Arginine promotes the activation of human lung fibroblasts independent of its metabolism

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 transforming growth factor-β (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 examined the role and regulation of arginine metabolism in lung fibroblasts activated with TGF-β. We found that TGF-β increases the expression of arginine biosynthetic and catabolic enzymes in lung fibroblasts. Surprisingly, using metabolic tracers of arginine and its precursors, we found little evidence of arginine synthesis or catabolism in lung fibroblasts treated with TGF-β. Despite this, arginine remained crucial for TGF-β-induced expression of collagen and α-smooth muscle actin. We found that arginine limitation leads to the activation of general control nonderepressible 2 (GCN2), while inhibiting TGF-β-induced mechanistic target of rapamycin complex 1 activation and collagen protein production. Extracellular citrulline could rescue the effect of arginine deprivation in an argininosuccinate synthase-dependent manner. Our findings suggest that the major role of arginine in lung fibroblasts is for charging of arginyl-tRNAs and promotion of signaling events which are required for fibroblast activation.

Keywords: arginine; fibroblast; metabolism; pulmonary fibrosis; transforming growth factor-β.

Figure 1:. TGF-β increases expression of arginine metabolic enzymes and intracellular levels of arginine in human lung fibroblasts (HLFs).

(A) Schematic illustration of arginine metabolism. Arginine is catabolized by arginase 1 and 2 (ARG1, ARG2) or by nitric oxide synthase (NOS), producing urea or nitric oxide, respectively. In cells with a functional urea cycle, ornithine is converted to citrulline by ornithine transcarbamylase. Citrulline is converted back to arginine through the combined actions of argininosuccinate synthase 1 (ASS1) and argininosuccinate lyase (ASL). (B) Intracellular levels of arginine, ornithine and citrulline in HLFs cultured in DMEM or Human Plasma-Like Medium (HPLM) and treated with TGF-β or left untreated for 48 hours. (C) Intracellular levels of arginine, ornithine, and citrulline from three clones of normal and three clones of IPF HLFs. Cells were cultured in HPLM and treated with TGF-β or left untreated for 48 hours. (D) Heatmap analysis showing the Kyoto Encyclopedia of Genes and Genomes (KEGG) arginine and proline metabolism pathway on RNA-seq data from three clones of normal and three clones of IPF HLFs (three biological replicates per clone). Cells were cultured in HPLM and treated with TGF-β or left untreated for 24 hours. (E) Western blot analysis showing protein expression of ASS1, ASL, ARG2, and NOS3 in three clones of normal and three clones of IPF HLFs. Cells were cultured in HPLM and treated with TGF-β for the indicated intervals.

Figure 2:. Single cell RNA-seq analysis of arginine metabolic enzymes in lung fibroblasts from patients with pulmonary fibrosis.

(A) Uniform Manifold Approximation and Projection (UMAP) projection of the subclustering of lung fibroblast populations from patients with pulmonary fibrosis and control donor lungs as defined by Habermann et al. [37] showing fibroblast, myofibroblast, PLIN2⁺ fibroblast, and HAS1^High fibroblasts. (B) Dot plot representation of the expression of arginine metabolic enzymes in lung fibroblast populations as defined in (A). (C) UMAP projection of the expression of ASS1 mRNA in lung fibroblasts from patient with pulmonary fibrosis and control donor lungs. (D) UMAP projection of the further subclustering of alveolar and myofibroblast populations from (A) as defined by Tsukui et al. showing alveolar fibroblasts, inflammatory fibroblasts, and fibrotic fibroblasts. (E) Dot plot representation of the expression of arginine metabolic enzymes in lung fibroblast populations as defined in (D). (F) UMAP projection of the expression of ASS1 mRNA in lung fibroblast populations as defined in (D). (G) Dot plot representation of the expression of proline and polyamine biosynthetic and catabolic enzymes in lung fibroblast populations as defined in (D).

Figure 3:. Metabolic tracing of arginine metabolism in human lung fibroblasts cultured in Human Plasma-Like Media (HPLM).

(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 guanido-¹⁵N₂ arginine. (D) Analysis of cellular arginine, argininosuccinate, and citrulline in HLFs after labeling with guanido-¹⁵N₂ arginine HPLM in the presence or absence of TGF-β. (E) Schematic representation of the metabolism of 4,4,5,5-D₄ citrulline. (F) 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. (G) Schematic representation of the metabolism of ¹⁵N₂ ornithine. (H) Analysis of cellular ornithine, citrulline, proline, and putrescine in HLFs after labeling with ¹⁵N₂ ornithine HPLM in the presence or absence of TGF-β. Data are normalized to untreated group and presented as mean ± SEM from four biologic replicates.

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

(A) Schematic representation of the metabolism of ¹³C₆ arginine. (B) Analysis of cellular arginine, argininosuccinate, citrulline, dimethylarginine, ornithine, proline, and putrescine in HLFs after labeling with ¹³C₆ arginine DMEM in the presence or absence of TGF-β. (C) Schematic representation of the metabolism of guanido-¹⁵N₂ arginine. (D) Analysis of cellular arginine, argininosuccinate, and citrulline in HLFs after labeling with guanido-¹⁵N₂ arginine DMEM in the presence or absence of TGF-β. (E) Schematic representation of the metabolism of ¹³C₅ glutamine. (F) Analysis of cellular glutamate, pyrroline-5-carboxylate, ornithine, proline, and putrescine in HLFs after labeling with ¹³C₅ glutamine DMEM in the presence or absence of TGF-β. Data are normalized to untreated group and presented as mean ± SEM from four biologic replicates.

Figure 5:. Arginine is required for TGF-β-induced signaling and gene expression in human lung fibroblasts (HLFs).

(A–B) Western blot analysis of collagen 1 and α-smooth muscle actin protein expression and S6-kinase and GCN2 phosphorylation in HLFs cultured in either (A) Human Plasma-Like Medium (HPLM) or (B) DMEM containing or lacking arginine. Cells were treated with TGF-β for the indicated intervals. (C–D) Western blot analysis of SMAD2/3 phosphorylation in HLFs cultured in either (C) HPLM or (D) DMEM containing or lacking arginine. Cells were treated with TGF-β for the indicated intervals. (E–F) qPCR analysis of COL1A1, ACTA2, CTGF, and SERPINE1 mRNA expression in HLFs cultured in the either (E) HPLM or (F) DMEM containing or lacking arginine. Cells were treated with TGF-β for 24 hours or left untreated. Data are normalized to Arg+ and no TGF-β treatment (Arg+/TGF-β-) group and presented as mean ± SEM from four biologic replicates. *P<0.05, **P<0.01, ***P<0.001 by two-way Analysis of Variance (ANOVA) using Tukey’s post-test.

Figure 6:. Metabolism of extracellular citrulline through ASS1 can rescue the effect of arginine depletion in human lung fibroblasts (HLFs).

(A–B) Western blot analysis of collagen 1 and α-smooth muscle actin (α-SMA) protein expression in HLFs cultured in Human Plasma-Like Medium (HPLM) either (A) containing ornithine and citrulline, or (B) lacking ornithine and citrulline. Cells were treated with TGF-β for the indicated intervals in the presence of the indicated concentrations of arginine. (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 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. (D) Intracellular levels of arginine, ornithine, and citrulline from cells cultured in HPLM as in (C). Cells were treated with TGF-β or left untreated for 48 hours. (E) Western blot analysis of collagen 1 and α-SMA 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. (G) Western blot analysis of collagen 1 and α-SMA protein expression in control and ASS1 knockdown HLFs cultured in HPLM that contains arginine (0.11 mM) and citrulline (0.1 mM) as indicated. Cells were treated with TGF-β for the indicated intervals. (H) Western blot analysis of collagen 1 and α-SMA protein expression in HLFs cultured in HPLM that contains arginine (0.11 mM) and citrulline (0.1 mM) as indicated. Cells were treated with TGF-β for 48 hours.