The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Liu, Y.-W.
Right arrow Articles by Chang, W.-C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liu, Y.-W.
Right arrow Articles by Chang, W.-C.
The Journal of Immunology, 2003, 171: 821-828.
Copyright © 2003 by The American Association of Immunologists

Functional Cooperation of Simian Virus 40 Promoter Factor 1 and CCAAT/Enhancer-Binding Protein {beta} and {delta} in Lipopolysaccharide-Induced Gene Activation of IL-10 in Mouse Macrophages1

Yi-Wen Liu*, Hui-Ping Tseng{dagger}, Lei-Chin Chen{dagger}, Ben-Kuen Chen{dagger} and Wen-Chang Chang2,{dagger}

* Graduate Institute of Biopharmaceutics, College of Life Science, National Chiayi University, Chiayi, Taiwan; and {dagger} Department of Pharmacology, Medical College, National Cheng Kung University, Tainan, Taiwan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies have revealed that LPS can activate transcription of the IL-10 gene promoter through an SV40 promoter factor 1 (Sp1) binding site in mouse macrophage RAW264.7. In this study, we determined that, in addition to Sp1, C/EBP{beta} and {delta} 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{beta} and {delta} 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{beta} and {delta} were increased by LPS treatment, but these results were not found for Sp1. Overexpression of C/EBP{beta} or C/EBP{delta}, 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{beta} and {delta}. These results suggested that the interaction between Sp1 and C/EBP{beta} and {delta} induced by LPS cooperatively activated expression of the IL-10 gene. The increase of C/EBP{beta} and {delta} proteins and the enhancement of transactivation activity of C/EBP{beta} and {delta} by LPS treatment, at least in part, explain the activation of IL-10 gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interleukin-10 is a pleiotropic cytokine that regulates a variety of functions of hemopoietic cells. The principal function of IL-10 appears to be inhibition of activities of Th1 cells (1). Based on this effect, IL-10 has been investigated in clinical trials, including treatment of chronic active Crohn’s disease (2, 3), rheumatoid arthritis (4), psoriasis (5, 6), and chronic hepatitis C infection (7, 8). In addition to human studies, experiments in IL-10 knockout mice also suggest that IL-10 has an important role in inhibiting innate and acquired responses induced by various bacteria (9, 10, 11). In addition to anti-inflammatory effects, IL-10 also impairs immune function because T cell responses are restored by the treatment of anti-IL-10 Abs in leprosy and HIV+ patients (12, 13). Besides these anti-inflammatory functions, many studies have demonstrated that an abnormal expression of IL-10 is associated with occurrence of some particular diseases. For example, a high expression of IL-10 has been suggested to play an exacerbating role in systemic lupus erythematosus patients (14, 15). Genetic predisposition to high IL-10 expression is also correlated with a higher rate of mortality in meningococcal disease (16). Two areas of multiple CA dinucleotide repeat polymorphisms in the 5' flanking region of human IL-10 are involved in genetic predisposition in human disease (17), indicating that abnormal regulation of IL-10 expression is related to some disease.

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{beta} and {delta} were also involved in LPS-induced gene expression of IL-10. Furthermore, we elucidated the physical protein-protein interaction between Sp1 and C/EBP{beta} and {delta} 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{beta} and {delta} in the regulation of the IL-10 gene promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

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). [{gamma}-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{beta}, and C/EBP{delta} 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 {beta}-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 {beta}-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({alpha}-1 acid glycoprotein (AGP)/EBP) and/or mouse sarcoma virus (MSV)/EBP{beta}. 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 [{gamma}-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{beta}, or C/EBP{delta} 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of IL-10 gene expression and promoter activation by LPS

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.



View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 1. Induction of IL-10 gene in the RAW264.7 cell line. A, RAW264.7 cells were treated with 5 µg/ml LPS in culture medium for 0 (lane 1) to 7 h (lane 5). Total RNA was reverse transcribed with an oligo(dT) primer, followed by PCR with either IL-10-specific primers or {beta}-actin-specific primers. Products were separated by 1% agarose gel, followed by ethidium bromide staining. B, After the transfection with 0.5 µg of IG553 luciferase plasmid, the cells were treated with 5 µg/ml LPS in culture medium for 0–24 h. The expression of luciferase activity and protein concentration was determined and normalized. Values are means ± SEM of three determinations.

 
Analysis of LPS-induced activation of the IL-10 gene promoter

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.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 2. Basal and transcriptional analysis of luciferase expression vectors bearing various lengths of IL-10 gene promoter. A, The 5'-deletion of the IL-10 promoter fragment was ligated into a luciferase plasmid as described in Materials and Methods. Numbers indicate the distance in base pairs from the transcription start site. The potential consensus binding sites in the 5'-flanking region are indicated. B, A total of 0.5 µg of each IG luciferase plasmid was transfected into RAW264.7 cells. The luciferase activity was normalized with protein concentration. Values are means ± SEM for three determinations. C, Plasmid transfection was performed as described above. The expression ratio of LPS-treated to control cells is indicated. The results shown represent the means ± SEM of 4–43 determinations.

 
Analysis of Sp1 binding site is required for basal expression and LPS response

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{triangleup}Sp and IG122{triangleup}Sp) 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 IG553{triangleup}Sp and IG122{triangleup}Sp 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 (IG122{triangleup}Sp) 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 (IG553{triangleup}Sp) 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.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 3. Basal and transcriptional analysis of Sp1-mutated vectors. A, Site-directed mutagenesis of Sp1 consensus sequences in the 5'-flanking region from -89 to -78 bp of the IL-10 gene. B, Appellation of wild-type or Sp1 site-directed mutant bearing luciferase expression plasmid. C, Plasmid transfection was performed as described in Materials and Methods. Values are means ± SEM for three determinations. D, Expression ratio of LPS-treated to control cells is indicated. The results shown represent the means ± SEM of 6–39 determinations.

 
Identification of NF binding to promoter region -422 to -383 bp

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{beta} or anti-C/EBP{delta} 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-{kappa}B-p50 Abs or by unrelated oligonucleotides with NF-{kappa}B binding oligonuleotides. These results indicated that the three retarded bands were due to the binding of C/EBP{beta} and {delta} 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 ({triangleup}C2), but to a lesser extent at -406/-402 bp ({triangleup}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{beta} and {delta} 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.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 4. Gel-shift analysis of the NFs binding to -422/-363 bp of the IL-10 gene promoter. A, [{gamma}-32P]ATP-labeled oligonucleotide covering the IL-10 promoter region from -422 to -363 bp was incubated with nuclear extracts from RAW264.7 cells treated with LPS for 0–6 h (lanes 1–4); three retarded bands (a, b, and c) are indicated. Binding reactions were performed with Abs against C/EBP{beta} (lanes 5 and 8), C/EBP{delta} (lanes 6 and 9), NF-{kappa}B-p50 (lane 12), C/EBP consensus oligonucleotide (lanes 7 and 10; 100-fold molar excess), or NF-{kappa}B consensus oligonucleotide (lane 13; 100-fold molar excess). B, Site-directed mutagenesis of C/EBP consensus sequences in the 5'-flanking region from -410 to -390 bp of the IL-10 gene. C, An EMSA experiment was performed with two [{gamma}-32P]ATP-labeled probes with different mutated sites of C/EBP in the 5'-flanking region from -410 to -390 bp of the IL-10 gene. The probes were incubated with cell nuclear extracts in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of LPS.

 
Functional analysis of C/EBP binding sites required for LPS response

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 IG533{triangleup}C1, which is a C/EBP site-directed mutant on -410/-399 bp, and another reporter vector IG553{triangleup}C2 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 (IG553{triangleup}C1) only slightly inhibited the LPS response by 30%, whereas mutation at -398/-385 bp (IG553{triangleup}C2) 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 (IG553{triangleup}C2-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.



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 5. Transcriptional analysis of C/EBP and/or Sp1-mutated vectors. A, Appellation of wild-type or C/EBP site-directed mutants bearing luciferase expression plasmid. B, Plasmid transfection was performed as described in Materials and Methods. Values are means ± SEM for three determinations. The expression ratio of LPS-treated to control cells is indicated. The results shown represent the means ± SEM of 11–43 determinations.

 
Overexpression of C/EBP{beta} and {delta} enhances promoter activation of IL-10

Expression vectors of C/EBP{beta} (pCMV-fl(AGP/EBP)) and/or C/EBP{delta} (MSV/EBP{delta}) 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{beta} or C/EBP{delta} expression vector for 6 h, followed by LPS stimulation. As shown in Fig. 6, transfection of either pCMV-fl(AGP/EBP) or MSV/EBP{delta} into cells dose-dependently enhanced promoter activation of IL-10. No additional effect was observed by coexpression of C/EBP{beta} and C/EBP{delta} in RAW264.7 cells. LPS treatment increased the promoter activity in C/EBP{beta}- or C/EBP{delta}-overexpressed cells by 1.4- and 3.5-fold, respectively. These results suggested that both C/EBP{beta} and {delta} are able to activate the IL-10 gene and that the transactivation effect of LPS on C/EBP{delta} seems to be more important than C/EBP{beta} is in RAW264.7 cells. Interestingly, when C/EBP{beta} and C/EBP{delta} were coexpressed in cells, LPS increased the promoter activity by ~1.5-fold, which is not as high as the effect on C/EBP{delta}-overexpressed cells. The above evidence suggested that C/EBP{delta} increased its transactivation activity considerably upon LPS stimulation, whereas C/EBP{beta} increased only slightly. Therefore, overexpression of C/EBP{beta} together with C/EBP{delta} attenuated the effect of LPS on C/EBP{delta}.



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 6. Overexpression of C/EBP{beta} and C/EBP{delta} activate promoter activity of IL-10. Plasmid transfection was performed as described in Materials and Methods. The results of luciferase activity were normalized by protein concentrations. Values are means ± SEM for three determinations. Statistical analysis was performed by Student’s t test. N.S., Not significant.

 
Additional activation of the IL-10 promoter by the cooperation of Sp1 and C/EBP{beta} and {delta} in SL2 cells

To verify whether Sp1 and C/EBP{beta} or C/EBP{delta} 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{beta}, and C/EBP{delta} expression vectors. As shown in Fig. 7, expression with 1 µg of C/EBP{beta} or C/EBP{delta} 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{beta} or C/EBP{delta} could not enhance the promoter activity compared with Sp1 only, but interestingly, when Sp1 and C/EBP{beta} and {delta} 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{beta} and C/EBP{delta} cooperated with the Sp1 protein in regulating the promoter activity of the mouse IL-10 gene.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 7. Overexpression of Sp1, C/EBP{beta}, and C/EBP{delta} in SL2 cells. Plasmid transfection was performed as described in Materials and Methods. The results of luciferase activity were normalized by protein concentrations. Values are means ± SEM for three determinations. Statistical analysis was performed by Student’s t test.

 
Increased physical interaction between Sp1 and C/EBP{beta} or C/EBP{delta} in LPS-treated cells

Expression of Sp1 and C/EBP{beta} and {delta} 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{beta} and {delta} expression was observed in cells treated with LPS for 6 h (Fig. 8A). Interaction between Sp1 and C/EBP{beta} and {delta} 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{beta} and {delta} increased in LPS-treated cells (Fig. 8B). An increase in coimmunoprecipitated C/EBP{beta} and {delta} suggested that, upon LPS treatment, the physical interaction of Sp1-C/EBP{beta} or Sp1-C/EBP{delta} is enhanced.



View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 8. Effect of LPS on the interaction between Sp1 and C/EBP{beta} and {delta} in vivo. RAW264.7 cell were treated with or without LPS in culture medium for 6 h. A, Nuclear extracts were prepared and subjected to Western blotting by using Abs specific for Sp1, C/EBP{beta}, or C/EBP{delta}. B, Nuclear extracts were immunoprecipitated with Abs against Sp1 bound to protein A-agarose. Immunoprecipitates were subjected to 10% SDS-PAGE followed by Western blotting with Abs against Sp1, C/EBP{beta}, or C/EBP{delta}.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the studies of LPS-induced promoter activation of the IL-10 gene, Brightbill et al. (22) previously reported the exclusive requirement of the Sp1 binding site residing on -89/-78 bp for LPS response. However, in the present study, we provide two pieces of evidence to indicate that a C/EBP binding site residing on -398/-385 bp is also required for LPS-induced promoter activation of the IL-10 gene. First, the LPS response was inhibited by ~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 (IG553{triangleup}C2) 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{beta}-induced IL-6 gene (25), and LPS-induced IL-1{beta} gene (26) and RANTES/chemokine (C-C motif) ligand 5 gene (27). Recently it was reported that C/EBP{alpha} and {beta} 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{beta} and {delta} (Fig. 4). LPS treatment enhanced the binding of C/EBP{beta} and {delta} 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{beta} and {delta}. 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{beta} and {delta} 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{beta} and C/EBP{delta} in LPS-induced promoter activation of the IL-10 gene. In the overexpression of C/EBP{delta} 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{beta} comparing LPS treatment with no treatment (Fig. 6). This result indicates that both C/EBP{beta} and {delta} proteins might be modified by LPS treatment to increase their transactivation activity on the IL-10 gene promoter and that C/EBP{delta} has a more important role upon LPS treatment. Taken together, the results showed not only the increase of C/EBP{beta} and {delta} proteins, but also that the post-translation modification of C/EBP{beta} and {delta} is involved in LPS-induced IL-10 gene activation. It was reported that phosphorylation of C/EBP{beta} 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{beta} and {delta} 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{beta} and {delta} 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{beta} and {delta} protein in intact cells. By using a coimmunoprecipitation method, we found that Sp1 protein could interact with C/EBP{beta} and {delta} protein, and the physical binding of Sp1-C/EBP{beta} or Sp1-C/EBP{delta} was increased after LPS treatment (Fig. 8B). This is the first evidence to identify the physical interaction between Sp1 and C/EBP{beta} and {delta} 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{beta} 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-{kappa}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{beta} and {delta} were all required for LPS-induced activation of the IL-10 gene promoter and that Sp1 could interact physically and functionally with C/EBP{beta} and C/EBP{delta} in mouse macrophages.


   Acknowledgments
 
We are greatly indebted to Dr. Sheng-Chung Lee (National Taiwan University, Taipei, Taiwan) and Dr. Steve Mcknight (University of Texas, Dallas) for providing plasmids pCMV-fl(AGP/EBP) and MSV/EBP{delta}, respectively. Thanks are also due to Johnson C. Hou and Dr. George. S. Roth for their critical reading of the manuscript.


   Footnotes
 
1 This work was supported in part by Grants NSC-89-B-041-2320-017 and NSC-90-B-041-2320-017 from the National Science Council of the Republic of China and by the Ministry of Education Program for Promoting Academic Excellence of Universities under Grant 91-B-FA09-1-4 of the Republic of China. Back

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 Back

3 Abbreviations used in this paper: Sp1, SV40 promoter factor 1; SL2, Schneider line 2; AGP, {alpha}-1 acid glycoprotein; MSV, mouse sarcoma virus. Back

Received for publication December 23, 2002. Accepted for publication May 6, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Fiorentino, D. F., M. W. Bond, T. R. Mosmann. 1989. Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J. Exp. Med. 170:2081.[Abstract/Free Full Text]
  2. van Deventer, S. J., C. O. Elson, R. N. Fedorak. 1997. Multiple doses of intravenous interleukin-10 in steroid-refractory Crohn’s disease: Crohn’s Disease Study Group. Gastroenterology 113:383.[Medline]
  3. Lindsay, J. O., H. J. Hodgson. 2001. Review article: the immunoregulatory cytokine interleukin-10—a therapy for Crohn’s disease. Aliment. Pharmacol. Ther. 15:1709.[Medline]
  4. Keystone, E., J. Wherry, P. Grint. 1998. IL-10 as a therapeutic strategy in the treatment of rheumatoid arthritis. Rheum. Dis. Clin. N. Am. 24:629.[Medline]
  5. Asadullah, K., W. Sterry, K. Stephanek, D. Jasulaitis, M. Leupold, H. Audring, H. D. Volk, W. D. Docke. 1998. IL-10 is a key cytokine in psoriasis. Proof of principle by IL-10 therapy: a new therapeutic approach. J. Clin. Invest. 101:783.[Medline]
  6. Asadullah, K., W. D. Docke, M. Ebeling, M. Friedrich, G. Belbe, H. Audring, H. D. Volk, W. Sterry. 1999. Interleukin-10 treatment of psoriasis: clinical results of a phase 2 trial. Arch. Dermatol. 135:187.[Abstract/Free Full Text]
  7. McHutchison, J. G., G. Giannelli, L. Nyberg, L. M. Blatt, K. Waite, P. Mischkot, S. Pianko, A. Conrad, P. Grint. 1999. A pilot study of daily subcutaneous interleukin-10 in patients with chronic hepatitis C infection. J. Interferon Cytokine Res. 19:1265.[Medline]
  8. Nelson, D. R., G. Y. Lauwers, J. Y. Lau, G. L. Davis. 2000. Interleukin-10 treatment reduces fibrosis in patients with chronic hepatitis C: a pilot trial of interferon nonresponders. Gastroenterology 118:655.[Medline]
  9. Dai, W. J., G. Kohler, F. Brombacher. 1997. Both innate and acquired immunity to Listeria monocytegenes infection are increased in IL-10 deficient mice. J. Immunol. 158:2259.[Abstract]
  10. Kullberg, M. C., J. M. Ward, P. L. Gorelick, P. Caspar, S. Hieny, A. Cheever, D. Jankovic, A. Sher. 1998. Helicobacter hepaticus triggers colitis in specific-pathogen-free interleukin-10 (IL-10)-deficient mice through an IL-12 and {gamma} interferon-dependent mechanism. Infect. Immun. 66:5157.[Abstract/Free Full Text]
  11. Brown, J. P., J. F. Zachary, C. Teuscher, J. J. Weis, R. M. Wooten. 1999. Dual role of interleukin-10 in murine Lyme disease: regulation of arthritis severity and host defense. Infect. Immun. 67:5142.[Abstract/Free Full Text]
  12. Sieling, P. A., J. S. Abrams, M. Yamamura, P. Salgame, B. R. Bloom, T. H. Rea, R. L. Modlin. 1993. Immunosuppressive roles for IL-10 and IL-4 in human infection: in vitro modulation of T cell responses in leprosy. J. Immunol. 150:5501.[Abstract]
  13. Landay, A. L., M. Clerici, F. Hashemi, H. Kessler, J. A. Berzofsky, G. M. Shearer. 1996. In vitro restoration of T cell immune function in human immunodeficiency virus-positive persons: effects of interleukin (IL)-12 and anti-IL-10. J. Infect. Dis. 173:1085.[Medline]
  14. Llorente, L., Y. Richaud-Patin, J. Couderc, D. Alarcon-Segovia, R. Ruiz-Soto, N. Alcocer-Castillejos, J. Alcocer-Varela, J. Granados, S. Bahena, P. Galanaud, D. Emilie. 1997. Dysregulation of interleukin-10 production in relatives of patients with systemic lupus erythematosus. Arthritis Rheum. 40:1429.[Medline]
  15. Lazarus, M., A. H. Hajeer, D. Turner, P. Sinnott, J. Worthington, W. E. Ollier, I. V. Hutchinson. 1997. Genetic variation in the interleukin 10 gene promoter and systemic lupus erythematosus. J. Rheumatol. 24:2314.[Medline]
  16. Westendorp, R. G., J. A. Langermans, T. W. Huizinga, A. H. Elouli, C. L. Verweij, D. I. Boomsma, J. P. Vandenbroucke. 1997. Genetic influence on cytokine production and fatal meningococcal disease. Lancet 349:170.[Medline]
  17. Eskdale, J., D. Kube, G. Gallagher. 1996. A second polymorphic dinucleotide repeat in the 5' flanking region of the human IL10 gene. Immunogenetics 45:82.[Medline]
  18. Platzer, C., E. Fritsch, T. Elsner, M. H. Lehmann, H. D. Volk, S. Prosch. 1999. Cyclic adenosine monophosphate-responsive elements are involved in the transcriptional activation of the human IL-10 gene in monocytic cells. Eur. J. Immunol. 29:3098.[Medline]
  19. Platzer, C., W. Docke, H. Volk, S. Prosch. 2000. Catecholamines trigger IL-10 release in acute systemic stress reaction by direst stimulation of its promoter/enhancer activity in monocytic cells. J. Neuroimmunol. 105:31.[Medline]
  20. Ma, W., W. Lim, K. Gee, S. Aucoin, D. Nandan, M. Kozlowski, F. Diaz-Mitoma, A. Kumar. 2001. The p38 mitogen-activated kinase pathway regulates the human interleukin-10 promoter via the activation of Sp1 transcription factor in lipopolysaccharide-stimulated human macrophages. J. Biol. Chem. 276:13664.[Abstract/Free Full Text]
  21. Benkhart, E. M., M. Siedlar, A. Wedel, T. Werner, H. W. L. Ziegler-Heitbrock. 2000. Role of stat3 in lipopolysaccharide-induced IL-10 gene expression. J. Immunol. 165:1612.[Abstract/Free Full Text]
  22. Brightbill, H. D., S. E. Plevy, R. L. Modlin, S. T. Smale. 2000. A prominent role for Sp1 during lipopolysaccharide-mediated induction of IL-10 promoter in macrophages. J. Immunol. 164:1940.[Abstract/Free Full Text]
  23. Tone, M., M. J. Powell, Y. Tone, S. A. J. Thompson, H. Waldmann. 2000. IL-10 gene expression is controlled by the transcription factors Sp1 and Sp3. J. Immunol. 165:286.[Abstract/Free Full Text]
  24. Wadleigh, D. J., S. T. Reddy, E. Kopp, S. Ghosh, H. R. Herschman. 2000. Transcriptional activation of the cyclooxygenase-2 gene in endotoxin-treated RAW 264.7 macrophages. J. Biol. Chem. 275:6259.[Abstract/Free Full Text]
  25. Hungness, E. S., G. J. Luo, T. A. Pritts, X. Sun, B. W. Robb, D. Hershko, P. O. Hasselgren. 2002. Transcription factors C/EBP-{beta} and -{delta} regulate IL-6 production in IL-1{beta}-stimulated human enterocytes. J. Cell. Physiol. 192:64.[Medline]
  26. Baldassare, J. J., Y. Bi, C. J. Bellone. 1999. The role of p38 mitogen-activated protein kinase in IL-1{beta} transcription. J. Immunol. 162:5367.[Abstract/Free Full Text]
  27. Fessele, S., S. Boehlk, A. Mojaat, N. G. Miyamoto, T. Werner, E. L. Nelson, D. Schlondorff, P. J. Nelson. 2001. Molecular and in silico characterization of a promoter module and C/EBP element that mediate LPS-induced RANTES/CCL5 expression in monocytic cells. FASEB J. 15:577.[Free Full Text]
  28. Brenner, S., S. Prosch, K. Schenke-Layland, U. Riese, U. Gausmann, C. Platzer. 2003. cAMP-induced interleukin-10 promoter activation depends on CCAAT/enhancer-binding protein expression and monocytic differentiation. J. Biol. Chem. 278:5597.[Abstract/Free Full Text]
  29. Caivano, M., B. Gorgoni, P. Cohen, V. Poli. 2001. The induction of cyclooxygenase-2 mRNA in macrophages is biphasic and requires both CCAAT enhancer-binding protein {beta} (C/EBP{beta}) and C/EBP{delta} transcription factors. J. Biol. Chem. 276:48693.[Abstract/Free Full Text]
  30. Nakajima, T., S. Kinoshita, T. Sasagawa, K. Sasaki, M. Naruto, T. Kishimoto, S. Akira. 1993. Phosphorylation at threonine-235 by a ras-dependent mitogen-activated protein kinase cascade is essential for transcription factor NF-IL6. Proc. Natl. Acad. Sci. USA 90:2207.[Abstract/Free Full Text]
  31. Saunders, M. A., L. Sansores-Garcia, D. W. Gilroy, K. K. Wu. 2001. Selective suppression of CCAAT/enhancer-binding protein {beta} binding and cyclooxygenase-2 promoter activity by sodium salicylate in quiescent human fibroblasts. J. Biol. Chem. 276:18897.[Abstract/Free Full Text]
  32. Zhu, Y., M. A. Saunders, H. Yeh, D. Wu-guo, K. K. Wu. 2002. Dynamic regulation of cyclooxygenase-2 promoter activity by isoforms of CCAAT/enhancer-binding proteins. J. Biol. Chem. 277:6923.[Abstract/Free Full Text]
  33. Lacroix, I., C. Lipcey, J. Imbert, B. Kahn-Perles. 2002. Sp1 transcriptional activity is up-regulated by phosphatase 2A in dividing T lymphocytes. J. Biol. Chem. 277:9598.[Abstract/Free Full Text]
  34. Schafer, D., B. Hamm-Kunzelmann, K. Brand. 1997. Glucose regulates the promoter activity of aldolase A and pyruvate kinase M2 via dephosphorylation of Sp1. FEBS Lett. 417:325.[Medline]
  35. Daniel, S., S. Zhang, A. A. DePaoli-Roach, K. H. Kim. 1996. Dephosphorylation of Sp1 by protein phosphatase 1 is involved in the glucose-mediated activation of the acetyl-CoA carboxylase gene. J. Biol. Chem. 271:14692.[Abstract/Free Full Text]
  36. Zutter, M. M., E. E. Ryan, A. D. Painter. 1997. Binding of phosphorylated Sp1 protein to tandem Sp1 binding sites regulates {alpha}2 integrin gene core promoter activity. Blood 90:678.[Abstract/Free Full Text]
  37. Milanini-Mongiat, J., J. Pouysségur, G. Pagès. 2002. Identification of two Sp1 phosphorylation sites for p42/p44 mitogen-activated protein kinases. J. Biol. Chem. 277:20631.[Abstract/Free Full Text]
  38. Lee, Y. H., S. C. Williams, M. Baer, E. Sterneck, F. J. Gonzalez, P. F. Johnson. 1997. The ability of C/EBP{beta} but not C/EBP{alpha} to synergize with an Sp1 protein is specified by the leucine zipper and activation domain. Mol. Cell. Biol. 17:2038.[Abstract]
  39. López-Rodriguez, C., L. Botella, A. L. Corbi. 1997. CCAAT-enhancer-binding proteins (C/EBP) regulate the tissue specific activity of the CD11c intergrin gene promoter through functional interactions with Sp1 proteins. J. Biol. Chem. 272:29120.[Abstract/Free Full Text]
  40. Khanna-Gupta, A., T. Zibello, C. Simkevich, A. G. Rosmarin, N. Berliner. 2000. Sp1 and C/EBP are necessary to activate the lactoferrin gene promoter during myeloid differentiation. Blood 15:3734.
  41. Aigueperse, C., P. Val, C. Pacot, C. Darne, E. Lalli, P. Sassone-Corsi, G. Veyssiere, C. Jean, A. Martinez. 2001. SF (steroidogenic factor-1), C/EBP{beta} (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.
  42. Dynan, W. S., R. Tjian. 1983. The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell 35:79.[Medline]
  43. Lee, J. S., K. M. Galvin, Y. Shi. 1993. Evidence for physical interaction between the zinc-finger transcription factors YY1 and Sp1. Proc. Natl. Acad. Sci. USA 90:6145.[Abstract/Free Full Text]
  44. Perkins, N. D., A. B. Agranoff, E. Pascal, G. J. Nabel. 1994. An interaction between the DNA-binding domains of RelA(p65) and Sp1 mediates human immunodeficiency virus gene activation. Mol. Cell. Biol. 14:6570.[Abstract/Free Full Text]
  45. Gregory, R. C., D. J. Taxman, D. Seshasayee, M. H. Kensinger, J. J. Bieker, D. M. Wojchowski. 1996. Functional interaction of GATA1 with erythroid Kruppel-like factor and Sp1 at defined erythroid promoters. Blood 87:1793.[Abstract/Free Full Text]
  46. Ding, H., A. M. Benotmane, G. Suske, D. Collen, A. Belayew. 1999. Functional interactions between Sp1 or Sp3 and the helicase-like transcription factor mediate basal expression from the human plasminogen activator inhibitor-1 gene. J. Biol. Chem. 274:19573.[Abstract/Free Full Text]
  47. Chen, B. K., W. C. Chang. 2000. Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase. Proc. Natl. Acad. Sci. USA 97:10406.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
M. E. Pennini, Y. Liu, J. Yang, C. M. Croniger, W. H. Boom, and C. V. Harding
CCAAT/Enhancer-Binding Protein beta and {delta} Binding to CIITA Promoters Is Associated with the Inhibition of CIITA Expression in Response to Mycobacterium tuberculosis 19-kDa Lipoprotein
J. Immunol., November 15, 2007; 179(10): 6910 - 6918.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
B. Csoka, Z. H. Nemeth, L. Virag, P. Gergely, S. J. Leibovich, P. Pacher, C.-X. Sun, M. R. Blackburn, E. S. Vizi, E. A. Deitch, et al.
A2A adenosine receptors and C/EBP{beta} are crucially required for IL-10 production by macrophages exposed to Escherichia coli
Blood, October 1, 2007; 110(7): 2685 - 2695.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
E. Y. Chang, B. Guo, S. E. Doyle, and G. Cheng
Cutting Edge: Involvement of the Type I IFN Production and Signaling Pathway in Lipopolysaccharide-Induced IL-10 Production
J. Immunol., June 1, 2007; 178(11): 6705 - 6709.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Z. Yang, D. M. Mosser, and X. Zhang
Activation of the MAPK, ERK, following Leishmania amazonensis Infection of Macrophages
J. Immunol., January 15, 2007; 178(2): 1077 - 1085.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
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]

Home page
 J. Biol. Chem.Home page
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]

Home page
 Infect. Immun.Home page
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]

Home page
 J. Immunol.Home page
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]

Home page
 J. Immunol.Home page
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]