CELL BIOLOGY AND METABOLISM
Cbfa1 Isoforms Exert Functional Differences in Osteoblast Differentiation*

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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.

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