Department of Burn and Plastic Surgery, Shanghai Jiao Tong University, School of Medicine, Rui Jin Hospital, Shanghai, China.
Mol Immunol. 2012 Feb;50(1-2):98-107. doi: 10.1016/j.molimm.2011.12.009. Epub 2012 Jan 4.
Secretion of proinflammatory cytokines by LPS activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in this process is not well understood. In the present study, we determined the roles of GEF-H1 (guanine-nucleotide exchange factor-H1)-RhoA signaling in LPS-induced interleukin-8 (IL-8, CXCL8) production in endothelial cells. First, we observed that GEF-H1 expression was upregulated in a dose- and time-dependent manner as consistent with TLR4 (Toll-like receptor 4) expression after LPS stimulation. Afterwards, Clostridium difficile toxin B-10463 (TcdB-10463), an inhibitor of Rho activities, reduced LPS-induced NF-κB phosphorylation. Inhibition of GEF-H1 and RhoA expression reduced LPS-induced NF-κB and p38 phosphorylation. TLR4 knockout blocked LPS-induced activity of RhoA, however, MyD88 knockout did not impair the LPS-induced activity of RhoA. Nevertheless, TLR4 and MyD88 knockout both significantly inhibited transactivation of NF-κB. GEF-H1-RhoA and MyD88 both induced significant changes in NF-κB transactivation and IL-8 synthesis. Co-inhibition of GEF-H1-RhoA and p38 expression produced similar inhibitory effects on LPS-induced NF-κB transactivation and IL-8 synthesis as inhibition of p38 expression alone, thus confirming that activation of p38 was essential for the GEF-H1-RhoA signaling pathway to induce NF-κB transactivation and IL-8 synthesis. Taken together, these results demonstrate that LPS-induced NF-κB activation and IL-8 synthesis in endothelial cells are regulated by the MyD88 pathway and GEF-H1-RhoA pathway.
内毒素激活的内皮细胞分泌促炎细胞因子在脓毒症发病机制中起重要作用。然而，这一过程中涉及的机制尚不清楚。在本研究中，我们确定了 GEF-H1（鸟嘌呤核苷酸交换因子-H1）-RhoA 信号在 LPS 诱导的内皮细胞白细胞介素-8（IL-8，CXCL8）产生中的作用。首先，我们观察到 GEF-H1 的表达与 TLR4（Toll 样受体 4）表达一致，呈剂量和时间依赖性上调，在 LPS 刺激后。随后，梭状芽胞杆菌毒素 B-10463（TcdB-10463），一种 Rho 活性抑制剂，减少 LPS 诱导的 NF-κB 磷酸化。抑制 GEF-H1 和 RhoA 的表达减少了 LPS 诱导的 NF-κB 和 p38 的磷酸化。TLR4 敲除阻断了 LPS 诱导的 RhoA 活性，而 MyD88 敲除并没有损害 LPS 诱导的 RhoA 活性。然而，TLR4 和 MyD88 敲除都显著抑制了 NF-κB 的转激活。GEF-H1-RhoA 和 MyD88 都引起了 NF-κB 转激活和 IL-8 合成的显著变化。GEF-H1-RhoA 和 p38 表达的共同抑制对 LPS 诱导的 NF-κB 转激活和 IL-8 合成产生了与单独抑制 p38 表达相似的抑制作用，从而证实了 p38 的激活对于 GEF-H1-RhoA 信号通路诱导 NF-κB 转激活和 IL-8 合成是必不可少的。总之，这些结果表明，LPS 诱导的内皮细胞 NF-κB 激活和 IL-8 合成受 MyD88 途径和 GEF-H1-RhoA 途径的调节。
