title: Osteogenic differentiation regulated by Rho-kinase in periodontal ligament cells.
abstract: The periodontal ligament is a multifunctional soft connective tissue, which functions not only as a cushion supporting the teeth against occlusal force, but is also a source of osteogenic cells that can regenerate neighboring hard tissues. Periodontal ligament cells (PDL cells) contain heterogeneous cell populations, including osteogenic cell progenitors. However, the precise mechanism underlying the differentiation process remains elusive. Cell differentiation is regulated by the local biochemical and mechanical microenvironment that can modulate gene expression and cell morphology by altering actin cytoskeletal organization mediated by Rho-associated, coiled-coil containing protein kinase (ROCK). To determine its role in PDL cell differentiation, we examined the effects of ROCK on cytoskeletal changes and kinetics of gene expression during osteogenic differentiation. PDL cells were isolated from human periodontal ligament on extracted teeth and cultured in osteogenic medium for 14 days. Y-27632 was used for ROCK inhibition assay. Osteogenic phenotype was determined by monitoring alkaline phosphatase (ALP) activity and calcium deposition by Alizarin Red staining. ROCK-induced cytoskeletal changes were examined by immunofluorescence analysis of F-actin and myosin light chain 2 (MLC2) expression. Real-time PCR was performed to examine the kinetics of osteogenic gene expression. F-actin and phospho-MLC2 were markedly induced during osteogenic differentiation, which coincided with upregulation of ALP activity and mineralization. Subsequent inhibition assay indicated that Y-27632 significantly inhibited F-actin and phospho-MLC2 expression in a dose-dependent manner with concomitant partial reversal of the PDL cell osteogenic phenotype. PCR array analysis of osteogenic gene expression indicated that extracellular matrix genes, such as fibronectin 1, collagen type I and III, and biglycan, were significantly downregulated by Y27632. These findings indicated crucial effects of ROCK in cytoskeletal reorganization and differentiation of PDL cells toward osteogenic cells. ROCK contributes to induction of osteogenic differentiation by synergistic increases in extracellular matrix gene expression in PDL cells.
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: 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: 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: Shinsuke
authors.initials: S
authors.lastname: Kochi
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: Hidetaka
authors.initials: H
authors.lastname: Ideguchi
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. Electronic address: stakashi@okayama-u.ac.jp.
authors.forename: Shogo
authors.initials: S
authors.lastname: Takashiba
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D002478:Cells, Cultured; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018994:Myosin Light Chains / Q000235:genetics / Q000378:metabolism; D010012:Osteogenesis; D010513:Periodontal Ligament / Q000166:cytology* / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000577:Amides; D016326:Extracellular Matrix Proteins; D018994:Myosin Light Chains; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C108830:Y 27632; D054460:rho-Associated Kinases; D000469:Alkaline Phosphatase
keywords: Actin cytoskeleton; Osteogenic differentiation; Periodontal ligament cells; Rho-associated coiled-coil containing protein kinase (ROCK); Y-27632