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Regulation of type I collagen mRNA in lung fibroblasts by cystine availability.

ABSTRACT: The steady-state level of alpha1(I) collagen mRNA is regulated by amino acid availability in human lung fibroblasts. Depletion of amino acids decreases alpha1(I) collagen mRNA levels and repletion of amino acids induces rapid re-expression of alpha1(I) mRNA. In these studies, we examined the requirements for individual amino acids on the regulation of alpha1(I) collagen mRNA. We found that re-expression of alpha1(I) collagen mRNA was critically dependent on cystine but not on other amino acids. However, the addition of cystine alone did not result in re-expression of alpha1(I) collagen mRNA. Following amino acid depletion, the addition of cystine with selective amino acids increased alpha1(I) collagen mRNA levels. The combination of glutamine and cystine increased alpha1(I) collagen mRNA levels 6.3-fold. Methionine or a branch-chain amino acid (leucine, isoleucine or valine) also acted in combination with cystine to increase alpha1(I) collagen mRNA expression, whereas other amino acids were not effective. The prolonged absence of cystine lowered steady-state levels of alpha1(I) collagen mRNA through a mechanism involving decreases in both the rate of gene transcription as assessed by nuclear run-on experiments and mRNA stability as assessed by half-life determination in the presence of actinomycin D. The effect of cystine was not mediated via alterations in the level of glutathione, the major redox buffer in cells, as determined by the addition of buthionine sulphoximine, an inhibitor of gamma-glutamylcysteine synthetase. These data suggest that cystine directly affects the regulation of alpha1(I) collagen mRNA.

SUBMITTER: Rishikof DC

PROVIDER: S-EPMC1219370 | biostudies-other | 1998 Apr

REPOSITORIES: biostudies-other

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Project description: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.

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Regulation of type I collagen mRNA in lung fibroblasts by cystine availability.

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