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 4

SLC38A2 mediates alanine uptake and myofibroblast differentiation.

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SLC38A2 mediates alanine uptake and myofibroblast differentiation. (A) N...
(A) Normalized transporter expression in lung cell types from the Human Lung Cell Atlas. (B) Fibroblast subset–specific expression in healthy lung samples. Dot color indicates mean expression; size indicates percentage of expressing cells. (C) Western blot analysis and quantification showing that TGF-β significantly upregulates SLC38A2 protein expression in NHLFs, independent of glutamine availability. (D) Western blot analysis and quantification showing that extracellular alanine deprivation promotes SLC38A2 protein expression in DMEM-cultured fibroblasts. (E) Western blot analysis and quantification showing that GPT2 knockdown reduces intracellular alanine levels and shows a trend toward increasing SLC38A2 protein expression in FBM. (F) Schematic overview of the short-term [U-13C3]-alanine-alanine and [15N1]-glutamine co-uptake assay in control and SLC38A2-knockdown fibroblasts and relative quantification of 13C3-alanine and [15N1]-glutamine uptake. (G) Western blot analysis and quantification showing that SLC38A2 knockdown reduces TGF-β–induced GPT2 expression in FBM. (H) Western blot analysis and quantification of α-SMA and COL1A1 expression in control and SLC38A2-knockdown fibroblasts cultured in DMEM for 48 hours with TGF-β stimulation, with or without AOA (1 mM) and/or alanine supplementation (2 mM). (I) Western blot analysis and quantification of α-SMA and COL1A1 expression in FBM-cultured fibroblasts after TGF-β stimulation and combined treatment with AOA and SLC38A2 knockdown for 48 hours. For Western blot analysis, individual data points represent biological replicates. For F, unpaired 2-tailed t test; for C–E and G–I, 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.