Phosphorylation-dependent Osterix Degradation Negatively Regulates Osteoblast Differentiation

<p>Mesenchymal stem cells (MSCs) are obtained from deciduous teeth noninvasively, attracting attention as a promising source for regenerative cell-based medicine. However, cellular heterogeneity of MSCs, such as differences in differentiation potential, is one of the main issues in its clinical applications. Therefore, establishing an efficient strategy inducing uniform cell differentiation is essential for efficient regenerative therapy. Since maintaining bone integrity is critical in preventing various oral diseases, we attempted to develop an effective approach of osteoblast differentiation from MSCs and preosteoblast cells. Previous studies demonstrated that proteasome inhibitors, a therapeutic agent for multiple myeloma, improve osteoclastic bone lesions and increase osteoblastic markers in patients with multiple myeloma. These studies prompted us to analyze proteasome-dependent regulation of osteoblast differentiation signals. To this end, we focused on Osterix (Osx/Sp7) protein, a transcription factor essential for osteoblast differentiation. We investigated the Osx protein degradation mechanism and its involvement in osteoblast differentiation using a pharmaceutical approach and obtained the following findings.</p><p>1. Simultaneous treatment of preosteoblasts and MSCs with low concentrations of bone morphogenetic protein (BMP) and proteasome inhibitors leads to synergistic enhancement of osteoblast differentiation and post-translational modification-dependent stabilization of Osx protein.</p><p>2. The Skp1-Cullin1-F-box protein (SCF)^Fbw7 complex serves as an E3 ligase for Osx protein degradation.</p><p>3. p38-mediated Osx phosphorylation facilitates the interaction between Osx and SCF^Fbw7.</p><p>4. p38 inhibitor-treated MSCs, Fbw7 knockdown MSCs, and Fbw7 knockout mouse-derived osteoblast progenitor cells show the accumulation of Osx protein and increased cell differentiation compared to control cells.</p><p>5. Introduction of p38 phosphorylation-deficient Osx mutant in Osx knockout cells significantly increases osteoblast differentiation compared to wild-type Osx.</p><p>These results suggest that regulating Osx protein stability through the Fbw7/p38 pathway may be critical in controlling bone metabolism by suppressing osteoblast differentiation.</p>