Role of Arginine and its Metabolism in TGF-β-Induced Activation of Lung Fibroblasts

Abstract

Arginine is a conditionally essential amino acid with known roles in protein production, nitric oxide synthesis, biosynthesis of proline and polyamines, and regulation of intracellular signaling pathways. Arginine biosynthesis and catabolism have been linked to TGF-β-induced activation of fibroblasts in the context of pulmonary fibrosis; however, a thorough study on the metabolic and signaling roles of arginine in the process of fibroblast activation has not been conducted. Here, we used metabolic dropouts and labeling strategies to determine how activated fibroblasts utilize arginine. We found that arginine limitation leads to activation of GCN2 while inhibiting TGF-β-induced mTORC1 activation and collagen protein production. Extracellular citrulline could rescue the effect of arginine deprivation in an ASS1-dependent manner. Using metabolic tracers of arginine and its precursors, we found little evidence of arginine synthesis or catabolism in lung fibroblasts treated with TGF-β. Extracellular ornithine or glutamine were the primary sources of ornithine and polyamines, not arginine. Our findings suggest that the major role for arginine in lung fibroblasts is for charging of arginyl-tRNAs and for promotion of mTOR signaling.

Keywords: Arginine; Fibroblast; Metabolism; Pulmonary Fibrosis.

Figure 1.. Arginine is required for TGF-β-induced signaling and gene expression in human lung fibroblasts.

(A) Western blot analysis of collagen 1 and α-smooth muscle actin protein expression and S6-kinase and GCN2 phosphorylation in HLFs cultured in DMEM that contains either 0.4mM arginine or no arginine. Cells were treated with TGF-β for the indicated intervals. (B) Western blot analysis of SMAD2/3 phosphorylation in HLFs cultured in DMEM that contains either 0.4mM arginine or no arginine. Cells were treated with TGF-β for the indicated intervals. (C) qPCR analysis of COL1A1, ACTA2, CTGF, and SERPINE1 mRNA expression in HLFs cultured in the DMEM that contains either 0.4mM arginine or no arginine. Cells were treated with TGF-β for 24 hours or left untreated. (D) Western blot analysis of collagen 1 and α-smooth muscle actin protein expression and S6-kinase and GCN2 phosphorylation in HLFs cultured in HPLM that contains either 0.11mM arginine or no arginine. Cells were treated with TGF-β for the indicated intervals. (E) Western blot analysis of SMAD2/3 phosphorylation in HLFs cultured in HPLM that contains either 0.11mM arginine or no arginine. Cells were treated with TGF-β for the indicated intervals. (F) qPCR analysis of COL1A1, ACTA2, CTGF, and SERPINE1 mRNA expression in HLFs cultured in the HPLM that contains either 0.11mM arginine or no arginine. Cells were treated with TGF-β for 24 hours or left untreated. *P<0.05, **P<0.01, ***P<0.001.

Figure 2.. Extracellular citrulline can rescue the effect of arginine depletion in HLFs.

(A) Western blot analysis of collagen 1 and α-smooth muscle actin protein expression and S6-kinase and GCN2 phosphorylation in HLFs cultured in HPLM that 1) contains arginine, ornithine, and citrulline, 2) contains no arginine, ornithine, or citrulline, 3) contains just arginine, 4) contains just ornithine, 5) contains just citrulline. Cells were treated with TGF-β for the indicated intervals. (B) Western blot analysis of collagen 1 and α-smooth muscle actin protein expression and S6-kinase and GCN2 phosphorylation in HLFs cultured in HPLM containing ornithine, citrulline, and the indicated concentrations of arginine. Cells were treated with TGF-β for the indicated intervals. (C) Western blot analysis of collagen 1 and α-smooth muscle actin protein expression and S6-kinase and GCN2 phosphorylation in HLFs cultured in HPLM containing no ornithine or citrulline, with the indicated concentrations of arginine. Cells were treated with TGF-β for the indicated intervals. (D) Intracellular levels of arginine, ornithine, and citrulline from cells cultured in HPLM as in (A). Cells were treated with TGF-β or left untreated for 48 hours. (E) Western blot analysis of collagen 1 and α-smooth muscle actin protein expression and S6-kinase and GCN2 phosphorylation in HLFs cultured in HPLM containing no ornithine or arginine, with the indicated concentrations of citrulline. Cells were treated with TGF-β for the indicated intervals. (F) Intracellular levels of arginine from cells cultured in HPLM as in (E). Cells were treated with TGF-β or left untreated for 48 hours.

Figure 3.. ASS1 promotes de novo arginine production in normal and IPF HLFs.

(A) Intracellular levels of argininosuccinate from cells cultured in HPLM as in (2A). Cells were treated with TGF-β or left untreated for 48 hours. (B) Western blot analysis of collagen 1 and α-smooth muscle actin protein expression in HLFs cultured in HPLM that contains arginine (0.11mM) and citrulline (0.1mM) as indicated. Cells were treated with TGF-β for the indicated intervals. (C) Heatmap analysis showing the KEGG arginine and proline metabolism pathway on RNAseq data from 3 clones of normal and 3 clones of IPF HLFs. (E) Dot plot representation of the expression of arginine metabolic enzymes in fibroblast subpopulations from pulmonary fibrosis patients and control donor lungs and stratified by disease state as defined by Habermann et al [44]. (F-G) UMAP projections of fibroblast subpopulations and ASS1 mRNA expression in fibroblasts from pulmonary fibrosis patients. (H) Intracellular levels of arginine, ornithine, citrulline, and argininosuccinate from normal and IPF HLFs cultured in HPLM. Cells were treated with TGF-β or left untreated for 48 hours. (I) Western blot analysis of collagen 1 and α-smooth muscle actin protein expression in HLFs cultured in HPLM that contains arginine (0.11mM) and citrulline (0.1mM) as indicated. Cells were treated with TGF-β for the indicated intervals.

Figure 4.. Metabolic tracing of arginine metabolism in human lung fibroblasts.

(A) Schematic representation of the metabolism of ¹³C₆ arginine. (B) Analysis of cellular arginine, ornithine, citrulline, argininosuccinate, and dimethylarginine in HLFs after labeling with ¹³C₆ arginine HPLM in the presence or absence of TGF-β. (C) Schematic representation of the metabolism of 4,4,5,5-D₄ citrulline. (D) Analysis of cellular citrulline, argininosuccinate, and arginine in HLFs after labeling with 4,4,5,5-D₄ citrulline HPLM in the presence or absence of TGF-β. (E) Schematic representation of the metabolism of ¹⁵N₂ ornithine. (F) Analysis of cellular ornithine, citrulline, proline, and putrescine in HLFs after labeling with ¹⁵N₂ ornithine HPLM in the presence or absence of TGF-β. (G) Schematic representation of the metabolism of ¹³C₆ arginine. (H) Analysis of cellular arginine, ornithine, citrulline, argininosuccinate, and dimethylarginine in HLFs after labeling with ¹³C₆ arginine DMEM in the presence or absence of TGF-β. (I) Schematic representation of the metabolism of ¹³C₅ glutamine. (J) Analysis of cellular glutamate, pyrroline-5-carboxylate, ornithine, and putrescine in HLFs after labeling with ¹³C₅ glutamine DMEM in the presence or absence of TGF-β.