Essential role for TAX1BP1 in the termination of TNF-alpha-, IL-1- and LPS-mediated NF-kappaB and JNK signaling

Abstract

The NF-kappaB transcription factor is normally transiently activated by proinflammatory cytokines and bacterial lipopolysaccharide (LPS); however, persistent NF-kappaB activation is commonly observed in inflammatory disease and malignancy. The ubiquitin editing enzyme A20 serves an essential role in the termination of TNF-alpha- and LPS-mediated NF-kappaB signaling by inactivating key signaling molecules. However, little is known about how A20 is regulated and if other molecules play a role in the termination of NF-kappaB signaling. Here we demonstrate that Tax1-binding protein 1 (TAX1BP1) is essential for the termination of NF-kappaB and JNK activation in response to TNF-alpha, IL-1 and LPS stimulation. In TAX1BP1-deficient mouse fibroblasts, TNF-alpha-, IL-1- and LPS-mediated IKK and JNK activation is elevated and persistent owing to enhanced ubiquitination of RIP1 and TRAF6. Furthermore, in the absence of TAX1BP1, A20 is impaired in RIP1 binding, deubiquitination of TRAF6 and inhibition of NF-kappaB activation. Thus, TAX1BP1 is pivotal for the termination of NF-kappaB and JNK signaling by functioning as an essential regulator of A20.

Figure 1

TAX1BP1 is a negative regulator of NF-κB. (A) 293T cells were transfected with 60 pmol scrambled (control) or TAX1BP1 siRNA (Ambion) together with 600 ng pCMV4. After 72 h, total RNA was isolated and converted to cDNA for qRT–PCR using a TAX1BP1 TaqMan probe. Absolute values of TAX1BP1 mRNA are indicated. (B–E) 293T cells were transfected with control or TAX1BP1 siRNA, κB-TATA Luc (0.1 μg) and pRL-tk (0.01 μg). In addition, pCMV4-Tax (2 μg), myc-CD40 (0.5 μg) and HA-TLR4 (1 μg) was transfected in panels B, D and E respectively. Cells were treated with TNF-α (20 ng/ml) for 7 h in panel C. Cells were harvested and dual luciferase assays were performed. (F) Transfection of 293T cells was performed as described above except that the reporter construct used was ISRE-Luc (0.25 μg) and treatment was IFN-γ (100 ng/ml).

Figure 2

Generation of TAX1BP1 knockout mice by gene trapping. (A) Genomic organization of the TAX1BP1 locus. PCR primers for genotyping are indicated by arrows. (B) PCR genotyping strategy for mice. The expected sizes of the PCR fragments are 275 bp with primers F1 and R2, and 154 bp with primers F1 and R1. RT–PCR using two sets of primers was performed with mRNA isolated from tails of four pups born from litters of a TAX1BP1 male chimera bred with a female C57BL/6 mouse. (C) TAX1BP1^m/m embryos exhibit hemorrhaging at E13.5. Photographs of E13.5 TAX1BP1^m/m and TAX1BP1^+/m embryos. (D) The expression of TAX1BP1 and SV40 large T antigen (Tag) in TAX1BP1^+/m and TAX1BP1^m/m MEFs was determined by RT–PCR. The same primers (F1, R1 and R2) used in panel B were used to genotype MEFs. Primers specific to Tag were used to amplify a 500 bp fragment from Tag. (E) Whole-cell lysates from TAX1BP1^+/m and TAX1BP1^m/m MEFs were subjected to immunoblotting with polyclonal αTAX1BP1.

Figure 3

TAX1BP1 is required for the termination of IL-1- and TNF-α-mediated NF-κB activation. (A, B) TAX1BP1^m/m and TAX1BP1^+/m MEFs were stimulated with either IL-1 (20 ng/ml) in panel A or TNF-α (20 ng/ml) in panel B for the indicated times. Nuclear extracts were prepared and subjected to NF-κB EMSA. (C, E) TAX1BP1^m/m and TAX1BP1^+/m MEFs were stimulated with either IL-1 (panel C) or TNF-α (panel E) for the indicated times. Whole-cell extracts were subjected to immunoblotting with anti-IκBα and β-actin antibodies. (D, F) TAX1BP1^m/m and TAX1BP1^+/m MEFs were treated with IL-1 (panel D) and TNF-α (panel F) for the indicated times together with MG-132 (25 μM) at the 15 min time point. Whole-cell extracts were subjected to immunoblotting with anti-IκBα and β-actin antibodies. (G) TAX1BP1^m/m and TAX1BP1^+/m MEFs were treated with either IL-1 or TNF-α for 20 h. Cell supernatants were collected and subjected to an IL-6 ELISA. The error bars represent the standard error of the mean (s.e.m.) of triplicate samples.

Figure 4

TAX1BP1 is essential for the downregulation of IKK and JNK activation upon IL-1 and TNF-α stimulation. (A) TAX1BP1^m/m and TAX1BP1^+/m MEFs were stimulated with IL-1 (20 ng/ml) or TNF-α (20 ng/ml) for the indicated times. Lysates were immunoprecipitated with anti-IKKγ for an in vitro kinase assay using GST-IκBα 1–54 as substrate. The amount of immunoprecipitated IKKγ was determined by immunoblotting for IKKγ. IgH represents the immunoglobulin heavy chain. (B, C) TAX1BP1^m/m and TAX1BP1^+/m MEFs were stimulated with IL-1 or TNF-α for the indicated times. In panel B, lysates were immunoprecipitated with anti-JNK1 (Santa Cruz) for an in vitro kinase assay using GST-c-jun 1–79 as substrate. In panel C, lysates were immunoblotted with either anti-phospho-JNK or anti-JNK.

Figure 5

Retroviral-mediated gene transfer of wild-type TAX1BP1 restores transient NF-κB and JNK activation in TAX1BP1^m/m MEFs. (A) TAX1BP1^m/m MEFs were infected with recombinant retroviruses expressing either Flag-TAX1BP1 or pCLXSN empty vector. Cells were selected with G418 and stimulated with either IL-1 (20 ng/ml) or TNF-α (20 ng/ml) for the indicated times. Whole-cell lysates were subjected to immunoblotting with anti-Flag to detect exogenous TAX1BP1, anti-IκBα and β-actin. (B) NF-κB EMSA was performed with reconstituted TAX1BP1^m/m MEFs. Cells were stimulated with TNF-α for the indicated times and nuclear extracts were used for EMSA. (C) Reconstituted TAX1BP1^m/m MEFs were treated with IL-1 or TNF-α for the indicated times. Immunoblotting was performed with anti-JNK and anti-phospho-JNK antibodies.

Figure 6

TAX1BP1 inhibits LPS- and Tax-mediated NF-κB activation. (A) TAX1BP1^m/m and TAX1BP1^+/m MEFs were stimulated with LPS (1 μg/ml) for the indicated times. Whole-cell extracts were subjected to immunoblotting with anti-IκBα, p-JNK and β-actin antibodies. (B) TAX1BP1^m/m, TAX1BP1^+/m and TAX1BP1^m/m MEFs reconstituted with Flag-TAX1BP1 were infected with either pCLXSN or pCLXSN-Tax expressing retroviruses. After 4 days, the expression of Tax was confirmed by immunoblotting and nuclear extracts were subjected to an NF-κB EMSA.

Figure 7

TAX1BP1 promotes the deubiquitination of TRAF6 in an A20-dependent manner. (A) TAX1BP1^m/m and TAX1BP1^+/m MEFs were treated with IL-1 (20 ng/ml) for the indicated times. Cells were lysed and TRAF6 was immunoprecipitated with anti-TRAF6 followed by immunoblotting with anti-ubiquitin or anti-TRAF6. (B) TAX1BP1^m/m and TAX1BP1^+/m MEFs were treated with LPS (1 μg/ml) and TRAF6 ubiquitination was examined as described in panel A. (C) 293T cells were transfected with Flag-TRAF6 (1 μg) and HA-Ub (0.5 μg) together with Flag-TAX1BP1 (0.25, 0.5 and 1 μg), Flag-TAX1BP1 1–204 (0.25, 0.5 and 1 μg), Flag-A20 (0.1, 0.25 and 0.5 μg) or Flag-A20 C103A (0.1, 0.25 and 0.5 μg). After 36 h, cells were lysed and immunoprecipitated with anti-TRAF6 followed by immunoblotting with anti-HA. Lysates were examined for TRAF6, TAX1BP1 and A20 expression by immunoblotting with anti-Flag. (D) 293T cells were transfected with vector or Flag-A20 (1 μg) together with pSuper (2 μg) or pSuper A20 nos. 1 or 2 siRNAs (2 μg). After 36 h, cells were lysed and subjected to immunoblotting with anti-Flag. (E) 293T cells were transfected with Flag-TRAF6, HA-Ub and Flag-TAX1BP1 as above together with 2 μg of pSuper or pSuper A20 siRNAs. The IPs and immunoblotting were performed as in panel C. Lysates were examined for TRAF6 and TAX1BP1 expression by immunoblotting with anti-Flag. (F) TAX1BP1 promotes an interaction between A20 and TRAF6. 293T cells were transfected with Flag-A20 (1 μg) and Flag-TRAF6 (1 μg) together with pCAGGS-TAX1BP1 (0, 0.25, 0.5 and 1 μg). After 36 h, cells were lysed and TRAF6 was immunoprecipitated followed by immunoblotting with anti-Flag. Lysates were examined for A20, TRAF6 and TAX1BP1 expression.

Figure 8

TAX1BP1 interacts with RIP1 and negatively regulates RIP1 ubiquitination. (A) 293T cells were transfected with Flag-RIP1 (1 μg), Flag-TRAF6 (1 μg) and GFP-TAX1BP1 (1 μg). After 36 h, cells were lysed and immunoprecipitated with anti-GFP followed by immunoblotting with anti-Flag. Lysates were examined for TAX1BP1 expression by immunoblotting with anti-GFP. RIP1 and TRAF6 were detected by immunoblotting with anti-Flag. (B) TAX1BP1^m/m and TAX1BP1^+/m MEFs were treated with TNF-α (20 ng/ml) as indicated. Cells were lysed and RIP1 was immunoprecipitated with anti-RIP1 followed by immunoblotting with anti-ubiquitin or anti-RIP1. (C) 293T cells were transfected with Flag-A20 (1 μg) and HA-RIP1 (1 μg) together with pCAGGS-TAX1BP1 (0, 0.25, 0.5 and 1 μg). After 36 h, cells were lysed and RIP1 was immunoprecipitated with anti-HA followed by immunoblotting with anti-Flag. Lysates were examined for A20, RIP1 and TAX1BP1 expression by immunoblotting with anti-Flag, anti-HA and anti-TAX1BP1, respectively. (D) 293T cells were transfected with Flag-TAX1BP1 together with either control or TAX1BP1 siRNA. (E) 293T cells were transfected with control or TAX1BP1 siRNA. Cells were either untreated or treated with TNF-α (20 ng/ml) for the indicated times. Immunoprecipitations were performed with either anti-control Ig or anti-RIP1 followed by immunoblotting with anti-A20.

Figure 9

TAX1BP1 is essential for A20-mediated deubiquitination of TRAF6 and inhibition of NF-κB activation. (A) 293T cells were transfected with control siRNA or TAX1BP1 siRNA. The following day, the same cells were transfected with Flag-TRAF6 (1 μg) and HA-Ub (0.5 μg) together with Flag-A20 (0.25 μg) or Flag-A20 C103A (0.25 μg). After 36 h, cells were lysed and immunoprecipitated with anti-TRAF6 followed by immunoblotting with anti-HA. Lysates were examined for TRAF6 and A20 expression. (B) 293T cells were transfected with control or TAX1BP1 siRNA, κB-TATA Luc (0.1 μg), pRL-tk (0.01 μg), Flag-A20 or Flag-A20 C103A (10, 25 and 50 ng). Cells were harvested after 7 h treatment with TNF-α (20 ng/ml), and dual luciferase assays were performed. Lysates were immunoblotted with anti-Flag to detect ectopic A20. (C) TAX1BP1^+/m and TAX1BP1^m/m MEFs were transfected with 2 μg of pCMV4, Flag-A20 or Flag-A20 C103A together with 1 μg κB-TATA Luc and 0.1 μg pRL-tk. After 36 h, cells were stimulated with TNF-α (20 ng/ml), and dual luciferase assays were performed. Lysates were immunoblotted with anti-Flag to detect ectopic A20.