Stimulation of "stress-regulated" mitogen-activated protein kinases (stress-activated protein kinases/c-Jun N-terminal kinases and p38-mitogen-activated protein kinases) in perfused rat hearts by oxidative and other stresses.

"Stress-regulated" mitogen-activated protein kinases (SR-MAPKs) comprise the stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinases (JNKs) and the p38-MAPKs. In the perfused heart, ischemia/reperfusion activates SR-MAPKs. Although the agent(s) directly responsible is unclear, reactive oxygen species are generated during ischemia/reperfusion. We have assessed the ability of oxidative stress (as exemplified by H2O2) to activate SR-MAPKs in the perfused heart and compared it with the effect of ischemia/reperfusion. H2O2 activated both SAPKs/JNKs and p38-MAPK. Maximal activation by H2O2 in both cases was observed at 0.5 mM. Whereas activation of p38-MAPK by H2O2 was comparable to that of ischemia and ischemia/reperfusion, activation of the SAPKs/JNKs was less than that of ischemia/reperfusion. As with ischemia/reperfusion, there was minimal activation of the ERK MAPK subfamily by H2O2. MAPK-activated protein kinase 2 (MAPKAPK2), a downstream substrate of p38-MAPKs, was activated by H2O2 to a similar extent as with ischemia or ischemia/reperfusion. In all instances, activation of MAPKAPK2 in perfused hearts was inhibited by SB203580, an inhibitor of p38-MAPKs. Perfusion of hearts at high aortic pressure (20 kilopascals) also activated the SR-MAPKs and MAPKAPK2. Free radical trapping agents (dimethyl sulfoxide and N-t-butyl-alpha-phenyl nitrone) inhibited the activation of SR-MAPKs and MAPKAPK2 by ischemia/reperfusion. These data are consistent with a role for reactive oxygen species in the activation of SR-MAPKs during ischemia/reperfusion.

The p38-MAPK inhibitor, SB203580, inhibits cardiac stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs).

SB203580 is a recognised inhibitor of p38-MAPKs. Here, we investigated the effects of SB203580 on cardiac SAPKs/JNKs. The IC50 for inhibition of p38-MAPK stimulation of MAPKAPK2 was approximately 0.07 microM, whereas that for total SAPK/JNK activity was 3-10 microM. SB203580 did not inhibit immunoprecipitated JNK1 isoforms. Three peaks of SAPK/JNK activity were separated by anion exchange chromatography, eluting in the isocratic wash (44 kDa), and at 0.08 M (46 and 52 kDa) and 0.15 M NaCl (54 kDa). SB203580 (10 microM) completely inhibited the 0.15 M NaCl activity and partially inhibited the 0.08 M NaCl activity. Since JNK1 antibodies immunoprecipitate the 46 kDa activity, this indicates that SB203580 selectively inhibits 52 and 54 kDa SAPKs/JNKs.

Stimulation of multiple mitogen-activated protein kinase sub-families by oxidative stress and phosphorylation of the small heat shock protein, HSP25/27, in neonatal ventricular myocytes.

We investigated the activation of three subfamilies of mitogen-activated protein kinases (MAPKs), namely the stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs), the extracellularly responsive kinases (ERKs) and p38-MAPK, by oxidative stress as exemplified by H2O2 in primary cultures of neonatal rat ventricular myocytes. The 46 and 54 kDa species of SAPKs/JNKs were activated 5- and 10-fold, respectively, by 0.1 mM H2O2 (the maximally effective concentration). Maximal activation occurred at 15-30 min, but was still detectable after 2 h. Both ERK1 and ERK2 were activated 16-fold by 0.1 mM H2O2 with a similar time course to the SAPKs/JNKs, and this was comparable with their activation by 1 microM PMA, the most powerful activator of ERKs that we have so far identified in these cells. The activation of ERKs by H2O2 was inhibited by PD98059, which inhibits the activation of MAPK (or ERK) kinases, and by the protein kinase C (PKC) inhibitor, GF109203X. ERK activation was also inhibited by down-regulation of PMA-sensitive PKC isoforms. p38-MAPK was activated by 0.1 mM H2O2 as shown by an increase in its phosphorylation. However, maximal phosphorylation (activation) was more rapid (<5 min) than for the SAPKs/JNKs or the ERKs. We studied the downstream consequences of p38-MAPK activation by examining activation of MAPK-activated protein kinase 2 (MAPKAPK2) and phosphorylation of the MAPKAPK2 substrate, the small heat shock protein HSP25/27. As with p38-MAPK, MAPKAPK2 was rapidly activated (maximal within 5 min) by 0.1 mM H2O2. This activation was abolished by 10 microM SB203580, a selective inhibitor of certain p38-MAPK isoforms. The phosphorylation of HSP25/27 rapidly followed activation of MAPKAPK2 and was also inhibited by SB203580. Phosphorylation of HSP25/27 was associated with a decrease in its aggregation state. These data indicate that oxidative stress is a powerful activator of all three MAPK subfamilies in neonatal rat ventricular myocytes. Activation of all three MAPKs has been associated with the development of the hypertrophic phenotype. However, stimulation of p38-MAPK and the consequent phosphorylation of HSP25/27 may also be important in cardioprotection.

Activation of mitogen-activated protein kinases (p38-MAPKs, SAPKs/JNKs and ERKs) by the G-protein-coupled receptor agonist phenylephrine in the perfused rat heart.

We investigated the ability of phenylephrine (PE), an alpha-adrenergic agonist and promoter of hypertrophic growth in the ventricular myocyte, to activate the three best-characterized mitogen-activated protein kinase (MAPK) subfamilies, namely p38-MAPKs, SAPKs/JNKs (i.e. stress-activated protein kinases/c-Jun N-terminal kinases) and ERKs (extracellularly responsive kinases), in perfused contracting rat hearts. Perfusion of hearts with 100 microM PE caused a rapid (maximal at 10 min) 12-fold activation of two p38-MAPK isoforms, as measured by subsequent phosphorylation of a p38-MAPK substrate, recombinant MAPK-activated protein kinase 2 (MAPKAPK2). This activation coincided with phosphorylation of p38-MAPK. Endogenous MAPKAPK2 was activated 4-5-fold in these perfusions and this was inhibited completely by the p38-MAPK inhibitor, SB203580 (10 microM). Activation of p38-MAPK and MAPKAPK2 was also detected in non-contracting hearts perfused with PE, indicating that the effects were not dependent on the positive inotropic/chronotropic properties of the agonist. Although SAPKs/JNKs were also rapidly activated, the activation (2-3-fold) was less than that of p38-MAPK. The ERKs were activated by perfusion with PE and the activation was at least 50% of that seen with 1 microM PMA, the most powerful activator of the ERKs yet identified in cardiac myocytes. These results indicate that, in addition to the ERKs, two MAPK subfamilies, whose activation is more usually associated with cellular stresses, are activated by the Gq/11-protein-coupled receptor (Gq/11PCR) agonist, PE, in whole hearts. These data indicate that Gq/11PCR agonists activate multiple MAPK signalling pathways in the heart, all of which may contribute to the overall response (e.g. the development of the hypertrophic phenotype).

Activation of c-Jun N-terminal kinases and p38-mitogen-activated protein kinases in human heart failure secondary to ischaemic heart disease.

Three well-characterized mitogen-activated protein kinase (MAPK) subfamilies are expressed in rodent and rabbit hearts, and are activated by pathophysiological stimuli. We have determined and compared the expression and activation of these MAPKs in donor and failing human hearts. The amount and activation of MAPKs was assessed in samples from the left ventricles of 4 unused donor hearts and 12 explanted hearts from patients with heart failure secondary to ischaemic heart disease. Total MAPKs or dually phosphorylated (activated) MAPKs were detected by Western blotting and MAPK activities were measured by in gel kinase assays. As in rat heart, c-Jun N-terminal kinases (JNKs) were detected in human hearts as bands corresponding to 46 and 54 kDa; p38-MAPK(s) was detected as a band corresponding to approximately 40 kDa, and extracellularly regulated kinases, ERK1 and ERK2, were detected as 44- and 42-kDa bands respectively. The total amounts of 54 kDa JNK, p38-MAPK and ERK2 were similar in all samples, although 46-kDa JNK was reduced in the failing hearts. However, the mean activities of JNKs and p38-MAPK(s) were significantly higher in failing heart samples than in those from donor hearts (P<0.05). There was no significant difference in phosphorylated (activated) ERKs between the two groups. In conclusion, JNKs, p38-MAPK(s) and ERKs are expressed in the human heart and the activities of JNKs and p38-MAPK(s) were increased in heart failure secondary to ischaemic heart disease. These data indicate that JNKs and p38-MAPKs may be important in human cardiac pathology.

Pro-inflammatory cytokines stimulate mitogen-activated protein kinase subfamilies, increase phosphorylation of c-Jun and ATF2 and upregulate c-Jun protein in neonatal rat ventricular myocytes.

Pro-inflammatory cytokines may be important in the pathophysiological responses of the heart. We investigated the activation of the three mitogen-activated protein kinase (MAPK) subfamilies ¿c-Jun N-terminal kinases (JNKs), p38-MAPKs and extracellularly-responsive kinases (ERKs) by interleukin-1 beta (IL-1 beta) or tumour necrosis factor alpha (TNF alpha) in primary cultures of myocytes isolated from neonatal rat ventricles. Both cytokines stimulated a rapid (maximal within 10 min) increase in JNK activity. Although activation of JNKs by IL-1 beta was transient returning to control values within 1 h, the response to TNF alpha was sustained. IL-1 beta and TNF alpha also stimulated p38-MAPK phosphorylation, but the response to IL-1 beta was consistently greater than TNF alpha. Both cytokines activated ERKs, but to a lesser degree than that induced by phorbol esters. The transcription factors, c-Jun and ATF2, are phosphorylated by the MAPKs and are implicated in the upregulation of c-Jun. IL-1 beta and TNF alpha stimulated the phosphorylation of c-Jun and ATF2. However, IL-1 beta induced a greater increase in c-Jun protein. Inhibitors of protein kinase C (PKC) (Ro318220, GF109203X) and the ERK cascade (PD98059) attenuated the increase in c-Jun induced by IL-1 beta, but LY294002 (an inhibitor of phosphatidylinositol 3' kinase) and SB203580 (an inhibitor of p38-MAPK, which also inhibits certain JNK isoforms) had no effect. These data illustrate that some of the pathological effects of IL-1 beta and TNF alpha may be mediated through the MAPK cascades, and that the ERK cascade, rather than JNKs or p38-MAPKs, are implicated in the upregulation of c-Jun by IL-1 beta.

Up-regulation of c-jun mRNA in cardiac myocytes requires the extracellular signal-regulated kinase cascade, but c-Jun N-terminal kinases are required for efficient up-regulation of c-Jun protein.

Cardiac hypertrophy, an important adaptational response, is associated with up-regulation of the immediate early gene, c- jun, which encodes the c-Jun transcription factor. c-Jun may feed back to up-regulate its own transcription and, since the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein kinases (MAPKs) phosphorylate c-Jun(Ser-63/73) to increase its transactivating activity, JNKs are thought to be the principal factors involved in c- jun up-regulation. Hypertrophy in primary cultures of cardiac myocytes is induced by endothelin-1, phenylephrine or PMA, probably through activation of one or more of the MAPK family. These three agonists increased c- jun mRNA with the rank order of potency of PMA approximately endothelin-1>phenylephrine. Up-regulation of c- jun mRNA by endothelin-1 was attenuated by inhibitors of protein kinase C (GF109203X) and the extracellular signal-regulated kinase (ERK) cascade (PD98059 or U0126), but not by inhibitors of the JNK (SP600125) or p38-MAPK (SB203580) cascades. Hyperosmotic shock (0.5 M sorbitol) powerfully activates JNKs, but did not increase c- jun mRNA. These data suggest that ERKs, rather than JNKs, are required for c- jun up-regulation. However, endothelin-1 and phenylephrine induced greater up-regulation of c-Jun protein than PMA and phosphorylation of c-Jun(Ser-63/73) correlated with the level of c-Jun protein. Up-regulation of c-Jun protein by endothelin-1 was attenuated by inhibitors of protein kinase C and the ERK cascade, probably correlating with a primary input of ERKs into transcription. In addition, SP600125 inhibited the phosphorylation of c-Jun(Ser-63/73), attenuated the increase in c-Jun protein induced by endothelin-1 and increased the rate of c-Jun degradation. Thus whereas ERKs are the principal MAPKs required for c- jun transcription, JNKs are necessary to stabilize c-Jun for efficient up-regulation of the protein.

Peptide growth factors signal differentially through protein kinase C to extracellular signal-regulated kinases in neonatal cardiomyocytes

The extracellular signal-regulated kinases 1/2 (ERK1/2) are activated in cardiomyocytes by Gq protein-coupled receptors and are associated with induction of hypertrophy. Here, we demonstrate that, in primary cardiomyocyte cultures, ERK1/2 were also significantly activated by platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or fibroblast growth factor (FGF), but insulin, insulin-like growth factor 1 (IGF-1) and nerve growth factor (NGF) had relatively minor effects. PDGF, EGF or FGF increased cardiomyocyte size via ERK1/2, whereas insulin, IGF-1 or NGF had no effect suggesting minimum thresholds/durations of ERK1/2 signaling are required for the morphological changes associated with hypertrophy. Peptide growth factors are widely accepted to activate phospholipase C gamma1 (PLCgamma1) and protein kinase C (PKC). In cardiomyocytes, only PDGF stimulated tyrosine phosphorylation of PLCgamma1 and nPKCdelta. Furthermore, activation of ERK1/2 by PDGF, but not EGF, required PKC activity. In contrast, EGF substantially increased Ras.GTP with rapid activation of c-Raf, whereas stimulation of Ras.GTP loading by PDGF was minimal and activation of c-Raf was delayed. Our data provide clear evidence for differential coupling of PDGF and EGF receptors to the ERK1/2 cascade, and indicate that a minimum threshold/duration of ERK1/2 signaling is required for the development of cardiomyocyte hypertrophy.