On the Molecular Mechanism of DKK1 Inhibition of Osteoblast Differentiation in Multiple Myeloma.

Abstract

Abstract Lytic bone destruction is a severe complication of multiple myeloma growth in the bone. We have demonstrated that Dkk1, an antagonist of Wnt signaling which is essential for osteoblast growth and differentiation, is expressed by myeloma plasma cells leading to increased levels of this protein in serum. Myeloma serum can block osteoblast differentiation in-vitro and a neutralizing anti-DKK1 antibody can inhibit this. These data suggest that MM cells disrupt Wnt signaling and that this promotes uncoupled bone turnover. However, the molecular mechanism responsible for this processes remains to be elucidated. Here we investigated Wnt signaling in osteoblast differentiation and identified the molecular mechanism regulating this process. Using qRT-PCR and sequence confirmation we showed that multiple Frizzled (Fz) mRNAs and LRP5/6 co-receptors were expressed in C2C12 cells and three human osteoblast cell lines, hFOB1.19, MG63 and Saos-2. Furthermore, these cells had high levels of total beta-catenin an indicator of active Wnt signaling. Wnt3a conditioned medium (CM) and recombinant Wnt3a protein increased uncomplexed beta-catenin level as assessed by GST-E-cadherin binding assay. Blocking autocrine Wnt signaling with Dkk1 blocked Wnt-3a-induced increase in beta-catenin levels in dose-dependent fashion. Co-culturing of OPM-2 MM cells ectopically expressing Dkk1 inhibited the Wnt-3a induced beta-catenin stabilization in C2C12 cells. Transfection of C2C12 cells with a dominant negative beta-catenin (DNBC) completely blocked endogenous and Wnt-3a-induced TCF/LEF transcriptional activity as evidenced by TOPflash luciferase activity. Furthermore, BMP-2 induced increase in alkaline phosphatase (ALP) activity was completed abrogated in C2C12 cells expressing DNCB. Silencing LRP5/6 mRNA with small inference RNAs blocked BMP-2 induced increase in ALP expression, suggesting that canonical Wnt pathway contributes to BPM-2 induced OB differentiation. BMP-2 induced phosphorylation of Smad 1, 5 and 8 whereas Wnt-3a had no effect on phosphorylation of these Smads. Moreover, Dkk1 did not block BMP-2 induced phosphorylation of Smad-1, −5 and −8 and did not alter transcriptional activity of Cbfa1, an essential transcriptional factor for OB differentiation. Taken together, these data suggest that Wnt induction of OB cell differentiation through canonical Wnt-beta-catenin pathway is independent of the activation of the Smad pathway. Our results suggested that canonical Wnt signaling pathway plays an important role in osteoblastogenesis and that Dkk1 inhibits OB cell differentiation in a way that may contribute to MM bone lesions. New therapies targeting DKK1 in myeloma may represent a novel treatment strategy.