Sirtuin1-regulated lysine acetylation of p66Shc governs diabetes-induced vascular oxidative stress and endothelial dysfunction Santosh Kumara,b,1, Young-Rae Kima,b, Ajit Vikrama,b, Asma Naqvic, Qiuxia Lia,b, Modar Kassana,b, Vikas Kumard, Markus M. Bachschmidd, Julia S. Jacobsa,b, Ajay Kumarc, and Kaikobad Irania,b,1 a Division of Cardiovascular Medicine, Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, IA 52242; bAbboud Cardiovascular Research Center, University of Iowa, Iowa City, IA 52242; cCardiovascular Institute, University of Pittsburgh, Pittsburgh, PA 15213; and d Vascular Biology Section, Cardiovascular Proteomics Center, Boston University School of Medicine, Boston, MA 02118
Edited by Marc Montminy, The Salk Institute for Biological Studies, La Jolla, CA, and approved December 27, 2016 (received for review August 23, 2016)
The 66-kDa Src homology 2 domain-containing protein (p66Shc) is a master regulator of reactive oxygen species (ROS). It is expressed in many tissues where it contributes to organ dysfunction by promoting oxidative stress. In the vasculature, p66Shc-induced ROS engenders endothelial dysfunction. Here we show that p66Shc is a direct target of the Sirtuin1 lysine deacetylase (Sirt1), and Sirt1-regulated acetylation of p66Shc governs its capacity to induce ROS. Using diabetes as an oxidative stimulus, we demonstrate that p66Shc is acetylated under high glucose conditions and is deacetylated by Sirt1 on lysine 81. High glucose-stimulated lysine acetylation of p66Shc facilitates its phosphorylation on serine 36 and translocation to the mitochondria, where it promotes hydrogen peroxide production. Endothelium-specific transgenic and global knockin mice expressing p66Shc that is not acetylatable on lysine 81 are protected from diabetic oxidative stress and vascular endothelial dysfunction. These findings show that p66Shc is a target of Sirt1, uncover a unique Sirt1-regulated lysine acetylationdependent mechanism that governs the oxidative function of p66Shc, and demonstrate the importance of p66Shc lysine acetylation in vascular oxidative stress and diabetic vascular pathophysiology. p66Shc
| sirt1 | lysine acetylation | diabetes | oxidative stress
T
he 66-kDa Src homology 2 domain-containing protein (p66Shc) belongs to ShcA family of adaptor proteins. It is unique in this family because unlike the p46 and p52 isoforms, it impairs growth factor signaling and promotes oxidative stress (1). p66Shc contributes to aging (2), obesity (3, 4), atherosclerosis (5), and diabetes and aging-related vascular dysfunction (6, 7) in mice. In response to oxidative stimuli, p66Shc gets phosphorylated at serine 36 (S36) and translocates to mitochondria, where it produces reactive oxygen species (ROS) by oxidizing cytochrome C (8, 9). p66Shc is also phosphorylated on other residues that increase its half-life (10). In addition, p66Shc activates PKCBII, which in turn phosphorylates p66Shc, creating a positive feedback loop (11). The importance of p66Shc in human disease is supported by evidence that its expression increases in pathologies such as diabetes and atherosclerosis (12, 13). Sirt1 belongs to the class III NAD+-dependent histone deacetylases (HDACs). In lower organisms, it mediates longevity in response to caloric restriction (14). In addition to histones, it deacetylates many nonhistone proteins such as p53, FOXO, PGC1-alpha, LXR, and e-NOS (15–19). Sirt1 and p66Shc have opposing effects on vascular function (20). Unlike p66Shc, Sirt1 mitigates diabetes-induced vascular dysfunction (21). Moreover, down-regulation of Sirt1 in diabetes leads to epigenetic up-regulation of p66Shc (22). However, whether Sirt1 directly targets p66Shc for lysine deacetylation and whether dynamic lysine acetylation of p66Shc governs its oxidative function are not known. Here we show that acetylation of lysine 81 in p66Shc is obligatory for diabetic vascular dysfunction, and Sirt1 antagonizes this acetylation, thereby suppressing p66Shc-mediated oxidative stress. 1714–1719 | PNAS | February 14, 2017 | vol. 114 | no. 7
Results and Discussion Sirt1 Deacetylates p66Shc on Lysine 81. We first determined
whether the acetylation status of p66Shc is dynamic and governed by Sirt1. Knockdown of Sirt1 using siRNA increased lysine acetylation of ectopically expressed p66Shc in human embryonic kidney-293 (HEK 293) cells (SI Appendix, Fig. S1A) and led to hyperacetylation of endogenous p66Shc in human umbilical vein endothelial cells (HUVECs) (Fig. 1A and SI Appendix, Fig. S1B). Next, we investigated whether Sirt1 and p66Shc associate with each other. Endogenous p66Shc in HUVECs coprecipitated with endogenous Sirt1 in HUVECs (Fig. 1B), and immunoprecipitation of overexpressed p66Shc in HEK 293 cells pulled down overexpressed Sirt1 (SI Appendix, Fig. S1C). It is important to note that knockdown of Sirt1 also increased lysine acetylation of p46 and p52Shc (SI Appendix, Fig. S1B), suggesting that there may be common lysine residues in all three isoforms that are deacetylated by Sirt1. However, we focused our attention on lysine residues in the N-terminal collagen homology 2 (CH2) domain of p66Shc (2), which is not shared by p46 and p52Shc, as it is this CH2 domain that confers upon p66Shc its unique oxidative function. There are three conserved lysine residues in the CH2 domain at positions 7, 9, and 81 (SI Appendix, Fig. S2). To determine which, if any, of these three lysines is acetylated and targeted by Sirt1 for deacetylation, we first performed acetylation–deacetylation assays followed by mass spectrometry Significance Many oxidative stimuli engage the 66-kDa Src homology 2 domaincontaining protein (p66Shc) to induce reactive oxygen species (ROS). ROS regulated by p66Shc promotes aging and contributes to cancer, diabetes, obesity, cardiomyopathy, and atherosclerosis. Here we identify a fundamental mechanism that controls p66Shc and p66Shc-regulated ROS. We show that p66Shc is lysine acetylated when cells are faced with an oxidative stimulus (diabetes), and lysine acetylation of p66Shc is obligatory for p66Shc-induced ROS. In addition, lysine-acetylated p66Shc is deacetylated by the Sirtuin1 lysine deacetylase, and Sirt1-mediated deacetylation of p66Shc curtails ROS production. This intersection between p66Shc and Sirtuin1 adds a dimension to how p66Shc is regulated by certain stimuli and how Sirtuin1 suppresses oxidative stress promoted by such stimuli. Author contributions: S.K., M.M.B., and K.I. designed research; S.K., Y.-R.K., A.V., A.N., Q.L., M.K., V.K., J.S.J., and A.K. performed research; S.K., Y.-R.K., A.V., and M.M.B. analyzed data; and S.K. and K.I. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1
To whom correspondence may be addressed. Email: [email protected] or [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1614112114/-/DCSupplemental.
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Fig. 1. Sirt1 inhibits oxidative function of p66Shc by deacetylating it on lysine 81. (A) Immunoblot for acetylated p66Shc with Sirt1 knockdown in HUVECs. (B) Coimmunoprecipitation for endogenous Sirt1 and p66Shc in HUVECs. (C) Tandem mass spectrometry of recombinant CH2 domain of p66Shc acetylated in vitro by p300. Acetylated peptide shown has m/z 554.96. (Inset) Acetyl-lysine immunoblot of p300-acetylated and Sirt1-deacetylated CH2 domain used for mass spectrometry. (D and E) Immunoblots for acetylp66ShcK81 (D) and phospho-p66ShcS36 (E) in HUVECs with Sirt1 knockdown. (F and G) Immunofluorescence for acetyl-p66ShcK81 (F) and phospho-p66ShcS36 (G) in aortas of mice with conditional deletion of endothelial Sirt1 (e-SIRT1KO) and wild-type mice. Arrow indicates endothelial layer. (H) Quantification of endothelial acetyl-p66ShcK81 and phospho-p66ShcS36 from F and G. *P < 0.05, **P < 0.01; n = 3; Student’s t test. (I) Quantification of H2O2 (DCF fluorescence) in HUVECs expressing p66ShcWT or p66ShcK81R and treated with Sirt1 inhibitor NAM. ***P < 0.001; n = 3–7; Student’s t test. Data represent mean ± SEM. Immunoblots are representative of at least three independent experiments. vWF, von Willebrand factor.
on recombinant CH2. His-tagged CH2 was acetylated in vitro with p300 acetyltransferase followed by deacetylation by Sirt1. Immunoblotting showed that p300 induced lysine acetylation of CH2, which was reversed by Sirt1 (Fig. 1C, Inset). CH2 acetylated by p300 and deacetylated by Sirt1 was then subjected to mass spectrometry. Lysine 81 (K81) was identified as the most abundantly acetylated and deacetylated residue (Fig. 1C and SI Appendix, Fig. S3 A and B). We developed an antibody against K81-acetylated p66Shc (acetyl-p66ShcK81) and verified that it detects only p300-acetylated and not nonacetylated recombinant CH2 (SI Appendix, Fig. S4A). Moreover, using this antibody, we Kumar et al.
verified that Sirt1 deacetylates K81 in full-length p66Shc expressed in HEK 293 cells (SI Appendix, Fig. S4B). Further, using this antibody, we showed that knockdown of Sirt1 in HUVECs engenders hyperacetylation of K81 in endogenous p66Shc (SI Appendix, Fig. S4C). To further verify that manipulation of Sirt1 and p300 hyperacetylates K81 and that the acetyl-p66ShcK81 antibody has specificity for Ac-K81, we created a p66Shc mutant that is nonacetylatable on K81 (K81R). Knockdown of Sirt1 in HUVECs or overexpression of p300 in HEK 293 cells increased acetylation on K81 in p66ShcWT but not p66ShcK81R (Fig. 1D and SI Appendix, Fig. S4 D and E). PNAS | February 14, 2017 | vol. 114 | no. 7 | 1715
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Fig. 2. Endothelial p66Shc is acetylated on lysine 81 by high glucose and in diabetes and promotes high glucose-induced mitochondrial oxidative stress. (A and B) Immunofluorescence for acetyly-p66ShcK81 (A) and quantification of acetyl-p66ShcK81 in aortic endothelium of STZ-induced diabetic mice. Magnified images are shown in Inset. Arrow indicates an endothelial layer. ***P < 0.001; n = 4–7; Student’s t test. (C and D) Immunoblot for high glucose-stimulated acetyl-p66ShcK81 and phospho-p66ShcS36 in HUVECs expressing p66ShcWT or p66ShcK81R (C) and densitometric quantification of immunoblots (D). **P < 0.01, ***P < 0.001; n = 3; Student’s t test. (E and F) DCF fluorescence images (E) and quantification of fluorescence (F) of whole HUVECs expressing p66ShcWT or p66ShcK81R and incubated with high glucose. (G) Quantification of DCF fluorescence in mitochondria isolated from HUVECs expressing p66ShcWT or p66ShcK81R and incubated with high glucose. **P < 0.01, ***P < 0.001; n = 3–7; Student’s t test. (H and I) Immunoblots for p66Shc in mitochondrial and whole-cell lysates of HUVECs expressing p66ShcWT or p66ShcK81R and incubated with high glucose (H) and their densitometric quantification (I). **P < 0.01; n = 5; Student’s t test. Data represent means ± SEM. STZ, streptozotocin. Immunoblots are representative of at least three independent experiments.
Deficiency of Sirt1 Promotes S36 Phosphorylation of p66Shc and p66Shc-Induced ROS via K81 Acetylation. There is precedent for lysine
acetylation facilitating serine phosphorylation (23). Phosphorylation of p66Shc on S36 is essential for p66Shc-mediated ROS production (2). We therefore asked if phosphorylation of p66Shc 1716 | www.pnas.org/cgi/doi/10.1073/pnas.1614112114
on S36 is dependent on acetylation of K81. Sirt1 knockdown in HUVECs increased S36 phosphorylation of p66Shc (SI Appendix, Fig. S5A) and increased ROS (H2O2) levels in both HUVECs and HEK 293 cells (SI Appendix, Fig. S5 B and C). Sirt1 knockdown did not stimulate S36 phosphorylation of p66ShcK81R Kumar et al.
High Glucose-Stimulated K81 Acetylation Promotes Mitochondrial Transport of p66Shc and p66Shc-Mediated Mitochondrial Oxidative Stress. Diabetes promotes vascular oxidative stress via p66Shc
(6). We evaluated the role of K81 acetylation in p66Shc-mediated diabetic oxidative stress in the vascular endothelium. We first examined the acetylation status of vascular p66Shc in streptozotocin (STZ)-induced diabetic mice. STZ-induced diabetes led to hyperacetylation of endothelial p66Shc on K81 (Fig. 2 A and B). A similar increase in K81 acetylation was observed in HUVECs incubated in high glucose-containing medium (Fig. 2 C and D). High glucose also stimulated S36 phosphorylation of p66Shc in HUVECs (Fig. 2 C and D). Importantly, high glucose failed to induce K81 acetylation or S36 phosphorylation of p66ShcK81R. Thus, K81 is acetylated in endothelial cells by high glucose in vitro and diabetes in vivo and facilitates S36 phosphorylation. We then evaluated the role of K81 acetylation in high glucoseinduced ROS mediated by p66Shc. High glucose-stimulated H2O2 was blunted in HUVECs expressing p66ShcK81R compared with those expressing p66ShcWT (Fig. 2 E and F and SI Appendix, Fig. S6). Because of the role of p66Shc in mitochondrial oxidative stress, we next asked if K81 acetylation is obligatory for high glucose-stimulated mitochondrial ROS mediated by p66Shc. High glucose-stimulated H2O2 in isolated mitochondria was significantly blunted in HUVECs expressing p66ShcK81R compared with those expressing p66ShcWT (Fig. 2G). Thus, cells expressing p66ShcK81R are protected from high glucose-stimulated mitochondrial oxidative stress. In response to oxidative stimuli, a fraction of p66Shc translocates to the mitochondrial intermembrane space in mitochondria, where it oxidizes cytochrome c, resulting in oxidative stress and mitochondrial depolarization (8, 9). Given the importance of K81 acetylation in high glucose-stimulated ROS, we asked if K81 acetylation is also important for mitochondrial translocation of p66Shc. Incubation of HUVECs with high glucose led to an increase in both endogenous and overexpressed p66ShcWT in crude mitochondrial fraction (SI Appendix, Fig. S7 A and B and Fig. 2 H and I). In contrast to p66ShcWT, p66ShcK81R did not accumulate in the mitochondria in response to high glucose (Fig. 2 H and I). These findings suggest that K81 acetylation plays an important part in high glucosestimulated mitochondrial translocation of p66Shc. K81 Acetylation of p66Shc Promotes Diabetic Vascular Endothelial Dysfunction. Diabetic vascular dysfunction is, in part, mediated
by p66Shc (6). To determine if K81 acetylation is required for p66Shc-mediated diabetic vascular dysfunction, we first expressed p66ShcK81R using a recombinant adenovirus (Ad-p66ShcK81R) in mouse aortas ex vivo (SI Appendix, Fig. S8A). Compared with expression of p66ShcWT (Ad-p66ShcWT), expression of p66ShcK81R did not result in impairment of endothelium-dependent vasorelaxation (SI Appendix, Fig. S8B). Endothelium-independent vascular relaxation was not different between mouse aortas expressing p66ShcWT and p66ShcK81R (SI Appendix, Fig. S8C). Kumar et al.
We also examined the effect of expressing p66ShcK81R in aortas of db/db diabetic mice that have impaired endothelium-dependent vasorelaxation. Expression of p66ShcK81R rescued endotheliumdependent vasorelaxation, whereas expression of p66ShcWT worsened it (SI Appendix, Fig. S8 D and E). However, endotheliumindependent relaxation was not different between db/db aortas expressing p66ShcWT and p66ShcK81R (SI Appendix, Fig. S8F). To explore the in vivo role of p66Shc K81 acetylation in diabetic vascular dysfunction, we generated a transgenic mouse with endothelium-specific expression of p66ShcK81R (henceforth called e-p66ShcK81R) (SI Appendix, Fig. S9A). These mice are viable and healthy. Diabetes was induced by a single bolus injection of STZ (SI Appendix, Fig. S9B). Aortas of e-p66ShcK81R mice had improved endothelium-dependent relaxation both under nondiabetic and diabetic conditions, compared with their wild-type nontransgenic littermate controls (Fig. 3A). Moreover, endothelium-specific oxidative stress (measured as 8-hydroxy deoxyguanosine, 8-OHdG) (Fig. 3 B and C) and acetylation of p66Shc on K81 in the endothelium were diminished in diabetic e-p66ShcK81R transgenic mice compared with diabetic wild-type nontransgenic littermates (SI Appendix, Fig. S9 C and D). To confirm the role of K81 acetylation in diabetic vascular dysfunction, we generated mice with global knockin of p66ShcK81R (SI Appendix, Fig. S10). These mice are fertile, viable, and healthy. There was no difference in STZ-induced hyperglycemia between p66ShcK81R knockin and wild-type mice (SI Appendix, Fig. S11). Although basal endothelium-dependent vasorelaxation was similar in p66ShcK81R knockin and wild-type mice, p66ShcK81R knockin mice were protected from STZ-induced impairment of endothelium-dependent vasorelaxation (Fig. 3D). In addition, bioavailable vascular nitric oxide was higher in p66ShcK81R knockin mice compared with wild-type mice in both the diabetic and nondiabetic states (Fig. 3E). Further, p66ShcK81R knockin mice were protected from STZ-induced vascular oxidative stress (Fig. 3 F and G). By showing that the oxidative function of p66Shc is governed by lysine acetylation and acetylated p66Shc is a substrate for Sirt1, this work uncovers dynamic lysine acetylation as another rheostat for the complex posttranslational regulation of p66Shc. The intersection between lysine acetylation and serine phosphorylation is not unique to p66Shc. Lysine acetylation promotes protein kinase B-mediated phosphorylation of Foxo1 (23), whereas phosphorylation of Beclin1 is required for its subsequent lysine acetylation (24). Interdependence between serine phosphorylation and lysine acetylation may be explained on structural grounds of these posttranslational modifications (25). While our data indicate that K81 acetylation is required for high glucose/diabetes-induced S36 phosphorylation, further studies showed that this requirement is not universally applicable for all oxidative stimuli. This was borne out in studies examining the effect of vascular endothelial growth factor (VEGF) on S36 phosphorylation in p66ShcWT and p66ShcK81R. Although basal S36 phosphorylation in serum-starved HUVECs was lower in p66ShcK81R, VEGF induced S36 phosphorylation to a similar extent in both p66ShcWT and p66ShcK81R, without changing K81 acetylation (SI Appendix, Fig. S12). This stimulus-specific reliance of S36 phosphorylation on K81 acetylation (present in high glucose/ diabetes; absent in VEGF) could be explained by selective engagement of specific kinases by different oxidative stimuli, as phosphorylation of S36 is promiscuously induced by multiple kinases (26–28). An alternative explanation is that dependence of S36 phosphorylation on K81 acetylation may be determined by whether the stimulus affects Sirt1 expression. Diabetes down-regulates endothelial Sirt1 (SI Appendix, Fig. S13), whereas some other oxidative stimuli (such as VEGF) may not. Observations from clinical and preclinical studies suggest that glycemic control alone is not sufficient to prevent diabetic complications and persistent oxidative stress could be responsible for PNAS | February 14, 2017 | vol. 114 | no. 7 | 1717
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expressed in either HEK 293 cells or HUVECs (Fig. 1E and SI Appendix, Fig. S5 D and E). In addition, both K81 acetylation and S36 phosphorylation were increased in the aortic endothelium of mice with conditional deletion of endothelial Sirt1 (e-Sirt1 KO) (Fig. 1 F–H). We then examined the role of Sirt1-regulated K81 acetylation in p66Shc-induced ROS production. Inhibition of Sirt1 with nicotinamide (NAM) increased S36 phosphorylation in cells expressing p66ShcWT but not in cells expressing p66ShcK81R (SI Appendix, Fig. S5 F and G). Further, expression of p66ShcWT, but not p66ShcK81R, amplified NAM-stimulated H2O2 in HUVECs (Fig. 1I). These findings underscore that inhibition of Sirt1 stimulates p66Shc-mediated ROS production via K81 acetylation.
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Fig. 3. Acetylation of p66Shc on lysine 81 mediates diabetic vascular oxidative stress and endothelial dysfunction. (A) Endothelium-dependent vasorelaxation of aortas from nondiabetic and STZ-induced diabetic wild-type mice and transgenic mice with endothelial expression of p66ShcK81R (e-p66ShcK81R). ###P < 0.001 vs. wild type; *P < 0.05, ***P < 0.001 vs. wild-type STZ. Wild type (n = 12), e-p66ShcK81R (n = 18), wild-type STZ (n = 20), and e-p66ShcK81R STZ (n = 8). (B) Immunofluorescence for oxidative stress (8-OHdG; oxidative DNA adduct) in aortic sections of nondiabetic and STZ-induced diabetic wild-type and e-p66ShcK81R mice. (C) Quantification of endothelial 8-OHdG in B. vWF, von Willebrand factor. ***P < 0.001; n = 5–8; Student’s t test. (D) Endothelium-dependent vasorelaxation of aortas of control (nondiabetic) and STZ-induced diabetic wild-type and p66ShcK81R knockin mice. #P < 0.05, ###P < 0.001 vs. wild type. Wild type, n = 14; p66ShcK81R, n = 13; wild-type STZ, n = 15; and p66ShcK81R STZ, n = 21. (E) Nitric oxide bioavailability in aortas of control and STZ-induced diabetic p66ShcK81R knockin and wild-type mice. #P < 0.05, ##P < 0.01 vs. wild type; **P < 0.01 vs. wild-type STZ. Wild type, n = 14; p66ShcK81R, n = 13; wild-type STZ, n = 15; and p66ShcK81R STZ, n = 21. (F) Immunofluorescence for 8-OHdG in aortic sections of control and STZ-induced diabetic wild-type and p66ShcK81R knockin mice. (G) Quantification of endothelial 8-OHdG in F. Arrow indicates endothelial layer. ***P < 0.001; n = 8–10; Student’s t test. 8-OHdG, 8 hydroxy-deoxyguanosine; ACh, acetylcholine; PE, phenylephrine. n, number of aortic rings. All vascular reactivity data were analyzed by two-way ANOVA followed by Tukey’s post hoc analysis. Data represent mean ± SEM.
this hyperglycemic memory (29, 30). One proposition that has been forwarded as responsible for persistent vascular oxidative stress in diabetes is the epigenetic up-regulation of p66Shc. In this regard, it has been shown that Sirt1 suppresses p66Shc expression epigenetically, and genetic deletion of p66Shc protects mice against diabetic endothelial dysfunction and vascular hyperglycemic memory (11, 22). Distinct from this finding, our work identifies an alternative mechanism by which Sirt1 regulates p66Shc—through 1718 | www.pnas.org/cgi/doi/10.1073/pnas.1614112114
direct lysine deacetylation. Although acetylation of p66Shc is independent from Sirt1-mediated epigenetic regulation of p66Shc, our data suggest some functional interplay between the two. This is borne out by the finding that suppression of K81 acetylation of p66Shc in diabetic mice partially rescued vascular Sirt1 expression (SI Appendix, Fig. S13). Thus, p66Shc acetylation feeds back to inhibit Sirt1 expression, which in turn may also lead to up-regulation of p66Shc. Given that Sirt1 expression is governed by oxidative Kumar et al.
stress (31), acetylated p66Shc may down-regulate Sirt1 by promoting vascular ROS production (SI Appendix, Fig. S14). In conclusion, these findings identify p66Shc as a target for Sirt1 and show that Sirt1-mediated deacetylation of p66Shc has a vital part in determining tissue vascular oxidative stress and endothelial dysfunction in diabetes. Although the studies were restricted to diabetes/high glucose as the oxidative stimulus and to vascular cells and tissue, the molecular mechanism by which Sirt1-regulated lysine acetylation of p66Shc governs ROS may also be operative in other oxidant-driven pathophysiology.
City, and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (32).
Methods
Study Approval. All experimental animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Iowa, Iowa
ACKNOWLEDGMENTS. We thank T. Finkel and L. Terada for the p66Shc constructs and M. Joiner for CoxIV and Mitomix antibodies. K.I. was supported by the University of Iowa Endowed Professorship in Cardiovascular Medicine and by US Department of Veterans Affairs Grant 1I01BX002940; Q.L. was supported by NIH Grant T32 HL007344; and M.K. was supported by NIH Grant T32 HL007121.
1. Migliaccio E, et al. (1997) Opposite effects of the p52shc/p46shc and p66shc splicing isoforms on the EGF receptor-MAP kinase-fos signalling pathway. EMBO J 16(4): 706–716. 2. Migliaccio E, et al. (1999) The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature 402(6759):309–313. 3. Berniakovich I, et al. (2008) p66Shc-generated oxidative signal promotes fat accumulation. J Biol Chem 283(49):34283–34293. 4. Ranieri SC, et al. (2010) Mammalian life-span determinant p66shcA mediates obesityinduced insulin resistance. Proc Natl Acad Sci USA 107(30):13420–13425. 5. Napoli C, et al. (2003) Deletion of the p66Shc longevity gene reduces systemic and tissue oxidative stress, vascular cell apoptosis, and early atherogenesis in mice fed a high-fat diet. Proc Natl Acad Sci USA 100(4):2112–2116. 6. Camici GG, et al. (2007) Genetic deletion of p66(Shc) adaptor protein prevents hyperglycemia-induced endothelial dysfunction and oxidative stress. Proc Natl Acad Sci USA 104(12):5217–5222. 7. Yamamori T, et al. (2005) P66shc regulates endothelial NO production and endotheliumdependent vasorelaxation: Implications for age-associated vascular dysfunction. J Mol Cell Cardiol 39(6):992–995. 8. Orsini F, et al. (2004) The life span determinant p66Shc localizes to mitochondria where it associates with mitochondrial heat shock protein 70 and regulates transmembrane potential. J Biol Chem 279(24):25689–25695. 9. Giorgio M, et al. (2005) Electron transfer between cytochrome c and p66Shc generates reactive oxygen species that trigger mitochondrial apoptosis. Cell 122(2):221–233. 10. Khanday FA, et al. (2006) Rac1 leads to phosphorylation-dependent increase in stability of the p66shc adaptor protein: Role in Rac1-induced oxidative stress. Mol Biol Cell 17(1):122–129. 11. Paneni F, et al. (2012) Gene silencing of the mitochondrial adaptor p66(Shc) suppresses vascular hyperglycemic memory in diabetes. Circ Res 111(3):278–289. 12. Pagnin E, et al. (2005) Diabetes induces p66shc gene expression in human peripheral blood mononuclear cells: Relationship to oxidative stress. J Clin Endocrinol Metab 90(2):1130–1136. 13. Franzeck FC, et al. (2012) Expression of the aging gene p66Shc is increased in peripheral blood monocytes of patients with acute coronary syndrome but not with stable coronary artery disease. Atherosclerosis 220(1):282–286. 14. Cohen HY, et al. (2004) Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase. Science 305(5682):390–392. 15. Vaziri H, et al. (2001) hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase. Cell 107(2):149–159. 16. Brunet A, et al. (2004) Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 303(5666):2011–2015.
17. Rodgers JT, et al. (2005) Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1. Nature 434(7029):113–118. 18. Li X, et al. (2007) SIRT1 deacetylates and positively regulates the nuclear receptor LXR. Mol Cell 28(1):91–106. 19. Mattagajasingh I, et al. (2007) SIRT1 promotes endothelium-dependent vascular relaxation by activating endothelial nitric oxide synthase. Proc Natl Acad Sci USA 104(37):14855–14860. 20. Paneni F, Volpe M, Lüscher TF, Cosentino F (2013) SIRT1, p66(Shc), and Set7/9 in vascular hyperglycemic memory: Bringing all the strands together. Diabetes 62(6): 1800–1807. 21. Orimo M, et al. (2009) Protective role of SIRT1 in diabetic vascular dysfunction. Arterioscler Thromb Vasc Biol 29(6):889–894. 22. Zhou S, et al. (2011) Repression of P66Shc expression by SIRT1 contributes to the prevention of hyperglycemia-induced endothelial dysfunction. Circ Res 109(6): 639–648. 23. Matsuzaki H, et al. (2005) Acetylation of Foxo1 alters its DNA-binding ability and sensitivity to phosphorylation. Proc Natl Acad Sci USA 102(32):11278–11283. 24. Sun T, et al. (2015) Acetylation of Beclin 1 inhibits autophagosome maturation and promotes tumour growth. Nat Commun 6:7215. 25. Parker BL, et al. (2014) Structural basis for phosphorylation and lysine acetylation cross-talk in a kinase motif associated with myocardial ischemia and cardioprotection. J Biol Chem 289(37):25890–25906. 26. Shi Y, et al. (2011) Oxidized low-density lipoprotein activates p66Shc via lectin-like oxidized low-density lipoprotein receptor-1, protein kinase C-beta, and c-Jun Nterminal kinase kinase in human endothelial cells. Arterioscler Thromb Vasc Biol 31(9):2090–2097. 27. Guo J, Gertsberg Z, Ozgen N, Steinberg SF (2009) p66Shc links alpha1-adrenergic receptors to a reactive oxygen species-dependent AKT-FOXO3A phosphorylation pathway in cardiomyocytes. Circ Res 104(5):660–669. 28. Oshikawa J, et al. (2012) Novel role of p66Shc in ROS-dependent VEGF signaling and angiogenesis in endothelial cells. Am J Physiol Heart Circ Physiol 302(3):H724–H732. 29. Ceriello A, Kumar S, Piconi L, Esposito K, Giugliano D (2007) Simultaneous control of hyperglycemia and oxidative stress normalizes endothelial function in type 1 diabetes. Diabetes Care 30(3):649–654. 30. Ihnat MA, et al. (2007) Reactive oxygen species mediate a cellular ‘memory’ of high glucose stress signalling. Diabetologia 50(7):1523–1531. 31. Caito S, et al. (2010) SIRT1 is a redox-sensitive deacetylase that is post-translationally modified by oxidants and carbonyl stress. FASEB J 24(9):3145–3159. 32. National Research Council (2011) Guide for the Care and Use of Laboratory Animals (Natl Acad Press, Washington, DC), 8th Ed.
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PNAS | February 14, 2017 | vol. 114 | no. 7 | 1719
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Detailed methods are described in SI Appendix.
Statistics. All data met assumptions of the statistical test, and all statistical analysis was performed using GraphPad prism 6 unless specified. Data represent means ± SEM of at least three independent assays unless otherwise stated. Significance of difference between two groups was determined using two-tailed independent sample Student’s t test. All of the vascular reactivity data were analyzed by two-way ANOVA followed by Tukey’s post hoc analysis. Results were considered significant if P values were ≤ 0.05.
Edited by Marc Montminy, The Salk Institute for Biological Studies, La Jolla, CA, and approved December 27, 2016 (received for review August 23, 2016)
The 66-kDa Src homology 2 domain-containing protein (p66Shc) is a master regulator of reactive oxygen species (ROS). It is expressed in many tissues where it contributes to organ dysfunction by promoting oxidative stress. In the vasculature, p66Shc-induced ROS engenders endothelial dysfunction. Here we show that p66Shc is a direct target of the Sirtuin1 lysine deacetylase (Sirt1), and Sirt1-regulated acetylation of p66Shc governs its capacity to induce ROS. Using diabetes as an oxidative stimulus, we demonstrate that p66Shc is acetylated under high glucose conditions and is deacetylated by Sirt1 on lysine 81. High glucose-stimulated lysine acetylation of p66Shc facilitates its phosphorylation on serine 36 and translocation to the mitochondria, where it promotes hydrogen peroxide production. Endothelium-specific transgenic and global knockin mice expressing p66Shc that is not acetylatable on lysine 81 are protected from diabetic oxidative stress and vascular endothelial dysfunction. These findings show that p66Shc is a target of Sirt1, uncover a unique Sirt1-regulated lysine acetylationdependent mechanism that governs the oxidative function of p66Shc, and demonstrate the importance of p66Shc lysine acetylation in vascular oxidative stress and diabetic vascular pathophysiology. p66Shc
| sirt1 | lysine acetylation | diabetes | oxidative stress
T
he 66-kDa Src homology 2 domain-containing protein (p66Shc) belongs to ShcA family of adaptor proteins. It is unique in this family because unlike the p46 and p52 isoforms, it impairs growth factor signaling and promotes oxidative stress (1). p66Shc contributes to aging (2), obesity (3, 4), atherosclerosis (5), and diabetes and aging-related vascular dysfunction (6, 7) in mice. In response to oxidative stimuli, p66Shc gets phosphorylated at serine 36 (S36) and translocates to mitochondria, where it produces reactive oxygen species (ROS) by oxidizing cytochrome C (8, 9). p66Shc is also phosphorylated on other residues that increase its half-life (10). In addition, p66Shc activates PKCBII, which in turn phosphorylates p66Shc, creating a positive feedback loop (11). The importance of p66Shc in human disease is supported by evidence that its expression increases in pathologies such as diabetes and atherosclerosis (12, 13). Sirt1 belongs to the class III NAD+-dependent histone deacetylases (HDACs). In lower organisms, it mediates longevity in response to caloric restriction (14). In addition to histones, it deacetylates many nonhistone proteins such as p53, FOXO, PGC1-alpha, LXR, and e-NOS (15–19). Sirt1 and p66Shc have opposing effects on vascular function (20). Unlike p66Shc, Sirt1 mitigates diabetes-induced vascular dysfunction (21). Moreover, down-regulation of Sirt1 in diabetes leads to epigenetic up-regulation of p66Shc (22). However, whether Sirt1 directly targets p66Shc for lysine deacetylation and whether dynamic lysine acetylation of p66Shc governs its oxidative function are not known. Here we show that acetylation of lysine 81 in p66Shc is obligatory for diabetic vascular dysfunction, and Sirt1 antagonizes this acetylation, thereby suppressing p66Shc-mediated oxidative stress. 1714–1719 | PNAS | February 14, 2017 | vol. 114 | no. 7
Results and Discussion Sirt1 Deacetylates p66Shc on Lysine 81. We first determined
whether the acetylation status of p66Shc is dynamic and governed by Sirt1. Knockdown of Sirt1 using siRNA increased lysine acetylation of ectopically expressed p66Shc in human embryonic kidney-293 (HEK 293) cells (SI Appendix, Fig. S1A) and led to hyperacetylation of endogenous p66Shc in human umbilical vein endothelial cells (HUVECs) (Fig. 1A and SI Appendix, Fig. S1B). Next, we investigated whether Sirt1 and p66Shc associate with each other. Endogenous p66Shc in HUVECs coprecipitated with endogenous Sirt1 in HUVECs (Fig. 1B), and immunoprecipitation of overexpressed p66Shc in HEK 293 cells pulled down overexpressed Sirt1 (SI Appendix, Fig. S1C). It is important to note that knockdown of Sirt1 also increased lysine acetylation of p46 and p52Shc (SI Appendix, Fig. S1B), suggesting that there may be common lysine residues in all three isoforms that are deacetylated by Sirt1. However, we focused our attention on lysine residues in the N-terminal collagen homology 2 (CH2) domain of p66Shc (2), which is not shared by p46 and p52Shc, as it is this CH2 domain that confers upon p66Shc its unique oxidative function. There are three conserved lysine residues in the CH2 domain at positions 7, 9, and 81 (SI Appendix, Fig. S2). To determine which, if any, of these three lysines is acetylated and targeted by Sirt1 for deacetylation, we first performed acetylation–deacetylation assays followed by mass spectrometry Significance Many oxidative stimuli engage the 66-kDa Src homology 2 domaincontaining protein (p66Shc) to induce reactive oxygen species (ROS). ROS regulated by p66Shc promotes aging and contributes to cancer, diabetes, obesity, cardiomyopathy, and atherosclerosis. Here we identify a fundamental mechanism that controls p66Shc and p66Shc-regulated ROS. We show that p66Shc is lysine acetylated when cells are faced with an oxidative stimulus (diabetes), and lysine acetylation of p66Shc is obligatory for p66Shc-induced ROS. In addition, lysine-acetylated p66Shc is deacetylated by the Sirtuin1 lysine deacetylase, and Sirt1-mediated deacetylation of p66Shc curtails ROS production. This intersection between p66Shc and Sirtuin1 adds a dimension to how p66Shc is regulated by certain stimuli and how Sirtuin1 suppresses oxidative stress promoted by such stimuli. Author contributions: S.K., M.M.B., and K.I. designed research; S.K., Y.-R.K., A.V., A.N., Q.L., M.K., V.K., J.S.J., and A.K. performed research; S.K., Y.-R.K., A.V., and M.M.B. analyzed data; and S.K. and K.I. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1
To whom correspondence may be addressed. Email: [email protected] or [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1614112114/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1614112114
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Fig. 1. Sirt1 inhibits oxidative function of p66Shc by deacetylating it on lysine 81. (A) Immunoblot for acetylated p66Shc with Sirt1 knockdown in HUVECs. (B) Coimmunoprecipitation for endogenous Sirt1 and p66Shc in HUVECs. (C) Tandem mass spectrometry of recombinant CH2 domain of p66Shc acetylated in vitro by p300. Acetylated peptide shown has m/z 554.96. (Inset) Acetyl-lysine immunoblot of p300-acetylated and Sirt1-deacetylated CH2 domain used for mass spectrometry. (D and E) Immunoblots for acetylp66ShcK81 (D) and phospho-p66ShcS36 (E) in HUVECs with Sirt1 knockdown. (F and G) Immunofluorescence for acetyl-p66ShcK81 (F) and phospho-p66ShcS36 (G) in aortas of mice with conditional deletion of endothelial Sirt1 (e-SIRT1KO) and wild-type mice. Arrow indicates endothelial layer. (H) Quantification of endothelial acetyl-p66ShcK81 and phospho-p66ShcS36 from F and G. *P < 0.05, **P < 0.01; n = 3; Student’s t test. (I) Quantification of H2O2 (DCF fluorescence) in HUVECs expressing p66ShcWT or p66ShcK81R and treated with Sirt1 inhibitor NAM. ***P < 0.001; n = 3–7; Student’s t test. Data represent mean ± SEM. Immunoblots are representative of at least three independent experiments. vWF, von Willebrand factor.
on recombinant CH2. His-tagged CH2 was acetylated in vitro with p300 acetyltransferase followed by deacetylation by Sirt1. Immunoblotting showed that p300 induced lysine acetylation of CH2, which was reversed by Sirt1 (Fig. 1C, Inset). CH2 acetylated by p300 and deacetylated by Sirt1 was then subjected to mass spectrometry. Lysine 81 (K81) was identified as the most abundantly acetylated and deacetylated residue (Fig. 1C and SI Appendix, Fig. S3 A and B). We developed an antibody against K81-acetylated p66Shc (acetyl-p66ShcK81) and verified that it detects only p300-acetylated and not nonacetylated recombinant CH2 (SI Appendix, Fig. S4A). Moreover, using this antibody, we Kumar et al.
verified that Sirt1 deacetylates K81 in full-length p66Shc expressed in HEK 293 cells (SI Appendix, Fig. S4B). Further, using this antibody, we showed that knockdown of Sirt1 in HUVECs engenders hyperacetylation of K81 in endogenous p66Shc (SI Appendix, Fig. S4C). To further verify that manipulation of Sirt1 and p300 hyperacetylates K81 and that the acetyl-p66ShcK81 antibody has specificity for Ac-K81, we created a p66Shc mutant that is nonacetylatable on K81 (K81R). Knockdown of Sirt1 in HUVECs or overexpression of p300 in HEK 293 cells increased acetylation on K81 in p66ShcWT but not p66ShcK81R (Fig. 1D and SI Appendix, Fig. S4 D and E). PNAS | February 14, 2017 | vol. 114 | no. 7 | 1715
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Fig. 2. Endothelial p66Shc is acetylated on lysine 81 by high glucose and in diabetes and promotes high glucose-induced mitochondrial oxidative stress. (A and B) Immunofluorescence for acetyly-p66ShcK81 (A) and quantification of acetyl-p66ShcK81 in aortic endothelium of STZ-induced diabetic mice. Magnified images are shown in Inset. Arrow indicates an endothelial layer. ***P < 0.001; n = 4–7; Student’s t test. (C and D) Immunoblot for high glucose-stimulated acetyl-p66ShcK81 and phospho-p66ShcS36 in HUVECs expressing p66ShcWT or p66ShcK81R (C) and densitometric quantification of immunoblots (D). **P < 0.01, ***P < 0.001; n = 3; Student’s t test. (E and F) DCF fluorescence images (E) and quantification of fluorescence (F) of whole HUVECs expressing p66ShcWT or p66ShcK81R and incubated with high glucose. (G) Quantification of DCF fluorescence in mitochondria isolated from HUVECs expressing p66ShcWT or p66ShcK81R and incubated with high glucose. **P < 0.01, ***P < 0.001; n = 3–7; Student’s t test. (H and I) Immunoblots for p66Shc in mitochondrial and whole-cell lysates of HUVECs expressing p66ShcWT or p66ShcK81R and incubated with high glucose (H) and their densitometric quantification (I). **P < 0.01; n = 5; Student’s t test. Data represent means ± SEM. STZ, streptozotocin. Immunoblots are representative of at least three independent experiments.
Deficiency of Sirt1 Promotes S36 Phosphorylation of p66Shc and p66Shc-Induced ROS via K81 Acetylation. There is precedent for lysine
acetylation facilitating serine phosphorylation (23). Phosphorylation of p66Shc on S36 is essential for p66Shc-mediated ROS production (2). We therefore asked if phosphorylation of p66Shc 1716 | www.pnas.org/cgi/doi/10.1073/pnas.1614112114
on S36 is dependent on acetylation of K81. Sirt1 knockdown in HUVECs increased S36 phosphorylation of p66Shc (SI Appendix, Fig. S5A) and increased ROS (H2O2) levels in both HUVECs and HEK 293 cells (SI Appendix, Fig. S5 B and C). Sirt1 knockdown did not stimulate S36 phosphorylation of p66ShcK81R Kumar et al.
High Glucose-Stimulated K81 Acetylation Promotes Mitochondrial Transport of p66Shc and p66Shc-Mediated Mitochondrial Oxidative Stress. Diabetes promotes vascular oxidative stress via p66Shc
(6). We evaluated the role of K81 acetylation in p66Shc-mediated diabetic oxidative stress in the vascular endothelium. We first examined the acetylation status of vascular p66Shc in streptozotocin (STZ)-induced diabetic mice. STZ-induced diabetes led to hyperacetylation of endothelial p66Shc on K81 (Fig. 2 A and B). A similar increase in K81 acetylation was observed in HUVECs incubated in high glucose-containing medium (Fig. 2 C and D). High glucose also stimulated S36 phosphorylation of p66Shc in HUVECs (Fig. 2 C and D). Importantly, high glucose failed to induce K81 acetylation or S36 phosphorylation of p66ShcK81R. Thus, K81 is acetylated in endothelial cells by high glucose in vitro and diabetes in vivo and facilitates S36 phosphorylation. We then evaluated the role of K81 acetylation in high glucoseinduced ROS mediated by p66Shc. High glucose-stimulated H2O2 was blunted in HUVECs expressing p66ShcK81R compared with those expressing p66ShcWT (Fig. 2 E and F and SI Appendix, Fig. S6). Because of the role of p66Shc in mitochondrial oxidative stress, we next asked if K81 acetylation is obligatory for high glucose-stimulated mitochondrial ROS mediated by p66Shc. High glucose-stimulated H2O2 in isolated mitochondria was significantly blunted in HUVECs expressing p66ShcK81R compared with those expressing p66ShcWT (Fig. 2G). Thus, cells expressing p66ShcK81R are protected from high glucose-stimulated mitochondrial oxidative stress. In response to oxidative stimuli, a fraction of p66Shc translocates to the mitochondrial intermembrane space in mitochondria, where it oxidizes cytochrome c, resulting in oxidative stress and mitochondrial depolarization (8, 9). Given the importance of K81 acetylation in high glucose-stimulated ROS, we asked if K81 acetylation is also important for mitochondrial translocation of p66Shc. Incubation of HUVECs with high glucose led to an increase in both endogenous and overexpressed p66ShcWT in crude mitochondrial fraction (SI Appendix, Fig. S7 A and B and Fig. 2 H and I). In contrast to p66ShcWT, p66ShcK81R did not accumulate in the mitochondria in response to high glucose (Fig. 2 H and I). These findings suggest that K81 acetylation plays an important part in high glucosestimulated mitochondrial translocation of p66Shc. K81 Acetylation of p66Shc Promotes Diabetic Vascular Endothelial Dysfunction. Diabetic vascular dysfunction is, in part, mediated
by p66Shc (6). To determine if K81 acetylation is required for p66Shc-mediated diabetic vascular dysfunction, we first expressed p66ShcK81R using a recombinant adenovirus (Ad-p66ShcK81R) in mouse aortas ex vivo (SI Appendix, Fig. S8A). Compared with expression of p66ShcWT (Ad-p66ShcWT), expression of p66ShcK81R did not result in impairment of endothelium-dependent vasorelaxation (SI Appendix, Fig. S8B). Endothelium-independent vascular relaxation was not different between mouse aortas expressing p66ShcWT and p66ShcK81R (SI Appendix, Fig. S8C). Kumar et al.
We also examined the effect of expressing p66ShcK81R in aortas of db/db diabetic mice that have impaired endothelium-dependent vasorelaxation. Expression of p66ShcK81R rescued endotheliumdependent vasorelaxation, whereas expression of p66ShcWT worsened it (SI Appendix, Fig. S8 D and E). However, endotheliumindependent relaxation was not different between db/db aortas expressing p66ShcWT and p66ShcK81R (SI Appendix, Fig. S8F). To explore the in vivo role of p66Shc K81 acetylation in diabetic vascular dysfunction, we generated a transgenic mouse with endothelium-specific expression of p66ShcK81R (henceforth called e-p66ShcK81R) (SI Appendix, Fig. S9A). These mice are viable and healthy. Diabetes was induced by a single bolus injection of STZ (SI Appendix, Fig. S9B). Aortas of e-p66ShcK81R mice had improved endothelium-dependent relaxation both under nondiabetic and diabetic conditions, compared with their wild-type nontransgenic littermate controls (Fig. 3A). Moreover, endothelium-specific oxidative stress (measured as 8-hydroxy deoxyguanosine, 8-OHdG) (Fig. 3 B and C) and acetylation of p66Shc on K81 in the endothelium were diminished in diabetic e-p66ShcK81R transgenic mice compared with diabetic wild-type nontransgenic littermates (SI Appendix, Fig. S9 C and D). To confirm the role of K81 acetylation in diabetic vascular dysfunction, we generated mice with global knockin of p66ShcK81R (SI Appendix, Fig. S10). These mice are fertile, viable, and healthy. There was no difference in STZ-induced hyperglycemia between p66ShcK81R knockin and wild-type mice (SI Appendix, Fig. S11). Although basal endothelium-dependent vasorelaxation was similar in p66ShcK81R knockin and wild-type mice, p66ShcK81R knockin mice were protected from STZ-induced impairment of endothelium-dependent vasorelaxation (Fig. 3D). In addition, bioavailable vascular nitric oxide was higher in p66ShcK81R knockin mice compared with wild-type mice in both the diabetic and nondiabetic states (Fig. 3E). Further, p66ShcK81R knockin mice were protected from STZ-induced vascular oxidative stress (Fig. 3 F and G). By showing that the oxidative function of p66Shc is governed by lysine acetylation and acetylated p66Shc is a substrate for Sirt1, this work uncovers dynamic lysine acetylation as another rheostat for the complex posttranslational regulation of p66Shc. The intersection between lysine acetylation and serine phosphorylation is not unique to p66Shc. Lysine acetylation promotes protein kinase B-mediated phosphorylation of Foxo1 (23), whereas phosphorylation of Beclin1 is required for its subsequent lysine acetylation (24). Interdependence between serine phosphorylation and lysine acetylation may be explained on structural grounds of these posttranslational modifications (25). While our data indicate that K81 acetylation is required for high glucose/diabetes-induced S36 phosphorylation, further studies showed that this requirement is not universally applicable for all oxidative stimuli. This was borne out in studies examining the effect of vascular endothelial growth factor (VEGF) on S36 phosphorylation in p66ShcWT and p66ShcK81R. Although basal S36 phosphorylation in serum-starved HUVECs was lower in p66ShcK81R, VEGF induced S36 phosphorylation to a similar extent in both p66ShcWT and p66ShcK81R, without changing K81 acetylation (SI Appendix, Fig. S12). This stimulus-specific reliance of S36 phosphorylation on K81 acetylation (present in high glucose/ diabetes; absent in VEGF) could be explained by selective engagement of specific kinases by different oxidative stimuli, as phosphorylation of S36 is promiscuously induced by multiple kinases (26–28). An alternative explanation is that dependence of S36 phosphorylation on K81 acetylation may be determined by whether the stimulus affects Sirt1 expression. Diabetes down-regulates endothelial Sirt1 (SI Appendix, Fig. S13), whereas some other oxidative stimuli (such as VEGF) may not. Observations from clinical and preclinical studies suggest that glycemic control alone is not sufficient to prevent diabetic complications and persistent oxidative stress could be responsible for PNAS | February 14, 2017 | vol. 114 | no. 7 | 1717
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expressed in either HEK 293 cells or HUVECs (Fig. 1E and SI Appendix, Fig. S5 D and E). In addition, both K81 acetylation and S36 phosphorylation were increased in the aortic endothelium of mice with conditional deletion of endothelial Sirt1 (e-Sirt1 KO) (Fig. 1 F–H). We then examined the role of Sirt1-regulated K81 acetylation in p66Shc-induced ROS production. Inhibition of Sirt1 with nicotinamide (NAM) increased S36 phosphorylation in cells expressing p66ShcWT but not in cells expressing p66ShcK81R (SI Appendix, Fig. S5 F and G). Further, expression of p66ShcWT, but not p66ShcK81R, amplified NAM-stimulated H2O2 in HUVECs (Fig. 1I). These findings underscore that inhibition of Sirt1 stimulates p66Shc-mediated ROS production via K81 acetylation.
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Fig. 3. Acetylation of p66Shc on lysine 81 mediates diabetic vascular oxidative stress and endothelial dysfunction. (A) Endothelium-dependent vasorelaxation of aortas from nondiabetic and STZ-induced diabetic wild-type mice and transgenic mice with endothelial expression of p66ShcK81R (e-p66ShcK81R). ###P < 0.001 vs. wild type; *P < 0.05, ***P < 0.001 vs. wild-type STZ. Wild type (n = 12), e-p66ShcK81R (n = 18), wild-type STZ (n = 20), and e-p66ShcK81R STZ (n = 8). (B) Immunofluorescence for oxidative stress (8-OHdG; oxidative DNA adduct) in aortic sections of nondiabetic and STZ-induced diabetic wild-type and e-p66ShcK81R mice. (C) Quantification of endothelial 8-OHdG in B. vWF, von Willebrand factor. ***P < 0.001; n = 5–8; Student’s t test. (D) Endothelium-dependent vasorelaxation of aortas of control (nondiabetic) and STZ-induced diabetic wild-type and p66ShcK81R knockin mice. #P < 0.05, ###P < 0.001 vs. wild type. Wild type, n = 14; p66ShcK81R, n = 13; wild-type STZ, n = 15; and p66ShcK81R STZ, n = 21. (E) Nitric oxide bioavailability in aortas of control and STZ-induced diabetic p66ShcK81R knockin and wild-type mice. #P < 0.05, ##P < 0.01 vs. wild type; **P < 0.01 vs. wild-type STZ. Wild type, n = 14; p66ShcK81R, n = 13; wild-type STZ, n = 15; and p66ShcK81R STZ, n = 21. (F) Immunofluorescence for 8-OHdG in aortic sections of control and STZ-induced diabetic wild-type and p66ShcK81R knockin mice. (G) Quantification of endothelial 8-OHdG in F. Arrow indicates endothelial layer. ***P < 0.001; n = 8–10; Student’s t test. 8-OHdG, 8 hydroxy-deoxyguanosine; ACh, acetylcholine; PE, phenylephrine. n, number of aortic rings. All vascular reactivity data were analyzed by two-way ANOVA followed by Tukey’s post hoc analysis. Data represent mean ± SEM.
this hyperglycemic memory (29, 30). One proposition that has been forwarded as responsible for persistent vascular oxidative stress in diabetes is the epigenetic up-regulation of p66Shc. In this regard, it has been shown that Sirt1 suppresses p66Shc expression epigenetically, and genetic deletion of p66Shc protects mice against diabetic endothelial dysfunction and vascular hyperglycemic memory (11, 22). Distinct from this finding, our work identifies an alternative mechanism by which Sirt1 regulates p66Shc—through 1718 | www.pnas.org/cgi/doi/10.1073/pnas.1614112114
direct lysine deacetylation. Although acetylation of p66Shc is independent from Sirt1-mediated epigenetic regulation of p66Shc, our data suggest some functional interplay between the two. This is borne out by the finding that suppression of K81 acetylation of p66Shc in diabetic mice partially rescued vascular Sirt1 expression (SI Appendix, Fig. S13). Thus, p66Shc acetylation feeds back to inhibit Sirt1 expression, which in turn may also lead to up-regulation of p66Shc. Given that Sirt1 expression is governed by oxidative Kumar et al.
stress (31), acetylated p66Shc may down-regulate Sirt1 by promoting vascular ROS production (SI Appendix, Fig. S14). In conclusion, these findings identify p66Shc as a target for Sirt1 and show that Sirt1-mediated deacetylation of p66Shc has a vital part in determining tissue vascular oxidative stress and endothelial dysfunction in diabetes. Although the studies were restricted to diabetes/high glucose as the oxidative stimulus and to vascular cells and tissue, the molecular mechanism by which Sirt1-regulated lysine acetylation of p66Shc governs ROS may also be operative in other oxidant-driven pathophysiology.
City, and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (32).
Methods
Study Approval. All experimental animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Iowa, Iowa
ACKNOWLEDGMENTS. We thank T. Finkel and L. Terada for the p66Shc constructs and M. Joiner for CoxIV and Mitomix antibodies. K.I. was supported by the University of Iowa Endowed Professorship in Cardiovascular Medicine and by US Department of Veterans Affairs Grant 1I01BX002940; Q.L. was supported by NIH Grant T32 HL007344; and M.K. was supported by NIH Grant T32 HL007121.
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PNAS | February 14, 2017 | vol. 114 | no. 7 | 1719
PHYSIOLOGY
Detailed methods are described in SI Appendix.
Statistics. All data met assumptions of the statistical test, and all statistical analysis was performed using GraphPad prism 6 unless specified. Data represent means ± SEM of at least three independent assays unless otherwise stated. Significance of difference between two groups was determined using two-tailed independent sample Student’s t test. All of the vascular reactivity data were analyzed by two-way ANOVA followed by Tukey’s post hoc analysis. Results were considered significant if P values were ≤ 0.05.
