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.

Keywords: Free radicals; Hypoxia; Inflammation; Liver; TIFA; TLR4.

Fig. 1. TIFA is upregulated in mouse liver by HS-R

(A): Effect of HS/T and resuscitation on TIFA gene level in mouse liver, analyzed with Affymetrix and CodeLink microarray. (B): Effect of HS-R on TIFA mRNA expression in mouse liver, analyzed by qRT-PCR using primers for TIFA and β-actin (internal control). (C): Effect of HS-R on TIFA protein expression in mouse liver, analyzed by Western blotting and shown as the intensity ratio of TIFA to β-actin. Data expressed as mean ± SD, n= 5–6 samples/group. * p < 0.05 vs. control.

Fig. 2. Hypoxia-reoxygenation induced TIFA upregulation is TLR4 and MyD88-mediated

(A): Effect of hypoxia-reoxygenation on TIFA mRNA expression in hepatocytes, analyzed by qRT-PCR. (B): Effect of hypoxia-reoxygenation on TIFA protein expression in hepatocytes, analyzed by Western blotting. Primary hepatocytes isolated from WT, TLR4−/−, and MyD88−/− mice were treated with hypoxia-reoxygenation and the expression of TIFA protein was analyzed by Western blotting (C) or immunofluorescence (200× magnification) (D). Red, TIFA; green, actin; blue, Hoechst. H, hypoxia; R, reoxygenation. Data are expressed as mean ± SD, n= 5–6 samples/group. * p < 0.05 vs. control or hypoxia (−).

Fig. 3. TIFA associates with TRAF6 and IRAK-1 in mouse hepatocytes

Lysates isolated from mouse hepatocyte exposed to hypoxia-reoxygenation (+) or without hypoxia-reoxygenation (−) were immunoprecipitated (IP) in the presence of an anti-TIFA or control IgG antibody, followed by Western blotting (WB) with anti-TIFA, anti-TRAF6, or anti-IRAK-1 antibodies.

Fig. 4. NF-κB involved in TIFA-mediated HMGB1 expression and release

Hepatocytes were transient transfected with TIFA specific siRNA (siTIFA) and the expression of TIFA mRNA and protein were detected by qRT-PCR (A) and Western blotting (B). The effect of siTIFA transfection on HMGB1 expression and release in hepatocytes in response to hypoxia-reoxygenation were analyzed by ELISA in cell culture supernatant (C) and by Western blotting in cell lysates (D). The effect of siTIFA transfection on NF-κB activation in hepatocytes in response to hypoxia-reoxygenation was detected by EMSA (E). The effect of siTIFA transfection on IKK, IκBα and MAPKs activation in hepatocytes in response to hypoxia-reoxygenation was detected by Western blotting (F). Data are expressed as mean ± SD, n= 5–6 samples/group. * p < 0.05 vs. normoxia, # p < 0.05 vs. control siRNA.

Fig. 5

Proposed schematic model for TIFA signaling in response to hypoxia-reoxygenation