Elsevier

Brain Research

Volume 996, Issue 1, 16 January 2004, Pages 55-66
Brain Research

Research report
Inhibition of MEK/ERK 1/2 pathway reduces pro-inflammatory cytokine interleukin-1 expression in focal cerebral ischemia

https://doi.org/10.1016/j.brainres.2003.09.074Get rights and content

Abstract

It has been proposed that mitogen-activated protein kinase (MAPK) pathways may play a role in the regulation of pro-inflammatory cytokines, such as interlukine-1, during cerebral ischemia. Our previous study showed that extracellular-signal-regulated kinases 1 and 2 (ERK 1/2) were activated during focal cerebral ischemia in mice [J. Cereb. Blood Flow Metab. 20 (2000) 1320]. However, the effect of ERK 1/2 activation in focal cerebral ischemia is still unclear. In this study we reported that in vivo phospho-ERK 1/2 expression increased following 30 min of middle cerebral artery occlusion (MCAO) in the mouse brain in both the ischemic core and perifocal regions. Western blot analysis and immunohistochemistry demonstrated that pro-treatment with 1,4-diamino-2,3-dicyano-1,4-bis butadiene (U0126) [J. Biol. Chem. 273 (1998) 18623] could significantly inhibit mouse brain phospho-MEK 1/2 and phospho-ERK 1/2 expression after 1–2 h of MCAO (p<0.05). Compared to the control group of mice, brain infarct volume was significantly decreased after 24 h of MCAO in the U0126-treated mice (27±6 vs. 46±9 mm2, p<0.05). Inhibition of the MEK/ERK 1/2 pathway also prevented downstream kinase Elk-1 phosphorylation, and further reduced cytokine IL-1β mRNA, but not TNFα, IL-1α, or chemokine MIP-1α mRNA expression. Our data demonstrates that in vivo the close linking of MEK 1/2, ERK 1/2, Elk-1, and IL-1 mRNA expression in the cerebral ischemia animals suggests that ERK 1/2 pathway activation is important in pro-inflammatory cytokine IL-1β signaling, which induces an inflammatory response and exacerbates ischemic brain injury. Inhibiting the ERK 1/2 pathway may therefore provide a novel approach for the reduction of ischemia-induced IL-1β overexpression.

Introduction

Mitogen-activated protein kinase (MAPK) pathways have been extensively studied in the past few years. These pathways play a crucial role in the transduction of signals through both protein kinases and protein phosphatases [7]. The most extensively characterized MAPK pathways are extracellular signal-regulated kinase 1 and 2 (ERK 1/2). These serine/threonine kinases are unusual in requiring phosphorylation on both tyrosine and threonine residues within the signature sequence T-E-Y (Yhr-183 and Yhr-185 in mammalian ERK2) for activity [17]. The ERK 1/2 pathway contains important mediators of the signal transduction responsible for cell growth and proliferation [26]. Several in vitro studies have shown MAP kinases to be potentially important signaling events in the inflammatory response [24].

The relationship between the MAPK pathway activation and ischemic injury have been studied since the mid-1990s. Walton et al. [30] reported the effect of cerebral ischemia on MAPK pathway activation. Previously, we demonstrated that MAPKs, including phospho-ERK 1/2, p38, and JNK expression, were greatly increased after permanent middle cerebral artery occlusion (MCAO) in the mouse brain [32]. However, how signal transduction relates to the entry of leukocytes into the ischemic area has not yet been elucidated. Cytokines usually act as intracellular messengers because they are low molecular weight glycoproteins. Aikawa et al. [1] found in a cultured cardiac myocytes study that MAPK signals relate to cellular apoptosis. They demonstrated that ERK 1/2 pathway activation could play a role in protecting cells from apoptotic death. Alessandrini et al. [2] demonstrated that inhibition of MEK activation using PD98059 could reduce infarct volume and neurological deficit after focal ischemia in mice. These signal transduction mechanisms may contribute to structural rearrangement in the brain infarct region. Nevertheless, the role of the ERK 1/2 pathway during cerebral ischemia has not been well identified. The aim of this study is to demonstrate whether the ERK1/2 signal pathway is involved in ischemic brain injury, and if so, whether inhibiting ERK 1/2 pathway will diminish cytokine expression and will reduce ischemic brain injury. We examined in vivo phospho-MEK 1/2, phospho-ERK 1/2, and phospho-Elk-1 expression. Using 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), we further examined whether inhibiting the MEK/ERK pathway diminishes cytokine expression, reducing ischemic brain injury. We anticipate identifying the role that the MEK/ERK 1/2 pathway plays during cerebral ischemia in mice.

Section snippets

Mouse focal cerebral ischemia model

Procedures using laboratory animals were approved by the institutional animal care and use committee. One hundred eight adult male CD-1 mice (Charles River, Wilmington, MA) weighing 25–35 g were anesthetized with 1.5% isoflorane in 70% N2/30% O2 mixed gases through a facemask. A PE-10 catheter was introduced into a femoral artery for continuous monitoring of arterial blood pressure, and sampling for blood gas and pH analysis.

After anesthesia, mice were subjected to left MCAO using our

Results

Surface CBF was measured before and after MCAO to verify that MCAO was successful and constant in the U0126-treated and the control groups of mice. Surface CBF was reduced to 10–20% of baseline CBF in the ipsilateral hemisphere and maintained at ∼100% of baseline CBF in the contralateral hemisphere in all groups of mice. There was no statistical significance among groups (data not shown). The body temperature averaged 37±0.5 °C during experiments in both the U0126-treated and the control

Discussion

Our previous study showed phospho-ERK 1/2 protein expression was greatly increased after focal cerebral ischemia in mice [32]. Double-labeled fluorescent staining demonstrated that phospho-ERK 1/2 was not only expressed in neurons, but also in astrocytes. In this in vivo ischemia study, we have shown that (1) systemic administration of U0126 reduces ischemic brain injury, which is related to inhibition of brain phospho-MEK and phospho-ERK 1/2 expression. (2) Inhibition of MEK/ERK activation

Acknowledgements

These studies were supported by NIH grant #NS35089 (GYY). The authors thank Carroll Schreibman and Jenny Li for editorial assistance and the collaborative support of the staff of the Center for Cerebrovascular Research (http://avm.ucsf.edu/).

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  • Cited by (0)

    1

    Present address: Institute of Neurology, Hua Shan Hospital School of Medicine, Fudan University, Shanghai 200040, PR China.

    2

    Present address: Center for Cerebrovascular Research, Departments of Anesthesia and Neurosurgery, University of California, San Francisco, CA 94110, USA.

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