Activated PPARd Protects from Senescence Figure 7. Bcl6 plays a key role in cellular senescence. Bcl6 siRNA(siBcl6) significantly increases the number of SA-b-gal positive cells in both unstressed and in 0.1 mM doxorubicin-treated cells, and it completely abolishes the anti-senescent effect of pre-treatment with L-165041 (L-165). siPPARd completely abolishes the pro-senescent effect of 0.1 mM doxorubicin. H9c2 cells were transfected with Bcl6-, PPARd specific or control (siCT) siRNA for 48 h before treatment with or without L-165041 for 2 h followed by treatment with or without 0.1 mM doxorubicin (Dox 0.1) for 3 h. Cells were assayed for protein amount (after 24 h), premature senescence (after 3 days) and apoptosis (after 24 h). (A) Western blot analysis with Bcl6 and PPARd antibodies. (B) SA-b-gal activity (magnification 6200). (C) Bar graph illustrating the percentage of SA-b-gal positive cells. *p,0.05 versus corresponding siCT of the same treatment condition, (D) Bar graph illustrating the percentage of caspase-3 positive cells. doi:10.1371/journal.pone.0046126.g007 who found that PPARd activation enhances Akt signaling and protects the heart from ischemia/reperfusion injury in Zucker fatty rats. Figure 8. L-165041 protects H9c2 from doxorubicin-induced apoptosis through a Bcl6-independent mechanism. (A) Pre- treatment with L-165141decreases the number of doxorubicin-induced apoptotic cells. Bcl6 siRNA (siBcl6) does not modify the number of activated caspase-3 positive cells in any of the treatment groups i.e., untreated cells, 1 mM doxorubicin-treated cells, and in cells pretreated with L-165041 and then incubated with doxorubicin. 24 h after treatment, cells were assayed by immunocytochemistry for cleaved caspase-3. Bar graph illustrates the percentage of cleaved-caspase-3 positive cells. *P,0.05 versus ct, 1P,0.05 versus doxorubicin. (B) Effects of L-165041(L-165) and 1 mM doxorubicin (Dox1) on PPARd and Bcl6 protein expression and on PPARd and Bcl6 protein interaction. Doxorubicin increases PPARd levels and down-regulates the amount of total and PPARd bound Bcl6 (IP-PPARd WB-Bcl6). Pre-treatment with L-165041 does not modify the effects induced by doxorubicin. H9c2 cells were pre-treated with or without L-165041 for 2 h, then treated with or without 1 mM doxorubicin for 3 h and analyzed after 24 h by western blot. The bar graph shows the protein quantification expressed as a percentage. *p,0.05 versus control (ct). doi:10.1371/journal.pone.0046126.g008 We also found that pre-treatment with L-165041 prevents the doxorubicin-induced increase in pJNK and pAkt but not the doxorubicin-induced increase in pp38. It is possible that the protection provided by L-165041 through Akt and JNK signaling is able to prevent doxorubicin-induced stress so that doxorubicin does not cause any further activation of these survival pathways. Protection through the activation of p38 occurs with an initial increase in phosphorylation due to pre-treatment with L-165041, followed by a further increase in phosphorylation due to treatment with doxorubicin. Collectively, our data show that Bcl6 plays a main role in the protective effect exerted by L-165041 against doxorubicin-induced senescence: L-165041 increases Bc16 expression levels through p38, JNK and Akt mediated pathways and induces its release from PPARd thus allowing Bcl6 binding to its target genes to exert its anti-senescent actions. Although apoptosis was not the main issue of our study we repeated several experiments using doxorubicin 1 mM, i.e., a pro- apoptotic dose, to compare the role played by the PPARd agonist in senescence and apoptosis. We found that pre-treatment with the PPARd agonist L165041 is effective in preventing apoptosis induced by doxorubicin 1 mM. Even though Bcl6 was down- regulated by doxorubicin, RNA interference experiments docu- mented that it is neither implicated in the execution of doxorubicin-induced apoptosis nor in the anti-apoptotic effects exerted by pre-incubation with the PPARd agonist. Studies investigating the role of Bcl6 in apoptosis produced inconsistent results [43–48]. Since doxorubicin-induced apoptosis is largely reactive oxygen species mediated [49], we speculate that upon ligand binding, PPARd is enabled to induce transcription of genes encoding the antioxidant enzymes. This hypothesis is in agreement with previous studies by Pesant et al, who found that the PPARd agonist GW501516 protects H9c2 from H2O2-induced cell apoptosis. They also found that this protection is totally dependent on PPARd and is carried out through catalase up-regulation [26]. In addition, since it has been shown that PPARd agonists also enhance the physical interaction between PPARd and the p65 subunit of NF-kB, thus preventing its ability to induce gene transcription [29,50], it can be hypothesized that even this mechanism might contribute to protect cardiomyocytes from the pro-apoptotic effects of doxorubicin. It is also worthy of note that silencing Bcl6 in cells treated with doxorubicin 0.1 mM potentiated the cardiotoxic effects of doxo- rubicin by increasing its pro-senescent effects without inducing a switch to apoptosis. The fact that Bcl6 is crucial for senescence induced by doxorubicin 0.1 mM, but not for apoptosis induced by doxorubicin 1 mM confirms that senescence and apoptosis are two very distinct stress response cellular programs. Since the most functionally significant cell type in the heart is represented by post mitotic, terminally differentiated cardiomyo- cytes, the idea of investigating both anthracycline cardiotoxicity and PPARd activation cardioprotection by studying mechanisms of cellular senescence in dividing neonatal rat cardiomyocytes and H9c2 might seem, at first glance, odd. It must be said however that this model has been extensively used in the past and it has been considered a convenient approach for preliminary investigations PLOS ONE | www.plosone.org 10 September 2012 | Volume 7 | Issue 9 | e46126