Activated PPARd Protects from Senescence Figure 5. Effects of L-165041 and/or doxorubicin 0.1 mM on MAPKs and Akt phosphorylation. Both L-165041(L-165) and doxorubicin (Dox) 0.1 mM activate MAPKs and Akt. If cells are pre-treated with L-165, the doxorubicin-induced increase of pJNK and pAkt levels is inhibited, pERK levels are maintained sustained, while pp38 levels result higher than those induced by Dox 0.1 alone. (A) Time curve analysis of phosphorylated pp38, pJNK, pAKT, pERK1/2 and total p38, JNK, AKT, ERK1/2 evaluated by western blot. (B, C, D, E) Graphs showing values for pp38, pJNK, pAKT and pERK1/2 normalized to the amount of total enzyme. doi:10.1371/journal.pone.0046126.g005 in PPARd expression levels. This increase is not influenced by pre- treatment with the PPARd ligand L-165041.We also found that doxorubicin 1 mM causes a 50% reduction in both total and PPARd-co-immunoprecipitated Bcl6. Of note, these changes were not influenced by pre-treatment with the PPARd agonist (Fig. 8B). We then examined the effects of transfection with siRNA targeting Bcl6 on apoptosis. We observed that silencing Bcl6 did not increase the apoptosis rate in untreated cells and did not enhance apoptosis in cells treated with doxorubicin 1 mM. We also found that pre-treatment with the PPARd ligand L-165041 in cells exposed to doxorubicin 1 mM significantly decreased the number of apoptotic cells, and what is noteworthy is that this protective effect was not affected by Bcl6 knock-down (Fig. 8A). Discussion The present study is a step forward towards understanding the cellular mechanisms of doxorubicin-induced senescence and highlights the cardioprotective actions of PPARd activation. We showed, for the first time, that pre-treatment with the PPARd agonist L-165041 is highly effective in preventing doxorubicin-induced senescence in neonatal cardiomyocytes and H9c2 cells. Pre-treatment inhibited TRF2 downregulation and prevented cell cycle changes. It partially rescued cell proliferation blockage, significantly attenuated cytoskeletal remodeling and the early loss of plasma membrane integrity, and significantly reduced the number of cells that were positive for SA-b-gal activity. We found that both doxorubicin-triggered senescence and the anti-senescent effects of pre-treatment with the PPARd agonist L- 165041 involve the interferences with the Bcl6 repressor. In fact, while doxorubicin 0.1 mM increases the PPARd protein expression that sequesters the transcriptional repressor Bcl6 in unliganded PPARd, L-1650141 increases the expression of Bcl6, which upon ligand binding, is released from the PPARd and is then able to bind to its target genes. Experiments performed with siRNA analysis techniques very clearly show the key role of Bcl6 in the cellular senescence program. Silencing Bcl6 led to senescence in unstressed cells, potentiated the pro-senescent effects of 0.1 mM doxorubicin, and abolished the anti-senescent effects of pre-treatment with the PPARd ligand L-165041. By increasing the amount of free Bcl6, PPARd protein knock-down prevented the prosenescent effects of 0.1 mM doxorubicin. To the best of our knowledge, this is the first study demonstrating that the transrepressive mode of action of PPARd plays a key role in the control of cellular senescence. To date, there are very few data on PPARd, Bcl6 and senescence. By genetic screening, Shvarts et al [23] identified Bcl6 as a potent inhibitor of senescence since it rendered cells unresponsive to anti-proliferative signals from the p19ARF–p53 pathway. Kim et al [24] demonstrated that GW501516, a specific agonist of PPARd, up- regulates the transcription of antioxidant genes and significantly inhibits Ang II-induced premature senescence of vascular smooth muscle cells. They also found that siRNA-mediated down- regulation of PPARd markedly suppresses the anti-senescent effect of GW501516, thus suggesting that in their experimental model the agonist-induced PPARd effects occur without relocation of a repressor. Unlike the scarcity of data on senescence, there is a large body of evidence showing the role that PPARd and Bcl6 play in inflammation. PPARd has been shown to control an inflammatory switch through its ligand-dependent association with, and disso- ciation from, Bcl6 [15,17,27–30]. In fact, unliganded PPARd is pro-inflammatory, while activated PPARd exerts anti-inflamma- tory effects [17,18]. It is not surprising that PPARd and Bcl6 are involved in both senescence and inflammation since important relationships do exist between inflammation and senescence. It has been shown that Angiotensin II induces vascular inflammation and senescence both in vitro and in vivo [31]. Senescent cells show a pro-inflammatory phenotype called senescent-associated secretory phenotype because this phenotype is characterized by the secretion of a great deal of inflammatory cytokines which have a profound impact on tissue homeostasis [32]. A tight link between the process of cellular senescence and the IL-dependent inflam- matory network has been proven. Using microarray analysis, Shelton [33] et al. demonstrated that senescent fibroblasts present a strong inflammatory type response. Kuilman et al. [34] found that IL-6 is up-regulated in cell lines programmed to prematurely enter oncogene-induced senescence and demonstrated that when IL-6 or its receptor is suppressed, cells re-enter the cell cycle and proliferate. Moreover, clinical studies have documented that some biomarkers of cellular senescence in circulating leukocyte DNA, especially telomere attrition, correlate with incident or prevalent atherosclerotic cardiovascular diseases [35]. We found that p38, JNK and Akt are activated by both the cardioprotective agent, L-165041, and by the cardiotoxic agent, doxorubicin. While Akt activation is generally associated with a protective role [36], p38 and JNK have been identified as stress kinases because they are activated by stimuli that cause some kind of stress to cells which eventually lead to cell death [37]. However, while this assumption is correct in most cases, several studies suggest that activation of p38 [38] and JNK [39] by stress stimuli does not necessarily promote damage, but rather, it enhances cell survival. Whether MAPK activation executes stress induced damage or survival pathway activation depends on the cell type or type of stress or stimulus. Previous studies on the signal transduction pathway in doxorubicin cardiotoxicity demonstrated that p38 activation is crucial for the execution of doxorubicin-induced damage, while the concomitant JNK and Akt activation has to be viewed as part of a cardiomyocyte survival pathway which attempts to limit the damage caused by doxorubicin [4,8,40]. In the present study, we evaluated the mechanism through which agonist-induced PPARd activation may exert protective effects against doxorubicin-induced senescence. We found that pre-treatment with specific inhibitors of p38, JNK, and Akt prevents the effect of L-165041 on Bcl6 levels and on doxorubicin- induced SA-b-gal, and that pre-treatment with the Akt inhibitor also prevents the effect of L-165041 on the up-regulation of PPARd. We demonstrated that not only Akt, but also p38 and JNK activation are essential in order for PPARd activation to exert a protective effect. This is in agreement with both the study by Liang et al. who demonstrated that L-165041 inhibits C-reactive protein induced inflammation in cardiomyocytes and in H9c2 through p38 and JNK [41] and with the study by Yue et al [42] PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e46126