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The Journal of Immunology, 1998, 161: 5997-6004.
Copyright © 1998 by The American Association of Immunologists

Signaling Pathways Mediated by the TNF- and Cytokine-Receptor Families Target a Common cis-Element of the IFN Regulatory Factor 1 Promoter1

Sanjay Gupta, Dengfeng Xia, Man Jiang, Sangjin Lee and Alessandra B. Pernis2

Department of Medicine, Columbia University, New York, NY 10032


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD40 activation of B cells is strongly influenced by the presence of cytokines. However, the molecular basis for the interplay between these distinct stimuli is not clearly delineated. IFN regulatory factor 1 (IRF-1) is a transcription factor activated by either CD40 or cytokines. We have found that these different sets of signals target a common cis-acting element in the promoter of this gene, the IRF-1 gamma-activated site (GAS). Targeting of the IRF-1 GAS is not confined to activation via CD40 but extends to other stimuli that mimic the CD40 signaling cascade, like TNF-{alpha} and EBV. In contrast to induction of STATs by cytokines, the IRF-1 GAS-binding complex activated by CD40, TNF-{alpha}, or EBV contains Rel proteins, specifically p50 and p65. In this system, simultaneous exposure to CD40L together with either IL-4 or IFN-{gamma} does not lead to the activation of novel Rel/STAT complexes. Given the importance of IRF-1 in a variety of biologic functions from proliferation to apoptosis, our findings support the notion that modulation of IRF-1 levels may be a critical control point in B cell activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The interaction between CD40 and its ligand (CD40L3) plays a critical role in shaping the proliferative and differentiative outcomes of B cells (1, 2, 3, 4). Stimulation of B cells by CD40 cross-linking is significantly enhanced by the presence of cytokines, in particular IL-4. However, the molecular basis underlying the cross-talk between CD40- and cytokine-mediated signaling pathways has not been clearly delineated.

CD40 shares structural and functional features with members of the TNF family of receptors (5, 6). Despite significant sequence divergence within their intracytoplasmic domain, distinct members of this family of receptors signal through similar components (7). Specifically, many of these receptors associate with a family of proteins termed TRAFs (TNFR-associated factors) (8, 9). Discovery of this family of molecules has revealed that the ability of EBV to successfully immortalize human B cells may rely, in part, on the capacity of one of the viral proteins, Latent membrane protein 1, to associate with TRAF molecules and thus constitutively activate the CD40 signaling pathway (10).

In addition to the TRAFs, CD40 and other members of the TNF family of receptors also activate the NF-{kappa}B/Rel family of transcription factors (9, 11, 12, 13). NF-{kappa}B plays a pivotal role in the transcriptional regulation of many genes critical to immune and inflammatory responses (14, 15). The generation of mice lacking individual Rel family members has indicated that many Rel proteins serve specific nonredundant immunologic functions (14). For instance, B cells from the p50 knockout (ko) are unable to switch to specific isotypes, while their proliferative responses to anti-CD40 Abs are intact (16). In contrast, B cells from the RelB ko demonstrate defective proliferative responses to CD40 stimulation, but undergo normal Ig class switching (17). Thus, different Rel family members may be involved in distinct signaling pathways in response to CD40.

Recruitment of similar signaling components by different members of a family of receptors is also a well-known feature of the cytokine receptor superfamily, which includes IL-4R (18, 19). Many of these receptors signal by following the paradigm originally described for the IFN signal transduction pathway (20). This pathway involves the activation, via JAK kinases, of latent cytoplasmic proteins belonging to the STAT family of proteins. Tyrosine phosphorylation of the STATs allows them to associate in complexes that can then translocate into the nucleus where they modulate gene transcription by binding to distinct DNA elements termed GAS (gamma-activated sites).

Unlike other GAS elements, the GAS in the IFN regulatory factor 1 (IRF-1) promoter displays high-affinity binding for STAT complexes activated in response to a variety of cytokines (21, 22, 23). IRF-1 is a member of a growing family of transcription factors whose members bind a related enhancer but impart contrasting effects on the transactivation of genes (24). For example, the ratio of IRF-1 and another IRF member, IRF-2, has been shown to play a critical role in the regulation of cellular proliferation (25). Additionally, IRF-1 is involved in apoptosis (26, 27) as well as in the development and function of a variety of immune effector cells (28, 29, 30, 31, 32).

We have been interested in understanding the cross-talk between CD40 and cytokine signaling pathways in B cells. We have found that signals triggered by CD40, or by stimuli that mimic the CD40 signaling cascade, like TNF-{alpha} and EBV, activate complexes that can bind to the GAS element of the IRF-1 promoter. However, the IRF-1 GAS-binding complexes activated by CD40, TNF-{alpha}, and EBV do not contain known STAT complexes but employ members of the Rel/NF-{kappa}B family, specifically p50 and p65. Targeting of a common cis-acting element by different pathways may thus allow a cell to integrate multiple incoming signals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

The human B cell lines Ramos (obtained from Dr. Seth Lederman, Columbia University), BL-2, and BJAB cells (obtained from Dr. Riccardo Dalla-Favera, Columbia University) are EBV-negative Burkitt’s lymphomas. WIL-2–729HF2 (WIL-2; American Type Culture Collection, Manassas, VA) and JY (obtained from Dr. Riccardo Dalla-Favera, Columbia University) are EBV-transformed lymphoblastoid B cell lines. THP-1 (obtained from Dr. Kathryne Calame, Columbia University) is a monocytic cell line. All cells were grown in Iscove’s modified Dulbecco’s medium supplemented with 10% FCS (Atlanta Biologicals, Norcross, GA). 293 cells stably transfected either with CD40L (CD40L tx) or CD8 (CD8 tx) were a generous gift of Dr. Seth Lederman (Columbia University, NY). Their derivation and culture conditions have been previously described (8).

DNA-binding assays and cell extracts

The preparation and employment of DNA oligonucleotide probes for electrophoretic mobility shift assays (EMSAs) have been described previously (21). The IRF-1 GAS probe employed in these studies was as follows: 5'-gatcGATTTCCCGAAAT-3'. The ISG-15 interferon-stimulated response element probe was as follows: 5'-gatcCTCGGGAAAGGGAAACCGAAACTGAAGCC. Double-stranded oligonucleotides used as cold competitors were prepared from single-stranded oligonucleotides (Life Technologies, Gaithersburg, MD) with the following sequences: ß-CAS GAS (5'-gatcGACTTCTTGGAATT-3') (21), I{epsilon} GAS (-119 to -104) (5'-gatcAACTTCCCAAGAACAG-3') (21), ICAM-1 GAS (5'-gatcGGTTTCCGGGAAAGC-3') (33), CD23a GAS (5'-gatcCCAATTCTCTTACCTGAGAAATGGAG-3') (34), CD23b GAS (5'-gatcGGGTGAATTTCTAAGAAAGGGAC-3') (34), IL-2R{alpha} NF-{kappa}B (5'-gatcCAGGGGAATCTCCCT-3') (35), and HIV long terminal repeat NF-{kappa}B (5'-gatcCTGGGGACTTTCCAGG-3') (35).

STAT or Rel antisera were added (final dilution 1:20) for 30–60 min (at 4°C), before a standard 20 min (at 25°C) incubation of extracts with the DNA probe. Oligonucleotide competitions were performed by adding a 100-fold excess of unlabeled oligonucleotides for 15 min (at 25°C) before standard 20 min (at 25°C) incubation of extracts with the radiolabeled probe.

Whole cell extracts (WCE) were prepared as described previously (21). Extracts from IL-3-treated FDtrk and IL-6-treated HepG-2 were a kind gift of Dr. Chris Schindler (Columbia University).

Antibodies

Polyclonal rabbit antisera against Stat1 and Stat2 were a generous gift of Dr. Chris Schindler. Polyclonal rabbit antisera against human Stat3, Stat5, and Stat6 were purchased from Santa Cruz Biotechnology, Inc. Polyclonal rabbit antisera against the Rel proteins p50, p65, c-Rel, RelB, p52 were also purchased from Santa Cruz Biotechnology, Inc. Purified anti-CD40 mAb G28–5 was a generous gift of Dr. Seth Lederman. The isotype matched control mAb was purchased from PharMingen (San Diego, CA).

Cell culture

Ramos B cells (50–80 x 106) were added to plates containing CD40L tx or CD8 tx cells (20 x 106) and then incubated at 37°C for varying periods of times in a final volume of 10 ml. Coculture with other cell lines followed the same protocol. Cytokine treatments were conducted simultaneously in separate plates in a final volume of 10 ml utilizing the following cytokine concentrations: human IL-4 (100 U/ml; a kind gift of Dr. Paul Rothman, Columbia University), human IFN-{gamma} (10 ng/ml; PeproTech, Rocky Hill, NJ), human TNF-{alpha} (20 ng/ml; PeproTech), and human IFN-{alpha} (100 U/ml; a generous gift of Dr. Chris Schindler). In experiments where Ramos cells were stimulated with anti-CD40 mAb, 20 x 106 cells were activated with 1 µg/ml of Ab in a final volume of 2 ml. Treatment of Ramos cells with TPCK (N-tosyl-L-phenylalanine chloromethyl ketone) was performed as previously described (36). Briefly, 50 x 106 cells were incubated with 25 µg of TPCK for 1 h before incubation with either CD40L or CD8 tx for 2 h. Treatment with cycloheximide was performed as previously described (21).

UV cross-linking

UV cross-linking was performed as previously described (37) except that the gel shift reactions were conducted at 25°C and irradiated twice with 1000 mJ of UV administered with a Stratalinker (Ultra-violet Products, Cambridge, UK). Cross-linked DNA-protein complexes were immunoprecipitated using either a p65 or a Stat3 antiserum (Santa Cruz Biotechnology, Santa Cruz, CA). The immunoprecipitates were then fractionated on a 7% SDS-polyacrylamide gel, transferred onto a nitrocellulose membrane (Schleicher & Schuell, Keene, NH) and exposed to an x-ray film. Reactions not exposed to UV cross-linking were simultaneously conducted as negative controls.

Northern analysis

Total RNA was extracted by using the Ultra-Spec II kit (Biotecx Laboratories, Houston TX). Northern blot analysis was performed with 10 µg of total RNA according to standard protocols. The blot was probed with either a human IRF-1 cDNA (kindly provided by Dr. Richard Pine) or a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA radiolabeled with the Pharmacia DNA Labelling beads (-dCTP) kit (Pharmacia Biotech, Piscataway, NJ).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of an IRF-1 GAS-binding complex by CD40, TNF-{alpha}, or EBV

To investigate the effects of CD40 stimulation on cytokine signaling pathways, we cocultured cells from Ramos, an EBV-negative Burkitt’s lymphoma cell line, together with 293 cells stably transfected either with human CD40L (CD40L tx) or with CD8 (CD8 tx) as a negative control (8). After harvesting the cells, we prepared whole cell extracts and assayed them by EMSA using as a probe the GAS element from the IRF-1 promoter. We have previously used this site to detect the induction of a variety of STAT complexes in response to different cytokines (21). A functional CD40/CD40L interaction was confirmed by detecting CD23 up-regulation via FACS (data not shown). These experiments revealed that stimulation of Ramos cells with CD40L tx but not with CD8 tx led to the appearance of a novel DNA-binding complex (Fig. 1GoA). Similar results were obtained by using an Ab to CD40 (G28–5) rather than the CD40L transfectants, confirming that induction of this complex was a direct effect of the CD40/CD40L interaction (Fig. 1GoA). Activation of this CD40-inducible complex was observed in a variety of B cell lines (BL-2, BJAB) as well as in a monocytic cell line, THP-1, which is known to respond to CD40 activation (Fig. 1GoA and data not shown). Consistent with the finding that both TNF-{alpha} and EBV utilize signaling pathways similar to those triggered by CD40, a comparable IRF-1 GAS-binding complex was observed upon stimulation of Ramos cells with TNF-{alpha} or constitutively in EBV transformed B cell lines (WIL-2 and JY) (Fig. 1GoB).



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  		FIGURE 1. A, Induction of IRF-1 GAS-binding activities in Ramos, BL-2, or THP-1 cells by CD40 stimulation. WCE were prepared from Ramos or BL-2 with or without a 24-h stimulation at 37°C with CD40L transfectants (tx) vs control transfectants. Ramos cells were also separately stimulated with either an anti-CD40 mAb (G28–5) at 1 µg/ml or a control mAb. WCE from IL-4-treated Ramos cells were included as a control. THP-1 cells were cultured for 45 min with either CD40L or control tx. The WCE were examined by EMSA using a 32P-labeled IRF-1 GAS probe. B, Comparison of IRF-1 GAS-binding activities induced in Ramos cells by CD40 or TNF-{alpha} stimulation vs those constitutively detected in EBV-transformed B cell lines (WIL-2, JY). WCE were prepared from Ramos cells with or without a 24-h stimulation at 37°C with either an anti-CD40 mAb (G28–5) at 1 µg/ml, a control mAb, or human TNF-{alpha} (20 ng/ml). WCE were simultaneously obtained from untreated WIL-2 (W) or JY cells. The WCE were assayed as described above.

 
Kinetic experiments revealed that both CD40- and TNF-{alpha}-inducible complexes were activated within 15 min after the stimulation of Ramos cells and lasted up to 48 h. Coculturing Ramos cells with CD40L transfectants in the presence of cycloheximide enhanced the induction of the complex (data not shown). The addition of an antiphosphotyrosine Ab to the EMSA reactions did not significantly affect the appearance of this complex (data not shown). Taken together, these data are consistent with the notion that utilization of related signaling pathways by CD40, TNF-{alpha}, and EBV is reflected in the activation of similar DNA-binding complexes. Furthermore, the rapid activation of these factors as well as their resistance to protein synthesis inhibitors suggest that these complexes are composed of latent cytoplasmic factors.

The CD40-, TNF-{alpha}-, and EBV-inducible complexes binding to the IRF-1 GAS do not contain known STAT complexes

Although some GAS elements (e.g., the IRF-1 and ß-CAS GAS) can bind a variety of STAT complexes, other GAS sequences (e.g., the I{epsilon} GAS) exhibit exquisite specificity for distinct STAT complexes (21). To begin delineating the nature of the GAS-binding complex activated in response to CD40 or TNF-{alpha}, we performed EMSA experiments using a panel of different GAS elements (21) as cold competitors of the radiolabeled IRF-1 GAS probe (Table IGo). Although the I{epsilon} GAS and the ICAM-1 GAS are derived from the promoters of CD40- and/or TNF-{alpha}-responsive genes, neither of these elements competed for the complex induced by CD40 or TNF-{alpha} (Fig. 2GoA). As described previously (21), these elements adequately competed for the IL-4-inducible Stat6 homodimer (34, 38, 39, 40) and/or the IFN-{gamma}-inducible Stat1 homodimer (41) (Fig. 2GoA). The distinctive competition pattern displayed by the CD40-inducible complex raises the possibility that this complex might not contain known STAT complexes.


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  		Table I. Oligonucleotides used in competition analysis

 


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  		FIGURE 2. Comparison of IRF-1 GAS-binding activities induced by CD40, IFN-{gamma}, or IL-4 stimulation by GAS oligonucleotide competition. A, Ramos cells were stimulated with either CD40L tx, human IFN-{gamma} (10 ng/ml), or human IL-4 (100 U/ml). Extracts were then prepared and analyzed as described in Fig. 1Go. Oligonucleotide competition assays were performed either in the absence or in the presence of a 100-fold molar excess of cold GAS oligonucleotides added to the shift reaction as indicated. Competitors included I{epsilon}-GAS, ßCAS-GAS, ICAM1- GAS, or IRF-1 GAS. B, Ramos cells were cultured and assayed as described above. Competitors included the CD23a GAS and the CD23b GAS.

 
Another CD40 target gene, CD23, exists as two different isoforms, CD23a and CD23b, whose expression is controlled by separate promoters (42). Both of these promoters contain GAS elements. Interestingly, when these GAS elements were tested, only the oligonucleotide containing the CD23b GAS was able to compete with the CD40-inducible complex (Fig. 2GoB). In contrast to this selective effect, both the CD23a and CD23b GAS blocked the appearance of the IL-4-inducible Stat6 homodimer, while neither the CD23a GAS nor the CD23b GAS affected the appearance of the IFN-{gamma}-inducible Stat1 homodimer. Thus, binding of the CD40-inducible complex can be mediated only by a restricted subset of GAS-containing DNA regions.

To confirm the absence of STAT proteins in the CD40- and TNF-{alpha}-inducible complex, we analyzed these complexes by supershift experiments with a panel of antisera against STAT 1, 3, 5, and 6 (Fig. 3Go, A and B) and against Stat2 (Fig. 3GoC). In each case, the Ab supershifted complexes known to contain specific STATs, but none of the antisera affected the CD40- or the TNF-{alpha}-inducible complex. Similar results were obtained when different STAT antisera were utilized to supershift the constitutive complex detected in EBV-transformed B cells lines (data not shown).



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  		FIGURE 3. Comparison of IRF-1 GAS-binding activities induced by CD40, TNF-{alpha}, IFN-{gamma}, or IL-4 stimulation by STAT antisera reactivity. A, Ramos cells were stimulated with either anti-CD40 mAb, human TNF-{alpha} (20 ng/ml), human IFN-{gamma} (10 ng/ml), or human IL-4 (100 U/ml). Extracts were then prepared and examined as described in Fig. 1Go. Ab interference mobility shift assays were conducted by addition of either an antiserum against Stat1 or against Stat6 or a preimmune serum (control). All antisera were added at a final dilution of 1:20 for 30 min at 4°C before incubation with the IRF-1 GAS probe for 20 min at 25°C. B, Ramos cells were stimulated with anti-CD40 mAb. Extracts were then prepared and examined as described in Fig. 1Go. Ab interference mobility shift assays were conducted as above by addition of either an antiserum against Stat3 or Stat5 or a preimmune serum (control). IL-6-treated HepG2 extracts and IL-3-treated FDtrk extracts known to contain Stat3 and Stat5, respectively, were a kind gift of Dr. Chris Schindler. C, Ramos cells were stimulated with either anti-CD40 mAb or human IFN-{alpha} (100 U/ml). Extracts were then prepared and assayed with either an ISG-15 interferon-stimulated response element probe (to detect the Stat2 containing ISGF3 complex) or the IRF-1 GAS probe. Ab interference assays were conducted as above by addition of either an antiserum against Stat2 or a preimmune serum (control).

 
Other groups have employed immunoprecipitation assays to detect the activation of Stat3 and Stat6 in response to CD40 (43, 44). To confirm the lack of STAT activation in our system, we thus performed immunoprecipitations with either a Stat6 or a Stat3 antiserum on extracts from CD40-stimulated Ramos cells. Despite adequate STAT activation in response to appropriate cytokine stimulation, we failed to detect tyrosine phosphorylation of either Stat3 or Stat6 upon CD40 stimulation (data not shown). The lack of Stat6 phosphorylation in response to CD40 was also confirmed by immunoprecipitation of extracts from a different Burkitt’s lymphoma cell line, BL-41 (data not shown). These results, thus, suggest that the complex we detect in response to CD40, TNF-{alpha}, or EBV is likely to be composed of non-STAT proteins.

The CD40-inducible complex binding to the IRF-1 GAS contains NF-{kappa}B-related proteins

The rapid induction of the CD40-inducible complex as well as its resistance to cycloheximide treatment suggested the presence of a latent cytoplasmic factor within the complex. Given the known ability of CD40 cross-linking to activate NF-{kappa}B (11), and the partial overlap of the IRF-1 GAS element with an NF-{kappa}B consensus site (Table IGo), we entertained the idea that this inducible complex might indeed be composed of Rel family members. Oligonucleotide competitions revealed that known NF-{kappa}B sites (15) were able to effectively compete for this CD40-inducible complex but not for the known STAT complexes (Fig. 4Go). Ab supershifting experiments with a panel of commercially available Rel antisera confirmed this finding and demonstrated that the CD40-inducible complex contains both p50 and p65 (RelA) or antigenically related proteins (Fig. 5Go). In contrast, neither the IL-4- nor the IFN-{gamma}-inducible complexes were affected by these antisera (data not shown). The NF-{kappa}B complex binding to the IRF-1 GAS was not significantly affected by antisera recognizing p52 or RelB and only minimally by antisera recognizing two distinct c-Rel epitopes (Fig. 5Go and data not shown). An identical pattern of competition was also displayed by both the TNF-{alpha}-inducible complex and the constitutive complex detected in EBV-transformed B cell lines (Fig. 5Go). Consistent with these results, activation of the CD40-inducible complex was prevented by pretreatment with TPCK, a proteasome inhibitor known to block degradation of I{kappa}B-{alpha} and thus p50/p65 translocation and activation (data not shown) (36).



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  		FIGURE 4. Comparison of IRF-1 GAS-binding activities induced by CD40, IFN-{gamma}, or IL-4 stimulation by NF-{kappa}B oligonucleotide competition. Ramos cells were stimulated with either CD40L tx, human IFN-{gamma} (10 ng/ml), or human IL-4 (100 U/ml). Extracts were then prepared and examined as described in Fig. 1Go. Oligonucleotide competition assays were performed either in the absence or in the presence of a 100-fold molar excess of cold NF-{kappa}B oligonucleotides added to the shift reaction as indicated. Competitors included IL-2R{alpha} NF-{kappa}B, HIV long terminal repeat NF-{kappa}B, IRF-1 GAS.

 


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  		FIGURE 5. The CD40-inducible IRF-1 GAS-binding complex contains p50 and p65. Ramos cells were stimulated and assayed as described in Fig. 1Go. JY cells were unstimulated. Ab interference mobility shift assays were conducted by addition of either an antiserum against p50, p65, c-Rel, or a preimmune serum (control). Antisera were added as described in Fig. 3Go.

 
The induction of p65 binding to the IRF-1 promoter in both CD40L-treated Ramos cells as well as in an EBV-transformed B cell line (JY) was further confirmed by subjecting these DNA-binding complexes to UV cross-linking followed by immunoprecipitations with a p65 antiserum (See Fig. 6Go, upper panel). Reprobing of the filter with a p65 antiserum ensured for equal loading of the immunoprecipitates (Fig. 6Go, lower panel).



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  		FIGURE 6. Induction of p65 binding to the IRF-1 GAS is detected in response to CD40 stimulation in Ramos cells or constitutively in an EBV-transformed B cell line (JY) by UV cross-linking followed by p65 immunoprecipitation. Ramos cells were stimulated with either CD40L tx, control tx, or human IFN-{alpha} (100 U/ml). JY cells were unstimulated. Extracts prepared as described in Fig. 1Go, were incubated with a radiolabeled 5-bromodeoxyuridine substituted IRF-1 GAS probe (BUdR-IRF-1 GAS) in a standard shift reaction. DNA-protein complexes were then irradiated twice with 1000 mJ of UV and then immunoprecipitated with either a p65 or a Stat3 antiserum as control. The immunoprecipitates were resolved by 7% SDS-PAGE, transferred onto a nitrocellulose membrane, and then exposed to an x-ray film (upper panel). The blot was later stripped and probed with a p65 antiserum to insure equal loading of precipitates. No cross-linking was detected in the absence of UV irradiation.

 
Thus, these studies indicate that, at the IRF-1 promoter, induction of NF-{kappa}B by CD40 stimulation converges on the same regulatory region targeted by cytokine signals.

Simultaneous treatment of B cells with CD40 and cytokines does not lead to the formation of novel complexes at the IRF-1 promoter

Given the ability of the IRF-1 GAS to function as a common binding site for distinct transcription factors, we evaluated whether simultaneous treatment with CD40L and cytokines would lead to the formation of a novel complex between STATs and Rel proteins. As shown in Fig. 7GoA, exposure of Ramos cells to CD40L transfectants together with either IL-4 or IFN-{gamma} did not lead to the induction of an additional STAT/Rel complex or to changes in the mobility of the individual complexes. Moreover, supershifting experiments with a Stat6 Ab (Fig. 7GoB) demonstrated that only the IL-4-inducible complex contains Stat6. These results suggest that, at the IRF-1 GAS, STAT and Rel proteins appear to be maintained as separate entities. Alternatively, these two distinct classes of transcription factors may bind this DNA element as alternate complexes.



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  		FIGURE 7. Simultaneous activation of Ramos cells with CD40L and IL-4 or CD40L and IFN-{gamma} does not lead to the formation of a STAT/Rel complex. A, Cells were stimulated with either CD40L tx, control tx, human IFN-{gamma} (10 ng/ml), or human IL-4 (100 U/ml) either alone or in combination. Extracts were then prepared and analyzed as described in Fig. 1Go. B, Ab interference mobility shift assays were conducted by addition of either an antiserum against Stat6 or a preimmune serum (control). Antisera were added as described in Fig. 3Go.

 
Functional consequences of NF-{kappa}B binding to the IRF-1 GAS

To determine whether CD40 stimulation could lead to the induction of IRF-1 in our system, we examined the RNA obtained from Ramos cells after stimulation for different lengths of time with CD40L tx, control tx, or TNF-{alpha}. Northern analysis of these samples revealed that stimulation of Ramos cells for 45 min with CD40L or TNF-{alpha} led, respectively, to a fivefold and threefold induction of IRF-1 expression as measured by densitometry (Fig. 8GoA). This induction was short-lived because it had diminished by 2 h and was undetectable at 24 h (data not shown). Exposure to IFN-{gamma} led to a stronger up-regulation of IRF-1 that lasted up to 24 h. In contrast, CD40 stimulation did not lead to IRF-1 up-regulation in a monocytic cell line THP-1, despite activating an NF-{kappa}B complex by EMSA and despite the ability of this cell line to up-regulate IRF-1 in response to IFNs (Fig. 8GoB). Thus, although CD40 and cytokine signals converge on similar target genes, utilization of distinct components by each cascade allows for varying levels of responsiveness. Moreover, cell type-specific constraints may modulate whether a response will take place.



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  		FIGURE 8. Induction of IRF-1 in response to CD40 or TNF-{alpha} stimulation in Ramos but not in THP-1 cells. A, Ramos cells were stimulated with either CD40L tx, control tx, human TNF-{alpha} (20 ng/ml), human IFN-{alpha} (100 U/ml), or human IFN-{gamma} (10 ng/ml) for either 45 min or 2 h. Total RNA was then extracted, and 10 µg of the RNA was assayed by Northern blotting as per standard protocols. The blot was then probed with either an IRF-1 cDNA (upper panel) or GAPDH cDNA radiolabeled by random hexamer priming. B, THP-1 cells were either unstimulated or stimulated with CD40L tx, control tx, human IFN-{alpha} (100 U/ml), or human IFN-{gamma} (10 ng/ml) for 45 min. Total RNA was then extracted and assayed as described in A.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our studies demonstrate that incoming signals provided by stimulation of B cells by the TNF- or the cytokine-receptor families target the GAS element of the IRF-1 promoter. In our experimental system, distinct classes of transcription factors are utilized by the different receptor families, Rel proteins in the case of CD40, EBV, or TNF-{alpha} and STATs in the case of cytokines. It is important to point out that among other GAS elements tested only an oligonucleotide containing the CD23b GAS appears to function as a composite NF-{kappa}B/STAT binding element (Fig. 2GoB). Given the fact that the DNA elements encompassing the IRF-1 and CD23b GAS differ in the location of potential {kappa}B sites (Table IGo), subtle differences in the interaction between NF-{kappa}B and STAT complexes at these GAS elements may, however, exist. Interestingly, the differential ability of oligonucleotides containing the CD23a and CD23b GAS to mediate NF-{kappa}B binding correlates with a differential regulation of the two CD23 isoforms in response to CD40 and/or IL-4 (our unpublished observation). Thus, the ability of a GAS element to mediate binding of both NF-{kappa}B and STATs may reflect specific regulatory requirements.

The simultaneous targeting of the IRF-1 GAS by Rel and STAT proteins suggests that regulation of the transcription factor IRF-1 may represent a critical control point in the progression of B cells toward proliferation and/or other differentiative pathways. Although only a modest induction of IRF-1 is detected in response to CD40, recent studies have demonstrated that small changes in the levels of transactivators may be translated into transcriptional synergy because of cooperative interactions within multiprotein enhancer complexes (45). Thus, activation of both NF-{kappa}B and IRF-1 by CD40 may lead to the formation of a transcriptional complex that then may target additional CD40-responsive genes. It must also be pointed out that IRF-1 can mediate growth inhibition (25), and overexpression of IRF-1 in B cells is deleterious to B cell development (46). Thus, the modest induction of IRF-1 in response to CD40 may allow for the activation of additional target genes without endangering the proliferative capability of B cells.

Consistent with this hypothesis, we have found that up-regulation of IRF-1 in response to CD40 is followed by a much stronger induction of the recently cloned IRF-4 (also termed ICSAT, Pip, LSIRF, or MUM1 (47, 48, 49, 50)) (S. Gupta et al., manuscript in preparation). IRF-4 is likely to play a key role in B cell biology as evidenced by the fact that IRF-4 ko mice display significant defects in mature B cell functions (51). An investigation of the IRF-4 promoter has revealed potential NF-{kappa}B- and IRF-1-binding sequences. Interestingly, CD40 stimulation of THP-1 cells, which leads to the induction of NF-{kappa}B but not of IRF-1, is not accompanied by the up-regulation of IRF-4.

The finding that similar IRF-1 GAS-binding complexes are activated in response to CD40, TNF-{alpha}, or EBV stimulation is further evidence that the biologic overlap demonstrated by these stimuli on B cells (2, 3, 52, 53) is reflected in the common usage of a similar set of signaling mechanisms. Given the ability of IRF-1 to act as a tumor suppressor gene and inhibit cell growth (25), modulation of IRF-1 levels by EBV may play an important role in the viral immortalization process. In addition, because IRFs play a key role in the complex transcriptional regulation of EBNA-1 (54, 55, 56), a viral protein critical for maintenance of EBV latency (53), the targeting of IRF-1 by EBV may reflect the virus’s own need to carefully control the balance of distinct IRFs within a B cell.

CD40 stimulation has been previously reported to activate Rel-containing complexes in both human and murine B cell lines (11, 57). However, these complexes differ from the one detected at the IRF-1 GAS with respect to the pattern of bands as well as Rel antisera competitions. Thus, the particular nucleotide sequence of the IRF-1 GAS allows for the differential recognition of this site by a specific Rel complex. Because the expression of Rel complexes changes with terminal B cell differentiation (58, 59, 60), the precise maturation stage of a B cell might modulate the ability of the CD40 signaling pathway to target this gene.

Preliminary studies with purified recombinant p50 and p65 have confirmed the ability of these proteins to bind to this element in the absence of additional factors (data not shown). However, the regulation of IRF-1 by CD40 is likely to be complex. Indeed, CD40 stimulation of a monocytic cell line, THP-1, albeit leading to the induction of an IRF-1 GAS-binding complex, is not accompanied by IRF-1 transactivation. In addition, transactivation of the IRF-1 gene in response to CD40 is short-lived, although binding of the p50/p65 heterodimer to the IRF-1 GAS can be detected even up to 48 h. Thus, additional factors are likely to modulate the transactivation potential of Rel family members at this promoter.

In contrast to published reports (43, 44), we have been unable to demonstrate the presence of known STAT proteins, in particular of Stat3 and Stat6, in response to CD40 or TNF-{alpha} stimulation as well as in the constitutive complexes found in EBV-transformed B cell lines. While these studies were in progress, two other groups also reported that, in non-lymphoid cells, TNF-{alpha} can activate an IRF-1 GAS-binding complex that contains Rel proteins (61, 62). The reason for our inability to detect activation of either Stat3 or Stat6 in response to CD40 is at present unclear. Potential explanations include the fact that different human cell lines as well as different culture conditions/stimulations were used. Because we can detect induction of IRF-1, CD23, ICAM-1, and Fas in response to CD40 (data not shown), our results suggest that, despite the intrinsic ability of CD40R to trigger multiple signaling pathways, activation of selected target genes may not require the full complement of signal-transducing factors.

The interaction between NF-{kappa}B/Rel proteins and STATs has been shown to have diametrically opposite effects in different experimental settings. While synergism between NF-{kappa}B and Stat6 was postulated at the murine germline epsilon promoter (57), antagonism between these two factors was found at the E-selectin promoter (63). Preliminary studies show that simultaneous targeting of Rel family members and STATs to the IRF-1 GAS does not appear to lead to either increased transcriptional activation or repression of the IRF-1 gene. However, the real in vivo significance for the Rel/STAT interaction at the IRF-1 GAS might require simultaneous exposure of cells to multiple stimuli. For instance, in the presence of IL-4, IFN-{gamma} does not inhibit but rather enhances B cell proliferation induced by CD40L (64). Given the involvement of IRF-1 in growth inhibition (25), the simultaneous presence of NF-{kappa}B and Stat6 at the IRF-1 GAS may prevent or modulate the ability of Stat1 to induce IRF-1. Thus, it appears that the precise context of the Rel/STAT interaction rather than the interaction per se will dictate the final functional outcome.


   Acknowledgments
 
We thank Rolf Freter for his help with the UV cross-linking experiments and Dimitris Thanos for the recombinant p50 and p65. We also thank G. Siu and C. Schindler for their constant support and helpful suggestions.


   Footnotes
 
1 This work was supported by a Grant-in Aid from the American Heart Association, the Silverberger Award, and the Arthur N. Saydman Investigatorship in Septicemia Research. In honor of Dr. Harold C. Neu Back

2 Address correspondence and reprint requests to Dr. Alessandra Pernis, Department of Medicine, Columbia University, 630 West 168th Street, New York, NY 10032. Back

3 Abbreviations used in this paper: CD40L, CD40 ligand; TRAF, TNFR-associated factor; ko, knockout; GAS, gamma-activated sites; IRF, IFN regulatory factor; tx, transfectant; EMSA, electrophoretic mobility shift assay; WCE, whole cell extract; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone. Back

Received for publication January 20, 1998. Accepted for publication August 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Grewal, I., R. Flavell. 1997. The CD40 ligand: at the center of the immune universe?. Immunol. Res. 16:59.[Medline]
  2. van Kooten, C., J. Banchereau. 1997. Functions of CD40 on B cells, dendritic cells and other cells. Curr. Opin. Immunol. 9:330.[Medline]
  3. Laman, J., E. Claassen, R. Noelle. 1996. Functions of CD40 and its ligand, gp39 (CD40L). Crit. Rev. Immunol. 16:59.[Medline]
  4. Stout, R., J. Suttles. 1996. The many roles of CD40 in cell-mediated inflammatory responses. Immunol. Today 17:487.[Medline]
  5. Smith, C., T. Farrah, R. Goodwin. 1994. The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death. Cell 76:959.[Medline]
  6. Beutler, B., C. van Huffel. 1994. Unraveling function in the TNF ligand and receptor families. Science 264:667.[Free Full Text]
  7. Baker, S., E. Reddy. 1996. Transducers of life and death: TNF receptor superfamily and associated proteins. Oncogene 12:1.[Medline]
  8. Cheng, G., A. Cleary, Z. Ye, D. Hong, S. Lederman, D. Baltimore. 1995. Involvement of CRAF1, a relative of TRAF, in CD40 signaling. Science 267:1494.[Abstract/Free Full Text]
  9. Rothe, M., V. Sarma, V. Dixit, D. Goeddel. 1995. TRAF2-mediated activation of NF-{kappa}B by TNF receptor 2 and CD40. Science 269:1424.[Abstract/Free Full Text]
  10. Mosialos, G., M. Birkenbach, R. Yalamanchili, T. vanArsdale, C. Ware, E. Kieff. 1995. The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the Tumor Necrosis Factor receptor family. Cell 80:389.[Medline]
  11. Berberich, I., G. Shu, E. Clark. 1994. Cross-linking CD40 on B cells rapidly activates Nuclear Factor-{kappa}B. J. Immunol. 153:4357.[Abstract]
  12. Lin, S., J. Stavnezer. 1996. Activation of NF-{kappa}B/Rel by CD40 engagement induces the mouse germ line immunoglobulin C{gamma}1 promoter. Mol. Cell. Biol. 16:4591.[Abstract]
  13. Hess, S., A. Rensing-Ehl, R. Schwabe, P. Bufler, H. Engelmann. 1995. CD40 function in nonhematopoietic cells. Nuclear Factor {kappa}B mobilization and induction of IL-6 production. J. Immunol. 155:4588.[Abstract]
  14. Baeuerle, P., D. Baltimore. 1996. NF-{kappa}B: ten years after. Cell 87:13.[Medline]
  15. Baeuerle, P., T. Henkel. 1994. Function and activation of NF-{kappa}B in the immune system. Annu. Rev. Immunol. 12:141.[Medline]
  16. Snapper, C., P. Zelazowski, F. Rosas, M. Kehry, M. Tian, D. Baltimore, W. Sha. 1995. B cells from p50/NF-{kappa}B knockout mice have selective defects in proliferation, differentiation, germ-line CH transcription, and Ig class switching. J. Immunol. 156:183.[Abstract]
  17. Snapper, C., F. Rosas, P. Zelawzoski, M. Moorman, M. Kehry, R. Bravo, F. Weih. 1996. B cells lacking RelB are defective in proliferative responses, but undergo normal B cell maturation to Ig secretion and Ig clas switching. J. Exp. Med. 184:1537.[Abstract/Free Full Text]
  18. Ihle, J., I. Kerr. 1995. Jaks and Stats in signaling by the cytokine receptor superfamily. Trends Genet. 11:69.[Medline]
  19. O’Shea, J. J.. 1997. Jaks, STATs, cytokine signal transduction, and immunoregulation: are we there yet?. Immunity 7:1.[Medline]
  20. Schindler, C., J. Darnell. 1995. Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu. Rev. Biochem. 64:621.[Medline]
  21. Pernis, A., S. Gupta, J. Yopp, H. Kashleva, C. Schindler, P. Rothman. 1995. {gamma} Chain-associated cytokine receptors signal through distinct transducing factors. J. Biol. Chem. 270:14517.[Abstract/Free Full Text]
  22. Rothman, P., B. Kreider, D. Levy, U. Wegenka, A. Eilers, T. Decker, F. Horn, H. Kashleva, J. Ihle, C. Schindler. 1994. Cytokines signal through tyrosine phosphorylation of a family of related transcription factors. Immunity 1:457.[Medline]
  23. Decker, T., P. Kovarik, A. Meinke. 1997. GAS elements: a few nucleotides with a major impact on cytokine-induced gene expression. J. Interferon Cytokine Res. 17:121.[Medline]
  24. Vaughan, P., A. van Wijnen, J. Stein, G. Stein. 1997. Interferon regulatory factors: growth control and histone gene regulation—it’s not just interferon anymore. J. Mol. Med. 75:348.[Medline]
  25. Harada, H., M. Kitagawa, N. Tanaka, H. Yamamoto, K. Harada, M. Ishihara, T. Taniguchi. 1993. Anti-oncogenic and oncogenic potentials of transcriptional regulators IRF-1 and -2. Science 259:971.[Abstract]
  26. Tanaka, N., M. Ishihara, M. Kiagawa, H. Harada, T. Kimura, T. Matsuyama, M. Lamphier, S. Aizawa, T. Mak, T. Taniguchi. 1994. Cellular commitment to oncogene-induced transformation or apoptosis is dependent on the transcription factor IRF-1. Cell 77:829.[Medline]
  27. Tamura, T., M. Ishihara, M. Lamphier, N. Tanaka, I. Oishi, S. Aizawa, T. Matsuyama, T. Mak, S. Taki, T. Taniguchi. 1995. An IRF-1-dependent pathway of DNA damage-induced apoptosis in mitogen-activated T lymphocytes. Nature 376:596.[Medline]
  28. Matsuyama, T., T. Kimura, M. Kitagawa, K. Pfeffer, T. Kawakami, N. Watanabe, T. Kundig, R. Amakawa, K. Kishihara, A. Wakeham, et al 1993. Targeted disruption of IRF-1 or IRF-2 results in abnormal type I IFN gene induction and aberrant lymphocyte development. Cell 75:83.[Medline]
  29. Kamijo, R., H. Harada, T. Matsuyama, M. Bosland, J. Gerecitano, D. Shapiro, J. Le, S. Koh, T. Kimura, S. Green, T. Mak, J. Vilcek. 1994. Requirement for transcription factor IRF-1 in NO synthase induction in macrophages. Science 263:1612.[Abstract/Free Full Text]
  30. Duncan, G., H. Mittrucker, D. Kagi, T. Matsuyama, T. Mak. 1996. The transcription factor interferon regulatory factor-1 is essential for natural killer cell function in vivo. J. Exp. Med. 184:2043.[Abstract/Free Full Text]
  31. Taki, S., T. Sato, K. Ogasawara, T. Fukuda, M. Sato, S. Hida, G. Suzuki, M. Mitsuyama, E. Shin, S. Kojima, T. Taniguchi. 1997. Multistage regulation of Th1-type immune responses by the transcription factor IRF-1. Immunity 6:673.[Medline]
  32. Lohoff, M., D. Ferrick, H. Mittrucker, G. Duncan, S. Bischof, M. Rollinghoff, T. Mak. 1997. Interferon regulatory factor-1 is required for a T helper 1 immune response in vivo. Immunity 6:681.[Medline]
  33. Caldenhoven, E., P. Coffer, J. Yuang, A. V. D. Stolpe, F. Horn, W. Kruijer, P. Vander Saag. 1994. Stimulation of the human intercellular adhesion molecule-1 promoter by interleukin-6 and interferon-{gamma} involves binding of distinct factors to a palindromic response element. J. Biol. Chem. 269:21146.[Abstract/Free Full Text]
  34. Kotanides, H., N. C. Reich. 1993. Requirement of tyrosine phosphorylation for rapid activation of a DNA binding factor by IL-4. Science 262:1265.[Abstract/Free Full Text]
  35. Cross, S., N. Halden, M. Lenardo, W. Leonard. 1989. Functionally distinct NF-{kappa}B binding sites in the immunoglobulin {kappa} and IL-2 receptor {alpha} chain genes. Science 244:466.[Abstract/Free Full Text]
  36. Wu, M., H. Lee, R. Bellas, S. Schauer, M. Arsura, D. Katz, M. FitzGerald, T. Rothstein, D. Sherr, G. Sonenshein. 1996. Inhibition of NF-{kappa}B/Rel induces apoptosis of murine B cells. EMBO J. 15:4682.[Medline]
  37. Freter, R., J. Alberta, G. Hwang, A. Wrentmore, C. Stiles. 1996. Platelet-derived growth factor induction of the immediate-early gene MCP-1 is mediated by NF-{kappa}B and a 90-kDa phosphoprotein coactivator. J. Biol. Chem. 271:17417.[Abstract/Free Full Text]
  38. Schindler, C., H. Kashleva, A. Pernis, R. Pine, P. Rothman. 1994. STF-IL4: a novel IL-4 induced signal transducing factor. EMBO J. 13:1350.[Medline]
  39. Kohler, I., E. Rieber. 1993. Allergy-associated I{epsilon} and Fc{epsilon} receptor II (CD23b) genes activated via binding of an interleukin-4-induced transcription factor to a novel responsive element. Eur. J. Immunol. 23:3066.[Medline]
  40. Hou, J., U. Schindler, W. Henzel, T. Ho, M. Brasseur, S. McKnight. 1994. An interleukin-4-induced transcription factor: IL-4 Stat. Science 265:1701.[Abstract/Free Full Text]
  41. Shuai, K., C. Schindler, V. Prezioso, J. E. Darnell. 1992. Activation of transcription by IFN-{gamma}: tyrosine phosphorylation of a 91-kD DNA binding protein. Science 258:1808.[Abstract/Free Full Text]
  42. Kikutani, H., T. Kishimoto. 1990. Molecular genetics and biology of two different species of Fc{epsilon}RII. Res. Immunol. 141:249.[Medline]
  43. Hanissian, S., R. Geha. 1997. Jak3 is associated with CD40 and is critical for CD40 induction of gene expression in B cells. Immunity 6:379.[Medline]
  44. Karras, J., Z. Wang, L. Huo, D. Frank, T. Rothstein. 1997. Induction of STAT protein signaling through the CD40 receptor in B lymphocytes: distinct STAT activation following surface Ig and CD40 receptor engagement. J. Immunol. 159:4350.[Abstract]
  45. Kim, T., T. Maniatis. 1997. The mechanism of transcriptional synergy of an in vitro assembled interferon-ß enhanceosome. Mol. Cell 1:119.[Medline]
  46. Yamada, G., M. Ogawa, K. Akagi, H. Miyamoto, N. Nakano, S. Itoh, J. Miyazaki, S. Nishikawa, K. Yamamura, T. Taniguchi. 1991. Specific depletion of the B-cell population induced by aberrant expression of human interferon regulatory factor 1 gene in transgenic mice. Proc. Natl. Acad. Sci. USA 88:532.[Abstract/Free Full Text]
  47. Eisenbeis, C., H. Singh, U. Storb. 1995. Pip, a novel IRF family member, is a lymphoid-specific, PU.1-dependent transcriptional activator. Genes Dev. 9:1377.[Abstract/Free Full Text]
  48. Iida, S., P. Rao, M. Butler, P. Corradini, M. Boccadoro, B. Klein, R. Chaganti, R. Dalla-Favera. 1997. Deregulation of MUM1/IRF4 by chromosomal translocation in multiple myeloma. Nat. Genet. 17:226.[Medline]
  49. Matsuyama, T., A. Grossman, H. Mittrucker, D. Siderovski, F. Kiefer, T. Kawakami, C. Richardson, T. Taniguchi, S. Yoshinaga, T. Mak. 1995. Molecular cloning of LSIRF, a lymphoid-specific member of the interferon regulatory factor family that binds the interferon-stimulated response element (ISRE). Nucleic Acids Res. 23:2127.[Abstract/Free Full Text]
  50. Yamagata, T., J. Nishida, T. Tanaka, R. Sakai, K. Mitani, T. Taniguchi, Y. Yazaki, H. Hirai. 1996. A novel interferon regulatory factor family transcription factor, ICSAT/Pip/LSIRF, that negatively regulates the activity of interferon-regulated genes. Mol. Cell. Biol. 16:1283.[Abstract]
  51. Mittrucker, H., T. Matsuyama, A. Grossman, T. Kundig, J. Potter, A. Shahinian, A. Wakeham, B. Patterson, P. Ohashi, T. Mak. 1997. Requirement for the transcription factor LSIRF/IRF4 for mature B and T lymphocyte function. Science 275:540.[Abstract/Free Full Text]
  52. Aggarwal, B., K. Natarajan. 1996. Tumor necrosis factors: developments during the last decade. Eur. Cytokine Netw. 7:93.[Medline]
  53. Liebowitz, D., E. Kieff. 1993. Epstein-Barr Virus. B. Roizman, and R. J. Whitley, and C. Lopez, eds. The Human Herpesviruses 107.-172. ,
  54. Schaefer, B., E. Paulson, J. Strominger, S. Speck. 1997. Constitutive activation of Epstein-Barr virus (EBV) nuclear antigen 1 gene transcription by IRF1 and IRF2 during restricted EBV latency. Mol. Cell. Biol. 17:873.[Abstract]
  55. Zhang, L., J. Pagano. 1997. IRF-7, a new interferon regulatory factor associated with Epstein-Barr virus latency. Mol. Cell. Biol. 17:5748.[Abstract]
  56. Nonkwelo, C., I. Ruf, J. Sample. 1997. Interferon-independent and-induced regulation of Epstein-Barr virus EBNA-1 gene transcription in Burkitt lymphoma. J. Virol. 71:6887.[Abstract]
  57. Iciek, L., S. Delphin, J. Stavnezer. 1997. CD40 cross-linking induces Ig{epsilon} germline transcripts in B cells via activation of NF-{kappa}B. J. Immunol. 158:4769.[Abstract]
  58. Liou, H., W. Sha, M. Scott, D. Baltimore. 1994. Sequential induction of NF-{kappa}B/Rel family proteins during B-cell terminal differentiation. Mol. Cell. Biol. 14:5349.[Abstract/Free Full Text]
  59. Miyamoto, S., M. Schmitt, I. Verma. 1994. Qualitative changes in the subunit composition of {kappa}B-binding complexes during murine B-cell differentiation. Proc. Natl. Acad. Sci. USA 91:5056.[Abstract/Free Full Text]
  60. Grumont, R., S. Gerondakis. 1994. The subunit composition of NF-{kappa}B complexes changes during B-cell development. Cell Growth Differ. 5:1321.[Abstract]
  61. Ohmori, Y., R. Screiber, T. Hamilton. 1997. Synergy between interferon-{gamma} and tumor necrosis factor-{alpha} in transcriptional activation is mediated by cooperation between signal transducer and activator of transcription 1 and nuclear factor {kappa}B. J. Biol. Chem. 272:14899.[Abstract/Free Full Text]
  62. Pine, R.. 1997. Convergence of TNF{alpha} and IFN{gamma} signaling pathways through synergistic induction of IRF-1/ISGF2 is mediated by a composite GAS/{kappa}B promoter element. Nucleic Acids Res. 25:4346.[Abstract/Free Full Text]
  63. Bennett, B., R. Cruz, R. Lacson, A. Manning. 1997. Interleukin-4 suppression of tumor necrosis factor {alpha}-stimulated E-selectin gene transcription is mediated by STAT6 antagonism of NF-{kappa}B. J. Biol. Chem. 272:10212.[Abstract/Free Full Text]
  64. Armitage, R., B. Macduff, M. Spriggs, W. Fanslow. 1993. Human B cell proliferation and Ig secretion induced by recombinant CD40 Ligand are modulated by soluble cytokines. J. Immunol. 150:3671.[Abstract]



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