Bone morphogenetic protein‐2 enhances Wnt/β‐catenin signaling‐induced osteoprotegerin expression

Abstract

Wnt/β-catenin signaling plays an important role in the developing skeletal system. Our previous studies demonstrated that Wnt/β-catenin signaling inhibits the ability of bone morphogenetic protein (BMP)-2 to suppress myotube formation in the multipotent mesenchymal cell line C2C12 and that this inhibition is mediated by Id1. In this study, we examined the role of intracellular signaling by Wnt/β-catenin and BMP-2 in regulating the expression of osteoprotegerin (OPG) and of the receptor activator of NFκB ligand (RANKL). OPG expression was induced by Wnt/ β-catenin signaling in C2C12 cells and osteoblastic MC3T3-E1 cells. Silencing of glycogen synthase kinase-3β also increased OPG expression. In contrast, R expression was suppressed by Wnt/β-catenin signaling. In a transfection assay, β-catenin induced the activity of a reporter gene, a 1.5 kb fragment of the 5′-flanking region of the OPG gene. Deletion and mutation analysis revealed that Wnt/β-catenin signaling regulates transcription of OPG via a promoter region containing two Wnt/β-catenin responsive sites. BMP-2 enhanced Wnt/β-catenin-dependent transcriptional activation of the OPG promoter. In response to BMP-2 stimulation, Smad 1 and 4 interacted with Wnt/β-catenin responsive sites. These results show that the regulation of OPG expression is mediated through two transcription pathways that involve the OPG promoter.

Regulation of CXCL12 expression by canonical Wnt signaling in bone marrow stromal cells

“…According to the model of canonical Wnt action, in cells lacking a Wnt signal, glycogen synthase kinase (GSK) -3β phosphorylates β-catenin, inducing rapid degradation of β-catenin via the ubiquitin-proteasome pathway (MacDonald et al, 2009, Sethi andVidal-Puig, 2010). Canonical Wnt signaling causes stabilization of β-catenin which then translocates to the nucleus, where it interacts with the transcription factors that regulate expression of several target genes including c-myc and osteoprotegerin (OPG) (MacDonald et al, 2009, Sato et al, 2009). …”

“…Loss of function of β-catenin in osteoblasts leads to low bone mass caused by increased numbers of osteoclasts resulting in increased bone resorption, indicating enhanced osteoclastogenesis from hematopoietic progenitor cells (Glass et al, 2005). Previously, we have shown that canonical Wnt signaling induces OPG expression and analysis of the murine OPG gene promoter revealed that constitutively active forms of β-catenin regulate transcription of OPG via a promoter region containing two responsive sites (Sato et al, 2009). In addition, canonical Wnt signaling reduces receptor activator of NFkB ligand (RANKL) expression (Spencer et al, 2006, Sato et al, 2009).…”

Regulation of neuropeptide Y Y1 receptor expression by bone morphogenetic protein 2 in C2C12 myoblasts

“…MC3T3-E1 cells were transfected with Silencer® select pre-designed siRNA for the Y1 receptor (Ambion, ID number s70765) or Silencer® negative control siRNA #1 (Ambion) at a concentration of 10 nM using Lipofectamine RNAiMAX (Invitrogen) as described previously [21,22]. Cells were harvested 48 h after transfection.…”

“…RT-PCR was performed as previously described [18] Quantitative RT-PCR (qRT-PCR) was performed using assay-on-demand TaqMan probes (Applied Biosystems) and the StepOne ® real time PCR system [21,22]. The relative level of gene expression was quantified using the comparative C T method with GAPDH as the endogenous control.…”

“…A ChIP assay was performed as described previously [21] using agarose gels and visualized with ultraviolet light. All ChIP assays were repeated at least two times.…”