title: Impaired TLR4 and HIF expression in cystic fibrosis bronchial epithelial cells downregulates hemeoxygenase-1 and alters iron homeostasis in vitro.
abstract: Hemeoxygenase-1 (HO-1), an inducible heat shock protein, is upregulated in response to multiple cellular insults via oxidative stress, lipopolysaccharides (LPS), and hypoxia. In this study, we investigated in vitro the role of Toll-like receptor 4 (TLR4), hypoxia-inducible factor 1α (HIF-1α), and iron on HO-1 expression in cystic fibrosis (CF). Immunohistochemical analysis of TLR4, HO-1, ferritin, and HIF-1α were performed on lung sections of CFTR-/- and wild-type mice. CFBE41o- and 16HBE14o- cell lines were employed for in vitro analysis via immunoblotting, immunofluorescence, real-time PCR, luciferase reporter gene analysis, and iron quantification. We observed a reduced TLR4, HIF-1α, HO-1, and ferritin in CFBE41o- cell line and CF mice. Knockdown studies using TLR4-siRNA in 16HBE14o- revealed significant decrease of HO-1, confirming the role of TLR4 in HO-1 downregulation. Inhibition of HO-1 using tin protoporphyrin in 16HBE14o- cells resulted in increased iron levels, suggesting a probable role of HO-1 in iron accumulation. Additionally, sequestration of excess iron using iron chelators resulted in increased hypoxia response element response in CFBE41o- and 16HBE14o-, implicating a role of iron in HIF-1α stabilization and HO-1. To conclude, our in vitro results demonstrate that multiple regulatory factors, such as impaired TLR4 surface expression, increased intracellular iron, and decreased HIF-1α, downregulate HO-1 expression in CFBE41o- cells.
authors.affiliation: Department of Medicine, Pulmonary Critical Care Philipps University, Marburg, Germany; Institute for Lung Research, Philipps-University, Marburg, Germany; Universities of Giessen and Marburg Lung Center (UGMLC), Giessen, Germany; Member of the German Center for Lung Research (DZL);
authors.forename: Shashi
authors.initials: S
authors.lastname: Chillappagari
authors.affiliation: Department of Internal Medicine, Justus-Liebig-University, Giessen, Germany; Universities of Giessen and Marburg Lung Center (UGMLC), Giessen, Germany; Member of the German Center for Lung Research (DZL);
authors.forename: Shalini
authors.initials: S
authors.lastname: Venkatesan
authors.affiliation: Department of Medicine, Pulmonary Critical Care Philipps University, Marburg, Germany; Institute for Lung Research, Philipps-University, Marburg, Germany; Universities of Giessen and Marburg Lung Center (UGMLC), Giessen, Germany; Member of the German Center for Lung Research (DZL);
authors.forename: Virajith
authors.initials: V
authors.lastname: Garapati
authors.affiliation: Department of Internal Medicine, Justus-Liebig-University, Giessen, Germany; Universities of Giessen and Marburg Lung Center (UGMLC), Giessen, Germany; Member of the German Center for Lung Research (DZL);
authors.forename: Poornima
authors.initials: P
authors.lastname: Mahavadi
authors.affiliation: Clinical Research Group 'Molecular Pathology of Cystic Fibrosis and Pseudomonas Genomics', Clinic for Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany; Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Germany;
authors.forename: Antje
authors.initials: A
authors.lastname: Munder
authors.affiliation: Department of Chemistry-Biochemistry, Philipps University, Marburg, Germany;
authors.forename: Andreas
authors.initials: A
authors.lastname: Seubert
authors.affiliation: Department of Medicine, Pulmonary Critical Care Philipps University, Marburg, Germany; Universities of Giessen and Marburg Lung Center (UGMLC), Giessen, Germany;
authors.forename: Gaurav
authors.initials: G
authors.lastname: Sarode
authors.affiliation: Department of Internal Medicine, Justus-Liebig-University, Giessen, Germany; Lung Clinic Waldhof-Elgershausen, Greifenstein, Germany; Universities of Giessen and Marburg Lung Center (UGMLC), Giessen, Germany; Member of the German Center for Lung Research (DZL);
authors.forename: Andreas
authors.initials: A
authors.lastname: Guenther
authors.affiliation: Institute for Lung Research, Philipps-University, Marburg, Germany; Universities of Giessen and Marburg Lung Center (UGMLC), Giessen, Germany; Member of the German Center for Lung Research (DZL);
authors.forename: Bernd T
authors.initials: BT
authors.lastname: Schmeck
authors.affiliation: Clinical Research Group 'Molecular Pathology of Cystic Fibrosis and Pseudomonas Genomics', Clinic for Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany; Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Germany;
authors.forename: Burkhard
authors.initials: B
authors.lastname: Tümmler
authors.affiliation: Department of Medicine, Pulmonary Critical Care Philipps University, Marburg, Germany; Universities of Giessen and Marburg Lung Center (UGMLC), Giessen, Germany; Member of the German Center for Lung Research (DZL); Pneumology, Asklepios Fachkliniken München-Gauting, Germany; Comprehensive Pneumology Center (CPC), Helmholtz Zentrum, Munich, Germany m.henke@asklepios.com.
authors.forename: Markus O
authors.initials: MO
authors.lastname: Henke
mesh_terms: D000818:Animals; D001980:Bronchi / Q000378:metabolism* / Q000473:pathology; D002460:Cell Line; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D015536:Down-Regulation; D004795:Enzyme Stability / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D051547:Heme Oxygenase-1 / Q000096:biosynthesis* / Q000235:genetics; D006706:Homeostasis; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis* / Q000235:genetics; D007501:Iron / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D020545:Respiratory Mucosa / Q000378:metabolism* / Q000473:pathology; D051197:Toll-Like Receptor 4 / Q000096:biosynthesis* / Q000235:genetics
chemical_list: C497442:HIF1A protein, human; C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008565:Membrane Proteins; C495345:TLR4 protein, human; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D007501:Iron; C496541:HMOX1 protein, human; D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse
keywords: Toll-like receptor 4; cystic fibrosis; hypoxia-inducible factor 1α; iron