cAMP-induced Interleukin-10 Promoter Activation Depends on CCAAT/Enhancer-binding Protein Expression and Monocytic Differentiation*

Susanne BrennerDagger, Susanna PröschDagger§, Katja Schenke-Layland, Ulrike Riese, Ulrike Gausmann, and Cornelia Platzer||

From the Institute of Anatomy II, Medical School, Friedrich Schiller University, D-07740 Jena, Germany, the § Department of Virology, Medical School Charité, Humboldt University, D-10098 Berlin, Germany, and the  Department of Genome Analysis, Institute of Molecular Biotechnology, D-07749 Jena, Germany

Received for publication, July 24, 2002, and in revised form, December 11, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The molecular mechanisms underlying the regulation of interleukin (IL)-10 transcription in monocytic cells by various stimuli during inflammation and the stress reaction are not fully understood. Recently, we provided evidence that stress-induced IL-10 promoter activation in monocytic cells is mediated by catecholamines via a cAMP-dependent signaling pathway including CREB/ATF (cAMP-responsive element binding protein/activating transcription factor) binding to two CRE motifs. However, the mutation of these sites diminished cAMP responsiveness by only 50%, suggesting a role for additional transcription factors and elements in the cAMP-dependent regulation of the human IL-10 promoter. Here, we analyze the functional role of one such factor, C/EBP, in two cell lines of myelomonocytic origin, THP-1 and HL-60, which are known to differ in their differentiation status and C/EBP protein content. We show that the level of basal as well as cAMP-stimulated IL-10 transcription depends on the expression of C/EBPalpha and beta  and their binding to three motifs in the promoter/enhancer region. The C/EBP5 motif, which is located between the TATA-box and the translation start point, is essential for the C/EBP-mediated constitutive and most of the cAMP-stimulated expression as its mutation nearly abolished IL-10 promoter activity. Our results suggest a dominant role of C/EBP transcription factors relative to CREB/ATF in tissue-specific and differentiation-dependent IL-10 transcription.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

An appropriate balance between pro- and anti-inflammatory cytokines during the immune response is critical in the resolution of many pathological conditions. In this context, interleukin (IL)1-10 is of special interest because of its anti-inflammatory and immunosuppressive properties. It is known that IL-10 synthesis in mononuclear cells can be induced or enhanced by various inflammatory stimuli and by sympathetic activation during the stress reaction or injury (1, 2), but the molecular mechanisms underlying the regulation of these processes are not fully understood. Recent reports demonstrate that lipopolysaccharide-induced IL-10 transcription may involve Sp1 activation by p38 mitogen-activated protein kinase (3) or the binding of Stat3 to the 5'-flanking promoter region of IL-10 (4). We showed previously that stress-induced IL-10 expression in monocytic cells is mediated by catecholamines via beta 2-adrenoreceptors linked to a cAMP-dependent signaling pathway (5), which activates CREB/ATF by phosphorylation. These transcription factors act through two cAMP-responsive elements (CRE), CRE1 and CRE4, located within 1308 bp upstream from the translation start codon of the IL-10 gene (6). Yet, site-directed mutagenesis of these two CRE sites resulting in loss of protein binding was not sufficient to abolish cAMP responsiveness with 50% activity remaining. However, we previously described an additional putative CRE site (CRE3) that binds proteins that do not belong to the CREB/ATF or the activating protein-1 families of transcription factors. Although mutation of this CRE3 motif had no effect on protein binding or on promoter stimulation by cAMP, deletion of CRE3 by 5'-exonuclease digestion resulted in a significant reduction in activity (6). Therefore, we concluded that other cAMP-responsive transcription factors bind to CRE3, even if mutated, or to hitherto unknown regulatory elements in this region during stress/cAMP-dependent activation of the IL-10 promoter. At first, we examined the involvement of three putative activating protein-2 sites exhibiting protein binding, but we were not able to confirm their specificity in supershift assays. We next investigated CCAAT/enhancer-binding proteins (C/EBP), because these transcription factors are shown to be involved in cAMP-dependent gene expression, e.g. of IL-6 and IL-1, as well as of enzymes of catabolic metabolism in the liver (7-9). It is well established that C/EBP proteins mediate cAMP responsiveness by indirect mechanisms, which include their increased expression and trans-location into the nucleus in response to elevated cAMP levels. Moreover, they possess domains that contain cAMP-inducible activities that are independent of direct phosphorylation by protein kinase A (PKA) (10, 11). Similar to CREB/ATF, C/EBP proteins belong to the group of basic region/leucine zipper transcription factors that have the potential to form heterodimers with each other and bind to motifs of either specificity (10, 12). C/EBP proteins are known to regulate adipocytic and monocytic differentiation, and their expression patterns vary depending on the state of cellular differentiation (13-15). Recently, we found that promonocytic THP-1 cells harbor significantly more C/EBPalpha than the myelomonocytic progenitor cell line HL-60 (16) and that THP-1 cells, unlike HL-60 cells, produce high levels of IL-10 protein after cAMP treatment (data not shown). To gain insight into the components involved in cAMP/stress-induced IL-10 expression, we asked whether C/EBP transcription factors contribute to promoter stimulation through the CRE3 site and four putative C/EBP binding sites, which were predicted by computer analysis.

Using THP-1 and HL-60 cells, we demonstrate that C/EBPalpha and beta  are critical in both basal and cAMP/stress-dependent regulation of IL-10 expression during monocytic differentiation. As targets, we identified three previously unknown C/EBP motifs in the promoter/enhancer of the IL-10 gene, one of which corresponds to the recently described CRE3 site.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Electrophoretic Mobility Shift and Assay (EMSA) and Supershift Assay-- Preparation of nuclear extracts and electrophoretic mobility shift assays were performed essentially as described elsewhere (16, 17). 10 µg of nuclear proteins extracted from THP-1 cells were incubated with 1 ng of radiolabeled oligonucleotides in a 20-µl reaction for 20 min at room temperature. The location of C/EBP binding motifs and the sequences of the used oligonucleotides are listed in Table I with the exception of CRE1 and CRE4, which we have described previously (6). For competition or supershifting, 100 ng of unlabeled oligonucleotide or 1.5 µl of the specific antiserum were added, respectively. The C/EBPalpha , beta , and delta  antisera and oligonucleotides corresponding to C/EBP and CRE consensus binding sites were obtained from Santa Cruz Biotechnology.

Plasmids-- Firefly luciferase reporter gene constructs containing 1308 bp (pGL2-1308) and 376 bp (pGL2-376) from the region upstream of the IL-10 translation start codon as well as the double CRE mutant of the 1308-bp fragment (pGL2-mutCRE1,4) have been described elsewhere (6). To obtain each single mutant pGL2-mutC/EBP1, pGL2-mutC/EBP3, and pGL2-mutC/EBP5, the plasmid pGL2-1308 was subjected to in vitro mutagenesis using the GeneEditor (Promega) and oligonucleotides as indicated in Table I. Successful mutagenesis was monitored by SpeI digestion, because the nucleotide exchange introduced an additional restriction site. The triple mutant pGL2-mutC/EBP1,3,5 was generated by cloning of a SacI fragment from pGL2-mutC/EBP3 into pGL2-mutC/EBP5 followed by digestion with Bsu36I and ligation of the resulting fragment into pGL2-mutC/EBP1. pGL2-AvrIImutC/EBP5 was made from pGL2-mutC/EBP5 by digestion with AvrII and SmaI followed by blunt-ended ligation after refilling of the 3' overhang and comprises 397 bp of DNA from upstream of the start codon. The sequences of triple mutated and AvrIImutC/EBP5 fragments were confirmed by sequence analysis (BigDye Terminator cycle sequencing kit, version 2.0, Applied Biosystems). The pMSV/EBPalpha and pMSV/EBPbeta expression plasmids that contain the respective cDNAs from rat under the control of the mouse sarcoma virus (MSV) long terminal repeat were kindly provided by S. McKnight (University of Texas Southwestern Medical Center, Dallas, TX). To obtain the MSV promoter control plasmid pMSV, the pMSV/EBPalpha plasmid was digested with NcoI followed by religation. The expression plasmids for rat p30, pcDNA3/p30, and CREB133 (pCMV/CREB) were supplied by A. Leutz (Max-Delbrück-Centrum, Berlin, Germany) and Clontech (BD Biosciences), respectively. The mock plasmid pcDNA3 was from Invitrogen and pCMV was obtained by digestion with XhoI and subsequent religation.

Cell Culture, Transfection, and Luciferase Assay-- THP-1 and HL-60 cells were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany) and maintained in RPMI 1640 medium with 10% fetal calf serum certified for low endotoxin and free of mycoplasma (Biochrom, Berlin, Germany). Transfections were performed by electroporation using 10 µg of reporter gene plasmid and 5 × 106 cells/0.25 ml in 0.4-cm electroporation cuvettes with a gene pulser (Gene zapper 450/2500, IBI, Cambridge, United Kingdom) at 1200 microfarads, 100 ohm, and 300 V. Transfection efficiencies were found to be similar in THP-1 and HL-60 cells as determined by the expression of the green fluorescent protein (pEGFP-C1, Clontech). Dot blot analysis also showed comparable results for different reporter gene constructs. In co-transfection experiments, 5 µg of expression plasmid were used unless stated otherwise. HL-60 cells transiently transfected with 5 µg of pMSV/EBPalpha were designated as HL-60/EBPalpha . To account for possible promoter interference of reporter gene and expression plasmid and to equalize DNA concentrations, the reaction mixture in the transfection cuvette was topped up with mock plasmid. After electroporation, 1 × 106 cells/ml/well were cultured in the absence or presence of 500 µM dibutyryl-cAMP (dbcAMP, Sigma) unless indicated otherwise. Cells were harvested 24 h after transfection and assayed for luciferase expression using the Promega luciferase assay kit and the Autolumat LB 953 (Berthold, Bad Wildbad, Germany). The expression level of C/EBPalpha in THP-1 and HL-60 was verified by Western blotting. Specific luciferase expression or promoter activity was expressed in arbitrary units (AU) and assessed by normalizing relative light units (raw data) for the protein content of the lysate measured by the Bradford protein assay (Sigma). The luciferase stimulation factor was calculated from the ratio of luciferase activity in dbcAMP-treated or co-transfected cells versus basal activity without dbcAMP treatment or transfected with the respective mock plasmid obtained in the same experimental setting. Conversely, the inhibition factor was evaluated as the ratio between the higher basal activity (AU with mock plasmid) and the reduced activity, resulting from expression of p30 or CREB133. An example for the calculation of the experimental results is given in Table II. Significance was estimated by the Man-Whitney-U test.

Computational Analysis of the IL-10 Promoter Region-- Human and murine genomic sequences were retrieved from the University of California, Santa Cruz Genome Browser (genome.ucsc.edu) datasets from April 2002 and February 2002, respectively. GenBankTM accession numbers for the other sequences are AF120030 (Marmota monax genomic IL-10 sequence), BG538741 (human EST), NM_000572 (human mRNA), Z30175 (human IL-10 promoter), NM_010548 (murine mRNA), and AF012909 (M. monax mRNA). Pairwise comparative analyses were performed with the program DNA Block Aligner (DBA) (18). The DBA input files were masked with Repeatmasker (ftp.genome.washington.edu/RM/RepeatMasker.html). The MatInspector Professional program (www.genomatix.de) was used to predict transcription factor binding sites (TFBS). All of the positions in the promoter/enhancer refer to the translation start site of IL-10.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

C/EBPalpha and beta  Bind to Three Motifs in the IL-10 Promoter/Enhancer Region-- In this study, we analyzed five putative C/EBP sites named C/EBP1 to C/EBP5 according to their position in the enhancer region upstream of the human IL-10 promoter (Table I). Four of these motifs (C/EBP2-5) were newly predicted by the MatInspector Professional program with core similarity of 0.7 and optimized matrix similarity. One motif, C/EBP1, is identical to the previously analyzed CRE3 site, which was shown not to be involved in CREB/ATF-induced promoter activation (6). Oligonucleotides harboring presumptive C/EBP motifs (Table I) were initially examined for their ability to compete with the consensus sequence using THP-1 nuclear extracts that are known to contain considerable amounts of C/EBP proteins. Unlabeled oligonucleotides consisting of C/EBP1, 3, 4, and 5 competed efficiently with the C/EBP consensus oligonucleotide for C/EBP protein binding (Fig. 1A, lanes 2, 4, 5, and 6), whereas C/EBP2 failed to compete effectively (lane 3). Supershift assays with labeled C/EBP1, 3, and 5 oligonucleotides revealed that DNA-protein complexes contained C/EBPalpha (Fig. 1B, lane 2, 4, and 9) and C/EBPbeta but not C/EBPdelta . Data obtained using C/EBP5 oligonucleotide are shown as a representative example for the reaction with antisera against C/EBPbeta and delta  (lanes 10 and 11). The proteins shifting the C/EBP4 oligonucleotide failed to react with C/EBPalpha or beta  antiserum (Fig. 1B, lanes 6 and 7). Since we found that the C/EBP1 motif, which is identical to the previously analyzed CRE3 site binds C/EBPalpha and beta , we wanted to exclude this possibility for the other CRE motifs. Competition experiments revealed that protein binding to labeled C/EBP3 does not interfere with unlabeled CRE consensus, CRE1 or CRE4 oligonucleotides (Fig. 1D, lanes 3, 4, and 6). Supershift analysis confirmed that protein complexes bound to CRE1 and CRE4 oligonucleotides do not contain C/EBPalpha or beta  (data not shown). The location of C/EBP binding sites in the 1308-bp IL-10 promoter/enhancer fragment is shown with respect to CRE motifs and the TATA box in Fig. 2.

                              
View this table:
[in this window]
[in a new window]
 
Table I
C/EBP sites, their positions in the 5'-untranslated region of the IL-10 gene and oligonucleotides used in EMSA and site-directed mutagenesis
Location of the elements is specified as the upstream position relative to translation start point. TFBS regions are underlined, and capital boldfaced letters indicate coincidence with the C/EBP consensus sequence in case of C/EBP1 with the CRE consensus sequence.


View larger version (61K):
[in this window]
[in a new window]
 
Fig. 1.   Protein binding to putative C/EBP motifs of the IL-10 promoter/enhancer. Radiolabeled oligonucleotides as indicated at the bottom were incubated with nuclear extracts from THP-1 cells and subjected to competition with a 100-fold molar excess of unlabeled oligonucleotide (panels A and C) or supershifted with specific antiserum (panel B). Competitor oligonucleotides and antibodies are shown at the top. The shifted DNA·C/EBP protein complex is indicated by one asterisk, and the supershifted complex is indicated by two asterisks. The same binding patterns with C/EBP1, C/EBP3, and C/EBP5 oligonucleotides were distinguishable. Therefore, the reaction of C/EBPbeta - and delta -specific antisera with C/EBP5 oligonucleotide is given as a representative example.


View larger version (7K):
[in this window]
[in a new window]
 
Fig. 2.   1308-bp fragment of the IL-10 promoter/enhancer region as cloned in pGL2-basic. The C/EBP and CRE motifs within the IL-10 promoter/enhancer region are delineated in relation to the TATA box (black box) and the IL-10 translation start (arrow) point. Gray circles and squares represent CRE and C/EBP motifs, respectively.

cAMP-stimulated IL-10 Promoter Activation in THP-1 Cells Involves C/EBPalpha -- To investigate the role of C/EBPalpha in cAMP-induced IL-10 promoter activation in THP-1 cells, we co-transfected increasing amounts of an expression plasmid for the 30-kDa truncated isoform of C/EBPalpha (pcDNA3/p30) along with the reporter plasmid pGL2-1308. This isoform acts as a partial C/EBPalpha antagonist by retaining its binding capacity but with significantly reduced trans-activation ability (19-21). Overexpression of p30 in THP-1 cells significantly reduced both the cAMP stimulation of the IL-10 promoter and its basal activity. The cAMP stimulation rates adjusted from the p30 inhibition of the basal promoter activity are shown in Fig. 3 (see also Table II). These results indicate that the binding of endogenous C/EBPalpha to the enhancer region and its trans-activation play a yet unknown role in constitutive and cAMP-stimulated IL-10 expression.


View larger version (7K):
[in this window]
[in a new window]
 
Fig. 3.   Influence of p30 overexpression on basal and cAMP-stimulated IL-10 promoter activity in THP-1 cells. Cells were co-transfected with pGL2-1308 along with increasing amounts of pcDNA3/p30 and incubated in the absence or presence of dbcAMP. Positive values indicate fold stimulation of the basal promoter activity obtained with the mock plasmid or the respective concentration of pcDNA/p30 by cAMP. Negative values represent the ratio (inhibition factor) between the basal and the reduced activity obtained with the mock plasmid and pcDNA/p30 in the absence of cAMP. Mean values ± S.D. of three independent experiments are shown.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Inhibition of basal and cAMP-stimulated promoter activity in THP-1 cells by p30
Results and the calculation of one typical co-transfection experiment using 10 µg of pGL2-1308 and 1 µg of p30 expression plasmid or the respective mock plasmid and 500 µM cAMP are shown. SF-stimulation factor is the ratio between promoter activity achieved by addition of cAMP and the basal activity obtained with the respective plasmid. IF-inhibition factor obtained by p30 expression is shown. SF in percent is determined on the premise that a factor of one means no stimulation (0%). Therefore, percentage from the reference in a given experiment is calculated from SF reduced by one. Basal activity in percent is calculated directly from arbitrary units.

cAMP-stimulated IL-10 Promoter Activation in THP-1 Cells Depends on the Integrity of C/EBP Binding Sites-- To understand the functional role of each individual C/EBP motif, we assessed the impact of C/EBP site mutations on cAMP-mediated IL-10 promoter activation in THP-1 cells. The oligonucleotides chosen for in vitro mutagenesis (Table I) were tested for loss of their C/EBP protein binding activity by EMSA (data not shown). In Fig. 4, A and B, the deleted or mutated promoter fragments are shown together with their residual promoter activity following cAMP-stimulation in THP-1 cells relative to the native fragment representing 100% activity. The mutation of C/EBP5, which lies closest to the translation start point, decreased cAMP-responsiveness to 22%, whereas the mutation of C/EBP3 and C/EBP1 reduced promoter activity to 42 and 57%, respectively. Subsequent experiments with the triple mutant pGL2-mutC/EBP1,3,5 revealed no further reduction compared with pGL2-mutC/EBP5. The truncated promoter/enhancer fragment comprising 376 bp upstream from the translation start point and lacking CRE and C/EBP motifs with the exception of C/EBP5 still showed 48% residual activity. Abolition of C/EBP5 in a deletion mutant of similar length (pGL2-AvrIImutC/EBP5 comprising 397 bp) reduced cAMP responsiveness further to 17%. These results emphasize a major functional role for the C/EBP5 motif in the trans-activation of the IL-10 gene by C/EBPalpha and beta . As outlined in panel C, the mutation of C/EBP motifs also reduced the basal promoter activity in THP-1 cells, suggesting that endogenous C/EBP proteins are involved in stimulated as well as in basal activity. Finally, as shown in Fig. 5, cAMP-induced promoter activity in THP-1 cells was almost completely abolished (residual activity 12%) when the binding of both C/EBPalpha and beta  as well as CREB/ATF was prevented by co-transfection of pGL2-mutCRE1,4 and pcDNA3/p30. Overexpression of p30 inhibited the cAMP effect on promoter activity of pGL2-1308 to the same degree as the destruction of all three C/EBP motifs (25 and 23% residual activity), but as expected, it had no effect on the activity of pGL2-mutC/EBP1,3,5 (Fig. 5).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   Involvement of C/EBP binding sites in basal and cAMP-stimulated IL-10 promoter activity in THP-1 cells and in C/EBPalpha -transfected HL-60 cells (HL-60/EBPalpha ). Luciferase reporter gene plasmids containing mutated or deleted promoter fragments (A) were transfected into THP-1 and into HL-60/EBPalpha cells. THP-1 cells were incubated in the presence or absence of dbcAMP. Promoter activity of the mutants is expressed as the percentage of cAMP-stimulated (B) and of basal (C) luciferase expression in THP-1 cells or as a fraction of C/EBPalpha -mediated luciferase expression obtained in HL-60/EBPalpha cells (D) with pGL2-1308 (representing 100%). Mean values ± S.D. of ten experiments are presented along. Gray circles and squares represent CRE and C/EBP motifs, respectively; blank symbols indicate mutated ones.


View larger version (8K):
[in this window]
[in a new window]
 
Fig. 5.   cAMP-induced IL-10 promoter activation is prevented by double mutation of CRE1 and CRE4 motifs and simultaneous overexpression of p30 in THP-1 cells. Cells transfected with pGL2-1308, pGL2-mutCRE1,4, or pGL2-mutC/EBP1,3,5 were either co-transfected along with pcDNA3/p30 or pcDNA3 and incubated in the presence of dbcAMP. Promoter activity is expressed as a percentage of cAMP-stimulated luciferase expression obtained with pGL2-1308 and pcDNA3. Results (mean values ± S.D.) of at least five independent experiments are shown.

C/EBPalpha Influences Constitutive IL-10 Promoter Activity in HL-60 and THP-1 Cells-- We recently showed that the pro-myelomonocytic cell line HL-60 contains significantly less endogenous C/EBPalpha compared with THP-1 cells (16). To study the influence of C/EBP on basal IL-10 promoter activity, we co-transfected pGL2-1308 along with increasing amounts of pMSV/EBPalpha in HL-60 cells. The data revealed that the expression of C/EBPalpha enhanced IL-10 promoter activity in a concentration-dependent manner up to 17-fold (Fig. 6). In contrast, C/EBPalpha expression over and above the high endogenous C/EBPalpha levels in THP-1 cells only increased IL-10 transcription approximately 3-fold (data not shown). Similar results were obtained by overexpression of C/EBPbeta (data not shown). The respective mock plasmid had no significant effect, indicating that the observed promoter activation resulted from C/EBP expression and was not the result of promoter interference of co-transfected plasmids. To examine the role of constitutively phosphorylated endogenous CREB in the C/EBP-mediated promoter activation, we co-transfected the expression plasmids for C/EBPalpha and CREB133 in HL-60 cells. This dominant negative CREB comprises a serine-to-alanine mutation, is therefore unable to be phosphorylated, and thus known to almost completely inhibit CREB-dependent trans-activation (22, 23). As shown in Fig. 7, the C/EBP-mediated IL-10 promoter activation was significantly impaired but not completely prevented by CREB133. Considering the reduced basal activity in the presence of CREB133, the genuine C/EBPalpha stimulation rate decreased from 9.6 (994/103) to 5.6 (208/37). The inhibitory effect of CREB133 was dependent on the molar concentration ratio of both co-transfected expression plasmids (data not shown). These data suggest that the phosphorylation of CREB is involved in basal as well as in C/EBPalpha -mediated promoter activation in HL-60 cells. To verify the role of C/EBPalpha in constitutive IL-10 promoter activity in HL-60 and THP-1 cells, we again co-transfected the p30 expression plasmid. As expected, p30 had a relatively minor influence on the basal promoter activity in original HL-60 cells because of their low endogenous C/EBP expression level. On the other hand, p30 significantly reduced constitutive IL-10 transcription in THP-1 cells in a manner comparable to HL-60/EBPalpha cells transiently expressing exogenous C/EBPalpha (Fig. 8). These results underline the role of C/EBPalpha in constitutive IL-10 expression.


View larger version (7K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of exogenous C/EBPalpha expression on basal IL-10 promoter activity in HL-60 cells. HL-60 cells were co-transfected with pGL2-1308 along with increasing amounts of pMSV/EBPalpha . Promoter activity obtained by supplying exogenous C/EBPalpha is expressed as multiples of the basal luciferase expression level in the presence of the mock plasmid. Results of at least three independent experiments are displayed.


View larger version (8K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of exogenous C/EBPalpha expression is inhibited by CREB133. Promoter activity (AU) from HL-60 cells transfected with pGL2-1308 and 5 µg of pMSV/EBPalpha or pMSV and additionally co-transfected with 5 µg of pCMV/CREB133 or pCMV is shown (mean values ± S.D. of seven independent experiments).


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 8.   Influence of p30 overexpression on basal IL-10 promoter activity in THP-1, HL-60, and HL-60/EBPalpha cells. pGL2-1308 was co-transfected into THP-1, HL-60, or HL-60/EBPalpha cells along with increasing amounts of pcDNA3/p30. Basal promoter activity is expressed as a percentage of the relative luciferase light units obtained with pGL2-1308 and pcDNA3 normalized for protein content. The mean values ± S.D. of at least three experiments are shown.

C/EBPalpha -dependent Constitutive IL-10 Promoter Activity Is Mediated Mainly through C/EBP5-- Our aim was to evaluate the role of each individual C/EBP motif in C/EBPalpha -dependent constitutive IL-10 expression. For this purpose, we used HL-60/EBPalpha cells because of the direct correlation between C/EBP expression and promoter activity. C/EBP5 had the greatest impact on C/EBP-dependent promoter stimulation, since its mutation nearly abolished promoter activity (residual activity 7%) while the mutation of C/EBP3 and C/EBP1 reduced it only to 38 and 56%, respectively (Fig. 4, panel D). Similar to cAMP-stimulated THP-1 cells, no further reduction was seen with the triple mutant pGL2-mutC/EBP1,3,5 compared with pGL2-mutC/EBP5 in HL-60/EBPalpha cells. The residual activity of the truncated promoter/enhancer fragment (pGL2-376) in HL-60/EBPalpha cells can be attributed to the effect of C/EBP5, because its mutation almost eliminated promoter activity. This confirms the essential role of C/EBP5 in the trans-activation of the IL-10 gene.

Efficient IL-10 Promoter Activation by cAMP Depends on C/EBPalpha -- From p30 overexpression experiments and functional analysis of C/EBP site mutations, we know that C/EBPalpha has a significant effect on cAMP stimulation in THP-1 cells. We now wanted to investigate whether promoter responsiveness is impaired in HL-60 cells because of the low C/EBP protein content in this cell line. In fact, comparing the cAMP responsiveness of the IL-10 promoter in both cell lines, we found that the elevation of intracellular cAMP levels had only a small effect in HL-60 cells, whereas in THP-1 cells, elevated cAMP levels induced a steep rise in promoter activity (Fig. 9). As already shown in Fig. 6, promoter activity was substantially increased, supplying HL-60 cells with exogenous C/EBPalpha . Starting from a higher level of constitutive promoter activity, it could be further stimulated by cAMP in HL-60/EBPalpha cells up to the same level as that achieved in THP-1 cells expressing endogenous C/EBPalpha (Fig. 9). Even though the cAMP stimulation rate remains nearly unchanged in HL-60 and HL-60/EBPalpha cells, together, our data indicate that for maximal promoter activity, both the presence of C/EBP protein and the phosphorylation of CREB are necessary.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 9.   The level of cAMP-stimulated IL-10 promoter activity depends on the expression of C/EBPalpha . pGL2-1308 was transfected into THP-1, HL-60, or HL-60/EBPalpha cells and incubated with increasing concentrations of dbcAMP. Promoter activity is expressed in AU assessed by normalizing relative luciferase light units for protein content. The graph represents results of ten independent experiments.

Comparative Analysis of the IL-10 Promoter/Enhancer Region-- The transcription start site of the human IL-10 gene has not yet been identified experimentally, but a human EST and 5' ends from mRNA of other species (Sus scrofa L20001, Ovis aries Z29362, and Trichosurus vulpecula AF026277) suggest that the transcription start site lies at position -59. Three IL-10 sequences were analyzed: human (hs), murine (mm), and the M. monax (ma) promoter/enhancer. All of the three sequences contain a TATA-box located at similar distances from the ATG: hs -89; mm -87; and ma -86. Pairwise comparison with DBA revealed two blocks of similarity that appeared in all of the three pairs: Block A at approximately -500 to -300 bp and Block B from -100 bp to the ATG start codon containing the TATA-box. These conserved sequences were analyzed by the MatInspector Professional program (core similarity 0.95, optimized matrix). Block A in human, mouse, and M. marmota contains one C/EBP binding site (binding site matrix CEBP.01 in hs at -439 to -452, in mm at -450 to -463, and in ma at -422 to -435) and one CREB binding site (binding site matrix ATF.01 in hs at -401 to -414, CREB.01 in mm at -415 to -422, and CREB.04 in ma at -385 to -396), corresponding to the C/EBP3 and CRE4 motifs analyzed in the human IL-10 promoter (Fig. 10). In Block B, another C/EPB site (C/EBP5 in the human sequence) was identified: in hs at -30 to -43 and in ma at -39 to -52 (both matched the C/EPB.01 matrix). Initially, in the murine sequence, this matrix was not detected, but after lowering the search parameters, the equivalent sequence was found at positions -39 to -52 (Fig. 10). The quality of local alignments (data not shown) and TFBS matrix similarity suggest a closer relationship of the human and the M. monax IL-10 gene versus human and mouse or mouse and M. monax.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 10.   Localization of DBA blocks and the predicted TFBS in the IL-10 promoter region. 1.5 kb upstream of the translation start codon of Homo sapiens (hsIL-10), Mus musculus (mmIL-10) and M. monax (maIL-10) were compared with the program DBA. The categories B, C, and D represent whole DBA block sequence identity (B, 70-80%; C, 80-90%; D, 90-100%). Boldface letters, nucleotides belonging to the whole TFBS region (gray boxes) identified by MatInspector; boldface capitalized letters, nucleotides of the TFBS core sequence defined by Genomatix. Only the parts of the blocks with the candidate TFBS are shown.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

With this study, we add new results to our previous findings concerning the important role of cAMP and cAMP-inducing agents in the trans-activation of the human IL-10 gene in monocytic cells. Here, we show that the binding of the transcription factors C/EBPalpha and beta  to three motifs in the IL-10 promoter/enhancer region contributes to basal activity of the IL-10 promoter and is essential for maximal cAMP stimulation in differentiated monocytic cells. Each of the individual C/EBP motifs appeared to have a different impact on the trans-activation of the IL-10 gene. The most important effector site seems to be C/EBP5. Because of the proximity of C/EBP5 to the TATA box, an interaction of C/EBPalpha bound to this motif with the basal transcription-initiation complex can be envisaged (24, 25). Conversely, this model explains the comparatively high trans-activating capacity of the 376-bp fragment lacking CRE1, CRE4, C/EBP1, and C/EBP3 sites but retaining C/EBP5. The C/EBP3 motif, close to the main CRE4 site, had a moderate influence, whereas C/EBP1 seems to be less important. C/EBP1 coincides with the CRE3 site that failed to bind CREB/ATF (6) despite its greater sequence similarity to the CRE rather than to the C/EBP consensus motif. The binding of C/EBP to non-consensus CRE sites and the functional substitution of CREB by C/EBP family members have been described previously (7, 26). It is not surprising that the functionally relevant motifs C/EBP3, CRE4, and C/EBP5 fall into regions that are evolutionary conserved between human, mouse, and M. monax, while the less important C/EBP1 and CRE1 sites are not conserved. These findings together with the fact that plasmids with single site mutation of C/EBP3 and CRE4 (6) retained similar cAMP-responsiveness as pGL2-376, bearing only the intact C/EBP5 motif hint at C/EBP-CREB interaction at multiple sites in a hierarchical and cooperative manner. We assume that the function of C/EBP3 requires prior occupation of the downstream C/EBP5 motif and the proximal CRE4 site, which implicates potential DNA looping and/or co-activator protein binding (19, 25, 27).

To study the role of C/EBPalpha in basal and cAMP-stimulated promoter activation, we used the truncated 30-kDa C/EBPalpha isoform (p30), which bears a conserved binding domain but lacks N-terminal regions required for its trans-activation and differentiation potential (19-21). Overexpressed p30 reduced the cAMP stimulation rate beyond the inhibition of basal activity and abolished the remaining promoter responsiveness of the double CRE mutant by competition with endogenous p42 C/EBPalpha in THP-1 cells. Data obtained from structure/function analysis of C/EBPalpha provide evidence that the mechanisms of constitutive and cAMP-inducible trans-activation are different and involve distinct albeit overlapping domains in the N-terminal portion of the protein (amino acids 55-86 and 52-124, respectively), which are both deleted in p30 (9, 19, 28, 29). The cAMP-mediated promoter stimulation was consistently stronger in THP-1 cells than in HL-60 cells, in agreement with their higher differentiation status and endogenous C/EBPalpha /beta expression levels. When supplementing HL-60 cells with exogenous C/EBPalpha , we found that basal/constitutive promoter activity rose in a concentration-dependent manner and the cAMP response in HL-60/EBPalpha cells reached similar levels as in THP-1 cells. However, given the substantially higher basal level, the cAMP stimulation rate seems to be independent of C/EBPalpha overexpression. The reason for this remains unclear. We suggest that the increasing stimulation of the basal activity in THP-1 cells in response to rising cAMP concentrations involves enhanced expression, trans-location, and the binding of C/EBPalpha /beta as well as phosphorylation of CREB, C/EBP and cofactors, and their interaction (11, 30-32). The model of transient overexpression of C/EBPalpha in HL-60 cells can only partially reproduce these processes. Co-transfection experiments with the dominant negative inhibitor of CREB in HL-60/EBPalpha cells revealed that trans-activation by C/EBPalpha to some extent depends on phosphorylated CREB being constitutively present in low amounts. Beyond this, C/EBPalpha overexpression seems to up-regulate the IL-10 promoter activity by itself. Taken together, our data from the mutation and co-expression experiments with p30 and CREB133 indicate a co-operative interaction of CREB and C/EBP transcription factors both in basal and in cAMP-stimulated promoter activation. In contrast to CREB, the cAMP-inducible domain of C/EBPalpha lacks a PKA phosphorylation site; thus, the mechanism of its activation by cAMP is yet unclear (33). It has been suggested that a co-activator could interact with the cAMP-inducible domain of C/EBP transcription factors only after it itself has been phosphorylated by PKA. This model is similar to that of CREB activation recruiting CREB-binding protein (CBP)/p300 after PKA phosphorylation with the exception that phosphorylation of the co-activator rather than the DNA-binding protein would regulate the interaction (9, 28, 31). In addition, CBP/p300 is a possible mediator between CREB and C/EBP. Indeed, CBP/p300 comprises a binding domain for C/EBPalpha /beta (amino acids 1752-1859), which is distinct from its CREB binding domain (amino acids 552-660) (34). On the basis of our experimental data, we conclude that CREB/ATF mediates a relatively low activating potential in the absence of C/EBPalpha /beta proteins, which are necessary for maximal IL-10 promoter activation. In our view, multiple enhancer binding proteins need to be available simultaneously in differentiated monocytes to achieve stable binding site occupancy required for maximally activated IL-10 transcription. This suggestion is in agreement with the observation that C/EBPalpha expression is tissue-specific and varies during differentiation, while CREB/ATF is ubiquitously expressed (35, 36). Therefore, C/EBP-mediated transcription of a gene, in particular IL-10, is regulated in a tissue-specific manner and depends on the state of differentiation of a given cell. Experimental data from a mouse model, which revealed immediate IL-10 production in response to lipopolysaccharide and cAMP-elevating agents by hepatocytes known to contain high amounts of C/EBPalpha , support this hypothesis (37, 38).

In contrast to pro-inflammatory cytokines, which are mainly regulated by transcription factors such as NFkB and activating protein-1, the transcription of the prototypic anti-inflammatory cytokine IL-10 in monocytic cells, as shown here, is activated by cAMP or cAMP-inducing agents, e.g. catecholamines during sympathetic activation through transcription factors C/EBP and CREB. The mechanism of their interaction remains to be investigated.

    ACKNOWLEDGEMENTS

We thank S. L. McKnight (University of Texas Southwestern Medical Center, Dallas, TX) for providing the expression plasmids for C/EBPalpha and beta , A. Leutz (Max-Delbrück-Centrum, Berlin, Germany) for pcDNA3/p30, O. Coutelle for critical reading, and M. Platzer for assistance in the preparation of this paper.

    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft Pl 163/4-3.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.

Dagger Both authors contributed equally to this work.

|| To whom correspondence should be addressed. Tel.: 49-3641-938570; Fax: 49-3641-938552; E-mail: cplatzer@mti-n.uni-jena.de.

Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M207448200

    ABBREVIATIONS

The abbreviations used are: IL, interleukin; CREB, cAMP-responsive element binding protein; ATF, activating transcription factor; AU, arbitrary units; CBP, CREB-binding protein; C/EBP, CCAAT/enhancer-binding protein; CRE, cAMP responsive element; dbcAMP, N6,2'-O-butyryl-cAMP; EMSA, electrophoretic mobility shift assay; MSV, mouse sarcoma virus; PKA, protein kinase A; TFBS, transcription factor binding site; DBA, DNA block aligner; hs, H. sapiens; mm, M. musculus; ma, M. monax.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Woiciechowsky, C., Asadullah, K., Nestler, D., Eberhardt, B., Platzer, C., Schoning, B., Glockner, F., Lanksch, W. R., Volk, H. D., and Döcke, W. D. (1998) Nat. Med. 4, 808-813[Medline] [Order article via Infotrieve]
2. Ziegler-Heitbrock, H. W. (1995) J. Inflamm. 45, 13-26[Medline] [Order article via Infotrieve]
3. Ma, W., Lim, W., Gee, K., Aucoin, S., Nandan, D., Kozlowski, M., Diaz-Mitoma, F., and Kumar, A. (2001) J. Biol. Chem. 276, 13664-13674[Abstract/Free Full Text]
4. Benkhart, E. M., Siedlar, M., Wedel, A., Werner, T., and Ziegler-Heitbrock, H. W. (2000) J. Immunol. 165, 1612-1617[Abstract/Free Full Text]
5. Platzer, C., Döcke, W., Volk, H., and Prösch, S. (2000) J. Neuroimmunol. 105, 31-38[CrossRef][Medline] [Order article via Infotrieve]
6. Platzer, C., Fritsch, E., Elsner, T., Lehmann, M. H., Volk, H. D., and Prösch, S. (1999) Eur. J. Immunol. 29, 3098-3104[CrossRef][Medline] [Order article via Infotrieve]
7. Tsukada, J., Saito, K., Waterman, W. R., Webb, A. C., and Auron, P. E. (1994) Mol. Cell. Biol. 14, 7285-7297[Abstract]
8. Grassl, C., Luckow, B., Schlondorff, D., and Dendorfer, U. (1999) J. Am. Soc. Nephrol. 10, 1466-1477[Abstract/Free Full Text]
9. Wilson, H. L., and Roesler, W. J. (2002) Mol. Cell. Endocrinol. 188, 15-20[CrossRef][Medline] [Order article via Infotrieve]
10. Akira, S., and Kishimoto, T. (1997) Adv. Immunol. 65, 1-46[Medline] [Order article via Infotrieve]
11. Berrier, A., Siu, G., and Calame, K. (1998) J. Immunol. 161, 2267-2275[Abstract/Free Full Text]
12. Vinson, C. R., Sigler, P. B., and McKnight, S. L. (1989) Science 246, 911-916[Medline] [Order article via Infotrieve]
13. Umek, R. M., Friedman, A. D., and McKnight, S. L. (1991) Science 251, 288-292[Medline] [Order article via Infotrieve]
14. Scott, L. M., Civin, C. I., Rorth, P., and Friedman, A. D. (1992) Blood 80, 1725-1735[Abstract]
15. Tenen, D. G., Hromas, R., Licht, J. D., and Zhang, D. E. (1997) Blood 90, 489-519[Free Full Text]
16. Prösch, S., Heine, A. K., Volk, H. D., and Krüger, D. H. (2001) J. Biol. Chem. 276, 40712-40720[Abstract/Free Full Text]
17. Prösch, S., Staak, K., Stein, J., Liebenthal, C., Stamminger, T., Volk, H. D., and Krüger, D. H. (1995) Virology. 208, 197-206[CrossRef][Medline] [Order article via Infotrieve]
18. Jareborg, N., Birney, E., and Durbin, R. (1999) Genome Res. 9, 815-824[Abstract/Free Full Text]
19. Erickson, R. L., Hemati, N., Ross, S. E., and MacDougald, O. A. (2001) J. Biol. Chem. 276, 16348-16355[Abstract/Free Full Text]
20. Lekstrom-Himes, J., and Xanthopoulos, K. G. (1998) J. Biol. Chem. 273, 28545-28548[Abstract/Free Full Text]
21. Lin, F. T., MacDougald, O. A., Diehl, A. M., and Lane, M. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9606-9610[Abstract]
22. Alberts, A. S., Arias, J., Hagiwara, M., Montminy, M. R., and Feramisco, J. R. (1994) J. Biol. Chem. 269, 7623-7630[Abstract/Free Full Text]
23. Roesler, W. J., Graham, J. G., Kolen, R., Klemm, D. J., and McFie, P. J. (1995) J. Biol. Chem. 270, 8225-8232[Abstract/Free Full Text]
24. Pedersen, T. A., Kowenz-Leutz, E., Leutz, A., and Nerlov, C. (2001) Genes Dev. 15, 3208-3216[Abstract/Free Full Text]
25. Nerlov, C., and Ziff, E. B. (1995) EMBO J. 14, 4318-4328[Abstract]
26. Bakker, O., and Parker, M. G. (1991) Nucleic Acids Res. 19, 1213-1217[Abstract]
27. Hernandez-Munain, C., Roberts, J. L., and Krangel, M. S. (1998) Mol. Cell. Biol. 18, 3223-3233[Abstract/Free Full Text]
28. Wilson, H. L., McFie, P. J., and Roesler, W. J. (2001) Mol. Cell. Endocrinol. 181, 27-34[CrossRef][Medline] [Order article via Infotrieve]
29. Roesler, W. J., Park, E. A., and McFie, P. J. (1998) J. Biol. Chem. 273, 14950-14957[Abstract/Free Full Text]
30. Metz, R., and Ziff, E. (1991) Genes Dev. 5, 1754-1766[Abstract]
31. Tae, H. J., Zhang, S., and Kim, K. H. (1995) J. Biol. Chem. 270, 21487-21494[Abstract/Free Full Text]
32. Roesler, W. J. (2001) Annu. Rev. Nutr. 21, 141-165[CrossRef][Medline] [Order article via Infotrieve]
33. Mahoney, C. W., Shuman, J., McKnight, S. L., Chen, H. C., and Huang, K. P. (1992) J. Biol. Chem. 267, 19396-19403[Abstract/Free Full Text]
34. Mink, S., Haenig, B., and Klempnauer, K. H. (1997) Mol. Cell. Biol. 17, 6609-6617[Abstract]
35. Roesler, W. J. (2000) Mol. Cell. Endocrinol. 162, 1-7[CrossRef][Medline] [Order article via Infotrieve]
36. Sieweke, M. H., and Graf, T. (1998) Curr. Opin. Genet. Dev. 8, 545-551[CrossRef][Medline] [Order article via Infotrieve]
37. Jilg, S., Barsig, J., Leist, M., Kusters, S., Volk, H. D., and Wendel, A. (1996) J. Pharmacol. Exp. Ther. 278, 421-431[Abstract]
38. Schrem, H., Klempnauer, J., and Borlak, J. (2002) Pharmacol. Rev. 54, 129-158[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.