The cardiotoxic effects of doxorubicin, a potent chemotherapeutic agent, have been linked to DNA damage, oxidative mitochondrial damage, and nuclear translocation of p53, but the exact molecular mechanisms causing p53 transactivation and doxorubicin-induced cardiomyopathy are not clear. The present study was carried out to determine whether extracellular signal-regulated kinases (ERKs), which are known to be activated by DNA damaging agents, are responsible for doxorubicin-induced p53 activation and oxidative mitochondrial damage in H9c2 cells. Cell death was measured by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling, annexin V-fluorescein isothiocyanate, activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase (PARP). We found that doxorubicin produced cell death in H9c2 cells in a time-dependent manner, beginning at 6 h, and these changes are associated decreased expression of Bcl-2, increases in Bax and p53 upregulated modulator of apoptosis-alpha expression, and collapse of mitochondria membrane potential. The changes in cell death and Bcl-2 family proteins, however, were preceded by earlier activation and nuclear translocation of ERKs, followed by increased phosphorylation at Ser15 and nuclear translocation of the phosphorylated p53. The functional importance of ERK1/2 and p53 in doxorubicin-induced toxicity was further demonstrated by the specific ERK inhibitor U-0126 and p53 inhibitor pifithrin (PFT)-alpha, which abrogated the changes in Bcl-2 family proteins and cell death produced by doxorubicin. U-0126 blocked the phosphorylation and nuclear translocation of both ERK1/2 and p53, whereas PFT-alpha blocked only the changes in p53. Doxorubicin and ERK inhibitors produced similar changes in ERK1/2-p53, PARP, and caspase-3 in neonatal rat cultured cardiomyocytes. Thus we conclude that ERK1/2 are functionally linked to p53 and that the ERK1/2-p53 cascade is the upstream signaling pathway responsible for doxorubicin-induced cardiac cell apoptosis. ERKs and p53 may be considered as novel therapeutic targets for the treatment of doxorubicin-induced cardiotoxicity.

Background-Myocyte apoptosis plays an important role in pathological cardiac remodeling and the progression of heart failure. cAMP signaling is crucial in the regulation of myocyte apoptosis and cardiac remodeling. Multiple cAMP-hydrolyzing phosphodiesterases (PDEs), such as PDE3 and PDE4, coexist in cardiomyocytes and elicit differential temporal/spatial regulation of cAMP signaling. However, the role of PDE3 and PDE4 in the regulation of cardiomyocyte apoptosis remains unclear. Although chronic treatment with PDE3 inhibitors increases mortality in patients with heart failure, the contribution of PDE3 expression/activity in heart failure is not well known. Methods and Results-In this study we report that PDE3A expression and activity were significantly reduced in human failing hearts as well as mouse hearts with chronic pressure overload. In primary cultured cardiomyocytes, chronic inhibition of PDE3 but not PDE4 activity by pharmacological agents or adenovirus-delivered antisense PDE3A promoted cardiomyocyte apoptosis. Both angiotensin II (Ang II) and the ␤-adrenergic receptor agonist isoproterenol selectively induced a sustained downregulation of PDE3A expression and induced cardiomyocyte apoptosis. Restoring PDE3A via adenovirus-delivered expression of wild-type PDE3A1 completely blocked Ang II-and isoproterenolinduced cardiomyocyte apoptosis, suggesting the critical role of PDE3A reduction in cardiomyocyte apoptosis. Moreover, we defined a crucial role for inducible cAMP early repressor expression in PDE3A reduction-mediated cardiomyocyte apoptosis. Conclusions-Our results suggest that PDE3A reduction and consequent inducible cAMP early repressor induction are critical events in Ang II-and isoproterenol-induced cardiomyocyte apoptosis and may contribute to the development of heart failure. Drugs that maintain PDE3A function may represent an attractive therapeutic approach to treat heart failure.

Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors that form a subfamily of the nuclear receptor gene family. Since both flow and PPAR␥ have atheroprotective effects and extracellular signal-regulated kinase 5 (ERK5) kinase activity is significantly increased by flow, we investigated whether ERK5 kinase regulates PPAR␥ activity. We found that activation of ERK5 induced PPAR␥1 activation in endothelial cells (ECs). However, we could not detect PPAR␥ phosphorylation by incubation with activated ERK5 in vitro, in contrast to ERK1/2 and JNK, suggesting a role for ERK5 as a scaffold. Endogenous PPAR␥1 was coimmunoprecipitated with endogenous ERK5 in ECs. By mammalian two-hybrid analysis, we found that PPAR␥1 associated with ERK5a at the hinge-helix 1 region of PPAR␥1. Expressing a hinge-helix 1 region PPAR␥1 fragment disrupted the ERK5a-PPAR␥1 interaction, suggesting a critical role for hinge-helix 1 region of PPAR␥ in the ERK5-PPAR␥ interaction. Flow increased ERK5 and PPAR␥1 activation, and the hinge-helix 1 region of the PPAR␥1 fragment and dominant negative MEK5␤ significantly reduced flow-induced PPAR␥ activation. The dominant negative MEK5␤ also prevented flow-mediated inhibition of tumor necrosis factor alpha-mediated NF-B activation and adhesion molecule expression, including vascular cellular adhesion molecule 1 and E-selectin, indicating a physiological role for ERK5 and PPAR␥ activation in flow-mediated antiinflammatory effects. We also found that ERK5 kinase activation was required, likely by inducing a conformational change in the NH 2 -terminal region of ERK5 that prevented association of ERK5 and PPAR␥1. Furthermore, association of ERK5a and PPAR␥1 disrupted the interaction of SMRT and PPAR␥1, thereby inducing PPAR␥ activation. These data suggest that ERK5 mediates flow-and ligand-induced PPAR␥ activation via the interaction of ERK5 with the hinge-helix 1 region of PPAR␥.Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors that form a subfamily of the nuclear receptor gene family. Among PPAR family members, the expression of PPAR␣ and PPAR␥ has been reported in endothelial cells (ECs). Recently, Pasceri et al. reported that PPAR␥ activators inhibit expression of vascular cellular adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) in activated ECs and significantly reduce monocyte/macrophage homing to atherosclerotic plaques (23). Mitogen-activated protein (MAP) kinase signaling pathways have been shown to phosphorylate PPAR␥ and to decrease PPAR␥ transcriptional activity (7, 13). The NH 2 -terminal domain of PPAR␥ contains a consensus MAP kinase site in a region conserved between PPAR␥1 and PPAR␥2 isoforms (7, 13). Phosphorylation of PPAR␥2 Ser112 (13) and PPAR␥1 Ser82 (7) significantly inhibits both ligand-independent and ligand-dependent transcriptional activation by PPAR␥. Phosphorylation-mediated transcriptional repression is due to a diminished ability of PPAR␥ to become transcriptionally a...