Volume 13 Issue 9 - May 7, 2010 PDF
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RNA-binding Protein HuR Interacts with Thrombomodulin 5 Untranslated Region and Represses Internal Ribosome Entry Site–mediated Translation under IL-1 beta Treatment
Chiu-Hung Yeh,1 Liang-Yi Hung,2,8 Chin Hsu,3,6 Shu-Yun Le,4  Pin-Tse Lee,5 Wan-Lin Liao,2 Yi-Tseng Lin,6 Wen-Chang Chang,1,8 and Joseph T. Tseng*7,8
1Department of Pharmacology, 5Institute of Basic Medical Sciences, College of Medicine, 2Institute of Biosignal Transduction, 7Institute of Bioinformatics, College of Bioscience and Biotechnology, and 8Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, Tainan 701, Taiwan; 3Graduate Institute of Physiology and Molecular Medicine, Department of Physiology, 6Graduate Institute of Medicine, School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; and 4Center for Cancer Research Nanobiology Program, National Cancer Institute (NCI) Center for Cancer Research, NCI, National Institutes of Health, Frederick, MD 21702
 
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There is ample evidence that inflammation and coagulation are intricately related processes, whereby inflammation not only leads to activation of coagulation, but coagulation also markedly affects inflammation activity (1). Inflammation-induced coagulation contributes to vascular thrombotic disease and is also the major consequence in the pathogenesis of microvascular failure and subsequent multiple organ failure in severe sepsis (2).

Thrombomodulin (TM) is an important anticoagulant protein present on the surface of vascular endothelial cells (3). TM forms a high-affinity complex with thrombin and results in approximately a 100-fold increase in the activation of protein C to execute anticoagulant effects. Recent studies have shown that TM also plays an important role in attenuation of the inflammatory response (4). Though TM plays an important role in modulating inflammation, unfortunately it appears that TM expression is reduced in inflammation. In vitro studies have demonstrated that endothelial TM expression is potently inhibited by bacterial endotoxin and inflammatory cytokines such as IL-1β and TNF-α (5). However, no further research has been conducted to study this mechanism.
Figure 1. The IRES element structure in the TM 5’UTR. The Y-shaped motif is denoted by Stem A, B and C.  The short 18S rRNA-complementary sequence is labeled by the character "*".

In this report, we firstly identified that the TM 5’UTR may contain the Y-shape structure which is the typical structure of internal ribosome entry site (IRES) element by using bioinformatics method (Fig. 1), and then demonstrated the 5’UTR really exhibited the IRES activity via bicistronic reporter assay. Using RNA probe pull-down assay, HuR was demonstrated to interact with the TM 5’UTR. Overexpression of HuR protein inhibited the activity of TM IRES, while on the other hand, reducing the HuR protein level reversed this effect. When cells were treated with TNF-α or IL-1β, the IRES activity was suppressed and accompanied by an increased interaction between HuR and TM 5’UTR. This phenomenon was also be observed in the animal model of sepsis. We found the TM protein expression level to be decreased while concurrently observing the increased interaction between HuR and TM mRNA in liver tissue (Fig. 2).

To sum up, we reported a novel translational regulation mechanism in the suppression of TM protein expression by proinflammatoy cytokines. Since TM plays an important role in anticoagulation and anti-inflammation response after severe inflammation, especially in severe sepsis, prevention of the decrease of TM protein could be a good way to increase the quantity of activated protein C and prevent the microvascular thrombosis. Therefore, blocking the interaction between HuR and TM 5’UTR through inhibiting the HuR activity will provide a potential new therapeutic strategy in treating severe sepsis.
Figure 2. Interaction between HuR and TM mRNA is increased in septic rat. (A) Immunolocalization of TM and HuR in the liver tissue of septic rat. Rat liver section was prepared from normal rats or 6 hours after cecal ligation and puncture (CLP) to induce sepsis. The intracellular localization patterns of HuR and TM were visualized by indirect immunofluorescence. The nuclei were identified after staining with DAPI. (B) TM protein expression was reduced in septic rat. Rat liver protein extract and total RNA were prepared from normal rats or CLP induced sepsis for 3 and 6 hours. (C) Increase of the interaction between HuR and TM mRNA. Rat liver extract was prepared from normal or CLP-6 hour rats, and the protein extracts were immunoprecipitated with HuR antibody. The mRNAs bound with HuR were extracted and TM mRNA expression levels were detected by RT-PCR. The protein expression levels of HuR and tubulin were shown as internal controls.

References
  1. Esmon, C. T. (2005). The interactions between inflammation and coagulation. Br. J. Haematol. 131, 417–430.
  2. Diehl, J. L., and Borgel, D. (2005). Sepsis and coagulation. Curr. Opin. Crit. Care 11, 454–460.
  3. Dittman WA, Majerus PW. (1990) Structure and function of thrombomodulin: a natural anticoagulant. Blood 75, 329-336.
  4. Van De Wouwer M, Conway EM. (2004) Novel functions of thrombomodulin in inflammation. Crit Care Med. 32, S254-S261.
  5. Moore KL, Andreoli SP, Esmon NL, Esmon CT, Bang NU. (1987) Endotoxin enhances tissue factor and suppresses thrombomodulin expression of human vascular endothelium in vitro. J Clin Invest. 79, 124-130.
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