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Science 3 August 2007:
Vol. 317. no. 5838, pp. 675 - 678
DOI: 10.1126/science.1142953
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Reports

Negative Regulation of Toll-Like Receptor Signaling by NF-{kappa}B p50 Ubiquitination Blockade

Ruaidhrí J. Carmody, Qingguo Ruan, Scott Palmer, Brendan Hilliard and Youhai H. Chen*

Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.


Figure 1 Fig. 1. Bcl-3 inhibits cytokine gene expression and controls NF-{kappa}B dimer exchange at gene promoters. (A) Bcl3–/– macrophages are hyperresponsive to LPS. Bone marrow–derived (BMD) macrophages were stimulated with LPS, and cytokine mRNA was quantified by real-time polymerase chain reaction (PCR) (25). Error bars indicate ±SEM. (B) Bcl3–/– dendritic cells are hyperresponsive to LPS. BMD dendritic cells were stimulated with LPS, and gene expression was measured by real-time PCR. (C) Enhanced proliferation of Bcl3–/– B cells to LPS. Splenic B cells were stimulated with LPS, and 3H-thymidine incorporation was measured as count per minute (cpm). (D) Enhanced gene expression in Bcl3–/– B cells. B cells were stimulated with LPS, and TNF{alpha} gene expression was measured by real-time PCR. (E) Bcl3–/– macrophages have reduced p50 DNA binding. Nuclear extracts from BMD macrophages were tested by EMSA with the consensus NF-{kappa}B–binding sequence and indicated antibodies. Arrow indicates p50 complexes. (F) Altered NF-{kappa}B dimer loading and exchange at gene promoters in Bcl3–/– macrophages. BMD macrophages were treated with LPS, and ChIP was performed with antibodies to the indicated factors. [View Larger Version of this Image (51K GIF file)]
 

Figure 2 Fig. 2. Bcl-3 inhibits p50 ubiquitination and degradation. (A) Bcl-3 increases p50 homodimer binding to DNA without altering its affinity. Human embryonic kidney (HEK) 293T cells were transfected with p50 and Bcl-3 expression plasmids. EMSA was performed with increasing amounts of unlabeled NF-{kappa}B consensus oligonucleotides (cold probe) (left), and p50 homodimer DNA binding was measured by densitometry (middle). Relative protein levels were determined by immunoblotting of whole-cell extracts (right). WB, Western blot. (B) Increased p50 homodimer DNA binding is associated with increased amounts of p50 protein. HEK 293T cells were transfected with XP-p50 and increasing amounts of myc-Bcl-3 plasmids. p50 and Bcl-3 levels were measured in whole-cell lysates by immunoblotting (top), and p50 homodimer DNA binding was measured by EMSA (bottom). Empty, empty vector alone. (C) Bcl-3 increases the half-life of p50. HEK 293T cells were cotransfected with XP-p50 and empty vector or myc-Bcl-3, and the half-life (t1/2) of proteins was determined (25). IP, immunoprecipitation. (D) p50 undergoes Lys48 (K48) polyubiquitination. HEK 293T cells were transfected with XP-p50 and expression vectors encoding either WT, Lys48->Arg48 (K48R) mutant, Lys63->Arg63 (K63R) mutant, or lysine-less (KØ) HA-tagged ubiquitin (Ub). Lysates were immunoprecipitated with antibody against XP and immunoblotted with antibody against HA. (E) Bcl-3 inhibits p50 ubiquitination. HEK 293T cells were transfected with XP-p50 and HA-ubiquitin with or without myc-Bcl-3. Ubiquitination was determined as in (D). [View Larger Version of this Image (41K GIF file)]
 

Figure 3 Fig. 3. Bcl3–/– macrophages have increased p50 ubiquitination and degradation. (A) Increased ubiquitination of p50 in Bcl3–/– macrophages. BMD macrophages were treated with (+) or without (–) LPS for 16 hours. Equal amounts of protein were immunoprecipitated with antibody against p50 and immunoblotted with antibody against ubiquitin. (B) Reduced p50 half-life in Bcl3–/– macrophages. BMD macrophages were treated with LPS, pulse-labeled with 35S-methionine cysteine, and tested as in Fig. 2C. (C) Generation of a p50 mutant that does not bind to DNA. HEK 293T cells were transfected with XP-p50 or an XP-p50 mutant containing Lys57->Ala57 (Y57A) and Gly60->Asp60 (G60D) substitutions in the DNA binding domain. EMSA was performed with the consensus NF-{kappa}B binding sequence. (D) The p50 mutant is resistant to ubiquitination. Cells were transfected with HA-ubiquitin plus XP-p50, XP-p50Y57A,G60D, or empty vector. Ubiquitination was determined as in Fig. 2D. (E) The p50 mutant has a significantly increased half-life. HEK 293T cells were co-transfected with XP-p50 or XP-p50Y57A,G60D, and protein half-life was determined (25). [View Larger Version of this Image (38K GIF file)]
 

Figure 4 Fig. 4. Bcl3 deficiency in mice and macrophages abolishes LPS tolerance. (A) Lack of LPS tolerance in Bcl3–/– macrophages. BMD macrophages were pretreated with (+) or without (–) LPS for 24 hours. After 1 hour of resting, cells were restimulated with LPS for an additional hour. mRNA levels were determined by real-time PCR. (B) Reduced p50 homodimer binding to TNF{alpha} promoter in Bcl3–/– macrophages under tolerizing conditions. BMD macrophages were treated as in (A). ChIP was performed with antibodies to p50, p65, and c-Rel. (C) Bcl3 deficiency in hematopoietic cells renders mice hypersensitive to septic shock. WT mice were lethally irradiated and reconstituted with either WT or Bcl3–/– bone marrow (BM) cells (n from 3 to 5) (25). Eight weeks later, chimeric mice were tolerized with two consecutive injections of low dose LPS (5 mg/kg on day –5 and 10 mg/kg on day –3) and then challenged with three high doses of LPS (15 mg/kg on day 0, 30 mg/kg on day 6, and 90 mg/kg on day 9 as indicated by arrows). Data shown are survival curves of the two groups. The difference between the two groups is statistically significant (P < 0.01). Error bars indicate ± SEM. [View Larger Version of this Image (38K GIF file)]
 

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