The early autophagic pathway contributes to osteogenic differentiation human periodontal ligament stem cells

Madoka Yasunaga, Hiroshi Kajiya, Takuya Toshimitsu, Hiroki Nakashima, Sachio Tamaoki, Hiroyuki Ishikawa, Hidefumi Maeda, Jun Ohno

Research output: Contribution to journalArticle

Abstract

This study aimed to investigate the role of autophagic stages through the interplay of AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Beclin-1 signaling pathways in osteogenic differentiation of human periodontal ligament stem cells (HPLSCs) using a combination of pharmacological inhibition and genetic knockdown approaches. The activation or inactivation of these factors and autophagy was evaluated by western blot and immunocyto-chemical assays, while osteogenic differentiation was analyzed by alkaline phosphatase staining. Enhancement of osteogenic differentiation of HPLSCs was correlated with the increased expression of the autophagy markers Beclin-1 and microtubule-associated protein 1 light chain 3-II (LC3-II), and phosphorylation of AMPK, consistent with reduced expression of phosphorylated p70S6 kinase, mTOR substrate. The RNA interference-mediated silencing of AMPK and Beclin-1 (marker of early autophagy) and the pharmacological inhibitors of AMPK (compound C) and early autophagy (3-methylad-enine) suppressed acceleration of osteogenic differentiation and autophagy. mTOR inhibition increased both osteogenic differentiation and autophagy. Inhibition of the late stage of autophagy by LC3 siRNA and chloroquine resulted in no changes in the osteogenic differentiation of HPLSCs. These findings suggest that the early stages of autophagy through the AMPK/ mTOR/Beclin-1 signaling pathway may be required for the enhancement of osteogenic differentiation of HPLSCs.

Original languageEnglish
Pages (from-to)63-70
Number of pages8
JournalJournal of Hard Tissue Biology
Volume28
Issue number1
DOIs
Publication statusPublished - Jan 1 2019

Fingerprint

Periodontal Ligament
AMP-Activated Protein Kinases
Ligaments
Autophagy
Stem cells
Sirolimus
Stem Cells
Proteins
Phosphorylation
Microtubule-Associated Proteins
Chloroquine
Phosphatases
Small Interfering RNA
Pharmacology
Alkaline Phosphatase
Assays
RNA
Phosphotransferases
Chemical activation
RNA Interference

All Science Journal Classification (ASJC) codes

  • Medicine (miscellaneous)
  • Biochemistry
  • Biomaterials
  • Orthopedics and Sports Medicine
  • Dentistry(all)
  • Cell Biology

Cite this

Yasunaga, M., Kajiya, H., Toshimitsu, T., Nakashima, H., Tamaoki, S., Ishikawa, H., ... Ohno, J. (2019). The early autophagic pathway contributes to osteogenic differentiation human periodontal ligament stem cells. Journal of Hard Tissue Biology, 28(1), 63-70. https://doi.org/10.2485/jhtb.28.63

The early autophagic pathway contributes to osteogenic differentiation human periodontal ligament stem cells. / Yasunaga, Madoka; Kajiya, Hiroshi; Toshimitsu, Takuya; Nakashima, Hiroki; Tamaoki, Sachio; Ishikawa, Hiroyuki; Maeda, Hidefumi; Ohno, Jun.

In: Journal of Hard Tissue Biology, Vol. 28, No. 1, 01.01.2019, p. 63-70.

Research output: Contribution to journalArticle

Yasunaga, M, Kajiya, H, Toshimitsu, T, Nakashima, H, Tamaoki, S, Ishikawa, H, Maeda, H & Ohno, J 2019, 'The early autophagic pathway contributes to osteogenic differentiation human periodontal ligament stem cells', Journal of Hard Tissue Biology, vol. 28, no. 1, pp. 63-70. https://doi.org/10.2485/jhtb.28.63
Yasunaga, Madoka ; Kajiya, Hiroshi ; Toshimitsu, Takuya ; Nakashima, Hiroki ; Tamaoki, Sachio ; Ishikawa, Hiroyuki ; Maeda, Hidefumi ; Ohno, Jun. / The early autophagic pathway contributes to osteogenic differentiation human periodontal ligament stem cells. In: Journal of Hard Tissue Biology. 2019 ; Vol. 28, No. 1. pp. 63-70.
@article{1d5d3f1fbbd24a7d9a821906fcfbb8ff,
title = "The early autophagic pathway contributes to osteogenic differentiation human periodontal ligament stem cells",
abstract = "This study aimed to investigate the role of autophagic stages through the interplay of AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Beclin-1 signaling pathways in osteogenic differentiation of human periodontal ligament stem cells (HPLSCs) using a combination of pharmacological inhibition and genetic knockdown approaches. The activation or inactivation of these factors and autophagy was evaluated by western blot and immunocyto-chemical assays, while osteogenic differentiation was analyzed by alkaline phosphatase staining. Enhancement of osteogenic differentiation of HPLSCs was correlated with the increased expression of the autophagy markers Beclin-1 and microtubule-associated protein 1 light chain 3-II (LC3-II), and phosphorylation of AMPK, consistent with reduced expression of phosphorylated p70S6 kinase, mTOR substrate. The RNA interference-mediated silencing of AMPK and Beclin-1 (marker of early autophagy) and the pharmacological inhibitors of AMPK (compound C) and early autophagy (3-methylad-enine) suppressed acceleration of osteogenic differentiation and autophagy. mTOR inhibition increased both osteogenic differentiation and autophagy. Inhibition of the late stage of autophagy by LC3 siRNA and chloroquine resulted in no changes in the osteogenic differentiation of HPLSCs. These findings suggest that the early stages of autophagy through the AMPK/ mTOR/Beclin-1 signaling pathway may be required for the enhancement of osteogenic differentiation of HPLSCs.",
author = "Madoka Yasunaga and Hiroshi Kajiya and Takuya Toshimitsu and Hiroki Nakashima and Sachio Tamaoki and Hiroyuki Ishikawa and Hidefumi Maeda and Jun Ohno",
year = "2019",
month = "1",
day = "1",
doi = "10.2485/jhtb.28.63",
language = "English",
volume = "28",
pages = "63--70",
journal = "Journal of Hard Tissue Biology",
issn = "1341-7649",
publisher = "Society of Hard Tissue Regenerative Biology",
number = "1",

}

TY - JOUR

T1 - The early autophagic pathway contributes to osteogenic differentiation human periodontal ligament stem cells

AU - Yasunaga, Madoka

AU - Kajiya, Hiroshi

AU - Toshimitsu, Takuya

AU - Nakashima, Hiroki

AU - Tamaoki, Sachio

AU - Ishikawa, Hiroyuki

AU - Maeda, Hidefumi

AU - Ohno, Jun

PY - 2019/1/1

Y1 - 2019/1/1

N2 - This study aimed to investigate the role of autophagic stages through the interplay of AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Beclin-1 signaling pathways in osteogenic differentiation of human periodontal ligament stem cells (HPLSCs) using a combination of pharmacological inhibition and genetic knockdown approaches. The activation or inactivation of these factors and autophagy was evaluated by western blot and immunocyto-chemical assays, while osteogenic differentiation was analyzed by alkaline phosphatase staining. Enhancement of osteogenic differentiation of HPLSCs was correlated with the increased expression of the autophagy markers Beclin-1 and microtubule-associated protein 1 light chain 3-II (LC3-II), and phosphorylation of AMPK, consistent with reduced expression of phosphorylated p70S6 kinase, mTOR substrate. The RNA interference-mediated silencing of AMPK and Beclin-1 (marker of early autophagy) and the pharmacological inhibitors of AMPK (compound C) and early autophagy (3-methylad-enine) suppressed acceleration of osteogenic differentiation and autophagy. mTOR inhibition increased both osteogenic differentiation and autophagy. Inhibition of the late stage of autophagy by LC3 siRNA and chloroquine resulted in no changes in the osteogenic differentiation of HPLSCs. These findings suggest that the early stages of autophagy through the AMPK/ mTOR/Beclin-1 signaling pathway may be required for the enhancement of osteogenic differentiation of HPLSCs.

AB - This study aimed to investigate the role of autophagic stages through the interplay of AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Beclin-1 signaling pathways in osteogenic differentiation of human periodontal ligament stem cells (HPLSCs) using a combination of pharmacological inhibition and genetic knockdown approaches. The activation or inactivation of these factors and autophagy was evaluated by western blot and immunocyto-chemical assays, while osteogenic differentiation was analyzed by alkaline phosphatase staining. Enhancement of osteogenic differentiation of HPLSCs was correlated with the increased expression of the autophagy markers Beclin-1 and microtubule-associated protein 1 light chain 3-II (LC3-II), and phosphorylation of AMPK, consistent with reduced expression of phosphorylated p70S6 kinase, mTOR substrate. The RNA interference-mediated silencing of AMPK and Beclin-1 (marker of early autophagy) and the pharmacological inhibitors of AMPK (compound C) and early autophagy (3-methylad-enine) suppressed acceleration of osteogenic differentiation and autophagy. mTOR inhibition increased both osteogenic differentiation and autophagy. Inhibition of the late stage of autophagy by LC3 siRNA and chloroquine resulted in no changes in the osteogenic differentiation of HPLSCs. These findings suggest that the early stages of autophagy through the AMPK/ mTOR/Beclin-1 signaling pathway may be required for the enhancement of osteogenic differentiation of HPLSCs.

UR - http://www.scopus.com/inward/record.url?scp=85061730294&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85061730294&partnerID=8YFLogxK

U2 - 10.2485/jhtb.28.63

DO - 10.2485/jhtb.28.63

M3 - Article

AN - SCOPUS:85061730294

VL - 28

SP - 63

EP - 70

JO - Journal of Hard Tissue Biology

JF - Journal of Hard Tissue Biology

SN - 1341-7649

IS - 1

ER -