Toll-like receptor 4 coupled Gi protein signaling pathways regulate extracellular signal-regulated kinase phosphorylation and AP-1 activation independent of NFκB activation

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-G(alphai) interaction, or transfected with dominant negative Galpha(i3) (Galpha(i3)dn) or Galpha(i2) minigene (an inhibitory carboxyl terminus of Galpha(i2)) 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 Galpha(i3)dn or Galpha(i2) minigene also reduced TLR4ca-induced ERK 1/2 phosphorylation (34 +/- 10% and 33 +/- 5%, respectively, P < 0.05). Constitutively active Galpha(i2) and Galpha(i3) 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 Galpha(i)-coupled signaling pathways are activated by TLR4. The TLR4-activated Galpha(i) signaling pathway activates ERK 1/2 phosphorylation and AP-1 activation independently of TLR4-mediated signaling to NFkappaB activation.