Title: Notch1 augments NF-κB activity by facilitating its nuclear retention
Abstract: Article1 December 2005free access Notch1 augments NF-κB activity by facilitating its nuclear retention Hyun Mu Shin Hyun Mu Shin Molecular and Cellular Biology Program, University of Massachusetts/Amherst, Amherst, MA, USA Search for more papers by this author Lisa M Minter Lisa M Minter Department of Veterinary and Animal Sciences, University of Massachusetts/Amherst, Amherst, MA, USA Search for more papers by this author Ok Hyun Cho Ok Hyun Cho Department of Veterinary and Animal Sciences, University of Massachusetts/Amherst, Amherst, MA, USA Search for more papers by this author Sridevi Gottipati Sridevi Gottipati Molecular and Cellular Biology Program, University of Massachusetts/Amherst, Amherst, MA, USA Search for more papers by this author Abdul H Fauq Abdul H Fauq Department of Neuroscience, Mayo Clinic, Mayo Clinic College of Medicine, Jacksonville, FL, USA Search for more papers by this author Todd E Golde Todd E Golde Department of Neuroscience, Mayo Clinic, Mayo Clinic College of Medicine, Jacksonville, FL, USA Search for more papers by this author Gail E Sonenshein Gail E Sonenshein Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Barbara A Osborne Corresponding Author Barbara A Osborne Molecular and Cellular Biology Program, University of Massachusetts/Amherst, Amherst, MA, USA Department of Veterinary and Animal Sciences, University of Massachusetts/Amherst, Amherst, MA, USA Search for more papers by this author Hyun Mu Shin Hyun Mu Shin Molecular and Cellular Biology Program, University of Massachusetts/Amherst, Amherst, MA, USA Search for more papers by this author Lisa M Minter Lisa M Minter Department of Veterinary and Animal Sciences, University of Massachusetts/Amherst, Amherst, MA, USA Search for more papers by this author Ok Hyun Cho Ok Hyun Cho Department of Veterinary and Animal Sciences, University of Massachusetts/Amherst, Amherst, MA, USA Search for more papers by this author Sridevi Gottipati Sridevi Gottipati Molecular and Cellular Biology Program, University of Massachusetts/Amherst, Amherst, MA, USA Search for more papers by this author Abdul H Fauq Abdul H Fauq Department of Neuroscience, Mayo Clinic, Mayo Clinic College of Medicine, Jacksonville, FL, USA Search for more papers by this author Todd E Golde Todd E Golde Department of Neuroscience, Mayo Clinic, Mayo Clinic College of Medicine, Jacksonville, FL, USA Search for more papers by this author Gail E Sonenshein Gail E Sonenshein Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Barbara A Osborne Corresponding Author Barbara A Osborne Molecular and Cellular Biology Program, University of Massachusetts/Amherst, Amherst, MA, USA Department of Veterinary and Animal Sciences, University of Massachusetts/Amherst, Amherst, MA, USA Search for more papers by this author Author Information Hyun Mu Shin1, Lisa M Minter2, Ok Hyun Cho2, Sridevi Gottipati1, Abdul H Fauq3, Todd E Golde3, Gail E Sonenshein4 and Barbara A Osborne 1,2 1Molecular and Cellular Biology Program, University of Massachusetts/Amherst, Amherst, MA, USA 2Department of Veterinary and Animal Sciences, University of Massachusetts/Amherst, Amherst, MA, USA 3Department of Neuroscience, Mayo Clinic, Mayo Clinic College of Medicine, Jacksonville, FL, USA 4Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA *Corresponding author. Department of Veterinary and Animal Sciences, 311 Paige Laboratory, University of Massachusetts/Amherst, 161 Holdsworth Way, Amherst, MA 01003, USA. Tel.: +1 413 545 4882; Fax: +1 413 545 1446; E-mail: [email protected] The EMBO Journal (2006)25:129-138https://doi.org/10.1038/sj.emboj.7600902 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Notch1 specifically upregulates expression of the cytokine interferon-γ in peripheral T cells through activation of NF-κB. However, how Notch mediates NF-κB activation remains unclear. Here, we examined the temporal relationship between Notch signaling and NF-κB induction during T-cell activation. NF-κB activation occurs within minutes of T-cell receptor (TCR) engagement and this activation is sustained for at least 48 h following TCR signaling. We used γ-secretase inhibitor (GSI) to prevent the cleavage and subsequent activation of Notch family members. We demonstrate that GSI blocked the later, sustained NF-κB activation, but did not affect the initial activation of NF-κB. Using biochemical approaches, as well as confocal microscopy, we show that the intracellular domain of Notch1 (N1IC) directly interacts with NF-κB and competes with IκBα, leading to retention of NF-κB in the nucleus. Additionally, we show that N1IC can directly regulate IFN-γ expression through complexes formed on the IFN-γ promoter. Taken together, these data suggest that there are two 'waves' of NF-κB activation: an initial, Notch-independent phase, and a later, sustained activation of NF-κB, which is Notch dependent. Introduction Notch proteins are a family of large (300 kDa) single-pass type I transmembrane receptors, activated by regulated intramembrane proteolysis (Schroeter et al, 1998). They function as cell surface receptors and direct regulators of gene transcription, and are involved in a variety of cellular events. In vertebrates, there are four Notch genes, which encode receptors (Notch1–4) for at least five different Notch ligands (Jagged1/Serrate1, Jagged2/Serrate2, Delta1, Delta2 and Delta3) (Artavanis-Tsakonas et al, 1999). Notch is activated through binding of appropriate ligands on neighboring cells to the extracellular domain of the Notch receptors (Artavanis-Tsakonas et al, 1999). This culminates in proteolytic cleavage by γ-secretase, release and nuclear translocation of the Notch intracellular domain (NIC) from the membrane, leading to the production of several downstream proteins (Artavanis-Tsakonas et al, 1999; Robey and Bluestone, 2004). The role of Notch signaling in early T- and B-cell development has been studied extensively, but its contribution to mature T-cell function is not fully understood. In mammals, the NF-κB family of transcription factors contains five members: NF-κB1 (p105/p50), NF-κB2 (p100/p52), RelA (p65), RelB and c-Rel. NF-κB1 and NF-κB2 are synthesized as large polypeptides that are post-translationally cleaved to generate the DNA binding subunits p50 and p52, respectively (Caamano and Hunter, 2002; Ghosh and Karin, 2002). Members of the NF-κB family are characterized by the presence of a Rel homology domain, which contains a nuclear localization sequence, and are involved in sequence-specific DNA binding, dimerization and interaction with the inhibitory IκB proteins. Dimeric NF-κB complexes are associated with specific responses to different stimuli and differential effects on transcription. NF-κB1 (p50) and NF-κB2 (p52) lack transcriptional activation domains, and their homodimers are thought to act as repressors (Lee et al, 1995; Li and Verma, 2002) unless complexed with Bcl-3, a nuclear binding partner possessing a transactivational domain (Bours et al, 1993; Fujita et al, 1993). In contrast, RelA, RelB and c-Rel carry transcriptional activation domains, and are able to form homo- and heterodimers with other NF-κB family members with the exception of RelB, which cannot homodimerize. NF-κB is held inactive in the cytoplasm by interaction with an inhibitor of NF-κB (IκB). NF-κB can be activated by cellular exposure to inflammatory cytokines such as TNF or IL-1, by-products of bacterial and viral infection, radiation or T-cell costimulation (Beinke and Ley, 2004). This results in activation of the IκB kinase complex, leading to phosphorylation and degradation of IκB proteins and release of NF-κB. Free NF-κB translocates to the nucleus, activating downstream target genes, including IκBα. Several studies suggest that newly synthesized IκBα enters the nucleus and binds nuclear NF-κB, mediating export of the complex to the cytosol (Arenzana-Seisdedos et al, 1997; Rodriguez et al, 1999; Hoffmann et al, 2002; Nelson et al, 2004). Super-repressor IκBα (SR-IκBα) encodes a mutant IκBα protein, unable to be phosphorylated or degraded, which complexes with NF-κB in the cytoplasm keeping it inactive and resulting in decreased IFN-γ secretion (Ferreira et al, 1999). The interaction between antigen-presenting cell (APC) and T cell also results in NF-κB activation in both cell types (Li and Verma, 2002). NF-κB activation is triggered in T cells by the engagement of the T-cell receptor (TCR) and the CD28 co-receptor with their respective ligands, the MHC class II-peptide complex and the costimulatory molecules CD80 and CD86, present on APCs (Schmitz et al, 2003). Signaling through the TCR and CD28 synergizes to induce the NF-κB-dependent genes required for T-cell activation and proliferation, such as IL-2, IL-2 receptor alpha chain and IFN-γ (Cross et al, 1989; Gerondakis et al, 1996; Sica et al, 1997; Zhou et al, 2002; Artis et al, 2003). Activated T cells, in turn, elicit NF-κB activation in APCs (Li and Verma, 2002). We have previously shown that Notch signaling results in the activation of NF-κB, IFN-γ secretion and cell proliferation in murine T cells (Palaga et al, 2003). A pharmacological inhibitor specific for γ-secretase inhibitor (GSI) is a useful drug to block proteolytic cleavage of Notch proteins by γ-secretase and prevents the activation of all Notch isoforms. Using GSI treatment to block proteolytic cleavage of Notch receptors or using transgenic mice expressing an antisense Notch1 construct (Notch1AS Mice), we previously demonstrated downregulation of NF-κB activity, IFN-γ secretion and cell proliferation, suggesting that, in concert with TCR signaling, Notch1 induces NF-κB activation. However, precisely how Notch1 regulated NF-κB activity in peripheral T cells remained unclear. Recent studies have suggested possible interactions between Notch and NF-κB (Oswald et al, 1998; Espinosa et al, 2003; Oakley et al, 2003). In this study, we initiated a series of experiments designed to more clearly define the relationship between Notch and NF-κB activity. Here, we show that GSI pretreatment abrogates the generation and nuclear translocation of active Notch1, leading to downregulation of NF-κB activity. Furthermore, pretreatment with GSI also attenuated the nuclear import and the sustained nuclear activity of NF-κB. Our data demonstrate that in splenocytes, IFN-γ is a direct target of Notch-regulated NF-κB activation. We present a model that suggests that Notch maintains NF-κB activity by direct interaction with p50/c-Rel in the nucleus. This interaction retains active NF-κB complexes in the nucleus, leading to sustained NF-κB activity over time and in the activation of NF-κB-regulated genes. Results Stimulating splenocytes with anti-CD3ε and anti-CD28 induces and sustains activation of NF-κB and upregulates p50 and c-Rel Our earlier studies demonstrated that active NIC increases NF-κB DNA binding activity following stimulation with anti-CD3ε and anti-CD28 antibodies (Palaga et al, 2003). To identify the nature of the NF-κB subunits induced, nuclear extracts were prepared from splenocytes from C57BL/6 mice that were left unstimulated or were stimulated with anti-CD3ε and anti-CD28 antibodies for 48 h. EMSA was performed using the oligonucleotide probes corresponding to the NF-κB binding sequence (5′-AGTTGAGGGGACTTTCCCAGGC-3′). To characterize the composition of the DNA-bound NF-κB complexes found in activated splenocytes, we performed supershift analysis with antibodies specific for each NF-κB family member. Antibody stimulation enhanced NF-κB DNA binding activity compared to unstimulated controls, as previously shown (Palaga et al, 2003). These NF-κB complexes were identified as p50 and c-Rel, based on the ability of anti-p50 or anti-c-Rel to recognize the complexes, whereas binding of anti-p65 was not detected (Figure 1A). It has been suggested that the p65 component of NF-κB complexes is involved in early time points following T-cell activation, whereas c-Rel-containing NF-κB complexes dominate the later time points (Himes et al, 1996). To assess the temporal responses of NF-κB, we performed supershift analysis of p50, c-Rel and p65 subunits at several time points following splenocyte stimulation. A supershifted band consisting of p50 steadily increased up to 48 h, whereas c-Rel activity peaked between 6 and 12 h and then diminished over time. In contrast, low levels of p65 activity could be detected only at 6 h (Figure 1B). These data indicate that NF-κB complexes present in stimulated splenocytes consist primarily of p50 and c-Rel. Figure 1.NF-κB expression during splenocyte activation. (A) Splenocytes isolated from C57BL/6 mice were stimulated with anti-CD3ε (1 μg/ml) and anti-CD28 (1 μg/ml) for 48 h. EMSAs were performed on nuclear extracts using radiolabeled oligonucleotides containing the NF-κB binding site and supershifted with specific NF-κB antibodies. The arrows indicate DNA-bound NF-κB complexes, and upper bands show specific antibody-bound DNA–NF-κB complexes. The lower band represents protein(s) present in the nuclear extracts that also bind the NF-κB consensus sequence of the oligonucleotides (Beverly and Capobianco, 2004), as addition of NF-κB-specific antibodies does not interfere or supershift this complex. (B) Splenocytes from C57BL/6 mice were stimulated as in panel A for the indicated time points, followed by EMSA, and supershifted with specific NF-κB antibodies (upper arrows). The lower arrows indicate DNA-bound NF-κB complexes and diamonds indicate competition with a 50-fold molar excess of unlabeled NF-κB oligonucleotides. Download figure Download PowerPoint During early splenocyte activation, temporal induction of phosphorylated IκBα leads to Notch-independent NF-κB activation To further investigate the temporal expression of NF-κB and IκBα in activated splenocytes, we analyzed the expression of these proteins by immunoblot and EMSA. Phosphorylation of IκBα increased 30 min after treatment and gradually returned to basal levels by 24 h (Figure 2A, upper panel). The data in the upper panel of Figure 2A were scanned and the relative intensity was plotted as a function of time (Figure 2A, lower panel). Over this same time course, expression of total IκBα, p65 and c-Rel remained constant, but p50 expression increased gradually following antibody stimulation (Figure 2B). We observed peak NF-κB binding activity 12–24 h following splenocyte activation and this was maintained over 48 h, even in the presence of high levels of endogenous IκBα expression and low levels of phosphorylated IκBα (Figure 2A and B). Recent data from our lab suggested that Notch1 specifically upregulates IFN-γ expression through NF-κB activation (Gottipati et al, in preparation); therefore, we also assessed the temporal induction of Notch1. Interestingly, Notch1 expression also peaked 12–24 h following stimulation; thus, it is unlikely that the early induction of NF-κB activity is mediated via Notch signaling. Furthermore, the fact that NF-κB binding activity was sustained at later time points, even when levels of IκBα were high, suggests a novel mechanism of maintaining NF-κB in activated T cells. Figure 2.Temporal expression of NF-κB components and Notch1. (A) Splenocytes from C57BL/6 mice were stimulated as described for indicated time periods, followed by EMSA to determine NF-κB DNA binding activity, and immunoblotted for Notch1 and phospho-IκBα; calculated densitometric analyses were made using ImageJ software V. 1.31 supported by Wayne Rasband, NIH. The loading control of p-IκBα and Notch1 immunoblots is the same as in panel B. (B) Immunoblots were performed on whole-cell lysates from stimulated splenocytes, using indicated antibodies. Download figure Download PowerPoint Blocking Notch signaling abrogates sustained but not initial NF-κB activation As daily treatment with GSI blocked NF-κB activity at 48 h in antibody-stimulated splenocytes (Palaga et al, 2003), we examined the effects of GSI on the kinetics of NF-κB activation during splenocyte activation. Splenocytes from C57BL/6 mice were pretreated with GSI or DMSO as control, and then stimulated with anti-CD3ε and anti-CD28 antibodies. At the indicated time points, fractionated cytosolic and nuclear extracts were prepared for EMSA and immunoblotting. Within the first 12 h of stimulation, we found comparable NF-κB binding regardless of pretreatment (Figure 3A). In contrast, GSI pretreatment essentially abrogated NF-κB binding at later time points, compared to control-treated cells (Figure 3A). These data indicate that using GSI to block Notch upregulation abolishes the later, sustained wave of NF-κB activation, but not its initial induction (Figure 3A). We used a supershift assay to examine the effects of GSI pretreatment on individual components of the NF-κB signaling complex using extracts prepared after 24 h of stimulation. GSI pretreatment blocked formation of the nuclear NF-κB complex, which was shown to supershift with antibodies against p50 and c-Rel (Figure 3B). The immunoblot analysis with the cytosolic and nuclear fractions shows decreased nuclear p50 and c-Rel in cells pretreated with GSI (Figure 3C). Stimulation with anti-CD3ε and anti-CD28 antibodies for 24 h increased expression of both p50 and its precursor, p105, and markedly enhanced nuclear translocation of p50, whereas nuclear expression of c-Rel was only slightly increased. In a reciprocal fashion, inhibition of p50 nuclear localization following GSI pretreatment was more robust than that of c-Rel at 24 h. Regardless of treatment conditions, IκBα was found primarily in the cytosol. Taken together, these findings indicate that Notch exerts little, if any, influence on the early NF-κB response, and that Notch signaling is involved in sustaining NF-κB activity at later time points. Furthermore, our data suggest that Notch may modulate NF-κB activity by preferentially enhancing p50 nuclear distribution, maintaining NF-κB activity and prolonging splenocyte activation. Figure 3.Blocking Notch signaling using GSI prevents sustained NF-κB activity. (A) Splenocytes from C57BL/6 mice were pretreated for 30 min with GSI (50 μM IL-CHO) or DMSO (vehicle control) before stimulation as previously described for the indicated time course. Nuclear extracts from treated splenocytes at each time point were harvested and analyzed by EMSA for DNA binding activity of NF-κB. The density of boxed bands (right upper panel) was calculated using ImageJ software. (B) Nuclear extracts from splenocytes were pretreated for 30 min with GSI (50 μM IL-CHO) or DMSO before stimulation as described for 24 h, followed by EMSA, and supershifted with p50 or c-Rel antibody. The arrow indicates DNA-bound NF-κB complexes and boxed bands show supershifted, antibody-bound DNA–NF-κB complexes. The diamond indicates competition with a 50-fold molar excess of unlabeled NF-κB oligonucleotides. (C) Splenocytes were pretreated for 30 min with GSI (50 μM IL-CHO) or DMSO before stimulation for 24 h, followed by immunoblotting of cytosolic and nuclear extracts with anti-cleaved Notch1, anti-p50, anti-c-Rel or anti-IκBα. Lack of expression of the nuclear protein PARP was used to assess purity of cytosolic fractions, whereas the lack of p105 (precursor of p50) in the nucleus indicated purity of the nuclear fraction. Download figure Download PowerPoint Direct interaction of NF-κB and N1IC promotes a synergistic signaling effect Although earlier reports showed that Notch directly interacts with NF-κB, it remains controversial as to whether this interaction represses (Guan et al, 1996; Wang et al, 2001) or enhances NF-κB activity (Bellavia et al, 2000; Cheng et al, 2001; Palaga et al, 2003). Additionally, ectopic expression of N1IC in the T-cell hybridoma line, DO11.10, which lacks endogenous Notch1 expression, increases NF-κB response and IFN-γ production (Gotipatti et al, in preparation). To confirm the effects of Notch signaling on NF-κB activity, NF-κB luciferase reporter constructs were transfected into the DO11.10 cells expressing N1IC or empty control. N1IC increased luciferase activity approximately four-fold above empty control (Figure 4A). To confirm physical interaction between Notch1 and components of the NF-κB complex, we performed co-immunoprecipitation experiments. Vectors expressing N1IC and either p50 or c-Rel were co-transfected into 293T cells and subjected to co-immunoprecipitation with antibodies against specific NF-κB subunits. Consistent with earlier reports, we detected direct interaction between N1IC and p50, as well as with c-Rel (Figure 4B). Figure 4.Synergistic effect of N1IC on NF-κB is mediated by their direct interaction. (A) NF-κB luciferase reporter plasmid (2 μg) and pRL-CMV (50 ng) plasmid of an internal control were transiently transfected in DO11.10 cells expressing N1IC or empty control and harvested 48 h later for dual luciferase assays. The relative luciferase values were calculated by dual luciferase assays as described in Materials and methods. Values shown are averages of at least two separate experiments. Harvested lysates were immunoblotted with anti-Notch1 and anti-GAPDH antibodies. (B) N1IC expression plasmids were transiently co-transfected with p50 or c-Rel expression plasmids and 293T cells were harvested 24 h later and subjected to co-immunoprecipitation as described in Materials and methods. 1/100 of input shown was immunoblotted with anti-Myc, anti-p50 and anti-c-Rel. (C) NF-κB luciferase reporter (400 ng) plasmid and pRL-CMV (100 ng) plasmid of an internal control were transiently transfected with the indicated plasmids into 293T cells. In the upper graph, the ratio of p50 to c-Rel expression plasmids was 1:1 (50 ng:50 ng) and in the lower graph, p50:c-Rel was 4:1 (200 ng:50 ng) with increasing amounts of N1IC expression plasmids (10, 50 and 100 ng). Transfected cells were incubated for 48 h and harvested for dual luciferase assays. The relative luciferase values were calculated by dual luciferase assays. Values shown are averages of at least three separate experiments. Download figure Download PowerPoint To further investigate the effects of Notch signaling on NF-κB activity, N1IC and p50 and c-Rel expression vectors were co-transfected into 293T cells along with an NF-κB luciferase reporter construct. Expression of p50 alone resulted in slightly increased reporter activity, suggesting possible interaction with endogenous c-Rel or p65. Coexpression of c-Rel and p50, increased luciferase activity approximately two-fold above non-transfected controls (Figure 4C). However, coexpression of N1IC increased NF-κB-dependent transactivation of the luciferase reporter gene in a dose-dependent manner, up to nearly 10-fold over controls, as shown in Figure 4C (upper panel). Based on the expression levels of nuclear p50 and c-Rel present in EMSAs (Figure 1A), we adjusted the ratio of p50 and c-Rel expression vectors (p50:c-Rel, 4:1). At these ratios, we observed similar synergistic effects of N1IC on NF-κB-dependent gene expression (Figure 4C, right panel), although the maximal fold induction was somewhat less than 1:1 ratios. N1IC induced NF-κB-dependent reporter activity in a dose-dependent manner up to a concentration of 100 ng. Together, these data suggest that N1IC can synergize with p50 and/or c-Rel to positively modulate NF-κB-dependent gene expression and this may be mediated by direct molecular interaction between these proteins. Cytosolic sequestering of NF-κB by IκBα is reversed by nuclear N1IC Given that N1IC can physically interact with p50 and c-Rel proteins and that increased N1IC expression correlated with sustained NF-κB activity in stimulated splenocytes even in the presence of high levels of non-phosphorylated IκBα, we asked whether N1IC might somehow prevent IκBα-mediated inhibition of NF-κB. SR-IκBα was co-transfected along with p50 and c-Rel expression constructs and an NF-κB reporter plasmid in the absence or presence of a vector expressing N1IC. As expected, SR-IκBα efficiently abrogated transactivation of the NF-κB reporter (Figure 5A). Remarkably, expression of N1IC restored NF-κB transcriptional activity in a dose-dependent manner (Figure 5A). To assess the influence of N1IC on the subcellular localization of NF-κB, we generated dsRed and GFP chimeras of p50 and c-Rel. The dsRed-p50 and GFP-c-Rel constructs were co-transfected into 293T cells in the absence or presence of vectors expressing SR-IκB-α or N1IC. As indicated in Figure 5B, transfected p50 and c-Rel were localized predominantly to the nucleus, but were sequestered in the cytosol in the presence of SR-IκBα. Addition of N1IC, however, completely abolished cytosolic sequestering of p50 by SR-IκBα and promoted its nuclear relocalization (Figure 5B). Similarly, cytosolic sequestration of c-Rel by SR-IκBα was also reversed when N1IC was coexpressed, although to a lesser extent (Figure 5B; also see Figure 2). Collectively, these data demonstrate that N1IC is capable of sustaining the nuclear activity of NF-κB through its direct interactions with p50 and c-Rel subunits, by increasing nuclear retention of NF-κB subunits. Figure 5.Notch1 rescues NF-κB activity from suppression by SR-IκBα. (A) NF-κB luciferase reporter (400 ng) plasmid and pRL-CMV (100 ng) of an internal control were transiently co-transfected with the indicated plasmids into 293T cells. Ratio of p50 expression plasmid to c-Rel expression plasmid was 4:1 (200 ng:50 ng) with SR-IκBα expression plasmid (50 ng) and increasing amounts of N1IC expression plasmids (10, 50, 100 and 500 ng). Transfected cells were incubated for 48 h and harvested for dual luciferase assays. The relative luciferase values were calculated as described. Values shown are averages of at least three separate experiments. (B) Indicated expression plasmids were transiently transfected into 293T cells. Transfected cells were incubated for 24 h and examined using confocal microscopy in green channel (488 nm), red channel (560 nm) and overlay. Scale bar represents 20 μm. Download figure Download PowerPoint Impaired nuclear localization of N1IC correlates with decreased transcriptional activity of NF-κB To further investigate the influence that subcellular localization of N1IC has on NF-κB signaling, GFP-N1IC constructs were modified so as to increase or decrease their nuclear retention. Specifically, GFP-N1IC was modified by inclusion of either an additional nuclear export signal to reduce nuclear levels (GFP-N1IC-NES) or an additional nuclear localization signal to increase its nuclear retention (GFP-N1IC-NLS) (Jeffries and Capobianco, 2000). These expression vectors were then used in fluorescence imaging to study their effects on localization of N1IC and on NF-κB reporter activity. When the GFP-N1IC-NES or GFP-N1IC-NLS expression vectors were transfected into 293T cells, addition of the NES sequence to GFP-N1IC promoted its cytosolic retention, whereas addition of the NLS sequence enhanced nuclear localization of GFP-N1IC (Figure 6A, upper panel). When an NF-κB reporter was co-transfected into 293T cells with these vectors, we found that enhanced nuclear localization of GFP-N1IC-NLS resulted in increased activity of the NF-κB luciferase reporter, whereas impaired nuclear localization, as seen with expression of GFP-N1IC-NES, reduced NF-κB reporter activity to basal levels (Figure 6A, lower panel). We next examined whether subcellular localization of N1IC could also influence cytosolic versus nuclear localization of p50. The dsRed-p50 vector was co-transfected with GFP-N1IC-NLS or GFP-N1IC-NES into 293T cells and analyzed by confocal microscopy. As expected, GFP-N1IC-NLS was predominantly located in the nucleus where it colocalized with dsRed-p50 (Figure 6B). In contrast, a large proportion of GFP-N1IC-NES was retained in the cytosol and also colocalized with dsRed-p50. These data strongly suggest that cellular distribution of Notch plays a critical role in its ability to sustain NF-κB transcriptional activity, by further influencing subcellular localization of NF-κB components. Figure 6.Cellular distribution of NF-κB p50 is regulated by Notch1. (A) 293T cells were transiently transfected with eGFP empty vector as a control, eGFP N1IC-NLS or eGFP N1IC-NES and examined 24 h after transfection using laser-scanning confocal microscopy. Composite panel, top row: images collected in green channel; center row: DIC images; bottom row: overlay. NF-κB luciferase reporter (400 ng) plasmid and pRL-CMV (100 ng) of an internal control were transiently transfected with the eGFP empty vector as a control, eGFP N1IC-NLS or eGFP N1IC-NES into 293T cells. Transfected cells were incubated for 24 h and harvested for dual luciferase assays. The relative luciferase values were calculated as described. Values shown are averages of at least three separate experiments. Scale bar represents 20 μm. (B) dsRed-p50 expression plasmids were transiently transfected with eGFP empty as a control, eGFP N1IC-NLS or eGFP N1IC-NES into 293T cells. Transfected cells were incubated for 24 h and examined using confocal microscopy. Images were captured in the green channel (488 nm), red channel (560 nm) and then merged in overlay. Scale bar represents 20 μm. Download figure Download PowerPoint N1IC can directly regulate IFN-γ expression through complexes for