Abstract
Pulmonary 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).
Primary HLFs were cultured in the presence (or absence) of TGF-β1 (5 ng/ml) under hypoxic (1% O
) or normoxic (21% O
) 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 (
H- NMR). Separate experiments were conducted to evaluate the effect of HuR on metabolic reprogramming using siRNA-mediated knock-down.
Hypoxia 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.
Hypoxia 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.