A discrete ubiquitin-mediated network regulates the strength of NOD2 signaling

Abstract

Dysregulation of NOD2 signaling is implicated in the pathology of various inflammatory diseases, including Crohn's disease, asthma, and sarcoidosis, making signaling proteins downstream of NOD2 potential therapeutic targets. Inhibitor-of-apoptosis (IAP) proteins, particularly cIAP1, are essential mediators of NOD2 signaling, and in this work, we describe a molecular mechanism for cIAP1's regulation in the NOD2 signaling pathway. While cIAP1 promotes RIP2's tyrosine phosphorylation and subsequent NOD2 signaling, this positive regulation is countered by another E3 ubiquitin ligase, ITCH, through direct ubiquitination of cIAP1. This ITCH-mediated ubiquitination leads to cIAP1's lysosomal degradation. Pharmacologic inhibition of cIAP1 expression in ITCH(-/-) macrophages attenuates heightened ITCH(-/-) macrophage muramyl dipeptide-induced responses. Transcriptome analysis, combined with pharmacologic inhibition of cIAP1, further defines specific pathways within the NOD2 signaling pathway that are targeted by cIAP1. This information provides genetic signatures that may be useful in repurposing cIAP1-targeted therapies to correct NOD2-hyperactive states and identifies a ubiquitin-regulated signaling network centered on ITCH and cIAP1 that controls the strength of NOD2 signaling.

Fig 1

Transcriptome analysis reveals cIAP1-specific pathways, biological functions, and TFs affected upon NOD2 activation. RNA-Seq analysis was performed with primary BMDMs from C57BL/6 mice treated with the cIAP1 inhibitor MEBS at 30 μM or with the vehicle for 30 min and left untreated or additionally stimulated with 10 μg/ml MDP for 4 h. (A) Canonical pathway analysis comparing differential gene expression in vehicle-plus-MDP-treated cells and MEBS-plus-MDP-treated cells indicates that, in the context of NOD2 activation, cIAP1 is involved in the processes of LTβR signaling, endocytic trafficking, and cytoskeletal signaling, aside from the well-described activation of the NF-κB and MAPK pathways. (B) Functional analysis of the same data set indicates that, in the context of NOD2 activation, cIAP1 is involved mainly in chemotaxis and phagocytosis of macrophages. (C) TF analysis of differentially expressed genes upon MDP stimulation and cIAP1 inhibition suggests that cIAP1 affects mainly the STAT1 and IRF1 TFs and their corresponding genes. TFs were ranked by z score, where the top 15 TFs induced by MDP treatment are shown. The regulation z score predicts the activation state of the TF, with the gene expression patterns of the TF and its downstream genes. An absolute z score of ≥2 is considered significant. A TF is activated if the z score is ≥2 and inhibited if the z score is ≤−2.

Fig 2

qRT-PCR and cIAP1 expression inhibition by siRNA validate the RNA-Seq analysis and pharmacologic data. (A) MEBS downregulates various inflammatory cell surface molecules, cytokines, chemokines, and enzymes induced by MDP. Primary BMDMs were generated and experimental treatments were performed as described in the legend to Fig. 1. The genes tested for validation were chosen from those determined by RNA-Seq analysis to be upregulated by MDP that were also inhibited by MEBS. FAS and CCL5 were chosen as controls, as they were upregulated by MDP but unaffected by MEBS treatment when analyzed by RNA-Seq. qRT-PCR showed that these transcripts tested by qRT-PCR behaved in a fashion similar to that seen in transcriptomic analysis. (B) To validate the specificity of MEBS, cIAP1 expression was inhibited by siRNA. WT C57BL/6 BMDMs were transfected with two different siRNAs against cIAP1 with Fugene. Twenty-four hours later, cells were treated with the vehicle or MDP and RNA was harvested as outlined above. Inhibition of cIAP1 expression via siRNA knockdown mirrored that seen with MEBS. (C) A second cell-permeating cIAP1 antagonist, smac n7, was used to verify the results in panel A. WT C57BL/6 BMDMs were pretreated with the vehicle or smac n7 for 30 min prior to MDP treatment, and RNA was harvested as outlined above. Inhibition of cIAP1 expression via smac n7 treatment mirrored that seen with MEBS.

Fig 3

cIAP1 promotes tyrosine autophosphorylation of RIP2. (A) RIP2 tyrosine autophosphorylation, a marker of RIP2 activation, is induced by cIAP1. HEK293 cells were transfected for 24 h with 3 μg of WT or kinase-inactive RIP2 (RIP2 K47A) in the absence or presence of cIAP1 or ligase-deficient cIAP1 (H588A). Five minutes before being harvested, cells were pretreated with pervanadate to irreversibly inhibit tyrosine phosphatases. Lysates were collected, and RIP2 was immunoprecipitated via its Omni tag and immunoblotted with an antiphosphotyrosine antibody. cIAP1 promoted tyrosine autophosphorylation of WT RIP2 but not RIP2 K47A. Such tyrosine autophosphorylation did not occur with ligase-deficient cIAP1. (B) RIP2 tyrosine phosphorylation is induced by cIAP1 in a dose-dependent manner. HEK293 cells were transfected for 24 h with 3 μg of RIP2 and increasing doses of cIAP1 (0, 1, 2, 3, and 4 μg). RIP2 was immunoprecipitated, and Western blotting was performed. Expression of cIAP1 enhanced RIP2 tyrosine phosphorylation in a dose-dependent manner. (C) The cIAP1-mediated increase in RIP2 tyrosine phosphorylation is reversed by siRNA knockdown of cIAP1. HEK293 cells were transfected for 24 h with 3 μg each of RIP2 and cIAP1 with or without 5 nM control siRNA (c) or two different siRNAs specific to cIAP1 (1 and 2). RIP2 was immunoprecipitated, and Western blotting was performed. Knockdown of cIAP1 decreases RIP2 tyrosine phosphorylation. (D) Competition with endogenous cIAP1 ligase activity can reduce NOD2-induced RIP2 tyrosine phosphorylation. HEK293 cells were transfected for 24 h with 0.5 μg NOD2, 3 μg RIP2, and increasing amounts of E3 ligase-defective cIAP1 H588A (0, 1, 2, and 3 μg). RIP2 was immunoprecipitated, and Western blotting was performed. NOD2-induced RIP2 tyrosine phosphorylation is inhibited by ligase-deficient cIAP1 in a dose-dependent manner, suggesting that NOD2-induced activation of RIP2 requires the ligase activity of cIAP1. (E) Prevention of cIAP1-mediated RIP2 ubiquitination also prevents RIP2 tyrosine phosphorylation. HEK293 cells were transfected for 24 h with 1 μg of ubiquitin, 3 μg each of either WT RIP2 or RIP2 K209R, and 3 μg each of either WT cIAP1 or cIAP1 H588A. RIP2 was immunoprecipitated via its Omni tag and immunoblotted with an antiphosphotyrosine antibody or an anti-HA (ubiquitin) antibody. Prevention of cIAP1-mediated RIP2 ubiquitination by using a ubiquitin conjugation-defective mutant form of RIP2 (K209R) or by using ligase-defective cIAP1 also prevented cIAP1-mediated RIP2 tyrosine phosphorylation. (F) The effects of cIAP1 on RIP2 tyrosine phosphorylation are nullified in the presence of ITCH. HEK293 cells were transfected for 24 h with 1 μg of ubiquitin and 3 μg each of RIP2, cIAP1, and ITCH. RIP2 was immunoprecipitated via its Omni tag and immunoblotted with an antiphosphotyrosine antibody or an anti-HA (ubiquitin) antibody. Both E3 ligases promote RIP2 ubiquitination, but only cIAP1 promotes RIP2 tyrosine phosphorylation. In the presence of both cIAP1 and ITCH, the RIP2 phosphorylation state resembles that of RIP2 in the presence of ITCH alone. rbt, rabbit; ms, mouse; gt, goat; Ub, ubiquitin.

Fig 4

ITCH ubiquitinates cIAP1, but cIAP1 does not ubiquitinate ITCH. (A) HEK293 cells were transfected for 24 h with 1 μg of ubiquitin and 3 μg each of ITCH, ITCH C830A, and cIAP1 H588A. cIAP1 H588A was immunoprecipitated under stringent washing conditions (1 M NaCl, 1% SDS) via its Myc tag and immunoblotted with an anti-HA (ubiquitin) antibody. cIAP1 is ubiquitinated in the presence of ligase-active but not ligase-defective ITCH. (B) cIAP1 does not ubiquitinate ITCH. HEK293 cells were transfected for 24 h with 1 μg of ubiquitin and 3 μg each of cIAP1, cIAP1 H588A, and ITCH C830A. ITCH H588A was immunoprecipitated under stringent washing conditions via its Flag tag and immunoblotted with an anti-HA (ubiquitin) antibody. ITCH is not ubiquitinated by cIAP1 despite the ability of cIAP1 to ubiquitinate RIP2 under similar conditions. (C, D) cIAP1 ubiquitination occurs endogenously and in an agonist-dependent manner. HT29 human intestinal epithelial cells stably transduced with NOD2 (C) or RAW 264.7 murine macrophages (D) were stimulated with MDP for 0, 30, 60, or 90 min. Endogenous cIAP1 was immunoprecipitated with an anti-cIAP1 antibody and immunoblotted with an antibody against ubiquitin (P4D1). cIAP1 undergoes endogenous agonist-dependent ubiquitination in both human and mouse cell lines. rbt, rabbit; ms, mouse; gt, goat; Ub, ubiquitin.

Fig 5

ITCH promotes lysosomal degradation of cIAP1, and loss of ITCH leads to increased levels of cIAP1. (A) HEK293 cells were transfected with 1 μg of ubiquitin and 3 μg each of cIAP1 H588A, ITCH, and ITCH C830A. cIAP1 H588A was immunoprecipitated via its Myc tag and immunoblotted with a K63 linkage-specific ubiquitin antibody (D7A11). (B) HEK293 cells were transfected with 1 μg of WT HA-tagged ubiquitin or with three different HA-tagged mutant forms of ubiquitin allowing only K29, K48, or K63 ubiquitin linkages and 3 μg each of cIAP1 H588A and ITCH. cIAP1 H588A was immunoprecipitated via its Myc tag and immunoblotted with an anti-HA antibody. The use of both a K63 linkage-specific antibody and a K63-only mutant form of ubiquitin confirms that ITCH-mediated cIAP1 ubiquitination occurs through K63 linkages. (C) ITCH decreases the half-life of cIAP1, and this is reversed by the use of ligase-deficient ITCH. HEK293 cells were transfected for 24 h with 1 μg ubiquitin; 2 μg Omni-cIAP1 H588A; and no ITCH, 3 μg WT ITCH, or 3 μg ligase-dead ITCH (ITCH C830A). Cells were treated for 0, 2, 4, or 6 h with the translational inhibitor CHX at 20 μg/ml to inhibit any further protein synthesis. The half-life of cIAP1 is decreased in the presence of ITCH, while it is prolonged in the presence of ligase-deficient ITCH. (D) ITCH promotes lysosomal degradation of cIAP1. HEK293 cells were transfected for 24 h with 1 μg of ubiquitin and 3 μg each of cIAP1 H588A and ITCH with overnight incubation after the addition of the vehicle, the lysosomal inhibitor chloroquine at 50 μM, or the proteasomal inhibitor MG132 at 10 μM at 6 h after transfection. Treatment with chloroquine but not MG132 considerably increased the levels of cIAP1, suggesting that ITCH is mediates the lysosomal degradation of cIAP1. (E) BMDMs from WT or Itchy (ITCH-deficient) mice were either left untreated or treated with MDP for 30 min before harvesting of the cells for analysis of cIAP1 levels. Levels of cIAP1 were increased in BMDMs deficient in ITCH. rbt, rabbit; ms, mouse.

Fig 6

ITCH competes with cIAP1 for binding to RIP2. (A, B) Reciprocal binding experiments with RIP2, ITCH, and cIAP1 suggest competition between ITCH and cIAP1 for binding to RIP2. HEK293 cells were transfected for 24 h with 2 μg N-terminal tandem affinity purification tag-labeled RIP2, 2 μg Omni-cIAP1, and 2 μg FLAG-ITCH, alone or in combination. RIP2 or cIAP1 was precipitated with streptavidin-agarose (Strep.) (A) or Omni antibody (B) and immunoblotted for RIP2, ITCH, or cIAP1. The presence of ITCH inhibited cIAP1-RIP2 binding. (C) ITCH can compete with cIAP1 for binding to RIP2. HEK293 cells were transfected for 24 h with 3 μg N-terminal tandem affinity purification tag-labeled RIP2 and 3 μg Omni-cIAP1. The complex was purified with streptavidin-agarose and incubated with increasing amounts of GST-tagged recombinant ITCH. GST alone was used to keep the recombinant protein levels constant between conditions. Western blotting showed that as the amounts of ITCH increased, the levels of cIAP1 bound to RIP2 decreased. (D) Model of cIAP1- and ITCH-mediated NOD2 activation and deactivation. Activation of NOD2 results in the recruitment of RIP2 and binding of cIAP1 for ubiquitination of RIP2 at lysine 209 (K209). Such ubiquitination results in the activation of RIP2, as evidenced by RIP2 tyrosine autophosphorylation. This then allows downstream signal transduction events to occur. Deactivation of NOD2 signaling is mediated by the recognition of activated, tyrosine-phosphorylated RIP2 by ITCH, which competes with cIAP1 for binding to RIP2. While in this complex, ITCH promotes the ubiquitination of both RIP2 and cIAP1, leading to cIAP1 lysosomal degradation and, ultimately, downregulation of NOD2 responses. rbt, rabbit; ms, mouse; gt, goat.

Fig 7

MEBS downregulates the hyperactive NOD2 response in ITCH^−/− macrophages. (A) Loss of ITCH increases the phosphorylation levels of both cIAP1 and RIP2 tyrosine. HEK293 cells were transfected with 1 μg HA-NOD2 to induce the activation of RIP2 in the absence or in the presence of a control siRNA or four different siRNAs against ITCH. Endogenous RIP2 or cIAP1 were immunoprecipitated to determine the effects of ITCH knockdown on RIP2 activity and cIAP1 levels. Decreasing ITCH through siRNA knockdown increases the tyrosine phosphorylation levels of both cIAP1 and RIP2 (activation). (B) MEBS downregulates cIAP1. WT C57BL/6 BMDM cells were treated overnight with increasing doses of MEBS (7.5, 15, 30, and 60 μM) or with smac n7, a second cIAP1 antagonist (15 μM). Endogenous levels of cIAP1 were downregulated by MEBS in a dose-dependent manner. (C) MEBS reduces the increased levels of inflammatory mediators in BMDMs from ITCH^−/− mice to WT levels. BMDMs were pretreated with 30 μM MEBS for 30 min prior to the administration of 10 μg/ml MDP for 4 h before RNA harvesting. RT-PCR assays of various inflammatory cell surface molecules, cytokines, chemokines, and enzymes identified by RNA-Seq analysis in Fig. 1 were performed. The experiments were performed concurrently with those in Fig. 2. In most of the genes tested, ITCH loss caused a 40 to 60% increase in MDP-induced genes and MEBS treatment reduced the exacerbated NOD2 inflammatory response in BMDMs from ITCH^−/− mice to WT levels. (D) To validate the specificity of MEBS, cIAP1 expression was inhibited by siRNA. Itchy BMDMs were transfected with two different siRNAs against cIAP1 with Fugene. Twenty-four hours later, cells were treated with the vehicle or MDP and RNA was harvested as outlined above. Inhibition of cIAP1 expression via siRNA knockdown corrected the exacerbated chemokine responses in the Itchy mice, mirroring the results seen with MEBS. rbt, rabbit; ms, mouse; gt, goat.