Lipid raft-dependent activation of dual oxidase 1/H2O2/NF-κB pathway in bronchial epithelial cells.

The present study addressed whether dual oxidase 1 (Duox1), a predominant isoform of NADPH oxidase in bronchial epithelial cells, is also activated through assembling of Duox1 and its partners such as p47phox due to lipid raft (LR) clustering. By gradient ultracentrifugation to isolate LR fractions in bronchial epithelial cells, it was found that Duox1 or p47phox was translocated into LR fractions when stimulated by tumor necrosis factor-α (TNF-α). Confocal microscopic analysis revealed that LRs were aggregated or clustered in the membrane, which were colocalized with Duox1 or p47phox. Ceramide, a hydrolysis product of sphingomyelin, was also found colocalized with Duox1 or p47phox upon stimulation. In the presence of the commonly used LR disruptor, methyl-β-cyclodextrin (MCD), or the acid sphingomyelinase (ASMase) inhibitor, desipramine (DES), TNF-α-stimulated aggregation, translocation, and colocalization of LR components and Duox1 or its partners was abolished. Functionally, TNF-α-stimulated H2O2 produ...