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(Circulation Research. 2007;101:663.)
© 2007 American Heart Association, Inc.

Molecular Medicine

Cytokine Activation of Nuclear Factor B in Vascular Smooth Muscle Cells Requires Signaling Endosomes Containing Nox1 and ClC-3

Francis J. Miller, Jr^*, Mohammed Filali^*, Gina J. Huss, Bojana Stanic, Ali Chamseddine, Thomas J. Barna, Fred S. Lamb

From the Departments of Medicine (F.J.M., B.S., A.C.)¹ and Pediatrics (M.F., G.J.H., T.J.B., F.S.L.), University of Iowa, Iowa City.

Correspondence Francis J. Miller Jr, Department of Internal Medicine, 200 Hawkins Dr, Rm E314-4 GH, Iowa City, IA 52242. E-mail francis-miller{at}uiowa.edu

Reactive oxygen species (ROS) are mediators of intracellular signals for a myriad of normal and pathologic cellular events, including differentiation, hypertrophy, proliferation, and apoptosis. NADPH oxidases are important sources of ROS that are present in diverse tissues throughout the body and activate many redox-sensitive signal transduction and gene expression pathways. To avoid toxicity and provide specificity of signaling, ROS production and metabolism necessitate tight regulation that likely includes subcellular compartmentalization. However, the constituent elements of NADPH oxidase-dependent cell signaling are not known. To address this issue, we examined cytokine generation of ROS and subsequent activation of the transcription factor nuclear factor B in vascular smooth muscle cells (SMCs). Tumor necrosis factor- and interleukin (IL)-1ß stimulation of SMCs resulted in diphenylene iodonium-sensitive ROS production within intracellular vesicles. Nox1 and p22^phox, integral membrane subunits of NADPH oxidase, coimmunoprecipitated with early endosomal markers in SMCs. ClC-3, an anion transporter that is primarily found in intracellular vesicles, also colocalized with Nox1 in early endosomes and was necessary for tumor necrosis factor- and interleukin-1ß generation of ROS. Cytokine activation of nuclear factor B in SMCs required both Nox1 and ClC-3. We conclude that in response to tumor necrosis factor- and interleukin-1ß, NADPH oxidase generates ROS within early endosomes and that Nox1 cannot produce sufficient ROS for cell signaling in the absence of ClC-3. These data best support a model whereby ClC-3 is required for charge neutralization of the electron flow generated by Nox1 across the membrane of signaling endosomes.

Key Words: smooth muscle cells • NAPDH oxidase • cell signaling • ion channels

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