mTOR signaling regulates multiple metabolic pathways in human lung fibroblasts after TGF-β and in pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis is a fatal disease characterized by the transforming growth factor (TGF-β)-dependent activation of lung fibroblasts, leading to excessive deposition of collagen proteins and progressive replacement of healthy lungs with scar tissue. We and others have shown that TGF-β-mediated activation of the mechanistic target of rapamycin complex 1 (mTORC1) and downstream upregulation of activating transcription factor 4 (ATF4) promotes metabolic reprogramming in lung fibroblasts characterized by upregulation of the de novo synthesis of glycine, the most abundant amino acid found in collagen protein. Whether mTOR and ATF4 regulate other metabolic pathways in lung fibroblasts has not been explored. Here, we used RNA sequencing to determine how both ATF4 and mTOR regulate gene expression in human lung fibroblasts following TGF-β. We found that ATF4 primarily regulates enzymes and transporters involved in amino acid homeostasis as well as aminoacyl-tRNA synthetases. mTOR inhibition resulted not only in the loss of ATF4 target gene expression but also in the reduced expression of glycolytic enzymes and mitochondrial electron transport chain subunits. Analysis of TGF-β-induced changes in cellular metabolite levels confirmed that ATF4 regulates amino acid homeostasis in lung fibroblasts, whereas mTOR also regulates glycolytic and TCA cycle metabolites. We further analyzed publicly available single-cell RNA-seq datasets and found increased expression of ATF4 and mTOR-regulated genes in pathologic fibroblast populations from the lungs of patients with IPF. Our results provide insight into the mechanisms of metabolic reprogramming in lung fibroblasts and highlight novel ATF4 and mTOR-dependent pathways that may be targeted to inhibit fibrotic processes.NEW & NOTEWORTHY Here, we used transcriptomic and metabolomic approaches to develop a more complete understanding of the role that mTOR, and its downstream effector ATF4, play in promoting metabolic reprogramming in lung fibroblasts. We identify novel metabolic pathways that may promote pathologic phenotypes, and we provide evidence from single-cell RNA-seq datasets that similar metabolic reprogramming occurs in patient lungs.

Keywords: ATF4; mTOR; metabolism; mitochondria; pulmonary fibrosis.

Figure 1.. TGF-β promotes metabolic reprogramming in human lung fibroblasts.

(A) Multidimensional scaling plot of differentially expressed genes (DEGs) in HLFs cultured in the presence or absence of TGF-β (1ng/mL). Cells were either transfected with a nontargeting siRNA or siRNA targeting ATF4. Alternatively, cells were cultured in the presence or absence of the mTOR kinase inhibitor TORIN1 (125nM) (B) Volcano plot of -Log₁₀(FDR) vs. Log₂ fold change for DEGs between untreated and TGF-β-treated HLFs (C) Significantly activated and suppressed Molecular Signatures Database (MSigDB) Hallmark pathways enriched in DEGs between untreated and TGF-β-treated HLFs. (D) Enrichment plots of significantly enriched metabolism-related Hallmark pathways regulated by TGF-β treatment in HLFs. The top 25 enriched genes in each pathway are indicated. (E) Transcription factor enrichment analysis using DoRothEA human regulon for TGF-β-regulated genes.

Figure 2.. ATF4 regulates the expression of amino acid transporters and biosynthetic enzymes in HLFs.

(A) Western blot analysis of ATF4 protein expression in HLFs transfected with siRNA targeting ATF4 or non-targeting control siRNA. (B) Heatmap analysis of significant DEGs (log₂FC≥0.5) between TGF-β-treated HLFs with either control or ATF4 knockdown. (C) Volcano plot of -Log₁₀(FDR) vs. Log₂ fold change for DEGs between control and ATF4 knockdown HLFs treated with TGF-β. (D) Significantly suppressed Reactome pathways enriched in DEGs between control and ATF4 knockdown HLFs treated with TGF-β. (E) CNET plot of Reactome pathways enriched in DEGs between control and ATF4 knockdown HLFs treated with TGF-β

Figure 3.. mTOR regulates the expression of genes encoding glycolytic enzymes and subunits of the mitochondrial respiratory chain.

(A) Heatmap analysis of significant DEGs (log₂FC≥0.5) between TGF-β-treated HLFs cultured in the presence or absence of TORIN1. (B) Volcano plot of -Log₁₀(FDR) vs. Log₂ fold change for DEGs between TGF-β-treated HLFs cultured in the presence or absence of TORIN1. (C) Significantly activated and suppressed MSigDB Hallmark pathways enriched in DEGs between TGF-β-treated HLFs cultured in the presence or absence of TORIN1. (D) Enrichment plots of significantly enriched metabolism-related Hallmark pathways regulated by mTOR inhibition in TGF-β-treated HLFs. The top 25 enriched genes in each pathway are indicated. (E-F) Transcription factor enrichment analysis using DoRothEA human regulon for TORIN1 (E) downregulated and (F) upregulated genes.

Figure 4.. ATF4 and mTOR regulate TGF-β-induced changes in cellular metabolite levels in HLFs.

(A) Relative cellular levels of the indicated amino acids in HLFs treated with TGF-β for 48 hours. Cells were transfected with either nontargeting siRNA or siRNA targeting ATF4 (B) Relative cellular levels of the lactate or the indicated TCA cycle intermediates in HLFs treated with TGF-β for 48 hours. Cells were transfected with either nontargeting siRNA or siRNA targeting ATF4 (C) Relative cellular levels of the indicated amino acids in HLFs treated with TGF-β for 48 hours. Cells were cotreated with TORIN1 as indicated. (D) Relative cellular levels of the lactate or the indicated TCA cycle intermediates in HLFs treated with TGF-β for 48 hours. Cells were cotreated with TORIN1 as indicated. Plots represent mean ± SD. *P<0.05, **P<0.01, ***P<0.001 by one-way (C, D) or two-way (A, B) ANOVA using Tukey’s post-test.

Figure 5.. ATF4 and mTOR target genes are expressed in pathologic fibroblast populations of pulmonary fibrosis patients.

(A) Enrichment plots of significantly enriched metabolism-related Hallmark pathways regulated by TGF-β treatment in HLFs. The top 25 enriched genes in each pathway are indicated. (B) Clustering of fibroblasts from 10 control lungs and 20 lungs from patients with pulmonary fibrosis into alveolar, inflammatory, and fibrotic populations as defined by Tsukui et al (46). (C-E) Dot plot and UMAP representation of the expression of genes involved in (C) amino acid biosynthesis, tRNA aminoacylation, and stress response, (D) glucose metabolism, and (E) mitochondrial respiration. (F) Dot plot and UMAP representation of the transcriptional activity of SMAD3, SMAD4, ATF4, HIF-1α, and YY1.

Figure 6.. ATF4 and mTOR target genes correlate negatively with pulmonary function in fibrosis patients.

(A-F) Pearson correlations for metabolism-related genes (A) SHMT2, (B) IARS, (C) DDIT4, (D) PFKP, (E) LDHA, (F) ATP13A3 gene expression with both FVC-pre-BD or DLCO from the same patients. (G-H) Pearson correlations of the fibrotic markers (G) CTHRC1 and (H) COL1A1 gene expression with FVC-pre-BD and DLCO.