Regulatory Role of GRK2 in the TLR Signaling-Mediated iNOS Induction Pathway in Microglial Cells.

Sailesh Palikhe,Hiromi Yamazaki,Kohshi Hattori,Takuya Sakamoto,Wakana Ohashi,Yuichi Hattori,Tsugunobu Andoh,Masaaki Kawakami

doi:10.3389/fphar.2019.00059

Abstract

G protein-coupled receptor kinase 2 (GRK2) is a ubiquitous member of the GRK family that restrains cellular activation by G protein-coupled receptor (GPCR) phosphorylation leading to receptor desensitization and internalization, but has been identified to regulate a variety of signaling molecules, among which may be associated with inflammation. In this study, we attempted to establish the regulatory role of GRK2 in the Toll-like receptor (TLR) signaling pathway for inducible nitric oxide synthase (iNOS) expression in microglial cells. When mouse MG6 cells were stimulated with the TLR4 ligands lipopolysaccharide (LPS) and paclitaxel, we found that interferon regulatory factor 1 (IRF1) protein expression and activation was upregulated, transcription of interferon-β (IFN-β) was accelerated, induction/activation of STAT1 and activation of STAT3 were promoted, and subsequently iNOS expression was upregulated. The ablation of GRK2 by small interfering RNAs (siRNAs) not only eliminated TLR4-mediated upregulation of IRF1 protein expression and nuclear translocation but also suppressed the activation of the STAT pathway, resulting in negating the iNOS upregulation. The TLR3-mediated changes in IRF1 and STAT1/3, leading to iNOS induction, were also abrogated by siRNA knockdown of GRK2. Furthermore, transfection of GRK2 siRNA blocked the exogenous IFN-β supplementation-induced increases in phosphorylation of STAT1 as well as STAT3 and abrogated the augmentation of iNOS expression in the presence of exogenous IFN-β. Taken together, our results show that GRK2 regulates the activation of IRF1 as well as the activation of the STAT pathway, leading to upregulated transcription of iNOS in activated microglial cells. Modulation of the TLR signaling pathway via GRK2 in microglia may be a novel therapeutic target for treatment of neuroinflammatory disorders.

Highlights

Lipopolysaccharide (LPS), a glycolipid constituent of the outer membrane of Gram-negative bacteria, initiates inflammatory signaling cascades in cells, including monocytes, macrophages, dendritic cells, and endothelial cells, leading to the upregulation of cytokines, chemokines, and other inflammatory mediators, such as inducible nitric oxide synthase andRole of GKR2 in Toll-like receptor (TLR) Signaling cyclooxygenase-2 (COX-2)
G protein-coupled receptor kinase 2 (GRK2) small interfering RNAs (siRNAs) transfection greatly reduced the poly(I:C)-induced upregulation of mRNA levels of IL-1β, IL-6, IP-10, and IRF7 (Supplementary Figure 4). These results suggest that GRK2 serves as a key player for TLR3 signaling to produce inducible nitric oxide synthase (iNOS) in microglial cells
We examined the role of GRK2 in signal transducers and activators of transcription 1 (STAT1)/3-mediated iNOS production in microglial cells supplemented with exogenous IFN-β

Summary

Introduction

Lipopolysaccharide (LPS), a glycolipid constituent of the outer membrane of Gram-negative bacteria, initiates inflammatory signaling cascades in cells, including monocytes, macrophages, dendritic cells, and endothelial cells, leading to the upregulation of cytokines, chemokines, and other inflammatory mediators, such as inducible nitric oxide synthase (iNOS) andRole of GKR2 in TLRs Signaling cyclooxygenase-2 (COX-2). As critical pattern recognition receptors for the first line of the host defense system against bacteria, viruses, fungi, and parasites, Toll-like receptors (TLRs) are widely found on the surface of those cells and play a key role in the innate immune system (Takeda and Akira, 2003; Beutler, 2009; Kawai and Akira, 2010; Satoh and Akira, 2016) Among these TLRs, TLR4 is activated by LPS, which is primarily associated with the accessory protein MD-2 and the co-receptor CD-14 to recognize LPS, resulting in transducing signals for activation of several transcription factors, such as nuclear factorκB (NF-κB), in cooperation with myeloid differentiation factor 88 (MyD88) (Beutler, 2009). It should be noted that the canonical NF-κB pathway responds to diverse stimuli, including TLR activation, is activated with the inducible degradation of IκBα (inhibitor of κBα) triggered through its site-specific phosphorylation by multisubunit IκB kinase complex, and participates in the induction of type I interferons (IFNs) and pro-inflammatory cytokines (Shin et al, 2015; Liu et al, 2017)

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