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*

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. Background: Phosphoinositides are involved in regulating TLR4 signaling. Results: PIP5Kα knockdown in BV2 microglial cells inhibits LPS-induced inflammatory responses, PIP 2 increase, and TIRAP translocation to the plasma membrane. Conclusion: PIP5Kα-derived PIP 2 facilitates TLR4-mediated microglial inflammatory responses through recruitment of TIRAP to the plasma membrane. Significance: Regulation of PIP5Kα-dependent PIP 2 pool may modulate TLR4-associated immune function in microglia.