NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells - PubMed (original) (raw)

. 2004 Dec;24(24):10703-17.

doi: 10.1128/MCB.24.24.10703-10717.2004.

Cécile Guichard, Véronique Ollivier, Fathi Driss, Michèle Fay, Céline Prunet, Jean-Claude Marie, Cécile Pouzet, Mohammad Samadi, Carole Elbim, Yvonne O'dowd, Marcelle Bens, Alain Vandewalle, Marie-Anne Gougerot-Pocidalo, Gérard Lizard, Eric Ogier-Denis

Affiliations

NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells

Eric Pedruzzi et al. Mol Cell Biol. 2004 Dec.

Abstract

The mechanisms involved in the cytotoxic action of oxysterols in the pathogenesis of atherosclerosis still remain poorly understood. Among the major oxysterols present in oxidized low-density lipoprotein, we show here that 7-ketocholesterol (7-Kchol) induces oxidative stress and/or apoptotic events in human aortic smooth muscle cells (SMCs). This specific effect of 7-Kchol is mediated by a robust upregulation (threefold from the basal level) of Nox-4, a reactive oxygen species (ROS)-generating NAD(P)H oxidase homologue. This effect was highlighted by silencing Nox-4 expression with a specific small interfering RNA, which significantly reduced the 7-Kchol-induced production of ROS and abolished apoptotic events. Furthermore, the 7-Kchol activating pathway included an early triggering of endoplasmic reticulum stress, as assessed by transient intracellular Ca(2+) oscillations, and the induction of the expression of the cell death effector CHOP and of GRP78/Bip chaperone via the activation of IRE-1, all hallmarks of the unfolded protein response (UPR). We also showed that 7-Kchol activated the IRE-1/Jun-NH(2)-terminal kinase (JNK)/AP-1 signaling pathway to promote Nox-4 expression. Silencing of IRE-1 and JNK inhibition downregulated Nox-4 expression and subsequently prevented the UPR-dependent cell death induced by 7-Kchol. These findings demonstrate that Nox-4 plays a key role in 7-Kchol-induced SMC death, which is consistent with the hypothesis that Nox-4/oxysterols are involved in the pathogenesis of atherosclerosis.

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Figures

FIG. 1.

FIG. 1.

7-Kchol stimulates Nox-4 mRNA expression in SMCs. (A) Subconfluent SMCs were incubated with 0 or 40 μg of 7-Kchol/ml for 16 h. Nox mRNA expression levels were quantified by real-time RT-PCR. The bars, expressed as the relative expression of Nox versus α-actin mRNA levels, are means ± the SD from three independent experiments. ∗, P < 0.01. (B) Subconfluent SMCs were treated with oxysterols (40 μg/ml) for various times and concentrations (inset), and the relative expression of Nox-4 mRNA was determined by real-time RT-PCR. (C) Western blot analysis, with an anti-Nox-4 antibody and an anti-α-actin antibody, were performed on untreated (time zero) and oxysterol-treated SMCs for 8 and 16 h. The bars correspond to the densitometric value of Nox-4 over α-actin, and results are representative of three separate experiments.

FIG. 2.

FIG. 2.

7-Kchol stimulates the intracellular production of ROS in SMCs. Cells were incubated with or without oxysterols (40 μg/ml) for 16 h, and ROS production was measured by chemiluminescence assay. Preincubation times were 5 min for the combination of PEG-SOD (10 μg/ml) plus PEG-catalase (10 U/ml) and 30 min for DPI (20 μM), respectively. The values, expressed as the relative chemiluminescence intensity, are means ± the SD of triplicate measurements from three separate experiments. ∗, P < 0.01.

FIG. 3.

FIG. 3.

ROS produced by 7-Kchol and Nox-4 exhibit paranuclear and nuclear localization in SMCs. (A) Cells were incubated in the presence or absence of 40 μg of 7-Kchol/ml for 16 h and then with the peroxide-sensitive fluorophore DCFH-DA for 20 min. DPI (20 μM) was added 10 min before and during DCFH-DA loading. Images were obtained by confocal laser-scanning microscopy. Fluorescence labeling (a, c, and e) and phase-contrast images (b, d, and f) are shown. Bars, 20 μm. (B) In the upper panels, SMCs were processed for indirect immunofluorescence with the anti-Nox-4 polyclonal antibody with or without preincubation with 10 μg of synthetic peptide/ml. Bars, 15 μm. In the lower panels, SMCs were processed for double indirect immunofluorescence with a mouse anticalnexin MAb (green) and the anti-Nox-4 polyclonal antibody (red). Note the colocalization of the proteins (Merge) in the paranuclear region of SMCs. Bar, 15 μm.

FIG. 4.

FIG. 4.

Silencing of Nox-4 expression by RNA interference in SMCs. (A) Cells were transfected with scrambled siRNA or Nox-4 siRNA duplexes. After incubation for 24 h in complete medium, SMCs were treated with 0 or 40 μg of 7-Kchol/ml for 16 h. Nox mRNA expression levels were then determined by real-time RT-PCR. Bars, indicating the relative expression of Nox versus α-actin mRNA levels, are means ± the SD from three independent experiments. ∗, P < 0.01. (B) SMCs transfected with scrambled or Nox-4 siRNAs were treated with 0 or 40 μg of 7-Kchol/ml for 16 h. The expression of Nox-4 was detected by Western blot analysis with the anti-Nox-4 antibody. The bars represent the mean densitometric value of Nox-4 over α-actin from three separate experiments for each of the experimental conditions tested. (C) ROS production (chemiluminescence assay) was measured on SMCs transfected with scrambled or Nox-4 siRNAs incubated with 0 or 40 μg of 7-Kchol/ml for 16 h. A combination of PEG-SOD (10 μg/ml) and PEG-catalase (10 U/ml) was added 5 min before the chemiluminescence assay. The chemiluminescence intensity values reported are means ± the SD from three separate experiments performed in triplicate. ∗, P < 0.01.

FIG. 5.

FIG. 5.

Silencing of Nox-4 expression prevents 7-Kchol-induced apoptosis in SMCs. (A) Flow cytometry analyses were performed on scrambled siRNA- or Nox-4 siRNA-transfected SMCs incubated with 0 or 40 μg of 7-Kchol/ml for 24 h. The plots of annexin V versus PI fluorescence shown correspond to three independent experiments. R1, apoptotic cells (annexin V+/PI−); R2, necrotic cells (annexin V+/PI+); R3, damaged cells (annexin V−/PI+); R4, viable cells (annexin V−/PI−). (B) The bars represent the percentage of annexin V+/PI−, annexin V+/PI+, and annexin V−/PI+ cells after exposure to 40 μg of 7-Kchol/ml for 24 h. The values reported are means ± the SD of three separate experiments. ∗, P < 0.001; ∗∗, P < 0.01; ∗∗∗, P < 0.05 (versus −7 Kchol values for each experimental condition tested). (C) 7-Kchol-induced apoptosis is sensitive to the pan caspase inhibitor Z-VAD-fmk. The ratio of floating to attached cells was determined on scrambled siRNA- or Nox-4 siRNA-transfected SMCs that had or had not been exposed to 7-Kchol for 24 h and 200 μM pancaspase inhibitor Z-VAD-fmk. The values reported are means ± the SD of triplicate counts from four independent experiments. ∗, P < 0.01. (D) Percentage of depolarized cells characterized by loss of mitochondrial transmembrane potential. SMCs transfected with scrambled or Nox-4 siRNAs were incubated with 0 or 40 μg of 7-Kchol/ml for 24 h. The percentage of cells with low mitochondrial potential was quantified by flow cytometry with DiOC6. The values reported are means ± the SD of four independent experiments. ∗, P < 0.01.

FIG. 6.

FIG. 6.

7-Kchol induces Ca2+ oscillations in SMCs. Untransfected and Nox-4 siRNA-transfected SMCs were loaded with Fluo-3/AM (5 μM, 1 h, 37°C) and placed in a culture chamber adapter to an inverted microscope. Then, 40 μg of 7-Kchol or 7-ketocholesteryl-3-oleate/ml were added 3 min after the start of the experiment. The images were generated by confocal microscopy every 10 s over a period of 20 min. The data are reported as the fluorescence intensity, reflecting changes in [Ca2+]i. The upper panel provides an illustration of real-time imaging generated by confocal microscopy.

FIG. 7.

FIG. 7.

Silencing of Nox-4 expression prevents the UPR induced by 7-Kchol. (A) Lysates of SMCs transfected with scrambled or Nox-4 siRNAs incubated with or without 40 μg of 7-Kchol/ml for 24 h were immunoblotted for CHOP/GADD 153, GRP78/Bip, and proapoptotic Bax and for antiapoptotic Bcl-2 and α-actin. (B) The relative protein content of samples was determined with an anti-α-actin antibody. The bars represent the densitometric value for each experimental condition and are representative of three separate experiments.

FIG. 8.

FIG. 8.

ER stress-dependent JNK activation is required for 7-Kchol-induced Nox-4 overexpression. (A) Western blot analyses with antibodies directed against anti-JNK phospho-specific Thr183 and Tyr185, anti- JNK, anti-c-jun phospho-specific Ser73, and anti-c-jun, were performed on untreated (time zero) and 7-Kchol-treated (times 1 and 4 h) SMCs. (B) Human monocytic THP-1 cells were transiently transfected with plasmids expressing the indicated luciferase _cis_-reporters (NF-κB or AP-1). Luciferase activities were measured after exposure for 4 h to the indicated concentrations of 7-Kchol. TNF-α (100 ng/ml) was used as a positive control. The bars are means ± the SD of relative luminescence intensity values from three separate experiments performed in triplicate. (C) SMCs were treated with 40 μg of 7-Kchol/ml for 16 h with or without 40 nM JNK inhibitor SP600125. When used, SP600125 was added 1 h before and maintained throughout the incubation period. After incubation, Nox-4 mRNA expression levels were determined by real-time RT-PCR. The bars are means ± the SD of the relative expression of Nox-4 versus α-actin mRNA levels from three separate experiments. (D) SMCs were exposed to 0 or 40 μg of 7-Kchol/ml for 24 h with 0 (control) or 40 nM JNK inhibitor SP600125. Lysates were then immunoblotted for Nox-4, CHOP/GADD 153, and GRP78/Bip, and α-actin was used as internal standard.

FIG. 9.

FIG. 9.

IRE-1 controls the 7-Kchol-induced expression of Nox-4. SMCs were transfected with scrambled siRNA or IRE-1 siRNA duplexes. After incubation for 24 h in complete medium, SMCs were exposed to 0 or 40 μg of 7-Kchol/ml for 16 h. The IRE-1 protein was detected by Western blot analysis with the anti-IRE-1 antibody, which recognizes both the wild-type (IRE-1) and the phosphorylated forms (IRE-1-P) of IRE-1. Cells lysates were analyzed by Western blotting with antibodies directed against JNK phospho-specific Thr183 and Tyr185, Nox-4, and α-actin. The Western blots shown are representative of three separate experiments.

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