Correction to: EGFR signaling augments TLR4 cell surface expression and function in macrophages via regulation of Rab5a activation

In the original publication the bands in Fig. 1J and Fig. 2B were not visible. The correct versions of Fig. 1J and Fig. 2B are provided in this correction.

Correction to: EGFR signaling augments TLR4 cell surface expression and function in macrophages via regulation of Rab5a activation

In the original publication the bands in Fig. 1J and Fig. 2B were not visible. The correct versions of Fig. 1J and Fig. 2B are provided in this correction.

Hypoxia Activates Toll-like Receptor 4 Signaling in Primary Mouse Hepatocytes Through the Receptor Clustering within Lipid Rafts

PURPOSE: 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 beta2-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.

Nitric oxide suppresses inducible nitric oxide synthase expression by inhibiting post-translational modification of I kappa B

The expression of inducible nitric oxide synthase (iNOS) is a critical factor in both normal physiological functions and the pathogenesis of disease. This study was undertaken to determine the molecular mechanism by which nitric oxide (NO) exerts negative feedback regulation on iNOS gene expression. Isolated rat hepatocytes stimulated with cytokines exhibited a marked increase in NO production as well as iNOS mRNA and protein levels, which were significantly reduced by pretreatment of the NO donors S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and V-PYRRO/NO. This effect of SNAP was inhibited when NO was scavenged using red blood cells. Pretreatment with oxidized SNAP, 8-Br-cGMP, NO2-, or NO3- did not suppress the cytokine-induced NO production. Moreover, LPS/ IFN-gamma-stimulated RAW264.7 cells, which produce endogenous NO, expressed lower levels of iNOS, IL-1beta, IL-6 and TNF-alpha mRNAs, without changes in their mRNA half-lives, than those in the presence of the iNOS inhibitor NG-monomethyl- L-arginine. The iNOS gene transcription rate exhibited an 18-fold increase after cytokine stimulation, which was significantly inhibited by SNAP pretreatment. SNAP also blocked cytokine- induced increase in NF-kappa B activation, iNOS promoter activity, nuclear translocation of cytosolic NF-kappa B p65 subunit, and I kappa B alpha degradation, which correlated with its inhibitory effect on phosphorylation and ubiquitination of I kappa B. These data indicate that NO down-regulates iNOS gene expression and NO production by inhibiting the post-translational processes of I kappa B alpha thereby preventing NF-kappa B activation. These results identify a novel negative feedback mechanism whereby NO down-regulates iNOS gene expression.