Abstract
Carfilzomib, the next generation of proteasome inhibitor, may increase osteoblast-related markers in patients with multiple myeloma, but the molecular mechanism of its effect on mesenchymal stem cell differentiation to osteoblasts remains unknown. Herein, we demonstrated that carfilzomib significantly promoted mesenchymal stem cell differentiation into osteoblasts. In osteoprogenitor cells and primary mesenchymal stem cells from patients with myeloma, carfilzomib induced increases in alkaline phosphatase activity, matrix mineralization, and calcium deposition via Wnt-independent activation of β-catenin/TCF signaling. Using affinity pull-down assays with immunoblotting analysis and immunofluorescence, we found that carfilzomib induced stabilization of both free and active forms of β-catenin in a time- and dose-dependent manner that was not associated with β-catenin transcriptional regulation. Nuclear translocation of β-catenin protein was associated with TCF transcriptional activity that was independent of the effects of GSK3β-activation and of signaling induced by 19 Wnt ligands, 10 Frizzled receptors, and LRP5/6 co-receptors. Blocking activation of β-catenin/TCF signaling by dominant negative TCF1 or TCF4 attenuated carfilzomib-induced matrix mineralization. Thus, carfilzomib induced osteoblast differentiation via Wnt-independent activation of the β-catenin/TCF pathway. These results provide a novel molecular mechanism critical to understanding the anabolic role of carfilzomib on myeloma-induced bone disease.
Highlights
Multiple myeloma (MM) is characterized by malignant plasma cells infiltrating bone marrow, where they closely interact with mesenchymal stem/stromal cells (MSCs), osteoblasts (OBs), and osteoclasts, triggering osteolytic bone lesions
To determine whether CFZ increases β-catenin protein levels in cell lines of OB progenitor cells and MSCs, we first used Ecadherin pull-down assays to separate the pool of free βcatenin from the pool of membrane-bound β-catenin; OBs and MSCs express abundant amounts of E-cadherin, which sequesters β-catenin in the membrane fraction [25] where it is unaffected by Wnt stimulation [9,10,26] or bortezomib treatment [24]
Wnt/β-catenin signaling is required for MSC differentiation to OBs and for bone formation [16], so we initially examined the effects of CFZ on activity of alkaline phosphatase (ALP), which is a marker of MSC differentiation to OBs
Summary
Multiple myeloma (MM) is characterized by malignant plasma cells infiltrating bone marrow, where they closely interact with mesenchymal stem/stromal cells (MSCs), osteoblasts (OBs), and osteoclasts, triggering osteolytic bone lesions. A number of studies have reported fewer OBs and decreased bone formation in MM patients with higher plasma cell infiltration [1]. DKK1-mediated suppression of Wnt/β-catenin signaling leads to positive regulation of RANKL expression and negative regulation of OB production of osteoprotegerin (OPG), resulting in enhanced osteoclast function [6,7] and in diminished differentiation of OBs from MSCs [8,9,10]. Preclinical in vivo studies have reported that increasing Wnt/β-catenin signaling by administering anti-DKK1 antibodies, Wnt3a, or LiCl suppresses MM-induced bone loss and MM cell growth [11,12,13,14,15]
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