Klotho modulates the stress response in human senescent endothelial cells Julia Carracedo a,b,c, *, Paula Buendı ´a a,c , Ana Merino a,c,e , Juan Antonio Maduen ˜o a,c , Esther Peralbo a , Alberto Ortiz c,d , Alejandro Martı ´n-Malo a,b,c , Pedro Aljama a,b,c , Mariano Rodrı ´guez a,b,c , Rafael Ramı ´rez a,c,f a Instituto Maimo ´nides de Investigacio ´n Biome ´dica de Co ´rdoba (IMIBIC)/Fundacio ´n de Investigaciones Biome ´dicas de Co ´rdoba (FIBICO), Reina Sofı´a University Hospital, Co ´rdoba, Spain b Nephrology Unit, Reina Sofı´a University Hospital, Co ´rdoba, Spain c REDinREN, Servicio de Nefrologı´a, Fundacio ´n para la Investigacio ´n Biome ´dica del Hospital Universitario La Paz, Instituto de Salud Carlos III, Fondos FEDER, Madrid, Spain d Unidad de Dia ´lisis, Fundacio ´n Jime ´nez Dı´az, Madrid, Spain e Nephrology Laboratory, Instituto de Investigacio ´n Biome ´dica de Bellvitge (IDIBELL), Barcelona, Spain f Physiology Department, Alcala de Henares University, Madrid, Spain 1. Introduction Klotho is recognized as an anti-aging protein. Lack of Klotho expression in mice produces premature aging and age-related diseases including vascular diseases. Animals that over-express Klotho live longer and do not present age-related diseases (Kuro-o et al., 1997; Takahashi et al., 2000). Klotho encodes a single-pass transmembrane protein (a- Klotho) with a long extracellular domain (130 kDa), and a short cytoplasmic tail (Kuro-o et al., 1997; Shiraki-Iida et al., 1998). This protein has a restricted distribution and is predominantly expressed in the distal convoluted tubules of kidneys, parathyroid cells and in the choroid plexus in the brain. Another form of Klotho protein, the secreted Klotho protein, has been described as an alternative mRNA splicing of Klotho derived from the type I protein, and has a molecular weight of approximately 65–70 kDa. Soluble Klotho plays an important role in inhibiting insulin/IGF-1 signals and inducing resistance to oxidative stress; this suggests that soluble Klotho may act as an ‘‘anti-aging’’ hormone (Shiraki- Iida et al., 1998; Li et al., 2004; Hayashi et al., 2007). A third Klotho protein is not present in the cell surface, but is expressed in the cytoplasm; its intracellular distribution mostly overlaps the endoplasmic reticulum and Golgi apparatus (Li et al., 2004; Hayashi et al., 2007), whose expression has been linked to the ability of cells to withstand stress conditions. Replicative cellular senescence can be studied in vitro and it serves as a model which helps to understand the mechanisms involved in cell aging (Cristofalo et al., 2004). In culture, the ability of normal human cells to divide is finite, and after a limited number of cycles of replication, cells enter a terminal state of arrested growth called replicative senescence. Furthermore, cells exposed to stressors that raise levels of reactive oxygen species (ROS), may undergo premature senescence via a process called stress-induced premature senescence (SIPS) (Toussaint et al., 2000; Serrano and Blasco, 2001). There are analogies between cells undergoing replicative aging and SIPS; some of the genes regulating the response against DNA damage are modified similarly under both conditions, cells express b-galactosidase (b-gal) activity and they Mechanisms of Ageing and Development 133 (2012) 647–654 A R T I C L E I N F O Article history: Received 2 December 2011 Received in revised form 5 September 2012 Accepted 7 September 2012 Available online 21 September 2012 Keywords: Klotho Human endothelial cells Cell senescence Stress-induced premature senescence A B S T R A C T Lack of Klotho expression in mice leads to premature aging and age-related diseases, including vascular diseases. The aim of this study was to determine how endothelial cell line senescence affects Klotho expression and whether intra- or extracellular Klotho has any effect on the response of senescent cells to oxidative stress. The study was performed using human endothelial cells (HUVEC); cell aging was obtained by prolongation of cell division to 42 population doublings (PD). Senescence was also obtained by exposure to TNFa, which causes cell changes resembling cellular senescence. The decline in Klotho preceded the manifestations of cell ageing: telomere shortening and b-galactosidase expression. Klotho was also reduced in cells exposed to the proinflammatory cytokine TNFa. The addition of exogenous Klotho to aging cells did not modify the proportion of cells with short telomeres or any other feature of cell aging; however, exogenous Klotho prevented the changes resembling premature cellular senescence associated with TNFa, such as the decrease in telomere length and the increase in b-galactosidase-positive cells. Likewise exogenous Klotho prevented the increases in reactive oxygen species (ROS) activity, mitochondrial potential and cell apoptosis induced by TNFa. ß 2012 Elsevier Ireland Ltd. All rights reserved. * Corresponding author at: Research Unit, Reina Sofı ´a University Hospital, Avda Menendez Pidal s/n., ES-14004 Cordoba, Spain. Tel.: +34 957 736541; fax: +34 957 010452. E-mail address: julia.carracedo.exts@juntadeandalucia.es (J. Carracedo). Contents lists available at SciVerse ScienceDirect Mechanisms of Ageing and Development jo ur n al ho mep ag e: www .elsevier .c om /lo cate/m ec hag ed ev 0047-6374/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mad.2012.09.002