Clerk A, Michael A, Sugden P H
NHLI Division (Cardiac Medicine), Royal Brompton Campus, Imperial College School of Medicine, Dovehouse Street, London SW3 6LY, UK.
Biochem J. 1998 Aug 1;333 ( Pt 3)(Pt 3):581-9. doi: 10.1042/bj3330581.
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.
我们研究了丝裂原活化蛋白激酶（MAPK）的三个亚家族，即应激激活蛋白激酶/c-Jun氨基末端激酶（SAPKs/JNKs）、细胞外反应激酶（ERKs）和p38-MAPK，在新生大鼠心室肌细胞原代培养物中，以过氧化氢（H2O2）为例的氧化应激作用下的激活情况。0.1 mM H2O2（最大有效浓度）分别使46 kDa和54 kDa的SAPKs/JNKs激活了5倍和10倍。最大激活发生在15 - 30分钟，但2小时后仍可检测到。ERK1和ERK2均被0.1 mM H2O2激活了16倍，其时间进程与SAPKs/JNKs相似，这与它们被1 microM佛波酯（PMA）激活的情况相当，PMA是我们目前在这些细胞中鉴定出的最强的ERK激活剂。H2O2对ERK的激活被PD98059抑制，PD98059可抑制MAPK（或ERK）激酶的激活，同时也被蛋白激酶C（PKC）抑制剂GF109203X抑制。ERK的激活也因对PMA敏感的PKC亚型的下调而受到抑制。0.1 mM H2O2使p38-MAPK激活，表现为其磷酸化增加。然而，最大磷酸化（激活）比SAPKs/JNKs或ERKs更快（<5分钟）。我们通过检测MAPK激活的蛋白激酶2（MAPKAPK2）的激活以及MAPKAPK2底物小热休克蛋白HSP25/27的磷酸化，研究了p38-MAPK激活的下游后果。与p38-MAPK一样，0.1 mM H2O2使MAPKAPK2迅速激活（5分钟内达到最大值）。10 microM SB203580（某些p38-MAPK亚型的选择性抑制剂）可消除这种激活。HSP25/27的磷酸化在MAPKAPK2激活后迅速发生，并且也被SB203580抑制。HSP25/27的磷酸化与其聚集状态的降低有关。这些数据表明氧化应激是新生大鼠心室肌细胞中所有三个MAPK亚家族的强大激活剂。所有三种MAPK的激活都与肥厚表型的发展有关。然而，p38-MAPK的刺激以及随之而来的HSP25/27的磷酸化在心脏保护中可能也很重要。
