[Transcriptional activation of interleukin-8 gene induced by shear stress in human umbilical vein endothelial cells].
OBJECTIVE
To examine the transcriptional activation of IL-8 gene induced by 4.2 dyne/cm2 shear stress in human vascular endothelial cells.


METHODS
The one-step RT-PCR was used for detection of IL-8 mRNAs expression in human umbilical vein endothelial cells (HUVECs) after shear stress for 0.5, 1, 2 hours. To construct IL-8 green fluorescent protein reporter gene plasmid pEGFP1-IL8USCS. The endothelial cells were transfected with the pEGFP1-IL8USCS, and stimulated with 4.2 dyne/cm2 shear stress for 3 hours. The green fluorescent protein expression was analyzed by flow cytometry. NF-kappaB nuclear translocation was observed by immunocytofluorescent staining in HUVECs stimulated by shear stress for 0.5, 1, 1.5, 2 hours. Western blotting was used to examine kappaB phosphorylation and degradation after shear stress for 10, 20, 30, 60 minutes respectively.


RESULTS
The results of RT-PCR showed that low laminar shear stress could induce IL-8 mRNA expression in HUVECs, its increased effect was time-dependent. 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 the pEGFP1-IL8USCS-transfected cells. NF-kappaB p65 immunocytofluorescent staining of HUVECs showed that flow shear stress could induce nuclear translocation of NF-kappaB. Flow shear stress could induce IkappaB phosphorylation and degradation in HUVECs detected by Western blotting.


CONCLUSIONS
These experiments suggested that the NF-kappaB signaling pathway would probably be involved in flow shear stress-induced IL-8 gene transcriptional activation, It may be involved in the development of atherosclerosis.
Liang
Feng
F
Cardiovascular Center, Beijing Tongren Hospital, Capital University of Medical Science, Beijing 100730, China.
Hu
Da-yi
DY
Wang
Bo-yao
BY
Huang
Ning
N
Wu
Qi
Q
Chen
Huai-qing
HQ
D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D016209:Interleukin-8 / Q000235:genetics* / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013314:Stress, Mechanical; D014158:Transcription, Genetic; D014471:Umbilical Veins / Q000166:cytology
D020672:I-kappa B Proteins; D016209:Interleukin-8; D016328:NF-kappa B; D012333:RNA, Messenger