title: Rho-kinase regulates extracellular matrix-mediated osteogenic differentiation of periodontal ligament cells.
abstract: The periodontal ligament (PDL) cells contain heterogeneous mesenchymal cell populations, which have the ability to differentiate into cells that produce adjacent mineralized tissues and abundant extracellular matrix (ECM). ECM is essential not only for the homeostasis of the periodontal tissue, but also for controlling the differentiation of the PDL cells. The process of differentiation involves mechanotransduction, which links the ECM to the cytoskeleton. The present study investigated the roles of Rho-associated coiled-coil containing protein kinase (ROCK) signaling, a crucial regulator of the cytoskeleton, during ECM-mediated osteogenic differentiation of PDL cells in vitro. The PDL cells were isolated from human periodontal ligaments of extracted teeth and cultured in osteogenic medium with or without Y-27632, a pharmacological inhibitor of ROCK. ECM-coated plates were used for ECM-mediated differentiation. The osteogenic phenotype was evaluated at different time points by real-time RT-PCR for the gene encoding alkaline phosphatase (ALP) and an ALP activity assay. The effects of ROCK on cytoskeletal changes and ECM synthesis were examined by immunofluorescence analysis. Y-27632 significantly inhibited ALP at the mRNA and protein activity levels in the late stage of differentiation; concomitantly, the actin filament content and the extracellular levels of collagen-I and fibronectin were markedly decreased by Y-27632. Exogenous collagen-I and fibronectin temporally increased ALP activity, with fibronectin showing a more pronounced effect. Importantly, ECM-mediated differentiation was almost completely inhibited by Y-27632. These findings indicated that ECM-mediated differentiation is dependent on ROCK signaling, and ROCK signaling contributes to the establishment of the ECM microenvironment for PDL cell differentiation.
authors.affiliation: Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ugawa
authors.affiliation: Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan.
authors.forename: Mari
authors.initials: M
authors.lastname: Kawamura
authors.affiliation: Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Yamashiro
authors.affiliation: Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Shimoe
authors.affiliation: Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Tomikawa
authors.affiliation: Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan.
authors.forename: Shoichi
authors.initials: S
authors.lastname: Hongo
authors.affiliation: Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Maeda
authors.affiliation: Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8525, Japan.
authors.forename: Shogo
authors.initials: S
authors.lastname: Takashiba
mesh_terms: D000577:Amides; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D040542:Mechanotransduction, Cellular; D059630:Mesenchymal Stem Cells / Q000166:cytology; D010012:Osteogenesis / Q000502:physiology; D010513:Periodontal Ligament / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D061251:Primary Cell Culture / Q000379:methods; D011725:Pyridines; D054460:rho-Associated Kinases / Q000378:metabolism* / Q000502:physiology*
chemical_list: D000577:Amides; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D011725:Pyridines; C108830:Y 27632; D054460:rho-Associated Kinases
keywords: Rho signaling; collagen; differentiation; fibronectin; periodontal ligament cells