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*

Yasunori Kanaho,Tu Thi Ngoc Nguyen,Sang Yoon Lee,Hiroshi Hasegawa,T Doohun Kim,Yong Min Kim,Ho Chul Kang,Oanh Thi Tu Le,Jae Jin Kim,Ilo Jou

doi:10.1074/jbc.m112.410126

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.

Highlights

Phosphoinositides are involved in regulating TLR4 signaling
We evaluated relative abundance of the type I PI 4-phosphate 5-kinase (PIP5K)␣, PIP5K␤, and PIP5K␥ expressed in BV2 microglial cells according to a quantitative real time PCR (qRT-PCR) standard curve method using known amounts of respective plasmid DNA templates and primer sets (Fig. 1A)
As a first step to investigate whether PIP5K␣ might play a role in microglial TLR4 activation, we developed stable PIP5K␣ KD cell lines of BV2 microglia

Summary

Introduction

Results: PIP5K␣ knockdown in BV2 microglial cells inhibits LPS-induced inflammatory responses, PIP2 increase, and TIRAP translocation to the plasma membrane. Conclusion: PIP5K␣-derived PIP2 facilitates TLR4-mediated microglial inflammatory responses through recruitment of TIRAP to the plasma membrane. Significance: Regulation of PIP5K␣-dependent PIP2 pool may modulate TLR4-associated immune function in microglia. PIP2 is known to mediate plasma membrane localization of the Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), required for the MyD88-dependent Tolllike receptor (TLR) 4 signaling pathway. A functional role for PIP5K and PIP2 in TLR4-dependent microglial activation remains unclear. PIP5K␣ knockdown attenuated signaling events downstream of TLR4 activation, including p38 MAPK and JNK phosphorylation, NF-␬B p65 nuclear translocation, and I␬B-␣ degradation. Complementation of the PIP5K␣ knockdown cells with wild type but not kinase-dead PIP5K␣ effectively restored the LPS-mediated inflammatory response.

Objectives

Results

Conclusion