Non-Canonical NF-κB Activation and Abnormal B Cell Accumulation in Mice Expressing Ubiquitin Protein Ligase-Inactive c-IAP2

Loss of c-IAP2 ubiquitin ligase activity, which occurs in the lymphoma-causing c-IAP2/MALT1 fusion protein, activates non-canonical NF-κB signaling and results in B cell abnormalities characteristic of MALT lymphoma

Correction: Non-Canonical NF-κB Activation and Abnormal B Cell Accumulation in Mice Expressing Ubiquitin Protein Ligase-Inactive c-IAP2

<p>Correction: Non-Canonical NF-κB Activation and Abnormal B Cell Accumulation in Mice Expressing Ubiquitin Protein Ligase-Inactive c-IAP2</p

Generation of c-IAP1^H582A mice.

<p>(A) Schematic representation of the c-IAP1 targeting construct and the recombination strategy. (B) c-IAP1^H582A mice were distinguished from WT littermates by LR-PCR and Spe I digestion. (C) c-IAP1 and c-IAP2 expression in splenocytes from WT, c-IAP1^H582A heterozygous and homozygous mice were determined by immunoblotting. (D) <i>Ciap1</i> and <i>Ciap2</i> mRNA expression was determined in WT and c-IAP1^H582A splenocytes by real-time PCR. Bars represent the fold increase over WT expression. (E) WT or c-IAP1^H582A splenocytes were lysed in sample buffer after 8 hr of incubation with 25 mM chloroquine (CHQ), 1∶200 of the Sigma protease inhibitor cocktail (PIs), 10 µM MG-132, or complete medium (−) and c-IAP1 expression was analyzed by immunoblotting.</p

c-IAP2 compensates for c-IAP1^H582A in non-canonical NF-κB inhibitory- and TNFR1 complexes.

<p>(A) Expression of p100 and p52 in MEFs was detected by immunoblotting. (B and F) MEFs were untreated (−) or transfected with non-targeting control (ctr) or c-IAP2 siRNA (c2) for 24 hr and lysed. p52 and c-IAP1/2 levels were determined by immunoblotting. IκBα expression was analyzed by immunoblotting of lysates of MEFs treated for the indicated times with 1 ng/ml of TNFα (C) or 15′ with the indicated doses of TNFα (D). (F) MEFs were untreated (-) or transfected with non-targeting control (ctr) or c-IAP2 siRNA for 24 hr, then treated with 1 ng/ml TNFα for 15′ and lysed. IκBα expression was analyzed by immunoblotting. Lanes were rearranged for clarity.</p

c-IAP1 and c-IAP2 are only partially redundant in T cells.

<p>(A) Purified T cells were cultured in vitro with the indicated amount of anti-CD3 in the absence (left panel) or presence (right panel) of 2 µg/ml of anti-CD28. After 48 hr, cells were pulsed with ³H-thymidine and harvested 18 hr later. The panel represents the average of four independent experiments and the error bars are the SEM. ***<i>p</i><0.005. (B) Expression of p100 and p52 in purified T cells was detected by immunoblotting. For each sample densitometry of p100 and p52 was performed and the results expressed as the ratio between each protein and β-actin. Expression of NIK (C) and c-IAP1/2 (D) in purified T cells was detected by immunoblotting. Values in C represent the ratio between NIK and β-actin. (E) p100/52 immunoblot of T cells freshly purified or stimulated for 24 hr with 1 µg/ml anti-CD3±2 µg/ml anti-CD28. (F) T cells were stimulated as in (E) and ELISA was performed on supernatants collected after 24 hr. One of two independent experiments is shown and the error bars are the SD of the duplicates. *<i>p</i><0.05.</p

Control of Borrelia burgdorferi-Specific CD4(+)-T-Cell Effector Function by Interleukin-12- and T-Cell Receptor-Induced p38 Mitogen-Activated Protein Kinase Activity

Infection with Borrelia burgdorferi, the causative agent of Lyme disease, results in a Th1 response and proinflammatory cytokine production. Mice deficient for MKK3, an upstream activator of p38 mitogen-activated protein (MAP) kinase, develop a lower Th1 response and exhibit an impaired ability to produce proinflammatory cytokines upon infection with the spirochete. We investigated the contribution of p38 MAP kinase activity in gamma interferon (IFN-γ) production in CD4(+) T cells in response to specific antigen through T-cell receptor (TCR)- and interleukin-12 (IL-12)-mediated signals. The specific inhibition of p38 MAP kinase in T cells and the administration of a pharmacological inhibitor of the kinase during the course of infection with the spirochete resulted in reduced levels of IFN-γ in the sera of infected mice. Our results also demonstrate that although p38 MAP kinase activity is not required for the differentiation of B. burgdorferi-specific CD4(+) T cells, the production of IFN-γ by Th1 effector cells is regulated by the kinase. Both TCR engagement and IL-12 induced the production of the Th1 cytokine through the activation of the p38 MAP kinase pathway. Thus, the inhibition of this pathway in vitro resulted in decreased levels of IFN-γ during restimulation of B. burgdorferi-specific T cells in response to anti-CD3 and IL-12 stimulation. These results clarify the specific contribution of the p38 MAP kinase in the overall immune response to the spirochete and its role in the effector function of B. burgdorferi-specific T cells

Posttranscriptional Downregulation of c-IAP2 by the Ubiquitin Protein Ligase c-IAP1 In Vivo

Inhibitor of apoptosis proteins (IAPs) c-IAP1 and c-IAP2 were identified as part of the tumor necrosis factor receptor 2 (TNFR2) signaling complex and have been implicated as intermediaries in tumor necrosis factor alpha signaling. Like all RING domain-containing IAPs, c-IAP1 and c-IAP2 have ubiquitin protein ligase (E3) activity. To explore the function of c-IAP1 in a physiologic setting, c-IAP1-deficient mice were generated by homologous gene recombination. These animals are viable and have no obvious sensitization to proapoptotic stimuli. Cells from c-IAP1(−/−) mice do, however, express markedly elevated levels of c-IAP2 protein in the absence of increased c-IAP2 mRNA. In contrast to reports implicating c-IAPs in the activation of NF-κB, resting and cytokine-induced NF-κB activation was not impaired in c-IAP1-deficient cells. Transient transfection studies with wild-type and E3-defective c-IAP1 revealed that c-IAP2 is a direct target for c-IAP1-mediated ubiquitination and subsequent degradation, which are potentiated by the adaptor function of TRAF2. Thus, the c-IAPs represent a pair of TNFR-associated ubiquitin protein ligases in which one regulates the expression of the other by a posttranscriptional and E3-dependent mechanism