Effects Of Protein Kinase C On Osteoblast Differentiation Of Mouse Bone Marrow-derived Mesenchymal Stem Cells

BackgroundThe osteoblast differentiation is a strictly regulated process which requires the involvement of many signaling pathways. It is well known that PKC plays an important role in the regulation of cell metabolism, growth, proliferation, and differentiation. Some reports have demonstrated that PKC is implicated in osteoblast differentiation of various cells. However, it remains largely unknown whether PKC plays a role in the regulation of osteoblast differentiation of bone marrow-derived mesenchymal stem cells (BMSCs).ObjectivesIn this study, we aimed to explore the potential role of different PKC isoforms in the osteoblast differentiation of BMSCs, in order to provide new therapeutic targets for clinical diseases such as osteoporosis, bone tumors, and bone injuries.Methods1. Osteogenic differentiation induction of BMSCs:When BMSCs at P3 became 80-90%, cells were induced with osteoblast differentiation medium (DM) for different time courses. DM contains 100 nM dexamethasone,50 pM L-ascrobic acid, and 10 mM β- glycerophosphate.2. Western blotting was utilized to examine the activation of different PKC isoforms in the osteoblast differentiation of BMSCs. We also determined the minimal dose of PKC blocker, GF109203X (GFX) by Western blotting analysis.3. ARS staining, qPCR and ALP activity assay were used to examine the osteoblast differentiation of BMSCs. Meanwhile, we also determined which PKC isoforms are involved in the regulation of osteogenic differentiation of BMSCs.Results1. Western blotting analysis showed that PKCβⅡ, PKC8, PKCμ, and PKCζ/λ are significantly activated in osteoblast differentiation of BMSCs. In contrast, the activation of other PKC isoforms including PKCa and PKCθ are not observed.2. PKC inhibition assay demonstrated that the minimal dose of PKC inhibitor GFX is 3 μM.3 μM GFX can significantly block the activation of PKCβ Ⅱ.3. The results of ARS staining, qPCR and ALP activity assay showed that the osteoblast differentiation of BMSCs is significantly elevated when PKCβ Ⅱ activity is blocked by GFX.ConclusionBased on existing laboratory condition and our data, it comes to a conclusion that PKCβⅡ activation might negatively regulate osteoblast differentiation of BMSCs. In summary, our data may provide a potential target for clinical therapy of osteoporosis, bone tumor, and bone injuries, by specific blocking on PKCβⅡ. Whether other PKC isoforms can effect the osteoblast differentiation of BMSCs or not, further researches need to be carried out under proper conditions.