View Abstract

A2205 - The Role of HuR in Myofibroblast Differentiation: Implication for Pulmonary Fibrosis
Author Block: F. F. AL-Habeeb1, P. K. Nair2, I. Azuelos3, C. Baglole4; 1Experimental medicine, McGill University, Montreal, QC, Canada, 2McMaster Univ, Hamilton, AB, Canada, 3McGill University Health Center, Montreal, QC, Canada, 4Meakins-Christie Laboratories, McGill University, Montreal, QC, Canada.
Introduction: Pulmonary fibrosis is an under-diagnosed lung disease characterized by progressive lung scarring. Extracellular matrix (ECM) deposition is a key event in disease pathogenesis. ECM proteins are excessively deposited due to increased differentiation of fibroblasts into the α-smooth muscle actin(α-SMA)-expressing myofibroblasts by transforming growth factor (TGF-β1). Fibrosis may be accompanied by metabolic reprogramming from aerobic respiration to aerobic glycolysis due to extensive tissue hypoxia, which enhances fibroblast differentiation via hypoxia inducible factor (HIF-1α). We predict that TGF-β1 promotes fibrosis via human antigen R (HuR), an RNA binding protein that may in turn increase levels of both TGF-β1 and HIF-1α. Hypothesis: HuR promotes the differentiation of myofibroblasts, which increases ECM proteins and a metabolic shift that stiffen the lungs. Methods: To explore the role of HuR in TGF-β1-treated human lung fibroblasts (HLFs), we first examined the effect of TGF-β1(5ng/ml) on the expression of HuR and fibrogenic (α-SMA, collagen and fibronectin) and metabolic (HIF-1α, lactate dehydrogenase A (LDH-A), hexokinase) markers. Then, HLFs were transfected with HUR siRNA or Control siRNA, treated with TGF-β1 and fibrotic/metabolic markers assessed. Actinomycin D (ActD)-chase experiments were performed to examine if HuR affects the stability of fibrogenic/metabolic transcripts. Immunofluorescence (IF) was performed to assess HuR localization in HLFs treated with TGF-β1. Seahorse XF96 used to assess metabolic activity in TGF-β1-treated HLFs. Results: Exposure of HLFs to TGF-β1 increased the total protein levels of HuR. The expression of metabolic and fibrogenic markers were also induced by TGF-β1 treatment. In addition, TGF-β1 caused the translocation of HuR from the nucleus to the cytoplasm- a feature consistent with HuR activation. siHuR-transfected cells showed a significant reduction in fibrogenic (α-SMA, collagen and fibronectin) and metabolic markers (HIF-1α,LDH-A) in response to TGF-β1 treatment compared to siControl. However, alterations in HuR levels did not affect mRNA stability of the markers. TGF-β1-treated HLF showed increase in acidification rate and oxygen consumption rate as compared to untreated cells. Conclusions: Our preliminary data show that during fibrotic stimuli, HuR increases the protein levels of key fibrogenic and metabolic markers. Thus, HuR could be a driving factor in the pathogenesis of pulmonary fibrosis.