2006 Fiscal Year Final Research Report Summary
STUDY OF BONE FORMATION SIGNALING IN WNT-LRP-DKK AND PROTEOSOME SYSTEM
| Project/Area Number |
16390529
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Functional basic dentistry
|
|---|
| Research Institution | Hokkaido University |
|---|
Principal Investigator |
TAMURA Masato HOKKAIDO UNIV., GRAD. SCH. DENT. MED., PROFESSOR, 大学院歯学研究科, 教授 (30236757)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
NASHIMOTO Masayuki NIIGATA UNIVERSITY OF PHARMACY AND APPLIED SCIENCES, DEPT. OF APPLIED LIFE SCIENCES, ASSOCIATE PROFESSOR, 応用生命科学部, 助教授 (30228069)
ISHISAKI Akira HOKKAIDO UNIV., GRAD. SCH. DENT. MED., ASSOCIATE PROFESSOR, 大学院歯学研究科, 助教授 (20356439)
|
|---|
| Project Period (FY) |
2004 – 2006
|
| Keywords | osteoblast / Wnt / BMP-2 / LRP / Dkk |
|---|
| Research Abstract |
Wnt/β-catenin signaling plays a role in the developing skeletal system. However, the exact mechanisms by which Wnt/β-catenin signaling regulates bone remodeling remain to be elucidated. In this study, we demonstrated that an interaction between β-catenin and Smad was identified and found to be crucial for Wnt-mediated regulation of the BMP-2 responsive gene expression, and BMP-2 up-regulated Wnt-induced lymphoid enhancing factor 1/T cell factor (Lef1/Tcf)-dependent TopFlash transcriptional activity in C2C12 cell line. Also, Wnt/β-catenin signaling regulates expression of osteoprotegerin (OPG) and receptor activator of NFkB ligand (RANKL) in osteoblasts. Moreover, we investigated the mechanism of how induces OPG expression by Wnt/β-catenin signaling in osteoblasts. OPG expression was induced by over-expression of Wnt3a or activated β-catenin in mesenchymal pluripotent C2C12 cells or osteoblastic MC3T3-E1 cells. In these cultures, BMP-2 synergistically enhanced their OPG expression. Sile
… More
ncing of glycogen synthase kinase-3β by siRNA or sgRNA (tRNaseZL-utilizing gene silencing method) also increased OPG levels of culture supernatant in C2C12 cells estimated by ELISA assay. To investigate the mechanisms of the Wnt/β-catenin signaling activated OPG gene transcription, we have cloned approximately 1.5-kilobase pair genomic DNA fragment corresponding to the 5'-flanking promoter region of the murine OPG gene. Activated β-catenin results in 10-15 fold increase in reporter gene transcription activity of the 1.5-kilobase fragment by transfection assay performed in C2C12 cells. Deletion analyses revealed that a proximal 253-base pair region in the promoter was required for Wnt/β-catenin responsiveness. In this region, we identified four putative Lef1/Tcf binding sites, which meet with a consensus sequence, reported to be recognized by Lef1/Tcf proteins. Using site-directed mutation analyses, functional Lef1/Tcf binding sites was identified in their region. Smad protein involved and interacted with their Wnt/β-catenin responsive element on the OPG promoter in response to BMP-2 stimulation. Chromatin IP assay indicated in viro evidence that β-catenin regulates transcription of OPG via promoter region of their site. These results show that OPG is a target gene for Wnt/β-catenin signaling and their induction is mediated novel Wnt/β-catenin response element of the OPG gene promoter. Less
|
