title: Involvement of PRIP (Phospholipase C-Related But Catalytically Inactive Protein) in BMP-Induced Smad Signaling in Osteoblast Differentiation.
abstract: Phospholipase C-related but catalytically inactive protein (PRIP) was first isolated as an inositol 1,4,5-trisphosphate binding protein. We generated PRIP gene-deficient mice which exhibited the increased bone mineral density and trabecular bone volume, indicating that PRIP is implicated in the regulation of bone properties. In this study, we investigated the possible mechanisms by which PRIP plays a role in bone morphogenetic protein (BMP) signaling, by analyzing the culture of primary cells isolated from calvaria of two genotypes, the wild type and a mutant. In the mutant culture, enhanced osteoblast differentiation was observed by measuring alkaline phosphatase staining and activity. The promoter activity of Id1 gene, responding immediately to BMP, was also more increased. Smad1/5 phosphorylation in response to BMP showed an enhanced peak and was more persistent in mutant cells, but the dephosphorylation process was not different between the two genotypes. The luciferase assay using calvaria cells transfected with the Smad1 mutated as a constitutive active form showed increased transcriptional activity at similar levels between the genotypes. The expression of BMP receptors was not different between the genotypes. BMP-induced phosphorylation of Smad1/5 was robustly decreased in wild type cells, but not in mutant cells, by pretreatment with DB867, an inhibitor of methyltransferase of inhibitory Smad6. Furthermore, BMP-induced translocation of Smad6 from nucleus to cytosol was not much observed in PRIP-deficient cells. These results indicate that PRIP is implicated in BMP-induced osteoblast differentiation by the negative regulation of Smad phosphorylation, through the methylation of inhibitory Smad6.
authors.affiliation: Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan.|Division of Orthodontics, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Miho
authors.initials: M
authors.lastname: Kotani
authors.affiliation: Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Miho
authors.initials: M
authors.lastname: Matsuda
authors.affiliation: Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Murakami
authors.affiliation: Division of Orthodontics, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Ichiro
authors.initials: I
authors.lastname: Takahashi
authors.affiliation: Division of Pathophysiology, Research Center for Genomic Medicine, Saitama Medical University, Hidaka, Saitama 350-1241, Japan.
authors.forename: Takenobu
authors.initials: T
authors.lastname: Katagiri
authors.affiliation: Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Hirata
mesh_terms: D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D002454:Cell Differentiation / Q000235:genetics*; D005786:Gene Expression Regulation; D008745:Methylation; D051379:Mice; D056918:Nuclear Receptor Coactivators / Q000235:genetics* / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism; D010012:Osteogenesis / Q000235:genetics*; D010766:Phosphorylation; D061251:Primary Cell Culture; D011401:Promoter Regions, Genetic; D015398:Signal Transduction / Q000235:genetics; D051898:Smad1 Protein / Q000235:genetics / Q000378:metabolism; D051902:Smad5 Protein / Q000235:genetics / Q000378:metabolism; D051905:Smad6 Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D019485:Bone Morphogenetic Proteins; C405683:Ncoa6 protein, mouse; D056918:Nuclear Receptor Coactivators; D051898:Smad1 Protein; D051902:Smad5 Protein; D051905:Smad6 Protein; C497803:Smad6 protein, mouse
keywords: BONE MORPHOGENETIC PROTEIN; KINASE; OSTEOBLAST; PHOSPHORYLATION; Smad