The deubiquitinase activity of A20 is dispensable for NF-κB signaling

Abstract

A20 has been suggested to limit NF-κB activation by removing regulatory ubiquitin chains from ubiquitinated substrates. A20 is a ubiquitin-editing enzyme that removes K63-linked ubiquitin chains from adaptor proteins, such as RIP1, and then conjugates them to K48-linked polyubiquitin chains to trigger proteasomal degradation. To determine the role of the deubiquitinase function of A20 in downregulating NF-κB signaling, we have generated a knock-in mouse that lacks the deubiquitinase function of A20 (A20-OTU mice). These mice are normal and have no signs of inflammation, have normal proportions of B, T, dendritic, and myeloid cells, respond normally to LPS and TNF, and undergo normal NF-κB activation. Our results thus indicate that the deubiquitinase activity of A20 is dispensable for normal NF-κB signaling.

Keywords: A20; K63‐linked ubiquitination; NF‐κB; deubiquitinase; signal transduction.

Figure 1. Generation of the A20-OTU/OTU mice

A Sequencing of genomic DNA from homozygous (A/A) and heterozygous littermates (C/A). B Genotyping strategy and photograph of wild-type, heterozygous, and homozygous littermates showing normal development and growth. C Similar induction of A20 protein upon stimulation of macrophages from C/C or A/A littermates, with TNF or LPS. D Histology of kidney, liver, spleen, thymus, lung, and heart of littermates.

Figure 2. Characterization of the cells of the immune system of C103A knock-in 6-week-old mice in the basal state

A Characterizing the monocytes, granulocytes, and B cells of the bone marrow. B Characterizing the CD4 and CD8 cell populations of the thymus. C Characterizing the myeloid cells, dendritic cells, T cells, and B cells of the spleen.

Figure 3. Activation of wild-type and mutant BMDM and BMDC in response to LPS and TNF in vitro; response of littermates to LPS shock in vivo

A TNF-α, IL-6, and IL-12 produced by BMDM in response to 1 μg/ml LPS were measured by ELISA. Error bars represent standard deviation representative of three independent experiments. B IL-6 and IL-12 produced by BMDM in response to 10 ng/ml TNF-α were measured by ELISA. Error bars represent standard deviation representative of three independent experiments. C Analysis of activation status of BMDMs by flow cytometry (black: wild-type; red: heterozygous; green: homozygous).

Figure 4. LPS shock of wild-type and homozygous littermates

A Age- and sex-matched mice (n = 5; representative of 3 independent experiments) were given intraperitonial injections of 50 μg/ml LPS, and survival was scored every 6 h for 72 h. B Serum cytokine levels of TNF-α, IL-6, and IL-12 in mice injected with 50 μg/ml LPS were measured by ELISA for indicated time points. Error bars represent standard deviation representative of three independent experiments.

Figure 5. Biochemical analysis of NF-κB activation in wild-type and homozygous BMDMs

A NF-κB binding to DNA in response to TNF-α stimulation was analyzed by electrophoretic mobility shift assay. EMSA was performed with nuclear extracts after stimulating BMDMs isolated from wild-type and homozygous littermates with 10 ng/ml of TNF-α for the indicated time points. The nuclear extract isolated at 30 min from wild-type BMDMs was used for the super-shift assay (with p65 and p50 antibodies) and analysis with the unlabeled κB probe. B BMDMs isolated from wild-type and homozygous littermates were stimulated with 10 ng/ml TNF-α for the indicated time points. The lysates were immunoprecipitated with RIP1 and immunoblotted with indicated antibodies. C EMSA was performed as in (A); cells stimulated with 1 μg/ml of LPS. D BMDMs isolated from wild-type and homozygous littermates were stimulated with 1 μg/ml of LPS for the indicated time points. The lysates were immunoprecipitated with TRAF6 and immunoblotted with indicated antibodies.