Inhibition of Doxorubicin-Induced Senescence by PPARd Activation Agonists in Cardiac Muscle Cells: Cooperation between PPARd and Bcl6 Paola Altieri1*., Paolo Spallarossa2., Chiara Barisione1, Silvano Garibaldi1, Anna Garuti3, Patrizia Fabbi1, Giorgio Ghigliotti2, Claudio Brunelli1,2 1 Research Centre of Cardiovascular Biology, University of Genova, Genova, Italy, 2 Division of Cardiology, IRCCS University Hospital San Martino, University of Genova, Genova, Italy, 3 Laboratory of Cellular Therapies, Department of Internal Medicine, University of Genova, Genova, Italy Abstract Senescence and apoptosis are two distinct cellular programs that are activated in response to a variety of stresses. Low or high doses of the same stressor, i.e., the anticancer drug doxorubicin, may either induce apoptosis or senescence, respectively, in cardiac muscle cells. We have demonstrated that PPARd, a ligand-activated transcriptional factor that controls lipid metabolism, insulin sensitivity and inflammation, is also involved in the doxorubicin-induced senescence program. This occurs through its interference with the transcriptional repressor protein B cell lymphoma-6 (Bcl6). Low doses of doxorubicin increase the expression of PPARd that sequesters Bcl6, thus preventing it from exerting its anti-senescent effects. We also found that L-165041, a specific PPARd activator, is highly effective in protecting cardiomyocytes from doxorubicin-induced senescence through a Bcl6 related mechanism. In fact, L-165041 increases Bcl6 expression via p38, JNK and Akt activation, and at the same time it induces the release of Bcl6 from PPARd, thereby enabling Bcl6 to bind to its target genes. L-165041 also prevented apoptosis induced by higher doses of doxorubicin. However, while experiments performed with siRNA analysis techniques very clearly showed the weight of Bcl6 in the cellular senescence program, no role was found for Bcl6 in the anti-apoptotic effects of L-165041, thus confirming that senescence and apoptosis are two very distinct stress response cellular programs. This study increases our understanding of the molecular mechanism of anthracycline cardiotoxicity and suggests a potential role for PPARd agonists as cardioprotective agents. Citation: Altieri P, Spallarossa P, Barisione C, Garibaldi S, Garuti A, et al. (2012) Inhibition of Doxorubicin-Induced Senescence by PPARd Activation Agonists in Cardiac Muscle Cells: Cooperation between PPARd and Bcl6. PLoS ONE 7(9): e46126. doi:10.1371/journal.pone.0046126 Editor: Gianfranco Pintus, University of Sassari, Italy Received March 9, 2012; Accepted August 28, 2012; Published September 25, 2012 Copyright: 2012 Altieri et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the University of Genova (cofinanziamento di Ateneo). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Paola.Altieri@unige.it . These authors contributed equally to this work. Introduction Anthracyclines are among the most effective anticancer treatments ever developed, but their clinical use is limited by their cumulative dose-related cardiotoxicity which may ulti- mately lead to a severe form of cardiomyopathy [1]. Despite solid evidence proving the induction of apoptosis in cardiomy- ocytes exposed to doxorubicin in vitro, there is controversy over whether apoptosis contributes to doxorubicin-induced cardio- toxicity in vivo [2]. It has recently been suggested that senescence may be a novel mechanism of cardiotoxicity induced by low doses of doxorubicin [3,4]. Senescence is a fundamental cellular program that contributes to the physiology of living tissues, the aging process, and diseases [5]. Stress-induced premature senescence is the result of changes in the expression levels of many proteins that regulate cell cycle, cytoskeletal function and cellular architecture, and it leads to the impairment of cell functions, including the regenerative capacity [6–8]. The signal transduction pathways of the anthracycline- induced senescence program are not fully understood. There is however convincing evidence that p38 activation and expression levels of Telomere Binding Factor 2 (TRF2) play an important role [4,9] Peroxisome proliferator-activated receptor d (PPARd) belongs to the nuclear hormone receptor superfamily together with PPARa and PPARc [10]. PPARd are ligand-activated transcriptional factors that regulate the expression of specific target genes involved in lipid metabolism, insulin sensitivity, energy homeostasis, obesity, and inflammation [11–15]. Activa- tion/repression of target genes occurs via two molecular mechanisms: transactivation and transrepression. In the trans- activation mode these nuclear receptors control gene expression by binding to a PPAR responsive element after heterodimeriza- tion with a retinoid X receptor. The transrepression activity of PPARs occurs through the physical interaction with other transcription factors. It has been shown that unliganded PPARd sequesters the transcriptional repressor protein B cell lympho- ma-6 (Bcl6) and prevents it from binding to the response elements in the promoter regions of its target genes. Following ligand binding, Bcl6 is released from PPARd and inhibits inflammatory signals [15–17]. Bcl6 inhibits chemokine gene transcription in most tissues and cell types [18], regulates cell cycle progression [19], and is involved in lymphocyte activation PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e46126