Dkk1 blocks Wnt-induced osteoprotegerin production in osteoblast progenitors in multiple myeloma

2050 Multiple myeloma (MM), a fatal malignancy of plasma cells growing systemically in bone, causes osteolytic bone lesions. Bone turnover is regulated by the coupled activity of osteoblasts (OB) and osteoclasts (OC). OC differentiation is positively modulated by the receptor activator of nuclear factor kappa B ligand (RANKL) and negatively regulated by the decoy receptor, osteoprotegerin (OPG). Although the RANKL/OPG axis appears to be deregulated in MM, little is known about how MM cells might promote this process. Wnt signaling promotes bone formation and OB differentiation and overexpression of an active form beta-catenin in OB leads to upregulation of OPG, indicating that Wnt signaling in OB regulates osteoclastogenesis. We have demonstrated that MM cells express Dkk1, a soluble antagonist of canonical Wnt signaling, and that increased levels of Dkk1 in serum from MM is linked to bone lesions and MM serum can inhibit OB differentiation in a Dkk1-specific manner. To better understand the molecular mechanisms of the osteolytic process in MM, we investigated the role of canonical Wnt signaling in regulating the RANKL/OPG axis in OB progenitors. By real time quantitative PCR (qPCR) and ELISA analysis, we demonstrate that Wnt3a significantly induced OPG in C2C12 cells, a pluripotential mesenchymal progenitor. This Wnt3a-induced increase in OPG was significantly inhibited by pretreatment of the cells with recombinant Dkk1 or conditioned medium from an MM cell line constitutively expressing Dkk1. Inhibition of canonical Wnt signaling by transfection with siRNA to Wnt co-receptors LRP5/6 and or a dominant negative beta-catenin abrogated both endogenous and Wnt3a-induced OPG relative to controls. Interestingly, Dkk2, which has been shown to promote OB differentiation, significantly blocked Wnt3a-induced increase in OPG. Finally, treatment of the cells with plasma from bone marrow of MM pateints with high concentrations (> 100 ng/ml) of Dkk1 inhibited both OPG mRNA and protein in C2C12 cells. While Wnt3a stimulated OPG, it had a negative effect on RANKL mRNA as accessed by qPCR. Constitutive expression of Dkk1 lead to increased production of RANKL. Our results suggest that canonical Wnt signaling plays an important role in regulating the RANKL/OPG axis and that Dkk1 secretion by MM cells directly inhibits osteoblast differentiation while indirectly promoting osteoclastogenesis. Restoring proper Wnt signaling in MM may prevent bone lesions and possibly disease progression.