Mitochondrial One-Carbon Metabolism is Required for TGF-β-Induced Glycine Synthesis and Collagen Protein Production

Abstract

ABSTRACTA hallmark of Idiopathic Pulmonary Fibrosis is the TGF-β-dependent activation of lung fibroblasts, leading to excessive deposition of collagen proteins and progressive scarring. We have previously shown that synthesis of collagen by lung fibroblasts requiresde novosynthesis of glycine, the most abundant amino acid in collagen protein. TGF-β upregulates the expression of the enzymes of thede novoserine/glycine synthesis pathway in lung fibroblasts through mTORC1 and ATF4- dependent transcriptional programs. SHMT2, the final enzyme of thede novoserine/glycine synthesis pathway, transfers a one-carbon unit from serine to tetrahydrofolate (THF), producing glycine and 5,10-methylene-THF (meTHF). meTHF is converted back to THF in the mitochondrial one-carbon (1C) pathway through the sequential actions of MTHFD2 (which converts meTHF to 10-formyl-THF), and either MTHFD1L, which produces formate, or ALDH1L2, which produces CO2. It is unknown how the mitochondrial 1C pathway contributes to glycine biosynthesis or collagen protein production in fibroblasts, or fibrosisin vivo. Here, we demonstrate that TGF-β induces the expression ofMTHFD2,MTHFD1L, andALDH1L2in human lung fibroblasts.MTHFD2expression was required for TGF-β-induced cellular glycine accumulation and collagen protein production. Combined knockdown of bothMTHFD1LandALDH1L2also inhibited glycine accumulation and collagen protein production downstream of TGF-β; however knockdown of either protein alone had no inhibitory effect, suggesting that lung fibroblasts can utilize either enzyme to regenerate THF. Pharmacologic inhibition of MTHFD2 recapitulated the effects ofMTHFD2knockdown in lung fibroblasts and ameliorated fibrotic responses after intratracheal bleomycin instillationin vivo. Our results provide insight into the metabolic requirements of lung fibroblasts and provide support for continued development of MTHFD2 inhibitors for the treatment of IPF and other fibrotic diseases.