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 G_i 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 G_i proteins are coupled to TLR4 activation of signaling pathways. To inhibit G_i protein function, human embryonic kidney (HEK) 293 cells or RAW 264.7 cells were pretreated with pertussis toxin (PTx), an inhibitor of receptor–Gα_i interaction, or transfected with dominant negative Gα_i3 (Gα_i3dn) or Gα_i2 minigene (an inhibitory carboxyl terminus of Gα_i2) plasmid. The cells were subsequently transfected with constitutively active TLR4 (TLR4ca) plasmid or TLR4ca together with an NFκB 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 NFκB 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 NFκB activation. Cotransfection of TLR4ca with Gα_i3dn or Gα_i2 minigene also reduced TLR4ca-induced ERK 1/2 phosphorylation (34 ± 10% and 33 ± 5%, respectively, P < 0.05). Constitutively active Gα_i2 and Gα_i3 plasmids potentiated TLR4ca-induced ERK 1/2 phosphorylation (27 ± 3% and 41 ± 6%, respectively, P < 0.05). βARK-ct plasmid, which inhibits the function of βγ 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 Gα_i-coupled signaling pathways are activated by TLR4. The TLR4-activated Gα_i signaling pathway activates ERK 1/2 phosphorylation and AP-1 activation independently of TLR4-mediated signaling to NFκB activation.