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Circulation Research. 2007;101:663-671
Published online before print August 2, 2007, doi: 10.1161/CIRCRESAHA.107.151076
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(Circulation Research. 2007;101:663.)
© 2007 American Heart Association, Inc.

Molecular Medicine

Cytokine Activation of Nuclear Factor {kappa}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


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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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 {kappa}B in vascular smooth muscle cells (SMCs). Tumor necrosis factor-{alpha} and interleukin (IL)-1ß 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-{alpha} and interleukin-1ß generation of ROS. Cytokine activation of nuclear factor {kappa}B in SMCs required both Nox1 and ClC-3. We conclude that in response to tumor necrosis factor-{alpha} 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


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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In response to diverse extracellular stimuli, intracellular signaling is dependent on generation of reactive oxygen species (ROS) by NADPH oxidase. The catalytic core of NADPH oxidase consists of a membrane-bound flavocytochrome b558 composed of a Nox (NADPH oxidase) (reviewed elsewhere1) subunit and p22phox. The prototypical model of NAPDH oxidase is found in phagosomes, where the orientation and biochemical properties of this heterodimer obligate reduction of oxygen to superoxide on the side of the membrane opposite from where NADPH and the cytosolic subunits of the oxidase bind.2 Based on this orientation, activation of NADPH oxidase in nonphagocytes should generate superoxide into the extracellular space or, following endocytosis, into intracellular vesicles. We hypothesized that extracellular stimuli activating the Nox1-based NADPH oxidase would produce superoxide in endocytotic vesicles.

The phagocyte NADPH oxidase is electrogenic, moving electrons from cytoplasmic NADPH through the enzyme into the phagosome to reduce oxygen to superoxide. Without charge compensation, this electron flux rapidly depolarizes the membrane (the voltage in the cytoplasm becomes positive relative to the phagosome) and inactivates the oxidase.3 It is expected that activation of NADPH oxidase in nonphagocytes is subject to a similar requirement for charge neutralization. However, this mechanism has never been described.

With 9 members, the CLC family is the largest identified family of mammalian Cl– channels and its members function in a surprisingly diverse set of biological roles, as revealed by human genetic diseases and murine knockout phenotypes.4 Fractionation studies aimed at determining ClC-3 function have localized it to the membrane of intracellular compartments such as endosomes and synaptic vesicles, where it has been proposed to facilitate acidification.5–8 The identification of ClC-4 and ClC-5, close structural relatives of ClC-3, as chloride-proton antiporters rather than anion channels has placed this role in question.9,10 We have previously demonstrated that ClC-3 is required for a normal oxidative burst in neutrophils.11 Based on these observations, we hypothesized that ClC-3 functions as a chloride–proton exchanger and is required for charge neutralization of the electron flow generated by NADPH oxidase in endosomes of nonphagocytes.

Our data show that in response to inflammatory cytokines, a Nox1-based NADPH oxidase generates ROS within intracellular vesicles of smooth muscle cells (SMCs). Nox1 and ClC-3 colocalize to early endosomes, and both are necessary for tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-1ß generation of ROS and subsequent activation of the transcription factor nuclear factor (NF)-{kappa}B. We propose that on receptor activation, a signaling endosome forms and compartmentalizes intracellular ROS production. ClC-3 provides charge neutralization of the electron flow generated by Nox1.


*    Materials and Methods
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 up arrowIntroduction
 *Materials and Methods
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Cultured Cells
Studies were performed in human aortic SMCs (Clonetics Corp, San Diego, Calif) and SMCs isolated from aorta of mice lacking the Clcn3 gene encoding ClC-3 chloride channels12 and wild-type littermate controls. Cells were isolated as previously described13 and grown in 10% FCS in DMEM containing 1% basal medium Eagle, 1% glutamine, 1% penicillin/streptomycin, 1% Eagle’s minimal essential medium 100x nonessential amino acids, and 2% HEPES. Studies were performed on 3 independent isolations of ClC-3–null SMCs at passages 4 to 9 and 70% to 90% confluence.

Detection of Endosomal ROS
The detection of endosomal generation of ROS was performed using the fluorogenic reagent OxyBURST Green H2HFF–BSA14 (Molecular Probes, Invitrogen). This reagent consists of BSA covalently linked to the oxidant-sensitive dihydro-2',4,5,6,7,7'-hexafluorofluorescein. Early endosomes develop an acidic internal pH of {approx}6.0, which facilitates dissociation of the ligand from its receptor. A limitation in the use of fluorophores to assess endosomal processes is the effect of pH on fluorescence intensity for many of these compounds. For example, as pH decreases, there is a significant reduction in fluorescence of oxidized 5(6)-carboxyfluorescein.15 In contrast, and essential to the interpretation of our data, there is no change in fluorescence intensity of oxidized H2HFF-BSA over the pH range of 4.5 to 7.5.15 Texas red–BSA (TXR-BSA) or –dextran conjugates were used as controls for endocytosis. Single-color cell fluorescence was analyzed by confocal microscopy or by flow cytometry. For details, see the expanded Materials and Methods section in the online data supplement at http://circres.ahajournals.org.

Cytochemical Assessment of NADPH Oxidase Activity
Cells were grown to 80% confluence, and intracellular localization of NADPH-derived ROS was detected using a method based on cerium cytochemistry,16 as detailed in the online data supplement.

Antibodies
Two rabbit polyclonal antibodies were raised against ClC-3 and affinity-purified. ClC-3a1 to the injected N-terminal peptides TYDDFHTIDWVREKC and CKDRERHRRINSKKKES (Research Genetics) and ClC-3a2 to a coinjected keyhole limpet hemocyanin (KLH)- and BSA-conjugated N-terminal peptide of an alternatively spliced form of ClC-3 (GenBank accession no. AF347689, PYDGGGDSIPLRELHKRGTHYTMTNC; Bio-Synthesis). Additional antibodies and their sources included Nox1, p22phox, and GAPDH (all from Santa Cruz Biotechnology), early endosome antigen (EEA)1 (BD Transduction Laboratory), {alpha}-actin (Sigma), Rab5 (Abcam), and p65 (Imgenex).

Immunostaining
Cells were grown on chamber slides, fixed in 2% paraformaldehyde for 15 minutes, and washed with PBS–Tween. Following blocking, SMCs were incubated with primary antibodies for ClC-3a2 (1:50) and p22phox (1:25) at room temperature for 2 hours. Secondary antibodies consisted of goat anti-rabbit Alexa 488 (1:200) and donkey anti-goat–biotin (1:500). Cells were then treated with streptavidin–Alexa fluor 568 and TO-PRO-3. Images were scanned sequentially and merged using the Bio-Rad LaserSharp software.

Immunoprecipitation
Antibody was cross-linked to protein G–Sepharose, and affinity chromatography of SMC extracts was performed as detailed in the online data supplement. Following elution, the collected fractions were subjected to SDS-PAGE followed by immunoblotting using specified antibodies.

NADPH Oxidase Activity
SMC lysates were immunoprecipitated with anti-Rab5 antibody, and superoxide levels were measured in PBS–lucigenin (5 µmol/L) by an FB12 luminometer. Samples were allowed 2 minutes of dark adaptation following the addition of NADPH (10 µmol/L), with or without diphenylene iodonium (DPI) (10 µmol/L) and relative light units per second normalized to milligrams of protein used in the immunoprecipitation.

Biotin–Streptavidin Pull-Down Assay
The NF-{kappa}B pull-down assay was performed as described in online data supplement. Briefly, double-stranded oligonucleotides containing the consensus sequence for NF-{kappa}B binding, and containing biotin on the 5'-nucleotide, were incubated with SMC lysate 4 hours after IL-1ß. Following Western blotting, membranes were probed with anti-p65 (Santa Cruz Biotechnology).

NF-{kappa}B Promoter Activity
NF-{kappa}B–mediated transcriptional induction was measured by infection of SMCs with replication-deficient adenovirus containing the luciferase reporter gene driven by NF-{kappa}B transcriptional activation.17 Luciferase activity (relative light units) was measured in reporter lysis buffer according to the protocol of the manufacturer (Promega Inc) and normalized to protein concentration. AdV12Rac1 and AdN17 Rac1 were obtained from the University of Iowa Vector Core, and the AdNox1 sense and antisense were from Kathy Griendling (Emory University, Atlanta, Ga); these have been characterized previously.18

Statistical Analysis
Results are expressed as means±SEM. Statistical comparisons were performed by Student’s 2-tailed t tests or ANOVA with the Tukey multiple comparison posttest as appropriate. A value of P<0.05 was considered significant.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
We assessed generation of ROS using a membrane impermeable oxidant-sensitive fluorescent probe (albumin-conjugated 2',4,5,6,7,7'-hexafluorofluorescein [H2HFF-BSA]). Intracellular punctate fluorescence thus indicates endocytosis of the fluorophore and production of ROS. In the presence of H2HFF-BSA, IL-1ß resulted within minutes in the appearance of numerous fluorescent vesicles in human SMCs (Figure 1A). Inhibition of fluorescence by superoxide dismutase (SOD) protein, which, like H2HFF-BSA, is endocytosed following stimulation with IL-1ß, confirmed that the fluorescence resulted from generation of superoxide in the endosome. Inhibition of fluorescence by DPI suggested NADPH oxidase as an enzymatic source of ROS. A similar pattern of H2HFF-BSA fluorescence was observed after stimulation of SMCs with TNF-{alpha}, which was also inhibited by SOD and DPI (data not shown).


Figure 1
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  		Figure 1. NADPH oxidase generates ROS in early endosomes. Human SMCs were incubated with H2HFF-BSA and stimulated for 10 minutes with IL-1ß (10 ng/mL) and imaged by confocal fluorescence microscopy. A, Vesicular ROS is observed 10 minutes after IL-1ß and inhibited when coincubated with SOD (1200 U/mL) or DPI (10 µmol/L). B, Transmission electron micrograph of NADPH-stimulated ROS in vesicles (arrows) of human SMCs by using methodology based on cerium cytochemistry.16 These ROS-generating structures did not represent mitochondria (open arrow). C, Western blot shows Nox1 coimmunoprecipitated with early endosomal markers EEA1 and Rab5. D, Addition of NADPH to Rab5-enriched SMC fractions stimulated a DPI-inhibitable superoxide production, as measured by lucigenin-enhanced chemiluminescence. *P<0.05 (n=3). Scale bars: 20 µm (A); 1 µm (B). RLU indicates relative light units.

The association of NADPH oxidase with intracellular vesicles was further investigated using a cytochemical method in which the reaction of cerium with ROS forms insoluble precipitates detectable by electron microscopy.16 ROS were produced within vesicular structures of human SMCs in response to NADPH (Figure 1B). These ROS-generating structures did not represent mitochondria and were not evident in SMCs treated with cerium-containing buffer without NADPH.

Further demonstrating an association of NADPH oxidase with endosomal vesicles, the NADPH oxidase subunit Nox1 coimmunoprecipitated with EEA1 and Rab5, both markers of early endosomes (Figure 1C). The addition of NADPH to Rab5-enriched SMC fractions resulted in DPI-inhibitable superoxide production (Figure 1D). Pretreatment of SMCs with IL-1ß for 15 minutes before isolation of Rab5-enriched fractions increased NADPH-stimulated superoxide production 1.5±0.1-fold as compared with unstimulated SMCs (P<0.05, n=3). Together, these data indicate that cytokine activation of SMCs results in NADPH oxidase–dependent generation of superoxide in early endosomes.

We have previously demonstrated that ClC-3 is required for a normal oxidative burst in neutrophils.11 Based on these observations, we examined the relationship between ClC-3 and NADPH oxidase in SMCs. A non–detergent-treated human SMC lysate was applied to a ClC-3 affinity column produced using antibodies specific for ClC-3 (Figure I in the online data supplement) and eluted with detergent-containing buffer. Membrane-associated NADPH oxidase subunits Nox1 and p22phox, in addition to early endosomal markers EEA1 and Rab5, were bound to the column (Figure 2A). Immunohistochemistry showed that ClC-3 colocalized with p22phox in SMCs (Figure 2B).


Figure 2
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  		Figure 2. ClC-3 colocalizes with NADPH oxidase in early endosomes. A, Non–detergent-treated human SMC lysate and murine brain lysate were applied to ClC-3 affinity columns and eluted with detergent-containing buffer. The 2 lanes on the left are total cell lysate; the 2 lanes on the right are bound proteins. Western blots were probed for the proteins indicated to the right. B, Human SMCs immunostained for ClC-3 and p22phox. Colocalization is evident by yellow staining. A magnified view is shown in the inset. Controls with no primary antibody showed no fluorescence labeling. Scale bar=20 µm.

Having confirmed an association of ClC-3 with Nox1 in early endosomes, we determined whether ClC-3 was required for NADPH oxidase activity. TNF-{alpha}–induced endocytosis and fluorescence of H2HFF-BSA in human SMCs was inhibited by the anion channel inhibitor niflumic acid (NFA) (Figure 3A). This inhibition was not caused by impairment of endocytosis because NFA had no effect on vesicular fluorescence of TXR-BSA following TNF-{alpha} stimulation (Figure 3A). Similarly, whereas stimulation of wild-type SMCs resulted in endocytosis and fluorescence of H2HFF-BSA, there was no detection of vesicular ROS in ClC-3–null cells after stimulation with TNF-{alpha} or IL-1ß (Figure 3B). In contrast, wild-type and ClC-3–null SMCs demonstrated similar vesicular fluorescence of dextran-conjugated Texas red following stimulation with TNF-{alpha} (Figure 3C). Flow-cytometric analysis confirmed that in response to TNF-{alpha}, H2HFF-BSA fluorescence was markedly blunted in ClC-3–null as compared with wild-type SMCs (Figure 3D), whereas TXR-BSA fluorescence was similar in ClC-3–null and wild-type SMCs (118±10 versus 120±26 normalized relative fluorescence units per cell; wild type [n=12] versus ClC-3–null [n=6]). The addition of SOD or DPI concurrently with TNF-{alpha} inhibited fluorescence of H2HFF-BSA in wild-type cells (Figure 3E). These data establish the association of ClC-3 with Nox1 in early endosomes and identify a role for ClC-3 in cytokine-dependent NADPH oxidase production of endosomal ROS.


Figure 3
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  		Figure 3. ClC-3 is necessary for cytokine-dependent generation of endosomal ROS. A, Human SMCs were incubated with H2HFF-BSA, stimulated for 10 minutes with TNF-{alpha} (10 ng/mL), and imaged by confocal fluorescence microscopy. The chloride channel inhibitor NFA (0.3 mmol/L) prevented TNF-{alpha}–dependent production of vesicular ROS. When SMCs were incubated instead with TXR-BSA, NFA had no effect on fluorescence, confirming that NFA did not inhibit endocytosis. B, Wild-type (WT) and ClC-3–null SMCs were incubated with H2HFF-BSA and stimulated with IL-1ß (10 ng/mL) or TNF-{alpha} (10 ng/mL). C, Wild-type and ClC-3–null SMCs were incubated with TXR-BSA and stimulated with TNF-{alpha}. Scale bars=20 µm. D, Flow-cytometric summary data of wild-type and ClC-3–null SMCs incubated for 10 minutes in H2HFF-BSA and TNF-{alpha} (wild type, n=15; null, n=8). *P<0.05 vs wild-type control. E, Flow-cytometric data of wild-type SMCs incubated for 10 minutes in H2HFF-BSA and TNF-{alpha}. SOD and DPI were added concurrently with TNF-{alpha} (n=3; data normalized to TNF-{alpha} stimulated). *P<0.05 vs TNF-{alpha} stimulated. RFU indicates relative fluorescence units.

ROS contribute to normal cell-signaling processes, including activation of many protein kinases, inhibition of protein phosphatases, and regulation of transcription factors. NADPH oxidase–mediated cell signaling has been extensively studied in vascular SMC, where ROS modulate gene expression associated with cell differentiation and growth.19,20 It was recently shown that following IL-1ß binding, endosomal generation of ROS facilitates assembly of adaptor proteins necessary for activation of NF-{kappa}B.14 In agreement with these observations, the antioxidant N-acetylcysteine prevented IL-1ß activation of NF-{kappa}B in SMCs (Figure 4A). In addition, inhibition of endosomal ROS by cytokine-mediated endocytosis of extracellular SOD,14 as demonstrated in Figures 1A and 3UpE, was sufficient to inhibit NF-{kappa}B activation (Figure 4B). This observation confirms that superoxide generated within early endosomes of SMCs participate in the activation NF-{kappa}B.


Figure 4
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  		Figure 4. NF-{kappa}B activation by IL-1ß and TNF-{alpha} is Nox1 dependent. NF-{kappa}B activation was measured by luciferase reporter assay. Luciferase activity was measured 6 hours after IL-1ß (10 ng/mL) or TNF-{alpha} (10 ng/mL) treatment. A, In response to IL-1ß and TNF-{alpha}, NF-{kappa}B activation in wild-type SMCs was inhibited by DPI (6 µmol/L) and by the antioxidant N-acetylcysteine (NAC) (30 mmol/L). B, NF-{kappa}B activation was inhibited whether SOD and TNF-{alpha} were washed from SMCs after 30 minutes or at the completion of the 6-hour incubation. C, Adenoviral-mediated expression of antisense to Nox1 inhibited NF-{kappa}B activation by IL-1ß in SMCs. D, Expression of dominant negative Rac1 (AdN17Rac1) inhibited activation of NF-{kappa}B by TNF-{alpha}. AdGFP served as a vector control. Results are averages±SEM of 3 to 5 experiments. For each experiment, values are normalized to the control value. *P<0.05 vs control, +P<0.05 vs AdGFP plus IL-1ß or TNF-{alpha}.

DPI also reduced NF-{kappa}B activation by IL-1ß and TNF-{alpha}, suggesting involvement of NADPH oxidase–derived ROS in this signal pathway (Figure 4A). Nox1 antisense inhibited activation of NF-{kappa}B in SMCs by IL-1ß (Figure 4C) and by TNF-{alpha} (data not shown). In addition, it was recently shown that Nox1 activation is dependent on Rac1.21 Consistent with this observation, dominant negative Rac1 (N17) inhibited the activation of NF-{kappa}B by TNF-{alpha} (Figure 4D) and IL-1ß (2.9-fold increase of AdGPF+IL-1ß versus 1.2-fold increase for AdN17Rac1+IL-1ß, as compared with AdGFP alone; n=3). These findings confirm Nox1-derived ROS are necessary for the activation of NF-{kappa}B by IL-1ß and TNF-{alpha} in SMCs.

Having established an association between Nox1 and ClC-3, and the dependence of NF-{kappa}B activation on Nox1-derived ROS, we hypothesized that ClC-3 is required for NADPH oxidase–dependent NF-{kappa}B signaling. SMCs isolated from the aorta of ClC-3–null mice had lower basal NF-{kappa}B activity than wild-type SMCs (1650±177 versus 3702±64 [n=14]; P<0.001) and failed to activate NF-{kappa}B in response to IL-1ß or TNF-{alpha}, independent of time or agonist concentration (Figure 5A through 5C). This observation was confirmed by pull-down assays using biotin-labeled oligonucleotides containing the consensus sequence for NF-{kappa}B binding (supplemental Figure II). Reconstitution of ClC-3 protein in ClC-3–null SMCs by gene transfer restored NF-{kappa}B activation by IL-1ß (Figure 5D) and TNF-{alpha} (not shown). Consistent with these observations, the anion channel blockers 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) and NFA inhibited NF-{kappa}B activation in wild-type SMCs (Figure 5E). Expression of NF-{kappa}B subunit p65 (RelA) was similar in ClC-3–null and wild-type SMCs (supplemental Figure II). Collectively, these data indicate that both Nox1 and ClC-3 are required for ROS-dependent activation of NF-{kappa}B by IL-1ß and TNF-{alpha} in SMCs.


Figure 5
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  		Figure 5. NF-{kappa}B activation by IL-1ß and TNF-{alpha} requires ClC-3. NF-{kappa}B activation was measured by luciferase reporter assay. TNF-{alpha} activation of NF-{kappa}B was markedly reduced in ClC-3–null SMCs independent of time (A) or concentration (B). C and D, IL-1ß activation of NF-{kappa}B was reduced in ClC-3–null SMCs (C) and rescued by adenoviral-mediated expression of ClC-3 (D). E, IL-1ß activation of NF-{kappa}B was inhibited by the chloride channel blockers NFA (0.3 mmol/L) and DIDS (0.3 mmol/L) in wild-type (WT) SMCs. Luciferase activity was measured 6 hours after IL-1ß (10 ng/mL) or TNF-{alpha} (10 ng/mL) treatment. Results are averages±SEM of 3 to 5 experiments. For each experiment, values are normalized to the control value. *P<0.05 vs control, +P<0.05 vs IL-1ß alone.

We next assessed the mechanism by which ClC-3 participates in Nox1-dependent activation of NF-{kappa}B. Having shown that activation of NF-{kappa}B by TNF-{alpha} is dependent on Rac1 (Figure 4D), we tested the ability of constitutively active Rac1 to rescue activation of NF-{kappa}B in ClC-3–null cells. Constitutively active Rac1 (V12) augmented the activation of NF-{kappa}B by TNF-{alpha} in wild-type SMCs, confirming the importance of Rac1 in this response (Figure 6A). In contrast, there was no effect of V12 Rac1 on the response of ClC-3–null SMCs to TNF-{alpha}. The failure of V12Rac1 to augment NF-{kappa}B activation in ClC-3–null SMCs suggests that ClC-3 participates in this signaling pathway after the activation of Nox1 by Rac1.


Figure 6
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  		Figure 6. Role of ClC-3 in NF-{kappa}B activation. A, Constitutively active Rac1 (AdV12Rac1) augmented the activation of NF-{kappa}B by TNF-{alpha} in wild-type (WT) SMCs but not ClC-3–null SMCs in response to TNF-{alpha}. B, In the absence of IL-1ß, addition of H2O2 did not activate NF-{kappa}B in ClC-3–null SMCs. However, following IL-1ß stimulation, H2O2 caused a concentration-dependent activation of NF-{kappa}B in ClC-3–null SMCs. Results are averages±SEM of 3 to 5 experiments. *P<0.05 vs control, +P<0.05 vs wild-type plus TNF-{alpha}.

Next, we determined whether NF-{kappa}B could be activated by ROS independent of ClC-3. In the absence of an agonist, addition of H2O2 did not activate NF-{kappa}B in either wild-type or ClC-3–null SMCs, similar to previous reports.22 However, following IL-1ß stimulation, H2O2 caused a concentration-dependent activation of NF-{kappa}B in ClC-3–null SMCs (Figure 6B). In wild-type SMCs, the addition of H2O2 augmented the activation of NF-{kappa}B by IL-1ß (data not shown). These observations suggest that NF-{kappa}B signaling requires both receptor activation and H2O2 for NF-{kappa}B activation and that ClC-3 is necessary to provide sufficient levels of H2O2 for activation of NF-{kappa}B.


*    Discussion
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 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
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Previous studies have identified a role for NAPDH oxidases in redox-dependent signaling of nonphagocytic cells.14,23–25 However, the specific location of superoxide generation by Nox1 following activation has not been described. Observations in nonphagocytic cells, including SMCs, support a membrane-associated flavocytochrome b558 composed of a Nox subunit and p22phox, similar to the well-characterized phagocyte NADPH oxidase. The biochemical properties of NADPH oxidase predict that reduction of oxygen to superoxide needs to occur in the extracellular space or within intracellular vesicles.2 Accordingly, in phagocytes, NADPH oxidase generates superoxide into phagosomes. Previous studies have implied that nonphagocytic NADPH oxidase generates extracellular superoxide; however, such a model would not be compatible with the proposed role of NADPH oxidase in intracellular signaling. In this study, we show for the first time that in response to extracellular activation by cytokines, a Nox1-based NADPH oxidase results in generation of ROS within early endosomes of SMCs. This observation is consistent with previous reports that occupied TNF-{alpha} and IL1-ß receptors are internalized through receptor-mediated endocytosis.14,26

We found that the activation of NF-{kappa}B by TNF-{alpha} and IL-1ß in SMCs is dependent on Nox1 activation by Rac1. Interestingly, neither the addition of constitutively active Rac1 nor H2O2 was sufficient to activate NF-{kappa}B in SMCs because they also required the addition of TNF-{alpha} or IL-1ß. Together, our data support the formation of signaling endosomes27 in SMCs on receptor binding, with subsequent generation of Nox1-derived ROS into the newly formed endosomes and assembly of adaptor proteins, both of which are necessary for activation of NF-{kappa}B.14

We also found that ClC-3 is colocalized to the early endosome with Nox1 and p22phox in SMCs and is necessary for the production of ROS and activation of NF-{kappa}B by cytokines. To produce superoxide, Nox1 moves electrons from intracellular NADPH across flavin adenine dinucleotide and 2 hemes to reduce oxygen within the endosome. Without charge compensation, this electron flux rapidly depolarizes the plasma membrane of neutrophils and inactivates the oxidase.3 Our data support a model whereby ClC-3 functions as a chloride–proton exchanger, necessary for charge neutralization of electron flow generated by Nox1 (Figure 7). The other members of the ClC family that are most homologous to ClC-3 have been shown to function as chloride–proton exchangers.9,10


Figure 7
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  		Figure 7. Proposed function of Nox1 and ClC-3 in the signaling endosome. Binding of IL-1ß or TNF-{alpha} to the cell membrane initiates endocytosis and formation of an early endosome (EEA1 and Rab5), which also contains NADPH oxidase subunits Nox1 and p22phox, in addition to ClC-3. Nox1 is electrogenic, moving electrons from intracellular NADPH through a redox chain within the enzyme into the endosome to reduce oxygen to superoxide. ClC-3 functions as a chloride–proton exchanger, required for charge neutralization of the electron flow generated by Nox1. The ROS generated by Nox1 result in NF-{kappa}B activation. Both ClC-3 and Nox1 are necessary for generation of endosomal ROS and subsequent NF-{kappa}B activation by IL-1ß or TNF-{alpha} in SMCs. In addition, redox-independent pathways not shown also contribute to the activation of NF-{kappa}B.

The ClC family of chloride channels includes 9 members, with ClC-3 being the most abundant in SMCs.28 Although the ClC-3 protein is highly conserved and ubiquitously expressed, its precise function is controversial. ClC-3 was first proposed as a swelling-activated Cl– channel in plasma membranes critical for volume regulation29; however, in recent studies, a primary intracellular localization has been proposed where ClC-3 participates in charge neutralization of the vacuolar ATPase (V-ATPase) during acidification of intracellular synaptic vesicles,5 insulin granules,6 and lysosomes.7 The complex phenotypic abnormalities of ClC-3–deficient mice denote a fundamental role for ClC-3 in cellular function.5,12

The ClC-3, -4, and -5 branch of the ClC family has been hypothesized to provide shunt conductance in membranes of intracellular organelles, permitting intraluminal acidification by the V-ATPase.4 This hypothesis is supported by impaired endosomal chloride accumulation and acidification in ClC-3–deficient mice.8 However, if ClC-3 functions as a Cl–/H+ antiporter as previously proposed,9,10 it may be relatively ill suited for direct charge compensation of the V-ATPase. ClC-4 and ClC-5 currents rectify very strongly in the outward direction, preferentially conducting Cl– to the cytoplasm from the extracellular space. Endocytosis of this type of channel situates the extracellular face within the endosome, whereby strong outward rectification favors the movement of Cl– out of, rather than into endosomes. Furthermore, for ClC-3 to function as a H+/Cl– antiporter and move current in the direction required to directly charge neutralize the V-ATPase (Cl– in, H+ out of the endosome), each cycle would require the removal of a proton from the endosome. Although the net charge movement of ClC-3 acting in this direction could still provide the required shunt current, this process would be energetically unfavorable, as the V-ATPase would need to transport twice the number of protons into the endosome to compensate for the simultaneous outward H+ flux via ClC-3. Direct compensation by ClC-3 only works efficiently if it is acting as a simple anion channel conducting Cl– into endosomes. The presence of NADPH oxidase in the endosome actually removes voltage constraints on vesicular acidification. ClC-3 is well suited to neutralize the movement of negative current into endosomes. The oxidase generates a rapid depolarization of >100 mV across the plasma membrane of neutrophils, and this effect may be even larger across phagosomal membranes.30 Therefore, the highly positive intravesicular voltages required to activate ClC-3 may exist across the endosomal membrane on activation of the NADPH oxidase. Furthermore, a net negative intravesicular potential would preclude the need to provide direct charge neutralization for the V-ATPase, because movement of protons into the lumen would be highly energetically favored. In this way, ClC-3 may provide indirect charge neutralization for the V-ATPase by facilitating the activity of the NADPH oxidase. Distinct from these theoretical issues, our data indicate that activation of V-ATPase is not necessary for ROS signaling, because inhibition of the protein with bafilomycin had no effect on activation of NF-{kappa}B by cytokines (supplemental Figure IV).

The inability of ClC-3–null SMCs to make endosomal ROS and activate NF-{kappa}B in response to TNF-{alpha} and IL-1ß suggests that redox-mediated signaling events may be regulated by modulation of ClC-3 activity. In support of this, when ClC-3 was overexpressed in wild-type SMCs, NF-{kappa}B activation markedly increased (AdGFP plus IL-1ß, 7.2±0.6-fold; AdClC-3 plus IL-1ß, 83.3±5.3-fold compared with unstimulated control [n=3]; P<0.05). This suggests that ClC-3 is not simply required for Nox1 activation, but rather the level of ClC-3 activity may regulate ROS production. Several mechanisms of ClC-3 regulation have been proposed, including its phosphorylation at a unique serine residue by protein kinase C29 and Ca2+/camodulin-dependent kinase II.13,31 The neutrophil NADPH oxidase (Nox2) is also dependant on ClC-3 for intracellular ROS production. ClC-3 colocalizes with the oxidase in secretory vesicles and is upregulated to phagosomes following uptake of opsonized zymosan.11 Therefore, regulation of this anion exchanger may represent a common mechanism for control of NADPH oxidase activity following activation.

The molecular mechanism by which endosomal ROS subsequently activates redox-dependent signals is not known. Superoxide has been shown to traverse both the plasma32 and mitochondrial33 membranes via anion channels to exert cytoplasmic effects. However, the pKa of the conjugate acid of superoxide, hydrogen superoxide (HO2·) (also known as hydroperoxyl radical) is 4.88.34 Therefore, the acidic environment of endosomes favors formation of HO2· from superoxide, which will react with a second proton to form H2O2. Both HO2· and H2O2 are uncharged and can diffuse to the cytosol to activate redox-dependent signal molecules. It is also possible that localized release of superoxide and H2O2 generate distinct and independent cytoplasmic signals.

There are multiple homologs of the Nox enzymes within cells.1 We have focused our evaluation on Nox1 because of previous reports identifying agonist-dependent activation of Nox1 and a subsequent role for Nox1-derived ROS in SMC signaling.18,20,25 We cannot exclude the possibility that activity of the other Nox homologs also are dependent on ClC-3 or related proteins. Similarly, it is not known whether other methods of Nox1 activation in SMCs involve ClC-3–dependent endosomal generation of ROS. These will be important areas of future research. Preliminary data from our laboratory indicate that our findings are not unique to vascular SMCs.

We describe NADPH oxidase–dependent generation of ROS in early endosomes and identify ClC-3 as a critical component and a novel intermediate in redox-dependent control of gene expression. Furthermore, ClC-3 may be a new target in the regulation of redox-dependent gene expression involved in vascular disease,35 cancer,36,37 and inflammation.11


*    Acknowledgments
 
We thank associates of the University of Iowa Roy J. and Lucille A. Carver College of Medicine Central Microscopy Research Facility, the Flow Cytometry Facility, and the Gene Transfer Vector Core Facility of the University of Iowa Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases, which is supported by National Institute of Diabetes and Digestive and Kidney Diseases/NIH grant P30 DK 54759.

Sources of Funding

This work was supported by NIH grants HL062483 (to F.S.L., F.J.M.) and HL081750 (to F.J.M.) and by the American Heart Association (F.S.L.).

Disclosures

None.


*    Footnotes
 
*Both authors contributed equally to this work. Back

Original received March 6, 2007; revision received July 3, 2007; accepted July 24, 2007.


*    References
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