title: AP1 binding site is another target of FGF2 regulation of bone sialoprotein gene transcription.
abstract: Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. We previously reported that fibroblast growth factor 2 (FGF2) regulates BSP gene transcription via FGF2 response element (FRE) in the proximal promoter of rat BSP gene. We here report that activator protein 1 (AP1) binding site overlapping with glucocorticoid response element (GRE) AP1/GRE in the rat BSP gene promoter is another target of FGF2. Using the osteoblastic cell line ROS17/2.8, we determined that BSP mRNA levels increased by 10 ng/ml FGF2 at 6 and 12 h. Runx2 protein levels increased by FGF2 (10 ng/ml) at 3 h. Treatment of ROS17/2.8 cells with FGF2 (10 ng/ml, 12 h) increased luciferase activities of constructs including -116 to +60 and -938 to +60 of the rat BSP gene promoter. Effects of FGF2 abrogated in constructs included 2 bp mutations in the FRE and AP1/GRE elements. Luciferase activities induced by FGF2 were blocked by tyrosine kinase inhibitor herbimycin A, src-tyrosine kinase inhibitor PP1 and MAP kinase kinase inhibitor U0126. Gel shift analyses showed that FGF2 increased binding of FRE and AP1/GRE elements. Notably, the AP1/GRE-protein complexes were supershifted by Smad1 and c-Fos antibodies, c-Jun and Dlx5 antibodies disrupted the complexes formation, on the other hand AP1/GRE-protein complexes did not change by Runx2 antibody. These studies demonstrate that FGF2 stimulates BSP gene transcription by targeting the FRE and AP1/GRE elements in the rat BSP gene promoter.
authors.affiliation: Department of Periodontology, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Takai
authors.forename: Shouta
authors.initials: S
authors.lastname: Araki
authors.forename: Masaru
authors.initials: M
authors.lastname: Mezawa
authors.forename: Dong-Soon
authors.initials: DS
authors.lastname: Kim
authors.forename: Xinyue
authors.initials: X
authors.lastname: Li
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.forename: Zhengyang
authors.initials: Z
authors.lastname: Li
authors.forename: Zhitao
authors.initials: Z
authors.lastname: Wang
authors.forename: Youhei
authors.initials: Y
authors.lastname: Nakayama
authors.forename: Yorimasa
authors.initials: Y
authors.lastname: Ogata
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001842:Bone and Bones / Q000378:metabolism*; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D017931:DNA Primers; D016222:Fibroblast Growth Factor 2 / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology*; D058574:Integrin-Binding Sialoprotein; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D012795:Sialoglycoproteins / Q000235:genetics*; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D017931:DNA Primers; C546873:Ibsp protein, rat; D058574:Integrin-Binding Sialoprotein; D012333:RNA, Messenger; C493717:Runx2 protein, rat; D012795:Sialoglycoproteins; D018808:Transcription Factor AP-1; D016222:Fibroblast Growth Factor 2