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. 2003 Feb;14(2):491-502.
doi: 10.1091/mbc.e02-07-0404.

IkappaBalpha and p65 regulate the cytoplasmic shuttling of nuclear corepressors: cross-talk between Notch and NFkappaB pathways

Lluís Espinosa  1 , Julia Inglés-Esteve, Alex Robert-Moreno, Anna Bigas
Affiliations

IkappaBalpha and p65 regulate the cytoplasmic shuttling of nuclear corepressors: cross-talk between Notch and NFkappaB pathways

Lluís Espinosa et al. Mol Biol Cell. 2003 Feb.

Abstract

Notch and NFkappaB pathways are key regulators of numerous cellular events such as proliferation, differentiation, or apoptosis. In both pathways, association of effector proteins with nuclear corepressors is responsible for their negative regulation. We have previously described that expression of a p65-NFkappaB mutant that lacks the transactivation domain (p65DeltaTA) induces cytoplasmic translocation of N-CoR leading to a positive regulation of different promoters. Now, we show that cytoplasmic sequestration of p65 by IkappaBalpha is sufficient to both translocate nuclear corepressors SMRT/N-CoR to the cytoplasm and upregulate transcription of Notch-dependent genes. Moreover, p65 and IkappaBalpha are able to directly bind SMRT, and this interaction can be inhibited in a dose-dependent manner by the CREB binding protein (CBP) coactivator and after TNF-alpha treatment, suggesting that p65 acetylation is modulating this interaction. In agreement with this, TNF-alpha treatment results in downregulation of the Hes1 gene. Finally, we present evidence on how this mechanism may influence cell differentiation in the 32D myeloid progenitor system.

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Figures

Figure 1
Figure 1
Stabilization of IκBα induces cytoplasmic translocation of NCoRs. (A and B) Subcellular localization of endogenous N-CoR protein in NIH-3T3 cells (A, upper panel) and nuclear staining with DAPI (A, lower panel) or transfected eGFP-p65 fusion protein (B) in cells incubated for 1 h in control media, LPS (1 μg/ml), mTNF-α (25 ng/ml), or PDTC (100 μM). α-N-CoR antibody was detected with FITC-conjugated secondary antibody. (C) NIH-3T3 cells were cotransfected with N-CoR (upper panels) or SMRT (lower panels) along with empty vector (left panels) or IκBα32–36 (right panels). Subcellular localization of the ectopic N-CoR and SMRT proteins was detected with α-flag antibody and FITC-conjugated secondary antibody. These are representative images of three independent experiments.
Figure 2
Figure 2
Stabilization of IκBα exerts a positive effect on the transcriptional activation of the Notch-dependent Hes1 promoter. (A) N1-IC (0.75 μg) was cotransfected along with either Hes1-luc or 2xκB-luc reporter in NIH-3T3 cells. Luciferase activity was measured in the cells after 12 h incubation in the presence or absence of 20 μM PDTC. (B) Hes1-luciferase activity of NIH-3T3 cells cotransfected with N1-IC (0.75 μg) and pCMV-flp65 (0.25 μg) plasmids and increasing amounts of the IκBα32–36 mutant. Luciferase activity is represented as the fold induction relative to cells transfected with empty vector. CMV-β-galactosidase was cotransfected as internal control in all experiments and equivalent amounts of N1-IC protein and increasing amounts of IκBα32–36 were confirmed by immunoblotting (unpublished data). The average and SD of duplicates from one of three independent experiments are presented.
Figure 3
Figure 3
p65-NFκB physically interacts with SMRT corepressor. (A and B) NIH-3T3 cells were cotransfected with the Gal4DBD-SMRT-D (5 ng) and (A) two different p65 constructs fused to the VP16 TA domain (0.5 μg) or (B) VP16-TAD-p65(1–551) in the presence or absence of 0.5 μg of the activated PKA catalytic subunit mutant (His87, Trp196) along with the 5xGal4-luc reporter. (C) Amino acids 444–455 of p65 are required for the p65/SMRT interaction. Cells were cotransfected with the indicated p65 constructs fused to VP16-TA (0.5 μg). Numbers in brackets correspond to the amino acids present in the p65 construct. p65 constructs are represented (RHD, rel homology domain; NLS, nuclear localization signal; NES, nuclear export signal; TAD, transactivation domain). Luciferase activity is represented as the fold induction relative to cells transfected with empty vector. The average and SD of duplicates from one representative of three independent experiments are presented. CMV-β-galactosidase was cotransfected as internal control in all experiments. Expression levels of the different p65 constructs were determined by Western blot using an α-Ha antibody (lower panels).
Figure 4
Figure 4
p65-NFκB and IκBα32–36 coimmunoprecipitates with SMRT. Whole-cell lysates from 293T cells cotransfected with the indicated plasmids were immunoprecipitated with the anti-myc antibody followed by immunoblotting with anti-Ha. 1/50 of the input is showing in the left panel. Lower panels show the same membrane stripped and reprobed with anti-myc to detect SMRT. (Ig H, immunoglobulin heavy chain)
Figure 5
Figure 5
Both p65-NFκB and IκBα are implicated in the cytoplasmic translocation of NCoRs. (A) Endogenous N-CoR subcellular localization was determined in p65−/− and p65+/+ MEFs after 2 h incubation in the presence or absence of PDTC (100 μM). α-N-CoR was detected with FITC-labeled secondary antibody (upper panels), and nuclei were visualized by DAPI staining (lower panels). (B) Expression of NFκB family members in p65−/− and p65+/+ MEFs. Cell lysates were electrophoresed and immunoblotted with anti-p65, anti-c-rel, and anti-IκBα antibodies. β-Actin was detected to ensure equal protein loading. (C) Endogenous N-CoR subcellular localization was determined in IκBα−/− and IκBα+/+ MEFs after 1 h incubation in the presence or absence of PDTC (100 μM). Cells were stained as above. (D) p65−/− and IκBα−/− MEFs were cotransfected with SMRT-flag and an empty vector or IκBα32–36 as indicated. SMRT protein was detected with α-flag antibody and FITC-conjugated secondary antibody. These are representative images of three independent experiments.
Figure 6
Figure 6
Binding of SMRT corepressor to p65 and IκBα is negatively regulated by CBP coactivator or TNF-α treatment. (A) CBP coactivator competes with SMRT for interacting with p65. NIH-3T3 cells were cotransfected with DBD-SMRT-D and VP16-TAD-p65(96–551) with increasing amounts (125, 250, and 500 ng) of the CBP coactivator. Lower panels show the immunoblot to confirm the expression of equivalent amounts of p65 and the increasing amounts of CBP. (B) DBD-SMRT-D and VP16-TAD-p65(96–551) interaction was assayed in the presence of 0.6 and 1.2 μM of the HDAC inhibitor TSA. (C) 293T cells were transfected with the indicated constructs and incubated with media alone, 0.6 μM TSA (4 h), or 40 ng/ml hTNF-α. Whole-cell lysates were immunoprecipitated with the anti-myc antibody followed by immunoblotting with anti-Ha. One-fiftieth of the input is showing in the left panel. Arrowheads indicate coimmunoprecipitated p65 (open) and IκBα (filled); IgH, immunoglobulin heavy chain. (D) Hes1-luc (left) and 6xκB-luc (right) activity measured in NIH-3T3 cells cotransfected with N1-IC (0.75 μg) and pCMV-flp65 (0.25 μg) when indicated, after 12 h incubation in medium alone (black bar) or 25 ng/ml mTNF-α (gray bar). Luciferase activity is represented as the fold induction, relative to cells transfected with empty vector. CMV-β-galactosidase was cotransfected as internal control. The average and SD of duplicates from one of three independent experiments are presented. (E) Activation by TNF-α or inhibition by PDTC of the NFκB pathway modifies the mRNA levels of different Notch-target genes. Northern blot of total RNA from 293T cells exposed to media, hTNF-α (40 ng/ml) or PDTC (100 μM) for 3 h. Membranes were hybridized with 32P-labeled DNA probes for the Notch-target genes Hes1 and Herp2. IκBα probe was used as a control for NFκB activity. Ethidium bromide–stained 18s rRNA is shown as loading control.
Figure 7
Figure 7
Stabilization of IκB by PDTC inhibits G-CSF–induced differentiation in N2-IC–expressing 32D cells. (A) N2-IC–expressing cells were induced to differentiate in the presence or absence of PDTC (10 μM). Graphs represent the percentage of blast-like undifferentiated cells (○) and band neutrophil-like differentiated cells (▪) during 6 d incubation in G-CSF. Cells with intermediate phenotype are not represented. Graphs show the average and SD of two different clones. Photographs of representative cells at day 6 of differentiation in the absence (left) or presence (right) of PDTC are shown in the lower panels. (B) Hes1p-eGFP activity was assayed in control and N2-IC-32D cells (used in A), after 24 h in G-CSF with or without PDTC. eGFP intensity was measured by flow cytometry and is represented relative to the control in the absence of PDTC for each cell type. One stably transfected Hes1p-eGFP clone for 32D wt and two clones for N2-IC-32D cells were evaluated. Values are the average and SD of three independent experiments. (C) N-CoR subcellular localization in 32D control cells or cells incubated 1 h with PDTC (100 μM). α-N-CoR was detected with a FITC-labeled secondary antibody (upper panels), and nuclei were visualized by DAPI staining (lower panels). (D) Model for Notch and NFκB cross-talk to regulate myeloid 32D cell differentiation. See DISCUSSSION for details.
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