Mechanically activated fyn utilizes mTORC2 to regulate RhoA and adipogenesis in mesenchymal stem cells

Authors

  • William R. Thompson,

    Corresponding author
    1. Department of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA
    • Correspondence: William R. Thompson, D.P.T., Ph.D., 5030 Burnett-Womack Building, Chapel Hill, North Carolina 27599, USA. Telephone: 919-966-6743; Fax: 919-966-4468; e-mail: wthomp@med.unc.edu

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    • Author contributions: W.T.: concept/design, collection/assembly of data, data analysis/interpretation, manuscript writing, final approval of manuscript; C.G., S.Y., B.S., G.U., M.S., N.C., and K.B.: data analysis/interpretation, final approval of manuscript; Z.X. and K.B.: collection/assembly of data; J.R.: concept/design, financial support, data analysis/interpretation, manuscript writing, final approval of manuscript.

  • Christophe Guilluy,

    1. Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, North Carolina, USA
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  • Zhihui Xie,

    1. Department of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA
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  • Buer Sen,

    1. Department of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA
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  • Kaitlyn E. Brobst,

    1. Department of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA
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  • Sherwin S. Yen,

    1. Department of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA
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  • Gunes Uzer,

    1. Department of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA
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  • Maya Styner,

    1. Department of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA
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  • Natasha Case,

    1. Department of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA
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  • Keith Burridge,

    1. Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, North Carolina, USA
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  • Janet Rubin

    1. Department of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA
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Abstract

Mechanical strain provides an anti-adipogenic, pro-osteogenic stimulus to mesenchymal stem cells (MSC) through generating intracellular signals and via cytoskeletal restructuring. Recently, mTORC2 has been shown to be a novel mechanical target critical for the anti-adipogenic signal leading to preservation of β-catenin. As mechanical activation of mTORC2 requires focal adhesions (FAs), we asked whether proximal signaling involved Src and FAK, which are early responders to integrin-FA engagement. Application of mechanical strain to marrow-derived MSCs was unable to activate mTORC2 when Src family kinases were inhibited. Fyn, but not Src, was specifically required for mechanical activation of mTORC2 and was recruited to FAs after strain. Activation of mTORC2 was further diminished following FAK inhibition, and as FAK phosphorylation (Tyr-397) required Fyn activity, provided evidence of Fyn/FAK cooperativity. Inhibition of Fyn also prevented mechanical activation of RhoA as well as mechanically induced actin stress fiber formation. We thus asked whether RhoA activation by strain was dependent on mTORC2 downstream of Fyn. Inhibition of mTORC2 or its downstream substrate, Akt, both prevented mechanical RhoA activation, indicating that Fyn/FAK affects cytoskeletal structure via mTORC2. We then sought to ascertain whether this Fyn-initiated signal pathway modulated MSC lineage decisions. siRNA knockdown of Fyn, but not Src, led to rapid attainment of adipogenic phenotype with significant increases in adipocyte protein 2, peroxisome proliferator-activated receptor gamma, adiponectin, and perilipin. As such, Fyn expression in mdMSCs contributes to basal cytoskeletal architecture and, when associated with FAs, functions as a proximal mechanical effector for environmental signals that influence MSC lineage allocation. Stem Cells 2013;31:2528–2537

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