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. 2006 Oct 30;203(11):2413-8.
doi: 10.1084/jem.20061166. Epub 2006 Oct 2.

Rescue of TRAF3-null mice by p100 NF-kappa B deficiency

Jeannie Q He  1 , Brian Zarnegar, Gagik Oganesyan, Supriya K Saha, Soh Yamazaki, Sean E Doyle, Paul W Dempsey, Genhong Cheng
Affiliations

Rescue of TRAF3-null mice by p100 NF-kappa B deficiency

Jeannie Q He et al. J Exp Med. .

Abstract

Proper activation of nuclear factor (NF)-kappaB transcription factors is critical in regulating fundamental biological processes such as cell survival and proliferation, as well as in inflammatory and immune responses. Recently, the NF-kappaB signaling pathways have been categorized into the canonical pathway, which results in the nuclear translocation of NF-kappaB complexes containing p50, and the noncanonical pathway, which involves the induced processing of p100 to p52 and the formation of NF-kappaB complexes containing p52 (Bonizzi, G., and M. Karin. 2004. Trends Immunol. 25:280-288). We demonstrate that loss of tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) results in constitutive noncanonical NF-kappaB activity. Importantly, TRAF3-/- B cells show ligand-independent up-regulation of intracellular adhesion molecule 1 and protection from spontaneous apoptosis during in vitro culture. In addition, we demonstrate that loss of TRAF3 results in profound accumulation of NF-kappaB-inducing kinase in TRAF3-/- cells. Finally, we show that the early postnatal lethality observed in TRAF3-deficient mice is rescued by compound loss of the noncanonical NF-kappaB p100 gene. Thus, these genetic data clearly demonstrate that TRAF3 is a critical negative modulator of the noncanonical NF-kappaB pathway and that constitutive activation of the noncanonical NF-kappaB pathway causes the lethal phenotype of TRAF3-deficient mice.

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Figures

Figure 1.
Figure 1.
TRAF3 deficiency results in constitutive processing of p100 in MEFs. (A) WT and TRAF3 −/− MEFs were stimulated with an anti-LTβR antibody for the indicated times. Processing of p100 to p52 in whole cell extract was detected by immunoblot. Total β actin is shown as a loading control. (B) Whole cell extracts from WT and TRAF3 −/− MEFs reconstituted with either pBABEpuro-TAP or pBABEpuro-TAP-TRAF3 were assessed for basal p100 processing to p52 by immunoblot. Reconstitution of TRAF3 expression in TRAF3 −/− MEFs was confirmed by immunoblot. Total β actin is shown as a loading control. *, endogenous TRAF3.
Figure 2.
Figure 2.
TRAF3-deficient B lymphocytes display ligand-independent activation. (A) WT and TRAF3 −/− B cells were stimulated with BAFF, anti-CD40 antibody, or LPS for 24 h, and processing of p100 to p52 was detected by immunoblot. β actin is shown as a loading control. (B) WT and TRAF3 −/− B cells were stimulated with BAFF or anti-CD40 antibody for 96 h, and cell death was measured by staining with propidium iodide. Error bars indicate ±1 SD between duplicate samples. WT and TRAF3 −/− B cells were stimulated with BAFF for 48 h, and (C) analysis of ICAM-1 protein expression was determined by FACS and (D) images of cells forming homotypic aggregates were taken (see In vitro B cell assays for details).
Figure 3.
Figure 3.
Enhanced NIK expression levels in TRAF3-deficient cells. (A) 150 μg of whole cell extracts from v-ABL–transformed WT and TRAF3 −/− B cells, 3T3s, and primary MEFs were assessed by immunoblotting for NIK. p100 processing to p52 in these cells was confirmed by immunoblot. *, NIK +/+ and NIK −/− 3T3s were treated with MG132 for 2 h to serve as a control. (B) WT and TRAF3 −/− MEFs were transfected with 150 nM control or NIK-siRNA. Whole cell extracts were obtained 48 h after transfection, and NIK and p100/p52 levels were detected by immunoblot. β actin is shown as a loading control.
Figure 4.
Figure 4.
Rescue of TRAF3-null phenotypes by combined deletion of the p100 gene. The rescue of the TRAF3-null phenotype was examined by body size (A), survival (B), spleen size and total splenocyte count (C; error bars indicate ±1 SD between three mice), and serum glucose and corticosterone concentration (D; error bars indicate ±1 SD between two and five mice).
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