Abstract
BackgroundPulmonary fibrosis is thought to be driven by recurrent alveolar epithelial injury which leads to the differentiation of fibroblasts into α-smooth muscle actin (α-SMA)-expressing myofibroblasts and subsequent deposition of extracellular matrix (ECM). Transforming growth factor beta-1 (TGF-β1) plays a key role in fibroblast differentiation, which we have recently shown involves human antigen R (HuR). HuR is an RNA binding protein that also increases the translation of hypoxia inducible factor (HIF-1α) mRNA, a transcription factor critical for inducing a metabolic shift from oxidative phosphorylation towards glycolysis. This metabolic shift may cause fibroblast differentiation. We hypothesized that under hypoxic conditions, HuR controls myofibroblast differentiation and glycolytic reprogramming in human lung fibroblasts (HLFs).MethodsPrimary HLFs were cultured in the presence (or absence) of TGF-β1 (5 ng/ml) under hypoxic (1% O2) or normoxic (21% O2) conditions. Evaluation included mRNA and protein expression of glycolytic and myofibroblast/ECM markers by qRT-PCR and western blot. Metabolic profiling was done by proton nuclear magnetic resonance (1H- NMR). Separate experiments were conducted to evaluate the effect of HuR on metabolic reprogramming using siRNA-mediated knock-down.ResultsHypoxia alone had no significant effect on fibroblast differentiation or metabolic reprogramming. While hypoxia- together with TGFβ1- increased mRNA levels of differentiation and glycolysis genes, such as ACTA2, LDHA, and HK2, protein levels of α-SMA and collagen 1 were significantly reduced. Hypoxia induced cytoplasmic translocation of HuR. Knockdown of HuR reduced features of fibroblast differentiation in response to TGF-β1 with and without hypoxia, including α-SMA and the ECM marker collagen I, but had no effect on lactate secretion.ConclusionsHypoxia reduced myofibroblasts differentiation and lactate secretion in conjunction with TGF-β. HuR is an important protein in the regulation of myofibroblast differentiation but does not control glycolysis in HLFs in response to hypoxia. More research is needed to understand the functional implications of HuR in IPF pathogenesis.
Highlights
Pulmonary fibrosis is thought to be driven by recurrent alveolar epithelial injury which leads to the dif‐ ferentiation of fibroblasts into α-smooth muscle actin (α-SMA)-expressing myofibroblasts and subsequent deposition of extracellular matrix (ECM)
We have recently shown that human antigen R (HuR) controls myofibroblast differentiation and ECM production in response to transforming growth factor-β (TGF-β) in primary human lung fibroblasts (HLFs) [20]
Hypoxia attenuates TGFβ1‐induced metabolic shift towards glycolysis To first establish the extent to which TGF-β causes changes in the expression of genes involved in metabolic reprogramming, HLFs were treated with TGF-β1 at varying concentrations for up to 48 h prior to analysis of gene expression
Summary
Pulmonary fibrosis is thought to be driven by recurrent alveolar epithelial injury which leads to the dif‐ ferentiation of fibroblasts into α-smooth muscle actin (α-SMA)-expressing myofibroblasts and subsequent deposition of extracellular matrix (ECM). HuR is an RNA binding protein that increases the translation of hypoxia inducible factor (HIF-1α) mRNA, a transcription factor critical for inducing a metabolic shift from oxidative phosphorylation towards glycolysis This metabolic shift may cause fibroblast differentiation. It is thought that damage to the alveolar epithelium drives the accumulation of myofibroblasts, which are α-smooth muscle actin (α-SMA)-expressing cells that produces copious amounts of extracellular matrix (ECM) proteins such as collagens (COL) and fibronectin (FN), which contribute to dysfunctional tissue remodeling [5]. TGF-β is produced as a latent protein that requires activation by factors such as mechanical stretch and changes in pH [6] This change in extracellular pH can be due to a switch from oxidative phosphorylation to aerobic glycolysis, a state in which predominant uptake/use of glucose for energy persists despite the presence of adequate oxygen for mitochondrial respiration [7, 8]. Excessive accumulation of myofibroblasts and ECM within the alveolar space creates hypoxic conditions
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