TOLL-LIKE RECEPTOR 4 COUPLED GI PROTEIN SIGNALING PATHWAYS REGULATE EXTRACELLULAR SIGNAL-REGULATED KINASE PHOSPHORYLATION AND AP-1 ACTIVATION INDEPENDENT OF NFκB ACTIVATION

Abstract

Previous studies have implicated heterotrimeric Gi proteins in signaling leading to inflammatory mediator production induced by lipopolysaccharide (LPS). TLR4 has recently been shown to play a central role in response to LPS activation. We hypothesized that Gi proteins are coupled to TLR4 activation of signaling pathways. To inhibit Gi protein function, human embryonic kidney (HEK) 293 cells or RAW 264.7 cells were pretreated with pertussis toxin (PTx), an inhibitor of receptor-Galphai interaction, or transfected with dominant negative Galphai3 (Galphai3dn) or Galphai2 minigene (an inhibitory carboxyl terminus of Galphai2) plasmid. The cells were subsequently transfected with constitutively active TLR4 (TLR4ca) plasmid or TLR4ca together with an NFkappaB or AP-1 reporter construct. TLR4ca transfection induced ERK 1/2 activation (157 +/- 14%, P < 0.01), AP-1 activation (4.0 +/- 0.2-fold, P < 0.01), and NFkappaB activation (8.1 +/- 0.4-fold, P < 0.01) compared with empty vector controls. Pretreatment with PTx inhibited TLR4ca-induced ERK 1/2 phosphorylation (30 +/- 7%, P < 0.05) and AP-1 activation (36 +/- 3%, P < 0.05) but did not inhibit NFkappaB activation. Cotransfection of TLR4ca with Galphai3dn or Galphai2 minigene also reduced TLR4ca-induced ERK 1/2 phosphorylation (34 +/- 10% and 33 +/- 5%, respectively, P < 0.05). Constitutively active Galphai2 and Galphai3 plasmids potentiated TLR4ca-induced ERK 1/2 phosphorylation (27 +/- 3% and 41 +/- 6%, respectively, P < 0.05). betaARK-ct plasmid, which inhibits the function of betagamma subunit of G protein, has no effect on TLR4ca-induced ERK 1/2 phosphorylation. These data support our hypothesis and provide the first evidence that Galphai-coupled signaling pathways are activated by TLR4. The TLR4-activated Galphai signaling pathway activates ERK 1/2 phosphorylation and AP-1 activation independently of TLR4-mediated signaling to NFkappaB activation.

Signaling through Gαi2 protein is required for recruitment of neutrophils for antibody-mediated elimination of larvalStrongyloides stercoralisin mice

“…This is in contrast to neutrophils recovered from TLR4-deficient mice, which were able to migrate to the parasite but were unable to kill the worms [24]. Although TLR4 and Gαi are linked in their activation and function [23,38], it can be concluded from the present study that Gαi2 protein is not required for TLR4-dependent neutrophil killing of the larvae. Neutrophils from TLR4-deficient mice cannot kill larvae [24], although they migrate to the site, whereas neutrophils from Gαi2-deficient mice can kill the larvae as efficiently as wild-type cells only if they are transported experimentally to the site of infection.…”

“…CXCR2 −/− mice have a defect in neutrophil recruitment and thus, a deficiency in the protective innate and adaptive immune response to larval S. stercoralis in mice [21]. Further study has shown that TLR4, which is linked to activation of Gαi-coupled signaling pathways [23], is required for activation of neutrophils [24]. Mice deficient in TLR4 fail to eliminate S. stercoralis infection, despite developing T and B cell immune responses and recruiting neutrophils to the parasite microenvironment [24].…”

Regulation of IL-1 and TNF Receptor Expression and Function by Endogenous Macrophage Migration Inhibitory Factor

“…Binding of TNF to the p55 TNFR leads to activation of a multiprotein signaling complex with release of silencer of death domains, recruitment of TNFR-associated death domain protein, and the adaptor proteins TNFR-associated factor 2 (TRAF2) and receptor-interacting protein, which ultimately activate MAPK and NF-B pathways involved in inflammation, proliferation, and apoptosis (53). Uncoupled receptor-dependent MAPK and NF-B signaling has been previously described for other TNFR superfamily members and TLR-4, either at the receptor level or through recruitment of other downstream components (54,55). Recent studies suggest that regulation of p55 TNFR and downstream components such as TRAF2 may also lead to divergent effects on MAPK and NF-B signaling.…”

Lysophosphatidic Acid Inhibits Bacterial Endotoxin-Induced Pro-Inflammatory Response: Potential Anti-Inflammatory Signaling Pathways

“…Heterotrimeric guanine nucleotidebinding regulatory (Gi) proteins modulate LPS signaling pathways and downstream pro-inflammatory gene expression (4)(5)(6)(7). In addition to toll-like receptor (TLR) 4, LPS also binds to a cluster of receptors in lipid rafts, some of which are Gi protein coupled (8).…”

“…Our previous studies demonstrated that LPS-induced inflammatory cytokines and chemokines were augmented in vitro and in vivo in Gi proteindeficient mice compared with WT mice, suggesting an anti-inflammatory role of Gi proteins (5,6). Gi protein-coupled ERK 1/2 signaling pathway mediates LPSinduced signaling (4). Activation of Gi protein with transforming growth factor-β (TGF-β) activates ERK 1/2, which can suppress NFκB and p38 signaling and consequently negatively regulate LPSinduced inflammatory responses (9,10).…”