title: Osteogenic differentiation of C2C12 myogenic progenitor cells requires the Fos-related antigen Fra-1 - a novel target of Runx2.
abstract: Bone formation is a developmental process requiring the differentiation of mesenchymal stem cells into osteoblasts. It is established that Runx2 tightly regulates osteoblast differentiation and bone formation. Fos-related antigen Fra-1 is an essential factor for bone formation. Current evidence does not support a relationship between Fra-1 and Runx2 in osteogenesis. Here, we explored the possibility that Runx2 regulates Fra-1 expression during osteogenic differentiation of C2C12 myogenic progenitor cells. Expression of Fra-1 was induced rapidly after activation of Runx2 in a Tet-on stable C2C12 cell-line (C2C12/Runx2(Dox) sub-line). Transient transfection assay showed that Runx2 activates Fra-1 promoter-reporter activity, suggesting that Fra-1 may be a direct target of Runx2. To determine the minimal region of the Fra-1 promoter that was activated by Runx2, a series of Fra-1 promoter deletion constructs were made. By transient transfection assay, we defined the minimal region to the proximal 342 bp (-84 to +258). Two potential Runx2-binding sites (at positions +139 and +208) were predicted within this region. Mutation of the Runx2 motif at position +208 significantly decreased Fra-1 promoter activity compared to wild type, whereas mutation of Runx2 at position +139 had no effect. Electrophoretic mobility shift assay (EMSA) demonstrated the existence of one atypical Runx2-binding element at position +208, and chromatin immunoprecipitation (ChIP) assay revealed that Runx2 bound to the native Fra-1 promoter in vivo via this site. Finally, forced expression of Fra-1 resulted in upregulation of alkaline phosphatase (ALP), a marker of early osteoblast differentiation. Together, these results indicate that Fra-1 is a direct target of Runx2 during osteogenic differentiation of C2C12 myogenic progenitor cells.
authors.affiliation: Institute of Bioengineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, Guangdong, PR China.
authors.forename: Shouhe
authors.initials: S
authors.lastname: Yu
authors.forename: Qianqian
authors.initials: Q
authors.lastname: Geng
authors.forename: Fenyong
authors.initials: F
authors.lastname: Sun
authors.forename: Yongchun
authors.initials: Y
authors.lastname: Yu
authors.forename: Qiuhui
authors.initials: Q
authors.lastname: Pan
authors.forename: An
authors.initials: A
authors.lastname: Hong
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation / Q000235:genetics; D047369:Chromatin Immunoprecipitation; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D018507:Gene Expression Regulation, Developmental; D051379:Mice; D008969:Molecular Sequence Data; D032448:Myoblasts, Skeletal / Q000166:cytology* / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology*; D010012:Osteogenesis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics*; D015854:Up-Regulation; D000099169:Fos-Related Antigen 1
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D016760:Proto-Oncogene Proteins c-fos; D000099169:Fos-Related Antigen 1; C493716:Runx2 protein, mouse