title: Mechanically activated Fyn utilizes mTORC2 to regulate RhoA and adipogenesis in mesenchymal stem cells.
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.
authors.affiliation: Department of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA.
authors.forename: William R
authors.initials: WR
authors.lastname: Thompson
authors.forename: Christophe
authors.initials: C
authors.lastname: Guilluy
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Xie
authors.forename: Buer
authors.initials: B
authors.lastname: Sen
authors.forename: Kaitlyn E
authors.initials: KE
authors.lastname: Brobst
authors.forename: Sherwin S
authors.initials: SS
authors.lastname: Yen
authors.forename: Gunes
authors.initials: G
authors.lastname: Uzer
authors.forename: Maya
authors.initials: M
authors.lastname: Styner
authors.forename: Natasha
authors.initials: N
authors.lastname: Case
authors.forename: Keith
authors.initials: K
authors.lastname: Burridge
authors.forename: Janet
authors.initials: J
authors.lastname: Rubin
mesh_terms: D050156:Adipogenesis / Q000502:physiology*; D018929:Cell Culture Techniques; D006801:Humans; D061986:MCF-7 Cells; D000076225:Mechanistic Target of Rapamycin Complex 2; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051076:Proto-Oncogene Proteins c-fyn / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D046912:Multiprotein Complexes; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D000076225:Mechanistic Target of Rapamycin Complex 2; D058570:TOR Serine-Threonine Kinases; D020742:rhoA GTP-Binding Protein
keywords: Adipogenesis; FAK; Fyn; Mesenchymal stem cells; RhoA; Src