Deubiquitinating enzyme CYLD negatively regulates the ubiquitin-dependent kinase Tak1 and prevents abnormal T cell responses

Abstract

The deubiquitinating enzyme CYLD has recently been implicated in the regulation of signal transduction, but its physiological function and mechanism of action are still elusive. In this study, we show that CYLD plays a pivotal role in regulating T cell activation and homeostasis. T cells derived from Cyld knockout mice display a hyperresponsive phenotype and mediate the spontaneous development of intestinal inflammation. Interestingly, CYLD targets a ubiquitin-dependent kinase, transforming growth factor-beta-activated kinase 1 (Tak1), and inhibits its ubiquitination and autoactivation. Cyld-deficient T cells exhibit constitutively active Tak1 and its downstream kinases c-Jun N-terminal kinase and IkappaB kinase beta. These results emphasize a critical role for CYLD in preventing spontaneous activation of the Tak1 axis of T cell signaling and, thereby, maintaining normal T cell function.

Figure 1.

Hyperresponsiveness of Cyld^−/− T cells. (A–C) Wild-type (+/+) and Cyld^−/− mesenteric lymph node T cells were either not treated (NT) or were stimulated with the indicated amounts of plate-bound anti-CD3 plus soluble anti-CD28. Cell proliferation (A) and cytokine production (B and C) were measured by thymidine incorporation and ELISA, respectively. Data are presented as means ± SD (error bars) of three independent experiments. (D and E) Purified naive and memory T cells were stimulated with plate-bound anti-CD3 (2.5 μg/ml for naive and 1 μg/ml for memory T cells) and anti-CD28 (2.5 μg/ml for naive and 1 μg/ml for memory T cells) for 48 h followed by measuring cell proliferation and IFN-γ production as in A and B.

Figure 2.

Spontaneous development of autoimmune symptoms and colonic inflammation in Cyld^−/− mice. (A–C) Hematoxylin-eosin staining of tissue sections of the liver (A) and the distal portion of the colon (B and C) from 8-wk-old control (+/+) and Cyld^−/− mice. Inflammatory cell infiltration in the periportal vein of the liver (A), colonic patches in the colon (B; arrows), and inflammatory cell infiltration in the colonic mucosa (C; arrow 1) are indicated. In C, arrows 2 and 3 indicate crypt damage and muscularis layer thickening, respectively. (D) Immunohistochemistry staining of colon sections with anti-CD4 showing massive CD4 T cell infiltration in the Cyld^−/− colon. Data in A–D are representative of four different experiments, each with four wild-type and four Cyld^−/− mice. (E) Histological scores of mucosal inflammation in wild-type and Cyld^−/− mice. Data were obtained from three wild-type and three Cyld^−/− mice (8 wks of age), each with two colon sections. Similar results were obtained from three additional experiments. (F) RNase protection assay showing the constitutive expression of several proinflammatory genes in the colon of Cyld^−/− mice (#1 and #3) but not wild-type mice (#2 and #4). The housekeeping genes L32 and Gapdh were included as loading controls. (G) Body weight of wild-type and Cyld^−/− mice showing the reduced body weights of male and female Cyld^−/− mice (8 wks old). *, P < 0.05. (H) Adjacent localization of Cyld and NOD2 genes on human chromosome 16. The nucleotide number was based on the sequence of Homo sapiens chromosome 16 clone RP11-327F22 (GenBank/EMBL/DDBJ accession no. AC007728). The map was drawn to scale, and the transcriptional direction of the genes is indicated by arrows. Note that the two genes have the same transcriptional direction and are separated by only 8,988 nucleotides. Error bars represent SD. Bars (A and D), 200 μm; (B) 500 μm; (C) 400 μm.

Figure 3.

Induction of autoimmunity and colitis by adoptively transferred Cyld^−/− T cells. RAG1^−/− mice (8 wks old) were intravenously injected with T cells isolated from the mesenteric lymph nodes of wild-type (+/+) or Cyld^−/− mice. (A–C) After 6 wk, the recipient mice were killed for flow cytometry analyses of transferred T cells in the spleen (A), histology analyses of lymphocyte infiltration in the liver (B), and inflammation of the colon (C). The numbers in A indicate the percentages of CD8 (top left quadrants) and CD4 (bottom right quadrants) T cells. An arrow in C (bottom) indicates a colonic patch detected in a recipient of Cyld^−/− T cells. Data are representative of three mice per group. (D) Histology scores measuring the degree of colonic inflammation in the recipient wild-type (+/+) and Cyld^−/− T cells. Data were obtained from three mice per group. (E) Body weights of recipient mice. Body weights were measured 6 wk after T cell transfer, showing the substantial weight loss in the recipients of Cyld^−/− T cells. (F) Image to compare the colons of recipients of wild-type (+/+) and Cyld^−/− T cells. Error bars represent SD. Bars (B), 200 μm; (C) 1 mm.

Figure 4.

Constitutive activation of JNK and NF-κB in Cyld^−/− T cells. (A) Purified naive lymph node T cells from control (+/+) and Cyld^−/− mice were stimulated with 1 μg/ml anti-CD3 and 1 μg/ml anti-CD28 for the indicated times. Immunoblotting (IB) assays were performed using the indicated phosphospecific (α-P) and pan-antibodies to determine the phosphorylation of ZAP-70, ERK, and JNK. (B) Hyperactivation of NF-κB in Cyld^−/− T cells. Wild-type (+/+) and mutant (−/−) total lymph node T cells were stimulated with 1 μg/ml of plate-bound anti-CD3 and 1 μg/ml of soluble anti-CD28 for the indicated times. Nuclear extracts were subjected to EMSA to determine the activity of NF-κB. Actin IB was included as a loading control. (C) Nuclear extracts were isolated from purified naive and memory T cells and were subjected to EMSA to determine the constitutive activity of NF-κB. An EMSA of NF-Y was included as a control. (D) EMSA was performed using the nuclear extract isolated from untreated Cyld^−/− T cells in the presence of the indicated antibodies. The antibody-shifted complexes are indicated by arrows. (E) Whole cell extracts were isolated from wild-type (+/+) and Cyld^−/− mesenteric T cells and subjected to IB assays to detect the indicated NF-κB members. (F) RT-PCR was performed to detect mRNA of relB and Gapdh using total RNA isolated from wild-type (+/+) or Cyld^−/− mesenteric lymph node cells.

Figure 5.

Cyld deficiency results in the constitutive activation of IKKβ and chronic degradation of IκBβ. (A and B) Chronic degradation and resynthesis of IκBα in Cyld^−/− T cells. Wild-type (+/+) and mutant (−/−) total T cells were subjected to IB assays (A) or RT-PCR (B) to detect the total and phosphorylated IκBα (P-IκBα) and the IκBα messenger RNA, respectively. (C) IκBα degradation in Cyld^−/− T cells is mediated by IKKβ. Wild-type and mutant T cells were incubated for 50 min in the absence (–) or presence (+) of 10 μg/ml cycloheximide (CHX). In lane 7, the cells were preincubated with 10 μM of the IKKβ inhibitor PS1145 for 60 min before the start of cycloheximide treatment. The expression of IκBα as well as CYLD and tubulin was analyzed by IB. (D) Chronic activation of IKKβ in Cyld^−/− T cells. IKKβ was isolated by immunoprecipitation (IP; using anti-IKKβ) from untreated wild-type (+/+) and Cyld ^−/− lymph node T cells and thymocytes, and the activity of IKKβ was measured by in vitro kinase assays using both GST-IκBα and GST-IKKβ substrates (top two panels). IB was performed to monitor the IKKβ protein level (bottom). (E) Whole cell extracts isolated from purified naive T cells (untreated) were subjected to IB. (F) Whole cell extracts isolated from untreated Jurkat-pSUPER and Jurkat-shCYLD cells were subjected to IKKβ kinase assay (KA; top) followed by IB to detect the IKKβ protein on the kinase assay membrane (second blot). Cell lysates were subjected to IB to detect the indicated proteins (third to fifth blots).

Figure 6.

CYLD physically interacts with Tak1 and inhibits the ubiquitination and autoactivation of Tak1. (A) Tak1 was isolated by IP from lymph node T cells, thymocytes, or Jurkat T cells followed by kinase assays (KA) using recombinant MKK6 as a substrate (top). The kinase assay membrane was further subjected to IB to detect the Tak1 protein (bottom). (B) The CYLD complex was isolated by IP from wild-type (+/+) or Cyld^−/− thymocytes followed by IB to detect CYLD and its associated Tak1. The Tak1 level in cell lysates was monitored by IB (bottom). (C) 293 cells were transfected with 150 ng CYLD either in the absence (–) or presence (+) of 100 ng Tak1. The Tak1 complex was isolated by IP followed by IB to detect Tak1 and associated CYLD. The CYLD expression level was analyzed by IB (bottom). (D) 293 cells were transfected with 100 ng Tak1 either in the absence (–) or presence (+) of CYLD (150 ng in lane 3 and 300 ng in lane 4) along with 200 ng hemagglutinin (HA)-tagged ubiquitin. Tak1 was isolated by IP followed by IB (with antihemagglutinin) to examine its ubiquitin conjugation. (E) 293 cells were transfected with 100 ng Tak1 in the absence (–) or presence (+) of two doses of CYLD (150 and 300 ng). The cells were also transfected with 100 ng of the Tak1 partner protein Tab1. Tak1 was isolated by IP followed by kinase assays using recombinant MKK6 as substrate (top). The kinase assay membrane was further subjected to IB to detect the precipitated Tak1 (middle). CYLD expression in lysates was monitored by IB (bottom). (F) Tak1 and IKKγ were isolated by IP from wild-type (+/+) and CYLD-deficient (–/–) T cells and were subjected to IB (using antiubiquitin) to detect the ubiquitinated Tak1 and IKKγ. The membrane was reprobed with anti-Tak1 to monitor the precipitated Tak1 protein (bottom). Because IKKγ comigrates with the Ig heavy chain, direct IB was performed to monitor its expression level (bottom).