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 (PIP2), generated by PI 4-phosphate 5-kinase (PIP5K), regulates many critical cellular events. PIP2 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 PIP2 in TLR4-dependent microglial activation remains unclear. Here, we knocked down PIP5K alpha, a PIP5K isoform, in a BV2 microglial cell line using stable expression of lentiviral shRNA constructs or siRNA transfection. PIP5K alpha knockdown significantly suppressed induction of inflammatory mediators, including IL-6, IL-1 beta, and nitric oxide, by lipopolysaccharide. PIP5K alpha knockdown also attenuated signaling events downstream of TLR4 activation, including p38 MAPK and JNK phosphorylation, NF-kappa B p65 nuclear translocation, and I kappa B-alpha degradation. Complementation of the PIP5K alpha knockdown cells with wild type but not kinase-dead PIP5K alpha effectively restored the LPS-mediated inflammatory response. Wefound that PIP5K alpha and TIRAP colocalized at the cell surface and interacted with each other, whereas kinase-dead PIP5K alpha rendered TIRAP soluble. Furthermore, in LPS-stimulated con- trol cells, plasma membrane PIP2 increased and subsequently declined, and TIRAP underwent bi-directional translocation between the membrane and cytosol, which temporally correlated with the changes in PIP2. In contrast, PIP5K alpha knockdown that reduced PIP2 levels disrupted TIRAP membrane targeting by LPS. Together, our results suggest that PIP5K alpha promotes TLR4-associated microglial inflammation by mediating PIP2-dependent recruitment of TIRAP to the plasma membrane.