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*Compound via MeSH
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*ESTRADIOL
Medline Plus Health Information
*Joint Disorders
*Rheumatoid Arthritis
The Journal of Immunology, 2007, 178: 3059-3066.
Copyright © 2007 by The American Association of Immunologists, Inc.

17beta-Estradiol Induces IL-1{alpha} Gene Expression in Rheumatoid Fibroblast-Like Synovial Cells through Estrogen Receptor {alpha} (ER{alpha}) and Augmentation of Transcriptional Activity of Sp1 by Dissociating Histone Deacetylase 2 from ER{alpha}1

Yuka Itoh*, Hidetoshi Hayashi*, Keiji Miyazawa{dagger}, Soichi Kojima{ddagger}, Tohru Akahoshi§ and Kikuo Onozaki2,*

* Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan; {dagger} Central Research Laboratories, Kissei Pharmaceutical Company, Azumino, Japan; {ddagger} Molecular Cellular Pathology Research Unit, Institute of Physical and Chemical Research, Wako, Japan; and § Departments of Laboratory Medicine, Kitasato University School of Medicine, Sagamihara, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Rheumatoid arthritis (RA) occurs four times more frequently in women than in men, although the mechanistic basis of the gender difference is unknown. RA is characterized by the overproliferation of synoviocytes producing proinflammatory cytokines such as IL-1, implicated in the pathogenesis of the disease. In this study we examined whether 17beta-estradiol (E2) induced IL-1{alpha} mRNA expression in the rheumatoid fibroblast-like cell line MH7A, as well as in primary synovial cells from RA patients, and investigated the underlying molecular mechanisms. E2 induced IL-1{alpha} mRNA expression in both cell types in an estrogen receptor-dependent manner. In MH7A cells ER{alpha} but not ERbeta mediated the effects of E2. Deletion and mutation analysis revealed that a GC-rich region within the IL-1{alpha} gene promoter was responsible for the response to E2. EMSAs showed that Sp1 and Sp3 bound to the GC-rich region and that the transcriptional activity of Sp1 was up-regulated by the treatment with E2. Sp1 and ER{alpha} interacted physically regardless of the presence of E2. Physical interaction was also observed between ER{alpha} and histone deacetylase 2 (HDAC2), and E2 induced the dissociation of HDAC2 from ER{alpha}. These results suggest that E2 induces the dissociation of corepressor HDAC2 from ER{alpha}, which leads to the augmentation of Sp1 transcriptional activity through the GC-rich region within the IL-1{alpha} gene promoter.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Rheumatoid arthritis (RA)3 is a chronic inflammatory disease characterized by an aberrantly proliferative synovium, leading to the formation of hyperplastic synovial tissue (pannus) that invades both cartilage and bone. Experimental and clinical studies have established the critical role of the cytokine network in mediating the inflammatory and destructive processes of RA. For example, a parallel improvement in synovial inflammation and decreased joint destruction has been demonstrated in RA patients following treatment with neutralizing an anti-TNF{alpha} Ab (1), a soluble TNFR (2), an IL-1R antagonist (IL-1Ra) (3), or a neutralizing anti-IL-6 Ab (4). However, the exact roles of these cytokines in the initial phase of the disease are not clear.

RA is a disease with higher incidence in women; the ratio of the incidence in women to men is ~4:1. It has been reported that physiological levels of estrogens stimulate immune responses, whereas androgens suppress inflammatory reactions (5). The ratio of estrogen to androgen in synovial fluid is elevated in both male and female RA patients, and the serum levels of estrogens, particularly estradiol, are altered in male RA patients (6, 7). However, the role(s) of estrogens in the disease are unclear. Estrogens such as 17beta-estradiol (E2) play critical roles in many physiological processes in both females and males, including normal growth, development, and cell type-specific regulation of gene expression in tissues of the reproductive tract, central nervous system, and skeleton. Although E2 works protectively against the pathogenesis of osteoporosis, Alzheimer’s disease, and cardiovascular disease, E2 plays a role in the development of breast cancer. The effects of E2 are mainly mediated through the estrogen receptors ER{alpha} and ERbeta, members of the nuclear receptor superfamily, which function as ligand-induced transcription factors (8). ER{alpha} and ERbeta regulate gene expression through binding directly to cognate sequences within the promoters of target genes, referred to as the estrogen response element (ERE), or through protein/protein interaction with other transcription factors.

IL-1 is an immunomodulatory and proinflammatory cytokine that exerts a wide spectrum of biological effects, including the stimulation of T and B lymphocytes, bone resorption, and pyrogenicity (9, 10, 11). IL-1 has also been implicated in the pathogenesis of chronic inflammatory joint diseases such as RA. In RA patients, elevated IL-1 levels have been identified in the synovial fluid, synovial membrane, and cartilage-pannus junction of arthritic joints (12). Synoviocytes derived from RA patients often produce IL-1{alpha} and IL-1beta. Indeed, IL-1 triggers synovial cell proliferation and induces matrix metalloproteinase production in vitro (13, 14), and intraarticular injections of IL-1 cause active synovitis and marked depletion of the cartilage matrix in several mammalian species in vivo (15, 16). Studies of collagen-induced arthritis in mice have revealed that IL-1 plays an important role in the process of joint destruction (17, 18), because blocking of IL-1 activity can reduce the severity of synovitis and prevent cartilage and bone destruction. Knockout mice of the IL-1Ra gene exhibit a spontaneous outcome of RA-like arthritis (19). There are two types of IL-1, {alpha} and beta, which exhibit several different characteristics (20, 21). Although they exert the same biological activities by binding to the same receptors (IL-1R), the precursor of IL-1{alpha} can bind to IL-1R and exert biological activities, whereas that of IL-1beta binds very weakly to IL-1R and cannot exert biological effects (22). Thus, IL-1beta works after proteolytic maturation following the release from its producing cells, while IL-1{alpha} can exert the biological activity even in the membrane-bound form (23). Both isoforms appear to play a pivotal role in cartilage destruction, while it has been reported that IL-1beta plays a more dominant role in established collagen-induced agonist based on evidence of the marked inhibitory effects of anti-IL-1beta treatment (18) or IL-1-converting enzyme inhibitor (24). IL-1{alpha} is reportedly predominant in the early phase of arthritis and participates in the inhibition of proteoglycan synthesis (25). Interestingly, membrane bound IL-1{alpha} in the synovial cells, but not soluble IL-1{alpha}, is important in the spontaneous outcome of the arthritis in IL-1{alpha} transgenic mice (23).

Because RA occurs more frequently in females than in males, we focused on the effect of E2 on IL-1{alpha} gene expression in synoviocytes from RA patients and a rheumatoid fibroblast-like synovial cell line. We showed that Sp1 binding to the GC-rich region within its promoter region of the IL-1{alpha} gene was critical and that E2 induced the transcriptional activity of Sp1 through ER{alpha} by removing histone deacetylase inhibitor (HDAC) 2 from the GC-rich region.


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

DMEM was purchased from Sigma-Aldrich, and FBS was from JRH Bioscience. The anti-ER{alpha} polyclonal Ab (clone HC-20), the anti-Sp1 polyclonal Ab (clone PEP-2), and the anti-Sp3 polyclonal Ab (clone H-225) were from Santa Cruz Biotechnology. The anti-HDAC1 mAb and anti-HDAC2 polyclonal Ab were purchased from Upstate; the anti-beta-actin mAb (clone AC-15), the anti-Flag mAb (M2), and E2 were purchased from Sigma-Aldrich; and ICI 182,780 was from Tocris Cookson.

Cell culture

Primary synovial cells from RA patients, as well as MH7A, an immortalized cell line established by stably transfecting rheumatoid synoviocytes with the SV40 T Ag gene (26), were cultured in DMEM without phenol red with 100 U/ml penicillin G, 100 mg/ml streptomycin, 4 mM L-glutamine, and 10% heat-inactivated FBS at 37°C in air containing 5% CO2. 293 cells were cultured in DMEM with 100 U/ml penicillin G, 100 mg/ml streptomycin, and 5% heat-inactivated FBS at 37°C in air containing 5% CO2. The day before transient transfection or stimulation, the medium was changed to phenol red-free DMEM with 100 U/ml penicillin G, 100 µg/ml streptomycin, 4 mM L-glutamine, and 10% heat-inactivated FBS pretreated with dextran-coated charcoal (Sigma-Aldrich).

Plasmids

The IL-1{alpha} promoter luciferase constructs pGL3-IL1a(–421), pGL3-IL1a(–103), pGL3-IL1a(–70), pGL3-IL1a(–51), and pGL3-IL1a(–31) were constructed using insertion oligonucleotides. pGL3-IL1a({Delta}CC) and pGL3-(IL1a-GC)3 were produced by PCR. pFR-Luc, which contained five tandem repeats of the GAL4 binding site, was purchased from Stratagene. The GAL4-Sp1 expression vector was constructed as described previously (27). Human ER{alpha} and ERbeta expression plasmids were provided by Dr. S. Kato (University of Tokyo, Tokyo, Japan). Flag-HDAC1 and Flag-HDAC2 expression plasmids were kindly provided by Dr. E. Seto (H. Lee, Moffitt Cancer Center and Research Institute, Tampa, FL).

Transient transfection and luciferase assays

The day before the transient transfection of MH7A cells or 293 cells, the culture medium was replaced by phenol red-free DMEM supplemented with 10% heat-inactivated FBS that had been pretreated with dextran-coated charcoal to remove endogenous estrogens. The IL-1{alpha} reporter plasmids, the hER{alpha} or hERbeta expression plasmid, and the pCMV-betagal plasmid (for normalization of transfection efficiency) were transiently transfected into MH7A cells using the calcium phosphate-DNA coprecipitation method. After 15 h of transfection, cells were incubated with E2 for an additional 24 h and harvested. Luciferase assays were performed with the luciferase reporter gene assay kit (Roche) according to the manufacturer’s instructions. The light emission was measured using the Arvo 1420 multilabel counter (Pharmacia). Luciferase activity was expressed after normalization with the beta-galactosidase value in the same sample.

Semiquantitive RT-PCR analysis

Total RNA from cells was extracted according to the method of Chomczynski and Sacchi (28). RT-PCR analysis was performed as described previously (29). The primers used for human ER{alpha} (5'-GTCTGAGGCTGCGGCGTTCGGCTCC-3' and 5'-ATTCCATAGCCATACTTCCCTTGTC-3'; 281-bp product) and human ERbeta (5'-GGCAACTACTTCAAGGTTTCGAGAG-3' and 5'-ACTCGCATGCCTGACGTGGGACAGG-3'; 265-bp product) were previously described (30, 31). For human IL-1{alpha} the primers were 5'-ATGGCCAAAGTTCCAGACATG-3' and 5'-CTACGCCTGGTTTTCCAGTATCTGAAAGTCAGT-3' (816-bp product), and for human GAPDH they were 5'-TGAAGGTCGGAGTCAACGGATTTGT-3' and 5'-CATGTGGGCCATGAGGTCCACCAC-3' (980-bp product).

EMSA

The preparation of nuclear extracts and EMSA was conducted as previously described (32, 33, 34). The oligonucleotides used for EMSA were as follows: probe 1, 5'-CGTAGCCACGCCTACTTAAG-3', which corresponds to IL-1{alpha} promoter –56/–37; probe 2, 5'-TAGCCACGCCTACTTAAGAC-3', which corresponds to IL-1{alpha} promoter –54/–35; probe 3, 5'-CCACGCCTACTTAAGACAAT-3', which corresponds to IL-1{alpha} promoter –51/–32; probe 4, 5'-ACGCACTTGTAGCCACGTAG-3', which corresponds to IL-1{alpha} promoter –71/–52; probe m1, 5'-CGTAGCCAATAATACTTAAG-3', which is a mutated version of probe 1 with a change in the four underlined bases; and probe m2, 5'-CGTATAAACGCCTACTTAAG-3', which is another mutated version of probe 1 with a change in the three underlined bases.

Immunoprecipitation and Western blot analysis

Cells were transiently transfected and treated as described in the figure legends. The cells were lysed in radioimmune precipitation assay (RIPA) buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, and 1% Triton X-100) supplemented with protease inhibitors and phosphatase inhibitors. The lysates were then subjected to immunoprecipitation with anti-Sp1 Ab or anti-Flag Ab. Coimmunoprecipitates were subjected to SDS-PAGE, transferred onto polyvinylidene difluoride membranes (Millipore), and probed with the Abs described in the figure legends. The immunoreactive proteins were visualized using ECL Western blotting detection reagents (Amersham Biosciences), and analyzed by a chemiluminescence image analyzer, LAS-1000 (Fuji Photo Film).

Promoter pull-down assay

By using PCR with a biotinylated primer and pGL3-IL1a(–70) plasmid or pGL3-IL1a(ATAA), the promoter was biotinylated at the 5'-end of the lower strand. MH7A cells were transiently transfected with pcDNA3-ER{alpha} and then incubated with 1 nM E2 for the indicated time. Cells were lysed in RIPA buffer supplemented with protease inhibitors. Cell lysates were supplemented with 0.2 mg/ml polydeoxyinosinic-deoxycytidylic acid and incubated with a 5 nM biotinylated IL-1{alpha} promoter fragment containing the 70 bp upstream from the transcriptional start site of the human IL-1{alpha} gene for 2 h at 4°C. Fifty milligrams of streptavidin-conjugated magnetic particles (Promega) were then added and incubated for an additional 1 h. The particles were washed four times and proteins were eluted with sample buffer and subjected to Western blot analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
17 beta-estradiol induces IL-1{alpha} mRNA expression in a manner dependent on ER{alpha}

To investigate the effect of E2 on IL-1{alpha} mRNA expression in RA synovial cells, two primary preparations of synovial cells from RA patients and synovial cell line MH7A were incubated with or without a physiological concentration of E2 and then RT-PCR analysis was performed (Fig. 1A). Physiological levels of E2 up to 100 nM are present in the circulation during the follicular phase of the menstrual cycle and in early and late pregnancy (35). E2 induced IL-1{alpha} mRNA in primary synovial cells (Fig. 1A, lanes 2 and 5), but not in MH7A cells (lane 8). A specific antagonist for ER, ICI 182,780, inhibited this effect (Fig. 1A, lanes 3 and 6), indicating that the effect of E2 is mediated through ER. E2 evokes many biological effects through ER{alpha} and/or ERbeta. As shown in Fig. 1B, primary synovial cells expressed the mRNAs of both ER{alpha} and ERbeta (lanes 1 and 2), whereas MH7A cells expressed only ERbeta mRNA (lane 3). These results suggested that the lack of E2 response in MH7A cells might have been due to the loss of ER{alpha} expression in this cell line. Consistent with this possibility, when we expressed ectopic ER{alpha} in MH7A cells, IL-1{alpha} mRNA expression was significantly increased by treatment with E2 (Fig. 1C, lane 2).


Figure 1
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  		FIGURE 1. Effects of E2 and ERs on IL-1{alpha} mRNA expression. A, Total RNA was extracted from primary synovial cells or synovial cell line MH7A incubated with 10 nM E2 in the presence or absence of 1 µM ICI 182,780 (ICI) for 4 h and IL-1{alpha} and GAPDH mRNA levels were determined by RT-PCR. B, Total RNA was extracted from each type of cell and ER{alpha}, ERbeta, and GAPDH mRNA levels were determined by RT-PCR. C, Total RNA was extracted from MH7A cells with ER{alpha}, and IL-1{alpha} and GAPDH mRNA levels were determined by RT-PCR.

 
E2 activates IL-1{alpha} transcription in a manner dependent on ER{alpha}

Next, we investigated the effects of E2 and ERs on IL-1{alpha} promoter activity to determine whether IL-1{alpha} mRNA is induced by E2 at the level of transcription. MH7A cells were transfected with pGL3-IL1a(–421), which contains 421 bp upstream from the transcriptional start site in the human IL-1{alpha} gene, upstream from a luciferase reporter cDNA together with the expression plasmid of ER{alpha} or ERbeta, and stimulated with E2, and then the luciferase activity in the cell lysates was determined (Fig. 2A). The promoter activity was increased by E2 when ER{alpha} was expressed, whereas ectopic expression of ERbeta did not affect the induction of the promoter activity by E2. We confirmed that the ERbeta overexpressed in this study was functionally active by using pERE3-tk-Luc, which contains estrogen responsive elements (data not shown). We also examined the dose dependency of the effect of E2 on the ER{alpha}-dependent IL-1{alpha} promoter activity. The promoter activity was up-regulated similarly by 1–100 nM E2 (data not shown). To confirm the effect of ERs on the IL-1{alpha} promoter activity, we treated the cells with ICI 182,780 with or without E2 and measured the luciferase activity (Fig. 2B). The IL-1{alpha} promoter activity induced by 1 nM E2 was blocked by 100 nM ICI182,780. The IL-1{alpha} promoter activity was increased by treatment with 10 nM ICI182,780 alone, and the level of activation did not change up to 1 µM ICI 182,780 (1.6- to 1.9-fold). These data indicate that E2 increases the IL-1{alpha} promoter activity through ER{alpha}.


Figure 2
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  		FIGURE 2. Effects of ERs and ICI on E2-induced IL-1{alpha} gene promoter activation in MH7A cells. A, Cells were transiently transfected with pcDNA3-ER{alpha} or pcDNA3-ERbeta, pGL3-IL1a(–421), and pCMV-betagal and then incubated with or without 1 nM E2 for 24 h. Luciferase activity was normalized by beta-galactosidase activity. B, Cells were transiently transfected with pcDNA3-ER{alpha}, pGL3-IL1a(–421), and pCMV-betagal and then incubated with or without 1 nM E2 in the presence of the indicated concentration of ICI for 24 h. Luciferase activity was normalized by beta-galactosidase activity.

 
GC-rich region is important for the ER{alpha}-dependent stimulation of transcription of IL-1{alpha} by E2

As shown in Fig. 3A, the 421-bp promoter region used in the reporter assays has no typical EREs but contains several transcriptional consensus sequences, including a glucocorticoid-responsive element-like sequence, an NF-{kappa}B binding site, a GC-rich sequence, a TATA-like sequence, and an AP-1 binding site. To analyze the promoter region(s) involved in the E2-dependent IL-1{alpha} promoter activation mediated through ER{alpha}, we constructed a series of 5'-end deletion mutants of the IL-1{alpha} gene promoter-luciferase chimera expression plasmids. These chimeric mutant genes were transfected into MH7A cells and the luciferase activity in the cell lysates was determined (Fig. 3B). Deletion of the nucleotides –421 to –70 caused almost no change in the augmentation by E2. However, further deletion to –51 led to a decrease in the transcriptional activity and loss of its enhancement by E2. These results suggest that the sequence between –70 and –51, containing a part of the GC-rich region, is critical for the enhancement by E2 of the IL-1{alpha} promoter activity. This was confirmed by experiments using a deletion mutant plasmid lacking part of the GC-rich region (pGL3-IL1a(–421) {Delta}CC). As shown in Fig. 3C, this mutant exhibited a marked decrease in the basal promoter activity and hardly any response to E2 as well, indicating that the GC-rich region is important for the E2-dependent IL-1{alpha} promoter activation in MH7A cells.


Figure 3
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  		FIGURE 3. Deletion and mutation analysis. A, Schematic diagram of IL-1{alpha} gene promoter and its various cis elements for transcription factors. GRE, Glucocorticoid-responsive element-like sequence. B, Deletion analysis of the IL-1{alpha} promoter-proximal region to examine the its ER{alpha}-dependent activation. Cells were transiently transfected with indicated IL-1{alpha} promoter constructs, pcDNA3-ER{alpha}, and pCMV-betagal, and then incubated with or without 1 nM E2 for 24 h. Luciferase activity was normalized by beta-galactosidase activity. C, Mutational analysis of pGL3-IL1a(–421). Cells were transiently transfected with pcDNA3-ER{alpha} and pGL3-IL1a(–421) or pGL3-IL1a(–421){Delta}CC (with deletion of part of the GC-rich region) and pCMV-betagal and then incubated with or without 1 nM E2 for 24 h. Luciferase activity was normalized by beta-galactosidase activity.

 
Sp1 and Sp3, but not ER{alpha}, bind to 10 bases (TAX4CGCC) of the GC-rich region within the IL-1{alpha} promoter

It is known that Sp1 and Sp3 bind to the GC-rich region in the promoter of a number of genes and regulate their promoter activities. We therefore investigated the binding of Sp1, Sp3, and ER{alpha} to the IL-1{alpha} promoter using EMSA (Fig. 4). There are several CG islands within the sequence between –70 and –31. The sequences of oligonucleotides used as probes are showed in Fig. 4A. As shown in Fig. 4B, three major DNA-nuclear protein complexes were observed using end-labeled oligonucleotide (–54 to –35, probe 2) (lane 1). The binding was not affected by overexpression of ER{alpha} alone (Fig. 4B, lane 2), and E2 treatment had little effect on the binding (lane 3); however, the binding was strongly attenuated in the presence of ICI 182,780 (lane 4). Supershift experiments using Abs against Sp1 and Sp3 indicated that the upper band was an Sp1-DNA complex (Fig. 4B, lane 5) and the lower two bands were Sp3-DNA complexes (lane 6), which was confirmed by an experiment using labeled probe 1(Fig. 4C, lanes 10 and 11). No supershifted band was observed using an ER{alpha} Ab even in the presence of E2 (Fig. 4C, lane 12), suggesting that ER{alpha} did not bind to the sequence directly or associated with it only weakly. To identify the sequence required for the binding of Sp1 and Sp3, competitive binding assays were performed using labeled probe 1 and unlabeled probes 1–4 as well as mutant oligonucleotides (probe m1 and probe m2) (Fig. 4C). The results indicated that the most downstream CG island (CGCC, –48 to –45) was critical for the Sp1/Sp3 binding and that the second downstream GC island (GCC, –52 to –50) was necessary for maximum binding (Fig. 4C, lanes 1~4).


Figure 4
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  		FIGURE 4. Binding of Sp1 and Sp3 in nuclear proteins from MH7A cells to the GC-rich region of the IL-1{alpha} gene promoter. A, Sequences of probes 1, 2, 3, and 4 and mutated oligonucleotides (m1 and m2) are presented. B, Effects of E2 and ICI 182,780 (ICI) on the binding of nuclear proteins to the GC-rich region of the IL-1{alpha} gene promoter. Cells were transiently transfected with pcDNA3-ER{alpha}, and then incubated with 1 nM E2 in the presence or absence of 1 µM ICI for 1 h. Nuclear extracts were obtained and EMSAs were performed with 32P-labeled probe 2 (–54/–35) or 32P-labeled probe 3 (–51/–32). Supershift analysis was performed using anti-Sp1 or anti-Sp3 Abs. The supershifted bands are indicated with the arrows on the right. C, Sp1- and Sp3-binding sequence in the GC-rich region in the IL-1{alpha} gene promoter. Cells were transiently transfected with pcDNA3-ER{alpha} and then incubated with or without 1 nM E2 for 1 h. EMSAs were performed with 32P-labeled probe 1 (–56/–37) and the nuclear extracts of MH7A cells in the presence of a 250-fold excess of each unlabeled probe, designated as probe 1, m1, m2, 2, 3, or 4 to identify the Sp1- and Sp3-binding sequences. The bands for specific binding and supershifted bands are indicated with the arrows on the right. Supershift analysis was performed using anti-Sp1, anti-Sp3, or anti-ER{alpha} Abs. comp, Competitor.

 
As shown in Fig. 4B, Sp1/Sp3 bound to probe 2, which contained the two CG islands; however probe 3, lacking a part of the CG island (–51,-50) but preserving the most downstream GCC sequence, bound to neither Sp1 nor Sp3. We also found that probe 2 completely blocked the binding of Sp1/Sp3 to probe1 but that probe 3 did not (Fig. 4C). These results suggest that not only CGCC (–48 to –45) but also the region upstream of the second downstream CG island (–54 and –53) was necessary for the stronger binding of Sp1/Sp3 to the IL-1{alpha} promoter.

E2 promotes the ER{alpha}-dependent transactivation of the IL-1{alpha} promoter via the Sp1 binding site and Sp1 activity

To determine whether ER{alpha} activated by E2 up-regulates the IL-1{alpha} promoter activity through the sequence critical for Sp1 and Sp3 binding, we performed the luciferase assay using a reporter gene (pGL3-(IL1a-GC)3) containing three tandem repeats of the putative sequence sufficient for Sp1 and Sp3 binding (TAGCCACGCC). As shown in Fig. 5A, E2 activated the promoter activity, indicating again that the sequence TAGCCACGCC is critical for the ER{alpha}-dependent transactivation of the IL-1{alpha} promoter by E2.


Figure 5
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  		FIGURE 5. Mutation analysis and effects of E2 and ICI on Sp1 activity. A, Mutational analysis of pGL3-IL1a(–421). Cells were transiently transfected with pcDNA3-ER{alpha}, pGL3-(IL1a-GC)3 (containing three tandem repeats of the GC-rich sequence in the IL-1{alpha} gene promoter), and pCMV-betagal and then incubated with or without 1 nM E2 in the presence or absence of 100 nM ICI 182,780 (ICI) for 24 h. Luciferase (Luc) activity was normalized by beta-galactosidase activity. B, MH7A cells were transiently transfected with a combination of pFR-Luc (containing five tandem repeats of the GAL4 binding site) and the GAL4-Sp1-expressing vector that expresses Sp1 fused with the DBD of GAL4 (GAL4-DBD) or pcDNA3-ER{alpha} and pCMV-betagal and then incubated with or without 1 nM E2 and/or 100 nM ICI for 24 h. Luciferase activity was normalized by beta-galactosidase activity.

 
Because ER{alpha} activated by E2 could promote the IL-1{alpha} promoter activity through the sequence TAGCCACGCC and because Sp1 bound to this sequence, ER{alpha} may augment the Sp1 transcriptional activity. To examine this possibility, we investigated the effects of ER{alpha} and E2 on the Sp1 transcriptional activity using the one-hybrid system using the GAL4-Sp1 plasmid, which expresses Sp1 in fusion with the DNA binding domain (DBD) of GAL4. As shown in Fig. 5B, the transcriptional activity of Sp1 was potently augmented by E2 in the presence of ER{alpha}, and this augmentation was completely abrogated by an ER antagonist ICI 182,780. These results suggest that ER{alpha} activated by E2 directly enhances the transcriptional activity of Sp1 to augment the IL-1{alpha} promoter activity through the Sp1 binding site.

HDAC2 but not HDAC1 plays a critical role in the E2-induced IL-1{alpha} promoter activity in a manner dependent on ER{alpha}

To analyze the mechanism by which E2/ER{alpha} activate the IL-1{alpha} promoter activity, we next focused on the interaction between Sp1 and ER{alpha}. As shown in Fig. 6A, Sp1 physically interacted with ER{alpha} regardless of the presence of E2 in MH7A cells. Next, we performed promoter pull-down assays by using a fragment containing the GC-rich region of the IL-1{alpha} gene promoter (–70 to +51). Sp1 bound to the promoter without ER{alpha} or E2 stimulation. ER{alpha} also bound to the promoter regardless of the presence of E2, consistent with the results of EMSA (Fig. 6B, lanes 1–4). We also performed promoter pull-down assays using a biotinylated IL-1{alpha} promoter in which CGCC (–48 to –45) was mutated to ATAA. We confirmed that Sp1 did not bind to this mutated promoter as revealed by EMSA assay (Fig. 4C). Neither Sp1 nor ER{alpha} bound to the mutated promoter (Fig. 6B, lane 7). These results indicate that without E2 stimulation Sp1 and ER{alpha} bind to the IL-1{alpha} promoter and that E2 regulates the transcription activity of Sp1 through ER{alpha}.


Figure 6
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  		FIGURE 6. Interactions between ER{alpha} and Sp1, ER{alpha}, and HDACs. A, Interaction between ER{alpha} and Sp1. MH7A cells were transiently transfected with pcDNA3-ER{alpha} and then incubated with or without 1 nM E2 for 24 h. The cell lysates were immunoprecipitated (IP) with anti-Sp1 Ab and immunoblotted (Blot) with anti-ER{alpha} Ab. Total lysates were immunoblotted with anti-Sp1 Ab or anti-ER{alpha} Ab. B, Effect of E2 on the binding of ER{alpha} to the GC-rich region in the IL-1{alpha} gene promoter. MH7A cells were transiently transfected with pcDNA3-ER{alpha} and then incubated with or without 1 nM E2 for 1 h. The cell lysates were subjected to promoter pull-down assays using the biotin-conjugated nucleotide containing the IL-1{alpha} gene promoter (–70) or the mutated promoter (CGCC located at –48 to –45 mutated to ATAA) (ATAA) and immunoblotted (IB) with anti-ER{alpha} or anti-Sp1 Abs. Total lysates were immunoblotted with anti-ER{alpha} or anti-Sp1 Abs. WT, wild type. C, Interactions between HDAC1, HDAC2, and ER{alpha}. MH7A cells were transiently transfected with pcDNA3-ER{alpha} and pME18s-HDAC1 or pME18s-HDAC2 and then incubated with or without 1 nM E2 for 24 h. The cell lysates were immunoprecipitated (IP) with anti-Flag Ab and immunoblotted (Blot) with anti-ER{alpha} Ab. Total lysates were immunoblotted with anti-Flag Ab or anti-ER{alpha} Ab. D, Effects of E2 on the binding of endogenous HDAC1 or HDAC2 to the GC-rich region of the IL-1{alpha} gene promoter. MH7A cells were transiently transfected with pcDNA3-ER{alpha} and then incubated with or without 1 nM E2 for 24 h. The cell lysates were subjected to promoter pull-down assays using the biotin-conjugated nucleotide containing the IL-1{alpha} gene promoter (–70) and immunoblotted (IB) with anti-HDAC1 or anti-HDAC2 Abs. Total lysates were immunoblotted with anti-HDAC1, anti-HDAC2, or anti-ER{alpha} Abs. E, Effect of E2 on the binding of Sp1 and HDAC2. 293 cells were transiently transfected with pcDNA3-ER{alpha} and pME18s-HDAC2 and then incubated with E2 for 24 h. The cells were immunoprecipitated (IP) with anti-Flag Ab and immunoblotted (Blot) with anti-Sp1 Abs. Total lysates were immunoblotted with anti-Flag, anti-Sp1, or anti-ER{alpha} Abs.

 
Recent studies have revealed that many transcription factors, including Sp1 and ER{alpha}, regulate transcriptional activity by recruiting coactivators or corepressors. It is known that corepressor complexes containing HDAC1 and HDAC2 interact with Sp1 and regulate its transcriptional activity (36). As shown in Fig. 6C, ER{alpha} and HDAC2 interacted with each other and this interaction was decreased by E2 treatment. (The relative density of ER{alpha} in the presence of E2 was 0.66 as compared with that in the absence of E2.) Interestingly, the expression level of the HDAC2 protein was also decreased by E2 treatment in the presence of ER{alpha}. (The relative density of HDAC2 in the presence of E2 was 0.43 as compared with that in the absence of E2.) ER{alpha} also interacted with HDAC1 even in the presence of E2, but the HDAC1 protein level did not change. To investigate whether endogenous HDAC1 or HDAC2 is involved in the IL-1{alpha} promoter activation, we performed promoter pull-down assays using a fragment containing the GC-rich region of the IL-1{alpha} gene promoter (–70 to +51) (Fig. 6D). Endogenous HDAC2 but not HDAC1 bound to the IL-1{alpha} promoter in the presence of ER{alpha}, and this binding was almost completely abrogated by E2 treatment. Sp1 also interacted with HDAC2 only in the presence of ER{alpha} and this interaction was clearly abolished by E2 treatment (Fig. 6E).

These results suggest that E2 induces dissociation of the corepressor HDAC2 from ER{alpha}, which leads to the augmentation of Sp1 transcriptional activity for the IL-1{alpha} gene through the GC-rich region within the IL-1{alpha} gene promoter.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we showed that E2 up-regulated IL-1{alpha} mRNA expression in primary synoviocytes from two independent RA patients. MH7A, a rheumatoid fibroblast like synovial cell line, did not express ER{alpha}. IL-1{alpha} mRNA was increased by E2 in MH7A/ER{alpha} cells expressing ectopic ER{alpha}, as in primary synoviocytes. As this E2 effect was inhibited by ICI 182,780, a specific antagonist of ER, the effect appeared to be mediated through ER. The primary synoviocytes were devoid of macrophages as a result of passage before their use in experiments, suggesting that these features are common to fibroblast-like synovial cells.

Because of their high transfection efficiency and lack of ER{alpha} expression, we analyzed the mechanism of the effect of E2 using MH7A cells. The induction of IL-1{alpha} mRNA by E2 appeared to be regulated at the transcriptional level through the ER. We then examined whether the effect of E2 was mediated through ER{alpha} or ERbeta. ERs are composed of six modular domains, A to F. The central C region is responsible for DNA sequence recognition and the cooperative binding of ER dimers to EREs consisting of a palindromic PuGGTCA motif separated by 3 bp (37). E2 lodges in a hydrophobic pocket defined by the C-terminal E domain (ligand binding domain (LBD)). The DBDs of ER{alpha} and ERbeta are highly homologous (96%), allowing both receptors to bind the same EREs. The LBDs are also conserved (58% homology). Compared with the DBD and the LBD, the N-terminal activation function-1 is not well conserved between ER{alpha} and ERbeta (28% homology). Previous studies have shown that the amino acid sequence differences between the activator function-1 regions of ER{alpha} and ERbeta can contribute to the different transcriptional activities of the two receptors in various cell types and promoter contexts, but these effects have not been fully investigated. In this study, the stimulatory effect of E2 on IL-1{alpha} promoter activity appeared to be mediated by ER{alpha} not by ERbeta. As ICI 182,780 inhibited the E2-mediated induction of IL-1{alpha} mRNA expression and IL-1{alpha} promoter activity, E2 appeared to induce IL-1{alpha} mRNA expression by augmenting its transcription through ER{alpha}.

There are no typical EREs in the IL-1{alpha} promoter used in this study, suggesting that some other region is responsible for the E2 effect. Deletion and mutation analyses indicated that the GC-rich region in the IL-1{alpha} promoter is required for the IL-1{alpha} promoter activity by E2 in a manner dependent on ER{alpha}. We found that Sp1 and Sp3 bound to the GC-rich region in the IL-1{alpha} promoter. We could not observe a slower migrating protein-DNA complex by EMSA upon the overexpression of ER{alpha}. Consistent with this result, we could not detect a supershifted band in the presence of the ER{alpha} Ab. As we observed the physical interaction of Sp1 and ER{alpha} with or without E2 in immunoprecipitation assays and also in promoter pull-down assays (Fig. 6, A and B), the ER{alpha}-Sp1 complex bound to DNA may not be stable enough for detection under the conditions used for EMSA. There are several CG islands in the GC-rich region and we found that the 10 bases (TAGCCACGCC) containing the most-downstream CG island (between –48 and –45 (CGCC)) was required for the Sp1 and Sp3 binding. We also found that the basal as well as ER{alpha}/E2-induced promoter activity was completely abolished when this region was mutated. Zaldivar et al. (38) reported that the GCC sequence present between –61 and –59 was required for the basal and stimulated IL-1{alpha} promoter activity in human epithelial (Hep-2) and erythroleukemia (HEL/DAMI) cell lines; however, they did not mention the sequence between –48 to –45. Therefore, we identified a novel E2/ER{alpha} regulatory element in the human IL-1{alpha} gene promoter in synovial cells in this study. We confirmed the E2/ER{alpha}-dependent transcriptional activity by using a reporter gene containing three tandem repeats of this core region (TAGCCACGCC) in the IL-1{alpha} gene promoter.

Sp1 and Sp3 are ubiquitously expressed members of the Sp family of transcription factors that are involved in the expression and regulation of many genes, including housekeeping genes, tissue-specific expressed genes, viral genes, and cell cycle-regulated genes (39, 40). The expression pattern, structure, and DNA-binding properties of Sp3 are very similar to those of Sp1. The physiological roles of Sp1 and Sp3, however, appear to be significantly different. Functional analysis of the transcription properties of Sp1 and Sp3 also revealed significant differences between these two transcription factors (41). Sp1 is known to be a strong transactivator, whereas Sp3 is generally inactive or acts only as a weak activator for many reporter constructs containing multiple Sp binding sites (42). Our study indicated that ER{alpha} interacted with Sp1 and activated the IL-1{alpha} promoter activity by E2 through the GC-rich region. E2 did not alter ER{alpha}-Sp1 interaction or ER{alpha}/Sp1 binding to the GC-rich region contained in the IL-1{alpha} promoter. Therefore, E2 increases the transcriptional activity of Sp1 by some mechanism other than the interaction between Sp1 and ER{alpha} and ER{alpha}/Sp1 binding to the GC-rich region.

HDAC negatively regulates the transcription of a number of genes by inducing conformational changes through removal of the acetyl group from the histones present in the nucleosome. It also regulates the activity of transcription factors, including p53 (43), GATA-1, Smad (44) and TFIIE, through deacetylation and does so by recruitment of corepressors such as m-Sin3A, N-CoR, and SMRT to Sp1 or NF-{kappa}B (36, 44, 45). Physical interaction was observed between ER{alpha} and HDAC1 or HDAC2 regardless of the presence of E2 in our study. We also observed an interaction between Sp1 and HDAC2 in the presence of ER{alpha} in 293 cells. We performed the same experiment in MH7A cells; however, it was difficult to detect the interaction between Sp1 and HDAC2, probably because of the low efficiency of transfection and/or lower Sp1 expression level. Promoter pull-down assays, however, revealed the binding of HDAC2 but not HDAC1 to the promoter region of the IL-1{alpha} gene. Interestingly, E2 in the presence of ER{alpha} caused dissociation of HDAC2 from ER{alpha}. In addition, the expression level of the HDAC2 protein was significantly decreased by treatment with E2 in the presence of ER{alpha}. In 293 cells, as well as MH7A cells, E2 decreased the expression of the HDAC2 protein. This reduction was not observed after pretreatment with MG132, a proteasome inhibitor, while the HDAC2 mRNA level was not changed after E2 treatment (data not shown). These data suggest the possibility that E2 promotes the degradation of HDAC2 protein in a proteasome-dependent manner. This effect of E2 was specific to HDAC2/ER{alpha}, because such effects were not observed in the case of HDAC1/ER{alpha} interaction and expression. These findings suggest that E2 may cause the specific dissociation of HDAC2 from ER{alpha} and consequently augment the transcriptional activity of Sp1 through the GC-rich region within the IL-1{alpha} gene promoter.

Experimental and clinical studies have established the critical role of the cytokine network in mediating the inflammatory and destructive processes in RA. Ab against TNF-{alpha}, soluble receptor for TNF-{alpha}, IL-1Ra, and Ab against IL-6 have been developed and their usefulness for the treatment of RA has been reported. However, fundamental issues regarding how RA is initiated and why the disease is more common in women have not been clarified. IL-1 has been implicated in the pathogenesis of RA. IL-1Ra gene knockout mice develop spontaneous arthritis (19). The effects of treatment with IL-1Ra or Abs against IL-1{alpha} or IL-1beta revealed that both IL-1{alpha} and IL-1beta appear to contribute to the arthritis caused by immunization with type II collagen or by an immune complex in the mouse (17). In type II collagen-induced arthritis, IL-1beta plays a major role in the outcome of arthritis (18, 46). In IL-1{alpha} transgenic mice, however, membrane-bound IL-1{alpha} from synoviocytes appeared to be critical for the induction of arthritis (23). Interestingly, the severity of arthritis was correlated with the expression level of membrane-bound IL-1{alpha} rather than serum IL-1{alpha} or IL-1beta. Our study showed that E2 induces the expression of IL-1{alpha} mRNA by augmenting its transcription in synovial cells, which provides at least a partial mechanism for this female-skewed disease.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by a Grant-in Aid for Scientific Research (B) from the Japan Society for the Promotion of Science; Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology; and Grants in-Aid for Scientific Research from Nagoya City University. Back

2 Address correspondence and reprint requests to Dr. Kikuo Onozaki, Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Japan. E-mail address: konozaki{at}phar.nagoya-cu.ac.jp Back

3 Abbreviations used in this paper: RA, rheumatoid arthritis; DBD, DNA binding domain; E2, 17beta-estradiol; ER, estrogen receptor; ERE, estrogen response element; HDAC, histone deacetylase; IL-1Ra, IL-1R agonist; LBD, ligand binding domain. Back

Received for publication December 29, 2005. Accepted for publication December 8, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Lipsky, P. E., D. M. van der Heijde, E. W. St. Clair, D. E. Furst, F. C. Breedveld, J. R. Kalden, J. S. Smolen, M. Weisman, P. Emery, M. Feldmann, et al 2000. Infliximab and methotrexate in the treatment of rheumatoid arthritis: anti-tumor necrosis factor trial in rheumatoid arthritis with concomitant therapy study group. N. Engl. J. Med. 343: 1594-1602. [Abstract/Free Full Text]
  2. Moreland, L. W.. 1998. Soluble tumor necrosis factor receptor (p75) fusion protein (ENBREL) as a therapy for rheumatoid arthritis. Rheum. Dis. Clin. N. Am. 24: 579-591. [Medline]
  3. Furst, D. E.. 2004. Anakinra: review of recombinant human interleukin-I receptor antagonist in the treatment of rheumatoid arthritis. Clin. Ther. 26: 1960-1975. [Medline]
  4. Choy, E. H., D. A. Isenberg, T. Garrood, S. Farrow, Y. Ioannou, H. Bird, N. Cheung, B. Williams, B. Hazleman, R. Price, et al 2002. Therapeutic benefit of blocking interleukin-6 activity with an anti-interleukin-6 receptor monoclonal antibody in rheumatoid arthritis: a randomized, double-blind, placebo-controlled, dose-escalation trial. Arthritis Rheum. 46: 3143-3150. [Medline]
  5. Cutolo, M., B. Seriolo, B. Villaggio, C. Pizzorni, C. Craviotto, A. Sulli. 2002. Androgens and estrogens modulate the immune and inflammatory responses in rheumatoid arthritis. Ann. NY Acad. Sci. 966: 131-142. [Abstract/Free Full Text]
  6. Tengstrand, B., K. Carlstrom, L. Fellander-Tsai, I. Hafstrom. 2003. Abnormal levels of serum dehydroepiandrosterone, estrone, and estradiol in men with rheumatoid arthritis: high correlation between serum estradiol and current degree of inflammation. J. Rheumatol. 30: 2338-2343. [Medline]
  7. Castagnetta, L. A., G. Carruba, O. M. Granata, R. Stefano, M. Miele, M. Schmidt, M. Cutolo, R. H. Straub. 2003. Increased estrogen formation and estrogen to androgen ratio in the synovial fluid of patients with rheumatoid arthritis. J. Rheumatol. 30: 2597-2605. [Medline]
  8. Mangelsdorf, D. J., C. Thummel, M. Beato, P. Herrlich, G. Schutz, K. Umesono, B. Blumberg, P. Kastner, M. Mark, P. Chambon, R. M. Evans. 1995. The nuclear receptor superfamily: the second decade. Cell 83: 835-839. [Medline]
  9. Lipsky, P. E., P. A. Thompson, L. J. Rosenwasser, C. A. Dinarello. 1983. The role of interleukin 1 in human B cell activation: inhibition of B cell proliferation and the generation of immunoglobulin-secreting cells by an antibody against human leukocyte pyrogen. J. Immunol. 13: 2708-2714.
  10. Gowen, M., D. D. Wood, E. J. Ihrie, M. K. B. McGuire, R. G. G. Russell. 1983. An interleukin 1 like factor stimulates bone resorption in vitro. Nature 306: 378-380. [Medline]
  11. Dinarello, C. A., S. M. Wolff. 1982. Molecular basis of fever in humans. Am. J. Med. 72: 799-819. [Medline]
  12. Chu, C. Q., M. Field, S. Allard, E. Abney, M. Feldmann, R. N. Maini. 1992. Detection of cytokines at the cartilage/pannus junction in patients with rheumatoid arthritis: implications for the role of cytokines in cartilage destruction and repair. Br. J. Rheumatol. 31: 653-661. [Abstract/Free Full Text]
  13. Mizel, S. B., J. M. Dayer, S. M. Krane, S. E. Mergenhagen. 1981. Stimulation of rheumatoid synovial cell collagenase and prostaglandin production by partially purified lymphocyte-activating factor (interleukin-1). Proc. Natl. Acad. Sci. USA 78: 2474-2477. [Abstract/Free Full Text]
  14. Dayer, J. M., B. de Rochemonteix, B. Burrus, S. Demczuk, C. A. Dinarello. 1986. Human recombinant interleukin 1 stimulates collagenase and prostaglandin E2 production by human synovial cells. J. Clin. Invest. 77: 645-648. [Medline]
  15. van de Loo, A. A. J., W. B. van den Berg. 1990. Effects of murine recombinant interleukin 1 on synovial joints in mice: measurements of patellar cartilage metabolism and joint inflammation. Ann. Rheum. Dis. 49: 238-245. [Abstract/Free Full Text]
  16. Pettipher, E. R., G. A. Higgs, B. Henderson. 1986. Interleukin 1 induces leukocyte infiltration and cartilage proteoglycan degradation in the synovial joint. Proc. Natl. Acad. Sci. USA 83: 8749-8753. [Abstract/Free Full Text]
  17. Wooley, P. H., J. D. Whalen, D. L. Chapman, K. A. Richard, D. G. Aspar, N. D. Staite. 1993. The effect of an interleukin-1 receptor antagonist protein on type II collagen-induced arthritis and antigen-induced arthritis in mice. Arthritis Rheum. 36: 1305-1314. [Medline]
  18. Joosten, L. A. B., M. M. A. Helsen, F. A. J. van de Loo, W. B. van den Berg. 1996. Anticytokine treatment of established type II collagen-induced arthritis in DBA/1 mice: a comparative study using anti-TNF, anti-IL-1/beta, and IL-1ra. Arthritis Rheum. 39: 797-809. [Medline]
  19. Horai, R., S. Saijo, H. Tanioka, S. Nakae, K. Sudo, A. Okahara, T. Ikuse, M. Asano, Y. Iwakura. 2000. Development of chronic inflammatory arthropathy resembling rheumatoid arthritis in interleukin 1 receptor antagonist-deficient mice. J. Exp. Med. 191: 313-320. [Abstract/Free Full Text]
  20. Dinarello, C. A.. 1991. Interleukin-1 and interleukin-1 antagonism. Blood 77: 1627-1652. [Abstract/Free Full Text]
  21. Dinarello, C. A.. 1996. Biologic basis for interleukin-1 in disease. Blood 87: 2095-2147. [Abstract/Free Full Text]
  22. Mosley, B., D. L. Urdal, K. S. Prickett, A. Larsen, D. Cosman, P. J. Conlon, S. Gillis, S. K. Dower. 1987. The interleukin-1 receptor binds to the human interleukin-1 precursor but not the interleukin-1 precursor. J. Biol. Chem. 262: 2941-2944. [Abstract/Free Full Text]
  23. Niki, Y., H. Yamada, T. Kikuchi, Y. Toyama, H. Matsumoto, K. Fujikawa, N. Tada. 2004. Membrane-associated IL-1 contributes to chronic synovitis and cartilage destruction in human IL-1 transgenic mice. J. Immunol. 172: 577-584. [Abstract/Free Full Text]
  24. Ku, G., T. Faust, L. L. Lauffer, D. J. Livingston, M. W. Harding. 1996. IL-1beta converting enzyme inhibition blocks progression of type II collagen-induced arthritis in mice. Cytokine 8: 377-386. [Medline]
  25. van den Berg, W. B.. 1998. Joint inflammation and cartilage destruction may occur uncoupled. Springer Semin. Immunopathol. 20: 149-164. [Medline]
  26. Miyazawa, K., A. Mori, H. Okudaira. 1998. Establishment and characterization of a novel human rheumatoid fibroblast-like synoviocyte line, MH7A, immortalized with SV40 T antigen. J. Biochem. 124: 1153-1162. [Abstract/Free Full Text]
  27. Suzuki, Y., J. Shimada, K. Shudo, M. Matsumura, M. P. Crippa, S. Kojima. 1999. Physical interaction between retinoic acid receptor and Sp1: mechanism for induction of urokinase by retinoic acid. Blood 93: 464-4276.
  28. Chomczynski, P., N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159. [Medline]
  29. Matsumura, T., A. Ito, T. Takii, H. Hayashi, K. Onozaki. 2000. Endotoxin and cytokine regulation of toll-like receptor (TLR) 2 and TLR4 gene expression in murine liver and hepatocytes. J. Interferon Cytokine Res. 20: 915-921. [Medline]
  30. Green, S., P. Walter, V. Kumar, A. Krust, J. M. Bornert, P. Argos, P. Chambon. 1986. Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature 320: 134-139. [Medline]
  31. Mosselman, S., J. Polman, R. Dijkema. 1996. ER beta: identification and characterization of a novel human estrogen receptor. FEBS Lett. 392: 49-53. [Medline]
  32. Hayashi, T., Y. Ueno, T. Okamoto. 1993. Oxidoreductive regulation of nuclear factor {kappa}B: involvement of a cellular reducing catalyst thioredoxin. J. Biol. Chem. 268: 11380-11388. [Abstract/Free Full Text]
  33. Okamoto, T., H. Ogiwara, T. Hayashi, A. Mitsui, T. Kawabe, J. Yodoi. 1992. Human thioredoxin/adult T cell leukemia-derived factor activates the enhancer binding protein of human immunodeficiency virus type 1 by thiol redox control mechanism. Int. Immunol. 4: 811-819. [Abstract/Free Full Text]
  34. Ohata, H., T. Tetsuka, H. Hayashi, K. Onozaki, T. Okamoto. 2003. 3-methylcholanthrene activates human immunodeficiency virus type 1 replication via aryl hydrocarbon receptor. Microbiol. Immunol. 47: 363-370. [Medline]
  35. Clarke, R., M. C. Liu, K. B. Bouker, Z. Gu, R. Y. Lee, Y. Zhu, T. C. Skaar, B. Gomez, K. O’Brien, Y. Wang, L. A. Hilakivi-Clarke. 2003. Antiestrogen resistance in breast cancer and the role of estrogen receptor signaling. Oncogene 22: 7316-7339. [Medline]
  36. de Ruijter, A. J., A. H. van Gennip, H. N. Caron, S. Kemp, A. B. van Kuilenburg. 2003. Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem. J. 370: 737-749. [Medline]
  37. Krust, A., S. Green, P. Argos, V. Kumar, P. Walter, J. M. Bornert, P. Chambon. 1986. The chicken oestrogen receptor sequence: homology with v-erbA and the human oestrogen and glucocorticoid receptors. EMBO J. 5: 891-897. [Medline]
  38. Zaldivar, F., D. J. Nugent, K. Imfeld, M. A. Berman. 2002. Identification of a novel regulatory element in the human interleukin 1 {alpha} (IL-1{alpha}) gene promoter. Cytokine 20: 130-135. [Medline]
  39. Philipsen, S., G. Suske. 1999. A tale of three fingers: the family of mammalian Sp/XKLF transcription factors. Nucleic Acids Res. 27: 2991-3000. [Abstract/Free Full Text]
  40. Suske, G.. 1999. The Sp-family of transcription factors. Gene 238: 291-294. [Medline]
  41. Bouwman, P., S. Philipsen. 2002. Regulation of the activity of Sp1-related transcription factors. Mol. Cell. Endocrinol. 195: 27-38. [Medline]
  42. Hagen, G., S. Müller, M. Beato, G. Suske. 1994. Sp1-mediated transcriptional activation is repressed by Sp3. EMBO J. 13: 3843-3851. [Medline]
  43. Juan, L. J., W. J. Shia, M. H. Chen, W. M. Yang, E. Seto, Y. S. Lin, C. W. Wu. 2000. Histone deacetylases specifically down-regulate p53-dependent gene activation. J. Biol. Chem. 275: 20436-20443. [Abstract/Free Full Text]
  44. Inoue, Y., Y. Itoh, K. Abe, T. Okamoto, H. Daitoku, A. Fukamizu, K. Onozaki, and H. Hayashi. 2006. Smad3 is acetylated by p300/CBP to regulate its transcription activitry. Oncogene In press.
  45. Doetzlhofer, A., H. Rotheneder, G. Lagge, M. Koranda, V. Kurtev, G. Brosch, E. Wintersberger, C. Seiser. 1999. Histone deacetylase 1 can repress transcription by binding to Sp1. Cell Biol. 19: 5504-5511.
  46. van den Berg, W. B., L. A. Joosten, M. Helsen, F. A. van de Loo. 1994. Amelioration of established murine collagen-induced arthritis with anti-IL-1 treatment. Clin. Exp. Immunol. 95: 237-243. [Medline]




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