HuR drives lung fibroblast differentiation but not metabolic reprogramming in response to TGF-ß and hypoxia.

BACKGROUND: 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-ß1) 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). METHODS: Primary HLFs were cultured in the presence (or absence) of TGF-ß1 (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. RESULTS: Hypoxia alone had no significant effect on fibroblast differentiation or metabolic reprogramming. While hypoxia- together with TGFß1- 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-ß1 with and without hypoxia, including α-SMA and the ECM marker collagen I, but had no effect on lactate secretion. CONCLUSIONS: Hypoxia reduced myofibroblasts differentiation and lactate secretion in conjunction with TGF-ß. 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.

Human antigen R promotes lung fibroblast differentiation to myofibroblasts and increases extracellular matrix production.

Idiopathic pulmonary fibrosis (IPF) is a disease of progressive scarring caused by excessive extracellular matrix (ECM) deposition and activation of α-SMA-expressing myofibroblasts. Human antigen R (HuR) is an RNA binding protein that promotes protein translation. Upon translocation from the nucleus to the cytoplasm, HuR functions to stabilize messenger RNA (mRNA) to increase protein levels. However, the role of HuR in promoting ECM production, myofibroblast differentiation, and lung fibrosis is unknown. Human lung fibroblasts (HLFs) treated with transforming growth factor ß1 (TGF-ß1) showed a significant increase in translocation of HuR from the nucleus to the cytoplasm. TGF-ß-treated HLFs that were transfected with HuR small interfering RNA had a significant reduction in α-SMA protein as well as the ECM proteins COL1A1, COL3A, and FN1. HuR was also bound to mRNA for ACTA2, COL1A1, COL3A1, and FN. HuR knockdown affected the mRNA stability of ACTA2 but not that of the ECM genes COL1A1, COL3A1, or FN. In mouse models of pulmonary fibrosis, there was higher cytoplasmic HuR in lung structural cells compared to control mice. In human IPF lungs, there was also more cytoplasmic HuR. This study is the first to show that HuR in lung fibroblasts controls their differentiation to myofibroblasts and consequent ECM production. Further research on HuR could assist in establishing the basis for the development of new target therapy for fibrotic diseases, such as IPF.

Utility and diagnostic accuracy of endobronchial ultrasound-guided transbronchial fine-needle aspiration cytology of mediastinal lesions: Saudi Arabian experience.

OBJECTIVE: The objective of this study is to evaluate the cytological accuracy of endobronchial ultrasound-guided transbronchial fine-needle aspiration (EBUS-TFNA) of the mediastinal mass/nodular lesions. STUDY DESIGN: Over 3½ years from inception at King Khalid University Hospital, a retrospective analysis of the cytological diagnoses of all the EBUS-TFNA procedures performed in 80 patients who had mediastinal mass/nodular enlargement. Cytology results were reviewed and correlated with the histologic follow-up. RESULTS: Of the 80 patients who underwent EBUS-TFNA, 15 cases (18.75%) were positive for malignancy, 48 cases (60%) negative for malignancy and 17 cases (21.25%) unsatisfactory. Of the 48 cases, which were negative for malignancy, 24 (50%) cases were of granulomatous inflammation. The overall diagnostic yield of our EBUS-TFNA specimen was 78.75%. Forty-seven cases (58.75%) of 80 cases had histological follow-up biopsies. Among them, 32 cases (68%) had the same cytological and histological diagnosis and 15 cases (31.09%) had discordance between the cytology and the follow-up histological diagnosis. The sensitivity, specificity, and positive and negative predictive values for diagnosing granulomas by EBUS-TFNA are 77%, 82%, 83%, and 75% and for diagnosing malignancy are 71%, 100%, 100%, and 82%, respectively. CONCLUSION: Preliminary results show that cytological samples obtained through EBUS-TFNA are accurate and specific in making a diagnosis of the mediastinal mass/nodular lesions. Its optimum use depends on the effective collaboration between the cytotechnologist, pathologist, and the bronchoscopist.