Abstract
Fibrotic diseases display mesenchymal cell (MC) activation with pathologic deposition of matrix proteins such as collagen. Here we investigate the role of mTOR complex 1 (mTORC1) and mTORC2 in regulating MC collagen expression, a hallmark of fibrotic disease. Relative to normal MCs (non-Fib MCs), MCs derived from fibrotic human lung allografts (Fib-MCs) demonstrated increased phosphoinositide-3kinase (PI3K) dependent activation of both mTORC1 and mTORC2, as measured by increased phosphorylation of S6K1 and 4E-BP1 (mTORC1 substrates) and AKT (an mTORC2 substrate). Dual ATP-competitive TORC1/2 inhibitor AZD8055, in contrast to allosteric mTORC1-specific inhibitor rapamycin, strongly inhibited 4E-BP1 phosphorylation and collagen I expression in Fib-MCs. In non-Fib MCs, increased mTORC1 signaling was shown to augment collagen I expression. mTORC1/4E-BP1 pathway was identified as an important driver of collagen I expression in Fib-MCs in experiments utilizing raptor gene silencing and overexpression of dominant-inhibitory 4E-BP1. Furthermore, siRNA-mediated knockdown of rictor, an mTORC2 partner protein, reduced mTORC1 substrate phosphorylation and collagen expression in Fib-, but not non-Fib MCs, revealing a dependence of mTORC1 signaling on mTORC2 function in activated MCs. Together these studies suggest a novel paradigm where fibrotic activation in MCs increases PI3K dependent mTORC1 and mTORC2 signaling and leads to increased collagen I expression via the mTORC1-dependent 4E-BP1/eIF4E pathway. These data provide rationale for targeting specific components of mTORC pathways in fibrotic states and underscore the need to further delineate mTORC2 signaling in activated cell states.
Highlights
Organ failure and death both before and after transplantation
Mesenchymal Cells Derived from Fibrotic Lung Allografts Demonstrate Mammalian target of rapamycin (mTOR) complex 1 (mTORC1)/mTORC2 Pathway Activation—Mesenchymal phenotype of cells isolated from lung allografts was confirmed by immunofluorescence staining for vimentin (Fig. 1A) and by realtime PCR demonstrating presence of N-cadherin and absence of E-cadherin (Fig. 1B)
Baseline protein expression in MCs derived from fibrotic lung allografts (FibMCs) were compared with mesenchymal cells derived from normal lung allografts
Summary
Scraped cell lysates which contains both intracellular and extracellular proteins were utilized for quantitating collagen I expression using an Anti-Human Collagen Type I, purified IgG (Polyclonal) (rabbit IgG) antibody (Cedarlane Laboratories, Ontario, Canada). This Collagen I antibody (CL50111AP) detects bands between molecular mass of 150 and 250 kDA, which correspond to detected epitopes located in the procollagen and collagen portions and the predominant band we detect at 200 kDA corresponds to collagen beta 1,1 and beta 1,2. Significance was assessed with a Student t test for comparisons of two groups, or with analysis of variance and a post hoc Neuman-Keuls test for three or more groups. p less than 0.05 were considered significant
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