte differentiation was severely disturbed in the rest of the skeleton. The expression of PTH/PTHrP receptor, Indian hedgehog, type X collagen, and BMP6 was not detected in humerus and femur, indicating that chondrocyte differentiation was blocked before prehypertrophic chondrocytes. These findings demonstrate that Cbfa1 is an important factor for chondrocyte differentiation. Dev Dyn 1999;214:279–290.

Cbfa1 isoforms exert functional differences in osteoblast differentiation

Cbfa1 is an essential transcription factor for osteoblast differentiation and bone formation. We investigated functional differences among three isoforms of Cbfa1: Type I (originally reported as Pebp2αA by Ogawa et al. (Ogawa, E., Maruyama, M., Kagoshima, H., Inuzuka, M., Lu, J., Satake, M., Shigesada, K., and Ito, Y. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6859–6863), Type II (originally reported astil-1 by Stewart et al. (Stewart, M., Terry, A., Hu, M., O’Hara, M., Blyth, K., Baxter, E., Cameron, E., Onions, D. E., and Neil, J. C. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8646–8651), and Type III (originally reported asOsf2/Cbfa1 by Ducy et al. (Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L., and Karsenty, G. (1997)Cell 89, 747–754). A reverse transcriptase-polymerase chain reaction analysis demonstrated that these isoforms were expressed in adult mouse bones. The transient transfection of Type I or Type IICbfa1 in a mouse fibroblastic cell line, C3H10T1/2, induced the expression of alkaline phosphatase (ALP) activity. This induction was synergistically enhanced by the co-introduction ofXenopus BMP-4 cDNA. In contrast, the transient transfection of Type III cDNA induced no ALP activity. In C3H10T1/2 cells stably transfected with each isoform ofCbfa1, the gene expression of ALP was also strongly induced in cells transfected with Type I and Type IICbfa1 but not in cells with Type III Cbfa1. Osteocalcin, osteopontin,and type I collagen gene expressions were induced or up-regulated in all of the cells stably transfected with each isoform of Cbfa1, and Type II transfected cells exhibited the highest expression level ofosteocalcin gene. A luciferase reporter gene assay using a 6XOSE2-SV40 promoter (6 tandem binding elements for Cbfa1 ligated in front of the SV40 promoter sequence), a mouse osteocalcinpromoter, and a mouse osteopontin promoter revealed the differences in the transcriptional induction of target genes by eachCbfa1 isoform with or without its β-subunit. These results suggest that all three of the Cbfa1 isoforms used in the present study are involved in the stimulatory action of osteoblast differentiation, but they exert different functions in the process of osteoblast differentiation.

Regulation of bone development and extracellular matrix protein genes by RUNX2

RUNX2 is a multifunctional transcription factor that controls skeletal development by regulating the differentiation of chondrocytes and osteoblasts and the expression of many extracellular matrix protein genes during chondrocyte and osteoblast differentiation. This transcription factor plays a major role at the late stage of chondrocyte differentiation: it is required for chondrocyte maturation and regulates Col10a1 expression in hypertrophic chondrocytes and the expression of Spp1, Ibsp, and Mmp13 in terminal hypertrophic chondrocytes. It is essential for the commitment of pluripotent mesenchymal cells to the osteoblast lineage. During osteoblast differentiation, RUNX2 upregulates the expression of bone matrix protein genes including Col1a1, Spp1, Ibsp, Bglap, and Fn1 in vitro and activates many promoters including those of Col1a1, Col1a2, Spp1, Bglap, and Mmp13. However, overexpression of Runx2 inhibits osteoblast maturation and reduces Col1a1 and Bglap expression. The inhibition of RUNX2 in mature osteoblasts does not reduce the expression of Col1a1 and Bglap in mice. Thus, RUNX2 directs pluripotent mesenchymal cells to the osteoblast lineage, triggers the expression of major bone matrix protein genes, and keeps the osteoblasts in an immature stage, but does not play a major role in the maintenance of the expression of Col1a1 or Bglap in mature osteoblasts. During bone development, RUNX2 induces osteoblast differentiation and increases the number of immature osteoblasts, which form immature bone, whereas Runx2 expression has to be downregulated for differentiation into mature osteoblasts, which form mature bone. During dentinogenesis, Runx2 expression is downregulated, and RUNX2 inhibits the terminal differentiation of odontoblasts.

Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures

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.

Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling

Runx2 and phosphatidylinositol 3-kinase (PI3K)–Akt signaling play important roles in osteoblast and chondrocyte differentiation. We investigated the relationship between Runx2 and PI3K-Akt signaling. Forced expression of Runx2 enhanced osteoblastic differentiation of C3H10T1/2 and MC3T3-E1 cells and enhanced chondrogenic differentiation of ATDC5 cells, whereas these effects were blocked by treatment with IGF-I antibody or LY294002 or adenoviral introduction of dominant-negative (dn)–Akt. Forced expression of Runx2 or dn-Runx2 enhanced or inhibited cell migration, respectively, whereas the enhancement by Runx2 was abolished by treatment with LY294002 or adenoviral introduction of dn-Akt. Runx2 up-regulated PI3K subunits (p85 and p110β) and Akt, and their expression patterns were similar to that of Runx2 in growth plates. Treatment with LY294002 or introduction of dn-Akt severely diminished DNA binding of Runx2 and Runx2-dependent transcription, whereas forced expression of myrAkt enhanced them. These findings demonstrate that Runx2 and PI3K-Akt signaling are mutually dependent on each other in the regulation of osteoblast and chondrocyte differentiation and their migration.

Collagenase 3 is a target of Cbfa1, a transcription factor of the runt gene family involved in bone formation.

ABSTRACT Collagenase 3 (MMP-13) is a recently identified member of the matrix metalloproteinase (MMP) gene family that is expressed at high levels in diverse human carcinomas and in articular cartilage from arthritic patients. In addition to its expression in pathological conditions, collagenase 3 has been detected in osteoblasts and hypertrophic chondrocytes during fetal ossification. In this work, we have evaluated the possibility that Cbfa1 (core binding factor 1), a transcription factor playing a major role in the expression of osteoblastic specific genes, is involved in the expression of collagenase 3 during bone formation. We have functionally characterized a Cbfa motif present in the promoter region of collagenase 3 gene and demonstrated, by cotransfection experiments and gel mobility shift assays, that this element is involved in the inducibility of the collagenase 3 promoter by Cbfa1 in osteoblastic and chondrocytic cells. Furthermore, overexpression of Cbfa1 in osteoblastic cells unable to produce collagenase 3 leads to the expression of this gene after stimulation with transforming growth factor β. Finally, we show that mutant mice deficient in Cbfa1, lacking mature osteoblasts but containing hypertrophic chondrocytes which are also a major source of collagenase 3, do not express this protease during fetal development. These results provide in vivo evidence that collagenase 3 is a target of the transcriptional activator Cbfa1 in these cells. On the basis of these transcriptional regulation studies, together with the potent proteolytic activity of collagenase 3 on diverse collagenous and noncollagenous bone and cartilage components, we proposed that this enzyme may play a key role in the process of bone formation and remodeling.