RESUMO
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.
Assuntos
Brônquios/metabolismo , Fibrose Cística/metabolismo , Regulação para Baixo , Células Epiteliais/metabolismo , Heme Oxigenase-1/biossíntese , Homeostase , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Ferro/metabolismo , Proteínas de Membrana/biossíntese , Mucosa Respiratória/metabolismo , Receptor 4 Toll-Like/biossíntese , Animais , Brônquios/patologia , Linhagem Celular , Fibrose Cística/genética , Fibrose Cística/patologia , Estabilidade Enzimática/genética , Células Epiteliais/patologia , Heme Oxigenase-1/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mucosa Respiratória/patologia , Receptor 4 Toll-Like/genéticaRESUMO
BACKGROUND: The stress-regulated enzyme hemeoxygenase-1 (HO-1) contributes to the cell response towards inflammation and oxidative stress. We previously reported on curtailed HO-1 expression in cystic fibrosis (CF) bronchial epithelial (CFBE41o-) cells and CF-mice, but the molecular mechanisms for this are not known. Here, we compared healthy and CF bronchial epithelial cells for regulatory circuits controlling HO-1 protein levels. METHODS: In this study, we employed immunohistochemistry on CF and healthy lung sections to examine the BACH1 protein expression. Alteration of BACH1 protein levels in 16HBE14o- and CFBE41o- cells was achieved by using either siRNA-mediated knockdown of BACH1 or by increasing miRNA-155 levels. HO-1 luciferase reporter assay was chosen to examine the downstream affects after BACH1 modulation. RESULTS: Human CF lungs and cells showed increased levels of the HO-1 transcriptional repressor, BACH1, and increased miR-155 expression. Knockdown studies using BACH1 siRNA and overexpression of miR-155 did not significantly rescue HO-1 expression in CFBE41o- cells. Elevated BACH1 expression detected in CF cells was refractory to the inhibitory function of miR-155 and was instead due to increased protein stability. CONCLUSION: We observed defects in the inhibitory activities of miR-155 and BACH1 on HO-1 expression in CF cells. Thus various defective regulatory loops account for dysregulated BACH1 expression in CF, which in turn may contribute to low HO-1 levels.