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 2

Glutamine promotes TGF-β–induced GPT2 expression to support myofibroblast differentiation.

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Glutamine promotes TGF-β–induced GPT2 expression to support myofibroblas...
(A) Schematic overview of glutamine catabolism and alanine synthesis involving GPT1 and GPT2. (B and C) Western blot analysis and quantification of GPT2 (B) and GPT1 (C) in NHLFs treated with TGF-β in glutamine-deficient or glutamine-sufficient (2 mM) DMEM for 48 hours. (D and E) Western blot analysis and quantification of GPT2 in NHLFs treated with TGF-β in DMEM for 48 hours with glutaminase-1 inhibitors (CB-839, 10 μM; BPTES, 5 μM) (D) or with/without DM-α-KG (5 mM) under glutamine-deficient or -sufficient conditions (E). (F) Chemical structure of the pan-transaminase inhibitor AOA. (G) NHLFs were stimulated with TGF-β in FBM or DMEM, with or without AOA (1 mM) for 48 hours. α-SMA and COL1A1 expression were examined by Western blot. (H) Chemical structure of the GPT1/2 inhibitor CS. (I) NHLFs were stimulated with TGF-β in FBM or DMEM, with or without CS (100 μM) for 48 hours. α-SMA and COL1A1 expression were examined by Western blot. (J) Chemical structure of the GPT1/2 inhibitor BCA. (K) NHLFs were stimulated with TGF-β in FBM or DMEM, with or without BCA (100 μM) for 48 hours. α-SMA and COL1A1 expression were examined by Western blot. (L) 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 FBM or DMEM for 48 hours. GPT2-knockdown efficiency, α-SMA, and COL1A1 expression were examined by Western blot. Individual data points represent biological replicates. For B–E, 1-way ANOVA; G, I, K, and L, 1-way ANOVA versus TGF-β within each medium. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.