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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/EBPα and β 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. 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/EBPα and β 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. 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) 1The 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, N 6,2′-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 -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 (1Woiciechowsky C. Asadullah K. Nestler D. Eberhardt B. Platzer C. Schoning B. Glockner F. Lanksch W.R. Volk H.D. Döcke W.D. Nat. Med. 1998; 4: 808-813Google Scholar, 2Ziegler-Heitbrock H.W. J. Inflamm. 1995; 45: 13-26Google Scholar), 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 (3Ma W. Lim W. Gee K. Aucoin S. Nandan D. Kozlowski M. Diaz-Mitoma F. Kumar A. J. Biol. Chem. 2001; 276: 13664-13674Google Scholar) or the binding of Stat3 to the 5′-flanking promoter region of IL-10 (4Benkhart E.M. Siedlar M. Wedel A. Werner T. Ziegler-Heitbrock H.W. J. Immunol. 2000; 165: 1612-1617Google Scholar). We showed previously that stress-induced IL-10 expression in monocytic cells is mediated by catecholamines via β2-adrenoreceptors linked to a cAMP-dependent signaling pathway (5Platzer C. Döcke W. Volk H. Prösch S. J. Neuroimmunol. 2000; 105: 31-38Google Scholar), 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 (6Platzer C. Fritsch E. Elsner T. Lehmann M.H. Volk H.D. Prösch S. Eur. J. Immunol. 1999; 29: 3098-3104Google Scholar). 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 (6Platzer C. Fritsch E. Elsner T. Lehmann M.H. Volk H.D. Prösch S. Eur. J. Immunol. 1999; 29: 3098-3104Google Scholar). 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 (7Tsukada J. Saito K. Waterman W.R. Webb A.C. Auron P.E. Mol. Cell. Biol. 1994; 14: 7285-7297Google Scholar, 8Grassl C. Luckow B. Schlondorff D. Dendorfer U. J. Am. Soc. Nephrol. 1999; 10: 1466-1477Google Scholar, 9Wilson H.L. Roesler W.J. Mol. Cell. Endocrinol. 2002; 188: 15-20Google Scholar). 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) (10Akira S. Kishimoto T. Adv. Immunol. 1997; 65: 1-46Google Scholar, 11Berrier A. Siu G. Calame K. J. Immunol. 1998; 161: 2267-2275Google Scholar). 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 (10Akira S. Kishimoto T. Adv. Immunol. 1997; 65: 1-46Google Scholar, 12Vinson C.R. Sigler P.B. McKnight S.L. Science. 1989; 246: 911-916Google Scholar). C/EBP proteins are known to regulate adipocytic and monocytic differentiation, and their expression patterns vary depending on the state of cellular differentiation (13Umek R.M. Friedman A.D. McKnight S.L. Science. 1991; 251: 288-292Google Scholar, 14Scott L.M. Civin C.I. Rorth P. Friedman A.D. Blood. 1992; 80: 1725-1735Google Scholar, 15Tenen D.G. Hromas R. Licht J.D. Zhang D.E. Blood. 1997; 90: 489-519Google Scholar). Recently, we found that promonocytic THP-1 cells harbor significantly more C/EBPα than the myelomonocytic progenitor cell line HL-60 (16Prösch S. Heine A.K. Volk H.D. Krüger D.H. J. Biol. Chem. 2001; 276: 40712-40720Google Scholar) 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/EBPα and β 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. Preparation of nuclear extracts and electrophoretic mobility shift assays were performed essentially as described elsewhere (16Prösch S. Heine A.K. Volk H.D. Krüger D.H. J. Biol. Chem. 2001; 276: 40712-40720Google Scholar, 17Prösch S. Staak K. Stein J. Liebenthal C. Stamminger T. Volk H.D. Krüger D.H. Virology. 1995; 208: 197-206Google Scholar). 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 (6Platzer C. Fritsch E. Elsner T. Lehmann M.H. Volk H.D. Prösch S. Eur. J. Immunol. 1999; 29: 3098-3104Google Scholar). For competition or supershifting, 100 ng of unlabeled oligonucleotide or 1.5 μl of the specific antiserum were added, respectively. The C/EBPα, β, and δ antisera and oligonucleotides corresponding to C/EBP and CRE consensus binding sites were obtained from Santa Cruz Biotechnology.Table IC/EBP sites, their positions in the 5′-untranslated region of the IL-10 gene and oligonucleotides used in EMSA and site-directed mutagenesisSiteSite positionOligonucleotidesC/EBP1 (CRE3)−865/−852tgtacaggTGAtGTaAcatctctgmutC/EBP1........gac.agtc........C/EBP2−584/−574gggctgcTTGgGaAcIttgaggC/EBP3−452/−439caattaTTtCtCAATcccattmutC/EBP3......gactagtc.......C/EBP4−435/−418ttgtattctggaaTGgGCAATttgtcC/EBP5−43/−30cttgctcTTGCaaAAccaaaccmutC/EBP5.......gact.gtc.......C/EBPconstgcagaTTGCGCAATtctgcaCREconsagagattgccTGACGTCAgagagctgaLocation of the elements is specified as the upstream position relative to translation start point. TFBS regions are underlined, and with the C/EBP consensus in of with the CRE consensus in a of the elements is specified as the upstream position relative to translation start point. TFBS regions are underlined, and with the C/EBP consensus in of with the CRE consensus gene 1308 bp and bp from the region upstream of the IL-10 translation start codon as well as the CRE of the have described elsewhere (6Platzer C. Fritsch E. Elsner T. Lehmann M.H. Volk H.D. Prösch S. Eur. J. Immunol. 1999; 29: 3098-3104Google Scholar). To each and the was to in mutagenesis the and oligonucleotides as in Table mutagenesis was by because the an additional site. The was by of a from into by digestion with and of the resulting into was from by digestion with by after of the and bp of DNA from upstream of the start The sequences of and were by The and expression that contain the from the of the mouse sarcoma were provided by S. McKnight of To the promoter the was with by The expression for and were by A. and respectively. The was from and was obtained by digestion with and THP-1 and HL-60 cells were from and in with for and of were performed by 10 μg of gene and in with a gene at 100 and were found to be in THP-1 and HL-60 cells as by the expression of the protein showed results for gene In μg of expression were used HL-60 cells with μg of were as To for promoter of gene and expression and to DNA the reaction in the was with 1 were in the or of were after and for expression the and the The expression level of C/EBPα in THP-1 and HL-60 was by expression or promoter activity was in arbitrary and by relative for the protein of the by the protein The stimulation factor was from the of activity in or cells basal activity treatment or with the obtained in the the factor was as the between the basal activity with and the resulting from expression of or An for the of the results is in Table was by the of basal and cAMP-stimulated promoter activity in THP-1 cells by and the of one 10 μg of and 1 μg of expression or the and cAMP are factor is the between promoter activity by of cAMP and the basal activity obtained with the factor obtained by expression is in is on the that a factor of one no stimulation Therefore, from the in a is from by activity in is from arbitrary in a and the of one 10 μg of and 1 μg of expression or the and cAMP are factor is the between promoter activity by of cAMP and the basal activity obtained with the factor obtained by expression is in is on the that a factor of one no stimulation Therefore, from the in a is from by activity in is from arbitrary and sequences were from the of Santa Cruz from and respectively. for the other sequences are monax IL-10 IL-10 and monax were performed with the DNA E. R. 1999; Scholar). The were with The was used to transcription factor binding sites of the positions in the promoter/enhancer to the translation start site of In this we putative C/EBP sites to C/EBP5 to their position in the region upstream of the human IL-10 promoter of these motifs were predicted by the with of and motif, is to the previously CRE3 which was shown not to be involved in promoter activation (6Platzer C. Fritsch E. Elsner T. Lehmann M.H. Volk H.D. Prösch S. Eur. J. Immunol. 1999; 29: 3098-3104Google Scholar). C/EBP motifs were examined for their to with the consensus THP-1 nuclear extracts that are known to contain of C/EBP oligonucleotides of and with the C/EBP consensus oligonucleotide for C/EBP protein binding 1 and to assays with and oligonucleotides that C/EBPα 1 and and but not obtained C/EBP5 oligonucleotide are shown as a for the reaction with antisera and δ 10 The proteins the oligonucleotide to with C/EBPα or β antiserum 1 and we found that the motif, which is to the previously CRE3 site binds C/EBPα and β, we to this for the other CRE motifs. that protein binding to not with unlabeled CRE CRE1 or oligonucleotides 1 and that protein to CRE1 and oligonucleotides do not contain C/EBPα or β (data not shown). The location of C/EBP binding sites in the IL-10 promoter/enhancer is shown with to CRE motifs and the in of the IL-10 promoter/enhancer region as in The C/EBP and CRE motifs within the IL-10 promoter/enhancer region are in to the and the IL-10 translation start point. CRE and C/EBP To the role of C/EBPα in IL-10 promoter activation in THP-1 cells, we of an expression for the of C/EBPα with the as a C/EBPα by its binding but with significantly J. Biol. Chem. 2001; 276: Scholar, J. J. Biol. Chem. 1998; Scholar, U. S. A. 90: Scholar). of in THP-1 cells significantly both the cAMP stimulation of the IL-10 promoter and its basal activity. The cAMP stimulation from the of the basal promoter activity are shown in These results that the binding of C/EBPα to the region and its a unknown role in constitutive and cAMP-stimulated IL-10 To the functional role of each C/EBP motif, we the of C/EBP site on IL-10 promoter activation in THP-1 The oligonucleotides for in were for loss of their C/EBP protein binding activity by EMSA (data not shown). In A and the or promoter are shown with their promoter activity in THP-1 cells relative to the activity. The mutation of which to the translation start point, to the mutation of and promoter activity to and respectively. with the no reduction with The promoter/enhancer bp upstream from the translation start and CRE and C/EBP motifs with the exception of C/EBP5 showed activity. of C/EBP5 in a deletion of cAMP responsiveness to These results a functional role for the C/EBP5 motif in the of the IL-10 gene by C/EBPα and As in the mutation of C/EBP motifs the basal promoter activity in THP-1 cells, suggesting that C/EBP proteins are involved in as well as in basal activity. as shown in promoter activity in THP-1 cells was abolished activity the binding of both C/EBPα and β as well as CREB/ATF was by of and of the cAMP effect on promoter activity of to the as the of three C/EBP motifs and but as had no effect on the activity of IL-10 promoter activation is by mutation of CRE1 and motifs and of in THP-1 with or were either with or and incubated in the of activity is as a of cAMP-stimulated expression obtained with and of at independent are We recently showed that the cell line HL-60 significantly C/EBPα with THP-1 cells (16Prösch S. Heine A.K. Volk H.D. Krüger D.H. J. Biol. Chem. 2001; 276: 40712-40720Google Scholar). To the of C/EBP on basal IL-10 promoter we with of in HL-60 The that the expression of C/EBPα enhanced IL-10 promoter activity in a to In C/EBPα expression and the high C/EBPα levels in THP-1 cells only increased IL-10 transcription (data not shown). Similar results were obtained by of (data not shown). The had no significant that the promoter activation resulted from C/EBP expression and was not the of promoter of To the role of in the C/EBP-mediated promoter we the expression for C/EBPα and in HL-60 dominant a is to be and known to J. M. J. Biol. Chem. 1994; Scholar, W.J. R. J. Biol. Chem. 1995; Scholar). As shown in the C/EBP-mediated IL-10 promoter activation was significantly but not by the basal activity in the of the C/EBPα stimulation from to The effect of was on the of both expression (data not shown). These suggest that the phosphorylation of is involved in basal as well as in promoter activation in HL-60 To the role of C/EBPα in constitutive IL-10 promoter activity in HL-60 and THP-1 cells, we the expression As had a on the basal promoter activity in HL-60 cells because of their C/EBP expression the other significantly constitutive IL-10 transcription in THP-1 cells in a to cells C/EBPα These results the role of C/EBPα in constitutive IL-10 of C/EBP expression is by activity from HL-60 cells with and μg of or and with μg of or is shown of independent of on basal IL-10 promoter activity in HL-60, and was into HL-60, or cells with of promoter activity is as a of the relative obtained with and for protein content. The of at three are Our was to the role of each C/EBP motif in constitutive IL-10 For this we used cells because of the direct between C/EBP expression and promoter activity. C/EBP5 had the on promoter its mutation nearly abolished promoter activity activity the mutation of and only to and Similar to cAMP-stimulated THP-1 cells, no reduction was with the with in The activity of the promoter/enhancer in cells can be to the effect of because its mutation promoter activity. the essential role of C/EBP5 in the of the IL-10 and functional of C/EBP site we that C/EBPα a significant effect on cAMP stimulation in THP-1 We to whether promoter responsiveness is in HL-60 cells because of the C/EBP protein in this cell In the cAMP responsiveness of the IL-10 promoter in both cell we found that the of cAMP levels had only a effect in HL-60 cells, in THP-1 cells, elevated cAMP levels induced a in promoter activity As shown in promoter activity was HL-60 cells with from a level of constitutive promoter be by cAMP in cells to the level as that in THP-1 cells C/EBPα the cAMP stimulation nearly in HL-60 and cells, that for promoter both the of C/EBP protein and the phosphorylation of are The transcription start site of the human IL-10 gene not identified but a human and from of other and suggest that the transcription start site at position IL-10 sequences were human and the M. monax of the three sequences contain a TATA-box located at from the hs and ma with two of that in of the three A at to bp and from bp to the start codon the These sequences were by the A in and M. one C/EBP binding site site in hs at to at to and in ma at to and one binding site site at to in mm at to and in ma at to corresponding to the and motifs in the human IL-10 promoter In site in the human was in hs at to and in ma at to the in the this was not but after the the was found at positions to The of (data not and TFBS suggest a of the human and the M. monax IL-10 gene human and mouse or mouse this we results to the role of cAMP and in the of the human IL-10 gene in monocytic Here, we show that the binding of the transcription factors C/EBPα and β to three motifs in the IL-10 promoter/enhancer region to basal activity of the IL-10 promoter and is essential for cAMP stimulation in monocytic of the C/EBP motifs to have a on the of the IL-10 The most site to be of the of C/EBP5 to the an of C/EBPα to this motif with the basal can be E. A. C. 2001; Scholar, C. J. 1995; 14: Scholar). this the high of the CRE4, and sites but The motif, to the had a to be with the CRE3 site that to bind CREB/ATF (6Platzer C. Fritsch E. Elsner T. Lehmann M.H. Volk H.D. Prösch S. Eur. J. Immunol. 1999; 29: 3098-3104Google Scholar) its to the CRE than to the C/EBP consensus The binding of C/EBP to CRE sites and the functional of by C/EBP have described previously (7Tsukada J. Saito K. Waterman W.R. Webb A.C. Auron P.E. Mol. Cell. Biol. 1994; 14: 7285-7297Google Scholar, 1991; Scholar). It is not that the motifs CRE4, and C/EBP5 into regions that are between and M. the and CRE1 sites are not These with the that with site mutation of and (6Platzer C. Fritsch E. Elsner T. Lehmann M.H. Volk H.D. Prösch S. Eur. J. Immunol. 1999; 29: 3098-3104Google Scholar) as only the C/EBP5 motif at at sites in a and We that the of of the C/EBP5 motif and the which potential DNA protein binding J. Biol. Chem. 2001; 276: Scholar, C. J. 1995; 14: Scholar, C. Mol. Cell. Biol. 1998; Scholar). To the role of C/EBPα in basal and cAMP-stimulated promoter we used the C/EBPα which a binding but regions for its and differentiation potential J. Biol. Chem. 2001; 276: Scholar, J. J. Biol. Chem. 1998; Scholar, U. S. A. 90: Scholar). the cAMP stimulation the of basal activity and abolished the promoter responsiveness of the CRE by competition with C/EBPα in THP-1 obtained from of C/EBPα evidence that the mechanisms of constitutive and cAMP-inducible are and involve domains in the of the protein and which are both in H.L. Roesler W.J. Mol. Cell. Endocrinol. 2002; 188: 15-20Google Scholar, J. Biol. Chem. 2001; 276: Scholar, H.L. Roesler W.J. Mol. Cell. Endocrinol. 2001; Scholar, W.J. J. Biol. Chem. 1998; Scholar). The promoter stimulation was in THP-1 cells than in HL-60 cells, in with their differentiation status and expression levels. HL-60 cells with C/EBPα, we found that promoter activity in a and the cAMP response in cells levels as in THP-1 However, the basal the cAMP stimulation to be independent of C/EBPα The for this We suggest that the stimulation of the basal activity in THP-1 cells in response to cAMP enhanced expression, and the binding of as well as phosphorylation of CREB, C/EBP and and their A. Siu G. Calame K. J. Immunol. 1998; 161: 2267-2275Google Scholar, R. E. 1991; Scholar, Zhang S. J. Biol. Chem. 1995; Scholar, W.J. 2001; Scholar). The of of C/EBPα in HL-60 cells can only these with the dominant of in cells that by C/EBPα to depends on in C/EBPα to the IL-10 promoter activity by from the mutation and with and a of and C/EBP transcription factors both in basal and in cAMP-stimulated promoter In to CREB, the cAMP-inducible of C/EBPα a phosphorylation site; the of its activation by cAMP is J. McKnight S.L. J. Biol. Chem. 1992; Scholar). It that a with the cAMP-inducible of C/EBP transcription factors only after by is to that of activation CREB-binding protein after phosphorylation with the exception that phosphorylation of the than the protein regulate the H.L. Roesler W.J. Mol. Cell. Endocrinol. 2002; 188: 15-20Google Scholar, H.L. Roesler W.J. Mol. Cell. Endocrinol. 2001; Scholar, Zhang S. J. Biol. Chem. 1995; Scholar). In is a between and a binding for which is from its binding S. B. Mol. Cell. Biol. 1997; Scholar). the of we that CREB/ATF a activating potential in the of which are for IL-10 promoter In binding proteins to be in to binding site for IL-10 transcription. is in with the that C/EBPα expression is tissue-specific and during differentiation, CREB/ATF is W.J. Mol. Cell. Endocrinol. 2000; Scholar, M.H. T. 1998; Scholar). Therefore, C/EBP-mediated transcription of a gene, in is in a tissue-specific and depends on the state of differentiation of a from a mouse which IL-10 in response to and by known to contain high of C/EBPα, this S. J. M. S. Volk H.D. A. J. Scholar, H. J. J. 2002; Scholar). In to which are by transcription factors such as and activating the transcription of the anti-inflammatory IL-10 in monocytic cells, as shown is by cAMP or e.g. catecholamines during sympathetic activation through transcription factors C/EBP and The of their to be We S. McKnight of for the expression for C/EBPα and β, A. for for critical and M. Platzer for in the of this
Brenner et al. (Sat,) studied this question.