Cardiomyocytes in culture can survive low or mild doses of oxidants but later increase cell volume and protein content. To understand the mechanism, we determined the early signaling events of oxidative stress. With 200 M H 2 O 2 , the activity of p70 S6 kinase-1 (p70S6K1) increased at 30 min and reached a plateau at 90 min. Dose-response studies at the 60 min time point show that p70S6K1 activity reached its highest level with 150 M H 2 O 2 . Increased p70S6K1 activity correlated with phosphorylation of Thr389 and Thr421/Ser424 residues, suggesting the involvement of an upstream kinase. Phosphoinositide 3-kinase (PI3K) activity was elevated by 5 min, reached a plateau at 10 min, and remained more than 6-fold induced for at least 60 min after 200 M H 2 O 2 exposure. The dose-response studies at 10 min found that 150 M H 2 O 2 induced the highest PI3K activity. Increased PI3K activity correlated with tyrosine phosphorylation of the 85-kDa regulatory subunit. Inactivating PI3K with wortmannin prevented H 2 O 2 from inducing Thr389 phosphorylation and p70S6K1 activation. Wortmannin and rapamycin prevented H 2 O 2 from inducing increases in cell volume and protein content. The antineoplastic drugs doxorubicin and daunorubicin also induced significant enlargement of cardiomyocytes at 10 to 100 nM dose range. Although the glutathione synthesis inhibitor buthionine sulfoximine potentiated the effect of doxorubicin and H 2 O 2 , the antioxidant N-acetylcysteine prevented induction of cell enlargement. Our data suggest that oxidative stress induces activation of PI3K, which leads to p70S6K1 activation and enlargement of cell size.

FP prostanoid receptors are G-protein-coupled receptors whose physiological activator is prostaglandin-F 2␣ (PGF 2␣ ). PGF 2␣ has been implicated in wound healing and cardiac hypertrophy, which are both known to involve the induction of the immediate-early response gene, early growth response factor-1 (EGR-1). We hypothesized that activation of the human FP receptor by PGF 2␣ could induce the expression of EGR-1 and found that 1 M PGF 2␣ produced a time-dependent induction of both mRNA and protein expression for EGR-1. This FP receptor-mediated induction of EGR-1 expression involved activation of the small GTPase Ras followed by activation of C-Raf and the mitogen-activated protein (MAP) kinase kinases 1 and 2 (MEK1/2). Thus, induction of EGR-1 expression by PGF 2␣ was blocked using dominant-negative constructs of Ras and C-Raf and the Raf kinase inhibitor 4-(4-(3-(4-chloro-3-trifluoromethylphenyl)ureido)phenoxy)-pyridine-2-carboxyllic acid methyamide-4-methylbenzenesulfonate (BAY43-9006). Likewise, the MEK1/2 inhibitor 2Ј-amino-3Ј-methoxyflavone (PD98059) blocked the induction of EGR-1 expression by PGF 2␣ . FP receptor stimulation by PGF 2␣ induced the phosphorylation of C-Raf, MEK1/2, and extracellular signal-regulated kinases 1 and 2, consistent with the activation of a MAP kinase signaling cascade. PGF 2␣ was also found to induce the expression of EGR-1 in rat cardiomyocytes through the activation of endogenous FP receptors. This induction of EGR-1 expression in cardiomyocytes also involved the activation of Raf and MAP kinase signaling and was dependent on the activation of protein kinase C. This is the first report to show the regulation of EGR-1 expression after PGF 2␣ activation of FP receptors and suggests that this could be an early event involved in wound healing and cardiac hypertrophy.Prostaglandin F 2␣ (PGF 2␣ ) is one of five major prostanoids that are formed from the initial metabolism of arachidonic acid by the cyclooxygenases COX