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We investigated the role of AMP-activated protein kinase (AMPK), Akt, mammalian target of rapamycin (mTOR), autophagy and their interplay in osteogenic differentiation of human dental pulp mesenchymal stem cells. The activation of various members of AMPK, Akt and mTOR signaling pathways and autophagy was analyzed by immunoblotting, while osteogenic differentiation was assessed by alkaline phosphatase staining and real-time RT-PCR/immunoblot quantification of osteocalcin, Runt-related transcription factor 2 and bone morphogenetic protein 2 mRNA and/or protein levels. Osteogenic differentiation of mesenchymal stem cells was associated with early (day 1) activation of AMPK and its target Raptor, coinciding with the inhibition of mTOR and its substrate p70S6 kinase. The early induction of autophagy was demonstrated by accumulation of autophagosome-bound LC3-11, upregulation of proautophagic,beclin-1 and a decrease in the selective autophagic target p62. This was followed by the late activation of Akt/mTOR at days 3-7 of differentiation. The RNA interference-mediated silencing of AMPK, mTOR or autophagy-essential LC3 beta, as well as the pharmacological inhibitors of AMPK (compound C), Akt (10-DEBC hydrochloride), mTOR (rapamycin) and autophagy (bafilomycin A1, chloroquine and ammonium chloride), each suppressed mesenchymal stem cell differentiation to osteoblasts. AMPK knockdown prevented early mTOR inhibition and autophagy induction, as well as late activation of Akt/mTOR signaling, while Ala inhibition suppressed mTOR activation without affecting AMPK phosphorylation. Our data indicate that AMPK controls osteogenic differentiation of human mesenchymal stem cells through both early mTOR inhibition-mediated autophagy and late activation of Akt/mTOR signaling axis. (C) 2012 Elsevier Inc. All rights reserved. Ministry of Education and Science of the Republic of Serbia [41025, 173053, 175062]; UNESCO LOREAL National Scholarship Program For Women in Science [403F]