Chronic Inflammatory Disease Research Center, Neuroscience Graduate Program, Graduate School of Interdisciplinary Programs, Ajou University, Suwon, Gyeonggi 443-721, South Korea.
J Biol Chem. 2013 Feb 22;288(8):5645-59. doi: 10.1074/jbc.M112.410126. Epub 2013 Jan 7.
Phosphatidylinositol (PI) 4,5-bisphosphate (PIP(2)), generated by PI 4-phosphate 5-kinase (PIP5K), regulates many critical cellular events. PIP(2) is also known to mediate plasma membrane localization of the Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), required for the MyD88-dependent Toll-like receptor (TLR) 4 signaling pathway. Microglia are the primary immune competent cells in brain tissue, and TLR4 is important for microglial activation. However, a functional role for PIP5K and PIP(2) in TLR4-dependent microglial activation remains unclear. Here, we knocked down PIP5Kα, a PIP5K isoform, in a BV2 microglial cell line using stable expression of lentiviral shRNA constructs or siRNA transfection. PIP5Kα knockdown significantly suppressed induction of inflammatory mediators, including IL-6, IL-1β, and nitric oxide, by lipopolysaccharide. PIP5Kα knockdown also attenuated signaling events downstream of TLR4 activation, including p38 MAPK and JNK phosphorylation, NF-κB p65 nuclear translocation, and IκB-α degradation. Complementation of the PIP5Kα knockdown cells with wild type but not kinase-dead PIP5Kα effectively restored the LPS-mediated inflammatory response. We found that PIP5Kα and TIRAP colocalized at the cell surface and interacted with each other, whereas kinase-dead PIP5Kα rendered TIRAP soluble. Furthermore, in LPS-stimulated control cells, plasma membrane PIP(2) increased and subsequently declined, and TIRAP underwent bi-directional translocation between the membrane and cytosol, which temporally correlated with the changes in PIP(2). In contrast, PIP5Kα knockdown that reduced PIP(2) levels disrupted TIRAP membrane targeting by LPS. Together, our results suggest that PIP5Kα promotes TLR4-associated microglial inflammation by mediating PIP(2)-dependent recruitment of TIRAP to the plasma membrane.
磷脂酰肌醇（PI）4,5-二磷酸（PIP(2)）由 PI 4-磷酸 5-激酶（PIP5K）生成，调节许多关键的细胞事件。PIP(2)也已知介导 Toll/IL-1 受体域包含衔接蛋白（TIRAP）的质膜定位，该蛋白是 MyD88 依赖的 Toll 样受体（TLR）4 信号通路所必需的。小胶质细胞是脑组织中主要的免疫活性细胞，TLR4 对于小胶质细胞的激活很重要。然而，PIP5K 和 PIP(2)在 TLR4 依赖的小胶质细胞激活中的功能作用仍不清楚。在这里，我们使用稳定表达的慢病毒 shRNA 构建体或 siRNA 转染，在 BV2 小胶质细胞系中敲低 PIP5Kα，一种 PIP5K 同工型。PIP5Kα 的敲低显著抑制了脂多糖诱导的炎性介质的诱导，包括 IL-6、IL-1β 和一氧化氮。PIP5Kα 的敲低也减弱了 TLR4 激活下游的信号事件，包括 p38 MAPK 和 JNK 磷酸化、NF-κB p65 核易位和 IκB-α 降解。用野生型而非激酶失活型 PIP5Kα 补充 PIP5Kα 敲低细胞有效地恢复了 LPS 介导的炎症反应。我们发现 PIP5Kα 和 TIRAP 在细胞表面共定位并相互作用，而激酶失活型 PIP5Kα 使 TIRAP 可溶。此外，在 LPS 刺激的对照细胞中，质膜 PIP(2)增加，随后减少，TIRAP 在质膜和细胞质之间双向易位，这与 PIP(2)的变化时间相关。相比之下，降低 PIP(2)水平的 PIP5Kα 敲低破坏了 LPS 对 TIRAP 质膜靶向的作用。总之，我们的结果表明，PIP5Kα 通过介导 PIP(2)依赖的 TIRAP 向质膜募集，促进 TLR4 相关的小胶质细胞炎症。
