Rho‐kinase regulates extracellular matrix‐mediated osteogenic differentiation of periodontal ligament cells

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.

[1]  S. Takashiba,et al.  Osteogenic differentiation regulated by Rho-kinase in periodontal ligament cells. , 2014, Differentiation; research in biological diversity.

[2]  Mikaël M. Martino,et al.  Growth Factors Engineered for Super-Affinity to the Extracellular Matrix Enhance Tissue Healing , 2014, Science.

[3]  G. Romanos,et al.  PERIOSTIN: Role in Formation and Maintenance of Dental Tissues , 2013, Journal of cellular physiology.

[4]  V. Vogel,et al.  Mesenchymal Stem Cells Exploit Extracellular Matrix as Mechanotransducer , 2013, Scientific Reports.

[5]  D. Rhodes,et al.  Superconductivity with extremely large upper critical fields in Nb$_{2}$Pd$_{0.81}$S$_{5}$ , 2013 .

[6]  M. Wendel,et al.  Bone Repair Using Periodontal Ligament Progenitor Cell-seeded Constructs , 2012, Journal of dental research.

[7]  Brenton D. Hoffman,et al.  Dynamic molecular processes mediate cellular mechanotransduction , 2011, Nature.

[8]  J. Schwarzbauer,et al.  Fibronectins, their fibrillogenesis, and in vivo functions. , 2011, Cold Spring Harbor perspectives in biology.

[9]  M. Somerman.  Growth Factors and Periodontal Engineering , 2011, Journal of dental research.

[10]  Matthias P. Lutolf,et al.  Designing materials to direct stem-cell fate , 2009, Nature.

[11]  Richard O. Hynes,et al.  The Extracellular Matrix: Not Just Pretty Fibrils , 2009, Science.

[12]  David J. Mooney,et al.  Growth Factors, Matrices, and Forces Combine and Control Stem Cells , 2009, Science.

[13]  J. Helms,et al.  Role of Wnt signaling in the biology of the periodontium , 2009, Developmental dynamics : an official publication of the American Association of Anatomists.

[14]  Shelly R. Peyton,et al.  ECM Compliance Regulates Osteogenesis by Influencing MAPK Signaling Downstream of RhoA and ROCK , 2009, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[15]  R. Assoian,et al.  Growth control by intracellular tension and extracellular stiffness. , 2008, Trends in cell biology.

[16]  S. Sen,et al.  Matrix Elasticity Directs Stem Cell Lineage Specification , 2006, Cell.

[17]  G. Adler,et al.  Collagen type I induces disruption of E-cadherin-mediated cell-cell contacts and promotes proliferation of pancreatic carcinoma cells. , 2006, Cancer research.

[18]  J. Hubbell,et al.  Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering , 2005, Nature Biotechnology.

[19]  Stan Gronthos,et al.  Investigation of multipotent postnatal stem cells from human periodontal ligament , 2004, The Lancet.

[20]  C. Damsky,et al.  FAK promotes organization of fibronectin matrix and fibrillar adhesions , 2004, Journal of Cell Science.

[21]  I. Ishikawa,et al.  Novel isolation of alkaline phosphatase-positive subpopulation from periodontal ligament fibroblasts. , 2003, Journal of periodontology.

[22]  J. Aubin,et al.  Global amplification polymerase chain reaction reveals novel transitional stages during osteoprogenitor differentiation , 2003, Journal of Cell Science.

[23]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[24]  M. McKee,et al.  Role of physical forces in regulating the form and function of the periodontal ligament. , 2000, Periodontology 2000.

[25]  W. Kiosses,et al.  Regulation of the small GTP‐binding protein Rho by cell adhesion and the cytoskeleton , 1999, The EMBO journal.

[26]  Shuh Narumiya,et al.  Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension , 1997, Nature.

[27]  G. Stein,et al.  Progressive development of the rat osteoblast phenotype in vitro: Reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix , 1990, Journal of cellular physiology.

[28]  G. Hajishengallis.  Immunomicrobial pathogenesis of periodontitis: keystones, pathobionts, and host response. , 2014, Trends in immunology.

[29]  L. Kiemeney,et al.  Analysis of Relative Gene Expression Data Using Real-time Quantita- Tive Pcr and the 2 Preanalytic Error Tracking in a Laboratory Medicine Department: Results of a 1-year Experience , 2006 .

[30]  R. Franceschi,et al.  The developmental control of osteoblast-specific gene expression: role of specific transcription factors and the extracellular matrix environment. , 1999, Critical reviews in oral biology and medicine : an official publication of the American Association of Oral Biologists.