Phosphorylation of Mitogen- and Stress-Activated Protein Kinase-1 in Astrocytic Inflammation: A Possible Role in Inhibiting Production of Inflammatory Cytokines

Peipei Gong,Xiaojian Lu,Qingfeng Huang,Jian Chen,Wei Shi,Jinlong Shi,Lanchun Ni,Dekang Nie,Liang Xia,Xide Xu,Alexander A Mongin

doi:10.1371/journal.pone.0081747

Abstract

PurposeIt is generally accepted that inflammation has a role in the progression of many central nervous system (CNS) diseases, although the mechanisms through which this occurs remain unclear. Among mitogen-activated protein kinase (MAPK) targets, mitogen- and stress-activated protein kinase (MSK1) has been thought to be involved in the pathology of inflammatory gene expression. In this study, the roles of MSK1 activation in neuroinflammation were investigated.MethodsThe bacterial lipopolysaccharide (LPS)-induced brain injury model was performed on Sprague-Dawley rats. The dynamic expression changes and the cellular location of p-MSK1 in the brain cortex were detected by Western blot and immunofluorescence staining. The synthesis of inflammatory cytokines in astrocytes was detected by enzyme-linked immunosorbent assay (ELISA).ResultsPhosphorylated MSK1 (p-MSK1 Thr-581) was induced significantly after intracerebral injection of LPS into the lateral ventricles of the rat brain. Specific upregulation of p-MSK1 in astrocytes was also observed in inflamed cerebral cortex. At 1 day after LPS stimulation, iNOS, TNFα expression, and the astrocyte marker glial fibrillary acidic protein (GFAP) were increased significantly. Also, in vitro studies indicated that the upregulation of p-MSK1 (Thr-581) may be involved in the subsequent astrocyte inflammatory process, following LPS challenge. Using an enzyme-linked immunosorbent assay (ELISA), it was confirmed that treatment with LPS in primary astrocytes stimulated the synthesis of inflammatory cytokines, through MAPKs signaling pathways. In cultured primary astrocytes, both knock-down of total MSK1 by small interfering RNAs (siRNA) or specific mutation of Thr-581 resulted in higher production of certain cytokines, such as TNFα and IL-6.ConclusionsCollectively, these results suggest that MSK1 phosphorylation is associated with the regulation of LPS-induced brain injury and possibly acts as a negative regulator of inflammation.

Highlights

Emerging evidence indicates that the inflammatory response in the brain represents a potential pathogenic factor in many central nervous system (CNS) diseases, including chronic neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), ischemic brain injury (IBI), and even traumatic brain injury (TBI) [1]
Using Western blotting, we examined p-mitogenand stress-activated protein kinase 1 (MSK1) Thr-581, glial fibrillary acidic protein (GFAP), iNOS protein levels. iNOS levels began to increase at 0.01 mg/mL and gradually peaked at 1 mg/mL of LPS
Total MSK1 expression was almost constant among the different time groups (Fig. 4E, F). These results suggest that upregulation of pMSK1Thr-581, but not total MSK1 expression, correlated with astrocyte inflammatory response after LPS stimulation

Summary

Introduction

Emerging evidence indicates that the inflammatory response in the brain represents a potential pathogenic factor in many central nervous system (CNS) diseases, including chronic neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), ischemic brain injury (IBI), and even traumatic brain injury (TBI) [1]. Despite obvious differences in morphology and functional properties, these cells are regarded as immune active cells and in some instances, they share common innate immune responses. Both astrocytes and microglial cells have been shown to respond to proinflammatory cytokines and lipopolysaccharides (LPS) in the induction of iNOS and other inflammatory factors [6,7,8]. Increasing evidence points to the potential of reactive astrogliosis to play important roles in the pathological process of neuroinflammation [8,9]

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