Osteogenic differentiation regulated by Rho-kinase in periodontal ligament cells

Tadashi Yamamoto, Yuki Ugawa, Keisuke Yamashiro, Masayuki Shimoe, Kazuya Tomikawa, Shoichi Hongo, Shinsuke Kochi, Hidetaka Ideguchi, Hiroshi Maeda, Shogo Takashiba

Research output: Contribution to journalArticle

11 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)33-41
Number of pages9
JournalDifferentiation
Volume88
Issue number2-3
DOIs
Publication statusPublished - Sep 1 2014

Fingerprint

rho-Associated Kinases
Periodontal Ligament
Actins
Gene Expression
Extracellular Matrix
Alkaline Phosphatase
Cell Differentiation
Tooth
Biglycan
Bite Force
Phenotype
Collagen Type III
Collagen Type I
Fibronectins
Connective Tissue
Fluorescent Antibody Technique
Real-Time Polymerase Chain Reaction
Up-Regulation
Stem Cells
Down-Regulation

Keywords

  • Actin cytoskeleton
  • Osteogenic differentiation
  • Periodontal ligament cells
  • Rho-associated coiled-coil containing protein kinase (ROCK)
  • Y-27632

ASJC Scopus subject areas

  • Developmental Biology
  • Cell Biology
  • Molecular Biology
  • Cancer Research
  • Medicine(all)

Cite this

Osteogenic differentiation regulated by Rho-kinase in periodontal ligament cells. / Yamamoto, Tadashi; Ugawa, Yuki; Yamashiro, Keisuke; Shimoe, Masayuki; Tomikawa, Kazuya; Hongo, Shoichi; Kochi, Shinsuke; Ideguchi, Hidetaka; Maeda, Hiroshi; Takashiba, Shogo.

In: Differentiation, Vol. 88, No. 2-3, 01.09.2014, p. 33-41.

Research output: Contribution to journalArticle

Yamamoto, T, Ugawa, Y, Yamashiro, K, Shimoe, M, Tomikawa, K, Hongo, S, Kochi, S, Ideguchi, H, Maeda, H & Takashiba, S 2014, 'Osteogenic differentiation regulated by Rho-kinase in periodontal ligament cells', Differentiation, vol. 88, no. 2-3, pp. 33-41. https://doi.org/10.1016/j.diff.2014.09.002
Yamamoto, Tadashi ; Ugawa, Yuki ; Yamashiro, Keisuke ; Shimoe, Masayuki ; Tomikawa, Kazuya ; Hongo, Shoichi ; Kochi, Shinsuke ; Ideguchi, Hidetaka ; Maeda, Hiroshi ; Takashiba, Shogo. / Osteogenic differentiation regulated by Rho-kinase in periodontal ligament cells. In: Differentiation. 2014 ; Vol. 88, No. 2-3. pp. 33-41.
@article{b53116b7570e4ed38d0a5264d941851a,
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.",
keywords = "Actin cytoskeleton, Osteogenic differentiation, Periodontal ligament cells, Rho-associated coiled-coil containing protein kinase (ROCK), Y-27632",
author = "Tadashi Yamamoto and Yuki Ugawa and Keisuke Yamashiro and Masayuki Shimoe and Kazuya Tomikawa and Shoichi Hongo and Shinsuke Kochi and Hidetaka Ideguchi and Hiroshi Maeda and Shogo Takashiba",
year = "2014",
month = "9",
day = "1",
doi = "10.1016/j.diff.2014.09.002",
language = "English",
volume = "88",
pages = "33--41",
journal = "Differentiation",
issn = "0301-4681",
publisher = "Elsevier BV",
number = "2-3",

}

TY - JOUR

T1 - Osteogenic differentiation regulated by Rho-kinase in periodontal ligament cells

AU - Yamamoto, Tadashi

AU - Ugawa, Yuki

AU - Yamashiro, Keisuke

AU - Shimoe, Masayuki

AU - Tomikawa, Kazuya

AU - Hongo, Shoichi

AU - Kochi, Shinsuke

AU - Ideguchi, Hidetaka

AU - Maeda, Hiroshi

AU - Takashiba, Shogo

PY - 2014/9/1

Y1 - 2014/9/1

N2 - 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.

AB - 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.

KW - Actin cytoskeleton

KW - Osteogenic differentiation

KW - Periodontal ligament cells

KW - Rho-associated coiled-coil containing protein kinase (ROCK)

KW - Y-27632

UR - http://www.scopus.com/inward/record.url?scp=84911369443&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84911369443&partnerID=8YFLogxK

U2 - 10.1016/j.diff.2014.09.002

DO - 10.1016/j.diff.2014.09.002

M3 - Article

C2 - 25278479

AN - SCOPUS:84911369443

VL - 88

SP - 33

EP - 41

JO - Differentiation

JF - Differentiation

SN - 0301-4681

IS - 2-3

ER -