Lipopolysaccharide-induced cytokine expression in alveolar epithelial cells: Role of PKCζ-mediated p47phox phosphorylation

Chronic inflammation incited by bacteria in the saccular lung of premature infants contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). LPS-mediated type II alveolar epithelial cell (AEC) injury induces the expression of pro-inflammatory cytokines that trigger pulmonary neutrophil influx, alveolar matrix degradation and lung remodeling. We hypothesized that NADPH oxidase (Nox)-dependent mechanisms mediate LPS-induced cytokine expression in AEC. We examined the role of p47phox in mediating LPS-dependent inflammatory cytokine expression in A549 cells (which exhibit phenotypic features characteristic of type II AEC) and elucidated the proximal signaling events by which Nox is activated by LPS. LPS-induced ICAM-1 and IL-8 expression was associated with increased superoxide formation in AEC. LPS-mediated oxidative stress and cytokine expression was inhibited by apocynin and augmented by PMA demonstrating that Nox-dependent redox signaling regulates LPS-dependent pro-inflammatory signaling in AEC. In LPS-treated cells, p47phox translocated from the cytoplasm to the perinuclear region and co-localized with gp91phox. LPS also induced a temporal increase in p47phox serine304 phosphorylation in AEC. While inhibition of classical PKC and novel PKC with calphostin and rottlerin did not inhibit ICAM-1 or IL-8 expression, the myristolyated PKC zeta pseudosubstrate peptide (a specific inhibitor of PKC zeta) inhibited LPS-induced cytokine expression in AEC. Inhibition of PKC zeta also attenuated LPS-mediated p47phox phosphorylation and perinuclear translocation in AEC. Consistent with these data, LPS activated PKC zeta in AEC as evidenced by increased threonine410 phophorylation. We conclude that PKC zeta-mediated p47phox activation regulates LPS-dependent cytokine expression in AEC. Selective inhibition of PKC zeta or p47phox might attenuate LPS-mediated inflammation and alveolar remodeling in BPD. (C) 2010 Elsevier Ireland Ltd. All rights reserved.