Bojana Stanic

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

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 &kgr;B in vascular smooth muscle cells (SMCs). Tumor necrosis factor-&agr; and interleukin (IL)-1&bgr; stimulation of SMCs resulted in diphenylene iodonium-sensitive ROS production within intracellular vesicles. Nox1 and p22phox, 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-&agr; and interleukin-1&bgr; generation of ROS. Cytokine activation of nuclear factor &kgr;B in SMCs required both Nox1 and ClC-3. We conclude that in response to tumor necrosis factor-&agr; and interleukin-1&bgr;, 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.

Role for Nox1 NADPH oxidase in atherosclerosis

OBJECTIVE Examine the contribution of Nox1 NADPH oxidase to atherogenesis. METHODS AND RESULTS Male apolipoprotein E deficient mice (ApoE(-/-)) and male mice deficient in both apolipoprotein E and Nox1 (ApoE(-/-) Nox1(-/y)) received an atherogenic diet for 18 weeks. Mean blood pressures, body weights, and serum cholesterol levels were similar between the two groups of mice. Deficiency of Nox1 decreased superoxide levels and reduced lesion area in the aortic arch from 43% (ApoE(-/-)) to 28% (ApoE(-/-) Nox1(-/y)). The reduction in lesion size at the level of the aortic valve in ApoE(-/-)/Nox1(-/y) was accompanied by a decrease in macrophage infiltration as compared to ApoE(-/-) mice. Carotid artery ligation in ApoE(-/-) mice induced accelerated intimal hyperplasia with decreased cellular proliferation and increased collagen content in the neointima of vessels deficient in Nox1. CONCLUSIONS Nox1-derived ROS modify lesion composition and contribute to lesion size in a murine model of atherosclerosis.

A Differential Role for Endocytosis in Receptor-Mediated Activation of Nox1

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)-alpha or thrombin, SMCs increased cellular levels of ROS that was inhibited by shRNA to Nox1. Treatment of SMC with TNF-alpha 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-alpha 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.

An Oxidized Extracellular Oxidation-Reduction State Increases Nox1 Expression and Proliferation in Vascular Smooth Muscle Cells Via Epidermal Growth Factor Receptor Activation

Objective—To examine the effect of an oxidized extracellular oxidation-reduction (redox) state (Eh) on the expression of NADPH oxidases in vascular cells. Methods and Results—The generation of reactive oxygen species by NADPH oxidase (Nox)-based NADPH oxidases activates redox-dependent signaling pathways and contributes to the development of “oxidative stress” in vascular disease. An oxidized plasma redox state is associated with cardiovascular disease in humans; however, the cellular mechanisms by which the extracellular redox state may cause disease are not known. Aortic segments and cultured aortic smooth muscle cells were exposed to Eh between −150 mV (reduced) and 0 mV (oxidized) by altering the concentration of cysteine and its disulfide, cystine, the predominant redox couple in plasma. A more oxidized Eh increased the expression of Nox1 and resulted in Nox1-dependent proliferation of smooth muscle cells. Oxidized Eh rapidly induced epidermal growth factor receptor phosphorylation via shedding of epidermal growth factor-like ligands from the plasma membrane and caused extracellular signal-regulated kinase 1/2-dependent phosphorylation of the transcription factors activating transcription factor-1 and cAMP-response element-binding protein. Inhibition of epidermal growth factor receptor or extracellular signal-regulated kinase 1/2 activation, or addition of small interference RNA to activating transcription factor-1, prevented the increase in Nox1 expression. Conclusion—Our results identify a novel mechanism by which extracellular oxidative stress increases expression and activity of Nox1 NADPH oxidase and contributes to vascular disease.

A critical role for chloride channel-3 (CIC-3) in smooth muscle cell activation and neointima formation.

Objective—We have shown that the chloride-proton antiporter chloride channel-3 (ClC-3) is required for endosome-dependent signaling by the Nox1 NADPH oxidase in SMCs. In this study, we tested the hypothesis that ClC-3 is necessary for proliferation of smooth muscle cells (SMCs) and contributes to neointimal hyperplasia following vascular injury. Methods and Results—Studies were performed in SMCs isolated from the aorta of ClC-3-null and littermate control (wild-type [WT]) mice. Thrombin and tumor necrosis factor-&agr; (TNF-&agr;) each caused activation of both mitogen activated protein kinase extracellular signal-regulated kinases 1 and 2 and the matrix-degrading enzyme matrix metalloproteinase-9 and cell proliferation of WT SMCs. Whereas responses to thrombin were preserved in ClC-3-null SMCs, the responses to TNF-&agr; were markedly impaired. These defects normalized following gene transfer of ClC-3. Carotid injury increased vascular ClC-3 expression, and compared with WT mice, ClC-3-null mice exhibited a reduction in neointimal area of the carotid artery 28 days after injury. Conclusion—ClC-3 is necessary for the activation of SMCs by TNF-&agr; but not thrombin. Deficiency of ClC-3 markedly reduces neointimal hyperplasia following vascular injury. In view of our previous findings, this observation is consistent with a role for ClC-3 in endosomal Nox1-dependent signaling. These findings identify ClC-3 as a novel target for the prevention of inflammatory and proliferative vascular diseases.

Increased Epidermal Growth Factor–Like Ligands Are Associated With Elevated Vascular Nicotinamide Adenine Dinucleotide Phosphate Oxidase in a Primate Model of Atherosclerosis

Objective—To characterize the relationship between the expression of epidermal growth factor (EGF)-like ligands and vascular nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression and activity in a primate model of atherosclerosis. Methods and Results—Adult male Cynomolgus monkeys were fed a normal or atherogenic (AS) diet for 45 months, after which animals from the AS group were placed on a normal diet for 8 months (regression). The expression of membrane-associated EGF-like ligands was increased in arteries from animals on the AS diet and normalized in the regression group. EGF-like ligands were distributed throughout atherosclerotic vessels but predominantly colocalized with macrophages. Consistent with ligand shedding, circulating heparin-bound EGF was elevated in the plasma of AS monkeys but not in those on regression diet. Atherosclerosis was associated with the activation of EGF receptor signaling. Expression of NADPH oxidase subunits Nox1 and Nox2 but not Nox4 or Nox5 was increased in arteries from monkeys on the AS diet and returned to normal with regression. Levels of Nox1 and Nox2 positively correlated with EGF-like ligands. In cultured monkey smooth muscle cells, treatment with EGF-like ligands increased Nox1 expression and activity. Conclusion—These data identify EGF-like ligands as potential modulators of atherogenesis, resulting in part from increased vascular NADPH oxidase activity.

Activation of NADPH Oxidase 1 Increases Intracellular Calcium and Migration of Smooth Muscle Cells

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 ([Ca2+]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 [Ca2+]i, as measured by fura-2 fluorescence ratio imaging, in wild-type but not Nox1 null SMCs. The increase in [Ca2+]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 [Ca2+]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 Ca2+ entry, cells were either bathed in Ca2+-free medium or exposed to dihydropyridines to block L-type Ca2+ channel activity. Treatment with nifedipine or removal of extracellular Ca2+ reduced the thrombin-mediated increase of [Ca2+]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 [Ca2+]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 Ca2+ mobilization in SMCs, in part through regulation of Ca2+ influx, to thereby promote cell migration.

Phosphorylation of Nox1 Regulates Association With NoxA1 Activation Domain

Rationale: Activation of Nox1 initiates redox-dependent signaling events crucial in the pathogenesis of vascular disease. Selective targeting of Nox1 is an attractive potential therapy, but requires a better understanding of the molecular modifications controlling its activation. Objective: To determine whether posttranslational modifications of Nox1 regulate its activity in vascular cells. Methods and Results: We first found evidence that Nox1 is phosphorylated in multiple models of vascular disease. Next, studies using mass spectroscopy and a pharmacological inhibitor demonstrated that protein kinase C-beta1 mediates phosphorylation of Nox1 in response to tumor necrosis factor-&agr;. siRNA-mediated silencing of protein kinase C-beta1 abolished tumor necrosis factor-&agr;–mediated reactive oxygen species production and vascular smooth muscle cell migration. Site-directed mutagenesis and isothermal titration calorimetry indicated that protein kinase C-beta1 phosphorylates Nox1 at threonine 429. Moreover, Nox1 threonine 429 phosphorylation facilitated the association of Nox1 with the NoxA1 activation domain and was necessary for NADPH oxidase complex assembly, reactive oxygen species production, and vascular smooth muscle cell migration. Conclusions: We conclude that protein kinase C-beta1 phosphorylation of threonine 429 regulates activation of Nox1 NADPH oxidase.

51 CLC-3 IS REQUIRED FOR NADPH OXIDASE-DEPENDENT NUCLEAR FACTOR κB ACTIVATION BY SIGNALING ENDOSOMES.

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. Here we show that activation of NADPH oxidase by inflammatory cytokines generates ROS within early endosomes and requires ClC-3, a member of the chloride channel (ClC) family. Nox1, one of multiple membrane-bound catalytic subunits of NADPH oxidase, colocalizes with ClC-3 in early endosomes. Both Nox1 and ClC-3 are necessary for tumor necrosis factor α and interleukin-1β generation of ROS and subsequent activation of the transcription factor NF-κB. We propose that ClC-3 functions as a chloride-proton exchanger and thereby influences ROS production via charge neutralization of the electron flow generated by Nox1 in the endosome. These findings identify ClC-3 as a critical component of the signaling endosome and a novel intermediate in redox-dependent control of gene expression.