Nitric oxide maintains endothelial redox homeostasis through PKM 2 inhibition
2019; Springer Nature; Volume: 38; Issue: 17 Linguagem: Inglês
10.15252/embj.2018100938
ISSN1460-2075
AutoresMauro Siragusa, Janina Thöle, Sofia‐Iris Bibli, Bert Luck, Annemarieke E. Loot, Kevin de Silva, Ilka Wittig, Juliana Heidler, Heike Stingl, Voahanginirina Randriamboavonjy, Karin Kohlstedt, Bernhard Brüne, Andreas Weigert, Beate Fißlthaler, Ingrid Fleming,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
ResumoArticle22 July 2019free access Source DataTransparent process Nitric oxide maintains endothelial redox homeostasis through PKM2 inhibition Mauro Siragusa Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Search for more papers by this author Janina Thöle Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Search for more papers by this author Sofia-Iris Bibli Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Search for more papers by this author Bert Luck Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Search for more papers by this author Annemarieke E Loot Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Kevin de Silva Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Ilka Wittig German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Functional Proteomics, SFB 815 Core Unit, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Juliana Heidler German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Functional Proteomics, SFB 815 Core Unit, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Heike Stingl Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Search for more papers by this author Voahanginirina Randriamboavonjy Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Search for more papers by this author Karin Kohlstedt Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Bernhard Brüne Institute of Biochemistry I, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Andreas Weigert Institute of Biochemistry I, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Beate Fisslthaler Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Search for more papers by this author Ingrid Fleming Corresponding Author [email protected] orcid.org/0000-0003-1881-3635 Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Search for more papers by this author Mauro Siragusa Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Search for more papers by this author Janina Thöle Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Search for more papers by this author Sofia-Iris Bibli Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Search for more papers by this author Bert Luck Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Search for more papers by this author Annemarieke E Loot Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Kevin de Silva Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Ilka Wittig German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Functional Proteomics, SFB 815 Core Unit, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Juliana Heidler German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Functional Proteomics, SFB 815 Core Unit, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Heike Stingl Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Search for more papers by this author Voahanginirina Randriamboavonjy Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Search for more papers by this author Karin Kohlstedt Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Bernhard Brüne Institute of Biochemistry I, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Andreas Weigert Institute of Biochemistry I, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany Search for more papers by this author Beate Fisslthaler Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Search for more papers by this author Ingrid Fleming Corresponding Author [email protected] orcid.org/0000-0003-1881-3635 Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany Search for more papers by this author Author Information Mauro Siragusa1,2, Janina Thöle1,2, Sofia-Iris Bibli1,2, Bert Luck1,2, Annemarieke E Loot1, Kevin Silva1, Ilka Wittig2,3, Juliana Heidler2,3, Heike Stingl1,2, Voahanginirina Randriamboavonjy1,2, Karin Kohlstedt1, Bernhard Brüne4, Andreas Weigert4, Beate Fisslthaler1,2 and Ingrid Fleming *,1,2 1Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany 2German Center for Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany 3Functional Proteomics, SFB 815 Core Unit, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany 4Institute of Biochemistry I, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany *Corresponding author. Tel: +49 69 6301 6972; Fax: +49 69 6301 86880; E-mail: [email protected] EMBO J (2019)38:e100938https://doi.org/10.15252/embj.2018100938 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Decreased nitric oxide (NO) bioavailability and oxidative stress are hallmarks of endothelial dysfunction and cardiovascular diseases. Although numerous proteins are S-nitrosated, whether and how changes in protein S-nitrosation influence endothelial function under pathophysiological conditions remains unknown. We report that active endothelial NO synthase (eNOS) interacts with and S-nitrosates pyruvate kinase M2 (PKM2), which reduces PKM2 activity. PKM2 inhibition increases substrate flux through the pentose phosphate pathway to generate reducing equivalents (NADPH and GSH) and protect against oxidative stress. In mice, the Tyr656 to Phe mutation renders eNOS insensitive to inactivation by oxidative stress and prevents the decrease in PKM2 S-nitrosation and reducing equivalents, thereby delaying cardiovascular disease development. These findings highlight a novel mechanism linking NO bioavailability to antioxidant responses in endothelial cells through S-nitrosation and inhibition of PKM2. Synopsis Generation of nitric oxide (NO) by endothelial NO synthase (eNOS) is key for a healthy vasculature, but how protein S-nitrosation affects endothelial cell function is poorly understood. eNOS induces S-nitrosation and inactivation of pyruvate kinase M2, thereby triggering antioxidant responses in mice through the rewiring of endothelial cell metabolism. Under homeostatic conditions, PKM2 is part of the endothelial NO synthase (eNOS) signalosome, and is inhibited by S-nitrosation. Inhibition of PKM2 shifts glucose catabolism towards the pentose phosphate pathway, which generates NADPH and reduced glutathione. Knock-in mice carrying activated eNOS (Y656F-eNOS) show enhanced endothelium-dependent NO generation and are protected against angiotensin II-induced hypertension and endothelial dysfunction. Introduction Endothelial cells are situated at the interface between the blood and the vessel wall and control vascular tone and homeostasis. Nitric oxide (NO) derived from the endothelial NO synthase (eNOS) plays a crucial role in these processes and in the modulation of endothelial cell activation and vascular inflammation (Siragusa & Fleming, 2016; Jamwal & Sharma, 2018). The continued generation of NO by eNOS has long been associated with a healthy vasculature while decreased NO bioavailability, as a consequence of reduced eNOS activity or the reaction of NO with superoxide anions, has been linked with cardiovascular disease (Forstermann et al, 2017). Certainly, a genetic predisposition toward enhanced NO signaling is clearly linked with a reduced risk of developing coronary artery disease, peripheral artery disease, and stroke in humans (Emdin et al, 2018). Early studies have shown that, in addition to anti-hypertensive properties, eNOS-derived NO prevents leukocyte adhesion to the vascular endothelium and leukocyte migration into the vessel wall through inhibition of nuclear factor κB to prevent the upregulation of adhesion molecules (Niu et al, 1994; De et al, 1995; Gauthier et al, 1995; Khan et al, 1996; Tsao et al, 1996). However, it is clear that the impact of NO on endothelial cell function extends beyond nuclear factor κB and that numerous endothelial proteins are targeted by the NO generated by eNOS. A significant portion of the overall biological response to NO can be attributed to the oxidative modification of cysteine residues to form S-nitrosothiols in a process referred to as S-nitros(yl)ation (Stamler et al, 2001; Hess & Stamler, 2012). The co-localization of eNOS with specific target proteins, via either a direct interaction with eNOS or through scaffolding proteins, is an important mechanism to ensure efficient S-nitrosation (Stomberski et al, 2018). Indeed, the localization of eNOS to the Golgi apparatus or to the nucleus was shown to increase the S-nitrosation of specific target proteins in the two subcellular compartments (Iwakiri et al, 2006). Of note, although a large number of proteins were shown to be S-nitrosated in different cell systems, including endothelial cells, the functional consequences of this post-translational modification have seldom been investigated in detail. As the changes in NO bioavailability that characterize the development of cardiovascular diseases are bound to affect steady-state protein S-nitrosation, the aim of this study was to interrogate the eNOS interactome for novel binding partners and potential S-nitrosation targets that could contribute to cellular redox regulation and to the NO-mediated protection of the vascular wall against atherogenesis. Results Pyruvate kinase M2 interacts with active eNOS To identify proteins interacting with the active eNOS that may be novel S-nitrosation targets with relevance to vascular pathophysiology, eNOS complexes were immunoprecipitated from growth factor-stimulated endothelial cells expressing FLAG-tagged wild-type eNOS and subjected to mass spectrometry (Fig 1A, Appendix Table S1). This approach identified well-known eNOS-associated proteins, e.g., calmodulin (CaM) and heat-shock protein (Hsp) family members, proteins whose relationship with eNOS has not been investigated to date, e.g., nuclear and cytoskeletal proteins, and proteins reported to be S-nitrosated. One protein belonging to the latter group was a glycolytic enzyme, pyruvate kinase M2 (PKM2). Figure 1. PKM2 interacts with active eNOS A. Volcano plot highlighting proteins significantly enriched (open circles, FDR < 0.05) in eNOS-FLAG immunoprecipitates from growth factor-stimulated human endothelial cells; n = 3 independent cell batches. B. Nitrite in the cell supernatant of HEK293 cells expressing wild-type (WT), Y657F (YF), or Y657D (YD) eNOS and treated with solvent (Sol) or ionomycin (Io, 1 μmol/l, 15 min); n = 6 independent experiments (2-way ANOVA and Bonferroni). C. The FMN/FAD ratio measured in eNOS immunoprecipitates from cells expressing WT, YF, or YD eNOS; n = 6 independent experiments (1-way ANOVA and Newman–Keuls). D. The consequence of mutating Y657 on the growth factor-induced formation of eNOS complexes with Hsp90 and calmodulin (CaM); n = 6–19 independent cell batches (1-way ANOVA and Newman–Keuls). E, F. Effect of mutating Y657 on the formation of complexes between eNOS, Hsp90, and PKM2 in growth factor-stimulated human endothelial cells. Complex formation was assessed in eNOS-FLAG immunoprecipitates (E), or PKM2 immunoprecipitates (F); n = 4–8 independent cell batches (1-way ANOVA and Newman–Keuls). Data information: Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure. Source Data for Figure 1 [embj2018100938-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint To study the consequences of elevated versus attenuated NO production on PKM2 activity, experiments were performed in the presence of the wild-type, the Tyr657Phe (Y657F), or Tyr657Asp (Y657D) eNOS mutants. These mutations were selected as the phosphorylation of eNOS on Y657 by the redox-sensitive kinase proline-rich tyrosine kinase (PYK2) was previously shown to abrogate eNOS enzymatic activity and may therefore contribute significantly to the impaired endothelial function that characterizes cardiovascular diseases. Consistent with a previous report (Fisslthaler et al, 2008), cells expressing the non-phosphorylatable Y657F-eNOS mutant consistently generated more NO than the wild-type enzyme under basal conditions as well as following Ca2+ ionophore stimulation (Fig 1B). The phosphomimetic Y657D-eNOS mutant, on the other hand, was unable to generate NO, an observation attributable to the lack of FMN binding (Fig 1C). In endothelial cells, the enhanced activity of the Y657F-eNOS mutant was accompanied by an increased association with Hsp90 and CaM, compared to either the wild-type or Y657D-eNOS enzymes (Figs 1D and EV1A). In the same conditions, significantly more PKM2 associated with Y657F-eNOS than with the wild-type or Y657D-eNOS proteins (Figs 1E and EV1A). The association between eNOS and PKM2 appeared to be mediated by Hsp90, as both eNOS and Hsp90 could be detected in PKM2 immunoprecipitates from growth factor-stimulated endothelial cells (Fig 1F). In line with the fact that PKM2 has been identified as an Hsp90 client protein in other cellular systems (Subbaramaiah et al, 2016; Yang et al, 2016), PKM2 and Hsp90 also formed complexes in HEK293 cells in the absence eNOS (Fig EV1B). To determine whether or not the increased association of PKM2 with eNOS was a consequence of the higher eNOS activity, experiments were repeated using a S633D-eNOS mutant that also displays a higher NO output (Boo et al, 2003). However, the mutation of eNOS on S633 did not alter Hsp90, CaM, or PKM2 binding compared to the wild-type enzyme (Fig EV1C). Thus, the increased binding of Hsp90 and not an increase in the activity of eNOS per se appears to be instrumental for the increased binding of PKM2 to the Y657F-eNOS. Click here to expand this figure. Figure EV1. Consequence of eNOS mutation on the assembly of the eNOS signalosome The consequence of mutating Y657 on the growth factor-induced formation of eNOS complexes with Hsp90, CaM, and PKM2 in human endothelial cells as summarized in Fig 1. Human endothelial cells expressing GFP were included as control. Complex formation was assessed in eNOS-FLAG immunoprecipitates. Formation of complexes between PKM2 and Hsp90 in the absence or presence of eNOS in HEK293 cells. Similar results were obtained in three additional experiments. Effect of mutating Ser633 to Asp (SD) on the formation of complexes between eNOS, Hsp90, and PKM2 in growth factor-stimulated human endothelial cells. Complex formation was assessed in eNOS-myc immunoprecipitates; n = 4 independent cell batches. Data are presented as mean ± SEM. Source data are available online for this figure. Download figure Download PowerPoint Inhibition of PKM2 by S-nitrosation contributes to endothelial cell antioxidant responses In human endothelial cells, the activity of the PKM2 that associated with Y657F-eNOS or wild-type eNOS was significantly lower than that associated with the phosphomimetic Y657D mutant (Fig 2A). Also in eNOS-transfected HEK293 cells, PKM2 exhibited the highest activity when associated with Y657D-eNOS and its activity decreased progressively when bound to the wild-type, Y657F, and S633D eNOS enzymes generating increasing amounts of NO (Fig 2B). PKM2 can be S-nitrosated (Wu et al, 2010; Chung et al, 2015; Zhang et al, 2016), making it tempting to speculate that the inhibition of eNOS-associated PKM2 could be related to its S-nitrosation. Therefore, endothelial cells were adenovirally transduced to express wild type or Y657F-eNOS and treated with H2O2, to activate PYK2 and elicit eNOS tyrosine phosphorylation (Loot et al, 2009). Under these conditions, PKM2 S-nitrosation was significantly lower in cells containing tyrosine phosphorylated wild-type eNOS than in cells expressing the non-phosphorylatable Y657F-eNOS mutant (Fig 2C). Thiol oxidation of PKM2 on Cys358 was previously reported to inhibit its activity (Anastasiou et al, 2011), and while a pronounced S-nitrosation of wild-type PKM2 could be detected in Y657F-eNOS-expressing cells, no signal was detected in cells expressing a C358S-PKM2 mutant (Fig 2D). Neither the wild-type enzyme nor the mutant PKM2 was S-nitrosated in cells expressing Y657D-eNOS. Importantly, while the activity of the wild-type PKM2 that associated with Y657F-eNOS was low, eNOS-associated pyruvate kinase activity was significantly higher when the cells were transfected with the S-nitrosation insensitive C358S-PKM2 mutant (Fig 2E). That a complete rescue was not achieved can most probably be attributed to the fact that the HEK293 cells studied expressed relatively high levels of endogenous PKM2 that could compete with the C358S-PKM2 mutant for binding to eNOS (see Fig 2D). To assess the link between eNOS and PKM2 in more detail, PKM2 activity was assessed in eNOS immunoprecipitates from HEK293 cells expressing either Y657F- or Y657D-eNOS. In the presence of increasing concentrations of phosphoenolpyruvate, both the Vmax and the Km of the Y657F-eNOS-associated PKM2 were significantly lower than for Y657D-eNOS-associated PKM2: Vmax: 0.08 ± 0.004 versus 0.06 ± 0.003 U/min and Km: 0.09 ± 0.03 versus 0.03 ± 0.02 mmol/l for PKM2-Y657D-eNOS versus PKM2-Y657F-eNOS, respectively (Fig 2F). Figure 2. Inhibition of PKM2 by S-nitrosation facilitates the accumulation of reducing equivalents to attenuate oxidative stress Pyruvate kinase (PK) activity in FLAG immunoprecipitates from human endothelial cells expressing FLAG-tagged wild-type (WT), Y657F (YF), or Y657D (YD) eNOS; n = 6 independent cell batches (1-way ANOVA and Tukey). PK activity in eNOS immunoprecipitates from HEK293 cells expressing WT, YF, YD, or S633D (SD) eNOS; n = 5–9 independent experiments (1-way ANOVA and Bonferroni). S-Nitrosation of PKM2 in human endothelial cells expressing FLAG-tagged WT or YF-eNOS and treated with H2O2 (30 μmol/l) for 15 min; n = 8 independent cell batches (Unpaired Student's t-test). Dithiothreitol (DTT) was included to demonstrate specificity of the SNO signal. PKM2 S-nitrosation in HEK293 cells expressing YF or YD eNOS and either FLAG-tagged WT PKM2 or a C358S (CS) PKM2 mutant. SNO-FLAG-PKM2 was detected with an anti-FLAG antibody after biotin switch technique; n = 4 independent experiments (2-way ANOVA and Bonferroni). DTT treatment was included to demonstrate specificity of the SNO signal. PK activity in eNOS immunoprecipitates from HEK293 cells co-expressing YF or YD eNOS and either WT PKM2 or the CS PKM2 mutant; n = 6 independent experiments (2-way ANOVA and Bonferroni). PKM2 enzyme kinetics measured with increasing concentrations of phosphoenolpyruvate (PEP) in eNOS immunoprecipitates from HEK293 cells expressing YF or YD eNOS. The data were fit with the Michaelis–Menten equation to determine Vmax and Km; n = 4 independent experiments (2-way ANOVA and Bonferroni). Quantification of pentose phosphate pathway (PPP) intermediates; gluconate-6-P (G6P), ribulose-5-P (Rl5P), ribose-5-P (R5P), sedoheptulose-7-P (S7P), fructose-6-P (F6P) and erythrose-4-P (E4P) in human endothelial cells expressing YF or YD eNOS; n = 6–8 independent cell batches (Unpaired Student's t-test). Link between the pentose phosphate pathway, generation of NADPH, and reduction of GSSG to GSH. NADPH/NADP+ and GSH/GSSG ratios in human endothelial cells expressing YF or YD eNOS; n = 6–8 independent cell batches (Unpaired Student's t-test). NADPH/NADP+ and GSH/GSSG ratios in human endothelial cells treated with solvent (Sol) or the PKM2 inhibitor shikonin (SKN, 1 μmol/l) for 45 min; n = 4 independent cell batches (Unpaired Student's t-test). Data information: Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure. Source Data for Figure 2 [embj2018100938-sup-0005-SDataFig2.pdf] Download figure Download PowerPoint The inhibition of PKM2 by thiol oxidation is reported to shift glucose catabolism to the pentose phosphate pathway, thereby generating reducing equivalents required for the generation of reduced glutathione (GSH) and the detoxification of reactive oxygen species (Anastasiou et al, 2011). The eNOS-dependent S-nitrosation of PKM2 resulted in the same effect as the majority of the intermediates of the pentose phosphate pathway and cellular NADPH/NADP+ and GSH/GSSG ratios were significantly higher in endothelial cells expressing the Y657F-eNOS than the Y657D-eNOS mutant (Fig 2G–I). Treating endothelial cells with the PKM2 inhibitor shikonin (Chen et al, 2011) resulted in similar increases in the NADPH/NADP+ and GSH/GSSG ratios (Fig 2J). Enhanced NO bioavailability results in PKM2 S-nitrosation and inhibition in vivo To study the consequences of preserved NO bioavailability on PKM2 activity under physiological conditions, Tyr656 (mouse sequence; equivalent to Tyr657 in the human sequence) was mutated to phenylalanine to generate knock-in mice, hereafter referred to as YF-eNOS mice (Fig EV2). Under basal conditions, the YF-eNOS mice demonstrated a tendency to lower systolic blood pressure compared with wild-type littermates (Fig 3A). In isolated rings of endothelium-intact aortae, phenylephrine-induced contractions were consistently attenuated in the YF-eNOS group, a phenomenon that was not observed following the addition of a NOS inhibitor (Fig 3B). In the same samples, the acetylcholine-induced NO-dependent relaxation was slightly but significantly potentiated in aortic rings from YF-eNOS mice (Fig 3C), while responses to sodium nitroprusside were comparable in both groups (Appendix Fig S1). Acetylcholine-induced relaxations were also significantly greater in the isolated perfused hindlimb circulation of YF-eNOS compared with wild-type mice (Fig 3D), a difference no longer observed following NOS inhibition. These results indicate that the mutation of eNOS Tyr656 to Phe resulted in the generation of a mouse model with slightly improved endothelium-dependent NO generation compared with wild-type mice. Consistent with the in vitro findings, PKM2 S-nitrosation was significantly higher in cultured endothelial cells from YF-eNOS compared with wild-type mice (Fig 3E). Also, pyruvate kinase activity in eNOS immunoprecipitates from pulmonary endothelial cells (Fig 3F) and hearts (Fig 3G) was significantly lower in the YF-eNOS than in the wild-type group. Proximity ligation assays in murine endothelial cells revealed that the association between eNOS and PKM2 occurred mostly in the cytoplasm and in proximity of the Golgi (Fig 3H). Click here to expand this figure. Figure EV2. Generation of the YF-eNOS mouse Schematic representation of the murine eNOS gene and of the targeting vector used to introduce the Y656F mutation. Schematic representation of the Y656F eNOS knock-in allele. The positive selection cassette (puromycin) flanked by F3 sites was deleted by crossing with a ubiquitously expressing FLP1 recombinase mouse strain. The Y656F mutation generates a BsmI restriction site, allowing identification of the wild-type, heterozygous, and knock-in alleles by PCR and subsequent BsmI digestion. Genotyping PCR using the primers described in the Materials and Methods section. Download figure Download PowerPoint Figure 3. Enhanced NO bioavailability results in PKM2 S-nitrosation and inhibition in vivo A. Systolic blood pressure (SBP) in wild-type (WT) and YF-eNOS (YF) mice; n = 9 animals per group (Unpaired Student's t-test). B, C. Phenylephrine (PE)-induced contraction (B) and acetylcholine (Ach)-induced relaxation (C) of endothelium-intact aortic rings from WT and YF mice. Experiments were performed in the absence and presence of L-NAME (LN, 300 μmol/l); n = 6–10 animals per group (2-way ANOVA and Bonferroni). D. Acetylcholine (ACh)-induced vasodilatation of the buffer-perfused hindlimb in situ. Experiments were performed in the absence and presence of L-NAME (LN, 300 μmol/l); n = 12–13 animals per group (2-way ANOVA and Bonferroni). E. PKM2 S-nitrosation in pulmonary endothelial cells from WT and YF mice; n = 5–6 independent cell batches (Unpaired Student's t-test). Dithiothreitol (DTT) treatment was included to demonstrate specificity of the SNO signal. The blots show non-adjacent bands cropped from the same membranes. F. PK activity in eNOS immunoprecipitates from WT and YF pulmonary endothelial cells; n = 7–10 independent cell batches (Unpaired Student's t-test). G. PK activity in eNOS immunoprecipitates from hearts from WT and YF mice; n = 6 mice per group (Unpaired Student's t-test). H. Representative images of eNOS-PKM2 interaction (PLA foci, red) in mouse pulmonary endothelial cells; the Golgi apparatus and endosomes were stained with Golph4 (green), the plasma membrane was stained with CD144, and nuclei were highlighted with DAPI (gray). Only rare PLA foci were found in samples incubated with control mouse and rabbit IgGs, demonstrating the specificity of the reaction; n = 3 independent cell batches. Scale bars: 20 μm. Data information: Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant. Source data are available online for this figure. Source Data for Figure 3 [embj2018100938-sup-0006-SDataFig3.pdf] Download figure Download PowerPoint Preserved NO bioavailability attenuates angiotensin II-induced endothelial dysfunction through PKM2 S-nitrosation and enhanced antioxidant responses in vivo To study the link between eNOS tyrosine phosphorylation and PKM2 activity in a pathophysiological condition, mice were administered angiotensin II via osmotic minipumps, a procedure that increased the tyrosine phosphorylation of eNOS in wild-type mice (Fig 4A). After 28 days of angiotensin II, hypertension was manifest in wild-type mice (Fig 4B) and was accompanied by endothelial dysfunction evidenced by attenuated acetylcholine-induced, NO-dependent relaxations (Fig 4C, compare with Fig 3C). Both the increase in blood pressure and the change in vascular reactivity were significantly attenuated in YF-eNOS mice: −log EC50
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