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 3

Alanine is essential for myofibroblast differentiation.

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Alanine is essential for myofibroblast differentiation. (A) Venn diagram...
(A) Venn diagram depicting the number of differentially regulated metabolites across 4 treatment conditions compared with their respective control groups, with alanine as the only shared metabolite. Differential metabolites: fold-change ≥ 1.5 and P ≤ 0.05. (B) Intracellular alanine levels in NHLFs treated with TGF-β for 48 hours, with or without GPT2 knockdown or inhibition in DMEM. (C) Western blot analysis and quantification of α-SMA and COL1A1 expression in NHLFs treated with AOA (1 mM) and TGF-β for 48 hours in DMEM, with or without individual NEAA supplementation (2 mM each). (D) NHLFs were stimulated with TGF-β in DMEM for 48 hours, with or without CS (100 μM) and alanine supplementation (2 mM). α-SMA and COL1A1 expression were examined by Western blot. (E) NHLFs were stimulated with TGF-β in DMEM for 48 hours, with or without BCA (100 μM) and alanine supplementation (2 mM). α-SMA and COL1A1 expression were examined by Western blot. (F) NHLFs were transfected with control siRNA or GPT2 siRNA for 24 hours, followed by 24 hours of starvation. Cells were then stimulated with TGF-β in DMEM for 48 hours, with or without alanine supplementation. α-SMA and COL1A1 expression were examined by Western blot. For Western blot, individual data points represent biological replicates. B versus TGF-β and C–F versus knockdown/inhibitor plus TGF-β were analyzed using 1-way ANOVA. Data are presented as mean ± SEM. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.