Abstract
Vascular endothelial cell senescence is a leading cause of age-associated and vascular diseases. Mammalian target of rapamycin complex 2 (mTORC2) is a conserved serine/threonine (Ser/Thr) protein kinase that plays an important regulatory role in various cellular processes. However, its impact on endothelial senescence remains controversial. In this study we investigated the role and molecular mechanisms of mTORC2 in endothelial senescence. A replicative senescence model and H2O2-induced premature senescence model were established in primary cultured human umbilical vein endothelial cells (HUVECs). In these senescence models, the formation and activation of mTORC2 were significantly increased, evidenced by the increases in binding of Rictor (the essential component of mTORC2) to mTOR, phosphorylation of mTOR at Ser2481 and phosphorylation of Akt (the effector of mTORC2) at Ser473. Knockdown of Rictor or treatment with the Akt inhibitor MK-2206 attenuated senescence-associated β-galactosidase (β-gal) staining and expression of p53 and p21 proteins in the senescent endothelial cells, suggesting that mTORC2/Akt facilitates endothelial senescence. The effect of mTORC2/Akt on endothelial senescence was due to suppression of nuclear factor erythroid 2-related factor 2 (Nrf2) at the transcriptional level, since knockdown of Rictor reversed the reduction of Nrf2 mRNA expression in endothelial senescence. Furthermore, mTORC2 suppressed the expression of Nrf2 via the Akt/GSK-3β/C/EBPα signaling pathway. These results suggest that the mTORC2/Akt/GSK-3β/C/EBPα/Nrf2 signaling pathway is involved in both replicative and inducible endothelial senescence. The deleterious role of mTORC2 in endothelial cell senescence suggests therapeutic strategies (targeting mTORC2) for aging-associated diseases and vascular diseases.
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References
Feletou M, Vanhoutte PM. Endothelial dysfunction: a multifaceted disorder (The Wiggers Award Lecture). Am J Physiol Heart Circ Physiol. 2006;291:H985–1002.
Luna C, Alique M, Navalmoral E, Noci MV, Bohorquez-Magro L, Carracedo J, et al. Aging-associated oxidized albumin promotes cellular senescence and endothelial damage. Clin Interv Aging. 2016;11:225–36.
Donato AJ, Morgan RG, Walker AE, Lesniewski LA. Cellular and molecular biology of aging endothelial cells. J Mol Cell Cardiol. 2015;89:122–35.
Erusalimsky JD. Vascular endothelial senescence: from mechanisms to pathophysiology. J Appl Physiol (1985). 2009;106:326–32.
Allsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai EV, Futcher AB, et al. Telomere length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci USA. 1992;89:10114–8.
Minamino T, Miyauchi H, Yoshida T, Ishida Y, Yoshida H, Komuro I. Endothelial cell senescence in human atherosclerosis: role of telomere in endothelial dysfunction. Circulation. 2002;105:1541–4.
Erusalimsky JD, Kurz DJ. Cellular senescence in vivo: its relevance in ageing and cardiovascular disease. Exp Gerontol. 2005;40:634–42.
Deanfield JE, Halcox JP, Rabelink TJ. Endothelial function and dysfunction: testing and clinical relevance. Circulation. 2007;115:1285–95.
Favero G, Paganelli C, Buffoli B, Rodella LF, Rezzani R. Endothelium and its alterations in cardiovascular diseases: life style intervention. Biomed Res Int. 2014;2014:801896.
Naylor RM, Baker DJ, van Deursen JM. Senescent cells: a novel therapeutic target for aging and age-related diseases. Clin Pharmacol Ther. 2013;93:105–16.
Saxton RA, Sabatini DM. mTOR signaling in growth, metabolism, and disease. Cell. 2017;168:960–76.
Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H, et al. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell. 2002;110:163–75.
Kim DH, Sarbassov DD, Ali SM, Latek RR, Guntur KV, Erdjument-Bromage H, et al. GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR. Mol Cell. 2003;11:895–904.
Hara K, Maruki Y, Long X, Yoshino K, Oshiro N, Hidayat S, et al. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell. 2002;110:177–89.
Jacinto E, Loewith R, Schmidt A, Lin S, Ruegg MA, Hall A, et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol. 2004;6:1122–8.
Sarbassov DD, Ali SM, Kim DH, Guertin DA, Latek RR, Erdjument-Bromage H, et al. Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Curr Biol. 2004;14:1296–302.
Jacinto E, Hall MN. Tor signalling in bugs, brain and brawn. Nat Rev Mol Cell Biol. 2003;4:117–26.
Yang C, Pan S, Yan S, Li Z, Yang J, Wang Y, et al. Inhibitory effect of 14,15-EET on endothelial senescence through activation of mTOR complex 2/Akt signaling pathways. Int J Biochem Cell Biol. 2014;50:93–100.
Miller RA, Harrison DE, Astle CM, Fernandez E, Flurkey K, Han M, et al. Rapamycin-mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. Aging Cell. 2014;13:468–77.
Kaeberlein M, Powers RW 3rd, Steffen KK, Westman EA, Hu D, Dang N, et al. Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients. Science. 2005;310:1193–6.
Selman C, Tullet JM, Wieser D, Irvine E, Lingard SJ, Choudhury AI, et al. Ribosomal protein S6 kinase 1 signaling regulates mammalian life span. Science. 2009;326:140–4.
Astle MV, Hannan KM, Ng PY, Lee RS, George AJ, Hsu AK, et al. AKT induces senescence in human cells via mTORC1 and p53 in the absence of DNA damage: implications for targeting mTOR during malignancy. Oncogene. 2012;31:1949–62.
Yentrapalli R, Azimzadeh O, Sriharshan A, Malinowsky K, Merl J, Wojcik A, et al. The PI3K/Akt/mTOR pathway is implicated in the premature senescence of primary human endothelial cells exposed to chronic radiation. PLoS ONE. 2013;8:e70024.
Ming XF, Montani JP, Yang Z. Perspectives of targeting mTORC1-S6K1 in cardiovascular aging. Front Physiol. 2012;3:5.
Rajapakse AG, Yepuri G, Carvas JM, Stein S, Matter CM, Scerri I, et al. Hyperactive S6K1 mediates oxidative stress and endothelial dysfunction in aging: inhibition by resveratrol. PLoS ONE. 2011;6:e19237.
Camici GG, Steffel J, Amanovic I, Breitenstein A, Baldinger J, Keller S, et al. Rapamycin promotes arterial thrombosis in vivo: implications for everolimus and zotarolimus eluting stents. Eur Heart J. 2010;31:236–42.
Ming XF, Rajapakse AG, Carvas JM, Ruffieux J, Yang Z. Opposing and uncoupling effects of mTOR and S6K1 in the regulation of endothelial tissue factor expression. FEBS Lett. 2010;584:135–40.
Xiong Y, Yepuri G, Forbiteh M, Yu Y, Montani JP, Yang Z, et al. ARG2 impairs endothelial autophagy through regulation of MTOR and PRKAA/AMPK signaling in advanced atherosclerosis. Autophagy. 2014;10:2223–38.
Wang CY, Kim HH, Hiroi Y, Sawada N, Salomone S, Benjamin LE, et al. Obesity increases vascular senescence and susceptibility to ischemic injury through chronic activation of Akt and mTOR. Sci Signal. 2009;2:ra11.
Copp J, Manning G, Hunter T. TORC-specific phosphorylation of mammalian target of rapamycin (mTOR): phospho-Ser2481 is a marker for intact mTOR signaling complex 2. Cancer Res. 2009;69:1821–7.
Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science. 2005;307:1098–101.
Bachschmid MM, Schildknecht S, Matsui R, Zee R, Haeussler D, Cohen RA, et al. Vascular aging: chronic oxidative stress and impairment of redox signaling-consequences for vascular homeostasis and disease. Ann Med. 2013;45:17–36.
Ross SE, Erickson RL, Hemati N, MacDougald OA. Glycogen synthase kinase 3 is an insulin-regulated C/EBPalpha kinase. Mol Cell Biol. 1999;19:8433–41.
Datta J, Majumder S, Kutay H, Motiwala T, Frankel W, Costa R, et al. Metallothionein expression is suppressed in primary human hepatocellular carcinomas and is mediated through inactivation of CCAAT/enhancer binding protein alpha by phosphatidylinositol 3-kinase signaling cascade. Cancer Res. 2007;67:2736–46.
Beurel E, Grieco SF, Jope RS. Glycogen synthase kinase-3 (GSK3): regulation, actions, and diseases. Pharmacol Ther. 2015;148:114–31.
Saxton RA, Sabatini DM. mTOR signaling in growth, metabolism, and disease. Cell. 2017;169:361–71.
Gaubitz C, Prouteau M, Kusmider B, Loewith R. TORC2 structure and function. Trends Biochem Sci. 2016;41:532–45.
Baar EL, Carbajal KA, Ong IM, Lamming DW. Sex- and tissue-specific changes in mTOR signaling with age in C57BL/6J mice. Aging Cell. 2016;15:155–66.
Miyauchi H, Minamino T, Tateno K, Kunieda T, Toko H, Komuro I. Akt negatively regulates the in vitro lifespan of human endothelial cells via a p53/p21-dependent pathway. EMBO J. 2004;23:212–20.
Minamino T, Miyauchi H, Tateno K, Kunieda T, Komuro I. Akt-induced cellular senescence: implication for human disease. Cell Cycle. 2004;3:449–51.
Kim YY, Jee HJ, Um JH, Kim YM, Bae SS, Yun J. Cooperation between p21 and Akt is required for p53-dependent cellular senescence. Aging Cell. 2017;16:1094–103.
Nogueira V, Park Y, Chen CC, Xu PZ, Chen ML, Tonic I, et al. Akt determines replicative senescence and oxidative or oncogenic premature senescence and sensitizes cells to oxidative apoptosis. Cancer Cell. 2008;14:458–70.
Hybertson BM, Gao B, Bose SK, McCord JM. Oxidative stress in health and disease: the therapeutic potential of Nrf2 activation. Mol Asp Med. 2011;32:234–46.
Senthil KKJ, Gokila VM, Wang SY. Activation of Nrf2-mediated anti-oxidant genes by antrodin C prevents hyperglycemia-induced senescence and apoptosis in human endothelial cells. Oncotarget. 2017;8:96568–87.
Yang G, Zhao K, Ju Y, Mani S, Cao Q, Puukila S, et al. Hydrogen sulfide protects against cellular senescence via S-sulfhydration of Keap1 and activation of Nrf2. Antioxid Redox Signal. 2013;18:1906–19.
Kapeta S, Chondrogianni N, Gonos ES. Nuclear erythroid factor 2-mediated proteasome activation delays senescence in human fibroblasts. J Biol Chem. 2010;285:8171–84.
Volonte D, Liu Z, Musille PM, Stoppani E, Wakabayashi N, Di YP, et al. Inhibition of nuclear factor-erythroid 2-related factor (Nrf2) by caveolin-1 promotes stress-induced premature senescence. Mol Biol Cell. 2013;24:1852–62.
Jodar L, Mercken EM, Ariza J, Younts C, Gonzalez-Reyes JA, Alcain FJ, et al. Genetic deletion of Nrf2 promotes immortalization and decreases life span of murine embryonic fibroblasts. J Gerontol A Biol Sci Med Sci. 2011;66:247–56.
Valcarcel-Ares MN, Gautam T, Warrington JP, Bailey-Downs L, Sosnowska D, de Cabo R, et al. Disruption of Nrf2 signaling impairs angiogenic capacity of endothelial cells: implications for microvascular aging. J Gerontol A Biol Sci Med Sci. 2012;67:821–9.
Duan W, Zhang R, Guo Y, Jiang Y, Huang Y, Jiang H, et al. Nrf2 activity is lost in the spinal cord and its astrocytes of aged mice. Vitr Cell Dev Biol Anim. 2009;45:388–97.
Shih PH, Yen GC. Differential expressions of antioxidant status in aging rats: the role of transcriptional factor Nrf2 and MAPK signaling pathway. Biogerontology. 2007;8:71–80.
Acknowledgements
The study is supported by the National Natural Science Foundation of China (No. 81400359, 81473205, and 81673433), Guangdong Natural Science Foundation (S2013040014102), Specialized Research Fund for the Doctoral Program of Higher Education of China (20130171120048), Training Program for Young Teachers from Sun Yat-sen University (No.15ykpy03), and the National Engineering and Technology Research Center for New Drug Druggability Evaluation (Seed Program of Guangdong Province), and funding from Guangdong Provincial Engineering Laboratory of Druggability and New Drugs Evaluation, and Guangzhou Key Laboratory of Druggability Assessment for Biologically Active Compounds.
Author contributions
P.-q.L. and Z.-m.L. designed the research; H.-w.Y., H.-l.H., W.-w.L., C.-m.D., and X.-y.C. performed the research; H.-w.Y., H.-l.H., W.-w.L., L.-p.W., Q.L., and Z.-q.L. analyzed the data; H.-w.Y. and Z.-m.L. wrote the manuscript.
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Yang, Hw., Hong, Hl., Luo, Ww. et al. mTORC2 facilitates endothelial cell senescence by suppressing Nrf2 expression via the Akt/GSK-3β/C/EBPα signaling pathway. Acta Pharmacol Sin 39, 1837–1846 (2018). https://doi.org/10.1038/s41401-018-0079-6
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DOI: https://doi.org/10.1038/s41401-018-0079-6
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