1. PIP5Kα expression was increased in LPS-stimulated BV2 microglial cells. 17
2. Attenuated NF-κB activation and IκB-α degradation in PIP5Kα-knockdown BV2 cells. 19
3. PIP5Kα knockdown attenuated cytokine production in BV2 cells 24
4. Signaling pathway in which PIP5Kα regulated cytokine productions 26
5. Rescue of PIP5Kα expression increased cytokine production in PIP5Kα-knockdown cells. 28
6. Overexpression of PIP5Kα rescued the phosphorylation of signaling protein 30
7. PIP5Kα interacted with TIRAP to regulate cytokine production 32
IV. DISCUSSION 35
V. CONCLUSION 38
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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