GEF-H1/RhoA signalling pathway mediates lipopolysaccharide-induced intercellular adhesion molecular-1 expression in endothelial cells via activation of p38 and NF-κB.
The purpose of study is to investigate the effects of GEF-H1/RhoA pathway in regulating intercellular adhesion molecule-1 (ICAM-1) expression in lipopolysaccharide (LPS)-activated endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) to LPS induced GEF-H1 and ICAM-1 expression in dose- and time-dependent up-regulating manners. Pretreatment with Clostridium difficile toxin B-10463 (TcdB-10463), an inhibitor of Rho activity, reduced LPS-related phosphorylation of p65 at Ser 536 in a dose-dependent manner. Inhibition of TLR4 expression significantly blocked LPS-induced RhoA activity, NF-κB transactivation, GEF-H1 and ICAM-1 expression. Coimmunoprecipitation assay indicated that LPS-activated TLR4 and GEF-H1 formed a signalling complex, suggesting that LPS, acting through TLR4, stimulates GEF-H1 expression and RhoA activity, and thereby induces NF-κB transactivation and ICAM-1 gene expression. However, GEF-H1/RhoA regulates LPS-induced NF-κB transactivation and ICAM-1 expression in a MyD88-independent pathway because inhibition of MyD88 expression could not block LPS-induced RhoA activity. Furthermore, pretreatment with Y-27632, an inhibitor of ROCK, significantly reduced LPS-induced p38, ERK1/2 and p65 phosphorylation, indicating that ROCK acts as an upstream effector of p38 and ERK1/2 to promote LPS-induced NF-κB transactivation and ICAM-1 expression. What is more, the p38 inhibitor (SB203580) but not ERK1/2 inhibitor (PD98059) blocked LPS-induce NF-κB transactivation and ICAM-1 expression, which demonstrates that RhoA mediates LPS-induced NF-κB transactivation and ICAM-1 expression dominantly through p38 but not ERK1/2 activation. In summary, our data suggest that LPS-induced ICAM-1 synthesis in HUVECs is regulated by GEF-H1/RhoA-dependent signaling pathway via activation of p38 and NF-κB.
Department of Burn and Plastic Surgery, Shanghai Jiao Tong University, School of Medicine, Rui Jin Hospital, Shanghai 230022, China.
Feng
F
Guo
Zengding
Z
Zhou
Yi
Y
Dou
Jiajun
J
Tang
Chengjin
C
Gao
Jingning
J
Huan
D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D064067:Rho Guanine Nucleotide Exchange Factors; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D051996:Transcription Factor RelA / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
C577356:ARHGEF2 protein, human; D020662:Guanine Nucleotide Exchange Factors; D008070:Lipopolysaccharides; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D064067:Rho Guanine Nucleotide Exchange Factors; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051996:Transcription Factor RelA; D018799:Intercellular Adhesion Molecule-1; D004247:DNA; D054460:rho-Associated Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020742:rhoA GTP-Binding Protein