Báo cáo khoa học: Nucling interacts with nuclear factor-jB, regulating its cellular distribution potx

Nucling interacts with nuclear factor-jB, regulating its cellular distribution Li Liu1,*, , Takashi Sakai1,*, Nam Hoang Tran1, Rika Mukai-Sakai1,2, Ryuji Kaji2 and Kiyoshi Fukui1 Division of Enzyme Pathophysiology, Institute for Enzyme Research, University of Tokushima, Japan Institute of Health Biosciences, University of Tokushima Graduate School, Japan Keywords IjBa; nuclear factor-jB; nuclear translocation; Nucling; tumor necrosis factor-a Correspondence K Fukui, Institute for Enzyme Research, University of Tokushima, 3-18-15 Kuramotocho, Tokushima 770-8503, Japan Fax: +81 88 633 7431 Tel: +81 88 633 7430 E-mail: kiyo@ier.tokushima-u.ac.jp yPresent address Department of Physics, Graduate School of Science, Kyoto University, Japan *These authors contributed equally to this work (Received 11 April 2008, revised 30 December 2008, accepted January 2009) Nucling is an Apaf1-binding proapoptotic protein involved in apoptosomemediated apoptosis Luciferase assays have revealed that the activation of nuclear factor-jB induced by tumor necrosis factor-a, interleukin-1b and lipopolysaccharide is downregulated by the overexpression of Nucling in HEK293 cells Moreover, the expression of endogenous cyclooxygenase 2, tumor necrosis factor-a and galectin-3, the end-point molecules in the pathway for the activation of nuclear factor-jB, as well as nuclear factor-jB (p65) itself, is upregulated in Nucling gene-deficient mouse embryonic fibroblasts, suggesting that nuclear factor-jB is a target of Nucling Subsequent study has revealed that Nucling physically interacts with nuclear factor-jB (p65 and p50) and that the binding domain of Nucling is its amino-terminal region (amino acids 1–466) containing ankyrin repeats Overexpression of Nucling prevents the translocation of nuclear factor-jB into the nucleus In addition, the cytoplasmic retention of endogenous nuclear factor-jB in resting cells is not observed in Nucling gene-deficient mouse embryonic fibroblasts These results reveal a novel function of Nucling as a suppressor of nuclear factor-jB, mediated by its cytoplasmic retention through physical interaction doi:10.1111/j.1742-4658.2009.06888.x Nucling was originally isolated from murine embryonal carcinoma cells as a novel protein, the expression of which was upregulated during cardiac muscle differentiation [1] We have reported previously that Nucling, as a proapoptotic factor, is associated with the apoptosome pathway It has been shown that Nucling recruits the Apaf1/procaspase-9 complex for the induction of stress-induced apoptosis [2] In addition, Nucling acts as an important factor during 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity through activation of the apoptosome pathway [3] We have also reported that Nucling mediates apoptosis by inhibiting the expression of galectin-3, an antiapoptotic factor, through interference with nuclear factor-jB (NF-jB) signaling [4] In Nucling-deficient (Nucling)/)) mice, a high occurrence of inflammatory lesions of preputial glands was observed in males, and hepatocellular carcinoma arising from cholestatic hepatitis was frequently detected in aged mice Furthermore, we have confirmed that the activation of NF-jB is involved in these pathological changes (T Sakai, L Liu, X Teng, N Ishimaru, R Mukai-Sakai, N H Tran, N Sano, Y Hayashi, R Kaji & K Fukui, unpublished results) Although Abbreviations COX2, cyclooxygenase 2; EMSA, electrophoretic mobility shift assay; FITC, fluorescein isothiocyanate; IL-1b, interleukin-1b; LPS, lipopolysaccharide; MEF, mouse embryonic fibroblast; NF-jB, nuclear factor-jB; TK, thymidine kinase; TNFa, tumor necrosis factor-a FEBS Journal 276 (2009) 1459–1470 ª 2009 The Authors Journal compilation ª 2009 FEBS 1459 Nucling regulates cellular distribution of NF-jB L Liu et al 35 30 Relative activation these findings strongly indicate that Nucling constitutes a missing link between inflammation and cancer via the NF-jB pathway, the mechanism underlying the regulation of this pathway by Nucling remains to be elucidated The classic form of NF-jB is the heterodimer of the p50 and p65 subunits, which is sequestered in the cytoplasm as an inactive complex bound to its inhibitor protein IjB [5,6] NF-jB is activated by various stimuli, including tumor necrosis factor (TNF), interleukin-1 (IL-1) and lipopolysaccharide (LPS) [7,8] It is then transported to the nucleus, where it binds to specific sequences in the promoter or enhancer region of target genes Activated NF-jB can mediate NF-jB-regulated gene transcription, the products of which are involved in antiapoptosis, cell proliferation and invasion [9,10] In this study, we propose a new regulatory mechanism for the activation of NF-jB mediated by Nucling Nucling, as a regulatory factor for apoptosis, links the NF-jB pathway and mediates the nuclear translocation and activation of NF-jB Nucling consists of a polypeptide of 1411 amino acids, containing an ankyrin repeat, a leucine zipper motif and t-SNARE coiled-coil domains These domains may play some role in the formation of complexes Recently, we have reported that Nucling interacts and downregulates the expression of galectin-3, an antiapoptotic factor [4] In addition, Nucling has been found to upregulate Apaf1 expression and recruit the Apaf1/procaspase-9 complex for the induction of apoptosis following proapoptotic stress [2] Moreover, we have identified several positive clones by yeast two-hybrid screening for the protein interacting with Nucling using NuclingDN as ‘bait’ [4] Therefore, it is conceivable that Nucling is involved in several signaling pathways through interactions with possible regulatory factors We have observed previously that a deficiency of Nucling expression achieved using gene targeting increases the basal level of activated NF-jB in mouse embryonic fibroblasts (MEFs) [4] However, it is unclear whether or not Nucling itself is a regulator of the activation of NF-jB In this study, experiments were carried out to investigate the molecular mechanism underlying this regulatory role of Nucling in the NF-jB pathway 25 20 15 10 TNFα – + – – – + – – IL-1β – – + – – – + – LPS – – – + – – – + Mock Nucling Fig Ectopic Nucling inhibits the transcriptional activity of NF-jB following TNFa treatment HEK293 cells were cotransfected with the NF-jB reporter gene and Flag-Nucling or Flag empty vector (mock) At 16 h post-transfection, cells were stimulated by TNFa, IL-1b or LPS Then, the activation of NF-jB was assessed by measurement of luciferase activity Each sample was analyzed at least three times absence of transfected Nucling We introduced pFLAG-Nucling or pFLAG empty vector (mock) together with a reporter plasmid comprising three repeats of the NF-jB site upstream of a minimal thymidine kinase (TK) promoter and a luciferase gene into HEK293 cells The activation of NF-jB was evaluated with the dual-luciferase reporter assay system (Fig 1) In the mock transfection, the activation of NF-jB was upregulated during stimulation with TNFa (lane 2), IL-1b (lane 3) and LPS (lane 4) from the resting level (lane 1) However, the transfection of Nucling clearly reduced TNFa-, IL-1b- and LPS-induced NF-jB activity (lanes 5–8) Results Ectopic expression of Nucling downregulates the activation of NF-jB following cytokine treatment To test whether Nucling is involved in the activation of NF-jB, we first examined the NF-jB-mediated activation of a luciferase reporter gene in the presence or 1460 Nucling is involved in the regulation of NF-jB signaling We have reported that Nucling inhibits the expression of galectin-3, an NF-jB-regulated gene, by suppressing the activation of NF-jB [4] To elucidate the general role of Nucling in the NF-jB signaling pathway, we FEBS Journal 276 (2009) 1459–1470 ª 2009 The Authors Journal compilation ª 2009 FEBS L Liu et al attempted to investigate whether Nucling also affects other NF-jB-regulated genes Therefore, we compared the protein and RNA levels of TNFa and cyclooxygenase (COX2), as NF-jB-regulated genes, in MEFs isolated from wild-type (WT) and Nucling)/) mice Four independently prepared WT and Nucling)/) MEFs were investigated The results showed that TNFa was upregulated in its expression in Nucling)/) MEFs in every case (Fig 2A, panel 1; Fig 2B, panel 1; Fig S1) COX2 was also upregulated in Nucling)/) MEFs in two of four samples (Fig 2A, panel 2; Fig 2B, panel 2) No significant difference for COX2 between WT and Nucling)/) MEFs was observed in two remaining samples (Fig S1) COX2 is not usually expressed in most tissues [11] In the present study, a weak band of COX2 protein was observed in WT MEFs We considered that the expression of COX2 was induced by the presence of nuclear NF-jBp65 in WT MEFs in half of the four samples (Fig 2A, panel 6, lane 1) The upregulated expression of galectin-3 and the absence of Nucling were found in Nucling)/) MEFs as a positive control (Fig 2A, panels 3, 4) In addition, the nuclear translocation of NF-jBp65 was also confirmed in the nuclear fraction of Nucling)/) MEFs (Fig 2A, panel 6) Moreover, semiquantitative and real-time RT-PCR analyses revealed that the transcription of COX2 was not upregulated in Nucling)/) MEFs by TNFa (Fig 2C; Fig S1) This result indicates that the NF-jB signaling pathway is impaired in Nucling)/) MEFs Taken together with the results of the reporter assays in this study, we concluded that Nucling is important for the regulation of the NF-jB signaling pathway Nucling interacts with NF-jB Considering that Nucling downregulates the activation of NF-jB, we investigated whether the two physically interact COS7 cells were transfected with the expression vector for NF-jB-p50, NF-jB-p105 or NF-jBp65 with pFLAG-Nucling or a pFLAG vector At 24 h post-transfection, cell lysates were immunoprecipitated using a mouse IgG1 against FLAG peptide (anti-FLAG M5) Figure 3B,C illustrates that all three NF-jB subunits interact with the full-length Nucling and NuclingDC (amino acids 1–466, containing the ankyrin repeat region; a deletion mutant lacking the C-terminal region; Fig 3A) directly in COS7 cells Concerning NuclingDN (amino acids 814–1411; a deletion mutant lacking the N-terminal region; Fig 3A), a faint band was observed for p65, but no band was detected for p50 or p105 The faint band was considered to be nonspecific because it was very weak Nucling regulates cellular distribution of NF-jB A B C Fig Nucling is involved in the regulation of the NF-jB signaling pathway (A) Whole MEFs of Nucling+/+ and Nucling)/) mice were used to determine the levels of TNFa, COX2, galectin-3 and Nucling (panels 1–4) Nuclear proteins from Nucling+/+ and Nucling)/) MEFs were used to determine the level of NF-jB-p65 (panel 6) The equal loading of protein in each lane was confirmed using mouse IgG1 against b-actin (panels and 7) (B) RT-PCR was carried out to detect the transcripts of the TNFa and COX2 genes Bar graphs show the compiled means ± standard deviation for results of densitometric scanning from three experiments for TNFa and COX2, quantified by NIH-IMAGE software Data were normalized to the density of Nucling+/+ MEFs for the TNFa/b-actin and COX2/b-actin ratios, respectively The values for Nucling)/) MEFs were statistically compared with those for Nucling+/+ MEFs *P < 0.05 (C) RT-PCR was carried out to detect the transcripts of COX2 genes following TNFa stimulation compared with the level of NuclingDN However, no interaction occurred between IjBa and Nucling (Fig 3D) Thus, we concluded that Nucling interacts FEBS Journal 276 (2009) 1459–1470 ª 2009 The Authors Journal compilation ª 2009 FEBS 1461 Nucling regulates cellular distribution of NF-jB L Liu et al A B C D Fig Nucling interacts with NF-jB-p50 and NF-jB-p65 but not with IjBa (A) Primary structure of full-length Nucling and the deletion mutants (NuclingDC, amino acids 1–466; NuclingDN, amino acids 814–1411) used for coimmunoprecipitation analysis Ank, SN and LZ represent the ankyrin repeat region, t-SNARE coiled-coil domain and leucine zipper motif, respectively (B) COS7 cells were transiently cotransfected with Flag-Nucling/Flag-NuclingDC/Flag-NuclingDN/Flag-vector and HA-NF-jB-p50/HA-NF-jB-p105 Lysates were subjected to a coimmunoprecipitation assay with anti-Flag M5 Then, an immunoblot analysis was performed with rat IgG against HA The presence of Nucling/ NuclingDC/NuclingDN and NF-jB-p50/NF-jB-p105 in the same lysates was verified by immunoblotting with anti-Flag M5 and antibody against HA, respectively (C) COS7 cells were cotransfected with Flag-Nucling/Flag-NuclingDC/Flag-NuclingDN/Flag-vector and NF-jB-p65, respectively The same method as in (B) was used for coimmunoprecipitation assay Immunoprecipitates were subjected to western blotting using antiNF-jB-p65 The expression levels of NF-jB-p65 and Flag-Nucling/Flag-NuclingDC/Flag-NuclingDN in cellular lysates were confirmed by western blotting (D) COS7 cells were transfected with Flag-Nucling, Flag-NuclingDC or Flag-vector Lysates were prepared and incubated with the antiFlag M5 for coimmunoprecipitation assay The expression levels of endogenous IjBa in cellular lysates were confirmed by western blotting The expression of Flag-Nucling and Flag-NuclingDC was confirmed by western blotting with anti-Flag M5 1462 FEBS Journal 276 (2009) 1459–1470 ª 2009 The Authors Journal compilation ª 2009 FEBS L Liu et al Nucling regulates cellular distribution of NF-jB with NF-jB-p50, NF-jB-p105 and NF-jB-p65, possibly through its N-terminal domain containing ankyrin repeats To confirm the presence of a protein complex containing Nucling and NF-jB in primary cells, the cytosolic fraction of WT or Nucling)/) MEFs was separated under nondenaturing conditions Immunoblots of these native gels revealed Nucling or NF-jB-p50 immunoreactivity within several distinct complexes in fractions from TNFa-treated WT MEFs (Fig 4A) We identified five bands, which were reactive to both antibodies (Fig 4Aa–e) Although bands a and b were specifically detected in WT MEFs (arrowhead), bands c–e were detected in both WT and Nucling)/) MEFs Thus bands c–e were considered to be nonspecific As a next step, cytosolic proteins from TNFa-treated WT or Nucling)/) MEFs were fractionated into native complexes under nondenaturing conditions (first dimension), and subsequently separated into individual components (second dimension) by placing a native gel slice at a horizontal position as a stack above an SDSPAGE denaturing gel (Fig 4B) Second-dimension gels were also transferred and immunoblotted, confirming that Nucling (molecular mass, 160 kDa) was present in A B C f g h i j j’ Fig Nucling forms a complex with NF-jB-p50 in primary MEF cells (A) Nucling-containing or NF-jB-p50-containing complexes were observed in TNFa-treated WT MEFs, but not in Nucling)/) MEFs on native electrophoresis Fifty micrograms of the cytosolic fraction were subjected to nondenaturing electrophoresis (first dimension) Proteins were transferred and immunoblotted with a rabbit serum against Nucling (anti-mNucl.mid) The arrows beside the photographs indicate the positions of the bands common to mNucl.mid and anti-NF-kB1 immunoreactive membranes Common complexes in lanes 1, 3, and are marked with arrowheads (B) Cytosolic fractions prepared from WT MEFs (+/+) or Nucling)/) MEFs ()/)) were analyzed as the second dimension Immunoblotting of the second-dimension gel of cytosolic proteins from the TNFa-treated WT MEFs (left panel) or Nucling)/) MEFs (right panel) revealed the presence of Nucling (160 kDa) and p50 (50 kDa, h and i) in the complexes indicated as a, b and c in the native first-dimension gel of WT MEFs (A) Arrows a–e indicate the positions of bands a–e in (A) Some other high molecular mass dots were detected in the membrane of WT MEFs for p50 (f and g) Arrows in the lower panel for WT MEFs (left panel) indicate Nucling, which was not detected in Nucling)/) MEFs (right panel) p65 was detected in WT and Nucling)/) MEFs (arrows j and j¢) IjBa was also detected in both MEFs (arrows in IjBa panels) FEBS Journal 276 (2009) 1459–1470 ª 2009 The Authors Journal compilation ª 2009 FEBS 1463 Nucling regulates cellular distribution of NF-jB L Liu et al complexes a, b and c in Fig 4A (arrows in the bottom panel in Fig 4B) We also confirmed the presence of p50 in complexes a and c (arrows h and i in Fig 4B) We observed two spots immunoreactive to the p50 antibody at the position of band c (arrows f and g in Fig 4B) In addition, there was a spot for p65 in the lane for band c (arrow j in Fig 4B); however, it was observed in the same position in the membrane of Nucling)/) MEFs (arrow j¢) We could not detect IjBa in the lanes for bands a–c (Fig 4B) Thus, we concluded that Nucling interacts with NF-jB-p50 and possibly with NF-jB-p65 in primary cells To assess the potential of endogenous Nucling to regulate the nuclear translocation of endogenous NF-jB, we investigated the endogenous distribution of NF-jB (p65 and p50) in WT and Nucling)/) MEFs (Fig 5K–N,K¢–N¢) In WT MEFs, both p50 and p65 were found exclusively in the cytoplasm (Fig 5K, K¢,M,M¢) In contrast, in Nucling)/) MEFs, p50 and p65 were distributed throughout the cell, including the nucleus and cytoplasm (Fig 5L,L¢,N,N¢) Based on these observations, we concluded that Nucling physically interacts with and can regulate the intracellular localization of NF-jB Nucling influences the localization of NF-jB Nucling is important for the strict regulation of the classical NF-jB signaling pathway To verify the interaction between Nucling and NF-jB proteins in vivo, we next examined whether the ectopic expression of Nucling affects the distribution of p65 or p50, which are overexpressed in COS7 cells A double immunostaining assay was performed after the coexpression of Nucling/p50 (Fig 5A–D) or Nucling/p65 (Fig 5E–H) The transfection of NF-jB alone (p50 or p65) resulted in a nuclear localization (Fig 5A,E) However, cytoplasmic staining of NF-jB (p50 or p65) was observed in approximately 80–85% of cells when Nucling or NuclingDC was overexpressed by cotransfection (Fig 5B,C,F,G) However, when NuclingDN was coexpressed with NF-jB (p50 or p65), over 65% of cells showed nuclear staining of NF-jB (p50 or p65) (Fig 5D,H) The cytosolic or nuclear localization of NF-jB (p50 and p65) was quantified and analyzed statistically (Fig 5I,J) The results clearly showed that the ectopic expression of Nucling inhibits the nuclear localization of NF-jB in vivo In addition, the distribution of ectopic NF-jB apparently overlapped with that of Nucling (Fig 5B,F) or NuclingDC (Fig 5C,G), but not with that of NuclingDN (Fig 5D,H), suggesting that Nucling interacts with NF-jB through its N-terminal region, and the interaction is sufficiently strong to regulate the cellular localization of NF-jB To verify the physiological function of Nucling in the classical NF-jB signaling pathway, we investigated the changes in the expression of p65, p50 and IjBa in cytosolic and nuclear lysates of MEFs following TNFa treatment A western blot analysis and electrophoretic mobility shift assay (EMSA) indicated that the NF-jB signaling pathway in WT MEFs was functioning normally After TNFa treatment, a decrease in IjBa (lane in Fig 6A, bar for IjBa at 15 in Fig 6B) followed by the expression of IjBa (lane in Fig 6A, bar for IjBa at 30 in Fig 6B), increases in nuclear p50 and p65 (lane in Fig 6A, bar for p50 and p65 at 15 in Fig 6B) and an increase in NF-jB complexes (p65/p50 and p50/p50) (lane in Fig 6C) were evident in WT MEFs In Nucling)/) MEFs, a decrease in IjBa (lane in Fig 6A) was observed following TNFa treatment, but it was not as marked as that in WT MEFs (lane in Fig 6A) In addition, the nuclear translocation of p65 and p50 after TNFa treatment was impaired in Nucling)/) MEFs (lanes and in Fig 6A, bar for p50 and p65 at 15 in Fig 6B) Although the relative levels of p65 and p50 were lower in the nuclear lysate from Nucling)/) MEFs than in that from WT MEFs (Fig 6B), intense signals for p65 in the cytosol were Fig Nucling mediates the localization of NF-jB (A–D) COS7 cells were cotransfected with Flag-Vector/Flag-Nucling/Flag-NuclingDC/ Flag-NuclingDN and pHA-p50, respectively Flag-Nucling and the mutant of Flag-Nucling were detected with an FITC-conjugated monoclonal antibody against the Flag epitope (green) HA-p50 was detected with antibody against HA and Texas Red-conjugated secondary antibody (red) Although the transfected cells were cultured in appropriate media with the pan-caspase inhibitor (zVADfmk), some cells transfected with Flag-Nucling showed an apoptotic morphology (e.g A) (E–H) COS7 cells were cotransfected with Flag-Vector/Flag-Nucling/FlagNuclingDC/Flag-NuclingDN and the p65 expression vector, respectively Flag-Nucling and the mutant of Flag-Nucling were detected with an FITC-conjugated monoclonal antibody against the Flag epitope (green) NF-jB-65 was detected with p65 antibody and rhodamine-conjugated secondary antibody (red) Representative, double-stained images are shown Scale bars represent 20 lm (A–H, K–N) or 10 lm (K¢–N¢) (I, J) Bar graphs show the cytosolic (filled bar) or nuclear (open bar) localization of NF-jB-p50 (I) and NF-jB-p65 (J) The data are the means ± standard deviation for three independent experiments (K–N) Nucling+/+ and Nucling)/) MEFs were cultured and analyzed by immunofluorescent staining Endogenous NF-jB-p65 and NF-jB-p50 were detected with a primary antibody against p65 and p50, respectively, and an FITC-conjugated secondary antibody 1464 FEBS Journal 276 (2009) 1459–1470 ª 2009 The Authors Journal compilation ª 2009 FEBS L Liu et al Nucling regulates cellular distribution of NF-jB observed in Nucling)/) MEFs (lanes and in Fig 6A) There was little or no upregulation of NF-jB (p65/p50 and p50/p50) activity on TNFa treatment (Fig 6C) These results strongly suggest that Nucling is not just a suppressor of NF-jB in resting cells, but a regulator for the activation of NF-jB in stimulated cells A B C D E F G H I J K L M N K' L' M' N' FEBS Journal 276 (2009) 1459–1470 ª 2009 The Authors Journal compilation ª 2009 FEBS 1465 Nucling regulates cellular distribution of NF-jB A L Liu et al B TNFα: Nucling+/+ MEF Nucling-/- MEF Nucling: ** 420 50 TNFα: 15 1- ** 5- * ** 50 1466 30 90 IκBα in cytosol/total (%) p50/β-actin 5- 2- * 10 50 TNFα: 15 C 30 90 p65/β-actin ** TNFα: 15 IκBα/β-actin Nucleus p65 in nucleus /total (%) 5- p50 in nucleus/total (%) p50/β-actin 10- IκBα/β-actin Cytosol p65/β-actin 100 30 Cold FEBS Journal 276 (2009) 1459–1470 ª 2009 The Authors Journal compilation ª 2009 FEBS L Liu et al In resting Nucling)/) cells (Nucling)/) MEFs), the activation of NF-jB (p65/p50) was upregulated (Fig 6B) Then, we investigated the expression of IjBa in Nucling)/) and WT MEFs However, the level in Nucling)/) MEFs was almost the same as that in WT MEFs in the cytosol or in total, with a slight but significant difference in the nucleus (Fig 6A, lanes and 2, Fig 6B) A decrease in IjBa was observed in Nucling)/) MEFs treated with TNFa (Fig 6A, lane 4), indicating the degradation of IjBa However, the decrease was less marked in Nucling)/) MEFs than in WT MEFs As IjBa itself is regulated by NF-jB, IjBa expression may be induced following the degradation caused by TNFa, as we observed the expression of IjBa at 30 in Fig 6A (lane in cytosol) The activation of NF-jB in resting cells may suppress the dramatic decrease in the level of IjBa in Nucling)/) MEFs In addition, there was no difference between WT and Nucling)/) MEFs in the cellular expression of IjBa by immunocytochemistry (data not shown) A considerable amount of IjBa was detected in the nucleus of resting cells (Fig 5B, lanes and 2, and data not shown for immunocytochemistry) Discussion In this study, we have demonstrated that Nucling negatively regulates the activation of NF-jB The inhibitory function proposed for Nucling in Fig was based on several publications [12–14] We hypothesized that the inhibitory effect might be caused by the cytoplasmic retention of NF-jB via interaction with Nucling In order to check the importance of the interaction between Nucling and NF-jB, we performed a two-dimensional native SDS-PAGE analysis in addition to immunoprecipitation In this experiment, we confirmed that Nucling and NF-jB-p50 form a complex in primary cells (MEFs) The activation of NF-jB increases the expression of genes that encode proinflammatory mediators, called Nucling regulates cellular distribution of NF-jB cytokines, and activates genes that regulate the balance between cell proliferation and cell death COX2, as an NF-jB-regulated proinflammatory gene, is expressed essentially in inflammatory lesions and tumor tissues [11,15] TNFa is regarded as a critical mediator, bridging inflammation and tumorigenesis The inhibition of tumor and/or stromal TNFa may provide a novel therapeutic strategy for cancer [8,16] In this study, we demonstrated that the expression of not only galectin3, but also COX2 and TNFa, was mediated by Nucling via the inhibition of the activation of NF-jB Moreover, the upregulated expression of galectin-3, COX2 and TNFa may play an important role in the pathological changes in Nucling)/) mice We have observed several inflammatory conditions in some Nucling)/) mice, including preputial gland abscesses [4], liver dysfunction and hepatic diseases (data not shown) Further detailed studies are necessary to reveal precisely how these NF-jB-regulated mediators are involved in the inflammation and tumorigenesis induced by a deficiency of Nucling In this study, we confirmed the upregulation of TNFa in three independent clones of resting Nucling)/) MEFs However, the upregulation of COX2 in Nucling)/) MEFs was clone dependent Some redundant pathways for the regulation of COX2 may occasionally work in resting Nucling)/) MEFs However, impairment of the upregulation of TNFa and COX2 in Nucling)/) MEFs following treatment with TNFa was consistently observed in every case, indicating the importance of Nucling for the activation of the NF-jB pathway Nucling was originally characterized as an apoptosis-inducing factor [2] We observed that its overexpression led to apoptosis in several cell lines, including COS7, NIH3T3 and HEK293 in the absence of a caspase inhibitor ([2] and data not shown) We revealed that Nucling reduces the expression of galectin-3, an antiapoptotic molecule, and increases that of Apaf1, an apoptosis-activating factor [2] Here, we have shown that Nucling is also a key regulator for NF-jB Fig Nucling is important for the regulation of the NF-jB signaling pathway (A) WT MEFs (+/+) or Nucling)/) MEFs ()/)) were treated with TNFa for 0, 15 or 30 min, and cytosolic and nuclear cell lysates were prepared Western blotting was performed to check the expression levels of NF-jB-p65, NF-jB-p50, IjBa or b-actin in the lysates The levels of b-actin revealed that the amounts of applied cell lysates were nearly the same Relative protein levels are shown in a bar graph below each of the representative images Three individual trials were performed and analyzed Data are presented as the mean ± standard error of the mean *P < 0.05; **P < 0.005 (B) The bar charts show the relative protein levels of p65, p50 and IjBa in the nucleus/total (for p65 and p50) or in the cytosol/total (for IjBa) (total = cytosol + nucleus) Values relative to the level of b-actin were used for the analysis (C) NF-jB (p65/p50 and p50/p50) DNA-binding activity in nuclear extracts from WT MEFs (+/+) or Nucling)/) MEFs ()/)) with or without TNFa treatment Where indicated, lg of specific antibody (anti-p65 in lanes 10 and 11, and anti-p50 in lanes 12 and 13) were added to the reaction mixture to demonstrate the specific binding of the NF-jB-p65 and NF-jB-p50 proteins For competition experiments, 100 ng of double-stranded cold competitor (cold probe) was added to the reaction (lanes and 9) Results from one of two independent experiments are shown FEBS Journal 276 (2009) 1459–1470 ª 2009 The Authors Journal compilation ª 2009 FEBS 1467 Nucling regulates cellular distribution of NF-jB L Liu et al during TNFa treatment NF-jB (p65) was consistently activated in Nucling)/) cells However, the activation of p65 by TNFa was partly impaired in Nucling)/) cells (Fig 6) These observations indicate that Nucling is critical for the cellular distribution of NF-jB during its activation Contrary to the result for p65, the upregulation of p50 and p105 expression was not so dramatic in the nuclear fraction of resting Nucling)/) MEFs compared with WT MEFs (Fig 6A,B, and data not shown) However, we also observed the nuclear distribution of p50/p105 in Nucling)/) MEFs (Fig 5L) From the results of immunoprecipitation (Fig 3) and two-dimensional nondenaturing gel electrophoretic analysis (Fig 4), we consider the interaction between p50 and Nucling to be much stronger than that between p65 and Nucling In addition, Nucling usually distributes in the perinucleus, not the nucleus, and is harvested in the nuclear fraction On the basis of these observations, we speculate that a considerable amount of cytoplasmic p50 interacting with Nucling can be recovered in the nuclear fraction This may explain why there was no dramatic difference between Nucling)/) and WT MEFs with regard to the expression of p50 in the nuclear fraction Previously, we have also observed that the expression of Nucling is upregulated by several cellular stressors, including H2O2, UV irradiation and anoikis ([2] and data not shown) Nucling is particularly sensitive to TNFa As observed here, TNFa induces the activation of NF-jB The upregulation of Nucling expression may be important for the regulation of the activation of NF-jB following treatment with TNFa According to the standard model of classical NF-jB signaling, the activation of NF-jB is promoted by the degradation of the inhibitors of NF-jB, the IjBs This family of mainly cytoplasmic inhibitors currently has eight members: IjBa, IjBb, IjBc, IjBe, IjBf, Bcl-3, as well as p105 and p100 In unstimulated cells, NF-jB forms a complex with the IjBs and is thereby locked into the cytoplasm IjBa is the best-characterized IjB, and is also a target of NF-jB itself Thus, we focused on IjBa for further investigation We observed a decrease in IjBa following TNFa treatment in Nucling)/) MEFs, indicating that the degradation of IjBa is independent of Nucling We actually observed a change in the expression of IjBa in Nucling)/) MEFs during TNFa treatment, which can be explained by an impairment of the regulation of NF-jB signaling In conclusion, we propose that Nucling is important for the regulation of the distinct intracellular distribution of NF-jB in its resting and activated states 1468 Materials and methods Cell lines and culture conditions HEK293, MEF and COS7 cells were used The cells were maintained in DMEM with 100 unitsỈmL)1 penicillin, 100 lgỈmL)1 streptomycin, lm mercaptoethanol and 10% (v/v) fetal bovine serum They were cultured in plastic tissue culture plates at 37 °C in an atmosphere of 5% CO2/95% air in a humidified incubator For propagation, cultures were split, and the growth medium was replenished every 3–4 days NF-jB reporter assay HEK293 cells cultured on 24-well plates (3 · 105 cells) were transfected with a reporter plasmid comprising three repeats of the NF-jB site upstream of a minimal TK promoter and a firefly luciferase gene in the pGL-2 vector, and the vector phRL-TK comprising a TK promoter upstream of a renilla luciferase gene [17], together with the Nucling plasmid or a negative control vector PhRL-TK was used to exclude nonspecific global effects of the transfections on transcription After 16 h, the transfected cells were treated with or without TNFa (10 ngỈmL)1), IL-1b (5 ngỈmL)1) or LPS (100 ngỈmL)1) for h, and then harvested in a luciferase lysis buffer Luciferase assays were performed with the Dual-LuciferaseÒ Reporter Assay System (Promega, Tokyo, Japan) using a luminometer (Lumat LB 9507; Berthold Technologies, Wildbad, Germany) Subcellular fractionation and western blotting Subcellular fractionation was performed as described previously [1] In brief, semiconfluent cells were washed twice with mL of cold NaCl/Pi, harvested by scraping, divided into two tubes and centrifuged (100 g for min) Half of the cell pellets were harvested as whole-cell extracts The remaining cell pellets were resuspended in mL of cold hypotonic buffer (42 mm KCl, 10 mm Hepes and mm MgCl2) supplemented with protease inhibitor cocktail (0.1 mm phenylmethanesulfonyl fluoride, lgỈmL)1 leupeptin and aprotinin) and incubated for 30 on ice Nuclei were removed by centrifugation at 600 g for 10 (nuclear fraction) Cytosolic fractions were prepared as supernatants separated by additional centrifugation at 100 000 g for 90 Whole-cell and nuclear pellets were resuspended in cold extraction buffer (1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS in · NaCl/Pi) supplemented with protease inhibitor cocktail, and the pellets were broken by passing the suspension through a 26G needle Following incubation on ice for 30 min, cell extracts were obtained as supernatants by centrifugation at 100 000 g for The protein concentrations of the extracts were estimated using the BCA kit (Pierce, Rockford, IL, USA) Ten micrograms of protein were loaded on to an SDS-PAGE gel, transferred FEBS Journal 276 (2009) 1459–1470 ª 2009 The Authors Journal compilation ª 2009 FEBS L Liu et al onto an ImmobilonÔ Transfer Membrane (Millipore, Bedford, MA, USA), and western blotting was performed The antibodies used were an NF-jB p65 mouse monoclonal antibody (F-6) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), a rabbit polyclonal antibody against IjBa (C-21) (Santa Cruz Biotechnology), a rabbit polyclonal antibody against COX2 (Cayman Chemical, Ann Arbor, MI, USA) and an antibody against TNFa (R&D Systems, Minneapolis, MN, USA) Western blot analysis was carried out according to standard procedures using an ECL detection kit (Amersham-Pharmacia, Uppsala, Sweden) Coimmunoprecipitation COS7 cells were transiently cotransfected with plasmids using the NucleofectorÔ system (Amaxa, Koln, Germany) At 16 h ă post-transfection, cells were harvested and lysed in lysis buffer [50 mm Tris/HCl, pH 7.4, 150 mm NaCl, 0.25% deoxycholic acid, 1% NP-40, mm EDTA and CompleteÔ protease inhibitor cocktail (Roche Diagnostics, Tokyo, Japan)] Cell extracts were clarified by centrifugation at 12 000 g for 20 at °C, and the supernatant was immunoprecipitated with anti-Flag M2 affinity gel (Sigma-Aldrich, Tokyo, Japan) by rotating overnight at °C The beads were washed 10 times with lysis buffer and suspended in SDS sample loading buffer Subsequently, the lysates and coimmunoprecipitated samples were subjected to an immunoblot assay with antiFlag M2 horseradish peroxidase conjugate (Sigma) and anti-HA horseradish peroxidase conjugate (3F10) (Roche) Nucling regulates cellular distribution of NF-jB Confocal immunofluorescence microscopic analysis COS7 cells were plated on to Lab-Tek Chamber Slides (Nalge Nunc International, Rochester, NY, USA), transiently cotransfected with Flag-Nucling/HA-NF-jB-p50 or Flag-Nucling/NF-jB-p65, Flag-NuclingDC/HA-NF-jB-p50 or Flag-NuclingDC/NF-jB-p65, Flag-NuclingDN/HA-NFjB-p50 or Flag-NuclingDN/NF-jB-p65, or Flag-vector/HANF-jB-p50 or Flag-vector/NF-jB-p65 (Fig 1A), and then cultured overnight First, the cells were fixed with 4% paraformaldehyde in NaCl/Pi for 15 at room temperature and immersed in NaCl/Pi containing 0.1% Triton X-100 for on ice For blocking, the cells were incubated with 3% bovine serum albumin in NaCl/Pi for h Then, they were incubated with the anti-HA (3F10) or NF-jB-p65 antibody (F-6) overnight at room temperature, and the slides were incubated with Texas Red-conjugated anti-goat IgG (ICN Biomedicals, Inc., Aliso Viejo, CA, USA) or rhodamine-conjugated anti-mouse IgG (Chemicon International, Inc., Temecula, CA, USA) for h Next, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated antiFLAG M2 (Sigma) overnight at °C Finally, cells on the slides were mounted with anti-fade solution, sealed and examined using a confocal laser scanning microscope and software (Leica TCS NT, Heidelberg, Germany) Between each step, the slides were washed three times with NaCl/Pi buffer EMSA RT-PCR and real-time RT-PCR Total RNA from MEFs was extracted from Nucling+/+ and Nucling)/) mice using TRIZOLÒ Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) cDNA synthesis was performed using a Superscript Preamplification System for First Strand cDNA Synthesis Kit (Invitrogen) The following primers were used: for TNFa, 5¢-ATGAGCACAG AAAGCATGATCC-3¢ and 5¢-CCAAAGTAGACCTGCC CGGACTC-3¢; for COX2, 5¢-AAAACCGTGGGGAATGT ATGAGC-3¢ and 5¢-GATGGGTGAAGTGCTGGGCAA AG-3¢; for b-actin, 5¢-TTGTAACCAACTGGGACGATAT GG-3¢ and 5¢-GATCTTGATCTTCATG GTGCTAGG-3¢ PCR was performed as follows: for TNFa, 30 cycles (94 °C, min; 60 °C, 45 s; 72 °C, min); for COX2, 25 cycles (94 °C, 20 s; 60 °C, 40 s; 72 °C, 40 s); for b-actin, 25 cycles (95 °C, min; 57 °C, min; 72 °C, min) PCR was performed on a DNA thermal cycler (GeneAmp PCR system 9700; Applied Biosystems, Foster City, CA, USA) For realtime RT-PCR, transcript levels of TNFa, COX2 and b-actin were examined using a PTC-200 DNA Engine Cycler (MJ Research Incorporated, Waltham, MA, USA) with SYBR Premix Ex Taq (Takara, Kyoto, Japan) Results were calculated with the software of the DNA Engine Opicon System (Roche Molecular System, Inc., Alameda, CA, USA) The NF-jB oligonucleotide (5¢-AGTTGAGGGGACT TTCCCAGGC-3¢, Promega) was labeled with [c-32P]ATP (NEN Life Science Products, Inc., Boston, MA, USA) using T4 polynucleotide kinase MEF nuclear extracts (1 lgỈlL)1) were added to lL of gel shift binding buffer [20% (v/v) glycerol, mm MgCl2, 2.5 mm EDTA, 2.5 mm dithiothreitol, 250 mm NaCl, 50 mm Tris/HCl, pH 7.5, and 0.25 mgỈmL)1 poly(dI-dC)] For competition or gel shift experiments, binding was performed in the presence of lL (1.75 pmolỈlL)1) of unlabelled NF-jB oligonucleotide or lg of NF-jB-p65 antibody (F-6) (Santa Cruz Biotechnology) and NF-jB-p50 antibody (E-10) (Santa Cruz Biotechnology), respectively The mixture was incubated for 20 on ice before the addition of lL of 32P-labelled NF-jB oligonucleotide as probe The resulting complexes were resolved by electrophoresis on a 4% polyacrylamide gel Finally, the gel was dried and analyzed by autoradiography Two-dimensional native/denaturing PAGE of protein complexes WT or Nucling)/) MEF cell lysates were fractionated into a cytosolic fraction as described previously [1] The lysate was resolved onto 2–15% precast gels (Daiichi Pure Chemi- FEBS Journal 276 (2009) 1459–1470 ª 2009 The Authors Journal compilation ª 2009 FEBS 1469 Nucling regulates cellular distribution of NF-jB L Liu et al cals Co Ltd., Tokyo, Japan) For western blotting, the native gel was soaked in blot buffer (20 mm Tris-base, 150 mm glycine and 0.08% SDS) for 10 min, and denatured proteins were transferred to polyvinylidene difluoride membranes (Millipore) in the same buffer using the semidry blotting technique Immunodetection was carried out according to standard procedures and was visualized by the ELC method (Amersham-Pharmacia) For the twodimensional analysis, individual lanes were cut out from the first-dimension native gel and layered on top of a 15–25% gradient resolving gel, and a 7% stacking gel was poured over and around the native gel slice Acknowledgements We thank T Fujita for the plasmid containing NF-jBp65 and NF-jB-p50 cDNA, and T Takemori and M Matsumoto for the NF-jB-dependent luciferase pGL-2 vector This work was supported by a Grantin-Aid for Scientific Research and a Grant for the 21st Century COE Program from the Ministry of Education, Science, Sports and Culture of Japan for the Promotion of Science, and a Research Grant from the Ministry of Health, Labor and Welfare of Japan References Sakai T, Liu L, Shishido Y & Fukui K (2003) Identification of a novel, embryonal carcinoma cell-associated molecule, nucling, that is up-regulated during cardiac muscle differentiation J Biochem (Tokyo) 133, 429–436 Sakai T, Liu L, Teng X, Mukai-Sakai R, Shimada H, Kaji R, Mitani T, Matsumoto M, Toida K, Ishimura K et al (2004) Nucling recruits Apaf-1/pro-caspase-9 complex for the induction of stress-induced apoptosis J Biol Chem 279, 41131–41140 Teng X, Sakai T, Liu L, Sakai R, Kaji R & Fukui K (2006) Attenuation of MPTP-induced neurotoxicity and locomotor dysfunction in Nucling-deficient mice via suppression of the apoptosome pathway J Neurochem 97, 1126–1135 Liu L, Sakai T, Sano N & Fukui K (2004) Nucling mediates apoptosis by inhibiting expression of galectin-3 through interference with nuclear factor kappaB signalling Biochem J 380, 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Oncogene 18, 7908–7916 12 Park MY, Jang HD, Lee SY, Lee KJ & Kim E (2004) Fas-associated factor-1 inhibits nuclear factor-kappaB (NF-kappaB) activity by interfering with nuclear translocation of the RelA (p65) subunit of NF-kappaB J Biol Chem 279, 2544–2549 13 van Heel DA, Hunt KA, Ghosh S, Herve M & Playford RJ (2006) Normal responses to specific NOD1activating peptidoglycan agonists in the presence of the NOD2 frameshift and other mutations in Crohn’s disease Eur J Immunol 36, 1629–1635 14 Van Huffel S, Delaei F, Heyninck K, De Valck D & Beyaert R (2001) Identification of a novel A20-binding inhibitor of nuclear factor-kappa B activation termed ABIN-2 J Biol Chem 276, 30216–30223 15 Bauer MK, Lieb K, Schulze-Osthoff K, Berger M, Gebicke-Haerter PJ, Bauer J & Fiebich BL (1997) Expression and regulation of cyclooxygenase-2 in rat microglia Eur J Biochem 243, 726–731 16 Szlosarek P, Charles KA & Balkwill FR (2006) Tumour necrosis factor-alpha as a tumour promoter Eur J Cancer 42, 745–750 17 Kashiwada M, Shirakata Y, Inoue JI, Nakano H, Okazaki K, Okumura K, Yamamoto T, Nagaoka H & Takemori T (1998) Tumor necrosis factor receptor-associated factor (TRAF6) stimulates extracellular signal-regulated kinase (ERK) activity in CD40 signaling along a ras-independent pathway J Exp Med 187, 237–244 Supporting information The following supplementary material is available: Fig S1 Nucling is involved in the regulation of the NF-jB signaling pathway This supplementary material can be found in the online version of this article Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 276 (2009) 1459–1470 ª 2009 The Authors Journal compilation ª 2009 FEBS ... the distribution of ectopic NF-jB apparently overlapped with that of Nucling (Fig 5B,F) or NuclingDC (Fig 5C,G), but not with that of NuclingDN (Fig 5D,H), suggesting that Nucling interacts with. .. FEBS 1461 Nucling regulates cellular distribution of NF-jB L Liu et al A B C D Fig Nucling interacts with NF-jB-p50 and NF-jB-p65 but not with IjBa (A) Primary structure of full-length Nucling. .. of NF-jB-p65 and Flag -Nucling/ Flag-NuclingDC/Flag-NuclingDN in cellular lysates were confirmed by western blotting (D) COS7 cells were transfected with Flag -Nucling, Flag-NuclingDC or Flag-vector