TIFA upregulation after hypoxia-reoxygenation is TLR4- and MyD88-dependent and associated with HMGB1 upregulation and release.

TRAF-interacting protein with a forkhead-associated domain (TIFA) is a tumor necrosis factor receptor-associated factor 6 (TRAF6) binding protein that mediates IL-1 signaling. We recently reported that TIFA mRNA is significantly upregulated early in the liver after trauma and hemorrhagic shock. In this study, we sought to characterize the upregulation of TIFA by hypoxia–reoxygenation and investigate its role in hypoxia-induced signaling. TIFA expression was detected by qRT-PCR and Western blotting in both mouse hemorrhagic shock with resuscitation (HS-R) and hepatocytes exposed to hypoxia–reoxygenation. Involvement of TLR4 and MyD88 was assessed using cells from TLR4−/− and MyD88−/− mice. The interaction of TIFA with TRAF6 and IRAK-1 was investigated using coimmunoprecipitation in vitro. RNAi was performed to knock down the endogenous expression of the TIFA gene in hepatocytes. High-mobility-group box 1 protein (HMGB1) expression was detected by Western blotting and ELISA, and the activation of NF-κB and MAPK was measured with EMSA and Western blotting. The results showed that TIFA expression was upregulated after HS-R in vivo and hypoxia–reoxygenation in vitro. Further analysis revealed that hypoxia–reoxygenation-induced upregulation of TIFA was TLR4- and MyD88-dependent. Moreover, TIFA was found to associate with TRAF6 constitutively, whereas its association with IRAK-1 was seen only after hypoxia–reoxygenation. Suppression of TIFA by siRNA reduced NF-κB activation and HMGB1 upregulation and release after hypoxia–reoxygenation. Taken together, these data suggest that TIFA is involved in the regulation of cell signaling in hypoxia–reoxygenation. The increase in TIFA level appears to be a feed-forward mechanism involved in TLR4/MyD88-dependent signaling, leading to NF-κB activation and HMGB1 release.