Glutamine Metabolism Is Required for Collagen Protein Synthesis in Lung Fibroblasts

Abstract

Idiopathic pulmonary fibrosis (IPF) is characterized by the transforming growth factor (TGF)-β-dependent differentiation of lung fibroblasts into myofibroblasts, leading to excessive deposition of extracellular matrix proteins, which distort lung architecture and function. Metabolic reprogramming in myofibroblasts is emerging as an important mechanism in the pathogenesis of IPF, and recent evidence suggests that glutamine metabolism is required in myofibroblasts, although the exact role of glutamine in myofibroblasts is unclear. In the present study, we demonstrate that glutamine and its conversion to glutamate by glutaminase are required for TGF-β-induced collagen protein production in lung fibroblasts. We found that metabolism of glutamate to α-ketoglutarate by glutamate dehydrogenase or the glutamate-pyruvate or glutamate-oxaloacetate transaminases is not required for collagen protein production. Instead, we discovered that the glutamate-consuming enzymes phosphoserine aminotransferase 1 (PSAT1) and aldehyde dehydrogenase 18A1 (ALDH18A1)/Δ¹-pyrroline-5-carboxylate synthetase (P5CS) are required for collagen protein production by lung fibroblasts. PSAT1 is required for de novo glycine production, whereas ALDH18A1/P5CS is required for de novo proline production. Consistent with this, we found that TGF-β treatment increased cellular concentrations of glycine and proline in lung fibroblasts. Our results suggest that glutamine metabolism is required to promote amino acid biosynthesis and not to provide intermediates such as α-ketoglutarate for oxidation in mitochondria. In support of this, we found that inhibition of glutaminolysis has no effect on cellular oxygen consumption and that knockdown of oxoglutarate dehydrogenase has no effect on the ability of fibroblasts to produce collagen protein. Our results suggest that amino acid biosynthesis pathways may represent novel therapeutic targets for treatment of fibrotic diseases, including IPF.

Keywords: cellular metabolism; fibroblast; glutamine; pulmonary fibrosis.

Figure 1.

Glutamine (Gln) and glutaminase (GLS) are required for transforming growth factor (TGF)-β–induced collagen protein production in lung fibroblasts. (A) Western blot analysis of collagen I and α-SMA protein concentrations in normal human lung fibroblasts (NHLFs) treated with TGF-β for the indicated intervals. Cells were cultured in the presence or absence of Gln as indicated. (B) Quantification of collagen concentrations in A is normalized to TGF-β–induced protein concentrations in the presence of Gln (mean ± SEM; n = 3). (C) qRT-PCR measurement of mRNA expression of COL1A1 (collagen type 1, α1); ACTA2 (α-SMA gene); CTGF (connective tissue growth factor); and PAI1 (plasminogen activator inhibitor 1). NHLFs were treated with TGF-β for 24 hours or left untreated in the presence or absence of Gln (mean ± SEM; n = 3). (D) mRNA expression of GLS measured by qRT-PCR. NHLFs were treated with TGF-β for 24 hours or left untreated (mean ± SEM; n = 3). (E) Western blot analysis of GLS protein concentrations in NHLFs treated with TGF-β for the indicated intervals. (F) Quantification of collagen concentrations in E normalized to TGF-β–induced protein concentrations in untreated cells (mean ± SEM; n = 3). (G) Western blot analysis of collagen I, α-SMA, and GLS protein concentrations in control and GLS-knockdown NHLFs treated with TGF-β for the indicated intervals. (H) Quantification of collagen concentrations in G normalized to TGF-β–induced protein concentrations in the control knockdown cells (mean ± SEM; n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001. α-SMA = α-smooth muscle actin; ns = not significant; si Cont = non-targeting control siRNA.

Figure 2.

Glutamate dehydrogenase (GLUD), glutamate-pyruvate transaminases (GPTs), and glutamate-oxaloacetate transaminases (GOTs) are not required for TGF-β–induced collagen protein production in lung fibroblasts. (A) Schematic representation of the biochemical reactions performed by GLUD, GPT, and GOT while converting glutamate to α-ketoglutarate (α-KG). (B) mRNA expression of GLUD, GPT1, GPT2, GOT1, and GOT2 measured by qRT-PCR. NHLFs were treated with TGF-β for 24 hours or left untreated (mean ± SEM; n = 3). (C) Western blot analysis of GLUD, GPT2, GOT1, and GOT2 protein concentrations in NHLFs treated with TGF-β for the indicated intervals. (D–G) Western blot analysis of collagen I and α-SMA protein concentrations in control or GLUD- (D), GPT2- (E), GOT1- (F), and GOT2-knockdown NHLFs treated with TGF-β for the indicated intervals (G). (H) Quantification of collagen concentrations in D–G normalized to TGF-β–induced protein concentrations in the control knockdown cells (mean ± SEM; n = 3). **P < 0.01 and ***P < 0.001. Ala = alanine; Asp = aspartate; Glu = glutamate; OAA = oxaloacetate; Pyr = pyruvate.

Figure 3.

Phosphoserine aminotransferase 1 (PSAT1) is required for TGF-β–induced collagen protein production in lung fibroblasts. (A) Western blot analysis of collagen I and α-SMA protein concentrations in NHLFs treated with TGF-β for the indicated intervals in the presence or absence of aminooxyacetic acid (AOA; 2 mM). (B) Quantification of collagen concentrations in A normalized to TGF-β–induced protein concentrations in the absence of AOA (mean ± SEM; n = 3). (C) Schematic representation of the biochemical reaction performed by PSAT1 while converting glutamate to α-KG. (D) mRNA expression of PSAT1 measured by qRT-PCR. NHLFs were treated with TGF-β for 24 hours or left untreated (mean ± SEM; n = 3). (E) Western blot analysis of PSAT1 in NHLFs treated with TGF-β for the indicated intervals. (F) Western blot analysis of collagen I, α-SMA, and PSAT1 protein concentrations in control and PSAT1-knockdown NHLFs treated with TGF-β for the indicated intervals. (G) Quantification of collagen concentrations in F normalized to TGF-β–induced protein concentrations in the control knockdown cells (mean ± SEM; n = 3). **P < 0.01 and ***P < 0.001.

Figure 4.

De novo proline synthesis is required for collagen protein production in lung fibroblasts. (A) Schematic representation of the biochemical reactions required for de novo proline production from Glu. (B) mRNA expression of ALDH18A1 (aldehyde dehydrogenase 18A1) or pyrroline-5-carboxylate reductase (PYCR1, PYCR2, or PYCRL) measured by qRT-PCR. NHLFs were treated with TGF-β for 24 hours or left untreated (mean ± SEM; n = 3). (C) Western blot analysis of P5CS (Δ-1-pyrroline-5-carboxylate synthase), PYCR1, PYCR2, and PYCRL protein concentrations in NHLFs treated with TGF-β for the indicated intervals. (D) Western blot analysis of collagen I, α-SMA, and P5CS protein concentrations in control and ALDH18A1-knockdown NHLFs treated with TGF-β for the indicated intervals. (E) Quantification of collagen concentrations in D normalized to TGF-β–induced protein concentrations in the control knockdown cells (mean ± SEM; n = 3). (F) Western blot analysis of collagen I, α-SMA, and P5CS protein concentrations in control and ALDH18A1-knockdown NHLFs treated with TGF-β for the indicated intervals in the presence of the indicated concentrations of proline. (G) Quantification of collagen concentrations in F normalized to TGF-β–induced protein concentrations in the control knockdown cells (mean ± SEM; n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001. NADPH = reduced form of nicotinamide adenine dinucleotide phosphate.

Figure 5.

Glutaminolysis is not required to support mitochondrial oxygen consumption in lung fibroblasts. (A) Analysis of cellular oxygen consumption rate (OCR) in NHLFs treated with TGF-β for 48 hours or left untreated. OCR was measured in the presence or absence of Gln. Cells were treated with oligomycin A, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and rotenone/antimycin (R+A) as indicated. (B) Average basal and maximal OCR values from A. (C) Western blot analysis of collagen I, α-SMA, and oxoglutarate dehydrogenase (OGDH) protein concentrations in control and OGDH-knockdown NHLFs treated with TGF-β for the indicated intervals. (D) Quantification of collagen concentrations in C normalized to TGF-β–induced protein concentrations in the control knockdown cells (mean ± SEM; n = 3). (E–G) Metabolomic analysis of cellular concentrations of (E) Gln and Glu and metabolites downstream of (F) PSAT1 and (G) P5CS in NHLFs treated with TGF-β for 48 hours or left untreated. (F) 3-Phosphoserine (3P-Ser), serine (Ser), and glycine (Gly). (G) P5C and proline (Pro). *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 6.

PSAT1 and P5CS expression are elevated in fibrotic lungs. (A) Western blot analysis of PSAT1 and P5CS protein expression in lung homogenates from lungs of patients with idiopathic pulmonary fibrosis (IPF) and healthy donors. (B) Quantification of A normalized to healthy lung concentrations. (C) Western blot analysis of PSAT1 and P5CS protein expression in lung homogenates from mice 21 days after intratracheal instillation of either bleomycin or PBS as a vehicle control. (D) Quantification of C normalized to saline-treated mice. *P < 0.05 and **P < 0.01.