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The Journal of Immunology, 2007, 178, 7302 -7309
Copyright © 2007 by The American Association of Immunologists, Inc.
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A Novel RBP-J{kappa}-Dependent Switch from C/EBPbeta to C/EBP{zeta} at the C/EBP Binding Site on the C-Reactive Protein Promoter1

Prem Prakash Singh, Bhavya Voleti and Alok Agrawal2

Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Regulation of basal and cytokine (IL-6 and IL-1beta)-induced expression of C-reactive protein (CRP) in human hepatoma Hep3B cells occurs during transcription. A critical transcriptional regulatory element on the CRP promoter is a C/EBP binding site overlapping a NF-{kappa}B p50 binding site. In response to IL-6, C/EBPbeta and p50 occupy the C/EBP-p50 site on the CRP promoter. The aim of this study was to identify the transcription factors occupying the C/EBP-p50 site in the absence of C/EBPbeta. Accordingly, we treated Hep3B nuclear extract with a C/EBP-binding consensus oligonucleotide to generate an extract lacking active C/EBPbeta. Such treated nuclei contain only C/EBP{zeta} (also known as CHOP10 and GADD153) because the C/EBP-binding consensus oligonucleotide binds to all C/EBP family proteins except C/EBP{zeta}. EMSA using this extract revealed formation of a C/EBP{zeta}-containing complex at the C/EBP-p50 site on the CRP promoter. This complex also contained RBP-J{kappa}, a transcription factor known to interact with {kappa}B sites. RBP-J{kappa} was required for the formation of C/EBP{zeta}-containing complex. The RBP-J{kappa}-dependent C/EBP{zeta}-containing complexes were formed at the C/EBP-p50 site on the CRP promoter in the nuclei of primary human hepatocytes also. In luciferase transactivation assays, overexpressed C/EBP{zeta} abolished both C/EBPbeta-induced and (IL-6 + IL-1beta)-induced CRP promoter-driven luciferase expression. These results indicate that under basal conditions, C/EBP{zeta} occupies the C/EBP site, an action that requires RBP-J{kappa}. Under induced conditions, C/EBP{zeta} is replaced by C/EBPbeta and p50. We conclude that the switch between C/EBPbeta and C/EBP{zeta} participates in regulating CRP transcription. This process uses a novel phenomenon, that is, the incorporation of RBP-J{kappa} into C/EBP{zeta} complexes solely to support the binding of C/EBP{zeta} to the C/EBP site.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
C-reactive protein (CRP)3 is a host-defense protein. The serum concentration of CRP increases in chronic and acute inflammatory and in some noninflammatory states (1, 2, 3, 4). The synthesis of CRP in hepatocytes is regulated at the transcriptional level (5, 6). In hepatoma cells, IL-6 induces CRP expression by activating transcription factors STAT3 (7, 8, 9, 10) and C/EBPbeta (9, 11). IL-1beta synergistically enhances the effects of IL-6, in part through the activation of NF-{kappa}B (12, 13, 14). The first 157 bp on the CRP proximal promoter is sufficient for the synergistic action of IL-6 and IL-1beta (IL-6 + IL-1beta) in human hepatoma Hep3B cells (12). Three other transcription factors, hepatocyte nuclear factor (HNF)-1, HNF-3, and OCT-1, are involved in maintaining the basal expression of CRP (11, 14, 15). The hepatocytes produce CRP even when they are under stress, and such induction of CRP expression requires transcription factor CREBH whose binding site is not in the promoter region but is located in the 5' untranslated region of the CRP gene (4). Many transcription factors are involved in CRP expression through the proximal 157 bp of the promoter (see Fig. 1A). In this study, we report the participation of two additional transcription factors, C/EBP{zeta} and RBP-J{kappa}, in regulating CRP expression.


Figure 1
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  		FIGURE 1. The sequence of the CRP gene. A, The portion of the CRP gene between positions –115 and +107, including –115/–1 region of the promoter, is shown. Sequences of the known binding sites for transcription factors on the promoter and 5' untranslated region (UTR) are boxed. The sequences we investigate in this study and the overlapping C/EBP-binding and p50-binding nonconsensus {kappa}B sites are in a bold line box. B, Sequences of the oligonucleotides used in EMSA. Mutated bases are in bold plus underlined. Consensus sequences are underlined.

 
There are six members in the C/EBP family of transcription factors: C/EBP{alpha}, C/EBPbeta, C/EBP{gamma}, C/EBP{delta}, C/EBP{epsilon}, and C/EBP{zeta}. C/EBP{zeta} is also known as CHOP10 (C/EBP homologous protein 10) and GADD153 (growth arrest and DNA damage-inducible gene 153). These C/EBP proteins can homodimerize or heterodimerize with each other. Except C/EBP{zeta}-containing dimers, all other C/EBP dimers bind to the classical C/EBP-binding consensus sequence (16, 17, 18, 19, 20). C/EBP{zeta} is ubiquitously expressed at low levels in proliferating cells including Hep3B and can be induced by cellular stresses (21). Within the 157 bp of the CRP proximal promoter, a C/EBP binding site is centered at position –52 (see Fig. 1A). A second C/EBP site is located at –219 (11).

There are five members in the NF-{kappa}B family of transcription factors: p50, p52, p65, Rel B, and c-Rel. These Rel proteins can also homodimerize or heterodimerize with each other (22, 23). Rel dimers bind to {kappa}B sites that are typically composed of five purines followed by five pyrimidines. The heterodimer of p50 and p65, the classical NF-{kappa}B, binds to a {kappa}B site centered at position –69 on the CRP promoter (14, 24). This {kappa}B site overlaps the binding sites for HNF-1, HNF-3, and OCT-1 (14) (see Fig. 1A). A second {kappa}B site on the CRP promoter is located at –2652 (25). The homodimers of p50 also bind to a nonconsensus {kappa}B site centered at position –47 overlapping the C/EBP site (26, 27, 28). The overlapping C/EBP site and p50-binding nonconsensus {kappa}B site (C/EBP-p50 site) on the CRP promoter is critical for the induction of CRP transcription (26, 28) (Fig. 1A).

In addition to binding to NF-{kappa}B, some {kappa}B sites are also recognized by RBP-J{kappa} (also known as recombination signal-binding protein J{kappa}, CBF1, and CSL) (29, 30, 31, 32). RBP-J{kappa} is a ubiquitously expressed transcription factor in mammals. The homologs of RBP-J{kappa} are also described, known as SuH in Drosophila melanogaster and LAG-1 in the nematode Caenorhabditis elegans (33, 34, 35, 36). The consensus sequence for the binding of RBP-J{kappa} is CGTGGGAAA although RBP-J{kappa} can bind to several variants of the consensus sequence (37, 38, 39). RBP-J{kappa} regulates transcription of genes in response to Notch signaling (36, 40). In the absence of signaling, RBP-J{kappa} binds to its target site and represses transcription by recruiting corepressors. In response to signaling the corepressors are replaced by coactivators (33, 36). Notch-independent functions of RBP-J{kappa} involve its recruitment into complexes containing other transcription factors, its interaction with components of transcription preinitiation complex (41, 42), and its competition with NF-{kappa}B for binding to {kappa}B sites (37, 43, 44).

The formation of a complex of unknown composition at the C/EBP-p50 site on the CRP promoter in Hep3B nuclei was reported earlier (26). This complex did not contain NF-{kappa}B proteins and was formed only when the nuclear extracts were pretreated with an oligonucleotide containing the C/EBP-binding consensus sequence (26). In the current study, we identified the transcription factors present in this complex as C/EBP{zeta} and RBP-J{kappa}. For the formation of C/EBP{zeta}-containing complex at the C/EBP-p50 site on the promoter, the recruitment of RBP-J{kappa} into the complex was necessary. Our data suggest that a switch between C/EBP{zeta} and C/EBPbeta operates at the C/EBP site on the CRP promoter to regulate CRP transcription. This uniqueness of the C/EBP-p50 site of the CRP promoter might be contributing to the acute phase nature of the induction of CRP gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture, cytokine treatment, transfection, CRP promoter-luciferase reporter constructs, and luciferase transactivation assay

Hep3B cells provided by Dr. G. J. Darlington (Baylor College of Medicine, Houston, TX) were grown as previously described (26). Cells were cultured in serum-free medium overnight for cytokine treatments and transfections. The confluency of cells was ~60% at the time of treatments. IL-6 and IL-1beta (R&D Systems) were used at concentrations of 10 ng/ml and 1 ng/ml, respectively. The cells were treated with cytokines for 24 h for luciferase transactivation assays.

For transient transfections, Hep3B cells were plated into 6-well plates and transfected using FuGENE6 reagent (Roche) as previously described (27). CRP promoter-luciferase reporter constructs were used at 1 µg of plasmid per well, and the transcription factor expression vectors were used as described in each experiment. Total amount of plasmid DNA transfected was held constant using empty pCDNA3. The preparation of wild-type CRP promoter-luciferase (–157/+3) reporter construct and the construct containing the mutated {kappa}B site has been previously described (14, 28). Expression vectors for C/EBPbeta, C/EBP{zeta}, and RBP-J{kappa} were obtained from Drs. P. F. Johnson (National Cancer Institute, Frederick, MD), N. J. Holbrook and J. L. Martindale (National Institute of Aging, Bethesda, MD), and L. D. Vales (University of Medicine and Dentistry of New Jersey, Piscataway, NJ), respectively. After 16 h of transfection, the transfected cells were either treated with cytokines for 24 h or left untreated. After 40 h of transfection, luciferase transactivation assays were performed following the protocol supplied by the manufacturer (Promega), and the luciferase activity was measured in a luminometer (Molecular Devices) as previously described (14).

Preparation of nuclear extract and EMSA

Hep3B nuclear extracts were prepared using NE-PER Nuclear and Cytoplasmic Extract kit (Pierce) as reported earlier (14). Primary human hepatocytes were purchased from Cambrex Biosciences (catalog no. cc-2591). One ampoule of cryopreserved human hepatocytes (3–6 x 106 viable cells) was used to prepare 100 µl of nuclear extract using NE-PER Nuclear and Cytoplasmic Extract kit and 4 µl of this extract was used for each reaction in EMSA. In the EMSA using purified recombinant human NF-{kappa}B p50 (Promega), 0.6 gel shift unit of the protein was used for each reaction. EMSA on Hep3B nuclear extracts was conducted as previously described (13). Unless otherwise mentioned, the gel shift incubation buffer contained 16 mM HEPES (pH 7.9), 40 mM KCl, 1 mM EDTA, 2.5 mM DTT, 0.15% Nonidet P-40, 8% Histopaque, and 1 µg of polydeoxyinosinic-polydeoxycytidylic acid. The sequences of the oligonucleotides derived from the CRP promoter, consensus oligonucleotides, and mutated oligonucleotides used in EMSA are shown in Fig. 1B. The C/EBP-binding (45), RBP-J{kappa}-binding (38), and STAT3-binding (46) consensus oligonucleotides were designed according to published sequences. Oligonucleotides were obtained from Integrated DNA Technologies. To prepare the probes, complementary oligonucleotides were annealed and labeled with either [{alpha}-32P]CTP or [{gamma}-32P]ATP. In supershift experiments, Ab (2 µg) were added to the reaction mixture and incubated on ice for 15 min before addition of the probe. In competition experiments, 150 ng of unlabeled oligonucleotides were added to the binding reactions before addition of the Ab and probe. The Ab to C/EBPbeta (C19), C/EBP{zeta} (F168), HNF-1 (H205), HNF-3 (C20), and OCT-1 (C21) were purchased from Santa Cruz Biotechnology. A rat mAb to RBP-J{kappa} (clone K0043) was purchased from the Institute of Immunology Company. DNA-protein complexes were resolved in native 5% polyacrylamide gels containing 2.5% glycerol, unless otherwise mentioned. Gels were analyzed in a phosphor imager using Image-Quant software (GE Healthcare).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
In the absence of C/EBPbeta, a RBP-J{kappa}-dependent C/EBP{zeta}-containing complex is formed at the C/EBP-p50 site on the CRP promoter

To explore the composition of the complexes formed at the C/EBP-p50 site, we performed EMSA (Fig. 2) using Hep3B nuclear extracts and a 23-bp oligonucleotide (oligo B) derived from the CRP promoter as the probe. Four DNA-protein complexes, three specific and one nonspecific, were formed (Fig. 2, lanes 1 and 2). The specific complexes contained C/EBP proteins including C/EBPbeta (Fig. 2, lanes 3 and 4), indicating that some C/EBP proteins were constitutively active in Hep3B nuclei. Pretreatment (i.e., before the addition of radiolabeled probe) of nuclear extract with unlabeled C/EBP consensus oligonucleotide resulted in the appearance of a new intense complex co-migrating with the nonspecific complex (Fig. 2, lane 4), consistent with the previously reported observations (26). For clarity, we retain the use of the name "band D" for this new complex, as it was called earlier (26). Ab to RBP-J{kappa} diminished the intensity of band D, indicating that this complex contained RBP-J{kappa} (Fig. 2, lane 5). Ab to C/EBP{zeta} abolished band D, indicating that this complex contained C/EBP{zeta} (Fig. 2, lanes 6 and 7). When the nuclear extract was pretreated with RBP-J{kappa} consensus oligonucleotide to remove free active RBP-J{kappa}, the band D was not formed, confirming that the band D contained RBP-J{kappa}. In addition, this finding suggested that RBP-J{kappa} was required for the formation of RBP-J{kappa}-C/EBP{zeta} complexes at the C/EBP-p50 site (Fig. 2, lane 8).


Figure 2
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  		FIGURE 2. Formation of RBP-J{kappa}-dependent C/EBP{zeta}-containing complex at the C/EBP-p50 site on the oligonucleotide derived from the CRP promoter. A representative EMSA using oligo B as the probe is shown. Two batches of Hep3B nuclear extracts were used. The sequences of the oligonucleotides used in this and subsequent EMSA are shown in Fig. 1B. Arrows point to the complexes formed on the probe. The mobility of the free probe in this and subsequent EMSA is not shown.

 
Two different patterns of complex formation were seen on this probe (oligo B) in various batches of nuclear extracts (Fig. 2, extract 1 and extract 2). In the pattern for extract 2, band D was formed even without pretreatment of nuclei with the C/EBP consensus oligonucleotide, indicating that this complex can also be formed in the presence of C/EBPbeta (Fig. 2, lanes 9 and 10). The band D contained C/EBPbeta (Fig. 2, lane 11). Removal of active C/EBPbeta from the nuclear extract by pretreatment with C/EBP consensus oligonucleotide resulted in the formation of a much more intense band D (Fig. 2, compare lane 9 with lane 12). The anti-RBP-J{kappa} Ab did not affect the intensity of band D (Fig. 2, lane 13). However, as in the pattern for extract 1 (Fig. 2, lane 8), when the nuclear extract was treated with RBP-J{kappa} consensus oligonucleotide to remove free RBP-J{kappa}, the formation of band D was abolished (Fig. 2, lane 16), indicating that RBP-J{kappa} was present in the complex and was required for the formation of this complex. Anti-C/EBP{zeta} Ab abolished band D (Fig. 2, lanes 14 and 15), indicating that the complex contained C/EBP{zeta}. From these EMSA, we conclude that two types of C/EBP{zeta}-containing complexes can be formed at the C/EBP-p50 site on the CRP promoter: C/EBPbeta-C/EBP{zeta} and RBP-J{kappa}-C/EBP{zeta}. The formation of both types of complexes is dependent on the presence of RBP-J{kappa} and perhaps also on the binding of RBP-J{kappa} to the C/EBP-p50 site.

The formation of C/EBP{zeta}-containing complex is independent of the binding sites for OCT-1, HNF-1, and HNF-3 on the promoter

To determine whether the binding sites for OCT-1, HNF-1, and HNF-3 on the CRP promoter influence the formation of C/EBP{zeta}-containing complexes, we performed EMSA using a 41-bp long oligonucleotide (oligo A), derived from the CRP promoter, as the probe (Fig. 3). Four specific complexes were formed (Fig. 3, lanes 1 and 2). As was expected, three of the four complexes contained OCT-1 (Fig. 3, lanes 8 and 9), HNF-1 (Fig. 3, lanes 8 and 10), and HNF-3 (Fig. 3, lanes 8 and 11). Pretreatment of nuclear extract with C/EBP consensus oligonucleotide did not affect the formation of complexes containing OCT-1, HNF-1, and HNF-3 (Fig. 3, lanes 3–7), however, the intensity of the fastest migrating fourth complex (band D) was enhanced (Fig. 3, compare lane 1 with lane 3). Ab to RBP-J{kappa} alone did not affect the intensity of band D (Fig. 3, lane 4). Ab to C/EBP{zeta} substantially decreased the intensity of band D, indicating that this complex contained C/EBP{zeta} (Fig. 3, lane 5). The combination of the two Ab abolished the formation of band D, indicating the presence of both RBP-J{kappa} and C/EBP{zeta} in the complex (Fig. 3, lane 6). Importantly, the complex in band D was competed by unlabeled RBP-J{kappa} consensus oligonucleotide, indicating that this complex contained RBP-J{kappa} and its formation required RBP-J{kappa} (Fig. 3, lane 7), consistent with the data obtained from EMSA using probe oligo B. We conclude that the formation of C/EBP{zeta}-containing complex is independent of OCT-1, HNF-1, and HNF-3 sites on the CRP promoter.


Figure 3
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  		FIGURE 3. The effect of OCT-1, HNF-1, and HNF-3 sites on the formation of C/EBP{zeta}-containing complex is analyzed by EMSA using a 41-bp long oligonucleotide (oligo A) as the probe and nuclear extract from Hep3B cells. A representative EMSA is shown.

 
RBP-J{kappa} associates with C/EBP{zeta}, and both free RBP-J{kappa} and RBP-J{kappa}-C/EBP{zeta} bind to CRP promoter

Next, we identified RBP-J{kappa} and RBP-J{kappa}-containing complexes in Hep3B nuclei by using RBP-J{kappa} consensus oligonucleotide as the probe in EMSA. We also evaluated competition between RBP-J{kappa} consensus probe and the CRP promoter-derived oligonucleotides for binding to RBP-J{kappa} (complex II) and RBP-J{kappa}-containing complexes (complex I) (Fig. 4A). Specific complexes of two different compositions were formed on the RBP-J{kappa} consensus probe (Fig. 4A, lanes 1 and 2). Complex II, but not complex I, was supershifted by Ab to RBP-J{kappa}, indicating that complex II contained RBP-J{kappa} and it reacted with the anti-RBP-J{kappa} mAb (Fig. 4A, lanes 3 and 12). Because the supershifted complex II co-migrated with complex I (Fig. 4A, compare lane 1 with lane 3), we could not determine whether the Ab to RBP-J{kappa} abolished the formation of complex I, if not supershifted. Because the anti-RBP-J{kappa} Ab is a mAb, it is possible that this Ab recognizes free RBP-J{kappa} only and does not recognize RBP-J{kappa} when RBP-J{kappa} is complexed with other transcription factors. Because the probe in this EMSA was RBP-J{kappa} consensus oligonucleotide, complex I should contain RBP-J{kappa}. Complex I, but not complex II, was abolished by Ab to C/EBP{zeta}, indicating that complex I also contained C/EBP{zeta} (Fig. 4A, lane 4). Thus, in complex I, C/EBP{zeta} was associated with RBP-J{kappa}. These data indicate the presence of both free RBP-J{kappa} and preformed RBP-J{kappa}-C/EBP{zeta} complexes in the Hep3B nuclei.


Figure 4
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  		FIGURE 4. Association between RBP-J{kappa} and C/EBP{zeta}. Two different exposures of the representative EMSA with RBP-J{kappa} consensus oligonucleotide as the probe and nuclear extract from Hep3B cells are shown. DNA-protein complexes were resolved in native 6% polyacrylamide gels. A, Hep3B nuclei contain RBP-J{kappa}-C/EBP{zeta} complexes that bind to the oligonucleotides derived from the CRP promoter. An Ab supershift (ss) is represented. B, The effect of mutating the p50 sites on the binding of RBP-J{kappa}-C/EBP{zeta} complexes to oligo A is described in Results.

 
Competitor oligonucleotides (oligos A, B, and C) derived from the CRP promoter competed with the probe for binding to RBP-J{kappa}-C/EBP{zeta} in complex I, indicating that this complex was capable of binding to CRP promoter (Fig. 4A, compare lanes 6–8 with lane 1). The competitor oligonucleotides also competed with the probe for binding to RBP-J{kappa} in complex II, indicating that free RBP-J{kappa} was also capable of binding to CRP promoter (Fig. 4A, compare lanes 15–17 with lane 10). Oligo B was a poor competitor compared with oligo A and oligo C. Binding of RBP-J{kappa} to oligo C could be due to the presence of the {kappa}B site at position –69. Used as a control, the C/EBP consensus oligonucleotide competitor did not compete with any complex (Fig. 4A, lane 9), indicating that an RBP-J{kappa} binding site was necessary for binding of C/EBP{zeta}-containing complexes to the C/EBP site.

To determine the role of the p50 site in the formation of RBP-J{kappa}-C/EBP{zeta} complexes at the C/EBP-p50 site on oligo A, we mutated the p50 site. We mutated only those bases of the p50 site that do not overlap the C/EBP site. We also mutated the {kappa}B site. The mutated oligonucleotides (shown in Fig. 1B) were used as competitors in an EMSA with RBP-J{kappa} consensus oligonucleotide as the probe (Fig. 4B). Similar to that shown in Fig. 4A, RBP-J{kappa} (complex II) and RBP-J{kappa}-C/EBP{zeta} complexes (complex I) were formed on the probe (Fig. 4B, lane 1). Oligo A competed with the probe for binding to complex I (Fig. 4B, compare lane 1 with lane 3) and complex II (Fig. 4B, compare lane 7 with lane 9), indicating that both complexes were capable of binding to oligo A. Oligo A-m1 competed with the probe for binding to complex I (Fig. 4B, lanes 1 and 4) but competed less efficiently with the probe for binding to complex II (Fig. 4B, lanes 7 and 10), indicating that the mutation of the p50 site did not abolish the binding of RBP-J{kappa}-C/EBP{zeta} to the C/EBP-p50 site. Oligo A-m2 competed with the probe for binding to complex II (Fig. 4B, lanes 7 and 11), but competed less efficiently with the probe for binding to complex I (Fig. 4B, lanes 1 and 5), indicating that the {kappa}B site also participates in the formation of complex I on oligo A. Oligo A-m3 did not compete with the probe for binding to complex I (Fig. 4B, lanes 1 and 6) and complex II (Fig. 4B, lanes 7 and 12), indicating that at least one of the two p50 sites was required for the formation of complex I on oligo A. The complex I often appeared in two forms as two bands. The additional component present in one of the two bands is not yet identified. Taken together, these results suggest that a p50 site is necessary for the formation of RBP-J{kappa}-C/EBP{zeta} complexes at the C/EBP-p50 site and that the {kappa}B site may be cooperating with the C/EBP-p50 site in regulating CRP transcription.

The RBP-J{kappa}-C/EBP{zeta} complex was also formed on the promoter when the nuclear extracts were pretreated with STAT3 consensus oligonucleotide

To determine whether the formation of RBP-J{kappa}-C/EBP{zeta} complexes was stimulated in the nuclei pretreated only with the C/EBP consensus oligonucleotide that depletes the nuclei of C/EBPbeta, we performed a control EMSA in which the nuclear extracts were pretreated with STAT3 consensus oligonucleotide (Fig. 5A). The oligo A and oligo B from the CRP promoter were used as probes. We expected that the band D would not be formed on these probes because the STAT3 consensus oligonucleotide does not bind C/EBP and hence will not deplete the nuclei of C/EBPbeta. Surprisingly, the intensity of band D was enhanced when the nuclear extract was pretreated with the STAT3 consensus oligonucleotide (Fig. 5A, compare lane 1 with lane 3). The complex in band D was competed by unlabeled RBP-J{kappa} consensus oligonucleotide, indicating that this complex contained RBP-J{kappa} and its formation required RBP-J{kappa} (Fig. 5A, lane 4), consistent with the results shown in Fig. 2. The intensity of the complex was reduced by individual Ab to C/EBP{zeta} and RBP-J{kappa} and was abolished by the combination of both Abs, indicating that the complex contained both C/EBP{zeta} and RBP-J{kappa} (Fig. 5A, lanes 5–7). Similar results were obtained with oligo A as the probe (Fig. 5A, lanes 8–13).


Figure 5
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  		FIGURE 5. Formation of RBP-J{kappa}-C/EBP{zeta} complexes at the C/EBP-p50 site in the presence of C/EBPbeta. A, A representative EMSA using oligo B (lanes 1–7) and oligo A (lanes 8–13) as probes and nuclear extract from Hep3B cells is shown. B, Binding of p50 to STAT3 consensus oligonucleotide is represented by EMSA using purified p50 and the STAT3 consensus oligonucleotide as probe. The gel shift incubation buffer contained 16 mM HEPES (pH 7.9), 40 mM KCl, 1 mM EDTA, 1 mM DTT, and 8% Histopaque. DNA-protein complexes were resolved in native 6% polyacrylamide gels.

 
Because the STAT3 consensus oligonucleotide contains a polypyrimidine region, similar to that present in the p50-binding {kappa}B sites, we examined the ability of the STAT3 consensus oligonucleotide to bind to p50. As shown in the EMSA (Fig. 5B), purified p50 bound to STAT3 consensus oligonucleotide.

RBP-J{kappa}-dependent C/EBP{zeta}-containing complexes are formed at the C/EBP-p50 site in the nuclei of primary human hepatocytes

We next investigated the binding of RBP-J{kappa}-C/EBP{zeta} complexes to the C/EBP-p50 site of the CRP promoter in the nuclei of primary human hepatocytes. We performed an EMSA using nuclear extracts from untreated hepatocytes and oligo B as the probe (Fig. 6). Three specific complexes including a very faint complex were formed (Fig. 6, lanes 1 and 2). The faint complex probably contained C/EBPbeta because it was abolished by C/EBP consensus oligonucleotide (Fig. 6, lanes 3–7), indicating negligible presence of C/EBPbeta in the hepatocyte nuclei. Pretreatment of nuclear extract with the C/EBP consensus oligonucleotide resulted in enhancement of the intensity of the other two complexes (bands D) (Fig. 6, lane 3). Ab to RBP-J{kappa} did not diminish the intensity of bands D (Fig. 6, lane 4), consistent with the results shown in Fig. 2 (lane 13). Ab to C/EBP{zeta} abolished bands D, indicating that both complexes contained C/EBP{zeta} (Fig. 6, lanes 5 and 6). When the nuclear extract was treated with RBP-J{kappa} consensus oligonucleotide to remove free active RBP-J{kappa}, the formation of bands D was abolished, indicating that RBP-J{kappa} was present in bands D and was required for the formation of these complexes (Fig. 6, lane 7). The additional component in one of the two bands D is not yet identified. The difference between the effects of anti-RBP-J{kappa} Ab and RBP-J{kappa} consensus oligonucleotide might be due to the possible inability of anti-RBP-J{kappa} mAb to recognize RBP-J{kappa}-C/EBP{zeta} complexes. The RBP-J{kappa} consensus oligonucleotide, however, binds both RBP-J{kappa} and RBP-J{kappa}-C/EBP{zeta}. From these EMSA, we conclude that the RBP-J{kappa}-C/EBP{zeta} complexes can be formed at the C/EBP-p50 site of the CRP promoter even in the nuclei of primary human hepatocytes.


Figure 6
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  		FIGURE 6. The formation of RBP-J{kappa}-dependent C/EBP{zeta}-containing complexes at the C/EBP-p50 site on the CRP promoter in human hepatocytes. A representative EMSA using oligo B as the probe and nuclear extract from untreated primary human hepatocytes shows that the RBP-J{kappa}-C/EBP{zeta} complexes can be formed at the C/EBP-p50 site of the CRP promoter.

 
Overexpressed C/EBP{zeta} inhibits both (IL-6 + IL-1beta)-induced and C/EBPbeta-induced CRP expression

To evaluate the role of C/EBP{zeta} in CRP expression induced by combined cytokines IL-6 and IL-1beta, we performed luciferase transactivation assays. Overexpression of C/EBP{zeta} inhibited (IL-6 + IL-1beta)-induced CRP promoter-driven luciferase expression in a C/EBP{zeta} dose-dependent manner (Fig. 7A). Overexpression of RBP-J{kappa} with C/EBP{zeta} was not required for the inhibitory effects of C/EBP{zeta} on luciferase expression (Fig. 7B). Thus, the role of C/EBP{zeta} is to repress CRP expression.


Figure 7
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  		FIGURE 7. Loss of CRP promoter-driven luciferase expression in cells overexpressing C/EBP{zeta}. Representative luciferase transactivation assays are shown; three independent experiments exhibited similar pattern. A, Cells were transfected with the CRP promoter-luciferase construct and expression vector encoding C/EBP{zeta} (increasing doses). After 16 h, cells were treated with cytokines. After another 24 h, transcription was measured as luciferase activity. The percentage of inhibition of luciferase activity in the presence of overexpressed C/EBP{zeta} is plotted on the y-axis. B, Cells were transfected with the CRP promoter-luciferase construct and expression vector encoding C/EBP{zeta} as in A, except that 1 µg of each expression vector was used for transfection. Basal luciferase activity is shown as 1 and the luciferase activity in treated cells is plotted as fold induction over basal activity. C, Cells were transfected with the CRP promoter-luciferase construct, and expression vector encoding C/EBP{zeta} (10 ng) and C/EBPbeta (increasing doses). After 40 h, transcription was measured and represented as relative luciferase activity. D, Cells were transfected with the CRP promoter-luciferase construct, and expression vector encoding C/EBP{zeta} (1 µg), RBP-J{kappa} (1 µg), and 50 ng of C/EBPbeta plasmid were used for transfection. Basal luciferase activity is shown as 1 and the luciferase activity in treated cells is plotted as fold induction over basal activity.

 
To determine whether C/EBP{zeta} inhibited the transactivating effect of C/EBPbeta, luciferase transactivation assays were performed by transfecting cells with both C/EBPbeta and C/EBP{zeta}. We first investigated the competition between C/EBPbeta and C/EBP{zeta} to regulate CRP expression based on their relative levels in the nuclei. We performed transactivation assays by transfecting cells with increasing amounts of C/EBPbeta in the presence of a constant amount of C/EBP{zeta} (10 ng of expression vector) (Fig. 7C). Overexpression of C/EBPbeta alone induced luciferase expression in a dose-dependent manner. At lower levels of C/EBPbeta, overexpressed C/EBP{zeta} inhibited C/EBPbeta-induced luciferase expression. At higher levels of C/EBPbeta, overexpressed C/EBP{zeta} did not inhibit C/EBPbeta-induced luciferase expression. These results indicate that relative levels of C/EBPbeta and C/EBP{zeta} are crucial to determine the extent of CRP expression.

We then determined the inhibitory role of C/EBP{zeta} by transfecting cells with 1 µg of C/EBP{zeta} expression vector (Fig. 7D). Overexpression of C/EBPbeta alone induced luciferase expression. Overexpressed C/EBP{zeta} repressed C/EBPbeta-induced luciferase expression. The endogenous level of RBP-J{kappa} in the nuclei was found to be sufficient for C/EBP{zeta} to repress C/EBPbeta-induced luciferase expression because overexpression of RBP-J{kappa} was ineffective.

Overexpressed RBP-J{kappa} alone has no effect on CRP expression

Overexpression of RBP-J{kappa} alone did not influence basal, IL-6-induced, (IL-6 + IL-1beta)-induced, or C/EBPbeta-induced transactivation of the CRP promoter (Fig. 8A). We then investigated the effects of overexpressed RBP-J{kappa} on enhanced basal expression from the CRP promoter with the mutated {kappa}B site (Fig. 8B). Consistent with previously published results (13), basal transactivation of mutated CRP promoter was increased ~10-fold when compared with the basal transactivation of the wild-type promoter. Overexpression of RBP-J{kappa} had no effect on the enhancement of basal expression from the mutated promoter. These results further suggest that the endogenous level of RBP-J{kappa} in the Hep3B nuclei was sufficient to participate in regulating CRP expression.


Figure 8
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  		FIGURE 8. No change in the CRP promoter-driven luciferase expression in cells overexpressing RBP-J{kappa}. Data shown represent mean + SEM of five experiments. Mann-Whitney two-tailed U test was used to determine p values. A, Cells were transfected with the CRP promoter-luciferase construct and plasmids encoding C/EBPbeta (50 ng) and RBP-J{kappa} (1 µg). Basal luciferase activity is shown as 1 and the luciferase activity in treated cells is plotted as fold induction over basal activity. B, Two promoter constructs were used: wild-type (WT) promoter (–157/+3) and the mutant promoter (with the {kappa}B site at position –69 mutated). The luciferase activity of the wild-type promoter is shown as 1 and the luciferase activities of the mutant promoter in the presence and absence of RBP-J{kappa} are plotted as fold induction over wild-type promoter activity.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We investigated transcription factors interacting with the overlapping C/EBP-binding and p50 binding sites on the CRP promoter and their role in regulating CRP expression. Our major findings were 1) in the absence of C/EBPbeta, a complex containing C/EBP{zeta} and RBP-J{kappa} was formed at the C/EBP-p50 site in the nuclei of both Hep3B cells and primary human hepatocytes; 2) RBP-J{kappa} was required for the binding of C/EBP{zeta}-containing dimers to the C/EBP-p50 site; 3) the formation of C/EBP{zeta}-containing complexes was independent of the binding sites for OCT-1, HNF-1, and HNF-3 on the promoter; and 4) overexpressed C/EBP{zeta} repressed both (IL-6 + IL-1beta)-induced and C/EBPbeta-induced CRP expression. These findings suggest that the regulation of CRP transcription is partly determined by the relative levels of C/EBPbeta and C/EBP{zeta} in the nuclei. The C/EBP{zeta}-mediated repression of CRP transcription involves a novel action of RBP-J{kappa}. RBP-J{kappa} associates with C/EBP{zeta}-complexes to facilitate the binding of C/EBP{zeta} to the C/EBP site on the CRP promoter.

Previously (26), EMSA was performed using an oligonucleotide containing the C/EBP-p50 site as the probe and Hep3B nuclear extracts pretreated with C/EBP consensus oligonucleotide as the source of transcription factors. This EMSA revealed the formation of a complex called band D on the probe. This complex did not appear to contain either C/EBP or NF-{kappa}B proteins (26). We, however, hypothesized that the complex might contain C/EBP{zeta} because the C/EBP consensus oligonucleotide binds to and depletes the nuclear extract of all C/EBP proteins except C/EBP{zeta}. Our alternate hypothesis was that the complex might contain RBP-J{kappa} because it has been shown that RBP-J{kappa} binds to certain {kappa}B sites (30, 32). On some gene promoters, RBP-J{kappa} competes with NF-{kappa}B proteins to repress NF-{kappa}B-induced transcription (37, 38, 43). Interestingly, we found that the complex present in band D contained both C/EBP{zeta} and RBP-J{kappa}.

Unexpectedly, we observed formation of an intense RBP-J{kappa}-C/EBP{zeta} complex at the C/EBP-p50 site in the nuclear extract pretreated with STAT3 consensus oligonucleotide, suggesting that the formation of band D could occur even in the presence of active C/EBPbeta in the nuclei. This phenomenon can be explained by considering the sequence similarity between STAT3 consensus oligonucleotide and p50-binding nonconsensus {kappa}B site on the CRP promoter. Both sequences contain a polypyrimidine region (Fig. 1B). In addition to binding to STAT3, the STAT3 consensus oligonucleotide also binds p50. It was, therefore, likely that p50 was absorbed by the STAT3 consensus oligonucleotide in the pretreated nuclear extract, and no p50 was available to bind to p50 site on the probe in EMSA. A vacant p50 site would then be occupied by RBP-J{kappa} facilitating the formation of band D. Together, these data indicate that C/EBP{zeta}-containing complexes at the C/EBP-p50 site can be formed in two situations: when C/EBPbeta is absent and when C/EBPbeta is present but p50 is absent. RBP-J{kappa} is required in both situations. This interpretation also suggests that the binding of p50 to p50 site is a prerequisite for the binding of C/EBPbeta to the C/EBP site.

In some EMSA, the Ab to RBP-J{kappa} did not affect the intensity of band D even if the formation of band D was invariably found to be dependent on the presence of RBP-J{kappa}. We interpret these results to indicate that the complex in such a band D contained RBP-J{kappa}-C/EBP{zeta} dimers and that the mAb to RBP-J{kappa} did not recognize these dimers. Our data show the existence of RBP-J{kappa}-C/EBP{zeta} dimers in the nuclei and their capability to bind to RBP-J{kappa} consensus sequence, in addition to their binding to C/EBP-p50 site on the CRP promoter.

C/EBP{zeta} inhibits expression of other C/EBP-inducible genes such as transferrin gene, in Hep3B cells (21). However, the mechanism of inhibitory action of C/EBP{zeta} in transferrin gene expression involves a dominant regulatory effect. C/EBP{zeta} dimerizes with other C/EBP proteins to inhibit their binding to C/EBP sites in the promoter (21, 47). Binding of C/EBP{zeta}-containing dimers to a novel DNA target sequence, PuPuPuTGCAAT(A/C)CCC, has been reported earlier (48). However, the binding of C/EBP{zeta} to this DNA sequence did not require either RBP-J{kappa} or an adjacent RBP-J{kappa} binding site. On the CRP promoter, we found that the binding of C/EBP{zeta}-containing dimers to the C/EBP site required RBP-J{kappa}. This function of RBP-J{kappa} in facilitating the binding of C/EBP{zeta} to the C/EBP site is novel. Other mechanisms for the role of RBP-J{kappa} in gene expression have been described. For example, adjacent C/EBP-binding and RBP-J{kappa} binding sites are present on the IL-6 promoter and it has been shown that RBP-J{kappa} suppresses activation of the IL-6 promoter by NF-{kappa}B and C/EBPbeta (43, 49).

We observed two patterns of the formation of C/EBP{zeta}-containing band D: either the complex was not formed until C/EBPbeta was removed from the nuclear extract, or the complex was formed in the presence of C/EBPbeta and its intensity was increased when C/EBPbeta was removed. We also found that the inhibition of CRP expression by overexpressed C/EBP{zeta} was related to the levels of C/EBPbeta in the nuclei. These results suggest that the C/EBP-p50 site on the CRP promoter is a critical regulatory element and that the regulation occurs by C/EBP dimers containing either C/EBPbeta (activating) or C/EBP{zeta} (inhibitory). The formation of a specific dimer causing activation or inhibition of CRP expression may depend upon the relative levels of C/EBPbeta and C/EBP{zeta} in the nuclei. The role of the relative levels of two transcription factors in the nuclei in regulating gene expression through a common site on the promoter has been described earlier (50). Because C/EBPbeta induces synthesis of C/EBP{zeta} (17), and C/EBP{zeta} is also activated in acute phase response (51), our data suggest that C/EBP{zeta} is a critical regulator of CRP expression.

Fig. 9 summarizes our conclusions on the roles of C/EBP{zeta}, C/EBPbeta, RBP-J{kappa}, and p50 in regulating CRP expression through the C/EBP-p50 site on the promoter. Under basal conditions, there are five possible arrangements of transcription factors at the C/EBP-p50 site. First, the RBP-J{kappa}-C/EBP{zeta} dimer binds to C/EBP-p50 site (arrangement A). This is supported by the data that RBP-J{kappa}-C/EBP{zeta} formed on the RBP-J{kappa} consensus oligonucleotide was competed by CRP promoter-derived oligonucleotides (Fig. 4A, compare complex I in lane 1 with lanes 6–8). Second, RBP-J{kappa} binds to p50 site and supports binding of C/EBPbeta-{zeta} heterodimer to C/EBP site (arrangement B). This finding is supported by the data that, occasionally, RBP-J{kappa}, C/EBPbeta, and C/EBP{zeta} were all constituents of band D (Fig. 2, lanes 11, 14, and 16). Third, RBP-J{kappa} binds to p50 site and supports binding of C/EBP{zeta}-{zeta} homodimer to C/EBP site (arrangement C). This finding is supported by the data that the intensity of band D in most EMSA was diminished by individual Ab to RBP-J{kappa} and C/EBP{zeta}, and that the band D was abolished by the combination of both Abs. Fourth, RBP-J{kappa}-C/EBP{zeta} dimer binds to p50 site and supports the binding of C/EBPbeta-{zeta} heterodimer to C/EBP site (arrangement D). This plan is a modified arrangement B, with RBP-J{kappa} being associated with C/EBP{zeta}. Lastly, RBP-J{kappa}-C/EBP{zeta} dimer binds to p50 site and supports the binding of C/EBP{zeta}-{zeta} homodimer to C/EBP site (arrangement E). This arrangement is a modified arrangement C, with RBP-J{kappa} being associated with C/EBP{zeta}. In all five arrangements, RBP-J{kappa} was required because no complex was formed on the C/EBP-p50 site in the nuclei pretreated with RBP-J{kappa} consensus oligonucleotide. All these arrangements would cause repression of CRP expression and maintain basal expression. Under induced conditions, C/EBP{zeta}-containing dimers are replaced by C/EBPbeta-beta homodimers and this switch requires binding of p50 homodimers to the p50 site. The formation of C/EBPbeta-{zeta} heterodimers (arrangements B and D under basal conditions) could be a transitory stage between C/EBP{zeta}-{zeta} homodimers and C/EBPbeta-beta homodimers.


Figure 9
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  		FIGURE 9. A model showing the switch between RBP-J{kappa}-C/EBP{zeta} and p50-C/EBPbeta at the C/EBP-p50 site on the CRP promoter. Under basal conditions, there are five possible arrangements (A–E) of transcription factors C/EBP{zeta} and RBP-J{kappa} in the complex formed at the C/EBP-p50 site as described in Results. Under induced conditions, C/EBP{zeta}-containing dimers are replaced by C/EBPbeta-beta homodimers and this switch requires binding of p50 homodimers to p50 site. The formation of C/EBPbeta-{zeta} heterodimers could be a transitory stage (arrangement C to B and E to D) between C/EBP{zeta}-{zeta} homodimers and C/EBPbeta-beta homodimers.

 

   Acknowledgments
 
We thank Drs. Peter F. Johnson, Nikki J. Holbrook, Jennifer L. Martindale, and Lynne D. Vales for the recombinant plasmids, Drs. Neal D. Goldman, Irving Kushner, and David Samols for the wild-type CRP promoter-luciferase vector, and Dr. Gretchen J. Darlington for Hep3B cells.


   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 Grant R01-HL71233 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Alok Agrawal, Department of Pharmacology, P.O. Box 70577, East Tennessee State University, Johnson City, TN 37614. E-mail address: agrawal{at}etsu.edu Back

3 Abbreviations used in this paper: CRP, C-reactive protein; HNF, hepatocyte nuclear factor. Back

Received for publication September 15, 2006. Accepted for publication March 16, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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