Wang L, Zhen H, Yao W, Bian F, Zhou F, Mao X, Yao P, Jin S. Lipid raft-dependent activation of dual oxidase 1/H2O2/NF-kappa B pathway in bronchial epithelial cells. Am J Physiol Cell Physiol 301: C171-C180, 2011. First published March 9, 2011; doi:10.1152/ajpcell.00363.2010.-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 p47(phox) due to lipid raft (LR) clustering. By gradient ultracentrifugation to isolate LR fractions in bronchial epithelial cells, it was found that Duox1 or p47(phox) was translocated into LR fractions when stimulated by tumor necrosis factor-alpha (TNF-alpha). Confocal microscopic analysis revealed that LRs were aggregated or clustered in the membrane, which were colocalized with Duox1 or p47(phox). Ceramide, a hydrolysis product of sphingomyelin, was also found colocalized with Duox1 or p47(phox) upon stimulation. In the presence of the commonly used LR disruptor, methyl-beta-cyclodextrin (MCD), or the acid sphingomyelinase (ASMase) inhibitor, desipramine (DES), TNF-alpha-stimulated aggregation, translocation, and colocalization of LR components and Duox1 or its partners was abolished. Functionally, TNF-alpha-stimulated H2O2 production was also blocked by MCD and DES (194.6 +/- 15.4% vs. 90.6 +/- 15.9% and 148.8 +/- 20.4%), and the activation of the pivotal proinflammatory transcription factor, NF-kappa B, by TNF-alpha was reversed by MCD and DES as well as by small interfering RNAs of Duox1 or ASMase. Our results for the first time demonstrate that Duox1-mediated redox signaling in bronchial epithelial cells is associated with LR clustering dependent on the production of ceramide through ASMase.