title: Upregulation of phosphoinositide 3-kinase and protein kinase B in alveolar macrophages following ozone inhalation. Role of NF-kappaB and STAT-1 in ozone-induced nitric oxide production and toxicity.
abstract: Inhalation of toxic doses of ozone causes lung injury and inflammation in humans and experimental animals. Using a rodent model of ozone toxicity, we have previously demonstrated that macrophages recruited to the lung following exposure to this oxidant contribute to the pathogenesis of tissue injury. In the present studies we analyzed potential mechanisms regulating alveolar macrophage activity following ozone inhalation and the role of inflammatory mediators in toxicity. Treatment of mice with ozone (0.8 ppm, 3 h) resulted in increased expression of inducible nitric oxide synthase (iNOS) protein and production of nitric oxide (NO) and peroxynitrite by alveolar macrophages. In contrast, these effects were not observed in macrophages from transgenic mice with a targeted disruption of the gene for iNOS, or in mice overexpressing superoxide dismutase. Moreover, ozone toxicity, as measured by bronchoalveolar lavage protein levels and nitrotyrosine staining of the lung was prevented in both of these transgenic mouse strains. The promoter/enhancer region of the iNOS gene contains binding sites for the transcription factors NF-kappaB and STAT-1 which regulate the activity of the gene. Ozone inhalation resulted in a rapid and prolonged activation of NF-kappaB in alveolar macrophages. Phosphoinositide 3-kinase (PI 3-K) and its down stream target, protein kinase B (PKB), which are known to regulate NF-kappaB activity, also increased in alveolar macrophages following ozone inhalation. These data, together with our findings that inhibitors of PI 3-K block NO production, suggest that these proteins are important in controlling expression of iNOS. Furthermore, the fact that macrophages from NF-kappaB p50 knockout mice did not generate reactive nitrogen intermediates and that these mice were protected from ozone induced toxicity demonstrate the importance of the NF-kappaB signaling pathway in lung injury. We also found that STAT-1 nuclear binding activity and STAT-1 protein expression were upregulated in macrophages from ozone treated animals. Taken together, these data suggest that biochemical signaling pathways that control the expression of genes critical for the inflammatory process play a role in ozone toxicity.
authors.affiliation: Environmental and Occupational Health Sciences Institute, Rutgers University and University of Medicine and Dentistry of New Jersey, Piscataway, USA. laskin@eohsi.rutgers.edu
authors.forename: Debra L
authors.initials: DL
authors.lastname: Laskin
authors.forename: Ladan
authors.initials: L
authors.lastname: Fakhrzadeh
authors.forename: Diane E
authors.initials: DE
authors.lastname: Heck
authors.forename: Donald
authors.initials: D
authors.lastname: Gerecke
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Laskin
mesh_terms: D000280:Administration, Inhalation; D000818:Animals; D001992:Bronchoalveolar Lavage Fluid; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D016676:Macrophages, Alveolar / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D016328:NF-kappa B / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D010126:Ozone / Q000008:administration & dosage / Q000494:pharmacology / Q000633:toxicity*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D050794:STAT1 Transcription Factor; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D016328:NF-kappa B; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D010126:Ozone; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D050794:STAT1 Transcription Factor; D015534:Trans-Activators; C494080:Stat1 protein, mouse; C496317:Nos2 protein, mouse