Ceramidase is involved in metabolic reprogramming of fibroblasts under hypoxia conditions

Abstract

Physiological metabolic reprogramming is a cells strategy to resist/counteract stress to preserve homeostasis. Hypoxia is an important stress stimulus that can promote tissue remodeling and fibrosis, in which alterations in sphingolipid metabolism may contribute to fibroblast responses. Here, we investigated the potential role of acid ceramidase (ASAH1), a key enzyme of the sphingolipid salvage pathway, in fibroblast adaptation to hypoxic stress. Human fibroblasts were exposed to hypoxia for 24, 48, and 72 hours, and metabolic and redox changes were investigated following pharmacological inhibition of acid ceramidase with D-Nmappd. These findings were complemented by analyses of publicly available bulk and single-cell RNA sequencing datasets from idiopathic pulmonary fibrosis (IPF) lungs. Hypoxic fibroblasts showed metabolic remodeling involving glucose-, amino acid-, and redox-associated pathways, together with changes consistent with an adaptive antioxidant response, including alterations in the GSH/GSSG balance. Pharmacological inhibition of acid ceramidase under hypoxia was associated with changes in carnitine metabolism and long-chain fatty acid β-oxidation, reduced GSH and GSSG levels, and decreased expression of Nuclear Factor Erythroid 2-Related Factor 2 (NRF2), Superoxide dismutase 2 (SOD2), and 8-Oxoguanine DNA Glycosylase 1 (OGG1). In IPF lung tissue, ASAH1 expression was associated with glycolytic and redox-related genes. Single-cell analysis showed that ASAH1-expressing IPF fibroblasts were enriched in transcriptional programs related to hypoxic responses and oxidative stress regulation and displayed features associated with a less activated phenotype. Overall, these findings suggest that acid ceramidase/ASAH1 is associated with fibroblast metabolic and redox adaptation to hypoxic stress and may influence fibroblast activation in IPF.