Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1α

Abstract

Increased synthesis of NO during airway inflammation, caused by induction of nitric-oxide synthase 2 in several lung cell types, may contribute to epithelial injury and permeability. To investigate the consequence of elevated NO production on epithelial function, we exposed cultured monolayers of human bronchial epithelial cells to the NO donor diethylenetriaamine NONOate. At concentrations generating high nanomolar levels of NO, representative of inflammatory conditions, diethylenetriaamine NONOate markedly reduced wound closure in an in vitro scratch injury model, primarily by inhibiting epithelial cell migration. Analysis of signaling pathways and gene expression profiles indicated a rapid induction of the mitogen-activated protein kinase phosphatase (MPK)-1 and decrease in extracellular signal-regulated kinase (ERK)1/2 activation, as well as marked stabilization of hypoxia-inducible factor (HIF)-1␣ and activation of hypoxia-responsive genes, under these conditions. Inhibition of ERK1/2 signaling using U0126 enhanced HIF-1␣ stabilization, implicating ERK1/2 dephosphorylation as a contributing mechanism in NO-mediated HIF-1␣ activation. Activation of HIF-1␣ by the hypoxia mimic cobalt chloride, or cell transfection with a degradation-resistant HIF-1␣ mutant construct inhibited epithelial wound repair, implicating HIF-1␣ in NO-mediated inhibition of cell migration. Conversely, NO-mediated inhibition of epithelial wound closure was largely prevented after small interfering RNA suppression of HIF-1␣. Finally, NO-mediated inhibition of cell migration was associated with HIF-1␣-dependent induction of PAI-1 and activation of p53, both negative regulators of epithelial cell migration. Collectively, our results demonstrate that inflammatory levels of NO inhibit epithelial cell migration, because of suppression of ERK1/2 signaling, and activation of HIF-1␣ and p53, with potential consequences for epithelial repair and remodeling during airway inflammation.Inflammatory diseases of the respiratory tract are commonly associated with increased production of NO, because of induction and activation of the inducible isoform of nitric-oxide synthase (NOS2), 2 within inflammatory immune cells as well as within the respiratory epithelium (1, 2). Indeed, elevated concentrations of exhaled NO and increased epithelial expression of NOS2 are characteristic features of chronic inflammatory airway diseases such as asthma (1, 2). The biological roles of NOS2 within the airway epithelium include contribution to innate host defense (3, 4), regulation of epithelial ion transport (5), and maintenance of epithelial barrier integrity (1, 6, 7). In addition, our recent studies showed that physiological concentrations of NO can promote airway epithelial cell migration and repair in response to in vitro injury, which was associated with increased expression and activation of gelatinase B (matrix metalloproteinase-9; MMP-9) (8). In contrast to these salutary properties of constitutive airway NO production, increased epithelial NOS2 expression ha...

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Modulation of NF-κB and Hypoxia-Inducible Factor−1 byS-Nitrosoglutathione Does Not Alter Allergic Airway Inflammation in Mice

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Keratin 8 phosphorylation regulates keratin reorganization and migration of epithelial tumor cells

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