β-Arrestin-2-ERK1/2 cPLA₂α axis mediates TLR4 signaling to influence eicosanoid induction in ischemic brain

LPS has been shown to elicit neuroinflammation associated with the up-regulation of the eicosanoid pathway in animal models; however, the regulatory mechanisms of TLR4 in brain neuroinflammatory conditions remain elusive. β-Arrestins are key regulators of the GPCR signaling pathway and are involved in the leukotriene B4-induced leukocyte migration to initiate inflammatory response. However, the roles of β-arrestins in eicosanoid regulation and related diseases are not clear. To address this issue, we conducted a study to investigate the effect of TLR4 on the eicosanoid pathway in ischemic stroke brain and to explore the underlying molecular regulation mechanism. Cerebral ischemia was produced by occlusion of the middle cerebral artery, followed by reperfusion for 24 h. We demonstrated that knockout of TLR4 improves ischemic stroke brain associated with eicosanoid down-regulation. Interestingly, genetic disruption of β-arrestin-2 failed to decrease neuroinflammation in the damaged brain of TLR4^-/- mice, which indicates the requirement of β-arrestin-2 for TLR4 knockdown protection. Further study showed that the negative regulation of phosphorylated (phospho-)ERK1/2 and phospho-cytosolic phospholipase A₂ α (cPLA₂α) by TLR4 deficiency was eliminated by genetic disruption of β-arrestin-2. In addition, β-arrestin-2 deficiency reversed the reduction of colocalization of phospho-ERK1/2 with phospho-cPLA₂α in TLR4^-/- mice following ischemic stroke. Mechanistic studies indicated that β-arrestin-2 specifically colocalized and associated with ERK1/2 to prevent ERK1/2-dependent cPLA₂α activation following ischemic injury, and β-arrestin-2 deficiency blocked the negative regulation of phospho-ERK1/2, revived the association of phospho-ERK1/2 with phospho-cPLA₂α, and subsequently increased the prostaglandin E2 and thromboxane A2 production remarkably. Our findings may provide novel insights that β-arrestin-2 is responsible for ischemic brain improvement in TLR4^-/- mice via negative regulation of eicosanoid production.-Xiang, Y., Wei, X., Du, P., Zhao, H., Liu, A., Chen, Y. β-Arrestin-2-ERK1/2 cPLA₂α axis mediates TLR4 signaling to influence eicosanoid induction in ischemic brain.