Toward Defining the Functional Relevance of Wnt Signaling in Osteoclasts in Multiple Myeloma.

Disruption of Wnt signaling is thought to play a central role in the development of myeloma bone disease. Although Wnt signaling in osteoblasts (OB) is essential for their differentiation and Wnt signaling in OB indirectly regulates osteoclast (OC) development through the regulation of both OPG and RANKL, little is known about the direct role of Wnt signaling in osteoclastogenesis. In this study, we sought to characterize the Wnt signaling pathway and its functional role in OC formation using the macrophage cell line Raw 264.7, which can be induced to differentiate into OC, as well as human primal progenitor osteoclast cells isolated from bone marrow of normal donors and patients with MM. We first analyzed the pattern of Wnt signaling components in these cells by RT-PCR and sequence analysis. High level of expression of Wnt receptors Frizzled (Fz) Fz2, 3, 4, 5, 7, 8 and 9, and co-receptors LRP5, and LRP6, and transcription factors TCF1, TCF3, and LEF1 was observed in 10 of 10 OC cells from MM patients and 4 of 4 OC from normal donors and Raw264.7. Wnt3a conditioned medium and recombinant Wnt3a induced increased levels of total and un-phosphorylated beta-catenin in all cells. Pretreatment of OC cells with sFRP1 decreased Wnt3a induced increased beta-catenin. Pretreatment with DKK1, a specific inhibitor of the canonical Wnt pathway, blocked Wnt3a induced stabilization of beta-catenin. Additionally, the GSK3beta inhibitor lithium chloride induced stabilization of beta-catenin in OC and Raw264.7 cells in dose-dependent fashion. Wnt-3a induced TCF/LEF transcriptional activity in Raw264.7 cells transfected with TOPflash compared with Raw264.7 transfected with FOPflash, which possess mutant sequence for binding beta-catenin. These results suggest that Wnt signaling functions in primal MM OC cells, as well as OC cell lines. Finally, we sought to determine the biological function of Wnt signaling in OC cells. The results showed that Wnt-3a did not affect the proliferation and survival of OC cells, nor increased formation of TRAP positive cells. Wnt3a did not synergize with RANKL and M-CSF in induction of OC differentiation. Additional biological functional assays are underway to determine the direct role of Wnt signaling on OC. Identification of the role of Wnt signaling in osteoclastogenesis may improve our understanding of the bone lytic process in multiple myeloma.