Coordinated time-dependent modulation of AMPK/Akt/mTOR signaling and autophagy controls osteogenic differentiation of human mesenchymal stem cells

• Published in: Bone (New York, N.Y.) Vol. 52; no. 1; pp. 524 - 531
• Main Authors: Pantovic, Aleksandar, Krstic, Aleksandra, Janjetovic, Kristina, Kocic, Jelena, Harhaji-Trajkovic, Ljubica, Bugarski, Diana, Trajkovic, Vladimir
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 01-01-2013; Elsevier
• Subjects: Adenylate Kinase - metabolism; Akt; AKT protein; Alkaline phosphatase; Ammonium chloride; AMP-activated protein kinase; AMPK; Autophagy; Biological and medical sciences; Cell Differentiation - physiology; Chloroquine; Dental pulp; Differentiation; Female; Fundamental and applied biological sciences. Psychology; Humans; Immunoblotting; Mesenchymal stem cells; Mesenchyme; mTOR; Orthopedics; Osteoblasts; Osteocalcin; Osteogenesis; Phagocytosis; Phosphorylation; Polymerase chain reaction; Rapamycin; RNA-mediated interference; Signal Transduction; Stem cells; Stem Cells - metabolism; TOR protein; TOR Serine-Threonine Kinases - metabolism; Transcription factors; Vertebrates: anatomy and physiology, studies on body, several organs or systems; hDP-MSC; Runt-related transcription factor 2; Akt; Osteoblasts; Autophagy; BMP2; mammalian target of rapamycin; microtubule-associated protein 1 light-chain 3; mTOR; AMPK; adenosine monophosphate-activated protein kinase; bone morphogenetic protein 2; LC3; human dental pulp mesenchymal stem cells; Runx2; Mesenchymal stem cells; Human; Stem cell; Morphology; Osteoblast; Mesenchymal cell
• ISSN: 8756-3282; 1873-2763
• DOI: 10.1016/j.bone.2012.10.024

Abstract 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-II, 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 LC3β, 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 Akt 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.