title: TIFA upregulation after hypoxia-reoxygenation is TLR4- and MyD88-dependent and associated with HMGB1 upregulation and release.
abstract: 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.
authors.affiliation: Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213, USA. dingninggz@hotmail.com
authors.forename: Ning
authors.initials: N
authors.lastname: Ding
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Loughran
authors.forename: Qingde
authors.initials: Q
authors.lastname: Wang
authors.forename: Allan
authors.initials: A
authors.lastname: Tsung
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Billiar
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D005786:Gene Expression Regulation; D024243:HMGB1 Protein / Q000378:metabolism*; D000860:Hypoxia / Q000378:metabolism* / Q000473:pathology; D007375:Interleukin-1 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism*; D012771:Shock, Hemorrhagic / Q000378:metabolism / Q000473:pathology; D015398:Signal Transduction / Q000235:genetics; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D024243:HMGB1 Protein; D007375:Interleukin-1; C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; C499176:T2bp protein, mouse; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4
keywords: Free radicals; Hypoxia; Inflammation; Liver; TIFA; TLR4