ERK1/2 Activity Is Critical for the Outcome of Ischemic Stroke

Ischemic disorders are the leading cause of death worldwide. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are thought to affect the outcome of ischemic stroke. However, it is under debate whether activation or inhibition of ERK1/2 is beneficial. In this study, we report that the ubiquitous overexpression of wild-type ERK2 in mice (ERK2wt) is detrimental after transient occlusion of the middle cerebral artery (tMCAO), as it led to a massive increase in infarct volume and neurological deficits by increasing blood–brain barrier (BBB) leakiness, inflammation, and the number of apoptotic neurons. To compare ERK1/2 activation and inhibition side-by-side, we also used mice with ubiquitous overexpression of the Raf-kinase inhibitor protein (RKIPwt) and its phosphorylation-deficient mutant RKIPS153A, known inhibitors of the ERK1/2 signaling cascade. RKIPwt and RKIPS153A attenuated ischemia-induced damages, in particular via anti-inflammatory signaling. Taken together, our data suggest that stimulation of the Raf/MEK/ERK1/2-cascade is severely detrimental and its inhibition is rather protective. Thus, a tight control of the ERK1/2 signaling is essential for the outcome in response to ischemic stroke.

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Inhibition of Perforin-Mediated Neurotoxicity Attenuates Neurological Deficits After Ischemic Stroke

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2021.664312 ◽

2021 ◽

Vol 15 ◽

Author(s):

Yuhualei Pan ◽

Dan Tian ◽

Huan Wang ◽

Yushang Zhao ◽

Chengjie Zhang ◽

...

Keyword(s):

T Cells ◽

Ischemic Stroke ◽

Immune Cells ◽

Infarct Volume ◽

Γδ T Cells ◽

Neurological Deficits ◽

Wild Type ◽

Critical Function ◽

Ischemic Brain ◽

Potential Strategy

Perforin-mediated cytotoxicity plays a crucial role in microbial defense, tumor surveillance, and primary autoimmune disorders. However, the contribution of the cytolytic protein perforin to ischemia-induced secondary tissue damage in the brain has not been fully investigated. Here, we examined the kinetics and subpopulations of perforin-positive cells and then evaluated the direct effects of perforin-mediated cytotoxicity on outcomes after ischemic stroke. Using flow cytometry, we showed that perforin+CD45+ immune cells could be detected at 12 h and that the percentage of these cells increased largely until on day 3 and then significantly declined on day 7. Surprisingly, the percentage of Perforin+CD45+ cells also unexpectedly increased from day 7 to day 14 after ischemic stroke in Perforin1-EGFP transgenic mice. Our results suggested that Perforin+CD45+ cells play vital roles in the ischemic brain at early and late stages and further suggested that Perforin+CD45+ cells are a heterogeneous population. Surprisingly, in addition to CD8+ T cells, NK cells, and NKT cells, central nervous system (CNS)-resident immune microglia, which are first triggered and activated within minutes after ischemic stroke in mice, also secreted perforin during ischemic brain injury. In our study, the percentage of perforin+ microglia increased from 12 h after ischemic stroke, increased largely until on day 3 after ischemic stroke, and then moderately declined from days 3 to 7. Intriguingly, the percentage of perforin+ microglia also dramatically increased from days 7 to 14 after ischemic stroke. Furthermore, compared with wild-type littermates, Perforin 1–/– mice exhibited significant increases in the cerebral infarct volume, neurological deficits, and neurogenesis and inhibition of neurotoxic astrogliosis. Interestingly, the number of CD45+CD3+ T cells was significantly decreased in Perforin 1–/– mice compared with their wild-type littermates, especially the number of γδ T cells. In addition, Perforin 1–/– mice had lower levels of IL-17 than their wild-type littermates. Our results identified a critical function of perforin-mediated neurotoxicity in the ischemic brain, suggesting that targeting perforin-mediated neurotoxicity in brain-resident microglia and invading perforin+CD45+ immune cells may be a potential strategy for the treatment of ischemic stroke.

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Mechanism of Suppression of the Raf/MEK/Extracellular Signal-Regulated Kinase Pathway by the Raf Kinase Inhibitor Protein

Molecular and Cellular Biology ◽

10.1128/mcb.20.9.3079-3085.2000 ◽

2000 ◽

Vol 20 (9) ◽

pp. 3079-3085 ◽

Cited By ~ 252

Author(s):

Kam Yeung ◽

Petra Janosch ◽

Brian McFerran ◽

David W. Rose ◽

Harald Mischak ◽

...

Keyword(s):

Kinase Inhibitor ◽

Competitive Inhibitor ◽

Erk Pathway ◽

Inhibitor Protein ◽

Extracellular Signal Regulated Kinase ◽

Raf Kinase ◽

Binding Domains ◽

Inhibitory Function ◽

Raf Kinase Inhibitor Protein ◽

Extracellular Signal

ABSTRACT We have recently identified the Raf kinase inhibitor protein (RKIP) as a physiological endogenous inhibitor of the Raf-1/MEK/extracellular signal-regulated kinase (ERK) pathway. RKIP interfered with MEK phosphorylation and activation by Raf-1, resulting in the suppression of both Raf-1-induced transformation and AP-1-dependent transcription. Here we report the molecular mechanism of RKIP's inhibitory function. RKIP can form ternary complexes with Raf-1, MEK, and ERK. However, whereas MEK and ERK can simultaneously associate with RKIP, Raf-1 binding to RKIP and that of MEK are mutually exclusive. RKIP is able to dissociate a Raf-1–MEK complex and behaves as a competitive inhibitor of MEK phosphorylation. Mapping of the binding domains showed that MEK and Raf-1 bind to overlapping sites in RKIP, whereas MEK and RKIP associate with different domains in Raf-1, and Raf-1 and RKIP bind to different sites in MEK. Both the Raf-1 and the MEK binding sites in RKIP need to be destroyed in order to relieve RKIP-mediated suppression of the Raf-1/MEK/ERK pathway, indicating that binding of either Raf-1 or MEK is sufficient for inhibition. The properties of RKIP reveal the specific sequestration of interacting components as a novel motif in the cell's repertoire for the regulation of signaling pathways.

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Reduction in Raf Kinase Inhibitor Protein Expression Is Associated with Increased Ras-Extracellular Signal-Regulated Kinase Signaling in Melanoma Cell Lines

Cancer Research ◽

10.1158/0008-5472.can-03-3861 ◽

2004 ◽

Vol 64 (15) ◽

pp. 5186-5192 ◽

Cited By ~ 133

Author(s):

Marion M. Schuierer ◽

Frauke Bataille ◽

Suzanne Hagan ◽

Walter Kolch ◽

Anja-Katrin Bosserhoff

Keyword(s):

Protein Expression ◽

Cell Lines ◽

Melanoma Cell ◽

Kinase Inhibitor ◽

Inhibitor Protein ◽

Extracellular Signal Regulated Kinase ◽

Raf Kinase ◽

Raf Kinase Inhibitor Protein ◽

Extracellular Signal ◽

Melanoma Cell Lines

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Loss of Gucy1a3 Causes Poor Post-Stroke Recovery by Reducing Angiogenesis Via the HIF-1α/VEGFA Signaling Pathway in Mice

10.21203/rs.3.rs-1220217/v1 ◽

2022 ◽

Author(s):

Man Luo ◽

Dongcan Mo ◽

LiuYu Liu ◽

Jianli Li ◽

Jing Lin ◽

...

Keyword(s):

Ischemic Stroke ◽

Signaling Pathway ◽

Infarct Volume ◽

Hypoxia Inducible Factor ◽

Soluble Guanylyl Cyclase ◽

Stroke Recovery ◽

Neurological Deficits ◽

Wild Type ◽

Expression Levels ◽

Post Stroke

Abstract Ischemic stroke is a common and debilitating disease that can cause permanent neurological damage. Gucy1a3, which encodes the α1 subunit of soluble guanylyl cyclase, has been reported to be associated with functional recovery after ischemic stroke. However, the mechanism is still not well understood. In the present study, we investigated the effects of Gucy1a3 on (i) post-stroke recovery; (ii) vascular endothelial growth factor A (VEGFA) and hypoxia inducible factor 1 alpha (HIF-1α) expression; and (iii) angiogenesis after ischemic stroke. A permanent middle cerebral artery occlusion (pMCAO) model was established using wild-type and Gucy1a3 knockout C57BL/6J male mice. Neurological deficits, infarct volume, microvascular density, and VEGFA and HIF-1α expression levels of mice were evaluated. Our results suggest that loss of Gucy1a3 increased the infarct volume and aggravated neurological deficits after pMCAO. In addition, the Gucy1a3 knockout brains exhibited significantly lower microvessel densities and VEGFA and HIF-1α expression levels than the wild-type brains at 96 hours post-pMCAO. The study shows that the expression of GUCY1A3 after ischemic stroke may play a substantial role in neurological function recovery and is related to angiogenesis in the peri-infarct region. The beneficial effects of GUCY1A3 might be mediated through the HIF-1α/VEGFA signaling pathway.

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