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 κB in vascular smooth muscle cells (SMCs). Tumor necrosis factor-α and interleukin (IL)-1β 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-α and interleukin-1β generation of ROS. Cytokine activation of nuclear factor κB in SMCs required both Nox1 and ClC-3. We conclude that in response to tumor necrosis factor-α and interleukin-1β, 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.

Internalization of activated receptors to a compartment enriched with NAPDH oxidase and associated signaling molecules is expected to facilitate regulation of redox-mediated signal transduction. The aim of this study was to test the hypothesis that endocytosis is necessary for generation of reactive oxygen species (ROS) by Nox1 and for redox-dependent signaling in smooth muscle cells (SMCs). Within minutes of treatment with tumor necrosis factor (TNF)-α or thrombin, SMCs increased cellular levels of ROS that was inhibited by shRNA to Nox1. Treatment of SMC with TNF-α induced a dynamin-dependent endosomal generation of ROS, whereas thrombin-mediated ROS production did not occur within endosomes and was not prevented by dominant-negative dynamin (dn-dynamin), but instead required transactivation of the epidermal growth factor receptor (EGFR). Activation of the phosphatidylinositol 3-kinase (PI3K)-Akt-activating transcription factor-1 (ATF-1) pathway by TNF-α and thrombin were both Nox1- and dynamin-dependent. In conclusion, we show that formation of specific ligand–receptor complexes results in spatially distinct mechanisms of Nox1 activation and generation of ROS. These findings provide novel insights into the role of compartmentalization for integrating redox-dependent cell signaling. Antioxid. Redox Signal. 12, 583–593.

Redox-dependent migration and proliferation of vascular smooth muscle cells (SMCs) are central events in the development of vascular proliferative diseases; however, the underlying intracellular signaling mechanisms are not fully understood. We tested the hypothesis that activation of Nox1 NADPH oxidase modulates intracellular calcium ([Ca ²⁺ ] _i ) levels. Using cultured SMCs from wild-type and Nox1 null mice, we confirmed that thrombin-dependent generation of reactive oxygen species requires Nox1. Thrombin rapidly increased [Ca ²⁺ ] _i , as measured by fura-2 fluorescence ratio imaging, in wild-type but not Nox1 null SMCs. The increase in [Ca ²⁺ ] _i in wild-type SMCs was inhibited by antisense to Nox1 and restored by expression of Nox1 in Nox1 null SMCs. Investigation into potential mechanisms by which Nox1 modulates [Ca ²⁺ ] _i showed that thrombin-induced inositol triphosphate generation and thapsigargin-induced intracellular calcium mobilization were similar in wild-type and Nox1 null SMCs. To examine the effects of Nox1 on Ca ²⁺ entry, cells were either bathed in Ca ²⁺ -free medium or exposed to dihydropyridines to block l -type Ca ²⁺ channel activity. Treatment with nifedipine or removal of extracellular Ca ²⁺ reduced the thrombin-mediated increase of [Ca ²⁺ ] _i in wild-type SMCs, whereas the response in Nox1 null SMCs was unchanged. Sodium vanadate, an inhibitor of protein tyrosine phosphatases, restored the thrombin-induced increase of [Ca ²⁺ ] _i in Nox1 null SMCs. Migration of SMCs was impaired with deficiency of Nox1 and restored with expression of Nox1 or the addition of sodium vanadate. In summary, we conclude that Nox1 NADPH oxidase modulates Ca ²⁺ mobilization in SMCs, in part through regulation of Ca ²⁺ influx, to thereby promote cell migration.