GEF-H1-RhoA signaling pathway mediates LPS-induced NF-κB transactivation and IL-8 synthesis in endothelial cells.
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.
Department of Burn and Plastic Surgery, Shanghai Jiao Tong University, School of Medicine, Rui Jin Hospital, Shanghai, China.
Feng
F
Guo
Jiajun
J
Tang
Zengding
Z
Zhou
Yi
Y
Dou
Derek
D
Van Lonkhuyzen
Chengjin
C
Gao
Jingning
J
Huan
D001426:Bacterial Proteins / Q000494:pharmacology; D001427:Bacterial Toxins / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis* / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D064067:Rho Guanine Nucleotide Exchange Factors; D015398:Signal Transduction; D013997:Time Factors; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
C577356:ARHGEF2 protein, human; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D020662:Guanine Nucleotide Exchange Factors; D016209:Interleukin-8; D008070:Lipopolysaccharides; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D064067:Rho Guanine Nucleotide Exchange Factors; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; C031815:toxB protein, Clostridium difficile; C066737:RHOA protein, human; D020928:Mitogen-Activated Protein Kinases; D020742:rhoA GTP-Binding Protein