title: The Novel mTOR Complex 1/2 Inhibitor P529 Inhibits Human Lung Myofibroblast Differentiation.
abstract: Idiopathic pulmonary fibrosis is a progressive and deadly disorder with very few therapeutic options. Palomid 529 (8-(1-hydroxyethyl)-2-methoxy-3-(4-methoxybenzyloxy)-benzo[c]chromen-6-one; P529) is a novel dual inhibitor of mechanistic target of rapamycin complex 1/2 (mTORC1/2). In these studies, we investigated the effect of P529 on TGF-β-dependent signaling and myofibroblast differentiation. TGF-β-induced phosphorylation of the mTORC1 targets, p70 S6 kinase 1 (S6K1), and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), were both dose dependently inhibited by P529 in human lung fibroblasts with maximal inhibition occurring between 10 and 20 μM. mTORC2-mediated phosphorylation of Akt at the S473 site was partially inhibited with a similar dose dependency, as was TGF-β-induced myofibroblast differentiation. Protein levels of TGF-β-induced fibronectin and collagen were similarly decreased by P529. At this dose, there was also inhibition of mRNA transcript levels for Col1 and α-SMA, suggesting inhibition of transcriptional activation. However, there was no effect of P529 on canonical TGF-β-induced Smad signaling, as assessed by receptor-associated Smad2/3 phosphorylation, Smad2/3/4 translocation, or Smad-driven gene expression, as assessed by Smad-binding element driven luciferase. Conversely, activation of mTORC1/2 signaling was dependent on TGF-β type I receptor (ALK5) signaling and on Smad2/3 expression. P529 treatment disrupted TGF-β-induced actin stress fiber formation during myofibroblast differentiation, the deposition of new extracellular fibronectin matrix, and linear wound closure by fibroblasts. Likewise, mTOR knockdown inhibited TGF-β-induced myofibroblast differentiation. In conclusion, P529 inhibits TGF-β-induced myofibroblast differentiation, actin stress fiber formation, and matrix protein expression and deposition. Inhibition of mTORC1/2 by P529 may be a promising approach to inhibit in vivo fibrosis. J. Cell. Biochem. 118: 2241-2249, 2017. © 2017 Wiley Periodicals, Inc.
authors.affiliation: Division of Allergy, Pulmonary, and Critical Care Medicine, University of Wisconsin-Madison School of Medicine and Public Health, 600 Highland Ave, Madison, Wisconsin, 53792.
authors.forename: Keith T
authors.initials: KT
authors.lastname: Ferguson
authors.affiliation: Division of Allergy, Pulmonary, and Critical Care Medicine, University of Wisconsin-Madison School of Medicine and Public Health, 600 Highland Ave, Madison, Wisconsin, 53792.
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Torr
authors.affiliation: Division of Allergy, Pulmonary, and Critical Care Medicine, University of Wisconsin-Madison School of Medicine and Public Health, 600 Highland Ave, Madison, Wisconsin, 53792.
authors.forename: Ksenija
authors.initials: K
authors.lastname: Bernau
authors.affiliation: Division of Allergy, Pulmonary, and Critical Care Medicine, University of Wisconsin-Madison School of Medicine and Public Health, 600 Highland Ave, Madison, Wisconsin, 53792.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Leet
authors.affiliation: GenAdam Therapeutics, Inc, 37 Neillian Crescent, Jamaica Plain, Massachusetts, 02130.
authors.forename: David
authors.initials: D
authors.lastname: Sherris
authors.affiliation: Division of Allergy, Pulmonary, and Critical Care Medicine, University of Wisconsin-Madison School of Medicine and Public Health, 600 Highland Ave, Madison, Wisconsin, 53792.
authors.forename: Nathan
authors.initials: N
authors.lastname: Sandbo
mesh_terms: D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D001578:Benzopyrans / Q000494:pharmacology*; D015153:Blotting, Western; D018797:Cell Cycle Proteins; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D000076222:Mechanistic Target of Rapamycin Complex 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000076225:Mechanistic Target of Rapamycin Complex 2 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D058628:Myofibroblasts / Q000166:cytology / Q000187:drug effects*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D055533:Survival of Motor Neuron 1 Protein / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D001578:Benzopyrans; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; D005353:Fibronectins; D010750:Phosphoproteins; D018125:Receptors, Transforming Growth Factor beta; D055533:Survival of Motor Neuron 1 Protein; D016212:Transforming Growth Factor beta; D003094:Collagen; D000076222:Mechanistic Target of Rapamycin Complex 1; D000076225:Mechanistic Target of Rapamycin Complex 2; D017346:Protein Serine-Threonine Kinases; D038762:Ribosomal Protein S6 Kinases, 70-kDa; C448901:ribosomal protein S6 kinase, 70kD, polypeptide 1; D000077293:Receptor, Transforming Growth Factor-beta Type I; C000625841:TGFBR1 protein, human; C533469:palomid 529
keywords: ACTIN CYTOSKELETON; FIBRONECTIN DEPOSITION; MYOFIBROBLAST; P529; PULMONARY FIBROSIS; Smad; TGF-β; mTOR; mTORC1; mTORC2