Nucleolar sequestration of RelA (p65) regulates NF-kappaB-driven transcription and apoptosis

Abstract

The molecular mechanisms that regulate nuclear NF-kappaB to determine whether the stimulation of this pathway has a pro- or anti-apoptotic effect on cells have yet to be fully defined. Nuclear compartmentalization is increasingly recognized as an important mechanism for regulating the activity of transcription-related proteins and modulating cell growth and death. We have investigated whether such compartmentalization serves as a mechanism for regulating NF-kappaB transcriptional activity. We demonstrate that the RelA component of NF-kappaB is sequestered in the nucleolus in response to the proapoptotic NF-kappaB stimuli aspirin, serum withdrawal, and UV-C radiation. In contrast, RelA is excluded from the nucleolus in response to the cytokines tumor necrosis factor and TRAIL. We identify an N-terminal motif of RelA that is essential for the nucleolar localization of the protein and show that deleting this motif inhibits the translocation of RelA from the nucleoplasm to the nucleolus. We demonstrate that the nucleolar accumulation of RelA is paralleled by a decrease in basal levels of NF-kappaB transcriptional activity and by apoptosis. Furthermore, we show that the retention of RelA in the nucleoplasm inhibits this decrease in NF-kappaB-driven transcription and blocks apoptosis induced by aspirin and UV-C radiation. This work identifies a novel cellular mechanism for regulating NF-kappaB-driven transcription and apoptosis, involving the nucleolar sequestration of a key NF-kappaB subunit. These data contribute to the understanding of the complexities of NF-kappaB function and have considerable relevance to cancer prevention and therapy.

FIG. 1.

RelA accumulates in distinct nuclear bodies following aspirin treatment. (A) Immunomicrograph (magnification, ×63) showing the subcellular distributions of RelA in control and aspirin-treated (0.5 to 2 mM for 72 h or 3 to 5 mM for 16 h) SW480 cells. Nuclear accumulations of RelA are observed at pharmacologically relevant doses. Nuclei are stained with DAPI. Bars, 10 μm. (B) SW480 cells were transiently transfected with a GFP-RelA expression vector 24 h prior to aspirin (0 [−] or 5 [+] mM for 16 h) stimulation. Fluorescent and phase-contrast images (magnification, ×40) of adherent cells were captured using an Axiovert 100 inverted microscope. Arrows indicate nucleoli.

FIG. 2.

RelA localizes in the nucleolus in response to aspirin. (A) Immunocytochemical staining (magnification, ×63) showing that RelA colocalizes with the nucleolar proteins nucleolin and fibrillarin, but not with Ki67, when induced by aspirin. SW480 cells were unstimulated (control) or stimulated with aspirin (5 mM, 16 h) and then fixed, and immunocytochemistry was performed using the specified antibodies. DNA is stained by DAPI. Bars, 10 μm. (B) RelA is held within nucleolar bodies after aspirin treatment. The immunomicrographs (magnification, ×63) show the cellular localization of RelA in aspirin (Asp.)-treated (5 mM, 16 h) SW480 cells with (+) and without (−) permeabilization (Perm.) prior to fixation and immunocytochemical analysis. Nucleolin (C23) staining depicts nucleoli. Merged image shows DAPI-stained DNA (blue). (C) Aspirin induces a decrease in nucleoplasmic and an increase in nucleolar levels of RelA. Nontreated and aspirin (Asp.)-treated (5 mM, 16 h) SW480 cells were fractionated using sucrose gradients. Western blot analysis was then performed on protein extracted from the specified cell fractions using anti-RelA followed by anti-nucleolin (C23) antibodies.

FIG.3.

Specificity of stimuli causing nucleolar localization of RelA. (A) SW480 cells were unstimulated (control) or stimulated with TNF (10 ng/ml, 1 h) and then fixed, and immunocytochemistry was performed using specified antibodies. Nucleoli are stained with nucleolin. (B) Immunocytochemistry was performed on SW480 cells either untreated or treated with TRAIL (10 ng/ml) for 16 h. Fibrillarin was used to identify nucleoli. (C) SW480 cells were seeded in medium containing 10% serum and grown for 16 h. Medium was then replaced by fresh medium containing either 10% or 0% serum, and cells were grown for a further 96 h. Following fixation, immunocytochemistry was performed using antibodies to RelA and nucleolin as described above. (D) Immunocytochemistry, using anti-RelA and anti-nucleolin antibodies, was performed on SW480 cells 5 h after mock or UV-C (40 J/m²) irradiation. In all merged panels, DNA is stained by DAPI and appears blue.

FIG.4.

Mode of nucleolar accumulation of RelA. (A) Immunocytochemical staining showing localization of RelA in SW480 cells treated with aspirin (Asp; 5 mM for 16 h) in the presence (+) and absence (−) of the inhibitor of nuclear export, leptomycin B (LMB), and of the inhibitor of IκB degradation, PDTC. Nucleoli are stained with nucleolin (C23), and nuclei are stained with DAPI. (B) Expression of superrepressor IκB inhibits nucleolar accumulation of RelA. Parental HRT18 cells and two clones expressing mutant IκB, resistant to aspirin-induced degradation (IκB SR1 and SR28) were untreated (control) or treated with 5 mM aspirin for 16 h. The cellular localization of RelA was determined using immunocytochemical staining. (C) Nucleolar accumulation of RelA requires de novo protein synthesis. The immunomicrographs show the cellular localization of RelA and nucleolin following treatment with aspirin (Asp.) (5 mM, 16 h) in the presence and absence of 0.5 μg/ml actinomycin D (ActD) or 10μM cycloheximide (CHX). The arrow indicates the exclusion of RelA from remaining nucleoli in actinomycin D-treated cells. (D) Immunomicrographs showing the cellular localization of RelA in cells exposed to 0% serum for 96 h in the presence or absence of actinomycin D (ActD) or cycloheximide (CHX) as described above. DNA is stained by DAPI and appears blue in the merged panels. Bars, 10 μm.

FIG.5.

Aspirin-induced nucleolar translocation of RelA requires an N-terminal nucleolar localization motif. (A) Deletions of RelA were cloned into the PEGFP-C1 vector. RHD, Rel homology domain; NLS, nuclear localization signal; NES, nuclear export signal. The distributions of the respective deletions are shown. +, nucleolar; −, nucleoplasmic. (B) Deleting the N-terminal 50 aa of RelA blocks nucleolar translocation of the protein. SW480 cells were transfected with the specified GFP-tagged RelA expression constructs 24 h prior to treatment with 0 or 5 mM aspirin. The cellular distribution of GFP-tagged protein was determined in live/adherent cells using an Axiovert 100 inverted fluorescent microscope (magnification, ×40). (C) The N terminus of RelA contains a potential NoLS. Amino acids 1 to 50 of RelA were aligned to other published nucleolar localization signals (19, 47) using ClustalW. Each shaded area indicates a potential NoLS. (D) Deletion of aa 27 to 30 inhibits nucleolar translocation of RelA. SW480 cells were transfected with the RelA Δ27-30 expression construct 24 h prior to treatment with 0 or 5 mM aspirin. The cellular distribution of GFP-tagged protein was determined as described above. (E) Nucleolar localization of RelA does not involve posttranslational modification of lysine (K) 28. SW480 cells were transfected with a full-length GFP-RelA expression construct containing a K-to-R (arginine) mutation at residue 28 (RelA K28/R). The cellular distribution of this mutant was determined following aspirin treatment as described above.

FIG. 6.

Nucleolar localization of RelA is paralleled by a decrease in NF-κB-driven transcription. (A and B) Pharmacologically relevant doses of aspirin repress NF-κB transcriptional activity. SW480 cells were transfected with 6 μg of wild-type (3x κB ConA and HIV-LTR) NF-κB-dependent luciferase reporter constructs or with equivalent plasmids with κB sites deleted (HIVΔκB, ConAΔκB) along with 3 μg of pCMVβ control plasmid. Twenty-four hours after transfection, cells were either unstimulated or stimulated with aspirin for a further 16 (3 to 10 mM) or 72 (0 to 2 mM) hours. Results were normalized using β-galactosidase activity and are presented as the percentages of relative luciferase activity compared to basal levels (untreated controls). (C) Aspirin induces apoptosis. Annexin V-FITC apoptosis assays were performed on aspirin-treated (0 to 2 mM, 72 h) SW480 cells. Apoptotic cells were visualized using fluorescent microscopy, and the percentages of apoptotic cells within the total cell populations were calculated. (D) Aspirin-induced apoptosis is not enhanced in low-serum conditions. Annexin V-FITC apoptosis assays were performed on SW480 cells treated with aspirin (Asp.) (0 or 0.5 mm, 72 h) in low-serum (0.5% FCS) or normal-serum (10% FCS) conditions. (E) Aspirin induces degradation of IκB. Immunoblot showing cytoplasmic levels of IκBα and control protein (Cu/ZnSOD) in SW480 cells treated with 0 to 2 mM aspirin for 72 h or 3 to 10 mM aspirin for 16 h. (F) Aspirin induces nuclear translocation of RelA/NF-κB complexes. SW480 cells were either unstimulated (−) or stimulated (+) with aspirin (5 mM for 16 h) or TNF (10 ng/ml, 1 h). Nuclear extracts were prepared and analyzed by EMSAs using a κB oligonucleotide. The incubation of extracts with the RelA antibody prior to the assay resulted in a complete shift of theaspirin-induced complex, confirming the complex consists predominantly of this component. The arrow indicates the supershifted band. The aspirin-induced complex was also competed out with 100× molar excess of unlabeled κB oligonucleotide. (G) Phosphorylation of RelA serine 536 in response to aspirin. SW480 cells were treated with aspirin (0 to 5 mM, 16 h) or TNF (10 ng, 1 h), and then immunoblot analysis was performed on total-cell extract. Blots were initially probed with a RelA phospho 536-specific antibody (Calbiochem) and then stripped and reprobed for total RelA. (H) Expression of superrepressor IκB blocks aspirin-mediated repression of NF-κB transcriptional activity. HRT18 parental cells and clones IκBSR1 and SR28 (which are resistant to aspirin-induced nuclear and nucleolar translocation of RelA) were transfected with the κB (HIV-LTR) and pCMV-β reporter constructs prior to a 16-h treatment with 0 or 5 mM aspirin. Results shown are normalized using β-galactosidase activity and are presented as described above.

FIG. 7.

IκB degradation, nucleolar translocation of RelA, repression of NF-κB transcription, and apoptosis occur sequentially. (A) IκB degradation occurs 2 to 5 h after stimulation with aspirin. Cells were treated with aspirin (10 mM) for 0 to 16 h, and then cytoplasmic IκBα and control protein (CU/Zn SOD) levels were determined using Western blot analysis. (B and C) Nucleolar accumulation of RelA (p65) is apparent 5 to 8 h after aspirin stimulation. Immunocytochemistry was performed on SW480 cells treated as described above. DAPI staining depicts nuclei. Nucleoli are stained with nucleolin. Bars, 10 μm. The percentage of cells in the population showing nucleolar RelA (p65) was quantified. Data are the means of at least three experiments (± SE). (D) Decreased NF-κB-driven transcription occurs concurrently with nucleolar accumulation of RelA. SW480 cells were transfected with the NF-κB dependent 3xκB ConA-luc and the control pCMVβ reporter constructs. Twenty-four hours after transfection, cells were treated as described above. Values are presented as the percentages of luciferase activity at time zero and are the means (± SE) of three independent experiments after β-galactosidase normalization. (E) Aspirin-induced caspase-3 cleavage occurs 16 h after stimulation. SW480 cells were treated as described above and levels of uncleaved caspase-3 determined by Western blot analysis performed on cytoplasmic extracts. (F) Percentages of SW480 cells undergoing apoptosis, as determined by annexin V staining, following aspirin treatment as described above.

FIG.8.

Inhibiting nucleolar translocation of RelA blocks aspirin-induced repression of NF-κB-driven transcription and apoptosis. (A) Exogenally expressed RelA Δ27-30 blocks nucleolar translocation of endogenous RelA. Immunomicrographs (magnification, ×63) showing localization of all (RelA) or GFP-tagged (GFP) RelA in SW480 cells transfected with GFP-WT (a to f) or Δ27-30 (g to o) RelA and then treated with 0 (−) or 5 (+) mM aspirin (Asp) for 16 h. Panels m to o show RelA in nontransfected cells (confirmed by the lack of a GFP signal in panel n) adjacent to the RelA Δ27-30-transfected cells shown in panels j to l. Blue depicts DAPI staining of DNA. Bars, 10 μm. (B) Deletion of amino acids 27 to 30 of RelA inhibits aspirin effects on NF-κB-driven transcription. SW480 cells were transfected with the GFP-RelA WT or Δ27-30 expression vector, along with the 3x κB ConA-luc reporter plasmid. Following aspirin treatment (5 mM, 16 h), GFP-tagged cells were isolated using a fluorescence-activated cell sorter. Relative luciferase activity was calculated as the level of luciferase activity per GFP-expressing cell in the population. Results are presented as the reduction in relative luciferase activity compared to untreated controls and are the means of at least three experiments (± SE). The basal (12.3 × 10⁶; SE, 3.2 × 10⁶) levels of luciferase activity were comparable between cells expressing WT-RelA and those expressing Δ27-30-RelA (11 × 10⁶; SE, 1.55 × 10⁶). (C) Immunoblots showing GFP and control protein (Actin) expression levels in cells transfected with GFP-tagged WT or Δ27-30 RelA before and after aspirin (5 mM, 16 h) treatment. (D) Deletion of amino acids 27 to 30 of RelA inhibits aspirin effects on cell viability. SW480 cells were transfected with the indicated RelA expression vectors 24 h prior to stimulation with aspirin (5 mM, 16 h). Phase-contrast images of randomly selected fields are shown (magnification, ×10). (E) Deletion of amino acids 27 to 30 of RelA inhibits aspirin-induced apoptosis. SW480 cells were transfected with the specified vectors and treated with aspirin as described above. Annexin V-biotin staining, with a Texas Red-streptavidin conjugate, was used to identify apoptotic cells. The percentages of cells expressing GFP-tagged RelA undergoing apoptosis were determined by fluorescent microscopy in at least 250 transfected cells for each sample. The number of apoptotic cells in the nontransfected population was also determined. The results are the means of at least three independent experiments ± standard errors.

FIG. 9.

Amino acids 27 to 30 of RelA are essential for aspirin effects on NF-κB transcriptional activity and apoptosis in rela null MEFs. (A) rela null MEFs were transfected with 3 μg of GFP-RelA WT or -RelAΔ27-30. Cells were either unstimulated (−Asp.) or stimulated for 5 h with aspirin (10 mM) (+Asp.), and then the localization of GFP-tagged proteins was analyzed in fixed cells using fluorescent microscopy (magnification, ×63). (B and C) rela null MEFs were transfected with the above plasmids along with 3 μg of 3xκB ConA NF-κB dependent luciferase reporter constructs and 3 μg of pCMVβ control plasmid. Twenty-four hours after transfection, cells were either unstimulated, (B) stimulated with aspirin (10 mM) for the times specified, or (C) stimulated with TNF (10 ng/ml) for 5 h. Results were normalized using β-galactosidase activity and are presentedas the percentages of relative luciferase activity compared to basal levels (untreated controls). The basal luciferase values for cells expressing GFP-RelAWT (26.36 × 10⁶; SE, 8.6 × 10⁶) and those expressing GFP-RelA Δ27-30 (17.15 × 10⁶; SE, 5.9 × 10⁶) were not significantly different. (D) Western blot analysis showing expression levels of GFP-RelA WT and Δ27-30 in aspirin-treated and untreated rela null MEFs. (E) The NoLS of RelA is required for aspirin-induced apoptosis. SW480 cells were transfected with the specified vectors and treated with aspirin as shown. Annexin V-biotin staining, with a Texas Red-streptavidin conjugate, was used to identify apoptotic cells. The percentages of cells expressing GFP-tagged RelA undergoing apoptosis were determined by fluorescent microscopy in at least 250 transfected cells for each sample. The results are the means of at least three independent experiments ± standard errors.

FIG. 10.

Nucleolar localization of RelA is required for UV-C-induced apoptosis. (A) Deleting aa 27 to 30 inhibits UV-C-induced nucleolar translocation of RelA. SW480 cells were transfected with 6 μg of GFP, GFP-RelA WT, or RelA Δ27-30 24 h prior to UV-C treatment (40 J/m²). The localization of GFP-tagged proteins was determined in live cells using an Axiovert 100 inverted fluorescent microscope (magnification, ×40) 8 h after treatment. (B) Expression of RelA Δ27-30 inhibits UV-C effects on cell viability. SW480 cells were transfected and UV-C treated as described above. Phase-contrast images (magnification, ×20) taken of randomly selected fields 24 h after treatment are shown. (C) Expression of RelA Δ27-30 inhibits UV-C effects on apoptosis. Annexin V apoptosis assays were performed on SW480 cells transfected with the specified GFP-tagged vectors 24 h after UV-C (40 J/m²) treatment. The percentages of cells expressing GFP-tagged protein undergoing apoptosis were determined by fluorescent microscopy in at least 250 transfected cells for each sample. The results are the means of at least three independent experiments ± standard errors.

FIG. 11.

A model for the compartmental modulation of NF-κB transcriptional activity and apoptosis. In nonstimulated tumor cells, there are two pools of NF-κB, an inducible cytoplasmic pool and a basal pool that drives the transcription of antiapoptotic genes. Upon induction by aspirin, UV-C radiation, serum deprivation, and similar stimuli, inducible RelA/NF-κB complexes translocate into the nucleus, recruiting an additional cofactor (AC) to the basal pool. The induced RelA/cofactor complex does not activate transcription but complexes with basal RelA/NF-κB, and then all nuclear RelA translocates to the nucleolus. Once in the nucleolus, RelA is in a physical location different from that of its target promoters, resulting in a decrease in transcription of antiapoptotic genes and, consequently, apoptosis.