C/EBPγ Has a Stimulatory Role on theIL-6andIL-8Promoters

CCAAT/enhancer-binding protein γ (C/EBPγ) 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/EBPγ dramatically augments the activity of C/EBPβ in lipopolysaccharide induction of the interleukin-6 and interleukin-8 promoters in a B lymphoblast cell line. This activating role for C/EBPγ is promoter-specific, neither being observed in the regulation of a simple C/EBP-dependent promoter nor the TNFαpromoter. C/EBPγ 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/EBPβ and C/EBPγ as the critical structural feature required for C/EBPγ stimulatory activity. These findings suggest a unique role for C/EBPγ 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.

CCAAT/enhancer-binding protein ␥ (C/EBP␥) 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/EBP␥ dramatically augments the activity of C/EBP␤ in lipopolysaccharide induction of the interleukin-6 and interleukin-8 promoters in a B lymphoblast cell line. This activating role for C/EBP␥ is promoter-specific, neither being observed in the regulation of a simple C/EBP-dependent promoter nor the TNF␣ promoter. C/EBP␥ 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/EBP␤ and C/EBP␥ as the critical structural feature required for C/EBP␥ stimulatory activity. These findings suggest a unique role for C/EBP␥ 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.
CCAAT/enhancer-binding protein (C/EBP) 1 ␣, ␤, ␥, ␦, ⑀, and 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/EBP␣, ␤, ␦, and ⑀ can activate in vivo transcription from promoters that contain a consensus binding site: 5Ј-T(T/G)NNGNAA(T/G)-3Ј (2). At this time, the reported in vitro binding activities of C/EBP␣, ␤, ␥, ␦, and ⑀ 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/EBP␤ and C/EBP␦ 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-1␣, IL-1␤, IL-8, tumor necrosis factor ␣ (TNF␣), granulocyte-colony stimulating factor, inducible nitric-oxide synthase, lysozyme, hemopexin, haptoglobin, ␣ 1 -acid glycoprotein, serum amyloid A1, A2, A3, complement C3, and C-reactive protein all contain C/EBP binding motifs (3). Furthermore, C/EBP␤ and C/EBP␦ 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/EBP␣, ␤, ␦, and ⑀ 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)(6)(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/EBP␤ is overwhelmingly present as a heterodimer with C/EBP␥ in B lymphoblasts dependent upon C/EBP␤ for LPS-induced IL-6 expression (8,9). C/EBP␥ 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/EBP␥ 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/EBP␥ 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/EBP␣, ␤, ␦, and ⑀ exceeded a threshold. It has been proposed that the predominance of C/EBP␥ over C/EBP␤ in early B cells prevents transcription of C/EBP-dependent genes, whereas increased expression of C/EBP␤ in mature cells, or in cells stimulated by proinflammatory cytokines or LPS, is permissive for expression (12).
Contrary to the notion of C/EBP␥ as an inhibitor, there have been studies suggesting an activation function for C/EBP␥. An activating role for C/EBP␥ has been reported in transcription from immunoglobulin heavy chain promoters (13,14). C/EBP␥ has also been implicated in ␤-globin (15) and pp52 (16) gene expression. Whether C/EBP␥ functions as an activator or an inhibitor, both its lack of expression and overexpression have consequences in vivo. C/EBP␥-deficient mice have defects in natural killer cell cytotoxic activity and interferon ␥ production (17). Moderate erythroid overexpression of C/EBP␥ in transgenic mice increases ␥-globin expression relative to ␤-globin, while high level expression blocks erythropoiesis (18).
Our observation that heterodimers between C/EBP␤ and C/EBP␥ predominate in lymphoblasts dependent upon C/EBP␤ for LPS-induced IL-6 expression (8,9), as well as the widespread occurrence of C/EBP␤:␥ heterodimers (9), led us to further explore the role of C/EBP␥ in regulating IL-6 transcription. In this report, we have unexpectedly found that C/EBP␥ dramatically augments the activity of C/EBP␤ in LPS induction of IL-6 in a B lymphoblast cell line. This activating role for C/EBP␥ is promoter-specific, being observed for the IL-6 and IL-8 promoters, but neither for a simple C/EBP-dependent promoter nor the TNF␣ promoter. C/EBP␥ 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/EBP␤ and C/EBP␥ as the critical structural feature required for C/EBP␥ stimulatory activity. Our current findings suggest a unique role for C/EBP␥ 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.
Transfections-Transient transfections were conducted with 2 ϫ 10 6 cells, 4 g of DNA, and 8 l of DMRIE-C reagent (Invitrogen) in 1.2 ml of Opti-MEM I medium (Invitrogen). The DNA was comprised of 1 g of a promoter-reporter, C/EBP expression vector, and pMEX plasmid to total 4 g. 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 g/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 ␤-galactosidase activity by using the Luminescent ␤-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-B p65 was expressed form pRc/CMV (Invitrogen), which utilizes the cytomegalovirus promoter (from N. Rice, NCI-Frederick). C/EBP␤-GCN LZ has been described previously (22). C/EBP␥-⌬Nco was constructed by religating pMEX-C/EBP␥ after restriction digestion with NcoI. C/EBP␥-␤ LZ was constructed by introducing an XhoI site at nucleotide position 283 in the C/EBP␥ gene by site-directed mutagenesis. The XhoI-HindIII fragment bearing the leucine zipper was removed from this pMEX-C/EBP␥ plasmid and replaced with an analogous fragment (nucleotides 703-831) from a rat C/EBP␤ vector in which an XhoI site had been inserted between the basic region and leucine zipper. The forms of C/EBP␤ and C/EBP␥ used in this manuscript are depicted in Fig. 1.
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 5Ј-[␣-32 P]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 g) were adjusted to 1ϫ 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).
DNA binding reactions were performed at room temperature in a 25-l reaction mixture containing 6 l of nuclear extract (1 mg/ml in buffer C) and 5 l of 5ϫ 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

FIG. 1. Diagram of the major C/EBP isoforms and mutants used in this study.
to a final concentration of 0.1%, 1 g 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 1ϫ 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/EBP␥ and the amino terminus of C/EBP␥ were prepared against synthetic peptides corresponding to these sequences (9). Rabbit anti-C/ EBP␣ (14AA), rabbit anti-C/EBP␤ specific to the carboxyl terminus (C-19), rabbit anti-C/EBP␦ (C-22), rabbit anti-C/EBP⑀ (C-22) and normal rabbit IgG were purchased from Santa Cruz Biotechnology. Rabbit anti-C/EBP␤ specific to the amino terminus has been described (21).

C/EBP␤ Heterodimerizes with C/EBP␥ in B Cell Lines-In
our previous studies, we found C/EBP␤ to be predominantly in heterodimers with C/EBP␥ in P388 B cells that are dependent upon transfected C/EBP␤ expression for LPS induction of IL-6 and MCP-1 (Refs. 8 and 9; Fig. 2A). We also found both C/EBP␤ and C/EBP␦ to be in heterodimers with C/EBP␥ 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/EBP␤:␥ and C/EBP␦:␥ heterodimers (Fig. 2, B and C). In order to further test the entry of C/EBP␤ into C/EBP␤:␥ heterodimers, a C/EBP␤ 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/EBP␤:␥ heterodimers were the predominant binding species at all quantities tested (Fig. 3C). Apparently, C/EBP␤:␥ heterodimers formed at the expense of C/EBP␥ homodimers at lower quantities of vector ( Fig. 3C; 0.5, 1, 2 g). C/EBP␤ homodimers were observed only at higher vector quantities, where C/EBP␥ homodimers were no longer observable ( Fig. 3C; 2, 4, 6, 8, 12 g). The fact that the major C/EBP species observed with LPS-stimulation were C/EBP␤:␥ heterodimers is inconsistent with an inhibitory role for C/EBP␥ in the LPS induction of IL-6 expression.
C/EBP␥ Augments C/EBP␤-stimulated Transcription of the IL-6 Promoter-C/EBP␥ 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/EBP␥-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/EBP␤ vector with and without added expression of C/EBP␥ (Fig. 3A). C/EBP␥ augmented LPS-induced expression from the IL-6 promoter at all quantities of C/EBP␤ expression vector used. This is very surprising for a factor generally believed to be a transdominant inhibitor of C/EBP activators (10). If C/EBP␥ acted as an inhibitor, C/EBP␤ would be expected to induce less luciferase expression in the presence of added C/EBP␥, rather than more luciferase expression. In fact, 0.5 g of C/EBP␤ vector with 0.5 g of C/EBP␥ vector is twice as effective as 1 g of C/EBP␤ vector alone. This is consistent with C/EBP␤:␥ heterodimers being more potent activators than C/EBP␤ homodimers. Presumably, overexpression of C/EBP␥ drives more C/EBP␤ into heterodimers than would occur at
endogenous levels of C/EBP␥ expression. When EMSA was performed upon nuclear extracts prepared from P388 cells transiently transfected with C/EBP␤ expression vector with and without added C/EBP␥ expression vector, a higher ratio of C/EBP␤:␥ heterodimer to C/EBP␤ homodimer is indeed observed in cells transfected with C/EBP␥ expression vector (2.2 as opposed to 1.3) (Fig. 3E). To further test the ability of C/EBP␥ to promote formation of C/EBP␤:␥ heterodimers, a constant quantity of C/EBP␤ expression vector was transiently transfected into P388 cells with and without C/EBP␥ 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/EBP␤:␥ heterodimers became apparent and increased in abundance with increasing quantities of C/EBP␥ (Fig. 3D).
The stimulatory effects of C/EBP␥ were also observed in transient transfections where increasing amounts of C/EBP␥ expression vector were added to a constant amount of C/EBP␤ expression vector. These transfections were performed with LPS stimulation, and the expression vectors were cotransfected with an IL-6 promoter-reporter. C/EBP␥ clearly augmented the ability of C/EBP␤ to mediate LPS induction of the IL-6 promoter (Fig. 3B). C/EBP␥ activity was observed even when the C/EBP␥ vector was transfected at a 8-fold excess over C/EBP␤ vector, although C/EBP␥ by itself exhibited no activity (data not shown). Our results therefore suggest that C/EBP␥, rather than functioning as an inhibitor to low levels of C/EBP␤ activity, actually augments that activity on the IL-6 promoter.
In contrast to the stimulatory effects observed when C/EBP␥ was cotransfected with C/EBP␤ in LPS-induced IL-6 expression, C/EBP␥ actually inhibited the modest activation of the Arrows on the right also indicate supershifts. The C/EBP␤⅐␤ complex is supershifted by only C/EBP␤-specific antibody, the C/EBP␥⅐␥ complex by only C/EBP␥-specific antibody, and the C/EBP␤⅐␥ complex by both C/EBP␤-specific and C/EBP␥-specific antibodies. A weak, nonspecific background species co-migrating with C/EBP␤⅐␥ is evident in the 0-g lane. D, EMSA was performed using nuclear extracts of P388 cells transiently transfected with pMEX control vector, 0.25 g of pMEX-C/EBP␤, and 0.25 g of pMEX-C/EBP␤ with increasing quantities (0, 0.1, 0.25, 0.5, 1, 2, and 4 g) of pMEX-C/EBP␥. The EMSA of the 4-g pMEX-C/EBP␥ transfectants also was performed with binding reactions that included normal rabbit IgG (N), carboxyl terminus-specific anti-C/EBP␤ (␤), or carboxyl terminus-specific anti-C/EBP␥ (␥). Arrows labeled ␤:␥ and ␥:␥ indicate the positions of C/EBP⅐DNA complexes. Arrows on the right also indicate supershifts. The C/EBP␥⅐␥ complex is supershifted by only C/EBP␥-specific antibody and the C/EBP␤⅐␥ complex by both C/EBP␤-specific and C/EBP␥-specific antibodies. Two unidentified slower migrating species that are not modulated by transfection and are reactive with C/EBP␥-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 g of pMEX-C/EBP␤, or 2 g of pMEX-C/EBP␤ plus 0.5 g pMEX-C/EBP␥. Arrows labeled ␤:␤, ␤:␥, and ␥:␥ indicate the positions of C/EBP⅐DNA complexes. The radioactivity associated with C/EBP␤ homodimers and C/EBP␤⅐␥ heterodimers was quantitated using a Storm PhosphorImager (Molecular Dynamics), and the ratio of C/EBP␤⅐␥ to C/EBP␤⅐␤ is shown.
IL-6 promoter that can be observed by transfection of C/EBP␤ without LPS stimulation (Fig. 4). This inhibition was reversed by cotransfection with NF-B p65, allowing dosage-dependent C/EBP␥ stimulatory activity in the absence of LPS stimulation (Fig. 4). The lowest quantity of p65 vector used in the cotransfection (0.05 g) potentiated robust stimulation by C/EBP␥. These data support the notion that C/EBP␥ may play a key role in the synergy between C/EBP␤ and NF-B.
It is possible that the C/EBP␥ expressed from our expression vector differed from endogenous C/EBP␥ in its ability to stimulate IL-6 transcription. Furthermore, other investigators who found that C/EBP␥ 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/EBP␥ into a form capable of transactivation. To test these possibilities, transient transfections were performed with the C/EBP␥ 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/EBP␥ expression vector amounts comparable to that used in the transient transfections where C/EBP␥ stimulatory activity was observed (data not shown). Thus C/EBP␥ has no stimulatory activity by itself, even in the presence of LPS treatment.
C/EBP␥ 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/EBP␥, we performed transient transfections with DEI 4 (Ϫ35alb)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/EBP␥ expression suggesting that a more complex promoter is required for stimulatory activity. We then performed transient transfections with the TNF␣ and IL-8 promoters (Fig. 5A). These promoters, like IL-6, are in part regulated by NF-B and C/EBP. The TNF␣ promoter does not display synergy between NF-B and C/EBP␤ (34), while the IL-8 promoter shows strong synergy between these two factors (35)(36)(37). Consistent with a possible role in the synergy between NF-B and C/EBP␤, C/EBP␥ expression had little effect upon the TNF␣ 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/EBP␥, C/EBP␤ 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/EBP␤). Furthermore, C/EBP␥ stimulatory activity does not appear to be dependent upon differential binding of C/EBP␥ to differing C/EBP binding sites. Both the IL-6 and DEI C/EBP binding motifs bound C/EBP␥-containing species in EMSA performed upon nuclear extracts from P388 cells overexpressing C/EBP␤ (Fig. 5B), while neither the TNF␣ 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/EBP␥ 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/EBP␥ is thus promoter specific, requires a complex promoter to be observed, and may function in the synergistic activation of promoters by NF-B and C/EBP family members.
The fact that C/EBP␥ 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/EBP␤⅐DNA complexes from these cells are supershifted by anti-C/EBP␥ 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/EBP␥ expression vector were added to a constant amount of C/EBP␤ 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/EBP␥ clearly augmented the ability of C/EBP␤ to mediate LPS induction of the IL-6 promoter, C/EBP␥ had no effect on C/EBP␤ stimulation of LPSinduced IL-6 expression in P388D1(IL1) cells (data not shown). Thus in addition to promoter specificity, the stimulatory activity of C/EBP␥ shows cell-type specificity.
C/EBP␥ Stimulatory Activity Requires Heterodimerization with C/EBP␤-We next sought to test whether C/EBP␥ stimulatory activity in transfections with C/EBP␤ requires heterodimer formation between these two proteins. To that end, we performed transient transfections with a chimeric C/EBP␤ containing the leucine zipper of yeast GCN4. In comparison to intact C/EBP␤, C/EBP␤-GCN4 LZ (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/EBP␥ 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/EBP␤-GCN4 LZ , Western blot analysis and EMSA were performed using nuclear extracts of transiently transfected cells FIG. 4. C/EBP␥ inhibits C/EBP␤-induced IL-6 transcription in the absence of LPS treatment, while that inhibition is reversed by NF-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/EBP␥ expression vector. The data presented for cells receiving C/EBP␤ but no NF-B p65 expression vector are the means of three experiments Ϯ S.E. The data for the cells receiving both C/EBP␤ and NF-B p65 expression vectors are derived from one experiment carried out at various doses of p65 vector. (Fig. 6, A and B). Western analysis of nuclear extracts from P388 cells transfected with increasing quantities of C/EBP␤-GCN4 LZ expression vector detected increasing quantities of a C/EBP-related protein at the expected molecular mass of ϳ38 kDa (Fig. 6A). As can be seen in an EMSA of the same nuclear extracts, the overexpression of C/EBP␤-GCN4 LZ fails to drive C/EBP␥ into heterodimers (Fig. 6B), in contrast to C/EBP␤ (Fig. 3C). The major EMSA species associated with transfection of the C/EBP␤-GCN4 LZ expression vector could be supershifted with antibody specific to the amino terminus of C/EBP␤, but not with antibody specific to the carboxyl terminus of C/EBP␤ 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/EBP␥, indicating a lack of dimerization with C/EBP␥. Transient transfection of increasing amounts of C/EBP␥ expression vector with a constant amount of C/EBP␤-GCN4 LZ expression vector were carried out in comparison to increasing amounts of C/EBP␥ expression vector with a constant amount of C/EBP␤ expression vector (Fig. 6C). The ability of C/EBP␥ to augment C/EBP␤ activity was largely blocked by the GCN4 leucine zipper. This is consistent with C/EBP␥ stimulatory activity being dependent on its ability to dimerize with C/EBP␤. The fact that C/EBP␤-GCN4 LZ by itself supports LPS induction of the IL-6 promoter indicates that while C/EBP␥ can augment C/EBP␤ activity, formation of heterodimers containing C/EBP␥ is not necessary for C/EBP activity on the IL-6 promoter.
C/EBP␥ Stimulatory Activity Resides with Its Leucine Zipper Domain-We next initiated studies to determine the structural components of C/EBP␥ sufficient for its stimulatory ac-tivity. A form of C/EBP␥ deleted for the region amino-terminal to the bZIP domain ( Fig. 1; C/EBP␥-⌬Nco) was compared with intact C/EBP␥ in the same experimental regime as described for Fig. 3B, where increasing amounts of C/EBP␥ expression vector were added to a constant amount of C/EBP␤ expression vector. These transfections were performed with LPS stimulation, and the expression vectors were cotransfected with an IL-6 promoter-reporter. C/EBP␥-⌬Nco, although lacking the 57-residue amino terminus, had as much stimulatory activity as wild type C/EBP␥ (Fig. 7A). An EMSA species that increased in abundance with increasing quantities of the C/EBP␥-⌬Nco vector further indicated successful expression of C/EBP␥-⌬Nco (Fig. 7B). Thus, the amino terminus of C/EBP␥ is unnecessary for its stimulatory activity.
Since C/EBP␥ homodimers by themselves have no stimulatory activity (data not shown) and the ability of C/EBP␥ to heterodimerize with C/EBP␤ appears to be critical for its stimulatory activity (Fig. 6), we tested whether C/EBP␥ 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/EBP␥ amino-terminal and basic region, and a C/EBP␤ leucine zipper ( Fig. 1; C/EBP␥-␤ LZ ). As a control for C/EBP␥-␤ 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/EBP␥-␤ LZ expression vector (Fig. 8, A and  B). Western analysis with antibody specific to the carboxyl terminus of C/EBP␤ detected increasing quantities of a C/EBPrelated protein at the expected molecular mass of ϳ19 kDa  Fig. 3B. The data presented for the IL-8, TNF␣, and DEI 4 (Ϫ35alb) 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-C␤ 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/EBP␤ (␤), or carboxyl terminus-specific anti-C/ EBP␥ (␥). Arrows labeled ␤:␤, ␥:␥, and ␤:␥ indicate the positions of C/EBP⅐DNA complexes.
FIG. 6. C/EBP␥ stimulatory activity is dependent upon the formation of C/EBP␤:␥ heterodimers. The replacement of the C/EBP leucine zipper in C/EBP␤ with that of GCN4 blocked C/EBP␥ 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 g) of pMEX-C/EBP␤-GCN4 LZ . The primary antibody used in the detection of C/EBP␤-GCN4 LZ was amino terminus-specific anti-C/EBP␤. An arrow marks the position of C/EBP␤-GCN4 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 g) of pMEX-C/EBP␤-GCN4 LZ . The EMSA of the 12-g transfectants was also performed with binding reactions that included normal rabbit IgG (N), amino terminus-specific anti-C/EBP␤ (N␤), carboxyl terminus-specific anti-C/EBP␤ (C␤), or carboxyl terminus-specific anti-C/EBP␥ (C␥). Arrows labeled ␤-GCN4 LZ , ␤:␤, ␤:␥, and ␥:␥ indicate the positions of C/EBP⅐DNA complexes. Arrows on the right indicate supershifts. The C/EBP␤-GCN4 LZ complex is only supershifted by amino terminus-specific anti-C/EBP␤, while the C/EBP␤ complexes from P388-C␤ are supershifted by all of the specific antisera. Weak, nonspecific background species co-migrating with C/EBP␤:␤ and C/EBP␤:␥ are evident in the 0-g 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/EBP␤-GCN4 LZ ϩC/EBP␥ (beta-GCN4LZϩgamma) are the mean of four experiments Ϯ S.E. The data for C/EBP␤ϩC/EBP␥ (betaϩgamma) from Fig. 3B are presented for comparison. (Fig. 8A). A major EMSA species was detected in proportion to the amount of C/EBP␥-␤ LZ expression vector (Fig. 8B). That species was supershifted with antibodies specific to the carboxyl terminus of C/EBP␤, the amino terminus of C/EBP␥, and the carboxyl terminus of C/EBP␥, but not with antibody specific to the amino terminus of C/EBP␤ (Fig. 8B). This is consistent with a heterodimer between C/EBP␥-␤ LZ and endogenous C/EBP␥. We tested the ability of C/EBP␥-␤ LZ to support LPS induction of IL-6 with and without transfection of a vector expressing intact C/EBP␥ (Fig. 8C). Surprisingly, in LPStreated cells, the C/EBP␥-␤ LZ expression vector by itself could support as much as 10-fold induction of the IL-6 promoter and the addition of 0.5 g of C/EBP␥ expression vector enhanced that stimulatory activity to 20-fold induction. While the stimulatory activity of C/EBP␥-␤ LZ is less than that of intact C/EBP␤ (40-fold for 1 g of vector without C/EBP␥ and 100-fold with C/EBP␥; see Fig. 3A), the degree to which C/EBP␥ augmented C/EBP␥-␤ LZ activity was similar to its enhancement of C/EBP␤ activity (about 2.5-fold). This suggests that C/EBP␥ stimulatory activity resides in formation of a heterodimeric C/EBP␤:␥ leucine zipper. DISCUSSION The data presented in this study demonstrate an activating role for C/EBP␥ in transcription from the IL-6 and IL-8 promoters in B lymphoid cells. C/EBP␥, which in other contexts can inhibit activation by C/EBP family members (9, 10), was found to augment the C/EBP␤-dependent LPS stimulation of IL-6 and IL-8 promoter-reporters in P388 B lymphoblasts. This stimulatory activity of C/EBP␥ is dependent on its formation of heterodimers with C/EBP␤ and, indeed, C/EBP␤ is largely found in heterodimers with C/EBP␥ in P388 B cells that have gained the capacity for LPS-induced IL-6 expression upon transfection of a C/EBP␤ expression vector. Surprisingly, the critical structural feature for this stimulatory activity is the formation of a heterodimeric leucine zipper between C/EBP␤ and C/EBP␥. C/EBP␥ stimulatory activity was found to be promoter-specific with activity seen on IL-6 and IL-8 promoterreporters, and not on TNF␣ and albumin DEI promoter-reporters. C/EBP␥ 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/EBP␥ 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/EBP␥ found that immunodepletion of C/EBP␥ from an in vitro transcription assay inhibited the activity of the BCL1 immunoglobulin heavy chain and the Rous sarcoma virus promoters (13). Similarly, C/EBP␥ synergizes with Stat6 and NF-B p50/p65 to induce the germline gamma 3-immunoglobulin promoter in a B cell line (14). C/EBP␥ has also been found to enhance ␤-globin gene expression in collaboration with CP-1 (15). Another instance of a positive role for C/EBP␥ has been found in the expression of pp52, a leukocyte-specific phosphoprotein postulated to regulate cytoskeleton structure (16). Thus, the role of C/EBP␥ as a transcriptional activator does not seem unusual. It seems nei- EMSA of P388 cells and the 12-g transfectants also was performed with binding reactions that included normal rabbit IgG (N), amino terminus-specific anti-C/EBP␤ (N␤), carboxyl terminus-specific anti-C/EBP␤ (C␤), amino terminus-specific anti-C/EBP␥ (N␥), or carboxyl terminus-specific anti-C/EBP␥ (C␥). Arrows labeled ␥:␥ and ␥:␥-␤LZ indicate the positions of C/EBP⅐DNA complexes. Arrows on the right indicate supershifts. The C/EBP␥-␤ LZ complex (similar in mobility to the C/EBP␥ complex in P388 cells) is supershifted by carboxyl terminus-specific anti-C/EBP␤, in addition to the C/EBP␥-specific antisera. C, transient transfections were carried out in duplicate with and without 0.5 g of C/EBP␥ vector, with the microgram quantities of C/EBP␥-␤ 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 Ϯ S.E. ther 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/EBP␥ in transcription. Heterodimerization with C/EBP␥ has two effects on the ability of C/EBP␤ to activate the IL-6 promoter: it inhibits C/EBP␤ activity in the absence of LPS and enhances C/EBP␤ transactivation in LPS-stimulated cells. Therefore, we predict that in B cells the net effect of C/EBP␥ 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/EBP␥-deficient mice (17).
C/EBP␥ stimulatory activity was observed with the IL-6 and IL-8 promoter-reporters, but not with the TNF␣ or the DEI promoter-reporters. One distinguishing characteristic of the IL-6 and IL-8 promoters is synergistic regulation by C/EBP␤ and NF-B (35)(36)(37). It is tempting to propose a specific role for C/EBP␥ in promoting this synergy. While the experiments reported here do not provide a direct demonstration for such a mechanism, the findings that C/EBP␥ inhibits C/EBP␤ activation of the IL-6 promoter in the absence of LPS and that this inhibitory effect is converted to a stimulatory effect by NF-B p65 expression (Fig. 4) are consistent with this model. Furthermore, our previous studies found that the activity of C/EBP␤ on the IL-6 promoter was dependent on an intact NF-B site (8). It is, however, unlikely that the stimulatory role of C/EBP␥ is limited to promoters that exhibit synergy between C/EBP␤ and NF-B. Other promoters for which C/EBP␥ stimulatory activity have been suggested, including immunoglobulin heavy chain (13,14), ␤-globin (15), and pp52 (16), do not display synergistic regulation by C/EBP␤ and NF-B.
C/EBP␥ stimulatory activity displays cell-type specificity. This is also the case for the inhibitory activity of C/EBP␥ (9). Stimulatory activity was seen in P388 B cells, but not in their macrophage derivative, P388D1(IL-1) (data not shown). C/EBP␥ is normally a minor component of the C/EBP family members expressed in these macrophages, where C/EBP␤ forms heterodimers with another as yet unidentified protein (9). Perhaps, C/EBP␥ stimulatory activity in P388D1(IL-1) macrophages is precluded by the heterodimerization of C/EBP␤ with this other protein. The activity of C/EBP␥ 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/EBP␥ activity lead us to speculate that the ability of C/EBP␥ 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 TNF␣. This could be particularly important as a source of IL-6 in a T-independent B cell response. Perhaps C/EBP␥-deficient mice (17) will exhibit slower kinetics in their B cell response to Gram-negative bacteria.
While we have observed C/EBP␥ 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/EBP␥ expression and the autocrine production of these cytokines in myelomas.
Although C/EBP␥ is most abundantly expressed in immature B cells (10), we have found C/EBP␤:␥ and C/EBP␦:␥ 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/EBP␤:␥ 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/EBP␥ can stimulate target genes that are known to be positively regulated by C/EBP␤ in these cell-types and tissues.
We found that ectopic expression of C/EBP␤ in P388 cells led to the formation of C/EBP␤:␥ heterodimers at the expense of C/EBP␥ homodimers, while C/EBP␤ homodimers were observed only at the highest levels of C/EBP␤ 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/EBP␥ or unbound C/EBP␥ dimers being available for dimerization with C/EBP␤. If this were the case, C/EBP␥ homodimers would not be eliminated as they are by C/EBP␤ 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/EBP␥ with the leucine zipper of C/EBP␤ to stimulate the IL-6 promoter in cells that express only endogenous C/EBP␥ (Fig. 8). Since C/EBP␥ 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/EBP␤:␥ 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/EBP␤, ␦, or ␣ was sufficient to confer LPS inducibility to the IL-6 promoter in P388 cells (8). In those studies, we found that the C/EBP␤ bZIP domain was largely dimerized with C/EBP␥ and that activity required an intact NF-B binding site. We have now found that C/EBP␥ stimulatory activity is observed on two promoters that show synergy between C/EBP and NF-B and that C/EBP␥ expression actually becomes inhibitory in the absence of NF-B expression (Fig. 4). Our findings are consistent with the C/EBP leucine zipper being a critical determinant in facilitating the synergy between NF-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-1␤, serum amyloid A1, A2, A3, and ␣ 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/EBP␣ mediates cell-type specificity of albumin promoter activation (42) and phosphorylation of serine 276 in the leucine zipper of human C/EBP␤ confers calcium-regulated transcriptional stimulation to a promoter that contains binding sites for C/EBP␤ (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-B or with coactivators of transcription in a manner dependent upon leucine zipper dimerization partners.