title: LPS-mediated endothelial activation in pulmonary endothelial cells: role of Nox2-dependent IKK-β phosphorylation.
abstract: Lipopolysaccharide (LPS)-mediated endothelial activation contributes to lung inflammation and alveolar remodeling seen in premature infants with bronchopulmonary dysplasia (BPD). The mechanisms underlying LPS-mediated oxidative stress and proinflammatory signaling in human pulmonary microvascular endothelial cells (HPMEC) remain unclear. We hypothesized that NADPH oxidase (Nox) mediates LPS-induced endothelial activation in HPMEC by regulating phosphorylation of Toll-like receptor (TLR) pathway proteins. LPS-induced expression of intercellular adhesion molecule 1 (ICAM-1) was associated with increased 2-OH-E(+) (marker for superoxide formation) levels and was attenuated by apocynin and the Nox inhibitor, VAS2870. LPS triggered membrane translocation of p67phox, suggesting activation of Nox2. Silencing Nox2, but not Nox4, suppressed LPS-induced ICAM-1 expression in HPMEC. Immunoprecipitation studies showed that inhibitor of κ-B kinase-β (IKK-β) serine phosphorylation induced by LPS was inhibited by Nox2 silencing. We examined whether Nox2-dependent, LPS-mediated IKK-β phosphorylation was regulated by protein phosphatase 2A (PP2A) or TGF-β associated kinase-1 (TAK1) in HPMEC. LPS increased PP2A activity in HPMEC, and inhibition of PP2A did not alter LPS-mediated ICAM-1 expression but attenuated IKK-β phosphorylation. TAK1 inhibition decreased LPS-induced ICAM-1 expression in HPMEC, and Nox2 silencing attenuated LPS-mediated TAK1 phosphorylation (Thr184/187). We demonstrate that Nox2 regulates LPS-mediated endothelial activation in pulmonary endothelial cells by modulating phosphorylation of key kinases in the TLR signaling cascade. Our data support a novel mechanism by which Nox-dependent signaling regulates proinflammatory signaling in pulmonary endothelial cells. Inhibition of vascular Nox may potentially limit lung injury and alveolar remodeling caused by infections in BPD.
authors: Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA.
authors: Heather
authors: H
authors: Menden
authors: Everett
authors: E
authors: Tate
authors: Neil
authors: N
authors: Hogg
authors: Venkatesh
authors: V
authors: Sampath
mesh_terms: D002478:Cells, Cultured; D042783:Endothelial Cells / Q000201:enzymology* / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism*; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008168:Lung / Q000098:blood supply; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D055806:Microvessels / Q000166:cytology*; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D054648:Protein Phosphatase 2 / Q000378:metabolism / Q000502:physiology; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D013481:Superoxides / Q000378:metabolism; D000099151:MAP Kinase Kinase Kinase 7
chemical_list: D051550:I-kappa B Kinase; D018799:Intercellular Adhesion Molecule-1; D007527:Isoenzymes; D008070:Lipopolysaccharides; D020930:MAP Kinase Kinase Kinases; D008562:Membrane Glycoproteins; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; D010750:Phosphoproteins; D054648:Protein Phosphatase 2; D034741:RNA, Small Interfering; D013481:Superoxides; D000099151:MAP Kinase Kinase Kinase 7; C573420:ICAM1 protein, human; C079789:neutrophil cytosol factor 67K; C056558:CYBB protein, human; C498910:NOX4 protein, human