Regulation of MC3T3-E1 differentiation by actin cytoskeleton through lipid mediators reflecting the cell differentiation stage

Abstract Actin cytoskeleton is reported to be related in various functions of osteoblast, bone-forming cell. However the function of actin cytoskeleton in osteoblasts is not fully understood, since bone formation is derived from extracellular interactions of functional proteins produced from osteoblasts, including osteocalcin (Ocn), and it is a result of closely and complex organized sequence of biochemical events. In this study, we showed that actin cytoskeleton of MC3T3-E1 cells functioned in recognition of cell condition and regulation of extracellular matrix mineralization, bone formation. Maturation of MC3T3-E1 cells by 14 days of culture reduced F-actin filaments, while induced expression of Ocn mRNA known as late stage differentiation marker and matrix mineralization, terminal stage of cell differentiation. The disruption of actin cytoskeleton with Cyto D in immature MC3T3-E1 cells significantly increased expression of Ocn mRNA in 24 h. Both PTX-induced inhibition of signal transduction through GPCRs and celecoxib-induced suppression of lipid mediators in immature MC3T3-E1 cells reduced actin filaments and suppressed matrix mineralization. Furthermore, addition of lipid mediators extracted from culture mediums of differentiated MC3T3-E1 cells by Bligh-Dyer method induced actin cytoskeleton reorganization and matrix mineralization change in MC3T3-E1 cells. Taken together, our data suggest that actin cytoskeleton of MC3T3-E1 cells regulates activation of developmental pathway reflecting cell differentiation stages through lipid mediators. The function we identified is important for bone formation tightly regulated by mechanical stress, since actin cytoskeleton is also known as a mechanosensor of osteoblasts.