Phosphorylation Of Mitogen-and Stress-activated Protein Kinase-1in Astrocytic Inflammation:a Possible Role In Inhibiting Production Of Inflammatory Cytokines

Objective It 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 MSK1activation in neuroinflammation were investigated.Methods The model of brain inflammation was prepared in rats by the intracerebroventricular injection of LPS. The expression and the cellular location of p-MSK1protein level were assayed by Western Blot and immunofluorescent staining. In cell culture, primary rat cerebral cortex astrocytes were prepared from7-day-old Sprague-Dawley rats. Cultured astrocytes were stimulated by LPS. Expression of p-MSKl, astrocyte-specific glial fibrillary acidic protein (GFAP), iNOS and TNF a were detected by Western Blot analysis, and the secretion of TNF-α, IL1-β and IL-6was detected by enzyme-linked immunosorbent assay (ELISA). Furthermore, we employed siRNA or specific mutation of Thr-581plasmid to knock down the expression of MSK1or its phosphorylation site in astrocytes, then we detected the expression of these cytokines by ELISA assay. Results Phosphorylated MSK1(p-MSK1Thr-581) was induced significantly after intracerebral injection of LPS into the lateral ventricles of the rat brain. Specific upregulation of p-MSK1in astrocytes was also observed in inflamed cerebral cortex. At1day after LPS stimulation, iNOS, TNFa expression, and the astrocyte marker glial fibrillary acidic protein (GFAP) were increased significantly. Also, in vitro studies indicated that the upregulation of p-MSK1may be involved in the subsequent astrocyte inflammatory process, following LPS challenge. Using an 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 MSK1by small interfering RNAs (siRNA) or specific mutation of Thr-581resulted in higher production of certain cytokines, such as TNFa and IL-6.Conclusion Collectively, these results suggest that MSK1phosphorylation is associated with the regulation of LPS-induced brain injury and possibly acts as a negative regulator of inflammation.