Abstract
A characteristic of dysregulated wound healing in IPF is fibroblastic-mediated damage to lung epithelial cells within fibroblastic foci. In these foci, TGF-β and other growth factors activate fibroblasts that secrete growth factors and matrix regulatory proteins, which activate a fibrotic cascade. Our studies and those of others have revealed that Akt is activated in IPF fibroblasts and it mediates the activation by TGF-β of pro-fibrotic pathways. Recent studies show that mTORC2, a component of the mTOR pathway, mediates the activation of Akt. In this study we set out to determine if blocking mTORC2 with MLN0128, an active site dual mTOR inhibitor, which blocks both mTORC1 and mTORC2, inhibits lung fibrosis. We examined the effect of MLN0128 on TGF-β-mediated induction of stromal proteins in IPF lung fibroblasts; also, we looked at its effect on TGF-β-mediated epithelial injury using a Transwell co-culture system. Additionally, we assessed MLN0128 in the murine bleomycin lung model. We found that TGF-β induces the Rictor component of mTORC2 in IPF lung fibroblasts, which led to Akt activation, and that MLN0128 exhibited potent anti-fibrotic activity in vitro and in vivo. Also, we observed that Rictor induction is Akt-mediated. MLN0128 displays multiple anti-fibrotic and lung epithelial-protective activities; it (1) inhibited the expression of pro-fibrotic matrix-regulatory proteins in TGF-β-stimulated IPF fibroblasts; (2) inhibited fibrosis in a murine bleomycin lung model; and (3) protected lung epithelial cells from injury caused by TGF-β-stimulated IPF fibroblasts. Our findings support a role for mTORC2 in the pathogenesis of lung fibrosis and for the potential of active site mTOR inhibitors in the treatment of IPF and other fibrotic lung diseases.
Highlights
Idiopathic Pulmonary Fibrosis (IPF) is a devastating disease, which afflicts over 200,000 patients in the United States and Europe [1]
We showed previously that secreted protein acidic and rich in cysteine (SPARC) produced by IPF fibroblasts activates Akt by phosphorylation of serine 473 (Ser473) leading to inhibition of glycogen synthase kinase 3b (GSK-3b), which resulted in activation of the b-catenin pathway and inhibition of PLOS ONE | www.plosone.org mTOR complex 2 (mTORC2) in Lung Fibrosis apoptosis [12]
Akt is activated by TGF-b and has recently been shown to be a target of mTORC2, so we first examined if TGF-b activates mTORC2 in IPF lung fibroblasts
Summary
Idiopathic Pulmonary Fibrosis (IPF) is a devastating disease, which afflicts over 200,000 patients in the United States and Europe [1]. The pathogenesis is unknown but a dysregulated wound healing response to lung epithelial injury, which leads to progressive interstitial fibrosis, is a hallmark of the disease. Activated fibroblasts in fibroblastic foci secrete a variety of profibrotic proteins in response to TGF-b, such as type I and type III collagen, fibronectin (FN), and the matricellular family members, secreted protein acidic and rich in cysteine (SPARC) and connected tissue growth factor (CTGF) [2]. MTOR integrates both extracellular and intracellular signals and acts as a central regulator of cell metabolism, growth, proliferation and survival [4]. The mTORC1 complex consists of at least five components: (i) mTOR, the catalytic subunit of the complex; (ii) Raptor; (iii) mLS8; (iv) PRAS40; and (v) Deptor; mTORC1 phosphorylates the ribosomal S6K1 (protein S6 kinase 1) and 4E-
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