Extracellular matrix mineralization in the mouse osteoblast-like cell line MC3T3-E1 is regulated by actin cytoskeleton reorganization and non-protein molecules secreted from the cells themselves

Abstract

Bone tissue constantly undergoes turnover via bone formation by osteoblasts and bone resorption by osteoclasts. This process enables bone to maintain its overall shape while altering its local structure. However, the detailed mechanism of how osteoblast cell-signaling systems induce various structural changes in bone tissue have not yet been completely elucidated. In this study, we focused on the actin cytoskeleton as a regulatory system for bone formation and constructed an in vitro experimental system using the mouse osteoblast-like cell line MC3T3-E1. We found that, in MC3T3-E1 cells, the actin cytoskeleton had an important role in matrix mineralization via activation of specific developmental pathways and it was regulated by non-protein molecules secreted from MC3T3-E1 cells themselves. In MC3T3-E1 cells, we observed changes of actin cytoskeleton reorganization and accumulation of PIP2 related to actin filament convergences during cell differentiation, in the undifferentiated, early, middle and late stage. Actin cytoskeleton disruption with Cyto D, polymerization inhibitor of actin filament, in early and middle stage cells induced significant increase of osteocalcin mRNA expression normally expressed only in late stage, decrease of Alkaline phosphatase mRNA expression after 24h and abnormal matrix mineralization in MC3T3-E1 cells. Inhibition of Gi with PTX known to regulate actin cytoskeleton in middle stage induced changes in the actin cytoskeleton and PIP2 accumulation and suppression of matrix mineralization after 5 days. Furthermore, addition of non-protein molecules from culture medium of cells at various differentiation stage induced difference of PIP2 accumulation after 5 min, actin cytoskeleton in 20 min, and matrix mineralization after 5 days. These results not only provide new knowledge about the actin cytoskeleton function in bone-forming cells, but also suggest that cell signaling via non-protein molecules such as lipids plays important roles in bone formation.