C71-24 Ornithine Metabolism Promotes Polyamine-dependent Collagen Protein Production in TGF-β-stimulated Lung Fibroblasts

Abstract Rationale Ornithine is a non-proteinogenic amino acid which plays important roles in nitrogen metabolism and is also a precursor for synthesis of proline and polyamines. Ornithine is produced from arginine through arginase (ARG1 and ARG2) enzymes. In cells with an intact urea cycle, ornithine transcarbamylase (OTC) converts ornithine to citrulline. Ornithine is also metabolized by ornithine aminotransferase (OAT), producing proline, and by ornithine decarboxylase (ODC), producing the polyamine putrescine. Ornithine has been speculated to support collagen synthesis through its conversion to proline while polyamines are known to regulate chromatin conformation and gene transcription. A thorough analysis of ornithine metabolism and its role in regulating activation of lung fibroblasts has not been conducted. Here we sought to determine the role of ornithine metabolism in lung fibroblasts. Methods Normal human lung fibroblasts (NHLFs) or HLFs from patients with idiopathic pulmonary fibrosis (IPF) (IPF HLFs) were treated with TGF-β in media containing or lacking ornithine. Fibroblast activation was assessed by qPCR (mRNA) and Western blot (protein) for collagen 1 (COL1A1) and α-Smooth Muscle Actin (ACTA2) expression. Metabolism of ornithine was inhibited by knocking down either ODC or OAT using siRNA. Cellular metabolites were measured by LC/MS (liquid chromatography/mass spectroscopy). Metabolism of ornithine was assessed by labeling with 15N-labeled metabolites. Results Ornithine deprivation did not significantly affect cellular proline levels or collagen synthesis in TGF-β-treated HLFs. Metabolic labeling experiments showed that the primary fate of extracellular ornithine was putrescine synthesis. Knockdown of OAT had no effect on TGF-β-induced fibroblast activation; however, ODC knockdown reduced collagen mRNA and protein expression downstream of TGF-β. Conclusions Our results demonstrate that the primary role of ornithine metabolism in HLFs is for production of polyamines. Inhibition of polyamine synthesis reduced TGF-β-induced fibroblast activation, suggesting a profibrotic role of polyamines. Polyamine synthesis may represent a therapeutic target for reducing collagen production by lung fibroblasts. This abstract is funded by: NIH, DOD