Contrasting Metabolic Profiles of TGFβ2 and TNFα in the Induction of Retinal Epithelial‐Mesenchymal Transition (EMT)

Abstract

Epithelial‐mesenchymal‐transition (EMT) is a process whereby epithelial cells transdifferentiate into matrix‐producing mesenchymal cells. EMT of retinal pigment epithelial cells (RPE) plays a crucial role in severe ocular pathologies including retinal fibrosis. Emerging evidence implicates metabolic and mitochondrial dysfunction as key drivers of EMT. Both transforming growth factor‐beta 2 (TGFβ2) and tumor necrosis factor‐alpha (TNFα) have the capacity to induce EMT in RPE; however, little is known about their impact on mitochondrial metabolism. We hypothesized that TGFβ2 and TNFα should share similar metabolic effects, i.e. repressed mitochondrial function and enhanced glycolysis, to support EMT of RPE. Matured human primary RPE (H‐RPE) were treated with TGFβ2 or TNFα (both at 10 ng/ml) for up to 5 days for Seahorse metabolic assays (Mito and Glycolytic Stress Tests), qPCR and untargeted metabolomics analysis (West Coast Metabolomics Center) analyzed using MetaboAnalyst. Both TGFβ2 and TNFα induced the upregulation of mesenchymal genes (fibronectin and matrix metalloproteinase‐2) in H‐RPE. While TNFα significantly upregulated inflammatory genes (IL‐6, IL‐8, IL‐1β, MCP‐1), no changes were evident in TGFβ2‐treated cells. Metabolically, TGFβ2 induced the “Warburg effect” in H‐RPE with enhanced glycolysis and a suppression of oxidative phosphorylation capacity. Surprisingly, TNFα showed the opposite effect with enhanced oxidative phosphorylation (higher basal levels of oxygen consumption and maximal respiration) and reduced glycolysis and glycolytic capacity. Metabolomics profiling showed distinct metabolomics signatures associated with TGFβ2‐ and TNFα‐treated primary RPE cells. Of the 188 metabolites tested, 7 metabolites were significantly changed in TGFβ2‐treated cells and 26 in TNFα‐treated cells. Enrichment analysis depicted altered glutathione metabolism, proline metabolism, aminoacyl‐tRNA synthesis, nicotinamide metabolism, pentose phosphate pathway and arachidonic acid metabolism in TNFα‐treated cells whereas TGFβ2‐treated cells showed perturbations in the biosynthesis of fatty acids, tryptophan metabolism, linoleic acid metabolism and arachidonic acid metabolism. The divergent bioenergetic rewiring governing TGFβ2 and TNFα in their induction of EMT may be linked to their differential effect on inflammation. Elucidating the contributions of TGFβ2 and TNFα and their mechanistic differences will enable the development of more effective drug targets for combating retinal fibrosis.