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Cell Biology International (2012) 36, 475–481 (Printed in Great Britain)
Toll-like receptor 4 is involved in bacterial endotoxin-induced endothelial cell injury and SOC-mediated calcium regulation
Rongju Sun*1, Zhihong Zhu*1, Qin Su†1, Tanshi Li*2 and Qing Song*2
*Department of Emergency, General Hospital of PLA, Beijing, 100853, Peoples Republic of China, and †Department of Emergency, First affiliated Hospital, General Hospital of PLA, Beijing, 100048, Peoples Republic of China

Bacterial endotoxins may lead to vascular endothelial cell injury. Our study explored the role of TLR4 (Toll-like receptor 4) and STIM1 (stromal interaction molecule 1) in bacterial endotoxin-induced calcium overload and inflammatory reactions in HUVECs (human umbilical vein endothelial cells). It showed that under LPS (lipopolysaccharide) stimulation, LBP (LPS-binding protein) mRNA levels peaked at 24 h, TLR4 levels at 12 h and NF-κB (nuclear factor κB) levels at 6 h (all P<0.01). LBP levels increased gradually and peaked at 24 h of LPS treatment. TLR4 protein levels increased significantly at 1 h and peaked at 12 h. NF-κB protein levels markedly increased at 1 h and peaked at 6 h. Knockdown of STIM1 alone, TLR4 alone or both STIM1 and TLR4 together, markedly abolished LPS-induced increase in calcium influx into cells (P<0.05, P<0.01 and P<0.01 respectively). LBP–TLR4 and STIM–NF-κB interactions were detected without LPS treatment, enhanced by LPS stimulation, and markedly reduced by knocking down TLR4 and STIM respectively. Both the NF-κB inhibitor, PDTC (pyrrolidine dithiocarbamate) and TLR4 knockdown could block LPS induction of NF-κB, STIM, TNFα (tumour necrosis factor α) and IL-6 (interleukin 6). The data indicate LPS–LBP may activate TLR4 signalling and downstream transcription factor NF-κB, which further can activate STIM1 and eventually lead to calcium influx and injury of HUVECs. Inhibition of TLR4 effectively reverses LPS induction of inflammatory mediator generation and extracellular calcium influx mediated by STIM1.

Key words: calcium overload, endotoxin, inflammatory mediators, stromal interaction molecule 1 (STIM1), Toll like-receptor 4, vascular endothelial cell

Abbreviations: [Ca2+]i, intracellular Ca2+, HUVEC, human umbilical vein endothelial cell, IL-6, interleukin 6, LBP, LPS-binding protein, LPS, lipopolysaccharide, NF-κB, nuclear factor κB, PDTC, pyrrolidine dithiocarbamate, RT–PCR, reverse transcription–PCR, siRNA, small interfering RNA, SIRS, systemic inflammatory response syndrome, SOC, store-operated Ca2+, STIM1, stromal interaction molecule 1, TLR4, Toll-like receptor 4, TNFα, tumour necrosis factor α

1These authors contributed equally to this work.

2Correspondence may be addressed to either of these authors (email or

Received 21 September 2011/9 December 2011; accepted 31 January 2012

Published as Cell Biology International Immediate Publication 31 January 2012, doi:10.1042/CBI20110535

© The Author(s) Journal compilation © 2012 International Federation for Cell Biology

ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)