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
N1 - Funding Information:
We would like to thank our colleagues at Okayama University, Drs. Wataru Sonoyama and Junji Mineshiba, for their helpful suggestions, and Drs. Ayaka Goto, Chiaki Yoshihara, and Mari Kawamura for excellent technical support. This work was supported by a Grant-in-Aid for Scientific Research (C) (20592429) from the JSPS and partially supported by 2010 Research Award from the Ryobi Teien Foundation. Appendix A
Publisher Copyright:
© 2014 International Society of Differentiation.
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
SN - 0301-4681
VL - 88
SP - 33
EP - 41
JO - Differentiation
JF - Differentiation
IS - 2-3
ER -
