[Assessment of the role of TLR-4 in shear-stress-induced IL-8 gene transcription activation in vascular endothelial cells by gene mutation and gene transfection technology].
This study is to evaluate the role of TLR-4 in lower laminar shear stress-induced interleukin-8 gene transcription activation in vascular endothelial cells by using gene mutation and transfection techniques. RT-PCR, Northern hybridization and immunocytochemical fluorescent staining showed that TLR-4 was expressed in human umbilical vein endothelial cells(HUVEC). When stimulated with 4.2 dyne/cm2 shear stress for 1 hour, an increase of TLR-4 mRNA expression was observed in HUVECs detected by RT-PCR and Northern hybridization. The intracellular domain deletion mutant TLR-4 cDNA (lacking the 155 COOH terminal amino acids of the wild type (TLR-4) and -102 -61 bp DNA sequence in 5'-flanking region of IL-8 gene (IL-8USCS) were isolated from endothelial cells by RT-PCR and PCR. These two DNA fragments were cloned into pcDNA3 and pEGFP1 respectively to construct TLR-4 dominant negative mutant pcDNA3-mTLR4 and IL-8 reporter gene pEGFP1-IL8USCS. ECV304 cells were transfected with the pEGFP1-IL8USCS or co-transfected with pEGFP1-IL8USCS and pcDNA3-mTLR4 by Dosper liposome transfectional reagent and selected by G418, then stimulated with 4.2 dyne/cm2 shear stress for 3 hours. Flow cytometric analysis showed that when exposed to 4.2 dyne/cm2 shear stress for 3 hours, there was a marked increase in the green fluorescent protein expression in pEGFP1-IL8USCS-transfected ECV304 cells. In contrast, there was almost no change in the green fluorescent protein expression in the cells co-transfected with pEGFP1-IL8USCS and pcDNA3-mTLR4 after the stimulation, suggesting the TLR-4 mutant depressed TLR-4 signaling. These experiments suggest that the inflammatory TLR-4/NF-kappa B signaling pathway would probably be involved in flow shear stress-induced IL-8 gene expression in human vascular endothelial cells.
Liang
Feng
F
Research Unit of Infection and Immunity, West China Medical Center, Sichuan University, Chengdu 610041.
Huang
Ning
N
Wang
Boyao
B
Chen
Huaiqing
H
D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D005786:Gene Expression Regulation; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D011956:Receptors, Cell Surface / Q000235:genetics / Q000502:physiology*; D013314:Stress, Mechanical; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014158:Transcription, Genetic / Q000502:physiology*; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology
D016209:Interleukin-8; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors