CYLD and the NEMO Zinc Finger Regulate Tumor Necrosis Factor Signaling and Early Embryogenesis

Abstract

NF-κB essential modulator (NEMO) and cylindromatosis protein (CYLD) are intracellular proteins that regulate the NF-κB signaling pathway. Although mice with either CYLD deficiency or an alteration in the zinc finger domain of NEMO (K392R) are born healthy, we found that the combination of these two gene defects in double mutant (DM) mice is early embryonic lethal but can be rescued by the absence of TNF receptor 1 (TNFR1). Notably, NEMO was not recruited into the TNFR1 complex of DM cells, and consequently NF-κB induction by TNF was severely impaired and DM cells were sensitized to TNF-induced cell death. Interestingly, the TNF signaling defects can be fully rescued by reconstitution of DM cells with CYLD lacking ubiquitin hydrolase activity but not with CYLD mutated in TNF receptor-associated factor 2 (TRAF2) or NEMO binding sites. Therefore, our data demonstrate an unexpected non-catalytic function for CYLD as an adapter protein between TRAF2 and the NEMO zinc finger that is important for TNF-induced NF-κB signaling during embryogenesis.

Keywords: CYLD; NEMO; NF-κB (NF-KB); cell signaling; immunodeficiency; protein complex; tumor necrosis factor (TNF).

FIGURE 1.

CYLD and NEMO lysine 392 are required for embryonic development. A, genotyping results from CYLD^+/−/NEMO-KR heterozygous intercross. B, gross appearance of CYLD^+/+/NEMO-KR, CYLD^+/−/NEMO-KR, and CYLD^−/−/NEMO-KR embryos at day 12.5. C, PCR genotyping of genomic tail DNA of WT, CYLD-KO, NEMO-KR, TNFR1-KO, and CYLD-KO/NEMO-KR/TNFR1-KO mice. Mut, mutant. D, PCR genotyping of genomic DNA (left panel) and Western blot (WB) analysis of CYLD and NEMO protein expression (right panel) in WT, CYLD-KO (KO), NEMO-KR (KR), and DM MEFs. E, TNF-induced cell death. Cell viability was measured after treatment of WT, CYLD-KO, NEMO-KR, and DM MEFs with 10 ng/ml TNF-α (left panel) or the indicated TNF-α concentrations (right panel) for 16 h. Viability is expressed relative to that of untreated cells (100%). m/m, mutant/mutant; NS, non-specific. F, WT and DM MEFs were treated with TNF-α for 16 h alone or in combination with z-VAD (TNF/Z) or necrostatin (TNF/N). Data are means ± S.D. G, cleavage of caspase-3 and PARP following treatment with TNF-α. WT and DM MEFs were treated with the indicated reagents for 6 h followed by Western blotting of cytoplasmic extracts with antibodies specific for caspase-3 and PARP. The same membrane was reprobed with anti-β-actin antibody as a control to show equal protein loading. T, TNF-α; C, cycloheximide; T/C, TNF-α/cycloheximide; T/C/Z, TNF-α/cycloheximide/z-VAD.

FIGURE 2.

Defective NF-κB signaling in double mutant MEFs. A, WT, CYLD-KO (KO), NEMO-KR (KR), and DM MEFs were treated with 10 ng/ml TNF-α for the indicated times followed by Western blotting of cytoplasmic extracts with antibody specific for phospho-IκBα (P-IκBα) or phospho-IKKα/β (P-IKKα/β). The membrane was reprobed with antibodies against IKKβ and GAPDH as controls. B, impaired phosphorylation of IKKα/β in the NEMO complex of DM MEFs. Lysates were prepared from MEFs following treatment with 10 ng/ml TNF-α. The NEMO protein complex was analyzed by immunoprecipitation with an anti-NEMO antibody followed by immunoblotting with antibodies specific for phospho-IKKα/β, IKKβ, and NEMO. C, WT, CYLD-KO, NEMO-KR, and DM MEFs were treated with 10 ng/ml TNF-α for the indicated times followed by Western blotting of cytoplasmic extracts with antibody specific for phospho-JNK (P-JNK) or phospho-ERK (P-ERK). The membrane was reprobed with antibodies against JNK and ERK as controls. D, impaired TNF-induced KC production in DM MEFs. MEFs were stimulated with 10 ng/ml TNF-α for 20 h, and the KC concentration in culture supernatant was determined by ELISA. E, KC expression in WT and DM MEFs following a 20-h treatment with increasing concentrations of TNF-α. Data are means ± S.D.

FIGURE 3.

RIP ubiquitination and degradation are enhanced in DM MEFs in response to TNF-α stimulation. A, cell lysates were prepared from WT, CYLD-KO (KO), NEMO-KR (KR), and DM MEFs following treatment with 10 ng/ml TNF-α for 5 min. RIP was immunoprecipitated (IP) with anti-RIP antibody followed by immunoblotting (IB) with antibodies that recognize specific forms of linked ubiquitin (Ln Ub). Control lanes contain Lys-48-ubiquitin (K48 Ub), Lys-63-ubiquitin (K63 Ub), or tri-linear ubiquitin chains. WB, Western blotting. B, cell lysates prepared from MEFs stimulated with 1 μg/ml Fc-TNF-α were immunoprecipitated with protein G beads and subjected to immunoblotting with antibodies specific for RIP, CYLD, or TNFR1. Specific protein levels in cell lysates were determined by Western blotting as indicated (lower panels). C, cell lysates were prepared from WT and DM MEFs following stimulation with 10 ng/ml TNF-α, immunoprecipitated with an anti-FADD antibody, and immunoblotted with antibodies specific for RIP or FADD. The lower panels show specific protein levels in cell lysates determined by Western blot analysis. NS, non-specific. D, cell lysates were prepared from MEFs stimulated with 10 ng/ml TNF-α for the indicated times followed by immunoblotting with antibodies specific for RIP or β-actin. E, cell lysates were prepared from DM MEFs stimulated with increasing concentrations of TNF-α in the presence or absence of 25 μm MG-132 for 6 h followed by immunoblotting with antibodies specific for RIP or β-actin.

FIGURE 4.

NEMO from DM MEFs is not recruited into the TNFR1 platform. A, lysates were prepared from WT or DM MEFs following treatment with 1 μg/ml TNF-α. The TNFR1 complex was immunoprecipitated (IP) with TNFR1 antibody followed by immunoblotting (IB) with anti-NEMO antibody or IKKβ antibody. The lower panels show immunoblot analysis of lysates using the indicated antibodies. WB, Western blotting. B, NF-κB luciferase activities were measured after treatment with 10 ng/ml TNF-α in DM MEFs transfected with various CYLD plasmids (upper left panel), as confirmed by immunoblot analysis (upper right panel). CAP-Gly, cytoskeleton-associated protein-glycine-rich; UCH, ubiquitin C-terminal hydrolase. Data are means ± S.D. C, cell lysates were prepared from DM MEFs transfected with CYLD-H870N at the indicated time points after treatment with 10 ng/ml TNF-α followed by immunoblotting with antibodies specific for IκBα or β-actin. D, lysates were prepared from WT or DM MEFs treated with 10 ng/ml IL-1α for the indicated times and subjected to immunoblot analysis with antibodies specific for IκBα or β-actin. E, lysates were prepared from WT or DM MEFs following treatment with 10 ng/ml LPS for the indicated times and subjected to immunoblot analysis with antibodies specific for IκBα or β-actin.