In this study we confirmed that P-ERK1/2 levels transiently increase after reperfusion [23
], and the ERK1/2 inhibitor, U0126, given at the onset of stroke, reduces infarction by decreasing P-ERK1/2 levels [12
], suggesting a detrimental role of ERK1/2 activity in stroke. In addition, we found that the δPKC inhibitor, δV1-1, delivered at the onset of reperfusion tended to enhance the protective effect of U0126, although it increased P-ERK1/2 levels. Thus, the protective effect of δv1-1 was not achieved by reducing P-ERK1/2 levels. Conversely, treatment with the δPKC activator, ψδRACK, which decreased ERK phosphorylation, partly abolished the protection induced by U0126, a MEK inhibitor. These data suggest that the detrimental effect of δPKC activation by ψδRACK is independent of the ERK1/2 activity and phosphorylation.
P-ERK1/2 is implicated in ischemic damage or neuronal survival after stroke. Previous studies generally agree that P-ERK1/2 transiently increases after reperfusion in both global and focal ischemia [10
]. Our current study is consistent with these previous reports [11
]; we showed that P-ERK1/2 levels transiently increased from 1 to 4 h after reperfusion, and then returned to baseline at 24 and 48 h.
Whether increases in P-ERK1/2 levels are detrimental or protective is controversial, as an increase in P-ERK1/2 after stroke is linked with both detrimental [12
] and protective effects [22
]. Several lines of evidence support the protective effects of P-ERK1/2. First, P-ERK1/2 is expressed in the ischemic penumbra after focal ischemia [8
]. Second, P-ERK1/2 is expressed in the ischemic resistant region of dentate gyrus in the hippocampus after transient global ischemia [10
]. Third, P-ERK1/2 is enhanced by a number of neuro-protectants, including various growth factors; in addition, inhibition of the blood brain barrier permeability augments P-ERK1/2 [16
]. Alternatively, some previous studies also strongly support the detrimental effects of ERK1/2 activity. For instance, ERK1/2 inhibition reduces ischemic damage [12
] and P-ERK1/2 levels are increased by free radical products [23
], inflammatory response [20
] and hyperglycemia [7
], which exacerbate ischemic damage.
Neverthless, in the current study we found that injection of the ERK1/2 inhibitor, U0126, at ischemia onset robustly reduced infarct size while it had no effect when injected at reperfusion, suggesting a limited therapeutic time window. The underlying mechanisms responsible for such difference are not known, but our pilot study showed that ERK1/2 phosphorylation was increased as early as 10 min after ischemia onset (data not shown), indicating that ERK activity might have been triggered before reperfusion. U0126 delivered at reperfusion, thus, might not be able to attenuate infarct size as long as ERK activity started, which, however, needs further study.
Our current study using the δPKC inhibitor and agonist reinforces the complicated role of ERK1/2 after stroke. The interplay between δPKC and ERK/2 has been studied in the non-neuronal systems, in which δPKC activity increases P-ERK1/2 [1
]. Thus, one would expect that activating δPKC would increase P-ERK1/2 and inhibiting δPKC would decrease P-ERK1/2. Surprisingly, the δPKC activator, ψδRACK, which increases δPKC activity, actually attenuated protein levels of P-ERK1/2, and inhibiting δPKC did not block increases in P-ERK1/2 in our study. Thus, our results are not consistent with previous reports regarding interaction between δPKC activity and P-ERK1/2 levels [18
]. The ERK1/2 inhibitor, U0126, reduced infarct size and protein levels of P-ERK1/2, suggesting that reduction in P-ERK1/2 may be necessary for the protective effect of U0126. However, the δPKC inhibitor δV1-1, which we previously reported to block ischemic damage [4
], did not reduce P-ERK1/2 levels. Furthermore, the δPKC activator, ψδRACK, inhibited P-ERK1/2 levels, yet it abolished the protective effect of the ERK1/2 inhibitor, U0126. Thus, protein levels of P-ERK1/2 itself may not be as critical for determining the protective or detrimental effect of ERK1/2; some other factors may be responsible for the effects of ERK1/2. It is well known that stroke causes changes in a myriad of genes and proteins [15
]. Although U0126 is an ERK1/2 inhibitor, it may also affect many other proteins simultaneously. Likewise, P-ERK1/2 is not the only protein altered by the δPKC activator or inhibitor. Thus, P-ERK1/2 appears to have a complex role in mediating injury or survival of brain after stroke.
In conclusion, we demonstrated for the first time that inhibition of both ERK1/2 and the δPKC pathways offers greater protection than does inhibition of either alone, suggesting they act in parallel. Our results imply that the protein level of P-ERK1/2 is not a simple marker for cerebral neuroprotection or worsened injury after stroke.