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Originally published In Press as doi:10.1074/jbc.M206224200 on August 12, 2002

J. Biol. Chem., Vol. 277, Issue 41, 38827-38837, October 11, 2002
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C/EBPgamma Has a Stimulatory Role on the IL-6 and IL-8 Promoters*

Hongwei GaoDagger , Sara Parkin§, Peter F. Johnson§, and Richard C. SchwartzDagger

From the Dagger  Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824-4320 and the § Eukaryotic Transcriptional Regulation Section, Regulation of Cell Growth Laboratory, NCI-Frederick, Frederick, Maryland 21702-1201

Received for publication, June 21, 2002, and in revised form, August 9, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CCAAT/enhancer-binding protein gamma  (C/EBPgamma ) is an ubiquitously expressed member of the C/EBP family of transcription factors that has been shown to be an inhibitor of C/EBP transcriptional activators and has been proposed to act as a buffer against C/EBP-mediated activation. We have now unexpectedly found that C/EBPgamma dramatically augments the activity of C/EBPbeta in lipopolysaccharide induction of the interleukin-6 and interleukin-8 promoters in a B lymphoblast cell line. This activating role for C/EBPgamma is promoter-specific, neither being observed in the regulation of a simple C/EBP-dependent promoter nor the TNFalpha promoter. C/EBPgamma activity also shows cell-type specificity with no activity observed in a macrophage cell line. Studies with chimeric C/EBP proteins implicate the formation of a heterodimeric leucine zipper between C/EBPbeta and C/EBPgamma as the critical structural feature required for C/EBPgamma stimulatory activity. These findings suggest a unique role for C/EBPgamma in B cell gene regulation and, along with our previous observation of the ability of C/EBP basic region-leucine zipper domains to confer lipopolysaccharide inducibility of interleukin-6, suggest that the C/EBP leucine zipper domain has a role in C/EBP function beyond allowing dimerization between C/EBP family members.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CCAAT/enhancer-binding protein (C/EBP)1 alpha , beta , gamma , delta , epsilon , and zeta  comprise a family of basic region-leucine zipper (bZIP) transcription factors (reviewed in Ref. 1). These proteins dimerize through their leucine zippers and bind to DNA through their adjacent basic regions. C/EBPalpha , beta , delta , and epsilon  can activate in vivo transcription from promoters that contain a consensus binding site: 5'-T(T/G)NNGNAA(T/G)-3' (2). At this time, the reported in vitro binding activities of C/EBPalpha , beta , gamma , delta , and epsilon  are nearly identical, but the variety of C/EBP isoforms and their potential for heterodimer formation could provide a large repertoire of transcription factors with complex in vivo regulatory features.

C/EBPbeta and C/EBPdelta have been implicated in the regulation of proinflammatory cytokines as well as other gene products associated with the activation of macrophages and the acute phase inflammatory response (reviewed in Ref. 3). For example, the promoter regions of the genes for interleukin-6 (IL-6), IL-1alpha , IL-1beta , IL-8, tumor necrosis factor alpha  (TNFalpha ), granulocyte-colony stimulating factor, inducible nitric-oxide synthase, lysozyme, hemopexin, haptoglobin, alpha 1-acid glycoprotein, serum amyloid A1, A2, A3, complement C3, and C-reactive protein all contain C/EBP binding motifs (3). Furthermore, C/EBPbeta and C/EBPdelta have both been shown to activate a reporter gene controlled by the IL-6 promoter in transient expression assays (2, 4). We have previously demonstrated that the stable expression of C/EBPalpha , beta , delta , and epsilon  in a B lymphoblast cell line is sufficient to confer lipopolysaccharide (LPS) inducibility of IL-6 and monocyte chemoattractant protein 1 (MCP-1) expression (5-7). The basis for this redundancy among C/EBP isoforms lies with the requirement of only the well-conserved C/EBP bZIP domain for this activity (8).

We have found that C/EBPbeta is overwhelmingly present as a heterodimer with C/EBPgamma in B lymphoblasts dependent upon C/EBPbeta for LPS-induced IL-6 expression (8, 9). C/EBPgamma is most highly expressed in immature B cells, although its expression is rather ubiquitous (9, 10). Its binding specificity is similar to that of other C/EBP family members (10), but it has a truncated structure. C/EBPgamma lacks known activation domains and is essentially a C/EBP bZIP domain (11). Consistent with this structure, it has been shown to inhibit C/EBP transcriptional activators (9, 11) and has been proposed to act as a "buffer" for C/EBP activators (11). In this model, C/EBPgamma prevents the activation of C/EBP-dependent gene expression under conditions where the abundance of classical C/EBP activators is low. Activation of C/EBP-dependent genes would occur only when the abundance of C/EBPalpha , beta , delta , and epsilon  exceeded a threshold. It has been proposed that the predominance of C/EBPgamma over C/EBPbeta in early B cells prevents transcription of C/EBP-dependent genes, whereas increased expression of C/EBPbeta in mature cells, or in cells stimulated by proinflammatory cytokines or LPS, is permissive for expression (12).

Contrary to the notion of C/EBPgamma as an inhibitor, there have been studies suggesting an activation function for C/EBPgamma . An activating role for C/EBPgamma has been reported in transcription from immunoglobulin heavy chain promoters (13, 14). C/EBPgamma has also been implicated in beta -globin (15) and pp52 (16) gene expression. Whether C/EBPgamma functions as an activator or an inhibitor, both its lack of expression and overexpression have consequences in vivo. C/EBPgamma -deficient mice have defects in natural killer cell cytotoxic activity and interferon gamma  production (17). Moderate erythroid overexpression of C/EBPgamma in transgenic mice increases gamma -globin expression relative to beta -globin, while high level expression blocks erythropoiesis (18).

Our observation that heterodimers between C/EBPbeta and C/EBPgamma predominate in lymphoblasts dependent upon C/EBPbeta for LPS-induced IL-6 expression (8, 9), as well as the widespread occurrence of C/EBPbeta :gamma heterodimers (9), led us to further explore the role of C/EBPgamma in regulating IL-6 transcription. In this report, we have unexpectedly found that C/EBPgamma dramatically augments the activity of C/EBPbeta in LPS induction of IL-6 in a B lymphoblast cell line. This activating role for C/EBPgamma is promoter-specific, being observed for the IL-6 and IL-8 promoters, but neither for a simple C/EBP-dependent promoter nor the TNFalpha promoter. C/EBPgamma activity also shows cell type-specificity, with stimulatory activity in a B lymphoblast and no effect in a macrophage cell line. Studies with chimeric C/EBP proteins implicated the formation of a heterodimeric leucine zipper between C/EBPbeta and C/EBPgamma as the critical structural feature required for C/EBPgamma stimulatory activity. Our current findings suggest a unique role for C/EBPgamma in B cell gene regulation and, along with our previous observation of the ability of C/EBP bZIP domains to confer LPS inducibility of IL-6, suggest that the C/EBP leucine zipper domain has a role in C/EBP function beyond allowing dimerization between C/EBP family members.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells and Cell Culture-- P388 cells are murine B lymphoblasts (19) (American Type Culture Collection; CCL 46). P388-Cbeta cells and P388-Neo cells have been described previously by Hu et al. (6). WEHI-231 cells are murine B cells (20) (American Type Culture Collection; CRL 1702). P388D1(IL1) cells are macrophages (19) (American Type Culture Collection; TIB 63). P388 cells and their derivatives were cultured in RPMI 1640 medium supplemented with 5% fetal calf serum and 50 µM beta -mercaptoethanol. WEHI-231 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and 50 µM beta -mercaptoethanol. P388D1(IL1) cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum. All IL-6 inductions were conducted with LPS derived from Escherichia coli serotype 055:B5 (Sigma) added to 10 µg/ml.

Transfections-- Transient transfections were conducted with 2 × 106 cells, 4 µg of DNA, and 8 µl of DMRIE-C reagent (Invitrogen) in 1.2 ml of Opti-MEM I medium (Invitrogen). The DNA was comprised of 1 µg of a promoter-reporter, C/EBP expression vector, and pMEX plasmid to total 4 µg. The quantities of C/EBP expression vectors are as indicated in the figure legends. Cells were incubated in the transfection mixture for 5 h followed by the addition of RPMI 1640 medium supplemented to 15% with fetal calf serum. After 24 h, the medium of certain transfections was supplemented with 10 µg/ml LPS. After 4 h in the presence or absence of LPS, transfected cells were harvested, lysed, and analyzed for luciferase activity by using the Luciferase Reporter Gene Assay Kit (Roche Molecular Biochemicals) and for beta -galactosidase activity by using the Luminescent beta -galactosidase Genetic Reporter System II (Clontech).

Expression Vectors and Promoter-Reporters-- For transient transfections, C/EBPs were expressed from pMEX (21), which utilizes the Moloney murine sarcoma virus promoter. NF-kappa B p65 was expressed form pRc/CMV (Invitrogen), which utilizes the cytomegalovirus promoter (from N. Rice, NCI-Frederick). C/EBPbeta -GCNLZ has been described previously (22). C/EBPgamma -Delta Nco was constructed by religating pMEX-C/EBPgamma after restriction digestion with NcoI. C/EBPgamma -beta LZ was constructed by introducing an XhoI site at nucleotide position 283 in the C/EBPgamma gene by site-directed mutagenesis. The XhoI-HindIII fragment bearing the leucine zipper was removed from this pMEX-C/EBPgamma plasmid and replaced with an analogous fragment (nucleotides 703-831) from a rat C/EBPbeta vector in which an XhoI site had been inserted between the basic region and leucine zipper. The forms of C/EBPbeta and C/EBPgamma used in this manuscript are depicted in Fig. 1.


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  		Fig. 1.   Diagram of the major C/EBP isoforms and mutants used in this study.

The IL-6 promoter-reporter consists of the murine IL-6 promoter (23) (-250 to +1) inserted into the luciferase vector, pXP2 (24). DEI4(-35alb)LUC (21) is also derived from pXP2 (24) and contains four copies of the DEI element upstream of the albumin minimal promoter. The TNFalpha promoter-reporter contains sequences extending to -1260 of the TNFalpha promoter inserted into the luciferase vector, pXP1 (23). The IL-8 promoter-reporter contains sequences extending from +44 to -133 inserted into pGL3-basic (Promega) (25-27). The SV40 early promoter-reporter is a commercial product, pbeta gal-Control (Clontech), where the SV40 early promoter and enhancer sequences are cloned upstream and downstream, respectively, of the lacZ gene.

RNA Isolation and Analysis-- Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's directions. RNAs were electrophoresed through 1% agarose/formaldehyde gels. Transfers to membranes were hybridized and washed to high stringency in 40 mM sodium phosphate/1% SDS/1 mM EDTA at 65 °C. Hybridization probes were prepared with a random priming kit (Invitrogen) with the incorporation of 5'-[alpha -32P]dATP (3000 Ci/mmol; PerkinElmer Life Sciences). The IL-6 probe was a 0.65 kb murine cDNA (from N. Jenkins and N. Copeland, NCI-Frederick). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was a 1.3 kb rat cDNA (28).

Western Analysis-- Nuclear extracts were prepared as described below. The extracts (50 µg) were adjusted to 1× Laemmli sample buffer (29) and processed by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis. The gel was transferred to Protran membrane (Schleicher and Schuell), and antigen-antibody complexes were visualized with the Enhanced Chemiluminescence Kit (Amersham Biosciences).

Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear extracts were prepared as follows. Cells were washed in phosphate-buffered saline and lysed in 15 mM KCl, 10 mM HEPES (pH 7.6), 2 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 0.1% (v/v) Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml leupeptin, 5 µg/ml antipain, and 5 µg/ml aprotinin for 10 min on ice. Nuclei were pelleted by centrifugation at 14,000 × g for 20 s at 4 °C. Proteins were extracted from nuclei by incubation at 4 °C with vigorous vortexing in buffer C (420 mM NaCl, 20 mM HEPES (pH 7.9), 0.2 mM EDTA, 25% (v/v) glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml leupeptin, 5 µg/ml antipain, and 5 µg/ml aprotinin). Nuclear debris was pelleted by centrifugation at 14,000 × g for 15 min at 4 °C, and the supernatant extract was collected and stored at -70 °C.

The EMSA probes were double-stranded oligonucleotides containing an optimal C/EBP binding site (5'-GATCCTAGATATCCCTGATTGCGCAATAGGCTCAAAGCTG-3' annealed with 5'-AATTCAGCTTTGAGCCTATTGCGCAATCAGGGATATCTAG-3'), a murine IL-6 C/EBP binding site (5'-CTAAACGACGTCACATTGTGCAATCTTAATAAGGTT-3' annealed with 5'-TGGAAACCTTATTAAGATTGCACAATGTGACGTCGT-3'), and a murine albumin DEI binding site (5'-TCGACTATGATTTTGTAATGGGGC-3' annealed with 5'-TCGAGCCCCATTACAAAATCATAG-3'). These probes were labeled with the incorporation of 5'-[alpha -32P]dATP (3000 Ci/mmol; PerkinElmer Life Sciences) and Klenow DNA polymerase. Underlined sequences correspond to the C/EBP binding motifs.

DNA binding reactions were performed at room temperature in a 25-µl reaction mixture containing 6 µl of nuclear extract (1 mg/ml in buffer C) and 5 µl of 5× binding buffer (20% (w/v) Ficoll, 50 mM HEPES pH 7.9, 5 mM EDTA, 5 mM dithiothreitol). The remainder of the reaction mixture contained KCl to a final concentration of 50 mM, Nonidet P-40 to a final concentration of 0.1%, 1 µg of poly(dI-dC), 200 pg of probe (unless otherwise noted), bromphenol blue to a final concentration of 0.06% (w/v), and water to volume. For supershifts, nuclear extracts were preincubated with antibodies for 30 min at 4 °C prior to the binding reaction. Samples were electrophoresed through 5.5% polyacrylamide gels in 1× TBE (90 mM Tris base, 90 mM boric acid, 0.5 mM EDTA) at 160 V.

Antibodies-- Rabbit antibodies specific to the carboxyl terminus of C/EBPgamma and the amino terminus of C/EBPgamma were prepared against synthetic peptides corresponding to these sequences (9). Rabbit anti-C/EBPalpha (14AA), rabbit anti-C/EBPbeta specific to the carboxyl terminus (C-19), rabbit anti-C/EBPdelta (C-22), rabbit anti-C/EBPepsilon (C-22) and normal rabbit IgG were purchased from Santa Cruz Biotechnology. Rabbit anti-C/EBPbeta specific to the amino terminus has been described (21).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

C/EBPbeta Heterodimerizes with C/EBPgamma in B Cell Lines-- In our previous studies, we found C/EBPbeta to be predominantly in heterodimers with C/EBPgamma in P388 B cells that are dependent upon transfected C/EBPbeta expression for LPS induction of IL-6 and MCP-1 (Refs. 8 and 9; Fig. 2A). We also found both C/EBPbeta and C/EBPdelta to be in heterodimers with C/EBPgamma in WEHI-231, a B cell line that has been used in several studies of IL-6 expression (30-33). In these cells, LPS-induced IL-6 expression was associated with induction of C/EBPbeta :gamma and C/EBPdelta :gamma heterodimers (Fig. 2, B and C). In order to further test the entry of C/EBPbeta into C/EBPbeta :gamma heterodimers, a C/EBPbeta expression vector was transiently transfected into P388 cells over a range of quantities, including those that effectively transactivated the IL-6 promoter following LPS stimulation (see Fig. 3A). EMSA of nuclear extracts of the transfected cells revealed that C/EBPbeta :gamma heterodimers were the predominant binding species at all quantities tested (Fig. 3C). Apparently, C/EBPbeta :gamma heterodimers formed at the expense of C/EBPgamma homodimers at lower quantities of vector (Fig. 3C; 0.5, 1, 2 µg). C/EBPbeta homodimers were observed only at higher vector quantities, where C/EBPgamma homodimers were no longer observable (Fig. 3C; 2, 4, 6, 8, 12 µg). The fact that the major C/EBP species observed with LPS-stimulation were C/EBPbeta :gamma heterodimers is inconsistent with an inhibitory role for C/EBPgamma in the LPS induction of IL-6 expression.


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  		Fig. 2.   C/EBPbeta forms heterodimers with C/EBPgamma in B lymphoblasts. A, EMSA was performed using nuclear extracts of P388-Neo and P388-Cbeta cells. Binding reactions included normal rabbit IgG (N), carboxyl terminus-specific anti-C/EBPbeta (beta ), or carboxyl terminus-specific anti-C/EBPgamma (gamma ). Arrows labeled beta :beta , gamma :gamma , and beta :gamma indicate the positions of C/EBP·DNA complexes. Arrows on the right indicate supershifts. The major C/EBPbeta complex is shifted by both C/EBPbeta -specific and C/EBPgamma -specific antibodies. B, a Northern blot of RNA samples isolated from a time course of LPS treatment upon WEHI 231 B cells was successively hybridized for IL-6 and GAPDH. C, EMSA was performed using nuclear extracts of WEHI 231 cells that were untreated or LPS-treated for 24 h. Binding reactions included normal rabbit IgG (N), anti-C/EBPalpha (alpha ), carboxyl terminus-specific anti-C/EBPbeta (beta ), anti-C/EBPdelta (delta ), anti-C/EBPepsilon (epsilon ), carboxyl terminus-specific anti-C/EBPgamma (gamma ), or anti-C/EBPbeta and anti-C/EBPdelta (beta +delta ). Arrows labeled beta :gamma , delta :gamma , LIP:gamma and gamma :gamma indicate the positions of C/EBP·DNA complexes. Arrows on the right indicate supershifts. The C/EBPbeta and LIP (a truncated form of C/EBPbeta consisting of amino acids 132-276) complexes are supershifted by both C/EBPbeta -specific and C/EBPgamma -specific antibodies. The C/EBPdelta complex is supershifted by both C/EBPdelta -specific and C/EBPgamma -specific antibodies.


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  		Fig. 3.   C/EBPbeta is a more potent activator of LPS-induced IL-6 transcription under conditions of added C/EBPgamma expression, and C/EBPgamma stimulates LPS-induced IL-6 transcription when expressed with C/EBPbeta . Furthermore, C/EBPbeta preferentially forms heterodimers with C/EBPgamma . A, transient transfections were carried out in duplicate with (+gamma) and without (-gamma) 0.5 µg of C/EBPgamma vector, with the microgram quantities of C/EBPbeta vector and LPS treatment as indicated. Luminometer values were normalized for expression from a co-transfected SV40 early promoter beta -galactosidase reporter. These values were then normalized to a relative value of 1 for cells receiving neither a C/EBP expression vector nor LPS. The data presented are the mean of three experiments ± S.E. B, transient transfections of P388 cells were carried out in duplicate with microgram quantities of expression vectors and LPS treatment as indicated. Luminometer values were normalized for expression from a cotransfected SV40 early promoter-beta -galactosidase reporter. These values were then normalized to a relative value of 1 for the cells receiving C/EBPbeta expression vector and treated with LPS. The data presented are the mean of seven experiments ± S.E. C, EMSA was performed using nuclear extracts of P388 cells transiently transfected with increasing quantities (0, 0.5, 1, 2, 4, 6, 8, and 12 µg) of pMEX-C/EBPbeta . The EMSA of the 12-µg transfectants also was performed with binding reactions that included normal rabbit IgG (N), carboxyl terminus-specific anti-C/EBPbeta (beta ), or carboxyl terminus-specific anti-C/EBPgamma (gamma ). Arrows labeled beta :beta , beta :gamma , and gamma :gamma indicate the positions of C/EBP·DNA complexes. Arrows on the right also indicate supershifts. The C/EBPbeta ·beta complex is supershifted by only C/EBPbeta -specific antibody, the C/EBPgamma ·gamma complex by only C/EBPgamma -specific antibody, and the C/EBPbeta ·gamma complex by both C/EBPbeta -specific and C/EBPgamma -specific antibodies. A weak, nonspecific background species co-migrating with C/EBPbeta ·gamma is evident in the 0-µg lane. D, EMSA was performed using nuclear extracts of P388 cells transiently transfected with pMEX control vector, 0.25 µg of pMEX-C/EBPbeta , and 0.25 µg of pMEX-C/EBPbeta with increasing quantities (0, 0.1, 0.25, 0.5, 1, 2, and 4 µg) of pMEX-C/EBPgamma . The EMSA of the 4-µg pMEX-C/EBPgamma transfectants also was performed with binding reactions that included normal rabbit IgG (N), carboxyl terminus-specific anti-C/EBPbeta (beta ), or carboxyl terminus-specific anti-C/EBPgamma (gamma ). Arrows labeled beta :gamma and gamma :gamma indicate the positions of C/EBP·DNA complexes. Arrows on the right also indicate supershifts. The C/EBPgamma ·gamma complex is supershifted by only C/EBPgamma -specific antibody and the C/EBPbeta ·gamma complex by both C/EBPbeta -specific and C/EBPgamma -specific antibodies. Two unidentified slower migrating species that are not modulated by transfection and are reactive with C/EBPgamma -specific antibody are evident in control and experimental lanes. E, EMSA was performed using nuclear extracts of P388 cells transiently transfected with pMEX control vector, 2 µg of pMEX-C/EBPbeta , or 2 µg of pMEX-C/EBPbeta plus 0.5 µg pMEX-C/EBPgamma . Arrows labeled beta :beta , beta :gamma , and gamma :gamma indicate the positions of C/EBP·DNA complexes. The radioactivity associated with C/EBPbeta homodimers and C/EBPbeta ·gamma heterodimers was quantitated using a Storm PhosphorImager (Molecular Dynamics), and the ratio of C/EBPbeta ·gamma to C/EBPbeta ·beta is shown.

C/EBPgamma Augments C/EBPbeta -stimulated Transcription of the IL-6 Promoter-- C/EBPgamma by itself is clearly not an activator of the IL-6 promoter because its presence in P388 cells is not sufficient to allow LPS induction of IL-6. However, our observations suggested that C/EBPgamma -containing heterodimers might activate the IL-6 promoter in LPS-stimulated cells. To test this notion, we performed transient transfections of increasing quantities of C/EBPbeta vector with and without added expression of C/EBPgamma (Fig. 3A). C/EBPgamma augmented LPS-induced expression from the IL-6 promoter at all quantities of C/EBPbeta expression vector used. This is very surprising for a factor generally believed to be a transdominant inhibitor of C/EBP activators (10). If C/EBPgamma acted as an inhibitor, C/EBPbeta would be expected to induce less luciferase expression in the presence of added C/EBPgamma , rather than more luciferase expression. In fact, 0.5 µg of C/EBPbeta vector with 0.5 µg of C/EBPgamma vector is twice as effective as 1 µg of C/EBPbeta vector alone. This is consistent with C/EBPbeta :gamma heterodimers being more potent activators than C/EBPbeta homodimers. Presumably, overexpression of C/EBPgamma drives more C/EBPbeta into heterodimers than would occur at endogenous levels of C/EBPgamma expression. When EMSA was performed upon nuclear extracts prepared from P388 cells transiently transfected with C/EBPbeta expression vector with and without added C/EBPgamma expression vector, a higher ratio of C/EBPbeta :gamma heterodimer to C/EBPbeta homodimer is indeed observed in cells transfected with C/EBPgamma expression vector (2.2 as opposed to 1.3) (Fig. 3E). To further test the ability of C/EBPgamma to promote formation of C/EBPbeta :gamma heterodimers, a constant quantity of C/EBPbeta expression vector was transiently transfected into P388 cells with and without C/EBPgamma expression vector over a range of quantities including those that effectively transactivated the IL-6 promoter following LPS stimulation (see Fig. 3A). An EMSA of nuclear extracts of the transfected cells revealed that C/EBPbeta :gamma heterodimers became apparent and increased in abundance with increasing quantities of C/EBPgamma (Fig. 3D).

The stimulatory effects of C/EBPgamma were also observed in transient transfections where increasing amounts of C/EBPgamma expression vector were added to a constant amount of C/EBPbeta expression vector. These transfections were performed with LPS stimulation, and the expression vectors were cotransfected with an IL-6 promoter-reporter. C/EBPgamma clearly augmented the ability of C/EBPbeta to mediate LPS induction of the IL-6 promoter (Fig. 3B). C/EBPgamma activity was observed even when the C/EBPgamma vector was transfected at a 8-fold excess over C/EBPbeta vector, although C/EBPgamma by itself exhibited no activity (data not shown). Our results therefore suggest that C/EBPgamma , rather than functioning as an inhibitor to low levels of C/EBPbeta activity, actually augments that activity on the IL-6 promoter.

In contrast to the stimulatory effects observed when C/EBPgamma was cotransfected with C/EBPbeta in LPS-induced IL-6 expression, C/EBPgamma actually inhibited the modest activation of the IL-6 promoter that can be observed by transfection of C/EBPbeta without LPS stimulation (Fig. 4). This inhibition was reversed by cotransfection with NF-kappa B p65, allowing dosage-dependent C/EBPgamma stimulatory activity in the absence of LPS stimulation (Fig. 4). The lowest quantity of p65 vector used in the cotransfection (0.05 µg) potentiated robust stimulation by C/EBPgamma . These data support the notion that C/EBPgamma may play a key role in the synergy between C/EBPbeta and NF-kappa B.


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  		Fig. 4.   C/EBPgamma inhibits C/EBPbeta -induced IL-6 transcription in the absence of LPS treatment, while that inhibition is reversed by NF-kappa B p65 expression. Transient transfections of P388 cells were carried out in duplicate with the microgram quantities of expression vectors as indicated. Luminometer values were normalized as in Fig. 3B, except final values were normalized to a relative value of 1 for cells not receiving C/EBPgamma expression vector. The data presented for cells receiving C/EBPbeta but no NF-kappa B p65 expression vector are the means of three experiments ± S.E. The data for the cells receiving both C/EBPbeta and NF-kappa B p65 expression vectors are derived from one experiment carried out at various doses of p65 vector.

It is possible that the C/EBPgamma expressed from our expression vector differed from endogenous C/EBPgamma in its ability to stimulate IL-6 transcription. Furthermore, other investigators who found that C/EBPgamma acted as an inhibitor of C/EBP transactivation performed their studies in the absence of LPS stimulation. Perhaps, LPS leads to the modification of C/EBPgamma into a form capable of transactivation. To test these possibilities, transient transfections were performed with the C/EBPgamma expression vector by itself with the IL-6 promoter-reporter. No stimulation of the IL-6 promoter above that induced by LPS stimulation alone was observed over a range of C/EBPgamma expression vector amounts comparable to that used in the transient transfections where C/EBPgamma stimulatory activity was observed (data not shown). Thus C/EBPgamma has no stimulatory activity by itself, even in the presence of LPS treatment.

C/EBPgamma Stimulatory Activity Shows Both Promoter and Cell-type Specificity-- In order to test whether the presence of a C/EBP binding site is sufficient for the stimulatory activity of C/EBPgamma , we performed transient transfections with DEI4(-35alb)LUC, a promoter-reporter that contains four copies of a C/EBP binding site tandemly arrayed upstream of the albumin minimal promoter (Fig. 5A). This simple C/EBP reporter failed to show any stimulation by C/EBPgamma expression suggesting that a more complex promoter is required for stimulatory activity. We then performed transient transfections with the TNFalpha and IL-8 promoters (Fig. 5A). These promoters, like IL-6, are in part regulated by NF-kappa B and C/EBP. The TNFalpha promoter does not display synergy between NF-kappa B and C/EBPbeta (34), while the IL-8 promoter shows strong synergy between these two factors (35-37). Consistent with a possible role in the synergy between NF-kappa B and C/EBPbeta , C/EBPgamma expression had little effect upon the TNFalpha promoter, but displayed even more stimulation of the IL-8 promoter than was observed for the IL-6 promoter. In contrast to the promoter specificity observed for C/EBPgamma , C/EBPbeta was stimulatory for all of the promoters tested (Fig. 5A, compare control cells treated with LPS to cells treated with LPS and cotransfected with C/EBPbeta ). Furthermore, C/EBPgamma stimulatory activity does not appear to be dependent upon differential binding of C/EBPgamma to differing C/EBP binding sites. Both the IL-6 and DEI C/EBP binding motifs bound C/EBPgamma -containing species in EMSA performed upon nuclear extracts from P388 cells overexpressing C/EBPbeta (Fig. 5B), while neither the TNFalpha nor the IL-8 C/EBP binding motifs detectably bound any C/EBP species under the same conditions (data not shown). The ability of C/EBPgamma to stimulate transcription does not seem to correlate with its avidity for specific C/EBP binding motifs, but rather depends upon more complex aspects of promoter structure such as those that determine synergy between transcription factors. The stimulatory activity of C/EBPgamma is thus promoter specific, requires a complex promoter to be observed, and may function in the synergistic activation of promoters by NF-kappa B and C/EBP family members.


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  		Fig. 5.   C/EBPgamma stimulates LPS-induced transcription from the IL-8 promoter but is inactive for the TNFalpha promoter and a simple C/EBP-driven promoter. A, transient transfections of P388 cells were carried out in duplicate with the microgram quantities of expression vector and LPS treatment as indicated. Luminometer values were normalized as in Fig. 3B. The data presented for the IL-8, TNFalpha , and DEI4(-35alb) promoters are means of three, three, and five experiments, respectively, with S.E. The data for the IL-6 promoter from Fig. 3B are presented for comparison. B, EMSA was performed using nuclear extracts from P388-Cbeta cells and labeled binding site oligonucleotides corresponding to the C/EBP consensus binding site, the IL-6 promoter C/EBP binding site, and the DEI albumin C/EBP binding site. Binding reactions included normal rabbit IgG (N), carboxyl terminus-specific anti-C/EBPbeta (beta ), or carboxyl terminus-specific anti-C/EBPgamma (gamma ). Arrows labeled beta :beta , gamma :gamma , and beta :gamma indicate the positions of C/EBP·DNA complexes.

The fact that C/EBPgamma is most prominently expressed in cells of the B lymphoid lineage (10) led us to ask if its stimulatory activity was unique to that cell type or could be observed in another cell lineage that displays LPS-inducible IL-6 expression. To test this, we utilized P388D1(IL1) macrophages. This cell line is actually a derivative of the original P388 B lymphoblast tumor (19). Only a relatively low proportion of C/EBPbeta ·DNA complexes from these cells are supershifted by anti-C/EBPgamma in an EMSA (Ref. 9; data not shown). LPS is a potent inducer of IL-6 expression in this cell line (data not shown). Transient transfections were performed where increasing amounts of C/EBPgamma expression vector were added to a constant amount of C/EBPbeta expression vector. These transfections were performed with LPS stimulation, and the expression vectors were cotransfected with an IL-6 promoter-reporter. In contrast to P388 B cells where C/EBPgamma clearly augmented the ability of C/EBPbeta to mediate LPS induction of the IL-6 promoter, C/EBPgamma had no effect on C/EBPbeta stimulation of LPS-induced IL-6 expression in P388D1(IL1) cells (data not shown). Thus in addition to promoter specificity, the stimulatory activity of C/EBPgamma shows cell-type specificity.

C/EBPgamma Stimulatory Activity Requires Heterodimerization with C/EBPbeta -- We next sought to test whether C/EBPgamma stimulatory activity in transfections with C/EBPbeta requires heterodimer formation between these two proteins. To that end, we performed transient transfections with a chimeric C/EBPbeta containing the leucine zipper of yeast GCN4. In comparison to intact C/EBPbeta , C/EBPbeta -GCN4LZ (Fig. 1) can activate transcription at a reduced level from an albumin DEI site-driven reporter (22), as well as the IL-6 promoter-reporter in conjunction with LPS treatment (Fig. 6C; see controls), and is unable to heterodimerize with C/EBPgamma in vitro or in vivo (9). The heterologous leucine zipper prevents heterodimerization, but allows the chimeric protein to homodimerize. To verify expression, DNA binding, and the heterodimeriztion properties of C/EBPbeta -GCN4LZ, Western blot analysis and EMSA were performed using nuclear extracts of transiently transfected cells (Fig. 6, A and B). Western analysis of nuclear extracts from P388 cells transfected with increasing quantities of C/EBPbeta -GCN4LZ expression vector detected increasing quantities of a C/EBP-related protein at the expected molecular mass of ~38 kDa (Fig. 6A). As can be seen in an EMSA of the same nuclear extracts, the overexpression of C/EBPbeta -GCN4LZ fails to drive C/EBPgamma into heterodimers (Fig. 6B), in contrast to C/EBPbeta (Fig. 3C). The major EMSA species associated with transfection of the C/EBPbeta -GCN4LZ expression vector could be supershifted with antibody specific to the amino terminus of C/EBPbeta , but not with antibody specific to the carboxyl terminus of C/EBPbeta as would be expected for replacement of the carboxyl terminus (Fig. 6B). Furthermore, this EMSA species could not be supershifted with antibody specific to the carboxyl terminus of C/EBPgamma , indicating a lack of dimerization with C/EBPgamma . Transient transfection of increasing amounts of C/EBPgamma expression vector with a constant amount of C/EBPbeta -GCN4LZ expression vector were carried out in comparison to increasing amounts of C/EBPgamma expression vector with a constant amount of C/EBPbeta expression vector (Fig. 6C). The ability of C/EBPgamma to augment C/EBPbeta activity was largely blocked by the GCN4 leucine zipper. This is consistent with C/EBPgamma stimulatory activity being dependent on its ability to dimerize with C/EBPbeta . The fact that C/EBPbeta -GCN4LZ by itself supports LPS induction of the IL-6 promoter indicates that while C/EBPgamma can augment C/EBPbeta activity, formation of heterodimers containing C/EBPgamma is not necessary for C/EBP activity on the IL-6 promoter.


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  		Fig. 6.   C/EBPgamma stimulatory activity is dependent upon the formation of C/EBPbeta :gamma heterodimers. The replacement of the C/EBP leucine zipper in C/EBPbeta with that of GCN4 blocked C/EBPgamma activity. A, a Western blot was performed using nuclear extracts of P388 cells transiently transfected with increasing quantities (0, 0.5, 1, 2, 4, 6, 8, and 12 µg) of pMEX-C/EBPbeta -GCN4LZ. The primary antibody used in the detection of C/EBPbeta -GCN4LZ was amino terminus-specific anti-C/EBPbeta . An arrow marks the position of C/EBPbeta -GCN4LZ. The positions of protein standards are noted. B, EMSA was performed using nuclear extracts of P388 cells transiently transfected with increasing quantities (0, 0.5, 1, 2, 4, 6, 8, and 12 µg) of pMEX- C/EBPbeta -GCN4LZ. The EMSA of the 12-µg transfectants was also performed with binding reactions that included normal rabbit IgG (N), amino terminus-specific anti-C/EBPbeta (Nbeta ), carboxyl terminus-specific anti-C/EBPbeta (Cbeta ), or carboxyl terminus-specific anti-C/EBPgamma (Cgamma ). Arrows labeled beta -GCN4LZ, beta :beta , beta :gamma , and gamma :gamma indicate the positions of C/EBP·DNA complexes. Arrows on the right indicate supershifts. The C/EBPbeta -GCN4LZ complex is only supershifted by amino terminus-specific anti-C/EBPbeta , while the C/EBPbeta complexes from P388-Cbeta are supershifted by all of the specific antisera. Weak, nonspecific background species co-migrating with C/EBPbeta :beta and C/EBPbeta :gamma are evident in the 0-µg lane. C, transient transfections of P388 cells were carried out in duplicate with the microgram quantities of expression vectors and LPS treatment as indicated. Luminometer values were normalized as in Fig. 3. The data for C/EBPbeta -GCN4LZ+C/EBPgamma (beta-GCN4LZ+gamma) are the mean of four experiments ± S.E. The data for C/EBPbeta +C/EBPgamma (beta+gamma) from Fig. 3B are presented for comparison.

C/EBPgamma Stimulatory Activity Resides with Its Leucine Zipper Domain-- We next initiated studies to determine the structural components of C/EBPgamma sufficient for its stimulatory activity. A form of C/EBPgamma deleted for the region amino-terminal to the bZIP domain (Fig. 1; C/EBPgamma -Delta Nco) was compared with intact C/EBPgamma in the same experimental regime as described for Fig. 3B, where increasing amounts of C/EBPgamma expression vector were added to a constant amount of C/EBPbeta expression vector. These transfections were performed with LPS stimulation, and the expression vectors were cotransfected with an IL-6 promoter-reporter. C/EBPgamma -Delta Nco, although lacking the 57-residue amino terminus, had as much stimulatory activity as wild type C/EBPgamma (Fig. 7A). An EMSA species that increased in abundance with increasing quantities of the C/EBPgamma -Delta Nco vector further indicated successful expression of C/EBPgamma -Delta Nco (Fig. 7B). Thus, the amino terminus of C/EBPgamma is unnecessary for its stimulatory activity.


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  		Fig. 7.   The amino-terminal region of C/EBPgamma is not required for stimulatory activity. A, transient transfections of P388 cells were carried out in duplicate with microgram quantities of expression vectors and LPS treatment as indicated. Luminometer values were normalized as described in Fig. 3B. The data for C/EBPbeta +C/EBPgamma -Delta Nco (beta+gamma-Nco) are the mean of three experiments ± S.E. B, EMSA was performed using nuclear extracts of P388 cells transiently transfected with 0, 2, and 4 µg of pMEX-C/EBPgamma -Delta Nco. Binding reactions included normal rabbit IgG (N), amino terminus-specific anti-C/EBPgamma (Ngamma ), or carboxyl terminus-specific anti-C/EBPgamma (Cgamma ). Arrows labeled gamma :gamma -Nco and gamma :gamma indicate the positions of C/EBP·DNA complexes. Arrows on the right indicate supershifts. The C/EBPgamma -Delta Nco complex is supershifted by both amino- and carboxyl terminus-specific anti-C/EBPgamma because it is heterodimeric with wild type C/EBPgamma .

Since C/EBPgamma homodimers by themselves have no stimulatory activity (data not shown) and the ability of C/EBPgamma to heterodimerize with C/EBPbeta appears to be critical for its stimulatory activity (Fig. 6), we tested whether C/EBPgamma activity required the formation of a heterodimeric leucine zipper, a heterodimeric DNA binding domain, or both. To that end, we performed transient transfections with a vector expressing a chimeric C/EBP comprised of a C/EBPgamma amino-terminal and basic region, and a C/EBPbeta leucine zipper (Fig. 1; C/EBPgamma -beta LZ). As a control for C/EBPgamma -beta LZ expression and DNA binding, Western blot analysis and EMSA were performed using nuclear extracts of cells transiently transfected over a range of quantities of the C/EBPgamma -beta LZ expression vector (Fig. 8, A and B). Western analysis with antibody specific to the carboxyl terminus of C/EBPbeta detected increasing quantities of a C/EBP-related protein at the expected molecular mass of ~19 kDa (Fig. 8A). A major EMSA species was detected in proportion to the amount of C/EBPgamma -beta LZ expression vector (Fig. 8B). That species was supershifted with antibodies specific to the carboxyl terminus of C/EBPbeta , the amino terminus of C/EBPgamma , and the carboxyl terminus of C/EBPgamma , but not with antibody specific to the amino terminus of C/EBPbeta (Fig. 8B). This is consistent with a heterodimer between C/EBPgamma -beta LZ and endogenous C/EBPgamma . We tested the ability of C/EBPgamma -beta LZ to support LPS induction of IL-6 with and without transfection of a vector expressing intact C/EBPgamma (Fig. 8C). Surprisingly, in LPS-treated cells, the C/EBPgamma -beta LZ expression vector by itself could support as much as 10-fold induction of the IL-6 promoter and the addition of 0.5 µg of C/EBPgamma expression vector enhanced that stimulatory activity to 20-fold induction. While the stimulatory activity of C/EBPgamma -beta LZ is less than that of intact C/EBPbeta (40-fold for 1 µg of vector without C/EBPgamma and 100-fold with C/EBPgamma ; see Fig. 3A), the degree to which C/EBPgamma augmented C/EBPgamma -beta LZ activity was similar to its enhancement of C/EBPbeta activity (about 2.5-fold). This suggests that C/EBPgamma stimulatory activity resides in formation of a heterodimeric C/EBPbeta :gamma leucine zipper.


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  		Fig. 8.   The formation of a heterodimeric C/EBPbeta :gamma leucine zipper is sufficient for the stimulatory activity of C/EBPg. A, Western blot was performed using nuclear extracts of P388 cells transiently transfected with increasing quantities (0, 0.5, 1, 2, 4, 6, 8, and 12 µg) of pMEX-C/EBPgamma -beta LZ. The primary antibody used in the detection of C/EBPgamma -beta LZ was carboxyl terminus-specific anti-C/EBPbeta . An arrow marks the position of C/EBPgamma -beta LZ. The positions of protein standards are noted. B, EMSA was performed using nuclear extracts of P388 cells transiently transfected with increasing quantities (0, 0.5, 1, 2, 4, 6, 8, and 12 µg) of pMEX- C/EBPgamma -beta LZ. EMSA of P388 cells and the 12-µg transfectants also was performed with binding reactions that included normal rabbit IgG (N), amino terminus-specific anti-C/EBPbeta (Nbeta ), carboxyl terminus-specific anti-C/EBPbeta (Cbeta ), amino terminus-specific anti-C/EBPgamma (Ngamma ), or carboxyl terminus-specific anti-C/EBPgamma (Cgamma ). Arrows labeled gamma :gamma and gamma :gamma -beta LZ indicate the positions of C/EBP·DNA complexes. Arrows on the right indicate supershifts. The C/EBPgamma -beta LZ complex (similar in mobility to the C/EBPgamma complex in P388 cells) is supershifted by carboxyl terminus-specific anti-C/EBPbeta , in addition to the C/EBPgamma -specific antisera. C, transient transfections were carried out in duplicate with and without 0.5 µg of C/EBPgamma vector, with the microgram quantities of C/EBPgamma -beta LZ expression vector and LPS treatment as indicated. Luminometer values were normalized as described in Fig. 3A. The data are the mean of three experiments ± S.E.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The data presented in this study demonstrate an activating role for C/EBPgamma in transcription from the IL-6 and IL-8 promoters in B lymphoid cells. C/EBPgamma , which in other contexts can inhibit activation by C/EBP family members (9, 10), was found to augment the C/EBPbeta -dependent LPS stimulation of IL-6 and IL-8 promoter-reporters in P388 B lymphoblasts. This stimulatory activity of C/EBPgamma is dependent on its formation of heterodimers with C/EBPbeta and, indeed, C/EBPbeta is largely found in heterodimers with C/EBPgamma in P388 B cells that have gained the capacity for LPS-induced IL-6 expression upon transfection of a C/EBPbeta expression vector. Surprisingly, the critical structural feature for this stimulatory activity is the formation of a heterodimeric leucine zipper between C/EBPbeta and C/EBPgamma . C/EBPgamma stimulatory activity was found to be promoter-specific with activity seen on IL-6 and IL-8 promoter-reporters, and not on TNFalpha and albumin DEI promoter-reporters. C/EBPgamma stimulatory activity was also found to be cell-type specific, being observed in P388 B cells, but not in their P388D1(IL-1) macrophage derivative.

The stimulatory activity of C/EBPgamma was surprising, since it is generally accepted as being an inhibitor of C/EBP transcriptional activators (11, 12). However, the same investigators that first demonstrated the inhibitory activity of C/EBPgamma found that immunodepletion of C/EBPgamma from an in vitro transcription assay inhibited the activity of the BCL1 immunoglobulin heavy chain and the Rous sarcoma virus promoters (13). Similarly, C/EBPgamma synergizes with Stat6 and NF-kappa B p50/p65 to induce the germline gamma 3-immunoglobulin promoter in a B cell line (14). C/EBPgamma has also been found to enhance beta -globin gene expression in collaboration with CP-1 (15). Another instance of a positive role for C/EBPgamma has been found in the expression of pp52, a leukocyte-specific phosphoprotein postulated to regulate cytoskeleton structure (16). Thus, the role of C/EBPgamma as a transcriptional activator does not seem unusual. It seems neither inherently an activator nor an inhibitor. Rather, the identity of its promoter context and dimerization partner may be the overriding features that govern the specific role of C/EBPgamma in transcription. Heterodimerization with C/EBPgamma has two effects on the ability of C/EBPbeta to activate the IL-6 promoter: it inhibits C/EBPbeta activity in the absence of LPS and enhances C/EBPbeta transactivation in LPS-stimulated cells. Therefore, we predict that in B cells the net effect of C/EBPgamma is to greatly increase the index of LPS inducibility of the IL-6 promoter. This prediction could be tested in B lineage cells derived from C/EBPgamma -deficient mice (17).

C/EBPgamma stimulatory activity was observed with the IL-6 and IL-8 promoter-reporters, but not with the TNFalpha or the DEI promoter-reporters. One distinguishing characteristic of the IL-6 and IL-8 promoters is synergistic regulation by C/EBPbeta and NF-kappa B (35-37). It is tempting to propose a specific role for C/EBPgamma in promoting this synergy. While the experiments reported here do not provide a direct demonstration for such a mechanism, the findings that C/EBPgamma inhibits C/EBPbeta activation of the IL-6 promoter in the absence of LPS and that this inhibitory effect is converted to a stimulatory effect by NF-kappa B p65 expression (Fig. 4) are consistent with this model. Furthermore, our previous studies found that the activity of C/EBPbeta on the IL-6 promoter was dependent on an intact NF-kappa B site (8). It is, however, unlikely that the stimulatory role of C/EBPgamma is limited to promoters that exhibit synergy between C/EBPbeta and NF-kappa B. Other promoters for which C/EBPgamma stimulatory activity have been suggested, including immunoglobulin heavy chain (13, 14), beta -globin (15), and pp52 (16), do not display synergistic regulation by C/EBPbeta and NF-kappa B.

C/EBPgamma stimulatory activity displays cell-type specificity. This is also the case for the inhibitory activity of C/EBPgamma (9). Stimulatory activity was seen in P388 B cells, but not in their macrophage derivative, P388D1(IL-1) (data not shown). C/EBPgamma is normally a minor component of the C/EBP family members expressed in these macrophages, where C/EBPbeta forms heterodimers with another as yet unidentified protein (9). Perhaps, C/EBPgamma stimulatory activity in P388D1(IL-1) macrophages is precluded by the heterodimerization of C/EBPbeta with this other protein. The activity of C/EBPgamma in specific cell-types may be dependent upon the availability of an appropriate partner for heterodimeriztion. Our studies strongly suggest that heterodimerization is critical for stimulatory activity (Fig. 6C).

The promoter and cell-type specificity of C/EBPgamma activity lead us to speculate that the ability of C/EBPgamma to augment LPS stimulation of IL-6 transcription in B cells may provide a mechanism of autocrine IL-6 production to drive the maturation of B cells, while suppressing or having a neutral effect on other inflammatory cytokines such as TNFalpha . This could be particularly important as a source of IL-6 in a T-independent B cell response. Perhaps C/EBPgamma -deficient mice (17) will exhibit slower kinetics in their B cell response to Gram-negative bacteria.

While we have observed C/EBPgamma stimulatory activity on both the IL-6 and IL-8 promoters, it is interesting to note that no IL-8 orthologue exists in mouse and rat (38, 39). In humans, however, both IL-6 and IL-8 are autocrine factors in myeloma tumor progression (40, 41). It would be interesting to test whether a functional association exists between C/EBPgamma expression and the autocrine production of these cytokines in myelomas.

Although C/EBPgamma is most abundantly expressed in immature B cells (10), we have found C/EBPbeta :gamma and C/EBPdelta :gamma heterodimers to be the predominant form of C/EBP in LPS-stimulated WEHI 231 cells (Fig. 2C), a relatively mature, surface-IgM expressing B cell. The occurrence of C/EBPbeta :gamma heterodimers as a major species has also been observed in glioma, mammary tumor, and hepatoma cell lines, as well as in brain, pancreas, and ovary (9). It will be worthwhile to evaluate whether C/EBPgamma can stimulate target genes that are known to be positively regulated by C/EBPbeta in these cell-types and tissues.

We found that ectopic expression of C/EBPbeta in P388 cells led to the formation of C/EBPbeta :gamma heterodimers at the expense of C/EBPgamma homodimers, while C/EBPbeta homodimers were observed only at the highest levels of C/EBPbeta expression (Fig. 3C). This may indicate a preference for heterodimeriztion between these C/EBP family members. This result cannot be explained by large pools of either monomeric C/EBPgamma or unbound C/EBPgamma dimers being available for dimerization with C/EBPbeta . If this were the case, C/EBPgamma homodimers would not be eliminated as they are by C/EBPbeta expression. However, His-tagged recombinant forms of these proteins do not show preferential dimerization (9). It is possible that post-translational modifications of these C/EBP family members regulate their dimerization.

Perhaps, the most surprising result reported here is the ability of a chimeric C/EBP consisting of C/EBPgamma with the leucine zipper of C/EBPbeta to stimulate the IL-6 promoter in cells that express only endogenous C/EBPgamma (Fig. 8). Since C/EBPgamma by itself is unable to support LPS induction of the IL-6 promoter (data not shown), this result demonstrates that the formation of a C/EBPbeta :gamma heterodimeric zipper in the absence of any conventional activation domains to sufficient to support LPS induction of the IL-6 promoter. This is consistent with our earlier finding that expression of the bZIP domains of C/EBPbeta , delta , or alpha  was sufficient to confer LPS inducibility to the IL-6 promoter in P388 cells (8). In those studies, we found that the C/EBPbeta bZIP domain was largely dimerized with C/EBPgamma and that activity required an intact NF-kappa B binding site. We have now found that C/EBPgamma stimulatory activity is observed on two promoters that show synergy between C/EBP and NF-kappa B and that C/EBPgamma expression actually becomes inhibitory in the absence of NF-kappa B expression (Fig. 4). Our findings are consistent with the C/EBP leucine zipper being a critical determinant in facilitating the synergy between NF-kappa B and C/EBP family members that is observed for several genes encoding cytokines and class I acute phase proteins including IL-6, IL-8, IL-12, granulocyte-colony stimulating factor, IL-1beta , serum amyloid A1, A2, A3, and alpha 1-acid glycoprotein (3). Functions other than dimerization have been demonstrated for leucine zipper domains. In the C/EBP family, the leucine zipper of C/EBPalpha mediates cell-type specificity of albumin promoter activation (42) and phosphorylation of serine 276 in the leucine zipper of human C/EBPbeta confers calcium-regulated transcriptional stimulation to a promoter that contains binding sites for C/EBPbeta (43). Recently, the leucine zipper of transcription factor v-Myb has been found to regulate the commitment of hematopoietic progenitors (44). Mutation of the leucine zipper can alter the transforming potential of v-Myb from the macrophage lineage to the erythroid and granulocytic lineages. It is tempting to speculate that the leucine zipper of C/EBP family transcription factors interacts differentially with other transcription factors such as NF-kappa B or with coactivators of transcription in a manner dependent upon leucine zipper dimerization partners.

    FOOTNOTES

* This research was supported by Grant-in-aid 9950490N (to R. C. S.) from the American Heart Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824-4320. Tel.: 517-355-6463, extension 1527; Fax: 517-353-8957; E-mail: schwart9@msu.edu.

Published, JBC Papers in Press, August 12, 2002, DOI 10.1074/jbc.M206224200

    ABBREVIATIONS

The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; bZIP, basic region-leucine zipper; IL, interleukin; TNFalpha , tumor necrosis factor alpha ; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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