TGF-β coordinates alanine synthesis and import for myofibroblast differentiation in pulmonary fibrosis
Fei Li, Niv Vigder, David R. Ziehr, Mari Kamiya, Hung N. Nguyen, Diana E. Ferreyra Faustino, Aseel H. Khalil, Hilaire C. Lam, Matthew L. Steinhauser, Edy Y. Kim, William M. Oldham
Fei Li, Niv Vigder, David R. Ziehr, Mari Kamiya, Hung N. Nguyen, Diana E. Ferreyra Faustino, Aseel H. Khalil, Hilaire C. Lam, Matthew L. Steinhauser, Edy Y. Kim, William M. Oldham
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Research Article Cell biology Metabolism Pulmonology

TGF-β coordinates alanine synthesis and import for myofibroblast differentiation in pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease driven by aberrant fibroblast-to-myofibroblast differentiation, which requires metabolic reprogramming. Here, we identify alanine as an essential metabolite for myofibroblast differentiation. TGF-β increases intracellular alanine levels through enhanced synthesis and import in both normal and IPF lung fibroblasts. Alanine synthesis is primarily mediated by glutamate-pyruvate transaminase 2 (GPT2), whose expression is regulated by the glutamine/glutamate/α-ketoglutarate axis. Inhibition of GPT2 depletes alanine and suppresses TGF-β–induced α-SMA and COL1A1 expression, which are rescued by exogenous alanine. We also identify solute carrier family 38 member 2 (SLC38A2) as a transporter for both alanine and glutamine, upregulated by TGF-β or alanine deprivation. SLC38A2 and GPT2 form a coordinated regulatory axis sustaining intracellular alanine levels to support myofibroblast differentiation. Mechanistically, alanine deficiency impairs glycolytic flux and depletes tricarboxylic acid cycle intermediates, while alanine supplementation provides carbon and nitrogen for intracellular glutamate and proline biosynthesis, particularly under glutamine deprivation. Combined inhibition of alanine synthesis and uptake suppresses fibrogenic responses in fibroblasts and human precision-cut lung slices, highlighting dual metabolic targeting as a potential therapeutic strategy for fibrotic lung disease.

Authors

Fei Li, Niv Vigder, David R. Ziehr, Mari Kamiya, Hung N. Nguyen, Diana E. Ferreyra Faustino, Aseel H. Khalil, Hilaire C. Lam, Matthew L. Steinhauser, Edy Y. Kim, William M. Oldham

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Figure 1

Increased intracellular alanine levels during myofibroblast differentiation.

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Increased intracellular alanine levels during myofibroblast differentiat...
(A) Schematic overview of the experimental design for primary normal human lung fibroblasts (NHLFs) treated with TGF-β (2 ng/mL) in DMEM or FBM. Samples were collected and analyzed after 48 hours using LC/MS (n = 6 for each condition). (B) Principal component analysis (PCA) of the experimental groups. (C and D) Enriched metabolite sets after 48 hours of TGF-β treatment in DMEM (C) and FBM (D), based on differential metabolites with fold-change ≥ 1.5 and P ≤ 0.05. Enrichment ratio = hits / expected. (E) Comparison of intracellular amino acid changes induced by TGF-β treatment in DMEM versus FBM conditions. (F) Heatmap showing the intracellular levels of amino acids in fibroblasts cultured in DMEM or FBM with or without TGF-β stimulation for 48 hours. Each column represents a sample, and each row corresponds to an amino acid. Colors indicate z score–normalized abundance, with red and blue representing higher and lower relative levels, respectively. (G) Relative quantification of the intracellular abundances of alanine, aspartate, asparagine, proline, and glutamate. (H) Schematic overview of LC/MS-based stable isotope tracing with [U-13C6]-glucose (13C6-GLC), [U-13C5]-glutamine (13C5-GLN), or[α-15N1]-glutamine (15N1-GLN) for 48 hours. (I–K) Stable isotope label enrichment of alanine from 13C6-GLC (I), 13C5-GLN (J), or 15N1-GLN (K). (L) Schematic overview of short-term uptake assays using isotope tracing with [U-13C3]-alanine (13C3-ALA) for 1 hour (left). Signal intensity of intracellular ¹³C3-alanine (undetectable without tracer) (right). (M) Schematic overview of the experimental design for IPF human lung fibroblasts (IPF-LF) from 4 donors treated with TGF-β (2 ng/mL) in FBM for 48 hours (left). Relative quantification of intracellular alanine levels (right). For G, 1-way ANOVA; I–M, unpaired 2-tailed t test. Data are presented as mean ± SEM. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.