Each experiment was repeated at least three times unless stated otherwise. The statistical significance of the differences between treatments was assessed using t-test and a p value of less than 0.05 was considered significant. We first examined the patterns of AMPK, Akt, mTOR and autophagy activation during 7-day differentiation of hDP-MSC. Osteoblastic differentiation of hDP-MSC was confirmed by a significant increase in alkaline phosphatase activity and the mRNA and/or protein levels of osteogenesis markers osteocalcin, Runx2 and BMP2 (Figs. 1A, B). This was associated with rapid phosphorylation of AMPK and its direct downstream target Raptor, which peaked
at day 1 and then gradually declined (Figs. 1C, D). An inverse activation pattern was observed with mTOR and its substrate S6K, demonstrating an early inhibition at day 1 followed by activation PF-562271 chemical structure from
day CB-839 research buy 3 onwards (Figs. 1C, D). The increase in Akt phosphorylation slightly lagged behind that of AMPK, reaching its maximum at day 3 and remaining high during the rest of the differentiation period (Figs. 1C, D). The conversion of LC3-I to autophagosome-associated LC3-II, as a marker of autophagy, was increased at day 1, but then rapidly declined at later stages of differentiation (Figs. 1C, E). The changes in LC3 conversion were correlated with the extent of autophagic proteolysis, which increased early and declined late during differentiation, as reflected in the reduction and increase, respectively, of the intracellular levels of p62 (Figs. 1C, E), a selective autophagy target . In accordance with the early induction of autophagy, the intracellular concentration of the proautophagic protein beclin-1 reached its maximum 24 h after initiation
of differentiation (Figs. 1C, E). These data demonstrate a complex, time-dependent modulation of AMPK/Akt/mTOR signaling and autophagy during osteogenic differentiation of hDP-MSC, involving early activation of AMPK and transient induction of autophagy, followed by the late activation of Akt and Phosphoglycerate kinase mTOR. We next investigated the role of an early induction of AMPK and autophagy in osteogenic differentiation of hDP-MSC. Autophagy inhibitors bafilomycin, chloroquine and NH4Cl, which prevent autophagolysosome acidification and/or autophagosome–lysosome fusion  and , all blocked osteogenic differentiation of hDP-MSC, as confirmed by the reduction in alkaline phosphatase activity and expression of osteocalcin and Runx2 (Fig. 2A). Accordingly, the shRNA-mediated knockdown of the autophagy-essential LC3β blocked the increase of osteoblast differentiation markers in hDP-MSC (Figs. 2B, C). The efficiency of LC3β shRNA silencing was confirmed by reduced levels of both LC3-I and LC3-II in differentiating hDP-MSC at day 1 (Fig. 2D).