TNF-α Activates High-Mobility Group Box 1 - Toll-Like Receptor 4 Signaling Pathway in Human Aortic Endothelial Cells

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Background/Aims: Toll-like receptor 4 (TLR4) interacts with endogenous substances as well as lipopolysaccharide. We explored whether TLR4 is implicated in tumor necrosis factor-α (TNF-α) signal transduction in human aortic endothelial cells. Methods: The pathway was evaluated by transfection of siRNAs, immunoprecipitation and Western blot analysis. Results: TNF-α activated spleen tyrosine kinase (Syk) within 10 min, which led to endothelin-1 (ET-1) production. TLR4 was also rapidly activated by TNF-α stimulation, as shown by recruitment of interleukin-1 receptor-associated kinase 1 to TLR4 and its adaptor molecule, myeloid differentiation factor 88 (MyD88). siRNA depletion of TLR4 markedly attenuated TNF-α-induced Syk activation and ET-1 production. TLR4 inhibitor (CLI-095), TLR4-neutralizing antibody and siRNA depletion of MyD88 also attenuated TNF-α-induced Syk activation. Syk was co-immunoprecipitated with TLR4, and TNF-α activated Syk bound to TLR4. High-mobility group box 1 (HMGB1) was rapidly released and associated with TLR4 after TNF-α stimulation with a peak at 5 min, which was prevented by N-acetylcysteine, an antioxidant. Glycyrrhizin (HMGB1 inhibitor), HMGB1-neutralizing antibody and siRNA depletion of HMGB1 all suppressed TNF-α-induced Syk activation and ET-1 production. Conclusion: Upon TNF-α stimulation, TLR4 is activated by HMGB1 that is immediately released after the generation of reactive oxygen species, and plays a crucial role in the signal transduction.