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Rheumatology Research and Advanced Therapeutics, 272, NCMLS, Radboud University Medical Centre Nijmegen, P.O. Box 9101, Geert Grooteplein 26-28, 6500 HB Nijmegen, The Netherlands Department of Biomolecular Chemistry, IMM & NCMLS, Radboud University Nijmegen, 6500 HB Nijmegen, The Netherlands c Centre for Rheumatology, Department of Endocrinology, Diabetology and Rheumatology, Heinrich-Heine University of Düsseldorf, 40225 Düsseldorf, Germany d Deutsches Krebsforschungszentrum (DKFZ) Heidelberg, 69120 Heidelberg, Germany e The Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands b
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Article history: Received 27 June 2008 Available online xxxx
Nucleoporin 88 kDa (Nup88) is a tumor marker, overexpressed in various types of cancer. In Drosophila Nup88 (mbo) was reported to selectively mediate the nucleocytoplasmic transport of NF-jB, an ubiquitous transcription factor involved in immune responses, apoptosis, and cancer. We addressed the function of Nup88 in mammalian cells. Selective depletion of Nup88 by small interfering RNA (siRNA) inhibited NF-jB-dependent reporter gene activation and the nuclear translocation of NF-jB without affecting the upstream activation pathway in NIH3T3 cells. In contrast, nuclear translocation of glucocorticoid receptor was not reduced by the depletion of Nup88. In metastatic melanoma cells overexpressing Nup88, constitutive activation of NF-jB was found both in nucleus and cytoplasm. Nup88 depletion in these cells reduced TNF-induced nuclear accumulation of NF-jB subunits. We conclude that Nup88 regulates the activity of NF-jB at the level of nucleocytoplasmic transport. Overexpression of Nup88 in tumor cells may, thus be involved in the constitutive NF-jB activation. Ó 2008 Elsevier Inc. All rights reserved.
Keywords: Nuclear pore complex NF-jB Nucleocytoplasmic transport Nucleoporin Cancer cells Reporter gene assay
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Nucleoporin 88 kDa (Nup88) is a component of the nuclear pore complex (NPC) that mediates nucleocytoplasmic trafficking of macromolecules [1]. Nup88 is reported to be located at the cytoplasmic face of NPC in tight association with CAN/Nup214, another nucleoporin and a proto-oncogene implicated in leukemia [2–4]. It is a putative tumor marker highly expressed in cancer and embryonic tissues, and is postulated to play a role in oncogenesis and development [5,6]. Furthermore its expression levels highly correlate with the rate of metastasis and mortality of colon cancer [7] and with aggressiveness of breast cancer [8]. However, the functional consequences of Nup88 overexpression in cancer remain unknown to date. A study carried out in Drosophila reported that members only (mbo), an ortholog of Nup88 (26% homology to human Nup88), selectively regulated the nuclear translocation or export of dorsal, a member of the Rel protein family, which comprises NF-jB [9–11]. NF-jB is a ubiquitous transcription factor mediating the
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Nozomi Takahashi a,*,1, Jeroen W.J. van Kilsdonk b,1, Benedikt Ostendorf a,c, Ruben Smeets a, Sophia W.M. Bruggeman b,e, Angel Alonso d, Fons van de Loo a, Matthias Schneider c, Wim B. van den Berg a, Guido W.M. Swart b
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Tumor marker nucleoporin 88 kDa regulates nucleocytoplasmic transport of NF-jB
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* Corresponding author. Fax: +31 24 354 04 03. E-mail address:
[email protected] (N. Takahashi). 1 These authors contributed equally to this study.
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induction of a wide range of genes involved in immune response, inflammation, and apoptosis [12,13]. Constitutive activation of NF-jB is found in cancer, and its role in cancer is supported by a large body of evidence [14,15]. NF-jB, a dimer composed of subunits of the Rel family of proteins, is retained in the cytoplasm by its inhibitory partner, inhibitor of NF-jB (IjB). The paradigm of canonical NF-jB activation involves several critical steps and its signaling pathway is triggered by activation of IjB kinase (IKK) complex, subsequent phosphorylation and degradation of IjB allowing the release of an active dimer of NF-jB [12]. Little is known, however, about any particular regulation at the nuclear membrane. We postulated that mammalian Nup88 would exert a similar function as its Drosophila ortholog in regulating the nuclear localization of NF-jB, and that overexpression of Nup88 in cancer could affect NF-jB activity. In order to underscore a link between Nup88 and NF-jB signaling, we have depleted Nup88 by short interfering (si)RNA [16] and evaluated the functional consequences on NF-jB activity by reporter gene assay, immunofluorescence and electrophoretic mobility shift assay (EMSA).
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.06.128
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Cell culturing. NIH3T3 and BLM cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS. TNFa and dexametasone (DEX) were purchased from R&D systems (Minneapolis, MN) and Sigma (St. Louis, MO), respectively. siRNA. Sense and anti-sense ssRNA corresponding to the target sequences (Table 1A) were purchased as HPLC purified oligonucleotides (RNA-TEC, Leuven, Belgium) and annealed to generate dsRNA. The cells were transfected with dsRNA (100 nM) using oligofectamine (Invitrogen, Carlsbad, CA). RNA isolation and quantitative (Q)-RT-PCR. Cells were seeded at the density of 6 105 cells/well, and transfected with siRNA 24 h later. RNA was isolated 24 h after the transfection using RNeasy kit (Qiagen) and reverse-transcribed by standard methods. Q-PCR was performed by the ABI/Prism 7000 using SYBR Green Master mix (Both Applied Biosystems Foster City, CA). Primer sequences used for the analysis are enlisted in Table 1B. Luciferase reporter gene assay. NIH3T3 cells were transfected with a plasmid construct carrying five consensus NF-jB binding sites followed by a luciferase reporter gene, and a stable clone with the highest responses to TNFa and IL-1 was selected (NF-jB-luc/ 3T3). NF-jB-luc/3T3 cells were seeded in a crystal 96-well microtiter plate (Porvair) at the density of 3 104 cells/well 24 h before transfection. At 24 h after transfection the cells were stimulated with TNFa (10 ng/ml). After 6 h stimulation cells were harvested and the lysate was analyzed for luciferase activity using the Bright-Glo luciferase assay system (Promega, Madison, WI) and luminomeric detection according to the manufacturer’s instruction (Polarstar Galaxy, BMG LabTechnologies, Offenburg, Germany). Electrophoretic mobility shift assay (EMSA). Cells were grown in six-well plate, fractionated into cytoplasmic and nuclear fraction 1 h after TNF stimulation and were subjected to EMSA as previously described [17,18]. We have used the following probe representing the NF-jB binding site of IL-6 promoter: 50 -AGCTATGTGGGATTTTCCCATGAGC-30 (underlined: single jB motif). Ten micrograms of lysate was loaded on each lane. The supershift experiments were performed with anti-p65 and antip50 antibodies (Santa Cruz Biotechnology). Immunofluorescence (IF). NF-jB-luc/3T3 cells (4 104 cells/ well) or BLM cells (2 104 cells/well) were seeded in an eight-well Lab-TekII chamber slide (Porvair, Shepperton, UK) 24 h before the transfection. Twenty-four hours after the transfection, the cells were stimulated with TNF (50 ng/ml) for 1 h or with DEX (100 lM) for 1/2 h, washed with PBS and fixed with methanol at 20 °C. We used rabbit anti-NF-jBp65 antibody, rabbit anti-GR antibody, goat anti-NF-jBp50 antibody (Santa Cruz Biotechnology) and anti-Nup88 monoclonal antibody [6]. FITC or TXRD conjugated secondary antibodies (SouthernBiotech,
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Table 1 Sequences used Gene
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Nup88 gene knock-down by siRNA reduces NF-jB-dependent gene activation
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Birmingham, AL) were used. Nup88 antibody was labeled with Alexa 647 using a labeling kit (Molecular Probes). The cells were analyzed by confocal fluorescence microscopy using the Leica TCS NT (Leica GmbH, Germany). Statistical analysis. Data are represented as means ± SD. Level of significance for comparisons between two-independent samples were determined using Student’s t-test.
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Materials and methods
To assess the functional relationship between Nup88 and NF-
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(A) PCR primers GAPDH 50 -GGCAAATTCAACGGCACA-30 Nup88 NF-jBp65
50 -GTTAGTGGGGTCTCGCTCCTG30 50 -CATGTTGCACTTATCGGAAGTAAAG- 50 -CAATCGGGATGGTGCTACAAT30 30 50 -GCTACGGCGGCCTTCTG-30 50 -CAATC CGGTGGCGATCA-30 GenBank Accession No. Target sequence
(B) siRNA target sequences mNup88 AJ532593 mNF-jBp65 BC003818 hNup88 NM_002532 hNF-jBp65 NM_021975 hALCAM NM_001627
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jB, we applied siRNA, using NIH3T3 cells stably transfected with a luciferase reporter gene under control of NF-jB (NF-jB-luc/ 3T3) as an assay system. We used siRNA targeting NF-jBp65 as a
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positive control because of the non-redundant function of this subunit [19]. TNF stimulation induced NF-jB-dependent activation of the luciferase gene (Fig. 1A). Similar results were obtained with IL1 or LPS as stimuli (results not shown). When the cells were transfected with anti-NF-jBp65 siRNA 24 h before TNF stimulation, luciferase activity was reduced by 94% (Fig. 1A). Anti-Nup88 siRNA suppressed reporter gene activity with nearly equal efficacy (85%). Both siRNA targeting NF-jBp65 and Nup88 inhibited basal levels of luciferase activity as well (Fig. 1A). Two control siRNAs targeting human activated leukocyte cell adhesion molecule (hALCAM) and hNF-jBp65 with one mismatch [16] exerted no effect. Therefore, we concluded that Nup88 was indispensable for NF-jB-dependent gene activation stimulated with TNF. The efficacy of mRNA depletion by the siRNA duplexes used above was assessed by Q-RT-PCR analysis. The results showed selective suppression of Nup88 mRNA levels by anti-Nup88 siRNA varying between 65% and 90%, while other siRNA species had no effect (Fig. 1B). Similarly, gene knock-down of NF-jBp65 was highly sequence specific as only siRNA against mouse (m) NFjBp65 significantly downregulated the target mRNA and antihNF-jBp65 siRNA had no effect (Fig. 1C). The silencing efficiency varied between 60% and 70%.
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Nup88 gene knock-down by siRNA selectively prevents nuclear accumulation of NF-jB without inhibiting its activation
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We then examined the cellular localization of NF-jB after Nup88 gene knock-down in 3T3 cells by immunofluorescence (IF). Without stimulus NF-jB was predominantly cytoplasmic in all conditions (Fig. 2A: a, c, and e), but the total level was reduced in the cells transfected with anti-NF-jBp65 siRNA (Fig. 2Ac). This reduction in protein levels was in agreement with mRNA levels (60–70% reduction) (Fig. 1C). Upon TNF stimulation NF-jB translocated into the nucleus in mock-transfected cells and in cells transfected with anti-NF-jBp65 siRNA (Fig. 2Ab and d). The nuclear translocation induced by TNF at this dose was observed in virtually all cells. In contrast, NF-jB mainly stayed in the cytoplasm of cells treated with anti-Nup88 siRNA (Fig. 2Af). These results suggested that Nup88 mediated the nuclear translocation of NF-jB. Cells treated with anti-Nup88 siRNA grew normally (data not shown), precluding any gross defect in the general nucleocytoplasmic transport of macromolecules. To assess general nuclear transport, we examined ligand-induced nuclear translocation of the glucocorticoid receptor (GR). In the absence of ligand dexamethasone (DEX) GR localization was predominantly cytoplasmic as expected (Fig. 2Ba and c). Treatment with DEX induced ligand-dependent nuclear translocation of GR in both mock- and Nup88 siRNA-transfected cells (Fig. 2Bb and d). Thus we concluded that NF-jB suppression
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Fig. 1. Effect of Nup88 depletion on NF-jB-dependent reporter gene expression. NF-jB-luc/3T3 cells were transfected with siRNAs targeting hALCAM, hNF-jBp65, mNFjBp65, and mNup88. (A) The luciferase activity was measured 6 h later with or without TNF stimulation (10 ng/ml). This experiment is a representative of four-independent experiments. *P < 0.02, **P < 0.002, ***P < 0.0001. Gene knock-down of (B) Nup88 and (C) NF-jBp65. The levels of mRNA were determined by Q-RT-PCR and expressed as percentage compared to mock transfection. *P < 0.02.
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Constitutively active NF-jB in malignant melanoma cells
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We then addressed the consequences of Nup88 overexpression and nuclear translocation of NF-jB in tumor cells. Through screening we selected a metastatic melanoma cell line BLM which expressed very high levels of Nup88. BLM cells showed characteristic overexpression of Nup88 on the nuclear membrane extending to the cytoplasm as reported in other tumor cells (Fig. 3Ca). However, BLM cell populations appeared heterogeneous in regard to Nup88 distribution, showing sometimes nuclear and intermediate localization (Fig. 3Cc and b, respectively). Notably, this heterogeneity of cell populations varied within cell cultures, with passage number and cell density. A recent report described
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after Nup88 depletion was not due to an unspecific blockade of all nucleocytoplasmic transport. We then examined whether the observed effects after Nup88 depletion were due to a blockade of the upstream NF-jB activation. NF-jB-luc/3T3 cells were transfected with anti-Nup88 siRNA and subcellular fractions were analyzed by EMSA. Without TNF stimulus, low constitutive levels of activated NF-jB were found in the nucleus (Fig. 3A, lanes 1–4). Upon TNF stimulation high levels of activated NF-jB appeared in the nucleus in mock-transfected cells (Fig. 3A, lane 7). In contrast, the majority of activated NF-jB was cytoplasmic in cells transfected with Nup88 siRNA (Fig. 3A, lanes 6 and 8, respectively). These results indicated that NF-jB activation did occur in cells treated with Nup88 siRNA but the activated NF-jB remained cytoplasmic. Analysis of NF-jB by supershift assay using antibodies against individual subunits identified both p65/p50 heterodimer and p50 homodimers (Fig. 3B). The low constitutive level of NF-jB appears to be p50 homodimer (Fig. 3A, lane 3), TNF stimulation activated both homo- and heterodimer of NF-jB (Fig. 3B, lanes 4–6), and Nup88 gene knock-down resulted in cytoplasmic accumulation of both active complexes (Fig. 3B, lanes 1–3).
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an altered subcellular localization of Nup88 depending on its relative abundance to Nup214 and could explain our observation [11]. We then analyzed the NF-jB activation in these cells. Strong constitutive activation of NF-jB complexes was detected in the nucleus (and to a lesser extent in cytoplasm) with further enhancement after TNF stimulation (Fig. 4D). These complexes were identified as p65/p50 heterodimers and p50 homodimers by supershift assay (Fig. 4E). Both NF-jB dimers were thus constitutively active and displayed a dual cytoplasmic and nuclear distribution in BLM in association with high levels of Nup88 overexpression. The constitutive nuclear localization of NF-jB was in agreement with an earlier report on malignant melanoma [20], while the nuclear localization in 3T3 cells required activating stimuli (Fig. 3A). These results suggested that Nup88 overexpression in malignant cells could contribute to constitutive activation of NF-jB both in cytoplasm and in nucleus. We have examined another melanoma cell line 530 (Supplementary Fig. 4), but these cells were difficult to transfect and therefore excluded from further study.
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We then assessed the relationship between Nup88 depletion and localization of NF-jB subunits in BLM cells by immunofluorescence. After transfection with siRNA targeting Nup88, cells were stimulated with TNF and microscopically analyzed using antibodies against NF-jBp65 and Nup88 combined with appropriate secondary antibodies (Fig. 4: NF-jBp65: green, Nup88: blue). Without stimulus, NF-jBp65 predominantly localized in cytoplasm. Upon TNF stimulation NF-jBp65 translocated into the nucleus (Fig. 4Ab). After anti-Nup88 siRNA treatment hardly any Nup88 protein was detectable anymore (Fig. 4Bc and d) and NFjBp65 remained cytoplasmic upon TNF stimulation (Fig. 4Bb). These findings were in line with the observations in mouse 3T3
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Fig. 2. Selective effect of Nup88 depletion on NF-jB nuclear translocation. NF-jB-luc/3T3 cells were transfected with siRNAs targeting NF-kBp65 and/or Nup88 and analyzed by confocal microscopy. (A) The cells were stimulated with TNF (50 ng/ml) for 1 h, and NF-jB was detected by anti-NF-jBp65 and anti-mouse IgG-FITC (B) The cells were stimulated with DEX (100 lg/ml) for 1/2 h, and GR was visualized by anti-GR and anti-mouse IgG-TXRD.
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Led by a Drosophila study that identified an unexpected selective role of mbo (an ortholog of Nup88) in regulating nucelocytoplasmic transport of dorsal [9,10], we set out to investigate the possible link between Nup88 and NF-jB activation. NF-jB is a pivotal transcription factor governing innate and adaptive immunity as well as cancer progression [12]. The canonical activation of NF-jB involves a cascade of events, leading to the release of NF-
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cells, and supported the indispensable role of Nup88 in regulating the subcellular localization of NF-jB subunits. Similar to NF-jBp65 the nuclear translocation of NF-jBp50 subunit was induced by TNF and dependent on Nup88 In addition the subcellular distribution of NF-jBp50 seemed also modulated by the abundance of NF-jBp65 (Supplementary Fig. 4). We conclude that Nup88 regulate the nucleocytoplasmic trafficking of both subunit of NF-jB in malignant cells.
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jB from an inactive cytoplasmic complex and subsequent translo-
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cation to the nucleus [12]. While the cytoplasmic signaling is well characterized, the regulations at the nuclear membrane have attracted less attention. Our results provide the first evidence that the mammalian Nup88 may play a role in the transport of NF-jB similar to the observations in Drosophila [9,10]. The EMSA assays reveal that the nucleocytoplasmic transport as a main target of regulation by Nup88. Nucleocytoplasmic transport of proteins involves transport receptors that recognize nuclear import (NLS) or export signals (NES) on the cargo proteins. The complex is transported through NPC via sequential interactions with nucleoporins in a Ran GTPase-dependent manner [1], and distinct nucleoporins determine substrate specificity. Nup153 is involved in import of proteins mediated by importin-a/importin-b complexes [21], whereas Nup358/RanBP2 and Nup214 play a role in export of proteins dependent on CRM1 export complexes [22,23]. Recent report identified narrower specificity of Nup214 than all CRM1-depen-
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dent export [3,23]. All these reports point to certain level of subset specificity, most likely arising from the composition of cargo complex. Our data suggest a role for mammalian Nup88 in mediating nucleocytoplasmic transport of NF-jB similar to the report in Drosophila [9,10]. Total nuclear NF-jB reflects its dynamic shuttling: The nuclear import of NF-jB depends on importin-a/importin-b determined by a classical (c)NLS [24], and its export is CRM1dependent [25]. The depletion of Nup88 by siRNA did not affect ligand-induced nuclear accumulation of GR, which harbors the bipartite NLS that also binds to importin-a/importin-b transporter [26]. On the other hand, nuclear export of GR is CRM1-independent [27]. Therefore, these data suggest either certain selectivity in the role of Nup88 in importin-a/importin-b-dependent nuclear import, or support more in favor of export as the main target of action as reported in Drosophila [10,11]. We were not able to demonstrate a direct physical association between Nup88 and subcomponents of NF-jB by co-immunoprecipitation (unpublished data). This may be due to indirect associations involving other components, but the identity of other components remains elusive. Nup88 was never shown to directly bind to transport complex, whereas Nup214 does bind directly to CRM1[3], and it is highly likely that Nup88 associates with the transport cargo via Nup214. Recent study revealed multimeric protein complex containing Nup88, Nup153 and importin-a at specific condition [28]. It also remains to be elucidated which subclass of proteins besides NF-jB depends on Nup88 for nucleocytoplasmic transport. Nup214-Nup88 subcomplex is reported to mediate the nuclear export of CRM1-depen-
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Fig. 3. NF-jB activation in 3T3 cells and BLM cells. (A) NIH3T3 cells were transfected with siRNA targeting mNup88 (siRNA), cytoplasmic (Cp) and nuclear (Nc) fraction of the cells were isolated 1 h after the TNF stimulation (50 ng/ml), and analyzed by EMSA. (B) Cell extracts containing activated NF-jB signal after TNF stimulation (lanes 6 and 7 of A) were analyzed with supershift assay using antibodies against NF-jBp65 and/or NF-jBp50. (C) BLM cells from different culture and passages were visualized by anti-Nup88 and alexa 647. Expression at (a) the nuclear membrane (b) partially at the nuclear membrane and nucleoplasmic (c) nucleoplasmic (D) BLM cells with or without TNF stimulation (50 ng/ml) were analyzed with EMSA 1 h after the stimulation. (E) Supershift assay of the nuclear fraction of TNF-stimulated cell (lane 4 of D).
dent NFAT in human cells [3], whereas Drosophila studies demonstrated more inhibitory role of these subcomplex in nuclear export of NF-jB [9,10]. The discrepancy was thought to be due to different species, but our results rather support the role of Nup88 demonstrated in the Drosophila study. However, the interdependency between Nup214 and Nup88 [3,22] and varying subcellular localization [10,28] makes it difficult to distinguish the role of each component. We demonstrated the indispensable role of Nup88 in mediating the gene activation induced by NF-jB in normal (NIH3T3) and metastatic tumor cells (BLM). Coordinated degradation and synthesis of IjB isoforms regulate the activity of NF-jB both in cytoplasm and in nucleus [29]. Overexpressed Nup88 may serve as a protective shuttle and preserve a cytoplasmic pool of active NF-jB for continuous supply into the nucleus, resulting in perpetual NF-jB activity as observed here in BLM cells. EMSA assays detected constitutively activated NF-jB in both nucleus and cytoplasm of BLM cells, which express high levels of Nup88. While chronic NF-jB activation in tumor cells is frequently accompanied by constitutive activation of IKK [30], preservation of activated NF-jB would provide another mechanism favoring sustained NF-jB activation. Nup88 overexpression could effect perpetual NF-jB activity and ultimately fuel aberrant cell growth. Therefore, our findings suggest that Nup88 is not only a marker for malignant cells, but also a potential therapeutic target. Its prominent effects on NF-jB warrant further research.
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Fig. 4. Effect of Nup88 depletion in BLM cells overexpressing Nup88. BLM melanoma cells were transfected with or without siRNA, stimulated with TNF and analyzed by confocal microscopy at 1 h after the stimulation. (A) Mock, (B) siRNA depletion of hNup88. NF-jBp65 and Nup88 were detected by anti-NF-jBp65 and anti-rabbit IgG-FITC (green) and anti-Nup88 labeled with Alexa 647 (blue). The last column shows the overlay of all fluorescent channels.
Acknowledgments
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We thank Dr. W. Falk (Regensburg, Germany) for the gift of the NF-jB luciferase reporter plasmid, and L. van den Bersselaar for technical assistance. This research was supported by European Commission (QLRT 1999-02072), the Dutch Arthritis Association (Reumafonds) (01-1-304) and the Dutch Cancer Society (KUN2002-2757). B.O. was a holder of a research grant from the ‘‘Rheumatology Network” of the German Research Center for Rheumatology, Berlin.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2008.06.128.
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Please cite this article in press as: N. Takahashi et al., Tumor marker nucleoporin 88 kDa regulates nucleocytoplasmic ..., Biochem. Biophys. Res. Commun. (2008), doi:10.1016/j.bbrc.2008.06.128
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