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The Thyroid Hormone Receptor Induces DNA Damage and Premature

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The Thyroid Hormone Receptor Induces DNA Damage and Premature Published January 6, 2014 JCB: Article The thyroid hormone receptor induces DNA damage and premature senescence Alberto Zambrano,1 Verónica García-Carpizo,1 María Esther Gallardo,1,3 Raquel Villamuera,1 Maria Ana Gómez-Ferrería,1 Angel Pascual,1 Nicolas Buisine,2 Laurent M. Sachs,2 Rafael Garesse,1,3,4 and Ana Aranda1 1Instituto de Investigaciones Biomédicas “Alberto Sols”, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, 28029 Madrid, Spain 2Département Régulation, Développement et Diversité Moléculaire, Unité Mixte de Recherche 7221, Centre National de la Recherche Scientifique, Muséum National d’Histoire Naturelle, 75231 Paris, France 3Centro de Investigación Biomédica en Red, 28029 Madrid, Spain 4Instituto de Investigación Sanitaria Hospital 12 de Octubre, 28041 Madrid, Spain here is increasing evidence that the thyroid hormone factor 1) and THRB to the promoters of genes with a key (TH) receptors (THRs) can play a role in aging, can- role on mitochondrial respiration. Increased respiration T cer and degenerative diseases. In this paper, we leads to production of mitochondrial reactive oxygen spe- Downloaded from demonstrate that binding of TH T3 (triiodothyronine) to cies, which in turn causes oxidative stress and DNA dou- THRB induces senescence and deoxyribonucleic acid ble-strand breaks and triggers a DNA damage response (DNA) damage in cultured cells and in tissues of young that ultimately leads to premature senescence of suscepti- hyperthyroid mice. T3 induces a rapid activation of ATM ble cells. Our findings provide a mechanism for integrat- (ataxia telangiectasia mutated)/PRKAA (adenosine mo- ing metabolic effects of THs with the tumor suppressor jcb.rupress.org nophosphate–activated protein kinase) signal transduc- activity of THRB, the effect of thyroidal status on longevity, tion and recruitment of the NRF1 (nuclear respiratory and the occurrence of tissue damage in hyperthyroidism. on October 23, 2014 Introduction Normal cells only proliferate for a finite number of times until against cellular transformation and cancer progression (Collado they become senescent (Hayflick, 1965), a state of irreversible and Serrano, 2010). proliferative arrest as a consequence of the genomic damage The actions of the thyroid hormones (THs) are mediated caused by telomere erosion (d’Adda di Fagagna et al., 2003). by binding to nuclear TH receptors (THRs) that are encoded Senescence can also be induced prematurely as a result of a by two genes: THRA and THRB (Aranda and Pascual, 2001). persistent DNA damage response (DDR) secondary to oxidative One of the most prominent actions of THs is the regulation of stress that induces double-strand breaks (DSBs; Campisi and mitochondrial function (Oppenheimer et al., 1987). Mitochon- THE JOURNAL OF CELL BIOLOGY d’Adda di Fagagna, 2007) or to DNA replication stress induced dria are the major site of oxidative processes that lead to heat by oncogenes or drugs, which lead to DSB generation and DDR production and to generation of reactive oxygen species (ROS). activation (Serrano et al., 1997; Bartkova et al., 2006; Di Micco Although it has been known for more than 100 yr that THs in- et al., 2006). Cellular senescence not only plays a role in aging crease basal metabolism (Magnus-Levy, 1895) and are major and contributes to the appearance of age-related pathologies regulators of mitochondrial activity and oxygen consumption (Vijg and Campisi, 2008; Baker et al., 2011) but is also an im- (Tata et al., 1962), the molecular mechanisms underlying these portant antiproliferative process that acts as a strong barrier effects have not yet been fully defined. THRs can also play a role in aging. Thus, hyperthyroidism A. Zambrano and V. García-Carpizo contributed equally to this paper. accelerates aging (Ooka and Shinkai, 1986; Buffenstein and Correspondence to Ana Aranda: [email protected] Pinto, 2009), whereas longevity is associated with decreased Abbreviations used in this paper: 8-OH-dG, 8-hydroxy-2-deoxyguanosine; thyroid function (Atzmon et al., 2009; Rozing et al., 2010; ChIP, chromatin immunoprecipitation; Cr, cell respiration; DDR, DNA dam- age response; DSB, double-strand break; dUTP, deoxy-UTP; KO, knockout; MEF, mouse embryonic fibroblast; NAC, N-acetyl-L-cysteine; OIS, oncogenic V12 © 2014 Zambrano et al. This article is distributed under the terms of an Attribution– H-Ras –induced senescence; PDL, population doubling level; ROS, reactive oxy- Noncommercial–Share Alike–No Mirror Sites license for the first six months after the pub- gen species; ROX, residual oxygen consumption; SA-gal, senescence-associated lication date (see http://www.rupress.org/terms). After six months it is available under a -galactosidase; TdT, terminal deoxynucleotidyl transferase; TH, thyroid hor- Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, mone; THR, TH receptor. as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). Supplemental Material can be found at: http://jcb.rupress.org/content/suppl/2014/01/02/jcb.201305084.DC1.html The Rockefeller University Press $30.00 J. Cell Biol. Vol. 204 No. 1 129–146 www.jcb.org/cgi/doi/10.1083/jcb.201305084 JCB 129 Published January 6, 2014 Downloaded from jcb.rupress.org on October 23, 2014 Figure 1. T3 induces cellular senescence and DNA damage. (A) 5 nM T3 increases the number of SA-gal+ cells in primary MEFs at consecutive cell passages (P < 0.0001, n = 3). Representative SA-gal images are also shown. (B) T3 increases expression of CDKN2A. To control for the level of pro- tein in each sample, the same samples were run on a separate gel and detected for ACTB (-actin). (C) Percentage of SA-gal+ cells incubated with and 130 JCB • VOLUME 204 • NUMBER 1 • 2014 Published January 6, 2014 Gesing et al., 2012). In mammals, life span correlates with the (KO) mice but not from THRB-deficient mice (Fig. S1 K). On metabolic rate (Mookerjee et al., 2010), which is controlled by the other hand, Thra KO MEFs showed a higher DNA damage the THs. Therefore, an increase of ROS production and oxida- after H2O2 treatment than those obtained from ThrbKO mice tive stress may be involved in the acceleration of aging by the (Fig. S1 K), and both Thra and Thrb mRNAs were reduced THs. There is also increasing evidence that THRs play a role in after 6-d treatment with the DNA damaging agent camptothecin cancer. THRs can inhibit oncogenic proliferation, transforma- (Fig. S1 L). T3 and GC-1 also induced senescence and DNA tion, tumor growth, tumor invasion, and formation of metastasis damage in human hepatocyte HUH7 cells (Fig. 1 G), indicating (García-Silva and Aranda, 2004; Aranda et al., 2009; García- the relevance of this hormonal action. Silva et al., 2011). In addition, mutations or loss of expression T3 did not induce senescence in MEFs obtained from of THRs are common events in some tumors (Aranda et al., Thra/ThrbKO mice that were spontaneously immortalized in 2009; Kim and Cheng, 2013), suggesting that these receptors culture. However, expression of THRB (but not THRA) in these have tumor suppressor activity. Because THs accelerate aging cells restored the ability of T3 to induce proliferation arrest, and THRs suppress tumor growth, in this work, we have exam- senescence, and formation of DNA damage foci (Fig. 2, A–D). ined the possibility that they could induce cellular senescence In ThrKO MEFs cultured with a serum substitute, transduction and found that THRB, but not THRA, can mediate T3 (triiodo- of THRB, but not THRA, increased senescence in the absence thyronine)-dependent premature senescence in cultured cells of ligand, and this action was further increased by T3 and and in mice. GC-1 (Fig. 2 E). Therefore, the unliganded receptor has consti- tutive effects on senescence when expressed at high intracellular Downloaded from Results concentrations. The percentage of cells undergoing senescence upon THRB expression was lower than in normal MEFs, but T3 induces senescence and DNA damage most of the immortal cell population became senescent when in mouse embryonic fibroblasts (MEFs) cells were previously exposed to a H2O2 shock (Fig. 2 F). Incubation of MEFs with a physiological-relevant T3 concentra- To determine the functional domains involved in T3- tion (5 nM) under 20% oxygen increases the amount of cells dis- dependent induction of senescence, we used various THRB playing senescence-like features, such as senescence-associated mutants (Fig. 3, A and B). Mutation C102G in the DNA-binding jcb.rupress.org -galactosidase (SA-gal) activity (Fig. 1 A), and increases the domain abolishes binding to the receptor recognition motifs. levels of CDKN2A (Fig. 1 B), demonstrating that the hormone The AHT (Ala-His-Tyr) mutant is a triple substitution in the co- induces premature senescence. Increased senescence by T3 is repressor box that blocks binding of corepressors, and the muta- also observed under normoxic conditions (Fig. 1 C). MEFs ex- tion E452Q is in the ligand-dependent transcriptional activation press both THRA and THRB (Fig. S1 A), but THRB appears to domain (AF-2) and abolishes the recruitment of coactivators. mediate senescence because the THRB-specific ligand GC-1, Only binding of T3 to wild-type THRB inhibited proliferation on October 23, 2014 with similar affinity as T3 for the receptor (Baxter et al., 2004), and induced DNA damage and senescence (Fig. 3, C–E). It is induces cell growth arrest and senescence to the same extent as remarkable that THRB was able to induce senescence in
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