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

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.

G Protein-Coupled Receptor Kinase 2 as Novel Therapeutic Target in Fibrotic Diseases

“…GRK2 has been identified to regulate a variety of pro‐inflammatory secretions. The high expression of nitric oxide synthase (iNOS) in microglial cells is caused by GRK2, which significantly enhances the phosphorylation levels of STAT1 and STAT3 through regulating the toll like receptor ( 53 ). The upregulation of iNOS could promote the secretion of inflammatory cytokines and development of many fibrous diseases ( 54 ).…”

Serum-borne lipids amplify TLR-activated inflammatory responses

“…Palikhe et al. have shown that GPCR kinase‐2 regulates transcription of iNOS in microglial cells by regulating activation of IRF‐1 and the STAT pathway 36 . Our results suggest that GPCRs including G2A that sense circulating lipids such as LPC might play a vital role in amplifying TLR‐mediated inflammatory and innate immune responses.…”

“…34,35 Palikhe et al have shown that GPCR kinase-2 regulates transcription of iNOS in microglial cells by regulating activation of IRF-1 and the STAT pathway. 36 Our results suggest that GPCRs including G2A that sense circulating lipids such as LPC might play a vital role in amplifying TLR-mediated inflammatory and innate immune responses. These findings have significant implications not only for inflammatory responses produced during infections but these are also relevant to chronic inflammation as the presence of circulating TLR agonists such as LPS has been suggested to be a critical factor in the induction of inflammatory response in several experimental models of chronic inflammatory disorders.…”

CP‐25 exerts therapeutic effects in mice with dextran sodium sulfate‐induced colitis by inhibiting GRK2 translocation to downregulate the TLR4‐NF‐κB‐NLRP3 inflammasome signaling pathway in macrophages

“…The current study highlights the important role of GRK2 in mediating colitis and suggests that GRK2 might be a potential drug target for IBD. A previous study demonstrated that GRK2 played a direct role in regulating TLR4‐mediated inflammatory signaling in microglia cells 56 . TLR4 is the main pattern recognition receptor expressed on the cell surface of macrophages 57 .…”

“…A previous study demonstrated that GRK2 played a direct role in regulating TLR4-mediated inflammatory signaling in microglia cells. 56 TLR4 is the main pattern recognition receptor expressed on the cell surface of macrophages. 57 Membrane localization of GRK2 increases in inflammatory macrophages, and GRK2 has been shown to promote the polarization of macrophage phenotypes to antiinflammatory macrophages.…”