Critical role for the TGF-β1/mTORC1 signalling axis in defining the transcriptional identity of CTHRC1+ pathologic fibroblasts

Abstract

Fibrosis, defined as the abnormal deposition of extracellular matrix (ECM), represents the concluding pathological outcome in a number of inflammatory, immune-mediated and metabolic diseases. Recent single cell RNA-sequencing studies have highlighted the diversity and functional heterogeneity of fibroblast populations in multiple fibrotic conditions. These include a novel pathogenic population of high collagen-producing fibroblasts, characterised by expression of the secreted glycoprotein, collagen triple helix repeat containing 1 (CTHRC1). The cardinal pro-fibrotic mediator TGF-{beta}1 has been widely implicated in promoting fibrogenesis and is a potent inducer of CTHRC1 and COL1A1 expression. In addition to the canonical Smad signalling pathway, TGF-{beta}1-induced collagen I production is under critical regulatory control by the mTORC1/4E-BP1 signalling hub. Using pharmacological inhibition in combination with gene-editing approaches, we now demonstrate that the role of the mTORC1 axis extends to the regulation of over a third of all TGF-{beta}1 regulated matrisome genes. We provide further evidence that the global transcriptome of TGF-{beta}1-stimulated fibroblasts in vitro matches that of a subpopulation of the high collagen expressing CTHRC1+ pathological fibroblast population in the IPF lung. In contrast, the TGF-{beta}1 induced transcriptome of fibroblasts in which mTORC1 signalling is disrupted (by RPTOR gene editing using CRISPR-Cas9) does not map to any fibroblast population present in human control or fibrotic lung. Using the novel and selective mTORC1 inhibitor RMC-5552, we further demonstrate a direct functional link between mTORC1 signalling and the acquisition of key marker genes which define the CTHRC1+ fibroblast population in IPF. These data demonstrate, for the first time, a critical role for the mTORC1 signalling hub in determining the transcriptional identity of the CTHRC1+ pathological fibroblast population and provide strong scientific support for targeting mTORC1 as a therapeutic strategy in IPF and potentially other fibrotic conditions associated with dysregulated TGF-{beta}1 signalling in the fibrotic niche.