c-Jun N-Terminal Kinases Mediate Reactivation of Akt and Cardiomyocyte Survival After Hypoxic Injury In Vitro and In Vivo

Abstract

Abstract-Akt is a central regulator of cardiomyocyte survival after ischemic injury in vitro and in vivo, but the mechanisms regulating Akt activity in the postischemic cardiomyocyte are not known. Furthermore, although much is known about the detrimental role that the c-Jun N-terminal kinases (JNKs) play in promoting death of cells exposed to various stresses, little is known of the molecular mechanisms by which JNK activation can be protective. We report that JNKs are necessary for the reactivation of Akt after ischemic injury. We identified Thr450 of Akt as a residue that is phosphorylated by JNKs, and the phosphorylation status of Thr450 regulates reactivation of Akt after hypoxia, apparently by priming Akt for subsequent phosphorylation by 3-phosphoinositide-dependent protein kinase. The reduction in Akt activity that is induced by JNK inhibition may have significant biological consequences, as we find that JNKs, acting via Akt, are critical determinants of survival in posthypoxic cardiomyocytes in culture. Furthermore, in contrast to selective p38 -mitogen-activated protein kinase inhibition, which was cardioprotective in vivo, concurrent inhibition of both JNKs and p38 -mitogen-activated protein kinases increased ischemia/reperfusion injury in the heart of the intact rat. These studies demonstrate that reactivation of Akt after resolution of hypoxia and ischemia is regulated by JNKs and suggest that this is likely a central mechanism of the myocyte protective effect of JNKs. (Circ Res. 2006;98:111-118.)Key Words: Akt Ⅲ apoptosis Ⅲ c-Jun NH2-terminal kinase Ⅲ hypoxia Ⅲ ischemia Ⅲ signal transduction T he families of stress-activated protein kinases (SAPKs) consist of the c-Jun N-terminal kinase (JNK) family and the p38 -mitogen-activated protein kinase (MAPK) family. 1 They are potently activated by a number of cellular stresses and produce a number of biological responses that vary by the stimulus and the cell type. These kinases are activated by ischemia (especially p38-MAPKs 2 ) and by reperfusion of ischemic tissues (JNKs and to a lesser extent p38-MAPKs 3 ).Recently, a potent and relatively selective inhibitor of the ␣ and ␤ p38-MAPK isoforms has demonstrated reductions in ischemic injury in animal models of myocardial infarction and stroke. 4,5 In addition, overexpression of dominant negative mutants of components of the p38-MAPK pathway in transgenic mice protected hearts from ischemia/reperfusion (I/R) injury. 6 However, the roles played by the JNKs in ischemic injury are much less clear than those of p38-MAPKs. This is due in large part to the fact that potent and selective inhibitors of the JNKs have only very recently been developed, are not widely available, and, to our knowledge, have not been used to study I/R injury either in vivo or in cultured cardiomyocytes.In support of a deleterious role for JNKs in ischemic injury, studies in mice in which the JNK3 gene has been deleted and studies with a peptide inhibitor of JNKs demonstrated markedly reduced ischemic injury and excitotoxicity in th...

Small Heat-Shock Protein Hsp20 Attenuates β-Agonist–Mediated Cardiac Remodeling Through Apoptosis Signal–Regulating Kinase 1

“…We found that the phosphorylated levels of p38, SAPK/JNK, and ERK1/2 were significantly increased in both WT and Hsp20 TG hearts treated with various ISO doses; however, the degree of p38 and SAPK/JNK activation was significantly greater in WT hearts, whereas ERK was similarly activated in the 2 groups (Figure 4). Although a wealth of information indicates that Akt signaling plays a crucial role in regulation of cardiac remodeling and apoptosis, and recent findings showed that JNK mediates reactivation of Akt in cardiomyocytes, 25 we did not observe any differences in Akt activation triggered by ISO between WTs and TGs, as determined by immunoblotting for phosphorylation of either Thr308 or Ser473 ( Figure 4). Intriguingly, the endogenous Hsp20 levels were decreased by 36Ϯ3% in these acutely ISO-treated WT hearts (Figure 4), whereas prolonged ISO-perfusion of hearts ex vivo resulted in increased expression of Hsp20.…”

“…39 More recently, a study further indicates that dual JNK/p38 inhibition leads to increased ischemia/reperfusion-induced cardiomyocyte apoptosis; however, this report also shows that the proapoptotic effects of the dual JNK/p38 inhibitor V-150 are possibly attributable to suppression of JNK as opposed to p38 MAPK. 25 Nevertheless, these previous studies collectively suggest that although inhibition/activation of either p38MAPK or JNK signaling pathways produces the same cardiac phenotype, the temporal manifestation of the disease possibly depends on the overall extent of cellular signaling inhibition/activation, and especially the upstream molecules of JNK/p38MAPK. This view Figure 7.…”

SIRT1 modulates MAPK pathways in ischemic–reperfused cardiomyocytes

“…In other studies performed in cardiac myocytes, inhibition of JNK activity promoted apoptosis and stimulated caspase-3 [64] and caspase-9 [65] activity in an in vitro IR model. It has also been suggested that the cardioprotective effect of JNK may be partly due to Akt activation, a key pro-survival protein in post-ischemic cardiomyocytes [66]. The role of JNK signaling in IR thus remains unclear, possibly due to the complexity of the multistage, interconnected signaling cascades that underlie this process.…”

Protein phosphatase-1 regulates Akt1 signal transduction pathway to control gene expression, cell survival and differentiation

“…Recent studies have shown that JNK1/2 are involved in priming Akt1 by phosphorylating Thr-450 for further activation by PDK1 and other kinases during hypoxia/reoxygenationinduced injury of cardiomyocytes under both in vitro and in vivo conditions. 15,16 To confirm that JNKs also phosphorylate AKT1 at Thr-450, we treated human lens epithelial cells (HLECs) with 1 mM of SP600125 to inhibit JNK-1, JNK-2 and JNK-3. 30 As shown in Supplementary Figure 1A, after inhibition of JNKs for 48 h, the basic phosphorylation of AKT1 at Thr-450 was significantly decreased.…”

“…8,14 First, a recent study has shown that Akt1 is primed through phosphorylation at Thr-450 in the cytosol by the stress-activated protein kinase, JNK1/2, under certain conditions. 15,16 Second, after activation by the ligand-regulated receptor-tyrosine kinases, the phosphoinositide-3 kinase (PI3K) generates phosphoinositol-3,4,5-triphosphate (PIP3), the lipid secondary messenger that recruits phosphoinositide-dependent kinase 1 (PDK1) and Akt1 to the membrane through its PH domain. 8,9 PDK1 thus phosphorylates Akt1 at Thr-308, causing a confirmation change of Akt1 to expose its C-terminal hydrophobic motif.…”