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

  • Hyoin Kim , Dong Hyun Kim , Bohyeon Jeong , Ju-Hee Kim , Sun-Ryung Lee und Jong Kyung Sonn EMAIL logo
Veröffentlicht/Copyright: 30. Juni 2017
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Biologia
Aus der Zeitschrift Biologia Band 72 Heft 6

Abstract

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 blebbistatin-induced 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.

Acknowledgements

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2013R1A1A2009749).

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Abbreviations

BSA

bovine serum albumin

DAPI

4’,6-diamidino-2-phenylindole

DMSO

dimethyl sulfoxide

FBS

foetal bovine serum

mTOR

mammalian target of rapamycin

PI3K

phosphoinositide 3-kinase

PKD1

phosphoinositide-dependent protein kinase 1

p70S6K

p70 ribosomal protein S6 kinase

Received: 2017-5-12
Accepted: 2017-6-9
Published Online: 2017-6-30
Published in Print: 2017-6-27

©2017 Institute of Molecular Biology, Slovak Academy of Sciences

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