Abstract T P206: Mkp-1 Inhibition Increases Stroke Injury via JNK Pathway Activation

Abstract

Background: MKP-1, a negative modulator of several MAP kinases including JNK and p38, is a critical regulator of inflammation and apoptosis in the CNS. Inhibition of MKP-1 worsens stroke outcome in mice, suggesting that MKP-1 is an endogenous protective molecule, although the underlying mechanisms remain unclear. Here we investigated the downstream mediators of MKP-1’s effects in stroke. Methods: Stroke was induced by 60 min transient middle cerebral artery occlusion (tMCAO) in male mice. Levels of p-JNK and p-p38 in MKP-1 KO mice and WT littermates 6 hours after stroke were assessed by Western blots. In a second cohort, the selective JNK inhibitor (SP600125) was administered intraperitoneally to both MKP-1 KO and WT mice 30 min before stroke then infarcts were analyzed at 72 hours post ischemia. In a third cohort, WT mice were intraperitoneally administrated the inhibitor or vehicle 30 min before stroke and sacrificed at 6 hours for protein analysis. Results: MKP-1 deletion produced significantly higher levels of p-JNK 6 hours after stroke (0.71±0.12 WT vs. 1.32± 0.14 KO, p<0.05, n= 3 p/g); this was not seen for p-p38 (1.06± 0.19 WT vs. 1.30± 0.34 KO, n=3 WT, 2 KO). Furthermore, MKP-1 deletion significantly increased expression of JNK pathway related molecules such as p-c-jun/c-jun (0.57± 0.17 WT vs. 3.65±1.19 KO, p<0.05, n=3 p/g) and cleaved-caspase-3 (0.62 ± 0.09 WT vs. 1.81 ± 0.32 KO, p<0.05, n=3 p/g), with no changes in cleaved-caspase-8 (1.11 ± 0.09 WT vs. 1.02 ± 0.04 KO, p>0.05, n=3 p/g). There was no additive deleterious effects of pharmacological JNK inhibition in MKP-1 KO mice as measured by infarcts at 72 hours (cortex: 25.84±2.44%WT vs. 22.32±1.91%KO, striatum: 33.10±3.61% WT vs. 26.52±1.92% KO, total: 27.85±2.95% WT vs. 26.75±1.61% KO, p>0.05, n=6 p/g). In WT mice, JNK inhibition significantly decreased p-JNK (0.99 ± 0.14 vehicle vs. 0.44 ± 0.13 drug, p<0.05, n=3 p/g) 6 hours after stroke but had no effects on MKP-1 (2.36 ± 0.34 vehicle vs. 3.43 ± 0.45 drug, p>0.05, n=3 p/g). Conclusions: JNK is an important downstream mediator of MKP-1’s effects in stroke. In the ischemic brain, activating MKP-1 may restrain JNK activity, thereby producing neuroprotective effects.