Blebbistatin induces chondrogenesis of single mesenchymal cells via PI3K/PDK1/mTOR/p70S6K pathway

Rearrangement of the actin cytoskeleton plays an inductive role in chondrogenic differentiation. Our previous study showed that blebbistatin, an inhibitor of myosin II, removes actin stress fibres and induces chondrogenesis of mesenchymal cells in monolayer cultures. In the present study, we investigated signalling pathways implicated in the induction of chondrogenesis by dissolving actin stress fibres after blebbistatin treatment. Blebbistatin increased the activity of phosphoinositide 3-kinase (PI3K). Inhibition of PI3K with LY294002 blocked blebbistatin-induced chondrogenesis without affecting blebbistatin-induced reorganization of actin filaments. Blebbistatin also upregulated the phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK1), and inhibition of PDK1 with GSK2334470 suppressed blebbistatininduced chondrogenesis, indicating that removal of actin stress fibres by blebbistatin induced chondrogenesis by activating PI3K/PDK1. Although inhibition of Akt activity by Akt inhibitor IV blocked blebbistatin-induced chondrogenesis, phosphorylation of Akt was not affected by blebbistatin. Blebbistatin increased the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and p70 ribosomal protein S6 kinase (p70S6K). Inhibition of mTOR with rapamycin almost completely abolished the phosphorylation of p70S6K. Inhibition of mTOR complex 1 (mTORC1) and complex 2 (mTORC2) with pp242 diminished phosphorylation of Akt at Ser473, whereas inhibition of mTORC1 with rapamycin did not. However, blebbistatin did not affect the phosphorylation of mTOR at Ser2481. Taken together, the present results suggest that blebbistatin induces chondrogenesis by activating the PI3K/PDK1/mTOR/p70S6K pathway. Our data also indicate that Akt activity is essential for chondrogenesis but is regulated by mTORC2, which is independent of blebbistatin treatment.