Non-canonical NF-κB activation and abnormal B cell accumulation in mice expressing ubiquitin protein ligase-inactive c-IAP2

Abstract

Chromosomal translocations between loci encoding MALT1 and c-IAP2 are common in MALT lymphomas. The resulting fusion proteins lack the c-IAP2 RING domain, the region responsible for its ubiquitin protein ligase (E3) activity. Ectopic expression of the fusion protein activates the canonical NF-κB signaling cascade, but how it does so is controversial and how it promotes MALT lymphoma is unknown. Considering recent reports implicating c-IAP1 and c-IAP2 E3 activity in repression of non-canonical NF-κB signaling, we asked if the c-IAP2/MALT fusion protein can initiate non-canonical NF-κB activation. Here we show that in addition to canonical activation, the fusion protein stabilizes NIK and activates non-canonical NF-κB. Canonical but not non-canonical activation depended on MALT1 paracaspase activity, and expression of E3-inactive c-IAP2 activated non-canonical NF-κB. Mice in which endogenous c-IAP2 was replaced with an E3-inactive mutant accumulated abnormal B cells with elevated non-canonical NF-κB and had increased numbers of B cells with a marginal zone phenotype, gut-associated lymphoid hyperplasia, and other features of MALT lymphoma. Thus, the c-IAP2/MALT1 fusion protein activates NF-κB by two distinct mechanisms, and loss of c-IAP2 E3 activity in vivo is sufficient to induce abnormalities common to MALT lymphoma.

Figure 1. c-IAP2/MALT1 fusion protein activates canonical and non-canonical NF-κB signaling.

(A) Schematic of c-IAP2, MALT1, c-IAP2/MALT fusion protein, and other mutants used. * denotes the location of the inactivating mutations. (B, C, and D) c-IAP1/MALT fusion protein activates canonical and non-canonical NF-κB signaling pathways. Lysates prepared from 293T cells that had been transfected with the indicated cDNAs for 24 h were immunoblotted with anti-FLAG, anti-phospho-IκB, anti-NIK, and anti-p52 antibodies. Because of differences in molecular weight, one can use anti-FLAG to detect and distinguish between the NF-κB activating proteins (Activator: IKKβ-CA, c-IAP2/MALT1, c-IAP2^H570A, and MALT1) and IκB. Some lanes were reordered for clarity. β-actin expression was used as a loading control. * denotes a non-specific band.

Figure 2. Generation of c-IAP2^H570A/H570A mice.

(A) Site-directed mutagenesis of the Zn²⁺-coordinating histidine residue in the RING domain of c-IAP2. Lower and upper case letters denote the nucleotide and corresponding amino acid sequence, respectively, before and after mutagenesis. The bolded amino acids highlight the histidine to alanine replacement, and the italicized nucleotides highlight the nucleotide substitutions. The box depicts the novel Spe1 restriction endonuclease site. (B) Spe1 endonuclease digestion of PCR products amplified from wild type (+/+), c-IAP2^+/H570A (+/mut), and c-IAP2^H570A/H570A (mut/mut) tail DNA. SpeI digestion of the PCR fragment amplified from the wild type (+) allele generates 4.9 and 0.7 kb fragments and the c-IAP2^H570A (mut) allele 4.3, 0.7, and 0.6 kb fragments. (C) c-IAP expression in +/+, +/mut, and mut/mut splenocytes was determined by immunoblot using an antiserum that recognizes both c-IAP1 and c-IAP2. β-actin expression was used as a loading control.

Figure 3. Secondary lymphoid homeostasis in 6–7-mo-old c-IAP2^H570A/H570A mice.

Cellularity of pooled axial, brachial, superficial cervical, and inguinal lymph nodes (Pooled LNs) (A), mesenteric lymph node (MLN) (D), and spleen (G) in wild type (+/+; n = 3) and c-IAP2^H570A/H570A (mut/mut; n = 3) mice. The error bars represent the standard error of the mean. T (TCRβ⁺B220⁻), B (TCRβ⁻B220⁺), and non-T and non-B cell (TCRβ⁻B220⁻) distribution in pooled LNs (B), MLN (E), and spleen (H) of +/+ and mut/mut mice determined by flow cytometry. Numbers represent the percentage of cells in each quadrant. Absolute number of T, B, and non-T/non-B cells in pooled LNs (C), MLN (F), and spleen (I) of +/+ and mut/mut mice. For the pooled lymph node, the absolute number was divided by the total number of lymph nodes harvested. The error bars represent the standard error of the mean. (J) Distribution of immature (CD23⁻CD21⁻), follicular (CD23⁺CD21^int), and marginal zone (CD23⁻CD21⁺) B cells (gated on B220⁺ cells) in spleen (Spl), pooled LNs, and MLN. Numbers represent the percent positive in each gate.

Figure 4. Serum immunoglobulin levels in c-IAP2^H570/H570A mice.

Wild type (+/+) and c-IAP2^H570A/H570A (mut/mut) mice were 8–10-wk-old, each dot represents the titer for an individual mouse, and the horizontal lines indicate the mean titer for each genotype.

Figure 5. Lymphocytic infiltrates in the lungs and GALT hyperplasia in c-IAP2^H570A/H570A mice.

Sections of GALT (A) and lung (B) from wild type (+/+) and c-IAP2 knockin (mut/mut) mice stained with hematoxylin and eosin. Both tissues are presented at 8× magnification and the bar denotes 100 µm. Arrows highlight the areas containing the infiltrating lymphocytes in the lungs of c-IAP2 ^H570A/H570A mice. (C) B cell distribution in GALT of +/+ and mut/mut mice. (D) Distribution of immature (CD23⁻CD21⁻), follicular (CD23⁺CD21^int), and marginal zone (CD23⁻CD21⁺) B cells (gated on B220⁺ cells) in GALT of +/+ and mut/mut mice. Numbers represent the percent positive in each gate.

Figure 6. c-IAP2^H570A/H570A B cells are more sensitive to proliferative stimuli and survive longer in vitro.

(A) Splenocytes from wild type and c-IAP2^H570A/H570A mice were cultured for the indicated times and the percentage of viable B and T cells was quantitated by staining with anti-B220, anti-TCRβ, and 7AAD. The experiment was performed in triplicate and the error bars represent the standard error of the mean. (B) Purified B cells were incubated with or without BAFF or agonistic anti-CD40 for 66 h and cell viability was determined as in (B). For the assessment of BAFF sensitivity, the percent viability of untreated cells was subtracted from the percent viability at each concentration of BAFF and the results are displayed as the increase in percent viable cells. (C) IgM- and LPS-induced proliferative responses of wild type (+/+) and c-IAP2^H570A/H570A (mut/mut) B cells. Purified B cells were cultured in vitro with the indicated concentrations of anti-μ (Fab′)₂ or lipopolysaccharide (LPS) for 48 h, pulsed with ³H-thymidine, and harvested 18 h later. The experiment was performed in triplicate and the error bars represent the standard error of the mean. (D) Adoptive transfer of equal numbers of splenic wild type (wt; CD45.1⁺) and c-IAP2^H570A/H570A (mut; CD45.2⁺) B cells into RAG2-deficient mice. Forty-five days later splenocytes and lymphocytes were prepared and the ratio of mut/wt B cells recovered was calculated by dividing the percentage of mut (B220⁺CD45.2⁺) cells by the percentage of wt (B220⁺CD45.1⁺) cells.

Figure 7. Elevated non-canonical NF-κB activation in c-IAP2^H570A/H570A cells.

(A) NF-κB target gene expression in WT (+/+) and c-IAP2^H570A/H570AB cells determined by real-time PCR. Bars represent the mean increase of each mRNA in c-IAP2^H570A/H570A cells from three independent experiments. The error bars represent the standard error of the mean. (B) Immunoblotting of phospho-IκB and IκB in lysates of +/+ and c-IAP2^H570A/H570A (mut/mut) B cells. β-actin expression was used as a loading of control. (C) Immunoblotting of phospho-IκB and IκB levels in lysates of unstimulated +/+ and mut/mut MEFs as in (B). Lysates of cells stimulated with TNF-α (10 ng/ml for 5 min) were blotted in parallel as a positive control for the phospho-IκB antibody. (D) Immunoblotting of NIK and p52 in lysates prepared from +/+ and mut/mut B and T cells. β-actin expression was used as a loading of control. (E) Immunoblotting of NIK and p52 in lysates prepared from +/+ and mut/mut MEFs as in (D). (F) Knockdown of c-IAP2 in +/+ and mut/mut MEFs. Cells were transfected with non-targeting (NT) or c-IAP2 siRNA (C2) for 24 h, harvested, and lysed. c-IAP2 and p52 levels were determined by immunoblot. β-actin expression was used as a loading of control. The lanes have been reordered for clarity. (G) 293T cells were transfected with expression vectors containing the indicated cDNAs and immunoblotted for the respective peptide tags and β-actin.