©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CCAAT Box Enhancer Binding Protein (C/EBP-) Stimulates B Element-mediated Transcription in Transfected Cells (*)

(Received for publication, October 10, 1995)

Ilja Vietor (§) Igor C. Oliveira (¶) Jan Vilcek (**)

From the Department of Microbiology and Kaplan Cancer Center, New York University Medical Center, New York, New York 10016

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A construct comprising three tandemly repeated copies of the kappaB element from the interleukin-8 gene linked to chloramphenicol acetyltransferase (CAT) (3xNF-kappaBCAT) was transcriptionally activated in normal human FS-4 fibroblasts by co-transfection with expression vectors for NF-kappaB p50, p65, or p52. Unexpectedly, a significant activation of 3xNF-kappaBCAT was also seen upon its co-transfection with the expression vector for CCAAT box enhancer binding protein alpha (C/EBP-alpha) (but not C/EBP-beta or C/EBP-). Stimulation by C/EBP-alpha required some other factor(s) present in FS-4 cells because no transcriptional activation of 3xNF-kappaBCAT was seen after co-transfection with C/EBP-alpha in F9 mouse embryonic carcinoma cells, known to be deficient in several transcription factors. To determine whether transcriptional activation was the result of interaction with one of the major NF-kappaB proteins, we co-transfected C/EBP-alpha with NF-kappaB p50, p65, p50 + p65, or p52 into F9 or FS-4 cells. No cooperative interaction was seen; in fact, C/EBP-alpha reduced p65-stimulated transcription, especially in F9 cells. Electrophoretic mobility shift assay with a kappaB probe revealed that the addition of recombinant C/EBP-alpha protein to nuclear extracts from untreated FS-4 cells resulted in the appearance of four bands. Only one of these bands was supershifted by antibody to p50, whereas antibodies to p65 or other NF-kappaB proteins had no effect. Our findings show that C/EBP-alpha may cause activation of some kappaB element-containing genes lacking C/EBP binding sites.


INTRODUCTION

NF-kappaB proteins regulate the expression of many different genes, including genes encoding cytokines and acute phase proteins and some viral genes(1, 2, 3) . The NF-kappaB family includes p50 (NFKB1), p52 (NFKB2), p65 (RelA), RelB, v-Rel, c-Rel and Drosophila proteins Dorsal and Dif. The N-terminal portion of NF-kappaB proteins contains a 300-amino acid domain termed the Rel homology region. This domain is responsible for DNA binding and dimerization of NF-kappaB proteins. Less homology is found within the C-terminal region of NF-kappaB proteins that is known to serve as the activation domain. In their inactive state NF-kappaB proteins are usually sequestered in the cytoplasm in a complex with an inhibitory subunit termed I-kappaB(1) . NF-kappaB proteins are activated by phosphorylation of the I-kappaB subunit and its subsequent proteasome-driven degradation leading to the release from the NF-kappaBbulletI-kappaB complex(4) . NF-kappaB then translocates into the nucleus and rapidly activates gene expression. Another group of transcription factors important in the regulation of cytokine and acute phase protein genes is the CCAAT box enhancer binding protein (C/EBP) (^1)family(5, 6, 7) . A typical structural feature of C/EBP proteins is the presence of a domain comprising a region of basic amino acids and a leucine zipper region (b-ZIP domain)(8, 9) . The leucine zipper domain is responsible for dimerization, causing changes in the structural conformation of the protein that allow the binding of the basic region to specific DNA sequences. The C/EBP family includes C/EBP-alpha, C/EBP-beta (also termed IL-6 DBP or LAP; its human analogue is NF-IL6), C/EBP- (Ig/EBP-1), C/EBP-, and CHOP/GADD/53(7, 10, 11, 12) .

An important feature of NF-kappaB and C/EBP proteins is their ability to heterodimerize with each other and with members of other transcription factor families(13, 14, 15, 16, 17, 18) . NF-kappaBbulletC/EBP-alpha heterodimers are formed through the interaction of the Rel homology region of NF-kappaB proteins with leucine zipper domains within b-ZIP regions of C/EBP transcription factors. Heterodimerization is facilitated by the presence of adjacent kappaB and C/EBP binding sites in several genes encoding cytokines (IL-6, IL-8, G-CSF, neutrophil-activating peptide ENA-78, MGSA/GRO)(16, 19, 20, 21, 22, 23, 24) , acute phase proteins (angiotensinogen, serum amyloid A protein, alpha(1)-acid glycoprotein, TSG-14/PTX-3) (25, 26, 27, 28) or adhesion receptors (intercellular adhesion molecule-1)(29) . The presence of C/EBPbulletNF-kappaB protein heterocomplexes has been demonstrated in intact cells(17, 30) . Heterodimerization between NF-kappaB and C/EBP proteins can lead to both cooperative and antagonistic interactions. For example, in the IL-8 promoter NF-kappaB and C/EBP proteins were shown to mutually augment their binding to the adjacent kappaB and C/EBP binding sites, resulting in increased gene expression(20) . Similarly, C/EBP-alpha and C/EBP-beta increased NF-kappaB p50- or p65-driven expression of a SAA3-CAT gene construct(31) . On the other hand, it was found that C/EBP can inhibit promoters with kappaB binding sites. Thus, when NF-kappaB p65 and C/EBP-beta were co-transfected together with an expression vector driven solely by the kappaB element from the IL-8 gene, C/EBP-beta inhibited p65-stimulated transcription (20) .

In our present study we show that when overexpressed in human FS-4 fibroblasts, C/EBP-alpha activates transcription from a CAT reporter construct based on a trimeric repeat of the kappaB element from the enhancer region of the IL-8 gene(32) . However, C/EBP-alpha did not stimulate transcription of the same construct in mouse F9 embryonic teratocarcinoma cells, which fail to express endogenous NF-kappaB activity(33, 34) . Therefore, an interaction of C/EBP-alpha with NF-kappaB and/or other transcription factor(s) is likely to be involved in the transcriptional activation of the kappaB-driven construct in FS-4 cells. Our studies of transcriptional activation were confirmed by electrophoretic mobility shift assays (EMSAs), showing that in the presence of the kappaB probe nuclear extracts from unstimulated FS-4 cells formed specific complexes with recombinant C/EBP-alpha protein.


EXPERIMENTAL PROCEDURES

Cytokines, Transcription Factors, and Antibodies

Recombinant human and murine TNF-alpha were kind gifts from Masafumi Tsujimoto (Suntory Institute for Biomedical Research, Osaka, Japan). Recombinant human IL-1alpha was a gift from Peter Lomedico and Alvin Stern (Hoffmann La-Roche, Nutley, NJ). Recombinant murine IL-1beta was from Program Resources (National Cancer Institute, Frederick, MD). NF-kappaB proteins p50 and p65 were a generous gift from John Hiscott (Lady Davis Institute for Medical Research, Montreal, Canada). These recombinant proteins were prepared as glutathione S-transferase fusion proteins, enzymatically cleaved, and purified(35) . Recombinant C/EBP-alpha protein was a generous gift of Steven McKnight (Tularik Inc., South San Francisco, CA)(36) . Polyclonal rabbit antibodies specific for NF-kappaB proteins p50 (number 1141, raised against a synthetic peptide from the N terminus; number 1613, raised against a peptide that includes the nuclear localization signal), and p65 (number 1226, raised against a peptide from the C terminus) were a generous gift from Nancy Rice (NCI-Frederick Cancer Research and Development Center, Frederick, MD). The polyclonal antibody against C/EBP-alpha was a gift from Steven McKnight (Tularik, Inc., South San Francisco, CA).

Plasmid Construction and Oligonucleotides

The construction of reporter plasmids containing three tandemly repeated copies of the NF-kappaB binding site from the human IL-8 gene (3xNF-kappaBCAT), or three copies of the NF-kappaB binding site from the human HLA-B7 gene (3xNF-kappaB(HLA)CAT), linked to the minimal promoter of the IL-8 gene has been described elsewhere(19, 32) . (The core kappaB sequence from the IL-8 gene is TGGAATTTCC as compared with TGGGGATTCC for the HLA B-7 gene.) Expression vector for p50B (p52) (37) was a gift from Ulrich Siebenlist (Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD). Expression vectors pMSV-C/EBP-alpha (38) and pMSV-C/EBP- (11) were a kind gift from Steven McKnight (Tularik Inc., South San Francisco, CA). Plasmid pSVK3-IkappaB-alpha was a generous gift from John Hiscott (Lady Davis Institute for Medical Research, McGill University, Montreal, Canada) (39) . The pCMV4T (empty vector), pCMV-C/EBP-beta, pCMV-p50, and pCMV-p65 expression plasmids were kindly provided by Bernd Stein (Signal Pharmaceuticals, San Diego, CA)(14) . An oligonucleotide probe composed of three copies of the NF-kappaB element from the IL-8 gene was used in EMSAs(32) .

Cell Cultures

Human diploid FS-4 fibroblasts were maintained in Eagle's minimum essential medium (Life Technologies, Inc., Grand Island, NY) supplemented with 5% heat-inactivated (56 °C, 30 min) fetal bovine serum (Life Technologies, Inc.), 6 mM Hepes, 3 mM Tricine, 100 units/ml of penicillin, and 100 µg/ml of streptomycin. Cultures were seeded in 175-cm^2 flasks (2 times 10^6 cells/flask). Undifferentiated F9 mouse embryonal carcinoma cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, and 100 µg/ml of streptomycin. F9 cells were seeded in 60-cm^2 dishes (2.0 times 10^6 cells/dish) coated with 0.5% gelatin (Difco).

Transfections and CAT Assays

The transfection of human FS-4 fibroblasts was done by the calcium co-precipitation method with the aid of a commercial kit (5` to 3`, Boulder, CO), using a protocol recommended by the manufacturer with minor modifications. Briefly, 1 day before the experiment, confluent cultures of FS-4 cells were trypsinized, and cells were seeded at a density of 3.5-4.0 times 10^6 cells/150 cm^2 dish and incubated overnight at 37 °C. The cultures were replenished with fresh medium and kept at 37 °C for at least 4 h before transfection. The DNA-calcium precipitate containing a given amount of the appropriate plasmid DNA and 15 µg of pCMV-beta plasmid encoding beta-galactosidase (Clontech, Palo Alto, CA) was added into the culture medium, and the cells were reincubated for an additional 4 h. Each construct was transfected into two 150-cm^2 dishes. Four h later the cultures were glycerol-shocked and replenished with fresh medium. After 2-3 h, trypsin was added, the cells obtained from two 150-cm^2 dishes were subdivided into four 60 cm^2 dishes (1.8-2.0 times 10^6 cells/dish), and the resulting cultures were incubated overnight. This procedure eliminates differences in transfection efficiency seen when the same construct is used for transfection of separate cultures. Transfection of F9 cells was done by the calcium co-precipitation method without glycerol-shock. F9 cells were transfected when the monolayer reached 70% confluency. After overnight incubation the culture medium was replaced, and cells were incubated for additional 28 h. In some experiments the cultures were then treated with the appropriate cytokines for 5 h. Cell extracts were prepared by subjecting the cells to three cycles of freeze-thawing in 0.25 mM Tris-HCl, pH 7.8. Immediately thereafter, equal amounts of protein from the extracts were used for the CAT determinations(40) . The beta-galactosidase activity in the extracts was determined with the aid of a kit (Promega, Madison, WI), following instructions provided by the manufacturer. After thin layer chromatography, radioactivity was measured with the aid of the AMBIS Radioanalytic System (Ambis, Inc., San Diego, CA), and the percentage of conversion to acetylated chloramphenicol was calculated. Conversion rates were normalized to beta-galactosidase activity(32) . All transfection experiments were repeated at least five times.

EMSAs

Nuclear protein extracts were prepared essentially as described(41) . The binding reactions were performed with 8 µg of nuclear protein extract in 10 mM HEPES-KOH, pH 8.0, 40-60 mM KCl, 1 mM EDTA, 1 mM beta-mercaptoethanol, 12% glycerol, 2 µg of poly(dI-dC) in a final volume of 30 µl at room temperature for 20 min. A total of 100 ng of the oligonucleotide used as a probe was labeled by Klenow, and 1 ng (>10,000 cpm/µg) of the radiolabeled material was used in each binding reaction. For the competition assays a 100-fold molar excess of the cold oligonucleotide was added to the binding reaction and incubated for 10 min, before the addition of the probe. After gentle mixing, the reaction was allowed to proceed for 20 min at room temperature. In the ``supershift'' experiments 1 µl of the antibody was added to the binding reactions before the probe and poly(dI-dC) and incubated at 37 °C for 30 min. After the addition of both poly(dI-dC) and the probe, the reaction was gently mixed and further incubated for 20 min at room temperature. The samples were electrophoresed at 200 V (in a buffer containing 6.6 mM Tris-HCl, pH 7.9, 3.3 mM sodium acetate, pH 7.9, and 1.0 mM EDTA, pH 8.0) through a 5.5% native polyacrylamide gel. The gel was dried and exposed to Fuji RX medical x-ray film at -70 °C. Scanning software Adobe Photoshop 3.0 by Adobe Systems, Inc., and Canvas 3.5.2 by Deneba Software (Miami, FL) were used for the scanning of the autoradiograms.


RESULTS

Activation of NF-kappaB-responsive CAT Reporter Construct by C/EBP-alpha in Human FS-4 Fibroblasts

Previous studies demonstrated that expression of the IL-8 gene is cooperatively regulated by members of two transcription factor families, NF-kappaB and C/EBP, which bind to two distinct, adjacent cis-acting DNA elements in the IL-8 promoter(16, 19, 20) . We have examined the ability of several members of the NF-kappaB and C/EBP families to induce the transcriptional activation of a reporter construct comprising a trimerized NF-kappaB binding site from the human IL-8 gene linked to the minimal promoter of the IL-8 gene and the CAT gene (3xNF-kappaBCAT)(32) . While co-transfection of FS-4 cells with 3xNF-kappaBCAT and the NF-kappaB p50 expression plasmid stimulated CAT activity less than 5-fold, co-transfection with NF-kappaB p65 or with both p50 and p65 increased CAT activity approximately 50-fold over control levels (Fig. 1, A and D). Transcription from the 3xNF-kappaBCAT construct was not activated by co-transfection with C/EBP-beta and C/EBP- expression plasmids, but to our surprise, C/EBP-alpha produced an approximately 5-fold increase in CAT activity (Fig. 1, B and D). To determine whether activation of the 3xNF-kappaBCAT construct is kappaB sequence-specific, we also employed expression construct 3xNF-kappaB(HLA)CAT, comprising three copies of the kappaB element from the human HLA B-7 gene that differs from the IL-8 kappaB sequence in three positions(32) . The 3xNF-kappaB(HLA)CAT construct contains the same minimal promoter from the IL-8 gene as 3xNF-kappaBCAT. The 3xNF-kappaB(HLA)CAT construct was less responsive to stimulation by the p65 expression vector than the 3xNF-kappaBCAT construct containing the kappaB sequence from the IL-8 gene (Fig. 1C). Unlike the latter construct, 3xNF-kappaB(HLA)CAT was not stimulated by co-transfection with the C/EBP-alpha expression vector, indicating that the sequence specificity of the kappaB element affects the activation process.


Figure 1: Transactivation of NF-kappaB-responsive CAT constructs by co-transfection with various NF-kappaB or C/EBP proteins. FS-4 cells were transfected with 10 µg of reporter construct 3xNF-kappaBCAT comprising three tandemly repeated copies of the NF-kappaB site from the IL-8 gene, except for the experiment shown in panel C in which 10 µg of construct 3xNF-kappaB(HLA)CAT, comprising three copies of the NF-kappaB binding site from the HLA B-7 gene, was used in one half of the cultures, as indicated. Co-transfected with the reporter construct were expression vectors encoding various NF-kappaB or C/EBP family proteins, as indicated (1 µg of expression vector/culture). Cells were harvested 20 h later, and CAT activity was determined as described under ``Experimental Procedures.'' Fold induction represents the increase in percentage of acetylation over control cells transfected with the reporter construct alone. Values shown represent the means of three separate determinations and their standard deviations. Panel D shows an autoradiogram of a representative experiment in which FS-4 cells were co-transfected with the reporter construct 3xNF-kappaBCAT and expression vectors for p50, p65, and C/EBP-alpha, as indicated.



Lack of Demonstrable Cooperative Interactions between C/EBP-alpha and NF-kappaB Factors in the Transcriptional Activation of the 3xNF-kappaBCAT Reporter Gene

To examine a possible cooperation between C/EBP-alpha and NF-kappaB proteins in the transcriptional activation of 3xNF-kappaBCAT seen upon overexpression of C/EBP-alpha, we co-transfected FS-4 cells with the reporter construct 3xNF-kappaBCAT and the C/EBP-alpha vector alone or in combination with vectors encoding different members of the NF-kappaB family. Transfection of different concentrations of the C/EBP-alpha vector alone stimulated activation of the CAT construct dose-dependently, up to approximately 8-fold (Fig. 2A). Transfection with 1 µg of the p50 (Fig. 2B) or p52 (Fig. 2C) vector alone stimulated CAT activity only modestly. Co-transfection with different amounts of C/EBP-alpha and 1 µg of either the p50 or the p52 expression plasmids did not have a major effect on the induction of CAT activity when compared with the results obtained by transfection of C/EBP-alpha alone (Fig. 2, B and C). Hence, these data did not reveal striking cooperative or antagonistic interactions between p50 or p52 NF-kappaB transcription factors and C/EBP-alpha. A possible interaction between C/EBP-alpha and p65 was examined by co-transfecting into FS-4 cells different doses of the p65 expression vector and a constant dose of the C/EBP-alpha vector along with 3xNF-kappaBCAT (Fig. 3). In general, the increase in CAT activity seen after co-transfection with p65 and C/EBP-alpha was less than the sum of CAT activities produced with either expression vector alone, suggesting some degree of antagonism between C/EBP-alpha and the p65 NF-kappaB protein. This apparent antagonism was most pronounced with 0.25 µg of the p65 expression vector.


Figure 2: Co-transfection of C/EBP-alpha with NF-kappaB p50 or p52 proteins does not result in a cooperative stimulation of the 3xNF-kappaBCAT reporter construct. FS-4 cells were co-transfected with 10 µg of the 3xNF-kappaBCAT reporter construct and the indicated amounts of expression vector pMSV-C/EBP-alpha (A), or combinations of different amounts of pMSV-C/EBP-alpha with 1 µg of pCMV-p50 (B) or pCMV-p52 (C). pCMV4T is the ``empty'' vector.




Figure 3: Effects of co-transfection with C/EBP-alpha and NF-kappaB p65 on 3xNF-kappaBCAT activity. FS-4 cells were co-transfected with 10 µg of the 3xNF-kappaBCAT reporter construct and increasing amounts of the pCMV-p65 expression vector with or without 1 µg of pMSV-C/EBP-alpha expression vector. Numbers in parentheses indicate concentrations of expression vectors in µg of plasmid DNA/culture.



C/EBP-alpha Fails to Activate 3xNF-kappaBCAT in the F9 Embryonic Carcinoma Cell Line

Earlier studies have shown that F9 mouse embryonic carcinoma cells are deficient in NF-kappaB-dependent functions(33, 42, 43) . Thus, this cell line offered an opportunity to determine if C/EBP-alpha could produce activation of the 3xNF-kappaBCAT construct in the absence of functional NF-kappaB proteins. To confirm that F9 cells lack activable NF-kappaB proteins, we first transfected F9 cells with the 3xNF-kappaBCAT plasmid and then treated the transfected cultures with different agents known to activate gene transcription via induction of NF-kappaB. Neither cytokines (TNF or IL-1) nor the phorbol ester TPA induced CAT activity, confirming that F9 cells lack activable NF-kappaB (Fig. 4). In contrast, treatment with TNF or IL-1 caused a marked stimulation of CAT activity in FS-4 cells transfected with the same 3xNF-kappaBCAT reporter construct ( (32) and data not shown). We then examined the ability of the expression vectors for C/EBP-alpha or NF-kappaB proteins p50, p52, and p65 to activate the 3xNF-kappaBCAT reporter construct in F9 cells. In contrast to the results obtained in FS-4 cells, C/EBP-alpha failed to produce any activation of CAT activity (Fig. 5, A and B). p50 or p52 alone, or combinations of C/EBP-alpha with p50 or p52 also failed to stimulate CAT activity. However, CAT activity was strongly stimulated in F9 cells by co-transfection with p65 (Fig. 5C). Co-transfection of p50 further potentiated the stimulatory effects of p65 on CAT activity (Fig. 5D). The stimulatory effect of p65 alone or of the combination of p65 and p50 was reduced by co-transfecting C/EBP-alpha, indicating an antagonistic relationship (Fig. 5, C and D).


Figure 4: Lack of inducibility of the 3xNF-kappaBCAT reporter construct by cytokines or TPA in F9 cells. F9 mouse embryonic carcinoma cells were transfected with 10 µg of the 3xNF-kappaBCAT reporter construct. After an overnight incubation the medium was replaced and 24 h later cells were treated with murine TNF (muTNF) (10 ng/ml), human TNF (huTNF) (10 ng/ml), human IL-1alpha (huIL-1alpha) (2 ng/ml), murine IL-1beta (muIL-1beta) (2 ng/ml), or TPA (20 ng/ml) for 5 h. Cells were harvested, and CAT activity was determined.




Figure 5: Activation of 3xNF-kappaBCAT by co-transfection of C/EBP-alpha and/or NF-kappaB proteins in F9 cells. F9 cells were co-transfected with 10 µg of the 3xNF-kappaBCAT reporter plasmid and various expression plasmids, as indicated. Numbers in parentheses denote µg of plasmid DNA/culture. Cell lysates were prepared 48 h after transfection and analyzed for CAT activity. A, co-transfections were performed with expression plasmids pMSV-C/EBP-alpha alone, pCMV-p50 alone, or their combinations as indicated. B, co-transfection with 1 µg of the C/EBP-alpha expression plasmid and/or indicated amounts of expression plasmid for p52. C, co-transfection with expression plasmids for C/EBP-alpha and/or p65. D, co-transfection with expression plasmids for C/EBP-alpha and p50 or p65, and their combinations.



Effects of I-kappaB on the Activation of 3xNF-kappaBCAT by p65 or C/EBP-alpha

The inhibitory protein I-kappaB is known to block NF-kappaB-regulated gene expression by binding to the NF-kappaB complex in the cytoplasm, thus preventing nuclear translocation. Overexpression of I-kappaB can inhibit NF-kappaB-mediated gene activation(34, 39, 44) . We compared the effects of I-kappaB overexpression on the stimulation of the 3xNF-kappaBCAT reporter construct by transfection with either p65 or C/EBP-alpha in FS-4 cells (Fig. 6). Overexpression of I-kappaB significantly inhibited the stimulation of CAT activity by p65 but had a much less marked influence on the ability of C/EBP-alpha to activate the 3xNF-kappaBCAT reporter construct.


Figure 6: Effect of I-kappaB on NF-kappaB p65- or C/EBP-alpha-induced transcriptional activation. FS-4 cells were transfected with the 3xNF-kappaBCAT reporter plasmid (10 µg) and combinations of pCMV-p65 (1 µg) or pMSV-C/EBP-alpha (1 µg) with 10 µg of expression plasmid pSVK3-IkappaB-alpha. At 24 h after transfection cell lysates were prepared and analyzed for CAT activity. Control represents CAT activity in cells transfected with the reporter plasmid alone.



Nuclear Protein Binding to NF-kappaB Site from the IL-8 Promoter

To determine if increased CAT activity in FS-4 cells transfected with 3xNF-kappaBCAT and C/EBP-alpha is due to the direct binding of C/EBP-alpha to the kappaB DNA element, we examined binding of recombinant C/EBP-alpha protein by EMSAs with a synthetic oligonucleotide probe containing the same kappaB DNA sequence from the IL-8 gene as the 3xNF-kappaBCAT construct. Incubation of recombinant C/EBP-alpha protein alone with the labeled oligonucleotide probe did not lead to the formation of a detectable complex (Fig. 7A, lane 2). However, FS-4 nuclear extracts mixed with small amounts of recombinant C/EBP-alpha protein reproducibly gave rise to four distinct bands (marked C1, C2, C3, and C4) that were not formed by the FS-4 nuclear extract in the absence of C/EBP-alpha protein (Fig. 7A, lanes 1 and 3). Formation of all four complexes was competed by preincubation of the nuclear extracts with an excess of the unlabeled kappaB oligonucleotide, indicating that these interactions are specific (Fig. 7A, lane 4). The addition of C/EBP-alpha antibody to the binding reactions resulted in the formation of a very large supershifted complex, with a concomitant disappearance of C1, C2, C3, and C4, suggesting that C/EBP-alpha is involved in the formation of all four complexes (Fig. 7B). In contrast, nonimmune serum did not interfere with complex formation. We also attempted to determine whether complexes C1-C4 contained proteins reactive with antibodies against various NF-kappaB proteins. The addition of two different antibodies to p50 decreased the intensity of complex C4 without a reduction in any of the other three complexes (Fig. 7C). The addition of anti-p65 antibody to the binding reaction did not significantly decrease the intensity of the complexes. In other experiments we found that antibodies to p52, RelB, and c-Rel also failed to reduce the formation of any of the four complexes formed in EMSAs with recombinant C/EBP-alpha protein, nuclear proteins from unstimulated FS-4 cells, and the NF-kappaB probe (data not shown).


Figure 7: Formation of protein-DNA complexes with nuclear extracts from unstimulated FS-4 cells and recombinant C/EBP-alpha protein. NF-kappaB probe was used in EMSAs as described under ``Experimental Procedures.'' Complexes were resolved in 5.5% native polyacrylamide gels. Locations of specific complexes C1-C4 are indicated by arrows. A, 8 µg of nuclear protein extract from unstimulated FS-4 cells and/or 200 ng of recombinant C/EBP-alpha protein were mixed with 10,000 cpm of the P-labeled NF-kappaB oligonucleotide probe. For the competition assay a 100-fold molar excess of the same unlabeled oligonucleotide was added to the binding reaction. B, prior to adding the P-labeled oligonucleotide probe, the mixture of FS-4 nuclear extract and recombinant C/EBP-alpha protein was preincubated for 30 min at 37 °C with antibody against C/EBP-alpha or with nonimmune serum. Other conditions were as in panel A. C, polyclonal antibodies against different NF-kappaB proteins (as indicated in the legend) were added to the EMSA binding reactions and preincubated for 30 min at 37 °C before the addition of the P-labeled oligonucleotide probe. Other conditions were as in panel A.




DISCUSSION

In this study we have shown that C/EBP-alpha expression in human FS-4 fibroblasts stimulates transcriptional activity of the 3xNF-kappaBCAT reporter construct comprising three tandemly repeated copies of the NF-kappaB binding site from the human IL-8 gene. The stimulatory effect of C/EBP-alpha was kappaB sequence-specific, because C/EBP-alpha expression did not activate a similar construct, 3xNF-kappaB(HLA)CAT, comprising three copies of the NF-kappaB binding site from the human HLA B-7 gene (Fig. 1). Two lines of evidence indicate that stimulation of the 3xNF-kappaBCAT construct was not the result of a direct binding of C/EBP-alpha to the kappaB sequence. First, C/EBP-alpha failed to produce transcriptional activation of 3xNF-kappaBCAT in the murine F9 embryonic carcinoma cell line (Fig. 5) in which the active (nuclear) form of NF-kappaB is not inducible (33, 42) and which is deficient in some other transcription factors(43, 45) . Second, in EMSAs recombinant C/EBP-alpha protein failed to bind to a probe comprising the same kappaB binding sites from the IL-8 gene as the 3xNF-kappaBCAT expression construct (Fig. 7). Our data support the notion that complex formation of C/EBP-alpha with other protein factor(s), present in FS-4 but not in F9 cells, is necessary for the transcriptional activation of 3xNF-kappaBCAT. Direct evidence of the formation of specific complexes between recombinant C/EBP-alpha protein and proteins from the nuclei of unstimulated FS-4 cells was obtained in EMSAs (Fig. 7).

The stimulatory effect on the 3xNF-kappaBCAT construct was specific for C/EBP-alpha, because no transcriptional activation was seen on co-transfection with C/EBP-beta or - (Fig. 1B). C/EBP-alpha is primarily a regulator of genes involved in energy metabolism (46) and was recently shown to be critical for energy homeostasis in newborn mice(47) . C/EBP-alpha is abundant in the liver and adipose tissue(48) . In murine 3T3-L1 preadipocytes and various murine fibroblast lines C/EBP-alpha is both necessary and sufficient for the induction of adipogenesis(49, 50, 51) . The role of C/EBP-alpha in the acute phase response is a complex one. Although C/EBP-alpha was shown to transactivate the serum amyloid protein A3 gene promoter in hepatoma cells, cytokine treatment led to a down-regulation of C/EBP-alpha activity with a concomitant increase in C/EBP-beta and C/EBP- binding activities(27) . An inverse correlation between C/EBP-alpha levels and the acute phase response was also seen with respect to the regulation of the alpha(1)-acid glycoprotein gene(25, 52) . Besides the liver and adipose tissues, high levels of C/EBP-alpha were also found in myelomonocytic cells and in granulocytes, but its functional role in these cells has not been analyzed(53) .

Transactivation of 3xNF-kappaBCAT by C/EBP-alpha was demonstrated in FS-4 fibroblasts but not in F9 embryonic carcinoma cells (Fig. 5). Earlier studies have demonstrated the absence of NF-kappaB activity in undifferentiated F9 cells, as demonstrated by a lack of nuclear protein binding to kappaB DNA probes or failure of kappaB-mediated transcriptional activation(33, 42) . F9 cells were also found to be deficient in some other transcription factor activities, e.g. transcriptional activation through the cAMP-response element (CRE) was blocked in F9 cells due to the absence of functional protein kinase A activity(43, 45) . We confirmed the absence of activable NF-kappaB in F9 cells by demonstrating that a variety of stimuli failed to activate the transfected 3xNF-kappaBCAT construct (Fig. 4). FS-4 and F9 cells also differed in the transactivation of 3xNF-kappaBCAT by various exogenously provided NF-kappaB proteins: transfection with p50 or p52 alone activated this construct in FS-4 cells ( Fig. 1and Fig. 3), but not in F9 cells (Fig. 5). While p65 was about equally stimulatory in FS-4 and F9 cells, a cooperative effect of p50 with p65 was seen in F9 but not in FS-4 cells (Fig. 2, Fig. 3, and Fig. 5). In addition, co-transfection of C/EBP-alpha with p65 seemed to have a stronger inhibitory effect in F9 than in FS-4 cells. Hence, FS-4 and F9 cells differ in the makeup of endogenous factors that can affect the function of exogenously provided NF-kappaB and C/EBP proteins. Earlier we showed that EMSAs with extracts from unstimulated FS-4 cells gives rise to two NF-kappaB-specific bands, one composed of p50 homodimers and one comprising p50/p65 heterodimers(32) . The pattern seen in Fig. 7(especially panel A, lane 1) is in agreement with these earlier data.

The most likely interpretation of the observed transcriptional activation of the 3xNF-kappaBCAT construct by C/EBP-alpha in FS-4 cells is that C/EBP-alpha stimulates NF-kappaB-regulated expression through the formation of a heteromeric complex with nuclear protein factors preexisting in FS-4 cells. Alternatively, transfection with C/EBP-alpha stimulates de novo synthesis of protein(s) responsible for the transcriptional activation in FS-4 cells, but this notion is inconsistent with results of EMSAs, which show that nuclei of unstimulated FS-4 cells contain proteins capable of forming specific complexes with recombinant C/EBP-alpha in the presence of the NF-kappaB probe. One possible candidate for complex formation with C/EBP-alpha was p65 because a recent report showed that a heterodimer of p65 and C/EBP- acts as a potent activator of transcription from both NF-kappaB and C/EBP sites(17) . However, we found no evidence for a role of p65. First, there was only antagonism between p65 and C/EBP-alpha when the two factors were co-transfected into cells ( Fig. 3and Fig. 5). Second, no evidence for the presence of p65 protein in the complexes formed by FS-4 cell nuclear proteins with recombinant C/EBP-alpha could be obtained in EMSAs with antibody to p65 (Fig. 7C). On the other hand, results obtained with two different antibodies specific for the p50 NF-kappaB protein suggest the presence of p50 in one of the four complexes formed between recombinant C/EBP-alpha and nuclear proteins from FS-4 cells (Fig. 7C). Yet, co-transfection of p50 and C/EBP-alpha showed no cooperative interaction either in FS-4 (Fig. 2) or in F9 cells (Fig. 5). Failure of antibodies to p52, RelB, and c-Rel (data not shown) to interfere with the appearance of any of the four bands formed between recombinant C/EBP-alpha and nuclear proteins from unstimulated FS-4 cells suggests that other common NF-kappaB proteins are probably not present in these complexes. In addition, the failure of transfected I-kappaB to significantly inhibit the stimulatory effect of C/EBP-alpha (Fig. 6) and the failure of p52 to synergize with C/EBP-alpha in the activation 3xNF-kappaBCAT ( Fig. 2and Fig. 5B) also argue against the involvement of other NF-kappaB proteins.

The fact that four different complexes were formed in EMSAs when recombinant C/EBP-alpha was mixed with nuclear proteins from FS-4 cells (Fig. 7) suggests the presence of multiple interacting proteins. Besides NF-kappaB proteins, C/EBP proteins can heterodimerize with a variety of other transcription factors, including c-Fos, c-Jun(13) , C/ATF(54) , and the glucocorticoid receptor(18) . It is conceivable that complex formation between C/EBP-alpha and these or some other transcription factor(s) would alter the binding specificity of the resulting complex, which then could recognize ``nonclassical'' sites. This notion finds support in the recent demonstration that the transcription factor CHOP can dimerize with other C/EBP proteins and that the resulting dimers are directed away from classical C/EBP sites recognizing instead other specific DNA binding domains(12) . Also possibly related is the reported increase in the binding of upstream stimulatory factor (USF) to the USF element after overexpression of C/EBP-alpha (but not C/EBP-beta), implicated in the autostimulatory effect of C/EBP-alpha on its own promoter, which lacks any C/EBP binding domain (55) . Since there was no accompanying increase in USF synthesis, C/EBP-alpha is likely to increase USF binding by forming a complex with USF. In a somewhat similar manner, in our system C/EBP-alpha does not bind to 3xNF-kappaBCAT, yet it stimulates transcription from this construct, most likely by complexing with, and thereby altering the specificity and binding affinity of, some other nuclear factor(s) present in unstimulated cells. Our findings establish a mechanism whereby C/EBP-alpha may cause activation of some kappaB element-containing genes that lack C/EBP binding sites.


FOOTNOTES

*
This work was supported by NCI, National Institutes of Health, Grant R35-CA49731. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Permanent address: Institute of Experimental Endocrinology, Slovak Academy of Sciences, Vlarska 3, 833 06 Bratislava, Slovakia.

Present address: Laboratory of Plant Molecular Biology, Dept. of Biology, New York University, 100 Washington Square East, New York, NY 10003.

**
To whom correspondence should be addressed: Dept. of Microbiology, New York University Medical Center, 550 First Ave., New York, NY 10016. Tel.: 212-263-6756; Fax: 212-263-7933.

(^1)
The abbreviations used are: C/EBP, CCAAT box enhancer binding protein; IL-8, interleukin-8; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; TNF, tumor necrosis factor; IL, interleukin; TPA, 12-0-tetradecanoylphorbol-13-acetate; USF, upstream stimulatory factor.


ACKNOWLEDGEMENTS

We thank Naoko Tanese, Luiz F. L. Reis, Vito J. Palombella, Pernille Rorth, David Ron, Nancy R. Rice, and Sergei Nedospasov for helpful discussions, Angel Feliciano for technical assistance, and Ilene Totillo for preparation of the manuscript.


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