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)-1alpha and activation of hypoxia-responsive genes, under these conditions. Inhibition of ERK1/2 signaling using U0126 enhanced HIF-1alpha stabilization, implicating ERK1/2 dephosphorylation as a contributing mechanism in NO-mediated HIF-1alpha activation. Activation of HIF-1alpha by the hypoxia mimic cobalt chloride, or cell transfection with a degradation-resistant HIF-1alpha mutant construct inhibited epithelial wound repair, implicating HIF-1alpha 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-1alpha. Finally, NO-mediated inhibition of cell migration was associated with HIF-1alpha-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-1alpha and p53, with potential consequences for epithelial repair and remodeling during airway inflammation.
Highlights
JUNE 27, 2008 VOLUME 283 NUMBER 26 tion 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]
Our results suggest that elevated concentrations of NO inhibit epithelial cell migration and wound repair in large part by two interdependent mechanisms, including induction of the MAPK phosphatase MAPK phosphatase-1 (MKP-1) and consequent inhibition of ERK1/2 activation, as well as stabilization and activation of the transcription factors hypoxia-inducible factor (HIF)-1␣ and p53
The addition of the MEK1/2 inhibitor U0126 (10 M) or the PI3K inhibitor LY294002 (10 M) dramatically inhibited wound closure of injured HBE1 monolayers (Fig. 2C), illustrating the involvement of these signaling pathways in wound closure. These results suggest that NO-mediated inhibition of epithelial wound closure is mediated by inhibitory effects on ERK1/2 and Akt signaling pathways, because of cGMP-independent induction of the extracellular signal-regulated kinase (ERK) phosphatase MKP-1
Summary
NO to modulate cell signaling pathways and gene expression has been intensively studied and involves various diverse mechanisms (19 –22). HIF-1 mediates adaptive responses to hypoxia but is recognized to play important roles in inflammatory processes [31], and recent studies have demonstrated HIF-1␣ activation during chronic airway inflammation [32, 33], conditions typically associated with increased NOS2 expression. The present studies were undertaken to determine the impact of inflammatory concentrations of NO on bronchial epithelial wound repair and cell migration in vitro and to identify the major signaling pathways involved in these responses. Our results suggest that elevated concentrations of NO inhibit epithelial cell migration and wound repair in large part by two interdependent mechanisms, including induction of the MAPK phosphatase MKP-1 and consequent inhibition of ERK1/2 activation, as well as stabilization and activation of the transcription factors HIF-1␣ and p53. In addition to potential direct effects of these signaling changes on actin cytoskeletal organization, they were associated with NO-dependent changes in expression of a number of genes involved in epithelial migration and remodeling, such as MMP-9 and PAI-1
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