Microglia, the resident macrophages enriched in the brain, have essential roles in the immune surveillance of the central nervous system. Activation of Toll-like receptor 4 (TLR4), the primary transducers of innate immune system, is critical in microglial functions. Especially, activation of microglia by lipopolysaccharide (LPS), a ligand for TLR4, has been extensively studied. It was previously demonstrated that phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane lipid produced mainly by the type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family members, was necessary for TLR4 signaling. However, details of the PIP5K-mediated PIP2 production pathway and its direct regulatory effect on TLR4 signaling remain not well understood. Thus, in the present study, I have examined a potential role for PIP5K-alpha, an isoform of PIP5K, in TLR4-mediated microglia inflammation. PIP5K-alpha knockdown stable cell lines of BV2 microglia were developed using lentiviral short hairpin RNA (ShRNA) expression system. PIP5K-alpha ShRNA significantly reduced PIP5K-alpha protein and mRNA levels. PIP5K-alpha knockdown significantly suppressed LPS-induced production of inflammatory mediators, such as interleukin IL-6, IL-1beta, and nitric oxide. PIP5K-alpha knockdown also attenuated the signaling events downstream of TLR4 activation, including phosphorylation of p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and nuclear factor-kappa B (NF-kappaB) p65, and degradation of inhibitor kappaB-alpha. Consistent with these, transcriptional activity of nuclear factor-kappaB was reduced by the PIP5K-alpha knockdown. Complementation of the PIP5K-alpha knockdown cells with PIP5K-alpha effectively restored the induction of IL-6 and IL-1beta, and activation of NF-kappaB signaling pathways in response to LPS. Together, our results suggest that PIP5K-alpha-derived PIP2 generation may facilitate TLR4-dependent microglial inflammation.
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