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B, AP-1, and C/EBP1
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Departments of
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Microbiology and
Internal Medicine, University of Iowa, and
Veterans Affairs Medical Center, Iowa City, IA 52242; and
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B. The present study was designed to characterize CD40-mediated transcriptional control of the IL-6 gene in B cells. CD40 engagement on B lymphocytes activated the IL-6 promoter, and mutations in the putative binding sites for AP-1 and C/EBP transcription factors reduced this activation. Interestingly, a mutation in the putative NF-B binding site completely abrogated the basal promoter activity, thus also rendering the promoter unresponsive to CD40 stimulation, suggesting that this site is required for binding of NF-B constitutively present in the nucleus of mature B cells. The expression of dominant negative Fos or C/EBP
proteins, which prevent binding of AP-1 or C/EBP complexes to DNA, also reduced CD40-mediated IL-6 gene expression. Furthermore, CD40 stimulation led to phosphorylation of c-Jun on its activation domain, implicating CD40-mediated Jun kinase activation in the transcriptional regulation of IL-6 production. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD40 has also been suggested as a potential growth receptor in B cell malignancies (6). Because CD40-mediated IL-6 production may be involved in various diseases, understanding how CD40 regulates IL-6 gene expression is important. A comparison of the human and mouse IL-6 genes shows regions of high homology 
350 bp upstream of the transcription start site (7). Several potential transcriptional control elements, including glucocorticoid-responsive elements, AP-1-binding sites, a c-fos serum-responsive element (SRE),3 a cAMP-responsive element, a C/EBP binding site, and an NF-B-binding site, have been identified in this region. Within the SRE homology region is a 14-bp palindromic sequence that binds the transcription factor NF-IL6 (also known as C/EBP
), which was identified as the IL-1-responsive element (8). In addition, others have identified a 23-bp multiresponse element within the SRE homology region that is critical for IL-6 production mediated by IL-1, TNF, forskolin, and phorbol ester (9). The NF-B binding site has also been reported to be responsible for controlling IL-6 production in response to specific stimuli in certain cell types (10). In B lymphocytes, the transcription factor c-Jun is required for activating the IL-6 gene following mitogen stimulation (11). However, the transcription factors required for CD40-mediated B cell IL-6 expression are currently unknown.
We previously reported that CD40 engagement results in increased IL-6 mRNA levels in normal mouse splenic B cells and the mouse B cell line CH12.LX, and IL-6 secretion is independent of CD40-mediated increases in NF-B activation (3). The results presented here show that CD40 stimulation activates the mouse IL-6 promoter, and binding sites for NF-B, AP-1, and C/EBP are required for maximum CD40-induced promoter activity as well as for maximal IL-6 protein production. The NF-B binding site is required for binding of basal nuclear NF-B that is present in B cells. In contrast, CD40-mediated activation of c-Jun transcriptional activity (via phosphorylation of serine 63 in the activation domain of c-Jun), but not increased binding to the AP-1 site, is necessary for IL-6 gene transcription. The results indicate that CD40-mediated control of the IL-6 gene in B cells is complex and involves several transcription factors that appear to have distinct roles in the process.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CH12.LX, a µ+, 
+ mouse B cell line, expresses IgM specific for phosphatidylcholine (12, 13). Subclones of CH12.LX that stably express either a mutant IB protein (CH12.IBAA) or a truncated (dominant negative (DN)) TNF receptor-associated factor 6 (TRAF6) protein (CH12.DNT6) under control of an isopropyl--D-thiogalactopyranoside (IPTG)-inducible promoter have been described previously (14, 15). Splenic B cells were purified from C57BL/6 x 129/J (F2) mice (wild type (Wt) or p50-/-), as previously described (16). B cells were cultured in RPMI 1640 medium containing 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µl/ml streptomycin, and 10-5 M 2-ME (BCM-10). Mouse CD154-expressing CHO cells (CHO-mCD154) were generated in our laboratory and previously described (3). Sf9 insect cells infected with Wt baculovirus or a recombinant baculovirus expressing mCD154 have been previously described (17). Both CD154-expressing cell types have been previously shown to be effective at inducing B cell IL-6 secretion (3).
Antibodies
The hybridomas used in this study include the following: 1C10 (rat IgG1 anti-mouse CD40), provided by Dr. F. Lund (Trudeau Institute, Saranac Lake, NY); EM95.3 (rat IgG1 anti-mouse IgE), provided by Dr. T. Waldschmidt (University of Iowa, Iowa City, IA); 20F3.11 (rat IgG1 anti-mouse IL-6), and 32C11.4 (rat IgG2a anti-mouse IL-6) purchased from American Type Culture Collection (Manassas, VA). The following Abs were purchased from commercial sources: anti-C/EBP (H-7; mouse IgG2a), anti-C/EBP
(C-20; goat polyclonal Ab), and anti-c-Jun (H-79; rabbit polyclonal IgG; Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho-c-Jun (Ser63; rabbit polyclonal IgG) (Upstate Biotechnology, Lake Placid, NY); anti-rabbit IgG-HRP (goat IgG; Bio-Rad, Hercules, CA); anti-FLAG M2-biotin (mouse IgG2b; Sigma-Aldrich, St. Louis, MO); anti-c-Fos, anti-c-Jun, anti-FosB, anti-JunB, and anti-JunD (rabbit polyclonal Abs, AP-1 Family Nushift Kit; Geneka Biotechnology, Montreal, Canada).
Plasmid constructs and site-directed mutagenesis
The IL-6 promoter region was amplified from CH12.LX genomic DNA by PCR with the primers 5'-CGCGGATCCTGAGAGTGTGTTTTGTAA-3' and 5'-CCCAAGCTTCCAGAGCAGAATGAGCTA-3', which contain flanking BamHI and HindIII restriction endonuclease sites, respectively. The PCR product was ligated into the vector pGL2-basic (Promega, Madison, WI), and the sequence of the mouse IL-6 promoter region was verified (7). The Wt full-length mouse IL-6 promoter/luciferase reporter gene plasmid is designated pmIL-6.luc(-1277). The numbers in parentheses for this and the following plasmids indicate the length of the IL-6 promoter fragment upstream of the transcriptional start site. 5' truncations of the mouse IL-6 promoter were generated by restriction enzyme digestion to produce the following plasmids: pmIL-6.luc(-231), pmIL-6(-161), and pmIL-6.luc(-84), respectively. Mutations in the putative AP-1 (-61 to -55), C/EBP (-161 to -147), and/or NF-B (-73 to -64) binding sites in the mouse IL-6 promoter were generated using the QuikChange Site-Directed Mutagenesis kit from Stratagene (La Jolla, CA) according to the manufacturer's instructions using the primers listed in Table I. Mutations were verified by sequencing. Plasmids containing the cDNA of dominant negative Fos (A-Fos) or C/EBP (A-C/EBP) under the control of the CMV promoter or control empty vector (CMV-500) were previously generated and described (18, 19). Plasmids containing IPTG-inducible A-Fos or A-C/EBP were generated by subcloning the cDNA of the respective proteins into the inducible pOPRSVmsc1 vector (14, 20). The C/EBP-dependent luciferase reporter gene plasmid, which contains four copies of the mouse IL-6 C/EBP binding site positioned 5' upstream of a minimal thymidine kinase promoter, was a gift from Dr. E. Clark (University of Washington, Seattle, WA). The AP-1-dependent luciferase reporter gene plasmid (PathDetect cis-Reporting System Plasmid), which contains seven copies of the AP-1 binding site, was purchased from Stratagene (La Jolla, CA). The pRL-Renilla plasmid (Promega), which contains the cDNA encoding the Renilla luciferase from the marine organism Renilla reniformis, was used as a transfection efficiency control in luciferase reporter assays.
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cell lines
CH12.LX B cells stably expressing the Lac repressor protein (LacI) (14, 20) were transfected with DN A-Fos or A-C/EBP constructs in the inducible vector, pOPRSVmcs1. Stable transfectants were generated by electroporation and selected in G418-containing medium as previously described (21). Induction of the A-Fos or A-C/EBP proteins was achieved by overnight incubation of cells with 100 µM IPTG as previously described (14, 20).
Transient transfections
Transient transfection conditions for the analysis of reporter genes were as follows: CH12.LX B cells (4 x 107) were washed once in 1x PBS, resuspended in 400 µl of Cytomix buffer (22), and placed in an electroporation cuvette. Cells were transfected with a total of 80 µg of DNA (76 µg of reporter plasmid, 4 µg of pRL-Renilla) by electroporation at 200 V for 50 ms in an electroporator (BTX, San Diego, CA). For conditions that required transfection of the A-Fos or A-C/EBP plasmid, the amount of the reporter plasmid used was reduced accordingly. After electroporation, the cuvette was placed on ice for 10–15 min. Cells were then transferred to 30 ml of BCM-20 and incubated at 37°C for 6 h. Cells were washed once in RPMI, and cell viability was determined by trypan blue exclusion. Cells were stimulated as described below (luciferase assay conditions).
Transient transfection conditions for assessment of IL-6 production were as follows. CH12.LX B cells (4 x 107) were washed once in 1x PBS, resuspended in 400 µl of Cytomix, and placed in an electroporation cuvette. Cells were transfected with a total of 20 µg of mutant A-Fos or A-C/EBP plasmid DNA by electroporation at 225 V for 30 ms in a BTX electroporator. The control CMV-500 plasmid was used in some conditions to bring the total amount of DNA to 20 µg. After electroporation, the cuvette was placed on ice for 10–15 min. Cells were then transferred to 30 ml of BCM-20 and incubated overnight at 37°C. Cells were washed once in RPMI, and cell viability was determined by trypan blue exclusion. Cells were stimulated as described below (ELISA conditions).
IL-6 ELISA
B cells (1 x 105) were resuspended in 200 µl of BCM-10 with various stimuli in 96-well, flat-bottom microtiter plates. After 48 h, 100 µl of supernatant was harvested and assayed for secreted IL-6 as previously described (3). The IL-6 concentration in the supernatants was then determined against a standard curve of recombinant murine IL-6. Recombinant murine IL-6 was purchased from BD PharMingen (San Diego, CA).
Luciferase assay
Viable B cells (5 x 105) were resuspended in 2 ml of BCM-10 with various stimuli in triplicate wells of 24-well tissue culture plates and incubated for 24 h at 37°C. Cells were harvested by centrifugation, and cell pellets were lysed in 100 µl of passive lysis buffer (Promega) and stored at -20°C until use. Forty microliters of cell lysate was assayed for both firefly and Renilla luciferase activities using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions, and relative light units (RLU) were measured on a luminometer. RLU from the firefly luciferase was normalized for transfection efficiency to the Renilla luciferase RLU in each lysate (normalized RLU = RLUfirefly luciferase/RLURenillaluciferase). The promoter activity of a reporter construct following stimulation was calculated as follows: activity = normalized RLUstimulation - normalized RLUcontrol.
EMSA
B cells (1 x 107) were resuspended in 10 ml of BCM-10, treated with various stimuli, and incubated at 37°C. Nuclear extracts were isolated in the presence of the phosphatase inhibitor sodium fluoride (10 mM) as previously described (23). Oligonucleotide probes (Table II) were radiolabeled with bacteriophage T4 polynucleotide kinase (T4 PNK) and [32P]ATP. Ten micrograms of nuclear extract was incubated with 0.25 ng of radiolabeled probe, 1 µg/ml poly(dI-dC), and 1x AP-1 binding buffer (20 mM HEPES (pH 7.9), 5 mM spermidine, 5 mM KCl, 1 mM MgCl2, 2 mM EDTA, and 5% glycerol) for 30 min at room temperature. 40x unlabeled cold competitor probes were added before the addition of radiolabeled probe in some samples. Samples were then loaded onto a 5% 0.5x Tris-borate-EDTA (C/EBP) or 1x Tris-borate-EDTA (AP-1) native acrylamide gel and electrophoresed for 2.5 h at 20 mA. The gel was dried, and autoradiography was performed.
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Cells (1 x 106) were lysed in 100 µl of boiling 2x SDS-PAGE sample dye for 15 min. Ten microliters of lysates or 10 µg of nuclear extracts (diluted in an equal volume of 2x SDS-PAGE sample dye and heated to 95°C for 5 min) were subjected to SDS-PAGE and electroblotted to a polyvinylidene difluoride (PVDF) membrane, and Western blot analysis was performed as described below. For detection of FLAG-tagged DN transcription factors, the PVDF membrane was incubated with anti-FLAG M2-biotin for 1.5 h, followed by HRP-conjugated streptavidin. The peroxidase activity of the HRP-conjugated reagent was detected by ECL reagents (Pierce, Rockford, IL) and for all Western blots performed. For detection of phospho-c-Jun in nuclear extracts, immunoblotting was performed according to the manufacturer's protocol and was analyzed by ECL. The intensities of the anti-phospho-c-Jun and anti-c-Jun reactive bands on Western blots were quantitated on an LAS-1000 luminescent image analyzer (FujiFilm Science, Stamford, CT). The intensities of the luminescent bands per square millimeter were obtained, and ratios of phospho-c-Jun to c-Jun were calculated.
Ab secretion assay
To examine the effects of expression of mutant A-Fos or A-C/EBP on IgM secretion by CH12.LX, stable inducible transfectants (described above) were incubated with 100 µM IPTG for 18 h at a concentration of 1.5 x 103 cells/100 µl/well in BCM-10, in flat-bottom, 96-well microtiter plates. Following this induction of expression of the mutant interfering proteins, cells were incubated for an additional 48 h in the presence of Wt or mCD154-expressing Sf9 cells at a ratio of 8 B cells/1 Sf9 cell. Sf9 rather than CHO cells were used for these experiments, because cell counts are obtained at the end of the culture period, and while it could be difficult to discriminate between CHO and B cells, Sf9 cells normally proliferate only at 25°C, so by the end of the culture period the Sf9 cells exist only as membrane fragments. IgM-secreting cells in each culture were enumerated by direct plaque assay on a lawn of SRBC as previously described (24). This method can be used for CH12.LX, because SRBC membranes contain phosphatidylcholine, the Ag for which the IgM of CH12.LX is specific (13). Induction of DN proteins did not affect the viability or proliferation of the transfected cells (not shown).
Statistical analysis
Statistical comparisons of experimental groups were performed using a one-tailed paired Student's t test from the Microsoft EXCEL program (Redmond, WA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously demonstrated that IL-6 mRNA levels are increased following CD40 stimulation (3). To determine whether CD40 induces increased IL-6 gene activation, we generated a luciferase reporter gene plasmid containing the firefly luciferase gene under transcriptional control of the mouse IL-6 promoter (mIL-6.luc). CH12.LX B cells were transiently transfected with this plasmid and cultured with control and CD40 stimuli. A low level of luciferase activity was detected in lysates of cells cultured in medium alone or with control CHO cells (Fig. 1), which was increased 5-fold increase in B cells cultured with CHO-mCD154. CD40 stimulation via agonistic anti-CD40 Ab cross-linking did not lead to a significant increase in luciferase activity above isotype control Ab stimulation. Thus, membrane-bound CD154 is required for activating the IL-6 promoter in CH12.LX B cells, consistent with our previous finding that anti-CD40 Abs or soluble CD154 cannot effectively induce B cell IL-6 protein production (3).
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8% of the Wt promoter, which was still consistently above activity of the control promoterless reporter plasmid. Anti-CD40 or isotype control mAb stimulation did not activate any of the promoter constructs (data not shown). Thus, a 231-nt region immediately upstream of the transcription start site is critical to confer promoter responsiveness to CD40 stimulation by CD154.
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Within the 231-nt region critical for CD40 responsiveness, nt -231 to -161 account for 75% of the CD40-mediated promoter activity, as shown by in vitro reporter gene analysis (Fig. 2). A putative transcription factor binding site for CREB has been identified in this region. However, to our knowledge, CD40 has not been reported to activate the CREB/activating transcription factor (ATF) family of transcription factors. Consistent with this, experiments using mIL-6 promoter reporter gene analysis in which this site has been mutated showed that CREB is not needed for CD40-induced promoter activity (data not shown). The -231 to -161 region does not contain consensus sequences for other known transcription factors of B cells. Thus, we are not yet able to identify the factor(s) that account for the effect of this region on CD40 responsiveness. However, potential transcription factor binding sites for NF-B, AP-1, and C/EBP are found elsewhere within the 231-nt CD40-responsive region of the mouse IL-6 promoter as well as in the human IL-6 promoter. To determine whether these cis-acting elements are required for CD40-mediated gene activation in B cells, mutations in the DNA sequences of the putative binding sites were generated. A mutation in the AP-1 binding sequence reduced promoter by 30% following CD40 ligation; mutation in the C/EBP binding site reduced promoter activity by 40% (Fig. 3). A promoter with mutations in both the AP-1 and C/EBP binding sites lost 70% activity compared with the Wt promoter. Interestingly, a mutation in the NF-B binding site completely abrogated the basal promoter activity, thus also rendering the promoter unresponsive to CD40 stimulation.
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proteins that abrogate the function of all AP-1 or C/EBP family members, respectively (18, 19). Overexpression of A-C/EBP resulted in an inhibition of 50–75% promoter activity, while overexpression of A-Fos inhibited promoter activity by 75–90% following CD40 ligation (Fig. 5A). The specificity of these effects is confirmed in Fig. 5B, where it is shown that transient expression of the A-C/EBP mutant did not affect induced promoter activity if the C/EBP binding site was abrogated (middle set of bars). Similarly, the reporter plasmid with a mutated AP-1 binding site was not affected by expression of A-Fos (right set of bars). Whole-cell lysates were subjected to Western blot analysis to verify expression of the mutant proteins (Fig. 5C).
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or A-Fos also inhibited CD40-induced IL-6 protein secretion (Fig. 6A). The inhibition of CD40-mediated IL-6 production by the mutant proteins was not as dramatic as the inhibition of the IL-6 promoter, but this is somewhat misleading. Due to 30% transfection efficiency, only about one-third of the B cells are expressing the DN proteins, so untransfected B cells can still secrete IL-6 following CD40 stimulation and are detected in this assay. Expression of the mutant proteins was verified by Western blot analysis of whole cell lysates (data not shown). Thus, the activities of AP-1 and C/EBP transcription factor family members are required for optimal IL-6 promoter activity and protein secretion. While we have shown that CD40-mediated increases in nuclear NF-B are not needed to stimulate IL-6 production (3), the results shown in Fig. 3 indicated that without basal NF-B, activation of additional transcription factors could not be effective in stimulating IL-6 promoter activity. To determine whether basal NF-B is needed for optimal IL-6 production, we used a subclone of CH12.LX that stably and inducibly expresses a mutant form of IB (IBAA) that cannot be phosphorylated; its lack of degradation thus prevents the release of NF-B into the nucleus. We have shown previously that CD40-induced IgM secretion and surface molecule up-regulation are inhibited by IBAA expression (14). Fig. 6B shows that expression of this protein also inhibited CD40-induced IL-6 production in CH12.LX B cells. Although a mutation in the NF-B binding site of our IL-6 reporter gene abrogated all detectable promoter activity, IL-6 production was not completely eliminated by the expression of IBAA. However, it is difficult for B cells to remain viable and express enough of the mutant protein to completely abrogate all NF-B activation, so the maximal achievable expression of this protein does not completely block NF-B (14). To assess the NF-B dependence of CD40-mediated IL-6 production using another approach, we built upon our previous finding that CD40-mediated NF-B activation in B cells principally involves the p50, c-Rel, and p65 subunits (23). We thus measured CD40-induced IL-6 in cultures of splenic B cells from p50-deficient mice and their Wt littermates. The results, shown in Fig. 6C, demonstrate markedly defective IL-6 production by p50-/- B cells in response to a CD40 stimulus. The small amount of IL-6 that is induced in these cells may be mediated by complexes composed of additional subunits.
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We have previously demonstrated that activation of NF-B is required for CD40-mediated induction of IgM secretion in B cells (14). To determine whether the activation of additional transcription factors plays a role in CD40-induced Ab production, we examined the ability of CH12.LX B cells to secrete IgM in the presence of A-Fos or A-C/EBP proteins. Induced expression of stably transfected A-Fos or A-C/EBP protein by overnight incubation with IPTG resulted in a 40–60% reduction in IgM secretion compared with uninduced B cells, although cell viability was not affected (Fig. 6D). The partial inhibition of Ab secretion by overexpression of A-Fos or A-C/EBP protein is consistent with previous findings, showing that inhibiting the production of IL-6 or its binding to B cells reduces CD40-mediated IgM secretion by 50% (15, 25).
CD40 signals induce phosphorylation of serine 63 in the activation domain of c-Jun
Activation of AP-1-responsive genes can be mediated by signals that induce the synthesis of AP-1 proteins and subsequent binding of the newly synthesized proteins to AP-1-responsive promoters or the phosphorylation of pre-existing AP-1 subunits on various serine or threonine residues (reviewed in Ref. 26). To determine the mechanism by which CD40 induces AP-1 transcriptional activation, nuclear translocation of AP-1 complexes and binding to DNA were determined by EMSA. Nuclear extracts from CH12.LX B cells cultured in BCM-10 (data not shown) or with parent CHO cells for 1 h (Fig. 7A) contain proteins capable of binding an oligonucleotide probe containing an AP-1 binding sequence (Table II). Increased protein binding to the oligonucleotide probe was not seen in CHO-mCD154-stimulated B cells, although increased nuclear translocation of NF-B could be detected in the nuclear extracts of these stimulated B cells (data not shown). Protein binding to the AP-1 probe was abrogated by an excess of unlabeled Wt AP-1 probe, but not by an excess of unlabeled oligonucleotide probe containing mutations in the AP-1 DNA binding sequence, thus demonstrating binding specificity. Neither nuclear extracts from B cells cultured with CHO-mCD154 cells nor anti-mCD40 mAb (not shown) for 30 min to 6 h showed increased protein binding to the AP-1 oligonucleotide probe compared with controls (Fig. 7A). Examination of binding to a C/EBP EMSA probe (Table II) showed that B cells also contain several complexes specifically binding to this site, but again CD40 stimulation does not enhance this binding (Fig. 8). The AP-1 complex contains the c-Jun subunit as indicated by the supershifted band in Fig. 7B. Abs to the c-Fos, FosB, JunB, or JunD subunits did not affect the AP-1 complex in nuclear extracts of mCD154-stimulated B cells. Although we could not detect complexes containing Fos subunits, there have been reports that c-Jun can heterodimerize with the ATF2 transcription factor (27, 28, 29), and it has been shown that CD40 stimulation of the EBV-transformed human B cell line Daudi results in ATF2 phosphorylation (30). Thus, we performed supershift EMSA analysis using anti-ATF2 Ab to determine whether c-Jun is in complex with ATF2. However, we saw no evidence that ATF2 was present in the nuclear complexes binding to the AP-1 probe (not shown). The aforementioned studies used in vitro translated ATF2, and as yet no association between physiologic levels of endogenous ATF2 and c-Jun in B cell nuclear extracts has been reported.
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Nuclear extracts of CH12.LX B cells contain proteins that complexed with the C/EBP DNA probe to form three distinct bands. The two faster migrating bands bound specifically to the putative C/EBP consensus binding sequence, while the slowest migrating band contains proteins that bound outside the consensus binding sequence as demonstrated by competition with unlabeled Wt or mutant C/EBP oligonucleotides (Fig. 8A). The C/EBP-specific complexes contained the C/EBP and C/EBP subunits as indicated by the supershifted bands in Fig. 8B. Although C/EBP has been reported to be a transcriptional activator, C/EBP, which does not contain a trans-activation domain, has been reported to repress C/EBP-dependent transcription (32). In contrast, another group showed that C/EBP does not inhibit C/EBP activity on the IL-6 promoter in response to LPS (33). Because our mutant A-CEBP protein can bind both the and subunits and block their function, we cannot yet determine whether either or both are specifically required for C/EBP-mediated transcriptional activity in CD40-stimulated B cells.
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Discussion |
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B, AP-1, and C/EBP sites) in the mouse IL-6 promoter region that are required for maximal CD40-induced IL-6 gene activation. In addition, a 70-nt region upstream of the aforementioned cis-acting elements contributes to the CD40 responsiveness of the IL-6 promoter, although the factor or factors that may bind in this region are not identified. This indicates that the regulation of the IL-6 gene by CD40 is multifaceted, complex, and involves the activation of several transcription factors, each of which plays a distinct role in CD40-mediated IL-6 transcriptional control.
We have previously reported that anti-CD40 mAb stimulation, which activates NF-B in mouse B cells, cannot induce B cell IL-6 protein production (3). However, a membrane-bound CD154 stimulus, which also activates NF-B, is effective in inducing IL-6 protein secretion (3). Therefore, activation and increased nuclear translocation of NF-B are not required for CD40-mediated IL-6 secretion in B cells (3). Interestingly, CH12.LX, like normal B cells, have a basal level of NF-B in the nucleus even in the unstimulated state. Thus, our earlier findings did not preclude the possibility that this basal nuclear NF-B plays a role in regulating IL-6 gene expression. In the present study we show that the NF-B binding site is required for basal and CD40-induced IL-6 gene activation (Fig. 3) and optimal IL-6 protein production (Fig. 6). In this regard, Hu and colleagues (33) have also reported that regulation of the IL-6 gene by overexpression of C/EBP requires an intact NF-B site.
We thus do not believe that our present findings are in conflict with those of our earlier study (3), because we postulate that the NF-B binding site is required for the binding of basal nuclear NF-B that is present in B cells. This would permit the structural and functional interaction of NF-B with the AP-1 and C/EBP transcription factors, two additional factors that have been shown in this study to be required for IL-6 gene activation following CD40 stimulation. In support of this idea, earlier reports demonstrated the cooperativity between NF-B and C/EBP in transcriptional activation of the IL-6 gene by other stimuli in non-B cells (33, 34, 35).
An earlier study reported that CD40 cross-linking on B cells activates the transcription factor AP-1, as demonstrated by gel shift assay, but the identity of the complex was not determined (36). The AP-1 transcription complex is composed of homo- and heterodimers of the bZIP group of DNA binding proteins belonging to members of the Jun and Fos family (26). We show here that the protein complex that binds the canonical AP-1 site in the mouse IL-6 promoter consists only of the c-Jun subunit, but not the other AP-1 subunits (Fig. 7B). Additionally, we do not see evidence for involvement of the ATF2 protein. Thus, c-Jun homodimers, which are transcriptionally active (26), may mediate CD40-induced AP-1 activity. Interestingly, we do not see an increase in DNA binding activity of c-Jun following CD40 stimulation (Fig. 7A), suggesting that CD40 may activate c-Jun independently of DNA binding. For example, the phosphorylation of c-Jun on serine 63 increases its trans-activation potential regardless of increased DNA binding (37, 38). In this regard, we see increased phosphorylation of c-Jun on serine 63 following CD40 stimulation (Fig. 7, C and D), and this correlates with activation of an AP-1-dependent reporter gene (Fig. 4A). This is in agreement with previous reports that showed increased c-Jun phosphorylation, but no increase in DNA binding activity in UV or phorbol ester-stimulated macrophages (39, 40). The phosphorylation of c-Jun on serine 63 is mediated by the JNK/stress-activated protein kinase/MAPK family members (26). Although it has been widely reported that CD40 stimulation activates JNK, as demonstrated by an in vitro JNK kinase assay using an artificial GST-c-Jun peptide substrate (14, 30, 31, 41, 42), this is the first report to our knowledge demonstrating CD40-mediated phosphorylation of endogenous c-Jun protein.
Surprisingly, we see c-Jun phosphorylation following membrane-bound CD154, but not anti-CD40 mAb stimulation (Fig. 7, C and D), although both stimuli can activate JNK activity in CH12.LX cells (14, 31). The inability of anti-CD40 mAb treatment to induce c-Jun phosphorylation could be explained by a lack of efficient nuclear translocation of JNK following Ab stimulation. Because c-Jun generally appears in the nucleus constitutively bound to promoters of target genes (39, 40), nuclear translocation of JNK would be required to phosphorylate c-Jun. Upon UV stimulation, activated JNK is reported to translocate to the nucleus and phosphorylate the transcription factor ELK-1 (43). Perhaps CD40-stimulated B cells differentially regulate the localization of JNK depending on the type of CD40-stimulating reagent used. Evidence for distinct subcellular localization of another MAPK family, ERK1/2, in B cells stimulated through the B cell Ag receptor or CD40 receptor has been reported (44), lending support to the idea that JNK may be subcellularly regulated.
CD40-mediated signal transduction pathways are thought to be mediated via adapter molecules called TRAFs that bind the cytoplasmic domain of CD40. TRAF2 and -6 have been reported to activate JNK, and in B cells appear to be redundant in this function (reviewed in Ref. 1). Only TRAF6 is required for CD40-mediated IL-6 secretion by B cells, while TRAF2 and -3 are dispensable (3). Because CD40-mediated IL-6 gene activation correlates with phosphorylation of nuclear c-Jun, it is tempting to speculate that TRAF6-mediated activation of JNK may result in its nuclear translocation and subsequent c-Jun phosphorylation, while TRAF2-mediated JNK activation may preferentially result in cytoplasmic localization. An alternative hypothesis is that JNK selectively phosphorylates distinct substrates depending upon the activation signal.
In addition to the AP-1 site, we show that the C/EBP site in the IL-6 promoter is required for CD40-mediated IL-6 gene activation. Members of the C/EBP family of transcription factors include C/EBP, C/EBP (NF-IL6), C/EBP, C/EBP, crp1, d/CEBP, and CHOP, which bind as homo- and heterodimers to DNA; however, B cells have been reported to express only the C/EBP and C/EBP subunits (45). We were unable to detect any increase in protein binding to the C/EBP site in response to CD40 stimulation, but could detect activation of an C/EBP-dependent reporter gene (Fig. 4B). In contrast, an earlier report showed that anti-CD40 mAb stimulation of a murine B cell line, M12, is unable to activate a C/EBP-dependent reporter gene (46). We also found that anti-CD40 mAb stimulation was unable to activate the C/EBP-dependent reporter gene, but stimulation with membrane-bound CD154 was effective. Thus, CD154 stimulation provides the signal needed to activate C/EBP. Because we could not detect an enhancement of binding to DNA in response to CD40 stimulation, other mechanisms of activating C/EBP trans-activation potential may be involved. Phosphorylation of C/EBP on serine 105, threonine 235, or serine 276 by a protein kinase C-dependent pathway, a MAPK-dependent pathway, or a Ca2+-calmodulin-dependent protein kinase pathway have all been reported to enhance the trans-activation potential of C/EBP (47, 48, 49).
In addition to regulating IL-6 gene expression, we found that AP-1 and C/EBP control CD40-mediated B cell IgM secretion (Fig. 6C) and CD80 surface expression (S.-Y. Woo and G. A. Bishop, unpublished observations). We have previously shown that CD40-mediated IgM production is NF-B dependent (14), and our finding in the present study suggests additional transcriptional regulation of this CD40 effector function. It is likely that additional CD40-mediated B cell effector functions are regulated by the AP-1 and C/EBP transcription factors, and this will be a subject of future studies.
During the review of this manuscript, we became aware of a recent report that examined transcriptional regulation of the IL-6 gene in mouse dendritic cells (DC). In agreement with our present findings in B cells, a requirement for AP-1, NF-B, and NF-IL-6 was reported previously (50). However, in CD40-stimulated DC, both Jun homodimers and Jun/Fos heterodimers are induced, and increased nuclear AP-1 binding complexes were seen, as well as a dependence upon the TRAF2 adapter protein (50). In addition, others have reported that in non-B cells (epithelial and fibroblast cell lines), CD40 induces gene transcription and protein production of IL-6 in an NF-B-dependent manner (51, 52). Our finding that basal NF-B, but not CD40-stimulated increases in NF-B, is required for CD40 to induce IL-6 transcription in B cells most likely reflects specific differences in CD40 signaling pathways between B cells, DC, and other cell types. For example, CD40 ligation induces ERK phosphorylation in human DC, but not human B cells (53), and in both mouse and human B cells, it has been shown that JNK is preferentially phosphorylated (30, 42). It has also been reported that B cells regulate CD40-induced IL-10 production in a p38-dependent manner, whereas DC do not depend upon p38 (53). We have also recently found that TRAF2 and -6 show considerable redundancy in several CD40-mediated pathways in B cells, whereas other cell types do not show this (B. S. Hostager and G. A. Bishop, unpublished observations). Thus, it is likely that activation of CD40-mediated signaling pathways involves overlapping, but distinct, mechanisms in different cell types.
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2 Address correspondence and reprint requests to Dr. Gail A. Bishop, Department of Microbiology, 3-570 Bowen Science Building, 51 Newton Road, University of Iowa, Iowa City, IA 52242. E-mail address: gail-bishop{at}uiowa.edu
3 Abbreviations used in this paper: SRE, serum-responsive element; ATF, activating transcription factor; DC, dendritic cells; DN, dominant negative; IPTG, isopropyl--D-thiogalactopyranoside; JNK, c-Jun N-terminal kinase; m, mouse; MAPK, mitogen-activated protein kinase; PNK, polynucleotide kinase; PVDF, polyvinylidene difluoride; RLU, relative light unit; TRAF, TNF receptor-associated factor; Wt, wild type.
Received for publication July 15, 2002. Accepted for publication January 10, 2003.
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B independent B cell IL-6 production. Eur. J. Immunol. 29:3855.[Medline]
B transcription factor. Mol. Cell. Biol. 10:2327.B activation by a distinct subset of CD40-mediated effector functions in B lymphocytes. J. Immunol. 162:2804. inhibits type I collagen synthesis through c/EBPs: implications for matrix remodeling. Mol. Cell. Biol. 20:912.B activation in lymphocytes. J. Immunol. 159:4898.[Abstract]
B activator Act1 in CD40-mediated signaling in epithelial cells. Proc. Natl. Acad. Sci. USA 99:9386.) is a transdominant negative inhibitor of C/EBP family transcriptional activators. Nucleic Acids Res. 23:4371.B synergistically activate transcription of the inflammatory cytokines, IL-6 and IL-8. Proc. Natl. Acad. Sci. USA 90:10193., NF-B, and CBF1 in IL-1-induced IL-6 synthesis by human rheumatoid fibroblast-like synoviocytes. J. Biol. Chem. 273:7620.B, AP-1 and NF-AT during B cell stimulation through the CD40 receptor. Int. Immunol. 7:151.B. J. Immunol. 153:4357.[Abstract]
B and CBF1. J. Biol. Chem. 8:8.
B pathway involving TRAFs. Oncogene 14:2899.[Medline]
B mobilization and induction of IL-6 production. J. Immunol. 155:4588.[Abstract]
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), pmIL-6 mutC/EBP (point mutation in the putative C/EBP site; 
), or pmIL-6 mutAP-1 (point mutation in the putative AP-1 site; 
). Cells were cotransfected with 10 µg of the three transcription factor plasmids shown in Fig. 5
) for 48 h. IL-6 was measured as described in A. The results are the mean ± SE for triplicate samples. C, Purified splenic B cells from Wt (
) were cultured with control CHO-K1 cells (
