Roshini Sathiaseelan

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and typically fatal lung disease with a very low survival rate. Excess accumulation of fibroblasts, myofibroblasts and extracellular matrix creates hypoxic conditions within the lungs, causing asphyxiation. Hypoxia is, therefore, one of the prominent features of IPF. However, there have been few studies concerning the effects of hypoxia on pulmonary fibroblasts. In this study, we investigated the molecular mechanisms of hypoxia-induced lung fibroblast proliferation. Hypoxia increased the proliferation of normal human pulmonary fibroblasts and IPF fibroblasts after exposure for 3-6 days. Cell cycle analysis demonstrated that hypoxia promoted the G1/S phase transition. Hypoxia downregulated cyclin D1 and A2 levels, while it upregulated cyclin E1 protein levels. However, hypoxia had no effect on the protein expression levels of cyclin-dependent kinase 2, 4, and 6. Chemical inhibition of hypoxia-inducible factor (HIF)-2 reduced hypoxia-induced fibroblast proliferation. Moreover, silencing of Nuclear Factor Activated T cell (NFAT) c2 attenuated the hypoxia-mediated fibroblasts proliferation. Hypoxia also induced the nuclear translocation of NFATc2, as determined by immunofluorescence staining. NFAT reporter assays showed that hypoxia-induced NFAT signaling activation is dependent on HIF-2, but not HIF-1. Furthermore, the inhibition or silencing of HIF-2, but not HIF-1, reduced the hypoxia-mediated NFATc2 nuclear translocation. Our studies suggest that hypoxia induces the proliferation of human pulmonary fibroblasts through NFAT signaling and HIF-2.

Long non-coding RNAs (lncRNAs) are a new arm of gene regulatory mechanism as discovered by sequencing techniques and follow-up functional studies. There are only few studies on lncRNAs as related to gene expression regulation and anti-viral activity during influenza virus infection. We sought to identify and characterize lncRNAs involved in influenza virus replication. Using RNA sequencing analysis, we found that 1,912 lncRNAs were significantly changed in human lung epithelial A549 cells infected with influenza A/Puerto Rico/8/34. Gene ontology analysis on neighboring genes of these lncRNAs revealed that the genes involved in type I interferon signaling and cellular response were highly enriched. Seven selected up-regulated lncRNAs (AC015849.2, RP-1-7H24.1, PSMB8-AS1, CTD-2639E6.9, PSOR1C3, AC007283.5 and RP11-670E13.5) were verified by real-time PCR. These lncRNAs were also induced by other two influenza H1N1 virus strains (A/WSN/1933 and A/Oklahoma/3052/09) and interferon β1. Repression of PSMB8 antisense RNA 1 (PSMB8-AS1) using CRISPR interference reduced viral mRNA and protein levels as well as the release of progeny influenza virus particles. Our study suggests that lncRNA PSMB8-AS1 could be a new host factor target for developing antiviral therapy against influenza virus infection.

Abstract Abnormal activation of lung fibroblasts contributes to the initiation and progression of idiopathic pulmonary fibrosis (IPF). The objective of the present study was to investigate the role of fetal-lethal noncoding developmental regulatory RNA (FENDRR) in the activation of lung fibroblasts. Dysregulated long noncoding RNAs in IPF lungs were identified by next-generation sequencing analysis from the two online datasets. FENDRR expression in lung tissues from patients with IPF and mice with bleomycin-induced pulmonary fibrosis was determined by quantitative real-time PCR. IRP1 (iron-responsive element–binding protein 1), a protein partner of FENDRR, was identified by RNA pulldown-coupled mass spectrometric analysis and confirmed by RNA immunoprecipitation. The interaction region between FENDRR and IRP1 was determined by cross-linking immunoprecipitation. The in vivo role of FENDRR in pulmonary fibrosis was studied using adenovirus-mediated gene transfer in mice. The expression of FENDRR was downregulated in fibrotic human and mouse lungs as well as in primary lung fibroblasts isolated from bleomycin-treated mice. TGF-β1 (transforming growth factor-β1)–SMAD3 signaling inhibited FENDRR expression in lung fibroblasts. FENDRR was preferentially localized in the cytoplasm of adult lung fibroblasts and bound IRP1, suggesting its role in iron metabolism. FENDRR reduced pulmonary fibrosis by inhibiting fibroblast activation by reducing iron concentration and acting as a competing endogenous RNA of the profibrotic microRNA-214. Adenovirus-mediated FENDRR gene transfer in the mouse lung attenuated bleomycin-induced lung fibrosis and improved lung function. Our data suggest that FENDRR is an antifibrotic long noncoding RNA and a potential therapeutic target for pulmonary fibrosis.

Metabolic dysfunction underlies several chronic diseases, many of which are exacerbated by obesity. Dietary interventions can reverse metabolic declines and slow aging, although compliance issues remain paramount. 17α-estradiol treatment improves metabolic parameters and slows aging in male mice. The mechanisms by which 17α-estradiol elicits these benefits remain unresolved. Herein, we show that 17α-estradiol elicits similar genomic binding and transcriptional activation through estrogen receptor α (ERα) to that of 17β-estradiol. In addition, we show that the ablation of ERα completely attenuates the beneficial metabolic effects of 17α-E2 in male mice. Our findings suggest that 17α-E2 may act through the liver and hypothalamus to improve metabolic parameters in male mice. Lastly, we also determined that 17α-E2 improves metabolic parameters in male rats, thereby proving that the beneficial effects of 17α-E2 are not limited to mice. Collectively, these studies suggest ERα may be a drug target for mitigating chronic diseases in male mammals.