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.