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and 
in Lipopolysaccharide-Induced Gene Activation of IL-10 in Mouse Macrophages1
* Graduate Institute of Biopharmaceutics, College of Life Science, National Chiayi University, Chiayi, Taiwan; and
Department of Pharmacology, Medical College, National Cheng Kung University, Tainan, Taiwan
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Abstract |
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and 
were also involved in LPS-induced gene expression of IL-10. By transient transfection with 5'-deletion mutants of the IL-10 promoter, we found that there were two LPS-responsive elements in the promoter of the mouse IL-10 gene. Analysis of these two regions by gel shift assay suggested that Sp1 and C/EBP and were bound to these two regions, respectively. By site-directed mutagenesis, we found that disruption at both the Sp1 and C/EBP binding sites almost completely blocked the LPS response. By gel shift assay and Western blotting, we found that the DNA binding complex and protein expression of C/EBP and were increased by LPS treatment, but these results were not found for Sp1. Overexpression of C/EBP or C/EBP, respectively, activated the promoter of the IL-10 gene, and they were enhanced by LPS. Coimmunoprecipitation experiments in intact cells indicated that LPS stimulated interaction between Sp1 and C/EBP and . These results suggested that the interaction between Sp1 and C/EBP and induced by LPS cooperatively activated expression of the IL-10 gene. The increase of C/EBP and proteins and the enhancement of transactivation activity of C/EBP and by LPS treatment, at least in part, explain the activation of IL-10 gene expression. |
Introduction |
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It is reported that IL-10 is an inducible gene. Catecholamines trigger human IL-10 release in monocytes via a cAMP-dependent pathway, and cAMP response elements on the promoter region are required (18, 19). In the studies of LPS-induced gene expression of human IL-10, transcription factors STAT3 and SV40 promoter factor 1 (Sp1)3 were reported to be essential in this regulation (20, 21). In mouse monocytes/macrophages, the Sp1 binding site residing at -89 to -78 bp of the IL-10 promoter is also critical for its basal and LPS-induced expression (22, 23). Although the Sp1 site is essential for activation of the IL-10 gene, DNA binding activity of Sp1 is not changed after LPS treatment (22). The immutability of Sp1-DNA binding activity is insufficient to explain the mechanism of gene activation in mouse macrophages. In this study, we determined that, in addition to Sp1, C/EBP and were also involved in LPS-induced gene expression of IL-10. Furthermore, we elucidated the physical protein-protein interaction between Sp1 and C/EBP and by using a coimmunoprecipitation method. In the Sp1-dificient Drosophila Schneider line 2 (SL2) cell system, we further confirmed the functional cooperation of Sp1 and C/EBP and in the regulation of the IL-10 gene promoter.
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Materials and Methods |
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LPS (Salmonella typhosa) was from Sigma-Aldrich (St. Louis, MO). The murine IL-10 ELISA kit was purchased from BD PharMingen (San Diego, CA). Superfect was from Qiagen (Hilden, Germany). The luciferase assay system and pGL3-Basic plasmid were from Promega (Madison, WI). The NucleoBond endotoxin-free plasmid purification kit was from Clontech (Palo Alto, CA). [
-32P]ATP (5000 Ci/mmol) and the ECL kit were purchased from Amersham Biosciences (Buckinghamshire, U.K.). DMEM, Schneider’s Drosophila medium, and FBS were obtained from Life Technologies (Grand Island, NY). Protein A-agarose, agarose-conjugated Sp1, C/EBP consensus binding oligonucleotides, and Abs against Sp1, C/EBP, and C/EBP were from Santa Cruz Biotechnology (Santa Cruz, CA). All other reagents used were of the highest purity obtainable.
Cell culture and LPS treatment
Murine macrophage RAW264.7 cells were grown and subcultured at 37°C under 5% CO2 in 75-cm2 plastic flasks containing 12 ml of DMEM supplemented with 10% FBS. In this series of experiments, cells were treated with 5 µg/ml LPS in culture medium supplemented with 10% FBS. SL2 cells were grown at 25°C in a 10-cm plastic dish containing 10 ml of Schneider’s Drosophila medium supplemented with 10% FBS.
Detection of mouse IL-10 mRNA by RT-PCR
Total RNA was isolated from RAW264.7 cells. Reverse transcription was performed on 2 µg of total RNA by oligo(dT) primers and SuperScript-II (Invitrogen, Carlsbad, CA), then 1/20 volume of reaction mixture was pooled, followed by PCR with mouse IL-10 specific primers (5'-CGTCGGATCCGCCATGCCTGGCTCACCACTGCT-3', 5'-CGTCTCTAGATTAGCTTTTCATTTTGATCA-3') or -actin specific primers (5'-CCTAAGGCCAACCGTGAAAAG-3', 5'-TCTTCATGGTGCTAGGAGCCA-3'), and then the PCR products were analyzed by 1% agarose gel. The yielding lengths of RT-PCR product by IL-10 specific primers and -actin specific primers were 560 bp and 623 bp, respectively.
Construction of luciferase reporter vectors
The murine IL-10 promoter region (-553/+64 bp) was prepared by PCR amplification of RAW264.7 genomic DNA with specific primers (5'-CCCAAGCTTGGATAGTCTTGAATACGTGA-3' and 5'-ATTAAGCTTATGGAGCTCTCTTTTCTG-3'). The DNA fragments were inserted into luciferase plasmid pGL3-Basic to form plasmid IG553. Other promoter deletion constructs were amplified from IG553 by PCR and ligated into pGL3-Basic individually. All of the constructed plasmids were confirmed by DNA sequencing as defined by GenBank accession number M84340. Mutated murine IL-10 promoters were prepared by PCR amplification of wild-type plasmid DNA with site-directed mutated primers. The mutated DNA fragments were also ligated into pGL3-Basic. The mutation sites were confirmed by DNA sequencing. All of the plasmids for transfection were purified by using the NucleoBond endotoxin-free plasmid purification kit.
Transfection of RAW264.7 cells with Superfect
Cells were transfected with plasmids by lipofection using Superfect according to the manufacturer’s instructions, with a slight modification. Cells were replated 12 h before transfection at a density of 9 x 105 cells in 2 ml of fresh culture medium in a 6-well plastic dish. For use in transfection, Superfect was incubated with plasmids (2 µl of Superfect/1 µg of total plasmids) in 0.1 ml of serum-free medium for 15 min at room temperature. Variable amounts of expression plasmids were compensated for with the empty vector pcDNA3.1. One milliliter of culture medium (with 10% FBS) was added to the DNA/Superfect mix, then added dropwise to the cells, and then incubated at 37°C in a humidified atmosphere of 5% CO2 for 6 h. After the change of DNA/Superfect medium to 2 ml of fresh culture medium, cells were treated with LPS for 18 h, unless stated otherwise. The cell lysates were prepared for luciferase and protein concentration assays.
Transfection of SL2 cells by the calcium phosphate method
One day before transfection, SL2 cells were plated onto 6-cm plastic dishes at a density of 4.5 x 106 cells and were transfected by the calcium phosphate method. Every plate received 4.5 µg of DNA, including 0.5 µg of IG553 luciferase plasmid, 2 µg of CMV-Sp1, and 1 µg of pCMV-fl(
-1 acid glycoprotein (AGP)/EBP) and/or mouse sarcoma virus (MSV)/EBP. Variable amounts of expression plasmids were compensated for with the empty vector pcDNA3.1. Twenty-four hours after addition of DNA, the medium was replaced. After another 24 h, cells were washed twice with PBS and harvested for luciferase and protein concentration assays.
Assay of luciferase activity
Luciferase activity was measured by the luciferase assay system. Transfected cells grown in 6-well plastic dishes were washed with PBS and lysed, each with 150 µl of luciferase lysis reagent per well. After 15-min incubation at room temperature, the lysates were centrifuged at 7200 x g for 15 s, and the supernatant solutions were used as the cell lysate. Luciferase assay substrates in 100 µl were mixed with 30 µl of the cell lysate, and the luciferase activity was then measured by a Berthold Lumat LB 9506 luminometer (Berthold, Bad Wildbad, Germany). All of the relative luciferase activities were normalized to the same protein concentration.
Nuclear extract preparation
Cells from 10-cm dishes were washed twice with PBS and scraped in 1 ml of PBS. Cells were collected by centrifuging at 400 x g for 10 s and resuspending in 0.4 ml of buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, and 0.5 mM EDTA) at 4°C for 10 min. Buffer A and all buffers described below contained 0.5 mM PMSF, 1 mM orthovanadate, 2 µg/ml pepstatin A, and 2 µg/ml leupeptin. Nuclei were pelleted by centrifugation at 400 x g for 10 s. Pellets were resuspended in 0.1 ml of buffer B (20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 25% glycerol (v/v)) at 4°C for 20 min. The suspension was pelleted by centrifugation at 400 x g for 2 min. Supernatants were collected and stored at -80°C until use.
Gel shift assay
Purified PCR products of IL-10 promoter were end-labeled with [-32P]ATP and T4 polynucleotide kinase. The binding reaction was performed in a 15-µl reaction mixture containing 0.2 µg of poly(dI-dC) · poly(dI-dC), 20 mM HEPES (pH 7.9), 0.1 mM KCl, 2 mM MgCl2, 15 mM NaCl, 0.2 mM EDTA, 5 mM DTT, 10% (v/v) glycerol, 2% (w/v) polyvinyl alcohol, 3 µg of the cell nuclear extracts, and the radiolabeled probe (4 x 104 cpm) and with or without Abs as described in each experiment. The mixtures were incubated at room temperature for 30 min and loaded on a 4% (w/v) polyacrylamide gel. Electrophoresis was performed at a constant 150 V for 3 h. The gels were dried and autoradiographed.
Western blotting
Analytical 10% SDS-PAGE was performed. The cell nuclear extracts were prepared from control and LPS-treated cells, and 30 µg of protein of each was analyzed. For immunoblotting, proteins in the SDS gels were transferred to a polyvinylidene difluoride membrane by an Electroblot apparatus (Amersham Biosciences). Abs against Sp1, C/EBP, or C/EBP were used as the primary Abs. Immunoblot analysis was conducted with mouse or rabbit IgG Ab coupled with HRP. An ECL kit was used for detection.
Coimmunoprecipitation
Two hundred micrograms of nuclear extracts was incubated with 10 µl of anti-Sp1 Abs-agarose conjugate in 300 µl of immunoprecipitation buffer (20 mM HEPES (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol (v/v), 0.5 mM PMSF, 1 mM orthovanadate, 2 µg/ml pepstatin A, and 2 µg/ml leupeptin) under gentle shaking at 4°C overnight. Beads were pelleted at 7500 x g for 2 min and washed three times with RIPA buffer (50 mM Tris · HCl (pH 7.5), 1% IGE-PAL CA-630 (v/v), 150 mM NaCl, and 0.5% sodium deoxycholate). Protein was removed from the beads by boiling in sample buffer (120 mM Tris · HCl (pH 6.8), 10% glycerol, 3% SDS, 20 mM DTT, and 0.4% bromophenol blue) for 5 min and was subjected to SDS-PAGE on a 10% gel. Western blot analysis was conducted as described above.
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Results |
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RAW264.7 mouse monocyte cells were incubated with 5 µg/ml LPS for various periods of time. Treatment of cells with LPS induced a time-dependent increase in IL-10 mRNA (Fig. 1A) and protein (data not shown). Induction of IL-10 mRNA was observed in cells treated with LPS for 1 h and lasted at least up to 7 h of treatment. The effect of LPS on the kinetics of transcriptional activation was studied using cells transfected with a luciferase reporter vector IG553 bearing promoter region -553 to +64 bp of the IL-10 gene. LPS induced the promoter activation in a time-dependent manner (Fig. 1B). A sustained increase lasted for 18 h and decreased at 24 h after LPS stimulation. Ratios of luciferase activity between LPS-treated and control cells were 1.2-, 2-, 6.9-, and 3.7-fold for 6, 9, 18, and 24 h, respectively.
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Several potential response elements are present in the 553-bp promoter region of the mouse IL-10 gene (Fig. 2A). To study the transcriptional regulation of the IL-10 gene, luciferase reporter vectors bearing various lengths of 5'-flanking region of IL-10 were constructed as shown in Fig. 2A. Cells were transfected with various DNA constructs, and the basal expression of luciferase activities in these vectors was studied. The results are summarized in Fig. 2B. Although there was little change in vectors IG422 and IG363, no obvious change in reporter activity was observed in vectors on which the 5'-flanking region was deleted from -553 to -122 bp (IG553 to IG122). However, the basal expression of reporter activity disappeared when the promoter region was deleted from -122 to -66 bp (IG122 to IG66). These results indicate that the promoter region covering -122 to -66 bp, which contains one Sp1 and one potential AP-1 binding sequence, was required for the basal expression of the IL-10 gene. Requirement of transcription factor binding sites for LPS-induced promoter activation of the IL-10 gene was then studied by using reporter vectors bearing different lengths of promoter. The results of the LPS response are summarized in Fig. 2C. Two responsive regions, which span from -422 to -383 bp and from -122 to -66 bp, were identified for LPS-induced promoter activation of the IL-10 gene. When the promoter was deleted from -422 to -382 bp, 
65% of the LPS response was abolished, and the remaining 35% of the LPS response disappeared when the promoter was deleted from -122 to -66 bp. These results indicated that the promoter region from -422 to -383 bp contains two potential C/EBP binding sites that, together with one known Sp1 and one potential AP-1 binding site residing on -122/-66 bp, were required for LPS response.
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The requirement of the Sp1 binding site in the promoter region from -122 to -66 bp for basal expression and LPS response was studied by using site-directed mutants. The Sp1 binding site residing from -89 and -78 bp was mutated as shown in Fig. 3A. Luciferase reporter vectors (IG533
Sp and IG122Sp) bearing -553- and -122-bp promoter lengths with mutated Sp1 binding sequences were constructed, respectively (Fig. 3B). As shown in Fig. 3C, compared with reporter activity of wild expression vectors IG553 and IG122, the luciferase activity of expression vectors IG553Sp and IG122Sp decreased greatly, indicating that the Sp1 binding sequence residing from -89 to -78 bp played a pivotal role in basal expression of the IL-10 gene. In studying the functional role of the Sp1 binding site in LPS response, mutation at Sp1 binding sites (IG122Sp) completely blocked the luciferase activity of the promoter with -122 bp (IG122), induced by LPS (Fig. 3D). However, an Sp1 mutant for the promoter with -533 bp (IG553Sp) exhibited only 50% inhibition, compared with wild-type promoter (IG553) activity induced by LPS. These results demonstrate again that the Sp1 site was not the only factor in LPS-induced IL-10 activation, even though it played an important role in building up a basal transcriptional expression of the IL-10 gene.
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In the promoter region, from -422 to -383 bp, there are two potential binding sites for C/EBP (-410/-399 bp and -398/-385 bp). Using an oligonucleotide (-422/-363 bp) as a probe, three retarded bands were observed in gel shift assays (Fig. 4A). The binding intensities of all three retarded bands were enhanced by using cell nuclear extracts prepared from LPS-treated cells. All three bands were diminished when anti-C/EBP or anti-C/EBP Abs were added in gel shift assays. Bands b and c could be completely competed by C/EBP consensus binding oligonucleotides, and band a was partially competed. The retarded bands were also not affected by a nonspecific Ab with NF-
B-p50 Abs or by unrelated oligonucleotides with NF-B binding oligonuleotides. These results indicated that the three retarded bands were due to the binding of C/EBP and to their DNA binding sites located in the promoter from -422 to -363 bp. To determine which of these two potential C/EBP binding sites in the promoter region from -422 to -383 bp was responsible for the binding to C/EBP, the two consensus C/EBP binding sites were modified by site-directed mutagenesis (Fig. 4B). In the gel shift assay, the elimination of nuclear protein binding was mainly observed with the DNA probe with a mutation at the -395/-393-bp site (C2), but to a lesser extent at -406/-402 bp (C1) (Fig. 4C). Taken together, these results indicated that the C/EBP site located from -398 to -385 bp was the major DNA sequence for binding cell nuclear C/EBP and and that LPS treatment enhanced the binding of C/EBP proteins to the C/EBP consensus binding site on the gene promoter of IL-10.
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To study the functional role of C/EBP binding sites located at -410/-399 bp and -398/-385 bp on the IL-10 promoter, we constructed a reporter vector IG533C1, which is a C/EBP site-directed mutant on -410/-399 bp, and another reporter vector IG553C2 with a C/EBP site-directed mutation on -398/-385 bp (Fig. 5A). Results of gel shift assays have suggested that the downstream region -398/-385 bp was a major C/EBP binding site on the promoter and that the upstream region -410/-389 bp was a minor one (Fig. 4C). The functional role of C/EBP binding on the promoter in LPS-induced activation of IL-10 promoter was then studied by using luciferase reporters bearing promoter with C/EBP site-directed mutations. The results are summarized in Fig. 5B. Mutation at -410/-399 bp (IG553C1) only slightly inhibited the LPS response by 30%, whereas mutation at -398/-385 bp (IG553C2) significantly inhibited the LPS response by 72%. Results of the functional reporter assay also clearly indicate that the downstream C/EBP binding region -398/-385 bp was the major responsible C/EBP binding site in LPS-induced promoter activation of the IL-10 gene. Corequirement of C/EBP and Sp1 binding sites in LPS response was then studied. Disruption of the Sp1 binding site exhibited 50% inhibition of LPS response, and double mutations at C/EBP (-398/-385 bp) and Sp1 (-89/-78 bp) binding sites (IG553C2-Sp) almost completely inhibited the LPS response. These results clearly indicated that both C/EBP and Sp1 binding sites were essential for LPS-induced promoter activation of the IL-10 gene and that the C/EBP binding site seems to be more important than the Sp1 binding site in the LPS response.
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and enhances promoter activation of IL-10
Expression vectors of C/EBP (pCMV-fl(AGP/EBP)) and/or C/EBP (MSV/EBP) were used to determine whether these two transcription factors enhance the promoter activation of IL-10. Cells were transfected with a luciferase reporter gene and a C/EBP or C/EBP expression vector for 6 h, followed by LPS stimulation. As shown in Fig. 6, transfection of either pCMV-fl(AGP/EBP) or MSV/EBP into cells dose-dependently enhanced promoter activation of IL-10. No additional effect was observed by coexpression of C/EBP and C/EBP in RAW264.7 cells. LPS treatment increased the promoter activity in C/EBP- or C/EBP-overexpressed cells by 1.4- and 3.5-fold, respectively. These results suggested that both C/EBP and are able to activate the IL-10 gene and that the transactivation effect of LPS on C/EBP seems to be more important than C/EBP is in RAW264.7 cells. Interestingly, when C/EBP and C/EBP were coexpressed in cells, LPS increased the promoter activity by 1.5-fold, which is not as high as the effect on C/EBP-overexpressed cells. The above evidence suggested that C/EBP increased its transactivation activity considerably upon LPS stimulation, whereas C/EBP increased only slightly. Therefore, overexpression of C/EBP together with C/EBP attenuated the effect of LPS on C/EBP.
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and in SL2 cells
To verify whether Sp1 and C/EBP or C/EBP together activate the promoter activity of the IL-10 gene, vector IG553 was transfected into the Sp1-dificient Drosophila Schneider SL2 cell line either in the presence or in the absence of Sp1, C/EBP, and C/EBP expression vectors. As shown in Fig. 7, expression with 1 µg of C/EBP or C/EBP individually did not significantly change the activity of the IL-10 promoter. Expression of Sp1 stimulated the activity of the IL-10 promoter by 2.4-fold. Coexpression of Sp1 with C/EBP or C/EBP could not enhance the promoter activity compared with Sp1 only, but interestingly, when Sp1 and C/EBP and were coexpressed into SL2 cells together, the luciferase activity of the IL-10 promoter was enhanced by 5.5-fold. It was suggested that C/EBP and C/EBP cooperated with the Sp1 protein in regulating the promoter activity of the mouse IL-10 gene.
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or C/EBP in LPS-treated cells
Expression of Sp1 and C/EBP and in nuclear extracts prepared from RAW264.7 cells treated with LPS for 0 or 6 h was studied by using immunoblot analysis. No difference of Sp1 expression between control and LPS-treated cells was observed (Fig. 8A). Increase of C/EBP and expression was observed in cells treated with LPS for 6 h (Fig. 8A). Interaction between Sp1 and C/EBP and was then studied by coimmunoprecipitation by using Sp1-agarose Abs. No significant change in the immunoprecipitated Sp1 between control and LPS-treated cells was observed (Fig. 8B). The coimmunoprecipitated C/EBP and increased in LPS-treated cells (Fig. 8B). An increase in coimmunoprecipitated C/EBP and suggested that, upon LPS treatment, the physical interaction of Sp1-C/EBP or Sp1-C/EBP is enhanced.
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Discussion |
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70% when the promoter was deleted from -422 to -383 bp (Fig. 2C), indicating that this promoter region plays a functional role in mediating the LPS response. Second, mutation of this C/EBP binding site (IG553C2) blocked the response of LPS by 70% (Fig. 5B). This evidence clearly indicates that the C/EBP binding site residing on -398/-385 bp also is required for the LPS response. However, this C/EBP binding site is not required for the basal promoter activity (data not shown). In terms of the requirement for the Sp1 binding site residing on -89/-78 bp, we found that it was required not only for LPS response but also for basal promoter activity, which is in accordance with the findings of Brightbill et al. (22). Therefore, both C/EBP and Sp1 binding sites are corequired for LPS response. Because mutations at the C/EBP binding site exhibited a more significant inhibition of LPS response than did mutations at Sp1 binding site, the C/EBP binding site seems to play a more important role in the LPS response. According to related literature, it is known that C/EBP is important for some gene expressions, e.g., endotoxin-induced cyclooxygenase-2 gene (24), IL-1-induced IL-6 gene (25), and LPS-induced IL-1 gene (26) and RANTES/chemokine (C-C motif) ligand 5 gene (27). Recently it was reported that C/EBP and are critical in both basal and cAMP/stress-dependent induction of human IL-10 expression during monocytic differentiation (28). In the LPS-induced mouse IL-10 gene, we determined that the transcription factors bound to the C/EBP consensus site were C/EBP and (Fig. 4). LPS treatment enhanced the binding of C/EBP and to the C/EBP binding site in a time-dependant manner (Fig. 4A), whereas binding of nuclear Sp1 to the Sp1 binding site on the promoter was not changed upon LPS treatment (data not shown). Caivano et al. (29) reported that treatment of RAW 264.7 cells with LPS induced the protein biosynthesis of C/EBP and . In this study, we also confirmed this finding (Fig. 8A) and found that there was no change of Sp1 protein in cells upon LPS treatment (Fig. 8A).
The increase of C/EBP and proteins by LPS treatment (Fig. 8A), at least in part, explains the enhancement of IL-10 gene expression. Furthermore, we also indicate the different mechanism between C/EBP and C/EBP in LPS-induced promoter activation of the IL-10 gene. In the overexpression of C/EBP in RAW264.7 cells, LPS could enhance the luciferase activity of the IL-10 promoter by 3.5-fold (Fig. 6), but there was only a 0.4-fold increase in overexpression of C/EBP comparing LPS treatment with no treatment (Fig. 6). This result indicates that both C/EBP and proteins might be modified by LPS treatment to increase their transactivation activity on the IL-10 gene promoter and that C/EBP has a more important role upon LPS treatment. Taken together, the results showed not only the increase of C/EBP and proteins, but also that the post-translation modification of C/EBP and is involved in LPS-induced IL-10 gene activation. It was reported that phosphorylation of C/EBP enhances its transactivation activity in NIH 3T3 cells (30) and that it also enhances its binding activity to promoter in quiescent human fibroblasts (31). In addition, it was recently found that different constitutions of C/EBP isoforms dynamically regulate cyclooxygenase-2 promoter (32). It will be interesting to study the regulation mechanism of C/EBP and protein by LPS treatment in the regulation of the IL-10 gene in the future.
In the present study, we conclude that transcription factors Sp1 and C/EBP and are all required for LPS-induced gene expression of IL-10 in mouse macrophages, and we have confirmed that Sp1 played an essential role in keeping the basal gene expression of IL-10. Although no change in the level of Sp1 protein expression was observed, we still do not know whether the transactivation activity of Sp1 is regulated by post-translation modification upon LPS treatment. Because the LPS response was inhibited by 50% when the -89 to -78 bp of the Sp1 binding site were mutated (Fig. 3D), indicating that the Sp1 binding site did play a functional role in mediating LPS response. Post-translation modification of Sp1 such as dephosphorylation (33, 34, 35) and phosphorylation (36), resulting in the transactivation of Sp1, has been reported. Milanini-Mongiat et al. (37) recently found that p42/p44 mitogen-activated protein kinase directly phosphorylates Sp1 on threonines 453 and 739, which results in the enhancement of binding of Sp1 to the gene promoter of vascular endothelial growth factor. Because the binding of Sp1 to the gene promoter of IL-10 was not altered upon LPS treatment in our experimental system, the transactivation activity of Sp1, which is possibly modified by phosphorylation or dephosporylation in LPS-induced expression of the IL-10 gene, requires further studies.
One of the interesting discoveries of this research is the identification of protein-protein interaction between Sp1 and C/EBP and protein in intact cells. By using a coimmunoprecipitation method, we found that Sp1 protein could interact with C/EBP and protein, and the physical binding of Sp1-C/EBP or Sp1-C/EBP was increased after LPS treatment (Fig. 8B). This is the first evidence to identify the physical interaction between Sp1 and C/EBP and and that LPS enhances these binding complexes. It is still not clear whether the increased physical interaction between Sp1 and C/EBP proteins stems purely from the presence of more C/EBP proteins after LPS treatment or if it is also from changes in either Sp1 or C/EBP that allow stronger interaction. The underlying mechanism needs more exploration. It has been reported that the transcription factors C/EBP family and Sp1 were determined to be required and that they cooperatively activate the gene promoters of rat CYP2D5 (38), human CD11c (39), lactoferrin (40), and mouse aldose reductase-like gene (41) by using transfection and gel shift assay. However, the physical interaction between Sp1 and C/EBP proteins by coimmunoprecipitation was not proven. In the previous study of CYP2D5, Lee et al. (38) also found that glutamine- and serine/threonine-rich domains of Sp1 are required for cooperating with C/EBP in the presence of DNA. Sp1 belongs to a zinc finger family of transcription factors that recognizes the GC-rich DNA sequence (42), and it interacts with several other transcription factors that bind to their respective response elements in the regulation of responsive genes. For example, Yin and Yang 1 (43), the p65 subunit of NF-B (44), GATA1 (45), and helicase-like transcription factor (46) have been shown to interact functionally with the zinc domain of Sp1. We also previously found that Sp1 could interact with c-Jun that does not need its DNA binding site, and the transcription factor complex of Sp1 and c-Jun cooperatively activated the gene promoter of human 12(S)-lipoxygenase (47). In this study, we found that transcription factors Sp1 and C/EBP and were all required for LPS-induced activation of the IL-10 gene promoter and that Sp1 could interact physically and functionally with C/EBP and C/EBP in mouse macrophages.
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Acknowledgments |
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, respectively. Thanks are also due to Johnson C. Hou and Dr. George. S. Roth for their critical reading of the manuscript. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Wen-Chang Chang, Department of Pharmacology, Medical College, National Cheng Kung University, Tainan 701, Taiwan. E-mail address: wcchang{at}mail.ncku.edu.tw
3 Abbreviations used in this paper: Sp1, SV40 promoter factor 1; SL2, Schneider line 2; AGP, -1 acid glycoprotein; MSV, mouse sarcoma virus.
Received for publication December 23, 2002. Accepted for publication May 6, 2003.
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References |
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interferon-dependent mechanism. Infect. Immun. 66:5157. and - regulate IL-6 production in IL-1-stimulated human enterocytes. J. Cell. Physiol. 192:64.[Medline]
transcription. J. Immunol. 162:5367. (C/EBP) and C/EBP transcription factors. J. Biol. Chem. 276:48693. binding and cyclooxygenase-2 promoter activity by sodium salicylate in quiescent human fibroblasts. J. Biol. Chem. 276:18897.2 integrin gene core promoter activity. Blood 90:678. but not C/EBP to synergize with an Sp1 protein is specified by the leucine zipper and activation domain. Mol. Cell. Biol. 17:2038.[Abstract]
(CCAAT/enhancer binding protein), and ubiquitous transcription factors NF1 (nuclear factor 1) and Sp1 (selective promoter factor 1) are required for regulation of the mouse aldose reductase-like gene (AKR1B7) expression in adrenocortical cells. Mol. Endocrinol. 94:93.
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S. Cao, X. Zhang, J. P. Edwards, and D. M. Mosser NF-{kappa}B1 (p50) Homodimers Differentially Regulate Pro- and Anti-inflammatory Cytokines in Macrophages J. Biol. Chem., September 8, 2006; 281(36): 26041 - 26050. [Abstract] [Full Text] [PDF]
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Z. Qiu, F. Norflus, B. Singh, M. K. Swindell, R. Buzescu, M. Bejarano, R. Chopra, B. Zucker, C. L. Benn, D. P. DiRocco, et al. Sp1 Is Up-regulated in Cellular and Transgenic Models of Huntington Disease, and Its Reduction Is Neuroprotective J. Biol. Chem., June 16, 2006; 281(24): 16672 - 16680. [Abstract] [Full Text] [PDF]
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 J. Katz, P. Zhang, M. Martin, S. N. Vogel, and S. M. Michalek Toll-Like Receptor 2 Is Required for Inflammatory Responses to Francisella tularensis LVS. Infect. Immun., May 1, 2006; 74(5): 2809 - 2816. [Abstract] [Full Text] [PDF]
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J. Shoemaker, M. Saraiva, and A. O'Garra GATA-3 Directly Remodels the IL-10 Locus Independently of IL-4 in CD4+ T Cells J. Immunol., March 15, 2006; 176(6): 3470 - 3479. [Abstract] [Full Text] [PDF]
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Z. H. Nemeth, C. S. Lutz, B. Csoka, E. A. Deitch, S. J. Leibovich, W. C. Gause, M. Tone, P. Pacher, E. S. Vizi, and G. Hasko Adenosine Augments IL-10 Production by Macrophages through an A2B Receptor-Mediated Posttranscriptional Mechanism J. Immunol., December 15, 2005; 175(12): 8260 - 8270. [Abstract] [Full Text] [PDF]
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M. Riemann, R. Endres, S. Liptay, K. Pfeffer, and R. M. Schmid The I{kappa}B Protein Bcl-3 Negatively Regulates Transcription of the IL-10 Gene in Macrophages J. Immunol., September 15, 2005; 175(6): 3560 - 3568. [Abstract] [Full Text] [PDF]
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M. Saraiva{paragraph}, J. R. Christensen, A. V. Tsytsykova, A. E. Goldfeld, S. C. Ley, D. Kioussis, and A. O'Garra Identification of a Macrophage-Specific Chromatin Signature in the IL-10 Locus J. Immunol., July 15, 2005; 175(2): 1041 - 1046. [Abstract] [Full Text] [PDF]
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 J. Wilmanski, M. Siddiqi, E. A. Deitch, and Z. Spolarics Augmented IL-10 production and redox-dependent signaling pathways in glucose-6-phosphate dehydrogenase-deficient mouse peritoneal macrophages J. Leukoc. Biol., July 1, 2005; 78(1): 85 - 94. [Abstract] [Full Text] [PDF]
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M. Lucas, X. Zhang, V. Prasanna, and D. M. Mosser ERK Activation Following Macrophage Fc{gamma}R Ligation Leads to Chromatin Modifications at the IL-10 Locus J. Immunol., July 1, 2005; 175(1): 469 - 477. [Abstract] [Full Text] [PDF]
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 J.-M. Wang, J. T. Tseng, and W.-C. Chang Induction of Human NF-IL6{beta} by Epidermal Growth Factor Is Mediated through the p38 Signaling Pathway and cAMP Response Element-binding Protein Activation in A431 Cells Mol. Biol. Cell, July 1, 2005; 16(7): 3365 - 3376. [Abstract] [Full Text] [PDF]
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M. Shiohara, A. F. Gombart, Y. Sekiguchi, E. Hidaka, S. Ito, T. Yamazaki, H. P. Koeffler, and A. Komiyama Phenotypic and functional alterations of peripheral blood monocytes in neutrophil-specific granule deficiency J. Leukoc. Biol., February 1, 2004; 75(2): 190 - 197. [Abstract] [Full Text] [PDF]
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