AMPK / TSC 2 / mTOR pathway regulates replicative senescence of human vascular smooth muscle cells

Age-associated diseases, including vascular diseases, are on the rise with the increase in the aging population. However, the mechanisms of aging and age-associated vascular dysfunction remain to be fully elucidated. Replicative senescence of vascular smooth muscle cells (VSMCs) contributes to aging as well as age-associated vascular diseases. Rapamycin may delay aging-associated diseases via inhibition of the mammalian target of rapamycin (mTOR), but its role in VSMC aging has remained elusive. The present study investigated the involvement of mTOR signaling in replicative senescence of VSMCs. Replicative senescence was induced in human VSMCs by extended passages and identified by assessing the cell morphology, senescence‐associated β-galactosidase activity, and p53 and p21 protein expression. Protein expression and phosphorylation were determined by western blot analysis. Significant senescence of VSMCs was observed in cells subjected to extended passaging (until passage 15). Significant decreases in adenosine monophosphate-activated protein kinase (AMPK)/tuberous sclerosis complex 2 (TSC2) phosphorylation, but significant increases in mTOR/ribosomal protein S6 kinase 1 (S6K1) phosphorylation, were observed in cells with replicative senescence compared with those in young cells. Pre-treatment of VSMCs with AMPK activator and mTOR inhibitor delayed replicative senescence and reversed changes in AMPKα, TSC2, mTOR and S6K1 phosphorylation in senescent VSMCs. The AMPK/TSC2/mTOR/S6K1 signaling axis was found to have an important role in regulating replicative senescence of human VSMCs. Introduction Age-associated vascular diseases are accompanied by structural and functional changes in blood vessels. A body of evidence suggested that the behavior of vascular smooth muscle cells (VSMCs) is modified with ageing (1), and senescent VSMCs have been observed in atherosclerotic plaques of patients with atherosclerosis (2). Thus, senescent VSMCs contribute to ageing and age-associated cardiovascular diseases. Although permanent inhibition of cell proliferation is thought to be a traditional hallmark of cellular senescence, this procedure is accompanied by changes in the expression of replicative senescence-associated genes, such as p53, p21 and senescence-associated β-galactosidase (SA-β-gal) (3). Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase and its functions rely on forming two distinct complexes, mTOR complex 1 (mTORC1) and mTORC2 (4). Dysfunctional mTOR signaling has been considered a central integral mechanism linked to ageing (5). AMP-activated protein kinase (AMPK) is a highly conserved heterotrimeric kinase complex. Activation of AMPK leads to the inhibition of mTORC1 via a tuberous sclerosis complex 2 (TSC2)-dependent or -independent mechanism (6). In animal models with impaired mTORC1 signaling, AMPK is highly activated (7), suggesting a negative crosstalk between these two pathways. AMPK/TSC2/mTOR is a classic upstream pathway of mTORC1 signaling, which affects the phosphorylation of downstream factors, such as ribosome protein subunit 6 kinase 1 (S6K1), to regulate cell metabolism, proliferation, differentiation and autophagy. However, the role of mTOR and particularly that of its downstream effector S6K1 in age-associated vascular diseases has remained largely elusive. A previous study has demonstrated that inhibition of the mTORC1/S6K1 pathway by rapamycin or activation of AMPK by 5-aminoimidazole-4-carboxamide riboside (AICAR) negatively regulates mTORC1 signaling and attenuated pressure overload-induced cardiac hypertrophy in vivo (8). Conversely, a deficiency in AMPK expression was reported to enhance mTOR/S6K1 signaling and exacerbate myocardial hypertrophy in response to pressure overload (9). However, the role of the AMPK/mTOR signaling in VSMC senescence has remained elusive. The purpose of the present study was to explore the roles of AMPK/TSC2/mTOR signaling in the replicative senescence AMPK/TSC2/mTOR pathway regulates replicative senescence of human vascular smooth muscle cells JUN-KUN ZHAN, YAN-JIAO WANG, SHUANG LI, YI WANG, PAN TAN, JE-YU HE, YI-YIN CHEN, HUI-QIAN DENG, WU HUANG, XIAO LIN and YOU-SHUO LIU Geriatric Department, The Second Xiangya Hospital, Institute of Aging and Geriatrics, Central South University, Changsha, Hunan 410011, P.R. China Received August 31, 2016; Accepted May 19, 2017 DOI: 10.3892/etm.2018. 6767 Correspondence to: Dr You-Shuo Liu, Geriatric Department, The Second Xiangya Hospital, Institute of Aging and Geriatrics, Central South University, 139 Middle Renmin Road, Changsha, Hunan 410011, P.R. China E-mail: liuyoushuo@yeah.net