Regulation of osteoprotegerin and RANKL gene expression by Wnt/β-catenin and bone morphogenetic protein-2 in C2C12 cells

Abstract

Wnt/β-catenin signaling plays an important role in developing the skeletal system. However, the exact mechanisms by which Wnt/β-catenin signaling regulates bone remodeling remain to be elucidated. Our previous studies demonstrated that canonical Wnt signaling inhibited the ability of bone morphogenetic protein (BMP)-2 to induce myotubes in the C2C12 cell line, and that this inhibition was mediated by Id-1. We also showed that BMP-2 induced β-catenin-mediated lymphoid enhancer factor 1/T cell factor (Lef1/Tcf)-dependent transcription in this cell line. Here, we examined the role of intracellular signaling by Wnt/β-catenin and BMP-2 in the regulation of expression of osteoprotegerin (OPG) and receptor activator of NFκB ligand (RANKL) in osteoblasts. OPG expression was induced by overexpression of activated β-catenin in C2C12 cells and caused an increased OPG concentration in the culture supernatant. Silencing of glycogen synthase kinase-3β (GSK-3β) by sgRNA (tRNaseZL-utilizing gene silencing) also increased OPG concentration in the culture supernatant. BMP-2 acted synergistically with Wnt3a to enhance OPG expression. In contrast, Wnt3a suppressed RANKL expression. Transient transfection analyses using murine OPG gene promoter constructs revealed that activated β-catenin induced transcription activity, and that this induction might be mediated by the Lef1/Tcf response element in the OPG gene promoter. These results show that both Wnt/β-catenin and BMP-2 signaling regulate OPG and RANKL expression.