Abstract
In multiple myeloma (MM), the proteasome inhibitor bortezomib induces mesenchymal stem cells (MSC) toward osteoblast differentiation. However, it is full unclear about the mechanism(s) underlying bortezomib in this process. Wnt/beta-catenin pathway plays a pivotal role in osteoblast differenciation and bone development. We have demonstrated that inhibition of Wnt/beta-catenin signaling by MM-derived Dkk1 suppresses osteoblast progenitor cell differentiation into osteoblasts (Qiang et al, Bone 2008) and deregulate RANKL and OPG expression in osteoblast cells (Qiang et al Blood 2008a). Increase Wnt signaling by overexpression of Wnt3a in myeloma cells diminished MM-trigged bone lesion in mouse model (Qiang et al Blood 2008b). In the present study we revealed that bortezomib promotes MSC differentiation into osteoblast cells via Wnt-independent activation of beta-catenin/TCF signaling. E-cadherin pull-down assay and subsequently immunoblotting analysis demonstrated that bortezomib induced increases in both free and active forms of beta-catenin protein in cytoplasm and nuclear in bell-shaped dose- and time-dependent manner in mouse and human osteoblast progenitor cell lines including C2C12, C3H10T1/2, Saos-2 and MG63. Similar results were illustrated in primary human 2 cases of normal MSC and MSC from 8 cases of MM pateints. Bortezomib induced increase in ubiquitinated beta-catenin was evidenced by obvious seen slow migration bands of beta-catenin protein in SDS-PAGE gel analysis indicating that bortezomib increased beta-catenin protein by modification of proteasome-mediated degradation of beta-catenin. Increase in cytoplasm and nuclear beta-catenin protein response to bortezomib treatment in the osteoblast cell lines and 4 cases MM derived MSC was further confirmed by immunofluorescent analysis. RT-PCT analysis of TCF family revealed that abundant TCF1 and TCF4 mRNA were expressed in all tested cell lines and in a primary normal MSC, and MM-derived MSC. Bortezomib treatment also resulted in TCF transcriptional activity in bell-shaped, dose-dependent pattern as determined by luciferase activity in these cells transfected with TOPflash plasmid DNAs. Maximal responses to bortezomib were seen at 12.5 nM for both C2C12 (p<0.001) and MG63 (P<0.01), 25 nM for C3H10T1/2 (p<0.001) (p<0.00001) compared with non-stimulation control. These results suggest that transcriptional activation was a downstream effect of bortezomib in osteoblast progenitor cell lines and MM derived MSC. Bortezomib induced increases in beta-catenin protein and TCF transcriptional activity were independent of modification expression of extracellular 19 members of Wnt family ligands, 10 members of Frizzled receptor family, LRP5/6 co-receptors, and antagonists of 4 members of Dkk and sFRP family, respectively, as determined by RT-PCR analysis. Bortezomib did not increase intracellular Dvl-3 proteins, a downstream target of Wnt pathway. Lithium chloride, an inhibitor of GSK3beta did not synergized bortezomib induced increases in beta-catenin protein or TCF transcriptional activity indicating that bortezomib active beta-catenin/TCF signaling independent of activity of GSK3beta. Blocking the catenin/TCF signaling by expressing dominant–activation of beta- negative TFC attenuated bortezomib-induced matrix mineralization indicating that bortezomib induced MSC differentiation into osteoblast through activation of beta-catenin/TCF signaling. Data from experiments in comparison with biological effect of bortezomib with Wnt3a demonstrated that bortezomib did not have effect on OPG and RANKL in these cells, while Wnt3a induces OPG mRNA and protein, but inhibited RANKL expression indicating that bortezomib may have not effect on osteoclastogensis. These results provide insights into a clinically relevant mechanism of action of bortezomib and as such a rationale for its use in the treatment of diseases related to suppression of Wnt/beta-catenin/TCF signaling.
A structure-guided molecular chaperone approach for restoring the transcriptional activity of the p53 cancer mutant Y220C
