Ferguson Keith T, Torr Elizabeth E, Bernau Ksenija, Leet Jonathan, Sherris David, Sandbo Nathan
Division of Allergy, Pulmonary, and Critical Care Medicine, University of Wisconsin-Madison School of Medicine and Public Health, 600 Highland Ave, Madison, Wisconsin, 53792.
GenAdam Therapeutics, Inc, 37 Neillian Crescent, Jamaica Plain, Massachusetts, 02130.
J Cell Biochem. 2017 Aug;118(8):2241-2249. doi: 10.1002/jcb.25878. Epub 2017 Apr 18.
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.
特发性肺纤维化是一种进展性致命疾病，治疗选择非常有限。帕罗米德529（8-(1-羟乙基)-2-甲氧基-3-(4-甲氧基苄氧基)-苯并[c]色烯-6-酮；P529）是一种新型的雷帕霉素复合物1/2（mTORC1/2）的双重抑制剂。在这些研究中，我们研究了P529对转化生长因子-β（TGF-β）依赖性信号传导和成肌纤维细胞分化的影响。在人肺成纤维细胞中，P529剂量依赖性地抑制TGF-β诱导的mTORC1靶点p70核糖体蛋白S6激酶1（S6K1）和真核翻译起始因子4E结合蛋白1（4E-BP1）的磷酸化，最大抑制作用发生在10至20μM之间。mTORC2介导的Akt在S473位点的磷酸化以及TGF-β诱导的成肌纤维细胞分化也受到类似剂量依赖性的部分抑制。P529同样降低了TGF-β诱导的纤连蛋白和胶原蛋白的蛋白水平。在此剂量下，Col1和α-SMA的mRNA转录水平也受到抑制，提示转录激活受到抑制。然而，通过受体相关的Smad2/3磷酸化、Smad2/3/4易位或Smad结合元件驱动的荧光素酶评估的Smad驱动的基因表达来评估，P529对经典的TGF-β诱导的Smad信号传导没有影响。相反，mTORC1/2信号传导的激活依赖于TGF-βI型受体（ALK5）信号传导和Smad2/3表达。P529处理破坏了TGF-β诱导的成肌纤维细胞分化过程中的肌动蛋白应力纤维形成、新的细胞外纤连蛋白基质的沉积以及成纤维细胞的线性伤口闭合。同样，mTOR敲低抑制了TGF-β诱导的成肌纤维细胞分化。总之，P529抑制TGF-β诱导的成肌纤维细胞分化、肌动蛋白应力纤维形成以及基质蛋白的表达和沉积。P529对mTORC1/2的抑制可能是抑制体内纤维化的一种有前景的方法。《细胞生物化学杂志》118: 2241 - 2249, 2017。© 2017威利期刊公司
