Toll-like receptor 4 coupled Gi protein signaling pathways regulate extracellular signal-regulated kinase phosphorylation and AP-1 activation independent of NFκB activation
Toll-like receptor 4 coupled Gi protein signaling pathways regulate extracellular signal-regulated kinase phosphorylation and AP-1 activation independent of NFκB activation
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DOI：
10.1097/01.shk.0000129759.58490.d6
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发表时间：
2004-07-01
期刊：
影响因子：
3.1
通讯作者：
Cook, JA
中科院分区：
文献类型：
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作者：
Fan, HK;Peck, OM;Cook, JA
先前的研究表明异三聚体G(i)蛋白参与了导致脂多糖（LPS）诱导的炎症介质产生的信号传导。TLR4最近被证明在响应LPS激活中发挥核心作用。我们假设G(i)蛋白与TLR4激活信号通路偶联。为了抑制G(i)蛋白的功能，用百日咳毒素（PTx）预处理人胚胎肾（HEK） 293细胞或RAW 264.7细胞，PTx是受体-G（α）相互作用的抑制剂，或者用显性阴性Galpha(i3) （Galpha(i3)dn）或Galpha(i2) minigene （Galpha（i2）的抑制性羧基端）质粒转染。随后用组成型活性TLR4 （TLR4ca）质粒或TLR4ca连同NFkappaB或AP-1报告基因构建物转染细胞。与空载体对照相比，转染TLR4ca诱导ERK 1/2激活（157 +/- 14%,P < 0.01）， AP-1激活（4.0 +/- 0.2倍，P < 0.01）， NFkappaB激活（8.1 +/- 0.4倍，P < 0.01）。PTx预处理可抑制tlr4ca诱导的ERK 1/2磷酸化（30 +/- 7%,P < 0.05）和AP-1活化（36 +/- 3%,P < 0.05），但对NFkappaB活化无抑制作用。与Galpha(i3)dn或Galpha(i2) minigene共转染TLR4ca也降低了TLR4ca诱导的ERK 1/2磷酸化（分别为34 +/- 10%和33 +/- 5%，P < 0.05）。组成型活性Galpha（i2）和Galpha（i3）质粒增强了tlr4ca诱导的ERK 1/2磷酸化（分别为27 +/- 3%和41 +/- 6%，P < 0.05）。betaARK-ct质粒抑制G蛋白betagama亚基的功能，对tlr4ca诱导的ERK 1/2磷酸化无影响。这些数据支持了我们的假设，并首次证明了Galpha(i)偶联信号通路是由TLR4激活的。tlr4激活的Galpha(i)信号通路激活ERK 1/2磷酸化和AP-1激活，独立于tlr4介导的NFkappaB激活信号。
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.