STUDY OF BONE FORMATION SIGNALING IN WNT-LRP-DKK AND PROTEOSOME SYSTEM

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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