Article |
Address correspondence to Toshihisa Komori, Dept. of Molecular Medicine, Osaka University Medical School, 2-2 Yamada-oka Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-7590. Fax: 81-6-6879-7796. E-mail: komorit{at}imed3.med.osaka-u.ac.jp
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: Cbfa1; osteoblast; osteocyte; transgenic mice; osteopenia
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cbfa1 plays a pivotal role in osteogenesis (Komori and Kishimoto, 1998). Mice heterozygously mutated in the Cbfa1 locus show a phenotype similar to that of cleidocranial dysplasia in humans, in whom mutations of Cbfa1 have been found (Komori et al., 1997; Mundlos et al., 1997; Otto et al., 1997). A homozygous mutation of this gene in mice induced a complete lack of bone formation with arrest of osteoblast differentiation (Komori et al., 1997; Otto et al., 1997). However, the complete lack of osteoblasts and neonatal lethality makes it difficult to examine the postnatal function of Cbfa1 by using this knockout model. An alternative model, which expressed the dominant negative form of Cbfa1 (DN-Cbfa1), developed an osteopenic phenotype in mice and was used to indicate the indispensability of the gene for postnatal bone formation by regulating the functions of mature osteoblasts (Ducy et al., 1999). Furthermore, a large number of recent in vitro studies also implied that Cbfa1 is a positive regulator that can upregulate the expression of bone matrix genes, including type I collagen, osteopontin, bone sialoprotein, osteocalcin, and fibronectin. (Banerjee et al., 1997; Ducy et al., 1997; Sato et al., 1998; Harada et al., 1999; Xiao et al., 1999; Kern et al., 2001; Lee et al., 2000; Prince et al., 2001). All of these studies have indicated that Cbfa1 plays important roles in matrix formation and mineralization.
In the process of osteoblast differentiation, Cbfa1 seems to function in the commitment of the osteoblast lineage from multipotential mesenchymal cells because Cbfa1-deficient calvarial cells had the potential to differentiate into both adipocytes and chondrocytes but completely lacked the ability to differentiate into the osteoblastic lineage (Kobayashi et al., 2000). However, after cells commit to the osteoblastic lineage it remains to be clarified how Cbfa1 operates in the process of bone formation. To understand fully the functions of Cbfa1 in the processes of osteoblast differentiation, matrix production, and mineralization, we generated transgenic mice that overexpress Cbfa1 specifically in osteoblasts under the control of type I collagen promoter. Unexpectedly, Cbfa1 transgenic mice showed severe osteopenia and suffered from bone fractures within a few weeks after birth. Osteopenia and fragility of bone were caused by the inhibition of osteoblast maturation, and immature osteoblasts accumulated in the bone of adult mice. These data indicate that Cbfa1 inhibits the late stage of osteoblast maturation, restricting Cbfa1's positive function to the early differentiation stage in the process of osteoblast development.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
Cbfa1 failed to induce expression of the genes related to bone matrix, mineralization, and osteoclastogenesis
Expression of the genes related to bone matrix proteins, including type I collagen, osteopontin, bone sialoprotein, osteocalcin, and matrix metalloproteinase (MMP)13, is considered to be regulated by Cbfa1 (Yamaguchi et al., 2000). Alkaline phosphatase (ALP) is considered to be upregulated during osteoblast differentiation (Stein et al., 1990). To analyze the expression of these genes, Northern blot or reverse transcriptase (RT)-PCR analysis was performed using RNA from long bones of 1-mo-old wild-type and transgenic mice (Fig. 9). The expression of pro-1 type I collagen was decreased slightly in transgenic mice, whereas pro-
2 type I collagen expression was similar between wild-type and transgenic mice. The expression of osteopontin and bone sialoprotein was increased but that of ALP, osteocalcin, and MMP13 was decreased in transgenic mice. By using RT-PCR, we also examined the expression of receptor activator of NF-
B ligand (RANKL) and osteoprotegerin (OPG), which are important for osteoclastogenesis and osteoclast activity. In transgenic mice, OPG was decreased only slightly at 1 mo of age, but both RANKL and OPG were decreased clearly at 3 mo of age.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous in vitro and in vivo data suggested that Cbfa1 plays an important role in maturation of osteoblasts. However, our data showed that the late stage of osteoblast maturation was inhibited in transgenic mice as indicated by the accumulation of osteopontin-positive cells and the decrease of highly osteocalcin-positive cells, osteocytes, and ALP and osteocalcin expression. The accumulation of less mature osteoblasts in transgenic mice seemed to be caused not only by the maturational blockage of osteoblasts but also by acceleration of osteoblast differentiation at an early stage of cell development because osteoblasts were increased in number at neonatal stage (Fig. 8). Furthermore, the proliferation and apoptosis of less mature osteoblasts in transgenic mice have to be considered, although their analyses in vitro were unsuccessful because of the loss of the transgene expression in primary culture of calvaria-derived cells (unpublished data) as previously described (Krebsbach et al., 1993).
ALP activity is detected at an early stage of osteoblast differentiation and continues to increase during osteoblast maturation until the mineralization phase (Stein et al., 1990; Weinreb et al., 1990). In vitro experiments demonstrated that Cbfa1 transfection induced ALP activity in multipotential mesenchymal cells, C3H10T1/2 and C2C12 (Harada et al., 1999; Lee et al., 2000), indicating an important role for Cbfa1 in the induction of ALP activity. Although the difference in ALP expression level between transgenic and wild-type mice was not apparent at birth, it became evident during development (Fig. 9; unpublished data). This suggests that overexpression of Cbfa1 blocks osteoblast maturation at a certain stage in vivo.
Since osteocalcin expression is restricted to mature osteoblasts and odontoblasts, it is a convenient marker for fully differentiated osteoblasts (Mark et al., 1988; Stein et al., 1990). Cbfa1 induced osteocalcin expression in various cells in vitro including MC3T3-E1, C3H10T1/2, and skin fibroblasts (Ducy et al., 1997; Harada et al., 1999). Furthermore, Cbfa1 or related proteins bound osteocalcin promoter and strongly induced osteocalcin promoter activity in various cell lines, including C3H10T1/2 and nonosteoblastic cells, HeLa and F9, and Cbfa1 binding sites, were essential for osteocalcin expression (Geoffroy et al., 1995; Banerjee et al., 1996; Frendo et al., 1998; Javed et al., 1999; Xiao et al., 1999). These findings suggested that Cbfa1 is the most important factor for osteoblast-specific osteocalcin expression in vitro. However, a major population of Cbfa1 highly positive cells consisted of less mature osteoblasts in wild-type mice, and overexpression of Cbfa1 failed to upregulate osteocalcin expression in vivo (Figs. 8 and 9). These data indicate that other factors, which are induced at a late stage of osteoblast differentiation, are required for the regulation of osteocalcin expression or that some factors suppress osteocalcin expression at an immature stage of osteoblast differentiation in vivo. It has been shown that Groucho/TLE proteins repress Runx-dependent activation of tissue-specific gene transcription (Levanon et al., 1998; Javed et al., 2000), and TLE downregulates Cbfa1-mediated activation of osteocalcin expression (Javed et al., 2000). Further, Runx1 is known to interact with the corepressor mSin3A (Lutterbach et al., 2000). Thus, these repressors may play an important role in the transcriptional regulation of osteocalcin by repressing Cbfa1-dependent activation at an early stage of osteoblast differentiation.
Overexpression of DN-Cbfa1 at a late stage of osteoblast differentiation caused a decrease in the bone formation rate and decreased expression of the genes encoding main bone matrix proteins and resulted in osteopenia (Ducy et al., 1999). Surprisingly, our transgenic mice overexpressing Cbfa1 at both early and late stages of osteoblast differentiation also showed an osteopenic phenotype, although the mechanism for osteopenia was different. The two kinds of transgenic mice had a common feature because Cbfa1 function was suppressed in fully differentiated osteoblasts of DN-Cbfa1 transgenic mice, and the fully differentiated osteoblasts were diminished in our Cbfa1 transgenic mice. Therefore, both transgenic mice lacked at least Cbfa1-dependent function of fully differentiated osteoblasts, which finally caused osteopenia in both transgenic mice. It indicates that Cbfa1 inhibits osteoblast differentiation at a late stage, but some level of Cbfa1 is required for the expression of the genes encoding main bone matrix proteins. It also indicates that the transcriptional regulation of bone matrix genes by Cbfa1 is dependent on the maturational stage of osteoblasts as discussed about osteocalcin expression in the previous paragraph.
It was reported recently that RANKL is essential for osteoclastogenesis and that OPG inhibits osteoclastogenesis (Aubin and Bonnelye, 2000), and it was suggested that Cbfa1 is involved in the regulation of RANKL or OPG expression (Gao et al., 1998; Kitazawa et al., 1999; Thirunavukkarasu et al., 2000). However, both RANKL and OPG decreased gradually during development in transgenic mice (Fig. 9). It was suggested that osteoprogenitor cells have more potential to support osteoclast development than more differentiated cells (Manolagas, 2000). Therefore, the decrease of RANKL may reflect the relative decrease of osteoprogenitor cells because Cbfa1 seemed to accelerate the early stage of osteoblast differentiation (Fig. 8). In transgenic mice, cortical bone mass but not trabecular bone mass was reduced severely without acceleration of osteoclastogenesis. However, the enlarged bone marrow cavity and the presence of numerous osteoclasts in cortical bone showed that cortical bone loss was a result of osteolytic activity. This seemed to be caused by the immature composition of cortical bone, which contains abundant osteopontin with the small cell attachment motif Arg-Gly-Asp recognized by integrins and promotes the attachment of osteoclasts to the extracellular matrix (Fig. 5; Young et al., 1993). The expression of bone sialoprotein, which also has the Arg-Gly-Asp motif, was increased in Northern blot analysis (Fig. 9).
The drastic decrease of osteocytes that is a unique phenotype of the transgenic mice was caused by the inhibition of osteoblast maturation. Osteocytes are spaced regularly throughout the bone and communicate with each other and with osteoblasts and bone marrow stromal cells using their processes. Although the exact function of osteocytes remains unknown, they are considered to work as mechanosensors (Nijweide et al., 1996). Thus, the fragility of transgenic bone might be caused by the near absence of osteocytes, causing an inability to detect mechanical stress and microfractures.
Overexpression of Cbfa1 in osteoblasts increased osteoblast number but inhibited their terminal maturation, resulting in accumulation of less mature osteoblasts and osteopenia. Therefore, in an attempt to increase bone mass by Cbfa1 intermittent induction of Cbfa1 in osteogenic cells might permit a periodic increase of immature osteoblasts and their maturation. This concept might be related to the anabolic action on bone mass induced by the intermittent administration of parathyroid hormone (Dempster et al., 1993; Ishizuya et al., 1997; Jilka et al., 1999) because parathyroid hormone induces a protein kinase Adependent transactivation of Cbfa1 (Selvamurugan et al., 2000).
We demonstrated using Cbfa1 transgenic mice that Cbfa1 negatively regulates osteoblast differentiation at a late stage of osteoblast development. However, Cbfa1 seems to regulate osteoblast differentiation positively at an early stage. These opposite functions of Cbfa1, depending on the maturational stage of osteoblasts, may play an important role in the regulation of bone mass. Since Cbfa1 is an essential factor for osteoblast differentiation, many factors and substances that have an effect on bone mass will influence Cbfa1 expression or activation. Thus, our findings are expected to be of great benefit to future trials to increase bone mass.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Detection of ß-galactosidase activity
To confirm the activity of the promoter used in this study, we also subcloned the DNA fragment covering the 2.3-kb pro-1 (I) promoter region into the EcoRI site of pNASSß to direct the expression of the ß-galactosidase gene. The ß-galactosidase transgenic embryos were analyzed at different days postcoitum. Detection of ß-galactosidase activity was performed as described (Ueta et al., 2001). Stained embryos were embedded in paraffin and used to generate 7-µm sections, which were counterstained with eosin.
X-ray and pQCT analyses
Transgenic mice and their wild-type littermates were anesthetized and subjected to x-ray exposure in a micro-FX1000 (Fuji Film, Inc.). Long bones were dissected from killed mice and exposed to x-rays. In pQCT analysis, femurs were fixed with 10% buffered formalin for 24 h and measured using an XCT Research SA (Stratec Medizintechnik). Voxel size was 0.08 x 0.08 x 0.46 mm. The contour of the total bone was determined automatically by the pQCT software algorithm. The cortical and trabecular parameters were obtained at the diaphysis and 2 mm from distal epiphysis, respectively. The threshold values of 690 mg/cm3 for the cortical region and 395 mg/cm3 for the trabecular region were used in this experiment.
Histological analyses
For histological analyses, mice were killed at birth, 2 wk, 3 wk, 4 wk, 6 wk, 3 mo, 8 mo, and 1 yr of age. For the assessment of dynamic histomorphometric indices, mice were injected twice with calcein at a dose of 0.16 mg/10 g body weight and analyzed at 3 or 6 wk of age. The 3-wk group received dual injections at 6 and 3 d before sacrifice, and the 6-wk group received them at 8 and 1 d before sacrifice. Long bones were fixed with ethanol, and the undecalcified bones were embedded in glycolmethacrylate. 3-µm longitudinal sections from the proximal parts of tibiae and 20-µm cross sections from mid-diaphyses of femurs were stained with toluidine blue and analyzed using a semiautomated system (Osteoplan II; ZEISS). Nomenclature, symbols, and units used are those recommended by the Nomenclature Committee of the American Society for Bone and Mineral Research (Parfitt et al., 1987). Some of the sections were stained with TRAP. Bones from the other mice were fixed in 4% paraformaldehyde/0.1 M phosphate buffer. Decalcified and paraffin-embedded sections (5 µm thick) were used for the following analyses. To examine collagen fiber deposition, sections from tibiae were stained with hematoxylin and eosin and observed under polarized light (Bucay et al., 1998). To examine osteoblast markers, sections of tibiae at different ages were also used for in situ hybridization using probes for type I collagen, osteopontin, osteocalcin, and Cbfa1 as described previously (Inada et al., 1999).
Immunohistochemistry
Paraffin sections were blocked with 1% BSA containing 10% swine or rabbit serum at room temperature for 20 min and then incubated with rabbit antimouse osteopontin antibody (IBL Co., Ltd.) overnight at 4°C. Localization of the first antibody was visualized by incubation with biotinylated F(ab)2 fragments of swine antirabbit IgG antibody (Dako) at room temperature for 40 min and then treated with the ABC reagents (Vector Laboratories). Finally, sections were stained by DAB substrate and counterstained with methyl green.
Northern blot and RT-PCR
Total RNA was extracted from long bones without fracture from newborn and 4- and 11-wk-old transgenic and wild-type mice by lithium chloride. 20 Mg of total RNA was denatured with formamide, subjected to electrophoresis on 1.0% agarose gels, and transferred onto nylon membranes. Membranes were hybridized with 32P-labeled cDNA probes of pro-1 (I) collagen, osteocalcin, MMP13, ALP, osteopontin, bone sialoprotein, and glyceraldehyde-3-phosphate-dehydrogenase as described previously (Inada et al., 1999). For RT-PCR, cDNA was amplified by Amp Taq DNA polymerase (PerkinElmer) using the following primers: Pro-
2 (I) collagen, 5'-TGTGCTTCTGCAGGGTTCCA-3' and 5'-ACACGGAATTCTTGGTCAGC-3'; RANKL, 5'-GTCACTCTGTCCTCTTGGTAC-3' and 5'-TGAAACCCCAAAGTACGTCG-3'; OPG, 5'-CAGCTTCTTGCCTTGATGGAGA-3' and 5'-AAACAGCCCAGTGACCATTCCT-3'; hypoxanthine guanine phosphoribosyl transferase (HPRT), 5'-GCTGGTGAAAAGGACCTCT-3' and 5'-CACAGGACTAGAACAACTGC-3'. 18 (pro-
2 (I) collagen), 27 (RANKL), 28 (OPG), and 23 cycles (HPRT) of amplification were done with a Gene Amp PCR system 2400 (Perkin Elmer) (30 s at 94°C, 30 s at 55 or 60°C, and 30 s at 72°C).
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
This work was supported by grants from the Ministry of Education, Science, and Culture, Japan, and the Yamanouchi Foundation for Research on Metabolic Disorders.
Submitted: 9 May 2001
Revised: 25 July 2001
Accepted: 20 August 2001
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aubin, J.E., and E. Bonnelye. 2000. Osteoprotegerin and its ligand: a new paradigm for regulation of osteoclastogenesis and bone resorption. Osteoporos. Int. 11:905913.[Medline]
Banerjee, C., S.W. Hiebert, J.L. Stein, J.B. Lian, and G.S. Stein. 1996. An AML-1 consensus sequence binds an osteoblast-specific complex and transcriptionally activates the osteocalcin gene. Proc. Natl. Acad. Sci. USA. 93:49684973.
Banerjee, C., L.R. McCabe, J.Y. Choi, S.W. Hiebert, J.L. Stein, G.S. Stein, and J.B. Lian. 1997. Runt homology domain proteins in osteoblast differentiation: AML3/CBFA1 is a major component of a bone-specific complex. J. Cell Biochem. 66:18.[Medline]
Bucay, N., I. Sarosi, C.R. Dunstan, S. Morony, J. Tarpley, C. Capparelli, S. Scully, H.L. Tan, W. Xu, D.L. Lacey, et al. 1998. Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev. 12:12601268.
Dempster, D.W., F. Cosman, M. Parisien, V. Shen, and R. Lindsay. 1993. Anabolic actions of parathyroid hormone on bone. Endocr. Rev. 14:690709.[Medline]
Ducy, P., R. Zhang, V. Geoffroy, A.L. Ridall, and G. Karsenty. 1997. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell. 89:747754.[Medline]
Ducy, P., M. Starbuck, M. Priemel, J. Shen, G. Pinero, V. Geoffroy, M. Amling, and G. Karsenty. 1999. A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development. Genes Dev. 13:10251036.
Enomoto, H., M. Enomoto-Iwamoto, M. Iwamoto, S. Nomura, M. Himeno, Y. Kitamura, T. Kishimoto, and T. Komori. 2000. Cbfa1 is a positive regulatory factor in chondrocyte maturation. J. Biol. Chem. 275:86958702.
Frendo, J.L., G. Xiao, S. Fuchs, R.T. Franceschi, G. Karsenty, and P. Ducy. 1998. Functional hierarchy between two OSE2 elements in the control of osteocalcin gene expression in vivo. J. Biol. Chem. 273:3050930516.
Gao, Y.H., T. Shinki, T. Yuasa, H. Kataoka-Enomoto, T. Komori, T. Suda, and A. Yamaguchi. 1998. Potential role of Cbfa1, an essential transcriptional factor for osteoblast differentiation, in osteoclastogenesis: regulation of mRNA expression of osteoclast differentiation factor (ODF). Biochem. Biophys. Res. Commun. 252:697702.[Medline]
Geoffroy, V., P. Ducy, and G. Karsenty. 1995. A PEBP2 alpha/AML-1-related factor increases osteocalcin promoter activity through its binding to an osteoblast-specific cis-acting element. J. Biol. Chem. 270:3097330979.
Harada, H., S. Tagashira, M. Fujiwara, S. Ogawa, T. Katsumata, A. Yamaguchi, T. Komori, and M. Nakatsuka. 1999. Cbfa1 isoforms exert functional differences in osteoblast differentiation. J. Biol. Chem. 274:69726978.
Inada, M., T. Yasui, S. Nomura, S. Miyake, K. Deguchi, M. Himeno, M. Sato, H. Yamagiwa, T. Kimura, N. Yasui, et al. 1999. Maturational disturbance of chondrocytes in Cbfa1-deficient mice. Dev. Dyn. 214:279290.[Medline]
Ishizuya, T., S. Yokose, M. Hori, T. Noda, T. Suda, S. Yoshiki, and A. Yamaguchi. 1997. Parathyroid hormone exerts disparate effects on osteoblast differentiation depending on exposure time in rat osteoblastic cells. J. Clin. Invest. 99:29612970.
Javed, A., S. Gutierrez, M. Montecino, A.J. van Wijnen, J.L. Stein, G.S. Stein, and J.B. Lian. 1999. Multiple Cbfa/AML sites in the rat osteocalcin promoter are required for basal and vitamin D-responsive transcription and contribute to chromatin organization. Mol. Cell. Biol. 19:74917500.
Javed, A., B. Guo, S. Hiebert, J. Choi, J. Green, S. Zhao, M.A. Osborne, S. Stifani, J.L. Stein, J.B. Lian, et al. 2000. Groucho/TLE/R-esp proteins associate with the nuclear matrix and repress RUNX (CBFß/AML/PEBP2ß) dependent activation of tissue-specific gene transcription. J. Cell Sci. 113:22212231.
Jilka, R.L., R.S. Weinstein, T. Bellido, P. Roberson, A.M. Parfitt, and S.C. Manolagas. 1999. Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone. J. Clin. Invest. 104:439446.
Kern, B., J. Shen, M. Starbuck, and G. Karsenty. 2001. Cbfa1 contributes to the osteoblast-specific expression of type I collagen genes. J. Biol. Chem. 276:71017107.
Kitazawa, R., S. Kitazawa, and S. Maeda. 1999. Promoter structure of mouse RANKL/TRANCE/OPGL/ODF gene. Biochim. Biophys. Acta. 1445:134141.[Medline]
Kobayashi, H., Y.-H. Gao, C. Ueta, A. Yamaguchi, and T. Komori. 2000. Multilineage differentiation of Cbfa1-deficient calvarial cells in vitro. Biochem. Biophys. Res. Commun. 273:630636.[Medline]
Komori, T., and T. Kishimoto. 1998. Cbfa1 in bone development. Curr. Opin. Genet. Dev. 8:494499.[Medline]
Komori, T., H. Yagi, S. Nomura, A. Yamaguchi, K. Sasaki, K. Deguchi, Y. Shimizu, R.T. Bronson, Y.-H. Gao, M. Inada, et al. 1997. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. 89:755764.[Medline]
Krebsbach, P.H., J.R. Harrison, A.C. Lichtler, C.O. Woody, D.W. Rowe, and B.E. Kream. 1993. Transgenic expression of COL1A1-chloramphenicol acetyltransferase fusion genes in bone: differential utilization of promoter elements in vivo and in cultured cells. Mol. Cell. Biol. 13:51685174.[Abstract]
Lee, K.S., H.J. Kim, Q.L. Li, X.Z. Chi, C. Ueta, T. Komori, J.M. Wozney, E.G. Kim, J.Y. Choi, H.M. Ryoo, et al. 2000. Runx2 is a common target of transforming growth factor ß1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12. Mol. Cell. Biol. 20:87838792.
Levanon, D., R.E. Goldstein, Y. Bernstein, H. Tang, D. Goldernberg, S. Stifani, Z. Paroush, and Y. Groner. 1998. Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors. Proc. Natl. Acad. Sci. USA. 95:1159011595.
Lutterbach, B., J.J. Westendorf, B. Linggi, S. Isaac, E. Seto, and S.W. Hiebert. 2000. A mechanism of repression by acute myeloid leukemia-1, the target of multiple chromosomal translocations in acute leukemia. J. Biol. Chem. 275:651656.
Manolagas, S.C. 2000. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr. Rev. 21:115137.
Mark, M.P., W.T. Butler, C.W. Prince, R.D. Finkelman, and J.V. Ruch. 1988. Developmental expression of 44-kDa bone phosphoprotein (osteopontin) and bone -carboxyglutamic acid (Gla)-containing protein (osteocalcin) in calcifying tissues of rat. Differentiation. 37:123136.[Medline]
Mundlos, S., F. Otto, C. Mundlos, J.B. Mulliken, A.S. Aylsworth, S. Albright, D. Lindhout, W.G. Cole, W. Henn, J.H.M. Knoll, et al. 1997. Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell. 89:773779.[Medline]
Nijweide, P.J., E.H. Burger, J. Klein Nulend, and A. Van der Plas. 1996. The osteocyte. Principles of Bone Biology. J.P. Bilezikian, L.G. Raisz, and G.A. Rodan, editors. Academic Press, London, UK. 115126.
Otto, F., A.P. Thornell, T. Crompton, A. Denzel, K.C. Gilmour, I.R. Rosewell, G.W.H. Stamp, R.S.P. Beddington, S. Mundlos, B.R. Olsen, et al. 1997. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell. 89:765771.[Medline]
Parfitt, A.M., M.K. Drezner, F.H. Glorieux, J.A. Kanis, H. Malluche, P.J. Meunier, S.M. Ott, and R.R. Recker. 1987. Bone histomorphometry: standardization of nomenclature, symbols, and units. J. Bone Miner. Res. 2:595610.[Medline]
Prince, M., C. Banerjee, A. Javed, J. Green, J.B. Lian, G.S. Stein, P.V. Bodine, and B.S. Komm. 2001. Expression and regulation of Runx2/Cbfa1 and osteoblast phenotypic markers during the growth and differentiation of human osteoblasts. J. Cell Biochem. 80:424440.[Medline]
Rossert, J., H. Eberspaecher, and B. de Crombrugghe. 1995. Separate cis-acting DNA elements of the mouse pro-1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice. J. Cell Biol. 129:14211432.[Abstract]
Sato, M., E. Morii, T. Komori, H. Kawahata, M. Sugimoto, K. Terai, H. Shimizu, T. Yasui, H. Ogihara, N. Yasui, et al. 1998. Transcriptional regulation of osteopontin gene in vivo by PEBP2alphaA/CBFA1 and ETS1 in the skeletal tissues. Oncogene. 17:15171525.[Medline]
Selvamurugan, N., M.R. Pulumati, D.R. Tyson, and N.C. Partridge. 2000. Parathyroid hormone regulation of the rat collagenase-3 promoter by protein kinase A-dependent transactivation of core binding factor 1. J. Biol. Chem. 275:50375042.
Stein, G.S., J.B. Lian, and T.A. Owen. 1990. Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation. FASEB J. 4:31113123.[Abstract]
Thirunavukkarasu, K., D.L. Halladay, R.R. Miles, X. Yang, R.J. Galvin, S. Chandrasekhar, T.J. Martin, and J.E. Onyia. 2000. The osteoblast-specific transcription factor Cbfa1 contributes to the expression of osteoprotegerin, a potent inhibitor of osteoclast differentiation and function. J. Biol. Chem. 275:2516325172.
Ueta, C., M. Iwamoto, N. Kanatani, C. Yoshida, Y. Liu, M. Enomoto-Iwamoto, T. Ohmori, H. Enomoto, K. Nakata, K. Takada, et al. 2001. Skeletal malformations caused by overexpression of Cbfa1 or its dominant negative form in chondrocytes. J. Cell Biol. 153:8799.
Weinreb, M., D. Shinar, and G.A. Rodan. 1990. Different pattern of alkaline phosphatase, osteopontin, and osteocalcin expression in developing rat bone visualized by in situ hybridization. J. Bone Miner. Res. 5:831842.[Medline]
Xiao, Z.S., T.K. Hinson, and L.D. Quarles. 1999. Cbfa1 isoform overexpression upregulates osteocalcin gene expression in non-osteoblastic and pre-osteoblastic cells. J. Cell Biochem. 74:596605.[Medline]
Yamaguchi, A., T. Komori, and T. Suda. 2000. Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr. Rev. 21:393411.
Yoon, K., R. Buenaga, and G.A. Rodan. 1987. Tissue specificity and developmental expression of rat osteopontin. Biochem. Biophys. Res. Commun. 148:11291136.[Medline]
Young, M.F., K. Ibaraki, J.M. Kerr, and A.M. Heegaard. 1993. Molecular and cellular biology of the major noncollagenous proteins in bone. Cellular and Molecular Biology of Bone. M. Noda, editor. Academic Press, London, UK. 191234.
Related Article