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The Journal of Immunology, 2007, 178, 6777 -6788
Copyright © 2007 by The American Association of Immunologists, Inc.
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Distinct Roles of Different NF-{kappa}B Subunits in Regulating Inflammatory and T Cell Stimulatory Gene Expression in Dendritic Cells1

Junmei Wang*, Xingyu Wang*, Sofia Hussain*, Ye Zheng{dagger}, Shomyseh Sanjabi{ddagger}, Fatah Ouaaz§ and Amer A. Beg2,*

* Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL 33612; {dagger} Department of Immunology, University of Washington, Seattle, WA 98195; {ddagger} Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06520; and § Département de Biologie Cellulaire, Institut Cochin, Institut National de la Santé et de la Recherche Médicale Unité 567, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
TLRs play a critical role in inducing inflammatory and immune responses against microbial agents. In this study, we have investigated the role of NF-{kappa}B transcription factors in regulating TLR-induced gene expression in dendritic cells, a key APC type. The p50 and cRel NF-{kappa}B subunits were found to be crucial for regulating genes important for dendritic cell-induced T cell responses (e.g., CD40, IL-12, and IL-18) but not for genes encoding inflammatory cytokines (e.g., TNF-{alpha}, IL-1{alpha}, and IL-6). In striking contrast, the RelA subunit was crucial for expression of inflammatory cytokine genes but not T cell stimulatory genes. These novel findings reveal a fundamentally important difference in biological function of genes regulated by different NF-{kappa}B subunits. Focusing on RelA target gene specificity mechanisms, we investigated whether the {kappa}B site and/or the unique composition of RelA played the most crucial role. Surprisingly, studies of IL-6 expression showed that the {kappa}B site is not a primary determinant of RelA target gene specificity. Instead, a major specificity mechanism is the unique ability of RelA to interact with the transcriptional coactivator CREB-binding protein, a function not shared with the closely related cRel subunit. Together, our findings indicate novel and critically important overall roles of NF-{kappa}B in TLR-induced gene expression that are mediated by unique functions of distinct subunits.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Toll-like receptors have emerged as a crucial set of microbial pattern-recognition molecules that can confer host responsiveness to a multitude of pathogenic organisms (1, 2, 3). TLR engagement results in activation of both inflammatory and immune responses, which serve to limit infection and provide lasting immunity. Dendritic cells (DCs)3 and macrophages play an exceptionally important role in regulating TLR-induced responses to microbial agents (4). Notably, microbial agents such as LPS can induce a significant increase in cell surface expression of MHC class I, MHC class II, the costimulatory molecules B7-1 (CD80), B7-2 (CD86), CD40, and adhesion molecules such as ICAM-1 on DCs (5, 6). This process, commonly referred to as "maturation," greatly enhances the ability of DCs to activate T cells (5, 6). Upon interaction with microbial agents, DCs and macrophages also secrete T cell stimulatory cytokines (IL-12 and IL-18) and inflammatory cytokines (ICs) (TNF-{alpha}, IL-1{alpha} IL-1beta, and IL-6). These dual effects of TLRs on T cell and inflammatory responses provide robust protection from pathogenic microorganisms. These functions of TLRs are primarily mediated by changes in gene expression. However, specific roles of different transcription factors responsible for regulating TLR-induced gene expression have yet to be fully defined.

Several key adaptor molecules, protein kinases and transcription factors are thought to mediate TLR signaling (1, 7, 8). For example, the combined functions of the MyD88 and Trif adaptors are crucial for many TLR4-induced responses (9, 10). Among transcription factors, IFN regulatory factor and NF-{kappa}B family members are activated by engagement of virtually all TLRs (1, 11). NF-{kappa}B activation is mediated, at least in part, by the adaptor proteins MyD88 and TIRAP, as well as members of the TRAF family (1, 11, 12, 13, 14). NF-{kappa}B transcription factors exist as homodimers or heterodimers of five distinct proteins: p50, p52, RelA/p65, RelB, and cRel (15). NF-{kappa}B activation by the TLR4 ligand LPS occurs by nuclear translocation following inducible phosphorylation of inhibitory I{kappa}B proteins by the I{kappa}b kinase (IKK) beta complex (16, 17, 18). Three members of the NF-{kappa}B family, RelA, cRel, and RelB, contain transcriptional activation domains. Additionally, NF-{kappa}B-driven transcription in the nucleus can be potentiated by several distinct proteins, including IKK{alpha}, I{kappa}B{zeta}, GSK-3beta, and ELKS proteins (19, 20, 21, 22, 23). Mouse knockouts of NF-{kappa}B subunits and IKKs have helped identify key roles for these proteins in lymphocyte development, activation (15, 24, 25, 26), and, most recently, as a key link between inflammation and cancer (27, 28). DC development and survival is also controlled by different NF-{kappa}B subunits (29, 30). In particular, combined absence of p50 and RelA subunits severely impairs DC generation (29). Knockouts of individual NF-{kappa}B subunits display distinct phenotypes that likely reflect their ability to regulate different sets of target genes. One mechanism by which individual NF-{kappa}B subunits or particular dimers show specificity in regulating target gene expression has been linked to the sequence of {kappa}B sites (31, 32). Additional means for temporal control are provided by change in nature of dimers bound to specific {kappa}B sites and autocrine signaling pathways (33, 34, 35, 36, 37). For the cRel subunit, a distinct region of the DNA-binding domain allows cRel-specific expression of the IL-12p40 gene (32). In contrast, little is known about RelA specificity-determining mechanisms and whether mechanisms other than the sequence of {kappa}B sites are important.

Although DCs are known to play a crucial role in TLR-induced responses, key signaling and gene expression pathways in DCs are poorly characterized. In this study, we have investigated the specific functions of NF-{kappa}B subunits p50, cRel, and RelA in regulating TLR-induced gene expression in DCs. Importantly, virtually all LPS-induced genes we examined were regulated by one of these three NF-{kappa}B subunits. Examination of individual functions of these subunits led to three fundamentally important findings. First, different functional subsets of genes were regulated by distinct NF-{kappa}B subunits: while p50 and cRel specifically regulate expression of genes involved in T cell responses, the RelA subunit regulates genes encoding ICs. Second, studies of RelA specificity mechanisms revealed that the {kappa}B is not a primary determinant. Third, RelA target gene specificity is determined by unique interactions with the CREB-binding protein (CBP) coactivator. Thus, identification of different mechanisms for regulating genes involved in inflammation and T cell activation opens new possibilities for specific intervention of these responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and materials

C57BL/6 CD45.1 and C57BL/6 CD45.2 mice were purchased from The Jackson Laboratory and bred in the animal facility of H Lee Moffitt Cancer Center & Research Institute. RelA–/–, p50–/–, and cRel–/– mice have been described previously (29). E14 wild-type (WT) and RelA–/– fetal liver hemopoietic precursors (CD45.2) were adoptively transferred into lethally irradiated CD45.1 recipient mice as previously described (29). All experiments with mice were conducted in accordance with institutional guidelines. Escherichia coli LPS 0127:B8 was purchased from Sigma-Aldrich.

Cells

Bone marrow-derived dendritic cells (BMDCs) and bone marrow-derived macrophages (BMM{phi}) were cultured from mouse bone marrow precursors. Briefly, bone marrow cells were cultured in 24-well plates in the presence of 3% supernatant from J558L cells transduced with mouse GM-CSF at 1 x 106 cells/ml. BMM{phi} were cultured in 6-well plates in the presence of 30% supernatant from L929 cells containing M-CSF. DCs generated were >95% CD11c+; macrophages were >95% CD11b+CD11c–Gr-1–. To obtain peritoneal macrophages, mice were i.p. injected with 5% thioglycolate (2 ml/mouse) 3 days before euthanasia.

RNase protection assay (RPA), real-time PCR, and ELISA

Total RNA was prepared using TRIzol reagent for RPA (Ambion). For detecting mRNA expression of ICs, RPA probe sets mCK2b and mCK3b (BD Pharmingen) were used. For detecting mRNA of ICAM-1, H-2Kb (MHC class I), B7-1, B7-2, CD40, and beta-actin, specific RPA probes were designed and cloned as a new probe set. For real-time PCR, RNA was further purified using a Qiagen RNeasy Kit and then subjected to real-time PCR analysis by a standard curve method for relative quantitation. Standards were prepared by serial dilution of a pool of all samples. RNA samples and standards were reverse-transcribed and then subjected to real-time PCR analysis in an Applied Biosystems 7900HT Sequence Detection System with SYBR Green I dye using gene-specific primers. All samples were run in triplicate, with SDs indicated in the figures. Results for target genes are presented after normalizing to beta-actin. Production of mouse TNF-{alpha}, IL-6, and IL-12 was determined using ELISA kits from BioSource International according to manufacturer’s instructions.

FACS analysis

Cell surface expression of B7-1, B7-2, and CD40 was determined by staining live cells with FITC- or PE-coupled Abs from BD Pharmingen. FACS analysis was performed on a FACSCalibur cytometer (BD Biosciences), and data were acquired and analyzed using CellQuest software. Results are shown as histograms and mean fluorescence intensity.

Lentiviral vector construction and promoter assays

Mouse WT IL-6 promoter (–1290 to + 27) was amplified from C57BL/6J genomic DNA by using PfuUltra high-fidelity DNA polymerase (Stratagene) and inserted into a luciferase containing self-inactivating lentiviral vector (pSIN-luc) (provided by Dr. D. Baltimore, California Institute of Technology, Pasadena, CA). IL-6 {kappa}B site mutations and substitutions were performed using a QuikChange Site-directed Mutagenesis Kit (Stratagene). Thymidine kinase (TK) promoter was amplified from the pRL-TK vector (Promega) and inserted into pSIN-luc. All promoter constructs were verified by DNA sequencing.

Lentiviruses were prepared by transfecting HEK 293T cells with 4 µg of pSIN-luc vectors and 2 µg of each packaging vector VSVG, pMDLg/pRRE and RSC-Rev. BMDCs were spin-infected with viral supernatant at room temperature for 2 h on days 2 and 4 of BMDC culture. Infected BMDCs were harvested and stimulated with LPS on day 6. All samples were collected in triplicate and experiments were performed two or three times.

Generation of RelA/cRel chimera-expressing fibroblasts

Immortalized RelA–/– fibroblasts were spin-infected using retroviral supernatant produced by HEK 293T cells transfected with 6 µg of FLAG-tagged RelA, cRel or, RelA/cRel chimera-expressing MIG retroviral vectors (32) and 4 µg of helper vector pCL-Eco. Infected cells were sorted 3 days later based on GFP expression. Anti-FLAG M2 mAb (Sigma-Aldrich) was used to determine protein levels by Western blotting.

Chromatin immunoprecipitation (ChIP)

RelA- or cRel-expressing fibroblasts were plated at 2 x 106 per 6-cm dish, and 48 h later were treated with 1 µg/ml LPS for 0 or 1 h. Cells were fixed on plates in 1% formaldehyde (Sigma-Aldrich) for 10 min, washed twice with cold PBS, and removed with 20% trypsin. The nuclear lysate was sonicated and precleared with Pansorbin cells (Calbiochem) for 1 h at 4°C. Two micrograms of anti-RelA A-20 (Santa Cruz Biotechnology), anti-cRel N (Santa Cruz Biotechnology), or normal rabbit IgG (Upstate Biotechnology) with 500 µg of nuclear lysate was incubated overnight. The mixture was then incubated with 40 µl of Pansorbin cells for 1 h. Pansorbin cells were washed twice with each of the following: nuclear lysis buffer (50 mM HEPES (pH 7.9), 140 mM NaCl, 1 mM EDTA, and 1% Triton X-100), high salt wash buffer (50 mM HEPES (pH 7.9), 500 mM NaCl, 1 mM EDTA, and 1% Triton X-100), and 1x TE (10 mM Tris and 1 mM EDTA). The DNA was eluted from Pansorbin cells in a SDS buffer. Reverse cross-linking was performed at 65°C overnight. After proteinase K digestion, phenol/chloroform extraction, and precipitation with ethanol, DNA was dissolved in H2O and real-time PCR was performed with IL-6 primers: sense, 5'-CCCTTCCTAGTTGTGATTCTTTCGATGC-3' and antisense, 5'-CTCCAGAGCAGAATGAGCTACAGA-3'.

Reporter assay and immunoprecipitations

HEK 293T cells were infected with lentiviruses containing WT IL-6 promoter. Five x 104 cells were plated to each well of 24-well plates in duplicate and transfected with RelA, cRel, or RelA/cRel chimera-expressing vectors and Renilla luciferase expressing pRL-TK (Promega) with or without Rous sarcoma virus (RSV)-CBP provided by Dr. J. Harton (University of South Florida, Tampa, FL). Forty-eight hours later, the 293T cells were lysed and luciferase activity was measured using a dual-luciferase reporter system (Promega). Renilla luciferase activity was used for normalization. For immunoprecipitations, HEK 293T cells were transfected with 5 µg of RelA, cRel, or RelA/cRel chimera-expressing vector and 5 µg of RSV-CBP. Forty-eight hours later, whole cell extract was prepared and immunoprecipitations were performed as previously described.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Role of NF-{kappa}B factors in TLR-induced expression of costimulatory molecules in DCs

TLR stimulation of DCs results in induction of expression of several key cell surface-expressed proteins and secreted cytokines. Among the most crucial cell surface proteins are MHC and costimulatory molecules, which are critical for T cell activation. Previous studies have shown that DCs display high levels of multiple NF-{kappa}B subunits (29, 33, 38). To better understand pathways involved in regulating DC function, we investigated the role of NF-{kappa}B controlled transcriptional mechanisms in regulating expression of ICAM-1, H-2Kb (MHC class I), CD40, B7-2, and B7-1. mRNA expression of all five molecules was enhanced following LPS (TLR4 ligand) treatment of WT BMDCs (Fig. 1A). Interestingly, mRNA expression of ICAM-1, CD40, B7-2, and B7-1 was reduced in p50–/– DCs, while cRel–/– DCs showed reduction in ICAM-1 and CD40 expression (Fig. 1A). Compared with WT DCs, reduction in expression of these genes was also noticed in p50–/–cRel–/– DCs. However, no obvious reduction in H-2Kb mRNA expression was noticed in any of the knockout DCs, suggesting no general defect in inducible gene expression. Other than reduced expression of ICAM-1 and CD40 at 2 h, LPS-treated RelA–/– DCs showed elevated mRNA expression of CD40, B7-1, and B7-2 (Fig. 1B, 6- and 18-h time points). These results therefore indicate that LPS-induced mRNA expression of four key molecules known to participate in DC-induced T cell activation (ICAM-1, CD40, B7-1, and B7-2) is regulated by individual and combined function of p50+cRel but not RelA.


Figure 1
Figure 1
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  		FIGURE 1. Role of NF-{kappa}B factors in TLR-induced expression of costimulatory molecules in DCs. A, RPA analysis of mRNA expression of costimulatory molecules in WT, p50–/–, cRel–/–, and p50–/–cRel–/– BMDCs. B, RPA analysis of mRNA expression of costimulatory molecules in WT and RelA–/– BMDCs. C, FACS analysis of surface expression of costimulatory molecules in BMDCs shown as histograms and mean fluorescence intensity (MFI). Upper panel, WT and p50–/–cRel–/–; lower panel, WT and RelA–/–. All experiments were independently performed two or three times.

 
We next determined the effect of reduced mRNA levels of CD40, B7-1, and B7-2 on cell surface expression. For these studies, we used p50–/–cRel–/– DCs to allow determination of the combined roles of both of these subunits (studies of p50–/– or cRel–/– single knockout DCs revealed more substantial reduction in p50–/– DCs, consistent with RPA results; data not shown). WT and p50–/–cRel–/– DCs were left untreated or treated with LPS for 18 h. Consistent with significantly reduced mRNA levels, LPS-induced cell surface expression of CD40 was more significantly reduced than that of B7-1 and B7-2 in p50–/–cRel–/– DCs (Fig. 1C). Also consistent with the mRNA expression level, LPS-induced surface expression of H-2Kb was not reduced in p50–/–cRel–/– DCs (Fig. 1C). However, constitutive surface expression of H-2Kb, but not mRNA expression, was elevated p50–/–cRel–/– DCs, suggesting that these subunits may influence posttranscriptional MHC regulation pathways. Interestingly, total and intracellular levels of B7-1 and B7-2 were much more substantially reduced in p50–/–cRel–/– DCs than cell surface levels (data not shown). These results suggest that lower mRNA levels lead to less total protein, but this is partly compensated by increased delivery from intracellular pools. Thus, B7-1 and B7-2 cell surface expression in p50–/–cRel–/– DCs is only modestly lower than in WT DCs. Consistent with increased mRNA levels, a modest increase in cell surface expression of B7-1 and B7-2 was noticed in RelA–/– DCs (Fig. 1D). Together, these results suggest that 1) mRNA expression of CD40, B7-1, and B7-2 is significantly increased during LPS-induced maturation, which leads to enhancement of cell surface expression of these molecules, and 2) mRNA induction depends in part on combined functions of NF-{kappa}B subunits p50 and cRel but not RelA.

IC expression in DCs requires RelA but not p50 and cRel

TLR stimulation of DCs results in a robust increase in expression of secreted cytokines that regulate both T cell responses (e.g., IL-12 and IL-18) and inflammatory responses (TNF-{alpha}, IL-1{alpha}, IL-1beta, and IL-6). The aforementioned ICs can be differentiated from IL-12/IL-18 in their ability to induce inflammation independently of T cells. LPS-induced IL-12p40 subunit expression was abolished in p50–/–cRel–/– DCs at early time points and significantly reduced in p50–/– and cRel–/– DCs (Fig. 2A). IL-12 p35 subunit expression was undetectable in p50–/–, cRel–/–, or p50–/–cRel–/– DCs (Fig. 2A). Overall, these results are consistent with previous studies demonstrating important roles for cRel and p50 in regulating both IL-12 subunits (29, 32, 39, 40). In addition, mRNA expression of the IL-12 functional homolog IL-18 was absent in both p50–/– and cRel–/– DCs (Fig. 2A). However, mRNA expression of key ICs TNF-{alpha}, IL-1{alpha}, IL-1beta, and IL-6 was robustly induced in p50–/–, cRel–/–, or p50–/–cRel–/– DCs (Fig. 2A). Together with our above findings, it therefore appears that p50 and cRel specifically regulate mRNA expression of cell surface molecules (CD40, B7-1, B7-2, and ICAM-1) and cytokines (IL-12 and IL-18) that impact T cell activation responses.


Figure 2
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  		FIGURE 2. IC expression in DCs requires RelA but not p50 and cRel. A, RPA analysis of cytokine mRNA expression in WT, p50–/–, cRel–/–, and p50–/–cRel–/– BMDCs. B, Cytokine mRNA expression in WT and RelA–/– BMDCs. For IL-12p40 and p35, longer exposure is shown in a different panel. L.E., Longer exposure. All experiments were independently performed three times.

 
Unlike p50–/– and cRel–/– DCs, IL-12p40, p35, and IL-18 mRNA expression was induced in RelA–/– DCs after LPS treatment (Fig. 2B). In fact, p40 mRNA expression was substantially elevated in RelA–/– DCs after 18 h of LPS treatment (Fig. 2B). Although the underlying reason for this is unclear, it may reflect potentially enhanced p50+cRel binding to IL-12p40 {kappa}B sites in the absence of RelA. Strikingly, significant reduction in mRNA expression of ICs IL-1{alpha}, IL-1beta, and IL-6 was noticed in RelA–/– DCs (Fig. 2B). We also performed real-time PCR to quantify mRNA levels of IL-1beta, IL-6, and TNF-{alpha}. Consistent with RPA results, IL-1beta and IL-6 mRNA expression was substantially reduced relative to beta-actin levels at 2 h after LPS treatment, with IL-1beta showing an ~4-fold reduction and IL-6 a 3-fold reduction (data not shown). Also similar to RPA results, TNF-{alpha} expression in RelA–/– DCs was not substantially reduced after a 2-h LPS treatment (data not shown). These results therefore indicate that expression of T cell stimulatory molecules and ICs DCs is controlled by distinct NF-{kappa}B subunits.

RelA is crucial for regulating IC expression in multiple cell types

Previous studies have identified a specificity-determining Rel homology subdomain of cRel that is crucial for IL-12p40 expression (32). In contrast, RelA specificity-determining mechanisms are not known. To better understand how RelA specifically regulates ICs, we first determined whether RelA is also important for IC expression in other cell types. Focusing on IL-6 and TNF-{alpha} expression, WT BMM{phi} induced mRNA expression of both cytokines after 2- and 6-h LPS treatment (Fig. 3A). RelA–/– BMM{phi} showed reduced expression of both IL-6 and TNF-{alpha} after LPS treatment (Fig. 3A). Interestingly, and somewhat different from DCs, macrophages were completely dependent on RelA for LPS-induced TNF-{alpha} expression (Fig. 3A). Furthermore, neither TNF-{alpha} nor IL-6 expression was affected in the individual or combined absence of p50 and cRel in macrophages (data not shown). Together, these studies indicate a key requirement for RelA in regulating IC expression in macrophages.


Figure 3
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  		FIGURE 3. Regulation of IC expression by RelA in macrophages and fibroblasts. A, RPA analysis of IC mRNA expression in WT and RelA–/– BMM{phi}. B, ELISA analysis of production of TNF-{alpha}, IL-6, and IL-12p70 protein by BMDCs (upper panel) and TNF-{alpha} and IL-6 by BMM{phi} (lower panel). C, RPA analysis of mRNA expression of TNF-{alpha} and IL-6 in peritoneal macrophages. D, RPA analysis of mRNA expression of TNF-{alpha} and IL-6 in MEFs.

 
We next determined secreted levels of IL-6 and TNF-{alpha} in WT and RelA–/– DCs and macrophages. Secreted levels of both IL-6 and TNF-{alpha} were reduced in supernatants of RelA–/– DCs and macrophages (Fig. 3B). The reductions largely mirrored reduction in mRNA levels of these proteins, e.g., both TNF-{alpha} mRNA and protein levels were more significantly reduced in RelA–/– macrophages than in RelA–/– DCs. In contrast, IL-12p70 levels were not substantially reduced in RelA–/– DCs, while, compared with DCs, WT or RelA–/– macrophages produced too little IL-12p70 for reliable detection. Nonetheless, previous studies have shown that cRel, but not RelA, regulates IL-12p40 expression in macrophages (39). We next determined the role of RelA in regulating IL-6 and TNF-{alpha} mRNA expression in thioglycolate-elicited peritoneal macrophages. As shown in Fig. 3C, LPS-induced expression of both IL-6 and TNF-{alpha} was greatly reduced in RelA–/– peritoneal macrophages. Finally, mouse embryonic fibroblasts also showed a critical requirement for RelA in LPS-induced expression of IL-6, as previously reported (41), as well as TNF-{alpha} (Fig. 3D). These results therefore demonstrate a critical requirement for RelA in regulating IL-6 and TNF-{alpha} expression in multiple cell types.

{kappa}B site is not a primary determinant of RelA specificity

Because RelA was found to be crucial for IL-6 expression in all cell types we have tested, additional studies were performed to identify potential RelA specificity-determining mechanisms involved in IL-6 regulation. We were especially interested in determining whether the {kappa}B site and/or the unique composition of the RelA subunit itself played the most crucial role. To this end, we first determined the role of the {kappa}B site by using a recently described self-inactivating lentivirus reporter system (31) for analysis of IL-6 gene expression. Luciferase activity in this system is driven by the cloned promoter of interest, which, because of integration into the host genome is subject to chromatin regulation (31). An additional advantage of this system is that it allows efficient transduction of primary cell types, such as DCs. An ~1.3-kb region of regulatory sequence upstream of the IL-6 transcriptional start site was used for these studies. DCs were infected first with the IL-6 promoter-containing lentivirus (IL-6-WT). LPS treatment of these DCs resulted in robust IL-6 promoter-driven luciferase expression (Fig. 4A). In contrast, mutation of a key NF-{kappa}B site (42) (IL-6-{kappa}B.Mut), which is highly conserved between mice and humans, resulted in dramatically reduced LPS-induced IL-6 promoter activity (Fig. 4A). Thus, a single conserved NF-{kappa}B binding site is critical for LPS-induced IL-6 expression in DCs.


Figure 4
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  		FIGURE 4. {kappa}B site is not a primary determinant of RelA specificity. A, WT and {kappa}B mutated ({kappa}B.Mut) IL-6 promoter assay in WT BMDCs. Lentivirus infection of WT BMDCs was performed on days 2 and 4 of culture. Infected BMDCs were treated with LPS for the indicated periods, after which luciferase activity was determined. Error bars, SDs of triplicate samples. RLU, Relative luciferase unit. All experiments were independently performed three times. B, WT IL-6 promoter assay in WT-DC and RelA–/– BMDCs. C, WT IL-6 promoter assay in WT-DC and p50–/–cRel–/– BMDCs. D, BMDCs were infected with TK promoter-containing lentivirus. TK promoter activity in WT, RelA–/–, and p50–/–cRel–/– BMDCs. Luciferase activity was determined on day 6.

 
In key experiments, we next determined whether dependence on RelA and lack of dependence on p50+cRel were also observed using the lentivirus reporter system. IL-6-WT lentivirus was used to infect WT, RelA–/–, and p50–/–cRel–/– DCs. Importantly, IL-6 promoter activity was virtually abolished in RelA–/– DCs (Fig. 4B). In contrast, LPS treatment of p50–/–cRel–/– DCs led to slightly higher induction of IL-6 promoter activity compared with WT DCs (Fig. 4C). The TK promoter (TK-lenti) in contrast showed similar activity in WT, RelA–/–, and p50–/–cRel–/– DCs (Fig. 4D). These results therefore indicate that IL-6 regulatory regions used here are sufficient for both LPS induction and for conferring NF-{kappa}B subunit specificity.

We next determined whether NF-{kappa}B subunit specificity was controlled by the IL-6 {kappa}B site. To this end, we substituted the IL-6 {kappa}B site with {kappa}B sites from the IL-12p40 promoter. Previous studies, and results shown above, demonstrate a crucial role for p50+cRel in IL-12p40 expression. Furthermore, two p40 sites termed distal (d.{kappa}B) and proximal (p.{kappa}B) bind with high affinity to cRel (32). To examine the role of the IL-6 {kappa}B site in NF-{kappa}B subunit specificity, we substituted this site with p40 p.{kappa}B or d.{kappa}B sites (pIL-12 to IL-6 and dIL-12 to IL-6). First, either substitution significantly reduced LPS-induced IL-6 promoter activity (Fig. 5A). Second, reporter activity was reduced in p50–/–cRel–/– DCs compared with WT DCs (Fig. 5B). Third, and most important, pIL-12 to IL-6 and dIL-12 to IL-6 reporter activity was also reduced in RelA–/– compared with WT DCs (Fig. 5C). Taken together, these results suggest that although the IL-6 {kappa}B site is crucial for high level promoter activity it is not crucial for determining RelA specificity.


Figure 5
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  		FIGURE 5. IL-6 {kappa}B site substitution with IL-12 and CD40 {kappa}B sites. A, Promoter assay of WT IL-6 promoter (WT IL-6) and {kappa}B site-substituted IL-6 promoter (dIL12b–IL-6 and pIL12b– IL-6). {kappa}B site of WT IL-6 promoter was replaced with proximal (pIL12b–IL6) and distal (dIL12b–IL6) {kappa}B sites from the IL-12p40 promoter. BMDCs were treated as before. Error bars, SDs of triplicate samples. B, dIL12b–IL6 and pIL12b–IL6 promoter assay in WT and p50–/–cRel–/– BMDCs. C, dIL12b–IL6 and pIL12b–IL6 promoter assay in WT and RelA–/– BMDCs. D, Promoter assay of WT IL-6 and {kappa}B site-substituted IL-6 promoter (dCD40–IL6 and pCD40–IL6) in WT DCs. E, dCD40–IL6 and pCD40–IL6 promoter assay in WT and p50–/–cRel–/– BMDCs. F, dCD40–IL6 and pCd40–IL6 promoter assay in WT and RelA–/– BMDCs. All experiments were independently performed two or three times.

 
To further test the role of the IL-6 {kappa}B site, we substituted this site with {kappa}B sites from the CD40 promoter. There are at least five distinct {kappa}B sites in CD40 regulatory regions. CD40 expression is driven by p50+cRel (Fig. 1), suggesting that one or more CD40 sites may confer p50+cRel-driven expression. Two CD40 sites have been shown to be important for expression, CD40 p.{kappa}B and d.{kappa}B (43), which differ from the IL-6 site at three or two nucleotide positions, respectively. Unlike substitution of p40 {kappa}B sites, substitution with CD40 {kappa}B sites had a less significant affect on IL-6 reporter activity in WT DCs (Fig. 5D), likely because they are more similar to the IL-6 site. Interestingly, these substitutions increased reporter activity in p50–/–cRel–/– DCs compared with WT DCs (Fig. 5E). However, IL-6 promoter activity remained dependent on RelA, further demonstrating that the IL-6 {kappa}B site is not a primary determinant of RelA specificity (Fig. 5F).

Multiple domains of RelA are required for optimal IL-6 and TNF-{alpha} expression

Because the IL-6 {kappa}B site did not appear to be primarily responsible for RelA specificity, we next considered the possibility that unique composition of the RelA subunit determines specificity. To test this possibility, and identify specificity-determining RelA domains, we performed a comparative analysis with cRel using immortalized RelA–/– mouse embryonic fibroblasts (MEFs). RelA and cRel are highly similar, making cRel the most similar NF-{kappa}B subunit to RelA. RelA is the major transcription-activating NF-{kappa}B subunit in fibroblasts (44, 45). In WT-immortalized MEFs, IL-6 expression was maximal after a 2-h LPS treatment, whereas RelA–/–-immortalized MEFs showed virtually no IL-6 expression at any time point (data not shown; Figs. 3 and 6A). However, retrovirus-driven RelA expression in these cells rescued IL-6 and TNF-{alpha} expression (Fig. 6A). Similar expression of cRel in RelA–/– cells in contrast did not rescue IL-6 and very slightly induced TNF-{alpha} expression (Fig. 6B). Importantly, cRel was clearly functional in these cells because expression of ICAM-1 was strongly induced by cRel (and RelA) (Fig. 6B). Using RelA- and cRel-transduced cells, we next determined in vivo DNA binding of these subunits to the IL-6 promoter by ChIP. Interestingly, both RelA and cRel showed clear association with the IL-6 promoter (Fig. 6C). This result is not surprising because crystallographic studies have shown that RelA and cRel associate with {kappa}B sites in a similar fashion (46, 47). Thus, inability of cRel to induce IL-6 expression is not due to a failure to associate with IL-6 regulatory regions.


Figure 6
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  		FIGURE 6. Multiple domains of RelA are required for IL-6 and TNF-{alpha} expression. A, RPA analysis of RelA–/–- and RelA-expressing RelA–/– fibroblasts untreated or treated with LPS for 2 h. B, RPA of RelA-expressing and cRel-expressing RelA–/– fibroblasts. Cells were treated the same as in A. C, ChIP assay of RelA-expressing and cRel-expressing RelA–/– fibroblasts untreated or treated with LPS for 1 h. ChIP was performed by using anti-RelA for RelA cells, anti-cRel for cRel cells, and IgG as negative control. Real-time PCR was performed with IL-6 promoter primers. D, RPA of RelA-expressing, cRel-expressing, or RelA/cRel chimera-expressing RelA–/– fibroblasts. Cells were treated the same as in A. E, Western blotting analysis to determine expression levels of FLAG-tagged RelA, cRel, and Rel/cRel chimeras. Whole cell extracts were blotted with anti-FLAG Ab to detect FLAG-tagged proteins. All experiments were independently performed two or three times. F, Representation of RelA/cRel chimeric proteins. Amino acid boundaries of different domains are indicated. Rel homology domain (RHD) contains N and C domains. G, Real-time PCR analysis of IL-6 mRNA expression in samples from D. H, Real-time PCR analysis of TNF-{alpha} mRNA expression in samples from D.

 
The above results suggest that unique composition of the RelA subunit may be responsible for IL-6 and TNF-{alpha} expression. To identify specificity-determining RelA regions, we used a series of RelA/cRel chimeric molecules (32) (Fig. 6F). These chimeras consist of combinations of one or more of three key domains of RelA and cRel: 1) the Rel homology region N domain (responsible for DNA binding), 2) the Rel homology region C domain (responsible for dimerization), and 3) the transactivation domain (TD). mRNA expression was analyzed by RPA (Fig. 6D) and quantified by real-time PCR (Fig. 6, G and H). Interestingly, compared with WT RelA, RelA N and TD chimeras induced significantly less IL-6 expression (Fig. 6D). These results suggest that the RelA-specificity mechanism is distinct from that of cRel where the N domain is sufficient for conferring cRel-specific expression of IL-12p40 (32). However, RelA N-C substantially rescued IL-6 expression (Fig. 6D). In no case, however, was IL-6 expression induced as robustly as by WT RelA, despite similar expression levels of chimeras (Fig. 6E). Similarly, maximal TNF-{alpha} induction only took place following WT RelA expression (Fig. 6D). Also similar to IL-6, the RelA N domain did not substantially induce TNF-{alpha} expression, but interestingly RelA C-TD induced robust TNF-{alpha} expression. Together, these results indicate the following: 1) all three subdomains (N, C, and TD) of RelA are required for maximal IL-6 and TNF-{alpha} expression. Thus, along with the above results, these findings indicate that the unique composition of the RelA subunit, rather than the sequence of the {kappa}B site, is the key determinant of target gene specificity. 2) The C domain or TD of RelA can functionally cooperate with the RelA N domain in inducing IL-6 expression, while RelA C-TD can induce substantial TNF-{alpha} expression.

RelA interaction with the CBP coactivator is a critical determinant of target gene specificity

The above results indicate that the unique overall composition of RelA specifies target gene expression. We were therefore intrigued by the possibility that protein-protein interactions between RelA and transcriptional coactivators could be a key determinant of target gene specificity. Previous studies have identified CBP/p300 as potentially key regulators of transcriptional activation by RelA (48, 49, 50, 51, 52). We therefore determined whether RelA-CBP interactions could play a role in IL-6 induction. To this end, we infected HEK 293T cells with IL6-WT lentivirus (as with DCs) to allow integration of the reporter gene in chromosomal DNA. Subsequent individual expression of RelA, cRel, or CBP in these cells had no significant effect on reporter activity (Fig. 7A). However, coexpression of RelA and CBP resulted in greatly increased transcriptional activity (Fig. 7A). These findings therefore demonstrate that RelA and CBP can induce synergistic activation of the IL-6 promoter. In striking contrast, combined expression of cRel and CBP did not significantly activate the IL-6 promoter (Fig. 7A). Importantly, the two RelA/cRel chimeric molecules (containing RelA N-C or RelA N-TD domains) that substantially induced endogenous IL-6 expression also showed significant synergy with CBP. Thus, RelA and specific RelA domains that are crucial for endogenous IL-6 expression can also synergize with CBP in this reporter assay.


Figure 7
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  		FIGURE 7. RelA interaction with the CBP coactivator is a critical determinant of target gene specificity. A, HEK 293T reporter assay. HEK 293T cells were infected with WT IL-6 promoter lentivirus. Forty-eight hours later, cells were transfected with RelA, cRel, or RelA/cRel chimera constructs and pRL-TK in the presence or absence of RSV-CBP. Forty-eight hours later, luciferase activity was measured. The results were normalized with Renilla activity from pRL-TK. RLU, Relative luciferase unit. B, Coimmunoprecipitation of CBP with RelA and cRel. Flag-tagged RelA or cRel construct with RSV-CBP were transfected into HEK 293T cells. Forty-eight hours later, immunoprecipitation was performed to whole cell extracts using anti-CBP or normal rabbit IgG. The immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-FLAG. C, Coimmunoprecipitation of CBP with RelA, cRel, and RelA/cRel chimeras. HEK 293T cells were transfected with RelA, cRel, or RelA/cRel chimera constructs with RSV-CBP and analyzed as in B. D, HEK 293T cells were infected with IL-6 promoter pIL12b–IL6 (see Fig. 5B) or pCD40–IL6 (see Fig. 5E) lentiviruses as in A above. All experiments were independently performed two or three times.

 
We next determined whether transcriptional synergism was due to direct interaction between RelA and CBP. RelA showed strong in vivo interaction with CBP (Fig. 7B). In contrast, cRel showed virtually no interaction ability with CBP (Fig. 7B). These results therefore identify a critically important difference between RelA and cRel in mechanisms used for regulation of gene expression. Importantly, the two RelA/cRel chimeric molecules that best induced endogenous IL-6 expression (RelA N-C and RelA N-TD), and showed functional synergy with CBP, also displayed a strong ability to physically interact with CBP (Fig. 7C). Although previous studies have shown that RelA regions corresponding to C and TD mediate interaction with CBP/p300 (48, 50), our results indicate an additional key role for the RelA N domain.

A prediction of the inability of cRel to associate with CBP is that even for {kappa}B sites that allow cRel transactivation (e.g., IL-12p40 {kappa}B sites), cRel will not functionally synergize with CBP. To test this, we used IL-6 reporters described above with substitution of IL-12p40 or CD40 p.{kappa}B sites. Although overall reporter activity was reduced compared with the WT IL-6 promoter, only RelA, and not cRel, functionally synergized with CBP (Fig. 7D). These results are remarkably consistent with our studies on DCs (Fig. 5), which demonstrate exclusive dependence on RelA regardless of the sequence of the {kappa}B site. In conclusion, although RelA interaction with CBP has been previously documented, our results show for the first time that this interaction can play a critical role in determining RelA target gene specificity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
TLRs induce expression of genes that play key roles in inducing inflammation and T cell activation. DCs are among the most crucial mediators of TLR-induced responses. In this study, we specifically examined the roles of NF-{kappa}B p50, cRel, and RelA subunits in regulating key TLR-induced genes in DCs. Our results indicate that combined functions of p50 and cRel specifically regulate mRNA expression of cell surface molecules and secreted cytokines involved in T cell responses. In contrast, these subunits have no obvious role in regulating expression of ICs. Remarkably, the opposite is true for RelA, i.e., RelA is crucial for expression of ICs in DCs but not for genes involved in regulating T cell responses. Thus, different functional subsets of genes in DCs are regulated by distinct NF-{kappa}B subunits. Another intriguing aspect of our findings is that virtually all LPS-induced genes in DCs were regulated by one of the three NF-{kappa}B subunits we have studied. Thus, a substantial number of TLR-induced genes in DCs may be controlled by NF-{kappa}B family members.

We have shown that combined p50+cRel function is crucial for regulating several key T cell stimulatory genes. Cell surface expression of proteins correlates with mRNA levels, with CD40 showing the most significant reduction. Cell surface expression of B7-1 and B7-2 is reduced but still robustly induced in p50–/–cRel–/– DCs, consistent with previous studies (29). Importantly, RelB–/– DCs also show impaired up-regulation of CD40 expression (53), and hyperactivation of RelB in the absence of p100/p52 results in enhanced MHC and costimulatory molecule expression (54). Thus, RelB may be responsible for inducing remaining costimulatory molecule mRNA expression in p50–/–cRel–/– DCs. Interestingly, DNA microarray studies further showed impaired LPS-induced expression of key T cell stimulatory molecules (e.g., 4-1BBL, CD27L, and IL-2; A. A. Beg, unpublished results) in p50–/–cRel–/– DCs. Thus, these NF-{kappa}B subunits may regulate a substantial subset of additional T cell stimulatory genes. The exact mechanisms by which p50+cRel specificity is conferred in regulating T cell stimulatory genes are presently unknown. However, as discussed in more detail below, the sequence of the specific {kappa}B site may be a key determinant.

The {kappa}B site sequence is not a primary determinant of RelA specificity

A main focus of this study was to better understand RelA function in regulating IC gene expression. We have found that RelA is crucial for IC expression not only in DCs, but also in macrophages and fibroblasts. However, RelA is not the only activator of IC expression in DCs because modest expression was evident in the absence of RelA. We have investigated in detail how RelA uniquely regulates IL-6 expression, a gene controlled by RelA in all cell types examined. Using a lentivirus reporter system, we found that similar to the endogenous IL-6 gene, RelA but not p50+cRel controlled IL-6 promoter-driven reporter activity. By substituting the single crucial {kappa}B site in the IL-6 promoter with known cRel binding {kappa}B sites from the IL-12p40 promoter, we found that promoter activity was still dependent on RelA. Similarly, substitution of the IL-6 site with CD40 promoter binding sites also did not impact dependence on RelA. Thus, the IL-6 {kappa}B site is not likely to be a primary determinant of RelA specificity.

Interesting differences were noticed in p50–/–cRel–/– DCs following {kappa}B site substitutions in the IL-6 promoter. Substitution with p40 {kappa}B sites resulted in partial dependence on p50+cRel. In contrast, the WT IL-6 promoter and, in particular, substitution with CD40 {kappa}B sites resulted in elevated reporter activity in p50–/–cRel–/– DCs compared with WT DCs. These results indicate that the sequence of the {kappa}B site can have strong influence on p50+cRel dependence. Although these findings can be interpreted in different ways, we believe that one simple interpretation is that only binding to "permissive" sites (i.e., sites that allow binding and transactivation, such as IL-12p40 sites), but not "nonpermissive" sites (i.e., sites that allow binding but not transactivation, such as the IL-6 site), can result in p50+cRel-driven gene expression. For nonpermissive sites, p50+cRel deficiency may actually enhance expression, potentially through increased RelA binding. Importantly, a previous study has demonstrated a crucial role for {kappa}B sites in IFN-{gamma}-inducible protein 10 and MCP-1 promoters in determining which NF-{kappa}B dimers activate transcription (31). In this study, the {kappa}B sequence did not regulate association of specific dimers, but instead allowed Bcl-3 or IFN regulatory factor 3 binding, which confer coactivator activity (31). It is therefore possible that similar {kappa}B site-dependent mechanisms also control transcriptional activation by p50+cRel. In contrast, our results indicate that the sequence of the {kappa}B site is unlikely to be a key determinant of RelA specificity for IL-6. However, it is possible that an additional {kappa}B site in IL-6 regulatory regions can confer RelA-specific binding and synergy with the identified {kappa}B site. We, nonetheless, believe this is unlikely because a transcription factor binding site search of the LPS-inducible IL-6 regulatory region used here only identified the known {kappa}B site as a high-affinity NF-{kappa}B binding site. Interestingly, previously defined {kappa}B sites in TNF-{alpha} and IL-1beta control regions bear no obvious similarity to the IL-6 {kappa}B site or to each other. Furthermore, the DNA-binding RelA N domain was not sufficient for TNF-{alpha} expression (Fig. 6D), indirectly suggesting a lack of a key role for TNF-{alpha} {kappa}B sites for RelA specificity. Collectively, these findings further indicate that the sequence of the {kappa}B site may not be a key determinant of RelA target gene specificity.

Interaction with the CBP coactivator is a major determinant of RelA target gene specificity

Our collective findings indicate that the unique composition of the RelA subunit, rather than the sequence of the {kappa}B site, is the primary determinant of target gene specificity. We believe a main mechanism responsible for RelA specificity is the ability of RelA to physically and functionally interact with coactivators such as CBP. We were surprised to find that the cRel NF-{kappa}B subunit, which is highly similar to RelA, has virtually no ability to physically or functionally interact with CBP. Although RelA interaction with CBP has been previously shown, our results indicate that this interaction plays a crucial role in determining RelA subunit-specific gene expression. These findings thus reveal a critically important difference between RelA and cRel with important consequences for the transactivation functions of these two subunits. Specific interaction between RelA and CBP may also shed light on how overall expression levels of NF-{kappa}B target genes are controlled. Thus, RelA interaction with CBP may allow high-level target gene expression. In this respect, it is intriguing to note that all RelA target genes we have identified in DCs are expressed at vastly higher levels than p50+cRel target genes (Fig. 2). Thus, differential regulation of inflammatory and T cell stimulatory cytokines genes through different NF-{kappa}B subunits may also be pivotal in controlling gene expression levels.

Previous studies have shown that RelA regions corresponding to C and TD mediate interaction with CBP/p300 (48, 50). Our results, however, show that in vivo interaction between CBP and RelA is critically dependent on the N domain along with either the C or TD. Importantly, the C domain contains a key protein kinase A phosphorylation site (S276), which is required for phosphorylation-dependent CBP-RelA interaction (48). This interaction can also occur in unstimulated cells through phosphorylation of S276 by basal protein kinase A activity (48). The TD in contrast contains the phosphorylation-independent CBP interaction domain (48). Our results indicate that either one of these two domains can mediate interaction with CBP but only in the presence of the N domain. Notably, recent studies have shown that CBP/p300 induce acetylation of RelA, resulting in enhanced RelA function (55, 56). Thus, RelA acetylation following interaction with CBP may be crucial for IC expression. It will be interesting to determine whether in the context of full-length RelA, the N domain directly mediates CBP interaction or associates with other factors that bind CBP. It is important to note that endogenous IL-6 gene induction was maximal with WT RelA, and all substitutions with cRel domains significantly reduced IL-6 induction. In addition, RelA N-C, and N-TD induce IL-6 at a lower level compared with WT RelA, yet strongly associate with CBP. These results suggest that although interaction with CBP is crucial, full RelA transcriptional activity may be achieved through interactions with additional coactivators (52).

We believe our findings may have important implications in design of vaccines using microbial adjuvants. Although adjuvant properties of microbial agents greatly enhance T cell activation and memory responses, potent induction of inflammatory responses can limit their use in human vaccines. Our results showing that genes involved in inflammation and T cell activation are subject to regulation by distinct NF-{kappa}B subunits opens up possibilities for specific modulation of these two pathways. Thus, inhibition of RelA gene activation function may have therapeutic potential in vaccines using microbial agents as adjuvants because of potential reduction in expression of inflammatory mediators but not stimulators of T cell activation.


   Acknowledgments
 
We thank Dr. Steven Smale (University of California Los Angeles) for providing chimera constructs and Drs. T. Leung and D. Baltimore (CalTech) for providing the pSIN-luc vector. We thank members of the Flow Cytometry and Molecular Biology core facilities at the Moffitt Cancer Center for their assistance. J.W. is a Ph.D. candidate at Columbia University, NY.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant R01 AI059715 and institutional funds from Moffitt Cancer Center (to A.A.B.). Back

2 Address correspondence and reprint requests to Dr. Amer Beg, H. Lee Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612. E-mail address: amer.beg{at}moffitt.org Back

3 Abbreviations used in this paper: DC, dendritic cell; CBP, CREB-binding protein; MEF, mouse embryonic fibroblast; BMDC, bone marrow-derived DC; BMM{phi}, bone marrow-derived macrophage; RPA, ribonuclease protection assay; IC, inflammatory cytokine; TK, thymidine kinase; ChIP, chromatin immunoprecipitation; RSV, Rous sarcoma virus; TD, transactivation domain. Back

Received for publication January 24, 2007. Accepted for publication March 19, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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