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, 125 (3), 1299-310

Disturbed Flow-Activated p90RSK Kinase Accelerates Atherosclerosis by Inhibiting SENP2 Function

Disturbed Flow-Activated p90RSK Kinase Accelerates Atherosclerosis by Inhibiting SENP2 Function

Kyung-Sun Heo et al. J Clin Invest.

Abstract

Disturbed blood flow (d-flow) causes endothelial cell (EC) dysfunction, leading to atherosclerotic plaque formation. We have previously shown that d-flow increases SUMOylation of p53 and ERK5 through downregulation of sentrin/SUMO-specific protease 2 (SENP2) function; however, it is not known how SENP2 itself is regulated by d-flow. Here, we determined that d-flow activated the serine/threonine kinase p90RSK, which subsequently phosphorylated threonine 368 (T368) of SENP2. T368 phosphorylation promoted nuclear export of SENP2, leading to downregulation of eNOS expression and upregulation of proinflammatory adhesion molecule expression and apoptosis. In an LDLR-deficient murine model of atherosclerosis, EC-specific overexpression of p90RSK increased EC dysfunction and lipid accumulation in the aorta compared with control animals; however, these pathologic changes were not observed in atherosclerotic mice overexpressing dominant negative p90RSK (DN-p90RSK). Moreover, depletion of SENP2 in these mice abolished the protective effect of DN-p90RSK overexpression. We propose that p90RSK-mediated SENP2-T368 phosphorylation is a master switch in d-flow-induced signaling, leading to EC dysfunction and atherosclerosis.

Figures

Figure 9
Figure 9. Role of the p90RSK-SENP2 module in atherosclerotic plaque formation.
(A) Atherosclerotic lesions 4 weeks after partial left carotid artery (LCA) ligation in NLC/Ldlr–/– (n = 9), DN-p90rsk-ETg/Ldlr–/– (n = 10), and DN-p90rsk-ETg/Senp2+/–/Ldlr–/– (n = 7) mice are shown. Unligated right carotid artery (RCA) in each mouse is used as control. Representative images are shown after H&E staining. Scale bars: 50 μm. The intimal lesion area and the media area (B), and the intima/media ratio (C) were determined in cross-sections of ligated LCAs. Data represent mean ± SEM. *P < 0.05; **P < 0.01, by 1-way ANOVA followed by Bonferroni’s post hoc test. (D) Representative images of frozen sections of LCA and RCA after LCA ligation are shown with oil red O staining displaying lipid deposits. Scale bars: 50 μm. (E) Bar graph shows quantification of oil red O–stained areas in the intima of NLC/Ldlr–/– (n = 4), DN-p90rsk-ETg/Ldlr–/– (n = 5), and DN-p90rsk-ETg/Senp2+/–/Ldlr–/– (n = 5) mice. Data represent mean ± SEM. **P < 0.01, by 1-way ANOVA followed by Bonferroni’s post hoc test.
Figure 8
Figure 8. Role of the p90RSK-SENP2 module in d-flow–elicited inflammation and apoptosis.
(A) En face preparations of aortic arch of 7-week-old NLC, DN-p90rsk-ETg, or DN-p90rsk-ETg/Senp2+/– mice were double-stained with anti–VE-Cad and anti–VCAM-1. Scale bars: 20 μm. The graph shows anti–VCAM-1 staining intensities in d- and s-flow areas of the aortic arch. Data represent mean ± SEM. Right panel, n = 3 for each genotype. **P < 0.01, by 1-way ANOVA followed by Bonferroni’s post hoc test. (BD) MECs isolated from NLC, DN-p90rsk-ETg, or DN-p90rsk-ETg/Senp2+/– mice were stimulated with d-flow, and SENP2-T368 phosphorylation (B), expression of adhesion molecules as inflammation markers (C), and cleaved caspase-3 expression as an apoptosis marker (D) were determined by Western blotting. (B) Bar graph shows the quantification of SENP2-T368 phosphorylation levels after normalization with tubulin expression. Data represent mean ± SEM (n = 3 for each genotype). **P < 0.01, by 1-way ANOVA followed by Bonferroni’s post hoc test. (C and D) The blots shown represent 1 of 3 independent experiments.
Figure 7
Figure 7. Role of p90RSK in EC inflammation and apoptosis and atherosclerotic plaque formation.
SENP2-T368 phosphorylation, protein levels of adhesion molecules, cleaved caspase-3, and eNOS (A), and mRNA levels of adhesion molecules (B) in MECs isolated from WT-p90rsk-ETg mice are shown. Data represent mean ± SEM (n = 6). **P < 0.01, by Student’s t test. (C and D) NLC/Ldlr–/– and WT-p90rsk-ETg/Ldlr–/– mice were given a high-cholesterol diet for 16 weeks. WT-p90rsk-ETg/Ldlr–/– mice exhibited increased oil red O–stained atherosclerotic lesions in the whole aorta as well as increased Masson’s trichrome–stained atherosclerotic lesions in the aortic valve region. Scale bars: 100 μm. Quantified en face (C) and histology (D) data are shown. Data represent mean ± SEM (n = 14 for each genotype). P = 0.0067; P = 0.0014, by Student’s t test.
Figure 6
Figure 6. d-flow–induced SENP2 nuclear export is regulated by T368 phosphorylation.
(A) After transduction for 18 hours with Ad-myc–tagged SENP2-WT or Ad-myc–tagged SENP2-T368A, HUVECs were exposed to d-flow or kept under static conditions for the indicated times and immunostained with anti-Myc and DAPI. Images were recorded using a confocal microscope equipped with a Planapo ×60 1.42 NA oil objective lens. Scale bars: 20 μm. Bar graph indicates quantification of percentage of nuclear fluorescence intensity divided by total cell fluorescence intensity of anti-Myc staining. Data represent mean ± SEM (n ≥ 30 cells per each condition). **P < 0.01, compared with no-flow condition in the presence of Ad-SENP2-WT; ##P < 0.01, compared with each time point of Ad-SENP2-WT control by 1-way ANOVA followed by Bonferroni’s post hoc test. (B and C) En face preparations of the aortic arch of 7-week-old WT C57BL/6 mice were triple-stained with anti–VE-Cad (as an EC marker), anti-SENP2 (B), or anti–phospo–SENP2-T368 (C), and DAPI. Scale bars: 10 μm (left panels, B and C); 20 μm (right panel, B and C). Representative images from 3 independent experiments are shown.
Figure 5
Figure 5. p90RSK-SENP2 association is critical for p53 and ERK5 SUMOylation.
(A) HeLa cells were transfected with Myc-tagged SENP2 and Flag-tagged p90RSK and subjected to immunoprecipitation with anti-Myc or IgG (as a control) followed by Western blotting with anti-Flag or anti-Myc (first and second panel from top). (B) HUVECs were transfected with plasmids containing Gal4-p90RSK WT, indicated VP16-SENP2 fragments or empty vector, and Gal4-responsive luciferase reporter pG5-luc. After 24 hours of transfection, cells were stimulated with or without d-flow for 2 hours and luciferase activities were quantified. Data represent mean ± SEM (n = 3). *P < 0.05; **P < 0.01, by 1-way ANOVA followed by Bonferroni’s post hoc test. (C) HUVECs were transduced with 50 MOI of adenoviruses of indicated SENP2 fragment mutants for 18 hours and then stimulated by d-flow for 30 minutes followed by Western blotting with anti–phospho–SENP2-T368, anti-SENP2, and anti-tubulin. (D) HUVECs were transduced with 50 MOI of indicated adenoviruses for 18 hours and then stimulated by d-flow for 30 minutes. SENP2 was immunoprecipitated and immunoblotted with anti-p90RSK. SENP2 phosphorylation at T368 and expression of p90RSK, SENP2, and tubulin were examined using specific antibodies. (AD) The blots or quantified data (mean ± SEM) shown are representative of 3 independent experiments. (E and F) d-flow–induced p53 and ERK5 SUMOylation were detected after transduction of Ad-SENP2 Fr.1, Ad-SENP2 Fr.2, or Ad-LacZ as a control. Quantification of data represents mean ± SEM (lower panel, n = 3). **P < 0.01, by 1-way ANOVA followed by Bonferroni’s post hoc test.
Figure 4
Figure 4. SENP2-T368 phosphorylation by p90RSK causes EC apoptosis and inflammation.
After HUVECs were transduced with Ad-SENP2-WT or Ad-SENP2-T368A and exposed to d-flow as indicated, p53 and ERK5 SUMOylation (A and B), p53–Bcl-2 binding (A, middle panel), cleaved caspase-3 expression (C), and eNOS and adhesion molecule expression (D) were analyzed. (AC) Quantification of data represents mean ± SEM (lower panel, n = 3). **P < 0.01, compared with no-flow condition in the presence of Ad-SENP2-WT; ##P < 0.01, compared with each time point of Ad-SENP2-WT control by 1-way ANOVA followed by Bonferroni’s post hoc test.
Figure 3
Figure 3. p90RSK-mediated SENP2-T368 phosphorylation is critical for p53 SUMOylation.
(A) An LC-MS/MS analysis identified SENP2 phosphorylation sites by p90RSK at T35, S38, and T368 with a less than 0.05 error (upper panel). An LC-MS/MS plot of different peptide fragments shows T368 phosphorylation based on mass shift of phosphorylated threonine within H2O (686 Da, lower panel). (B and C) CHO cells were cotransfected with 4 plasmids, Flag-p53, HA-SUMO3, Xpress-p90RSK1, and an additional plasmid containing one of the following pCS-Myc–tagged plasmids encoding SENP2-WT, SENP2-T35A, SENP2-S38A, or SENP2-T368A. Control plasmid contained only Flag-p53 and HA-SUMO. p53 SUMOylation was determined as described in Methods. p53, SUMO3, p90RSK, and SENP2 expression were detected by Western blotting. (D) HUVECs were transduced with Ad-SENP2-WT or SENP2 phosphorylation mutants as indicated and stimulated by d-flow or no flow (–) for 3 hours, and SENP2-T368 phosphorylation was detected using anti–phospho–SENP2-T368. (BD) The blots shown are representative of 3 independent experiments. (E) After transduction of Ad-LacZ or Ad-DN-p90RSK, d-flow–induced SENP2-T368 phosphorylation was analyzed by Western blotting. Quantification of data represents mean ± SEM (lower panel, n = 3). **P < 0.01, compared with no-flow conditions in the presence of Ad-LacZ; ##P < 0.01, compared with each time point of Ad-LacZ control by 1-way ANOVA followed by Bonferroni’s post hoc test.
Figure 2
Figure 2. Depletion of p90RSK abolishes d-flow–induced p53 and ERK5 SUMOylation.
(A) HUVECs were transfected with control siRNA or a mixture of siRNAs against p90RSK isoforms 1 and 2 followed by d-flow stimulation for indicated times. To detect p53 SUMOylation, anti-p53 or IgG was immunoprecipitated and immunoblotted with anti-SUMO. p90RSK1 and -2, p53, and SUMO expression were detected by Western blotting. Quantified data represent mean ± SEM (n = 3). (B and C) HUVECs were pretreated with either vehicle or FMK-MEA and then transduced with Ad-LacZ or Ad-DN-p90RSK followed by d-flow stimulation. SUMOylation of p53 (B) and ERK5 (C) were detected (upper panel). p90RSK, p53, ERK5, and SUMO expression were detected by Western blotting. Quantification of data represents mean ± SEM (lower panel, n = 3). **P < 0.01, by 1-way ANOVA followed by Bonferroni’s post hoc test. siControl, control siRNA.
Figure 1
Figure 1. Role of p90RSK activation in d-flow–induced apoptosis and inflammation.
(AC) HUVECs were stimulated by d-flow (upper panel) or s-flow (lower panel) for indicated times, and p90RSK (A) and ERK5 (B) activation were examined by Western blotting using anti–phospho-p90RSK and anti–phospho-ERK5 (T218/Y220), respectively. (C) Quantification of d-flow–induced p90RSK and ERK5 activation is shown after normalization by total protein levels. Data represent mean ± SEM (0 minutes = 100, n = 3). (D) HUVECs were transduced with 50 MOI of Ad-LacZ, Ad-WT-p90RSK, or Ad-DN-p90RSK for 18 hours and then stimulated by d-flow or no flow for 36 hours, followed by TUNEL staining. Images were recorded as described in Methods after counterstaining with DAPI to visualize nuclei. Apoptotic nuclei are indicated by arrows (left panel). Quantification of apoptosis is shown as the percentage of TUNEL-positive cells (right panel). Data represent mean ± SEM, n = 5. Scale bars: 60 μm. (E and F) HUVECs were transduced with 50 MOI of Ad-LacZ, Ad-WT-p90RSK, or Ad-DN-p90RSK and then stimulated by d-flow for 24 hours. Expression of cleaved caspase-3 (E) or E-selectin, ICAM-1, VCAM-1, p90RSK, and tubulin (F) is shown. *P < 0.05; **P < 0.01, by 1-way ANOVA followed by Bonferroni’s post hoc test.

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