Hypoxia activates toll-like receptor 4 signaling in primary mouse hepatocytes through the receptor clustering within lipid rafts

Dong Hee Kim, Timothy R. Billiar

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


Transient hypoxia is an initial event that accentuates ischemia/reperfusion (I/R) injury in the liver. Hepatic ischemia/reperfusion (I/R) injury is largely related to innate immunity via Toll-like receptor 4 (TLR4) signaling. However, the mechanism by which hypoxia could lead to activate TLR4 signaling remains unclear. Therefore, the aim of this experimental study investigates how TLR4 signalling is activated by hypoxia. Methods: Hepatocytes were isolated from male wild-type (C57BL/6) mice (8∼12 weeks old) by an in situ collagenase (Type IV, Sigma-Aldrich) perfusion technique. In this study, using primary mouse hepatocytes in culture to 1% oxygen, detection of TLR4 translocation to the lipid rafts on the cell membrane by immunofluorescence staining and immunoblotting was saught. Results: Hypoxia caused TLR4/MD2 and β2-Integrin (CD11b/CD18) translocation to lipid rafts associated with CD14 in hepatocytes. The cholesterol sequestering agent, Nystatin and Filipin prevented hypoxia-induced TLR4/MD2 translocation to lipid rafts. Consistent with a role for oxidative stress in this effect, in vitro H2O2 treatment of hepatocytes similarly caused TLR4/MD2 translocation to lipid rafts. In addition, translocation of hypoxia-induced TLR4 complex was inhibited by N-acetylcysteine (NAC) demonstrating that the activation of TLR4 signaling is dependent on ROS. Further, the cholesterol sequestering agent, nystatin, prevented hypoxia-induced high mobility group box 1 (HMGB1) release in hepatocytes. Conclusion: These results suggest that ROS dependent TLR4 signaling is achieved following receptor translocation to the lipid raft in hepatocytes. We hypothesized that this mechanism is required for the release of HMGB1, an early mediator of injury and inflammation in hepatic I/R injury.

Original languageEnglish
Pages (from-to)194-203
Number of pages10
JournalJournal of the Korean Surgical Society
Issue number3
StatePublished - Mar 2011
Externally publishedYes


  • High-mobility group box 1 (HMGB1)
  • Ischemia/Reperfusion
  • Liver
  • Reactive oxygen species (ROS)
  • Toll-like receptor 4 (TLR4)


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