Bortezomib Induces Osteoblast Differentiation Via Wnt-Independent Activation of Beta-catenin/TCF Signaling

Bone loss in multiple myeloma (MM) and other conditions represents a serious clinical problem, and extensive research is being conducted to develop new drugs that target signaling cascades whose disruption is known to cause bone loss. Inhibition of Wnt/beta-catenin/TCF signaling induces proliferation of mesenchymal stem cells and/or suppresses their differentiation into osteoblasts. Osteolysis in MM and several other diseases has recently been linked to the suppression of Wnt/beta-catenin/TCF signaling. The antitumor activity of the proteasome inhibitor bortezomib in MM has been linked to its bone anabolic effects. However, the molecular basis of bortezomib’s action on bone is not well characterized. Here we show that bortezomib activates the beta-catenin/TCF pathway in murine and human osteoblast progenitors and human mesenchymal stem cells derived from bone marrow of healthy donors and patients with MM, leading to their subsequent differentiation in-vitro as assessed by matrix mineralization and calcium deposition. Pull-down assays followed by immunoblotting and immunofluorescence microscopy revealed that bortezomib induced stabilization of beta-catenin in both the cytoplasm and nucleus. Nuclear translocation of stabilized beta-catenin was associated with the activation of beta-catenin/TCF transcriptional activity as detected by luciferase activity from TOPFLASH reporter constructs. This activation was not dependent on the drug inducing Wnt ligand or receptor expression, the suppression of Wnt signaling inhibitors or change in disheveled protein levels. Synergy experiments showed that recombinant Wnt3a or LiCl-induced inactivation of GSK3beta did not enhance bortezomib-induced changes in TCF activity. Matrix mineralization was not accompanied by the induction of alkaline phosphatase or osteoprotegerin, which can be activated by Wnt3a in these cell types. Blocking the activation of beta-catenin/TCF signaling by dominant negative TCF attenuated bortezomib-induced matrix mineralization. These results add to the growing body of evidence demonstrating a critical role of the Wnt/beta-catenin pathway in bone biology and provide mechanistic insights into the recently recognized bone anabolic effects of bortezomib. As the effects of bortezomib completely bypass all known regulatory mechanisms governing Wnt signaling, these data also provide a strong rationale for the use of bortezomib in the treatment of diseases linked to suppression of Wnt/beta-catenin signaling.