Osteogenic differentiation regulated by Rho kinase in periodontal ligament cells

Periodontal ligament cells contain heterogeneous cell populations including mesenchymal stem cells that can differentiate to osteogenic cells, and contribute to wound healing and regeneration of periodontal tissue. However precise mechanism of the differentiation process remains elusive. We have previously reported that Rho kinase (ROCK) effects gene expression by mechanical forces in periodontal ligament cells. ROCK regulates varieties of biological functions, such as cell contraction, migration and differentiation by mediating stress fiber that consists of filamentous actin (F-actin) and myosin light chain (MLC). Objectives: We aim to examine the effect of ROCK in periodontal ligament cells during osteogenic differentiation, and to examine the kinetics of cytoskeletal and osteo-inductive molecules. Methods: Fibroblastic cells were isolated from human periodontal ligament on extracted teeth by enzymatic dissociation method. Cell colony clusters derived from a single-cell suspension were subjected to the experiment. Calcium accumulation was induced in osteogenic differentiation medium for 14-days. Osteogenic phenotype was confirmed by analysis of alkaline phosphatase and osteocalcin production. Subcellular localization of F-actin and MLC were examined by immunofluorescence. ROCK inhibition assay using Y-27632, specific inhibitor of ROCK activity, was conducted, and cell viability was monitored by MTS assay. Results: F-actin and phosphorylated MLC were induced during osteogenic differentiation of periodontal ligament cells. Subsequent inhibition assay revealed that Y-27632 inhibited the production of F-actin and phosphorylated MLC in a dose dependent manner while cell viability remained same level. Moreover, Y-27632 inhibited alkaline phosphatase activity and mineralized nodule formation with decreased amount of osteocalcin production. Conclusions: These findings demonstrated crucial effects of ROCK in stress fiber formation and differentiation process toward osteogenic cells. ROCK would contribute to induce osteogenic differentiation through the alteration of cytoskeletal components in periodontal ligament cells. Supported by Grant-in-Aid for Scientific Research (C) (20592429) from the JSPS