Harada H, Tagashira S, Fujiwara M, Ogawa S, Katsumata T, Yamaguchi A, Komori T, Nakatsuka M
Sumitomo Pharmaceuticals Research Center, Osaka 554-0022, Japan.
J Biol Chem. 1999 Mar 12;274(11):6972-8. doi: 10.1074/jbc.274.11.6972.
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 Pebp2alphaA 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 as til-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 as Osf2/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 II Cbfa1 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 of Xenopus 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 of Cbfa1, the gene expression of ALP was also strongly induced in cells transfected with Type I and Type II Cbfa1 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 of osteocalcin 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 osteocalcin promoter, and a mouse osteopontin promoter revealed the differences in the transcriptional induction of target genes by each Cbfa1 isoform with or without its beta-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.
Cbfa1是成骨细胞分化和骨形成所必需的转录因子。我们研究了Cbfa1三种亚型的功能差异：I型（最初由小川等人报道为Pebp2alphaA（小川，E.，丸山，M.，鹿儿岛，H.，犬冢，M.，陆，J.，佐竹，M.，重定，K.，和伊藤，Y.（1993年）美国国家科学院院刊90，6859 - 6863）），II型（最初由斯图尔特等人报道为til - 1（斯图尔特，M.，特里，A.，胡，M.，奥哈拉，M.，布莱思，K.，巴克斯特，E.，卡梅伦，E.，洋葱，D. E.，和尼尔，J. C.（1997年）美国国家科学院院刊94，8646 - 8651）），以及III型（最初由杜西等人报道为Osf2/Cbfa1（杜西，P.，张，R.，杰弗里，V.，里德尔，A. L.，和卡尔森蒂，G.（1997年）细胞89，747 - 754））。逆转录聚合酶链反应分析表明，这些亚型在成年小鼠骨骼中表达。在小鼠成纤维细胞系C3H10T1/2中瞬时转染I型或II型Cbfa1可诱导碱性磷酸酶（ALP）活性的表达。通过共导入非洲爪蟾BMP - 4 cDNA，这种诱导作用协同增强。相比之下，瞬时转染III型cDNA未诱导出ALP活性。在稳定转染了Cbfa1各亚型的C3H10T1/2细胞中，I型和II型Cbfa1转染的细胞中ALP的基因表达也被强烈诱导，而III型Cbfa1转染的细胞中则未被诱导。在所有稳定转染了Cbfa1各亚型的细胞中，骨钙素、骨桥蛋白和I型胶原基因的表达均被诱导或上调，且II型转染细胞中骨钙素基因的表达水平最高。使用6XOSE2 - SV40启动子（6个串联的Cbfa1结合元件连接在SV40启动子序列之前）、小鼠骨钙素启动子和小鼠骨桥蛋白启动子进行的荧光素酶报告基因分析揭示了每种Cbfa1亚型在有或没有其β亚基的情况下对靶基因转录诱导的差异。这些结果表明，本研究中使用的所有三种Cbfa1亚型都参与了成骨细胞分化的刺激作用，但它们在成骨细胞分化过程中发挥着不同的功能。
