The Early Autophagic Pathway Contributes to Osteogenic Differentiation of Human Periodontal Ligament Stem Cells

This study aimed to investigate the role of autophagic stages through the interplay of AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Beelin-I 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 immunocytochemical 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-methyladenine) 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.