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Comparative Study
. 2003 Jan;4(1):82-7.
doi: 10.1038/sj.embor.embor710.

Lymphotoxin and lipopolysaccharide induce NF-kappaB-p52 generation by a co-translational mechanism

Benjamin Mordmüller  1 , Daniel Krappmann, Meral Esen, Elmar Wegener, Claus Scheidereit
Affiliations
Comparative Study

Lymphotoxin and lipopolysaccharide induce NF-kappaB-p52 generation by a co-translational mechanism

Benjamin Mordmüller et al. EMBO Rep. 2003 Jan.

Abstract

The 'classical' NF-kappaB activation pathway proceeds via IkappaB kinase (IKK)-beta/gamma-mediated phosphorylation, induced ubiquitination and the degradation of small IkappaBs. An alternative, NF-kappaB-inducing kinase and IKK-alpha-dependent pathway, which stimulates the processing of NF-kappaB2/p100, has recently been suggested. However, no physiological stimulus has been shown to trigger the activation of this pathway. Here we demonstrate that persistent stimulation with lymphotoxin beta (LT-beta) receptor agonists or lipopolysaccharide (LPS), but not with interleukin-1beta, tumour necrosis factor-alpha or 12-O-tetradecanoylphorbol-13-acetate, induces the generation of p52 DNA-binding complexes by activating the processing of the p100 precursor. Induction of p52 DNA-binding activity is delayed in comparison with p50/p65 complexes and depends on de novo protein synthesis. p100 is constitutively and inducibly polyubiquitinated, and both ubiquitination and p52 generation are coupled to continuing p100 translation. Thus, both LT-beta receptor agonists and LPS induce NF-kappaB/p100 processing to p52 at the level of the ribosome.

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Figures

Figure 1
Figure 1
LT-βR ligation and LPS treatment induce p100 processing and generation of p52 DNA-binding complexes. (A) HeLa cells were stimulated with TNF-α or LT-β and extracts were tested for NF-κB DNA-binding complexes by EMSA and for p100/p52 protein in western blots. In the middle panel, anti-p100/p52 antibody was added to each DNA binding reaction (p52 sup., supershift). (B) HeLa and MCF-7 cells were stimulated for 30 min or 4 h with TNF-α, IL-1β, TPA, LT-α1/β2 or LIGHT, and extracts were analysed for NF-κB DNA-binding activity. Anti-p100/p52 antibody was added to each DNA binding reaction. (C) 70Z/3 cells were stimulated with LPS or TPA for the times indicated and extracts were analysed by EMSA for NF-κB DNA-binding activity and for expression of NF-κB/IκB proteins by western blotting, as indicated. (D) Subunit composition of LPS-induced NF-κB complexes (at 0, 2 and 24 h, as indicated) in 70Z/3 cells was determined by supershift analysis. (E) Immature dendritic cells treated with 1 μg/ml LPS (1 and 24 h, as indicated) were analysed for NF-κB activity and subunit composition. In D and E, supershifted NF-κB complexes are indicated by open circles. Specificities of p50 and p52 antibodies were evaluated by supershifting of overexpressed or in vitro-translated proteins; no antibody that was used showed significant cross-reactivity with other NF-κB family members (data not shown).
Figure 2
Figure 2
LT-β receptor ligation- and LPS-induced p100 processing depends on IKK/NF-κB activation and protein synthesis. (A) HeLa cells were stimulated with LIGHT for 4 and 8 h alone (lanes 2 and 3), or in the presence of the protein synthesis inhibitor CHX (lanes 4–7) or the proteasomal inhibitor ALLN (lanes 8–11). Extracts were analysed for p52 DNA-binding activity by EMSA, including anti-p52 antibodies (top panel). p100/p52 protein amounts were determined by western blotting (middle panel), and Oct1 DNA-binding activity was determined as an internal control. (B) HeLa cells were activated by either TNF-α (30 min) or LIGHT (6 h) in the presence or absence of CHX, and p52 DNA-binding activity was analysed by the addition of supershifting anti-p52 antibodies to the NF-κB EMSA reaction. (C) 70Z/3 and 1.3E2 cells, or 70Z/3 cells infected with control retrovirus (CV) or with FlagIκBαΔN-expressing retrovirus (FIκBαΔN), were stimulated with LPS or LPS/CHX as indicated, and tested for NF-κB DNA-binding activity (top panel) and for p100/p52, RelB, p65 and IκBα/IκBαΔN proteins (lower panels). As judged by trypan blue exclusion, CHX did not strongly affect the viability of cells during the time of incubation. In every case we observed more than 90% viable cells (data not shown). ns, non-specific. (D) Mock-transfected and IκBαΔN-transfected dendritic cells (DC) were treated with LPS and NF-κB DNA-binding activity; p100, p52 and IκBα protein levels were determined, as indicated.
Figure 3
Figure 3
Translation-dependent constitutive and induced p100 polyubiquitination. (A) MCF-7 cells were treated with LIGHT for 4 h in the absence or presence of CHX and proteasomal degradation was blocked by ALLN for the last hour of incubation. (B) 70Z/3 and 1.3E2 cells were treated with LPS for 5 h. CHX was added, where indicated, 2 h before lysis; ALLN was included for the last 1 h. (C) 70Z/3 cells were treated with LPS for 20 min after preincubation with CHX and/or ALLN, as indicated. (D) 70Z/3 cells were treated with LPS (5 h) and ALLN (1 h) before extraction. Extracts were immunoprecipitated (IP) either with anti-p100/p52, anti-IκBα or anti-IKK-α antibodies and western blotting (WB) of the input material and after immunoprecipitation was performed as indicated on both sides of the panels. ns, non-specific.
Figure 4
Figure 4
LT-β receptor ligation- and LPS-induced processing of p100 requires continuing protein synthesis. (A) Left panel, MCF-7 cells were treated with solvent or LIGHT for 3 h in the presence of [35S]methionine to pulse-label the cells. Right panel, HeLa cells were treated with solvent, LIGHT or TNF-α for 4 h. [35S]methionine was added to the cells during the last 1 h of stimulation (pulse). Samples were immunoprecipitated with an isotype control (lanes 1 and 2) or anti-p100/p52 antibody (lanes 3 and 4), and precipitates were analysed by SDS–PAGE followed by autoradiography. The band corresponding to p52 in lanes 4 and 6 is marked with an open circle. (B) HeLa cells were labelled with [35S]methionine for 1 h before stimulation and the chase was performed in the presence of cytokines, as indicated. Immuno-precipitation was performed as in A and analysed by SDS–PAGE followed by autoradiography. The migration of p100 and p52 is indicated. (C) 70Z/3 cells were treated with LPS for 4 or 8 h, as indicated (lanes 2–4). CHX was added during the last 4 h of stimulation with LPS or for 4 h alone (lanes 4 and 5). NF-κB DNA-binding activity was analysed by EMSA and NF-κB/IκB protein amounts were determined by western blotting. Enhanced p52 protein amounts in lane 2 are indicated by an open circle. ns, non-specific. (D) 70Z/3 cells were either left untreated or were stimulated with LPS for 4 h. During the last 1.5 h, cells were labelled with [35S]methionine. Cells were washed and incubated in complete medium again in the absence or presence of LPS for up to 2 h. After immunoprecipitation, p100/p52 and IκBα were analysed by SDS–PAGE followed by autoradiography. The positions of p100, p52 and IκBα are indicated.
Figure 5
Figure 5
Model for production of p50 and p52 from the NF-κB precursor molecules NF-κB1/p105 and NF-κB2/p100 after stimulation. On activation with TNF-α or LPS, NF-κB1/p105 is completely degraded post-translationally, leading to a release of p105-bound Rel molecules. LT-βR ligation and LPS rapidly activate canonical IKK/NF-κB signalling, which leads to upregulation of the nf-κB2/p100 gene. Both pathways also induce the delayed co-translational processing of p100 and thereby the generation of homodimeric or heterodimeric p52 complexes.
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