title: Phosphatidylinositol 4-phosphate 5-kinase α facilitates Toll-like receptor 4-mediated microglial inflammation through regulation of the Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) location.
abstract: 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.
authors.affiliation: Chronic Inflammatory Disease Research Center, Neuroscience Graduate Program, Graduate School of Interdisciplinary Programs, Ajou University, Suwon, Gyeonggi 443-721, South Korea.
authors.forename: Tu Thi Ngoc
authors.initials: TT
authors.lastname: Nguyen
authors.forename: Yong Min
authors.initials: YM
authors.lastname: Kim
authors.forename: T Doohun
authors.initials: TD
authors.lastname: Kim
authors.forename: Oanh Thi Tu
authors.initials: OT
authors.lastname: Le
authors.forename: Jae Jin
authors.initials: JJ
authors.lastname: Kim
authors.forename: Ho Chul
authors.initials: HC
authors.lastname: Kang
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hasegawa
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Kanaho
authors.forename: Ilo
authors.initials: I
authors.lastname: Jou
authors.forename: Sang Yoon
authors.initials: SY
authors.lastname: Lee
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007249:Inflammation; D007375:Interleukin-1 / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D017628:Microglia / Q000378:metabolism; D009569:Nitric Oxide / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000502:physiology*; D017472:Receptors, Interleukin-1 / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D051197:Toll-Like Receptor 4 / Q000378:metabolism*
chemical_list: D007375:Interleukin-1; D008562:Membrane Glycoproteins; D017472:Receptors, Interleukin-1; C434984:TIRAP protein, human; C499045:TIRAP protein, mouse; D051197:Toll-Like Receptor 4; D009569:Nitric Oxide; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase