title: Insulin-like growth factor-I increases bone sialoprotein (BSP) expression through fibroblast growth factor-2 response element and homeodomain protein-binding site in the proximal promoter of the BSP gene.
abstract: Insulin-like growth factor-I (IGF-I) promotes bone formation by stimulating proliferation and differentiation of osteoblasts. Bone sialoprotein (BSP), is thought to function in the initial mineralization of bone, is selectively expressed by differentiated osteoblast. To determine the molecular mechanism of IGF-I regulation of osteogenesis, we analyzed the effects of IGF-I on the expression of BSP in osteoblast-like Saos2 and in rat stromal bone marrow (RBMC-D8) cells. IGF-I (50 ng/ml) increased BSP mRNA levels at 12 h in Saos2 cells. In RBMC-D8 cells, IGF-I increased BSP mRNA levels at 3 h. From transient transfection assays, a twofold increase in transcription by IGF-I was observed at 12 h in pLUC3 construct that included the promoter sequence from -116 to +60. Effect of IGF-I was abrogated by 2-bp mutations in either the FGF2 response element (FRE) or homeodomain protein-binding site (HOX). Gel shift analyses showed that IGF-I increased binding of nuclear proteins to the FRE and HOX elements. Notably, the HOX-protein complex was supershifted by Smad1 antibody, while the FRE-protein complex was shifted by Smad1 and Cbfa1 antibodies. Dlx2 and Dlx5 antibodies disrupted the formation of the FRE- and HOX-protein complexes. The IGF-I effects on the formation of FRE-protein complexes were abolished by tyrosine kinase inhibitor herbimycin A (HA), PI3-kinase/Akt inhibitor LY249002, and MAP kinase kinase inhibitor U0126, while IGF-I effects on HOX-protein complexes were abolished by HA and LY249002. These studies demonstrate that IGF-I stimulates BSP transcription by targeting the FRE and HOX elements in the proximal promoter of BSP gene.
authors.affiliation: Department of Periodontology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan.
authors.forename: Youhei
authors.initials: Y
authors.lastname: Nakayama
authors.forename: Yu
authors.initials: Y
authors.lastname: Nakajima
authors.forename: Naoko
authors.initials: N
authors.lastname: Kato
authors.forename: Hideki
authors.initials: H
authors.lastname: Takai
authors.forename: Dong-Soon
authors.initials: DS
authors.lastname: Kim
authors.forename: Masato
authors.initials: M
authors.lastname: Arai
authors.forename: Masaru
authors.initials: M
authors.lastname: Mezawa
authors.forename: Shouta
authors.initials: S
authors.lastname: Araki
authors.forename: Jaro
authors.initials: J
authors.lastname: Sodek
authors.forename: Yorimasa
authors.initials: Y
authors.lastname: Ogata
mesh_terms: D000818:Animals; D016227:Benzoquinones; D001665:Binding Sites; D001854:Bone Marrow Cells / Q000166:cytology*; D002070:Butadienes / Q000494:pharmacology; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002999:Clone Cells; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000235:genetics* / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D058574:Integrin-Binding Sialoprotein; D047029:Lactams, Macrocyclic; D009154:Mutation; D009570:Nitriles / Q000494:pharmacology; D012516:Osteosarcoma / Q000473:pathology; D011401:Promoter Regions, Genetic; D011809:Quinones / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020218:Response Elements / Q000235:genetics*; D017828:Rifabutin / Q000031:analogs & derivatives; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D016227:Benzoquinones; D002070:Butadienes; D004791:Enzyme Inhibitors; D018398:Homeodomain Proteins; C546871:IBSP protein, human; C546873:Ibsp protein, rat; D058574:Integrin-Binding Sialoprotein; D047029:Lactams, Macrocyclic; D009570:Nitriles; D011809:Quinones; D012333:RNA, Messenger; D012795:Sialoglycoproteins; C113580:U 0126; D016222:Fibroblast Growth Factor 2; D017828:Rifabutin; D007334:Insulin-Like Growth Factor I; C020754:herbimycin