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J. Biol. Chem., Vol. 276, Issue 32, 30208-30215, August 10, 2001

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p65-activated Histone Acetyltransferase Activity Is Repressed by Glucocorticoids

MIFEPRISTONE FAILS TO RECRUIT HDAC2 TO THE p65-HAT COMPLEX^*

Kazuhiro Ito, Elen Jazrawi, Borja Cosio, Peter J. Barnes, and Ian M. Adcock^§

From the Thoracic Medicine, Imperial College School of Medicine at the National Heart and Lung Institute, Dovehouse St., London SW3 6LY, United Kingdom

Received for publication, April 23, 2001, and in revised form, June 5, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Glucocorticoids acting through their specific receptor can either enhance or repress gene transcription. Dexamethasone represses interleukin-1-stimulated histone acetylation and granulocyte-macrophage colony-stimulating factor expression through a combination of direct inhibition of p65-associated histone acetyltransferase (HAT) activity and by recruiting histone deacetylase 2 (HDAC2) to the p65-HAT complex. Here we show that mifepristone, a glucocorticoid receptor partial agonist, has no ability to induce gene expression but represses interleukin-1-stimulated histone acetylation and granulocyte-macrophage colony-stimulating factor release by 50% maximally. Mifepristone was able to inhibit p65-associated HAT activity to the same extent as dexamethasone but failed to inhibit the natural promoter to an equal extent due to an inability to recruit HDAC2 to the p65-associated HAT complex. These data suggest that the maximal repressive actions of glucocorticoids require recruitment of HDAC2 to a p65-HAT complex. These data also suggest that pharmacological manipulation of specific histone acetylation status is a potentially useful approach for the treatment of inflammatory diseases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Actively transcribed genes are associated with an unwinding of the previously closed DNA structure, allowing accessibility to DNA-binding proteins, thereby allowing modulation of gene transcription (1, 2). DNA is packaged into chromatin, a highly organized and dynamic protein-DNA complex. The fundamental subunit of chromatin, the nucleosome, is composed of an octamer of four core histones; an H3/H4 tetramer, and two H2A/H2B dimers, surrounded by 146 bp¹ of DNA (1, 3). The nucleosome therefore acts as a barrier to the initiation of transcription by preventing the access of transcription factors and RNA polymerase II to their cognate recognition sequences on DNA (4). The N-terminal tails of histones contain lysine residues that are the sites for post-transcriptional acetylation. This is a dynamic process that occurs on actively transcribed chromatin only (5). In addition, core histones may be modified by phosphorylation, methylation, ADP-ribosylation, or ubiquitinization of a specific amino acid residue (6).

Increased gene transcription is associated with an increase in histone acetylation, whereas hypoacetylation is correlated with reduced transcription or gene silencing (2, 7). Targeted acetylation of histone H4 tails plays an important role in allowing regulatory proteins to access DNA and is likely to be a major factor in the regulation of gene transcription (8-10).

Glucocorticoids are the most effective therapy for the treatment of inflammatory diseases such as asthma, a chronic inflammatory disease of the airway (11). Functionally, they act partly by inducing some anti-inflammatory genes such as secretory leukocyte proteinase inhibitor (SLPI) (12), lipocortin-1 (13), and IL-1 receptor antagonist (14) but mainly by repression of inflammatory genes, such as cytokines, adhesion molecules, inflammatory enzymes, and receptors (11). They act by binding to a cytosolic glucocorticoid receptor (GR), which upon binding is activated and rapidly translocates to the nucleus. Within the nucleus, GR either induces gene transcription by binding to specific DNA elements in the promoter/enhancer regions of responsive genes or reduces gene transcription by transrepression (15). GR reduces gene transcription by a functional interaction with proinflammatory transcription factors such as AP-1 (Fos-Jun heterodimers) and NF-B (p65-p50 heterodimers) (15-17). We have recently shown that GR represses NF-B-mediated HAT activity and GM-CSF release by a combination of direct inhibition of CBP-associated HAT activity, but not that of CBP itself, and by recruitment of HDACs to the NF-B activation complex (18).

Many of the anti-inflammatory effects of corticosteroids may be mediated by repression of transcription factors (transrepression), whereas the endocrine and metabolic effects of corticosteroids are mediated via GRE binding (transactivation). This has led to a search for novel corticosteroids that selectively transrepress, thus reducing the risk of systemic side effects. A separation of transactivation and transrepression has been demonstrated using reporter gene constructs in transfected cells using selective mutations of GR (19). Furthermore, some corticosteroids, such as RU24858, RU486 (mifepristone), and ZK98299, have a greater transrepression than transactivation effect (19, 20). These corticosteroids, including RU24858 and RU40066, have anti-inflammatory effects in vivo (21). These studies rely on the overexpression of components of these pathways, which could lead to problems in interpretation. We have therefore examined the role of CBP and associated HATs in the regulation of glucocorticoid functions in nontransfected cells.

We have investigated the ability of dexamethasone and mifepristone to suppress expression of the inflammatory cytokine, granulocyte-macrophage colony stimulation factor (GM-CSF), and induce SLPI and to regulate histone acetylation and deacetylation in A549 cells. We have demonstrated that mifepristone, unlike dexamethasone, is unable to induce histone H4 acetylation but is able partially to repress IL-1-stimulated histone acetylation without recruitment HDAC2 to the p65 complex. This results in a partial reduction of acetylated Lys⁸ associated with the GM-CSF promoter and reduced GM-CSF mRNA production. The mechanism of mifepristone repression of IL-1-stimulated histone H4 Lys⁸ acetylation was by direct inhibition of p65-associated HAT activity with no effect on recruitment of HDAC2 to the p65-HAT complex.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- A549 cells, a human lung adenoma cell line (ATCC designation CCL185) and a good model for human lung epithelial cells, were grown to 50% confluence in Dulbecco's modified Eagle's medium containing 10% fetal calf serum before incubation for 48-72 h in serum-free media. Cells were stimulated by IL-1 (1 ng/ml), and the effects of dexamethasone, mifepristone, and the histone deacetylase inhibitor trichostatin A (TSA) (Sigma, Poole, UK) (22) on GM-CSF and SLPI release were measured.

GM-CSF and SLPI ELISAs-- Determination of GM-CSF expression was measured by sandwich ELISA (R&D Systems Europe, Abingdon, UK) according to the manufacturer's instructions. For immunoassay of SLPI, polystyrene microtiter plates were coated overnight at 4 °C with sample diluted with hydroxycarbonate (pH 9.6). Plates were blocked for 2 h at room temperature with 5% ovalbumin in phosphate-buffered saline. Antibodies against SLPI (R&D Systems Europe) were diluted 1:2000 and added to each plate. After 1 h at room temperature, plates were washed sequentially with 0.1% Tween 20/phosphate-buffered saline and incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (Dako, Cambridge, UK) for 1 h. Detection was performed following R&D instructions. Recombinant human SLPI (R&D Systems Europe) was used as a standard.

RNA Isolation and Reverse Transcription-PCR-- PCR primers were as follows, with annealing temperature and product size in parentheses: GM-CSF sense 5'-CCC AAT gAA gCC TCA CCg AAT-3' and antisense 5'-TCg gAg Cgg gTA gTT AAC AgC-3' (67 °C, 604 bp); SLPI sense 5'-ATg AAg TCC AgC ggC CTC TT-3' and antisense 5'-ATg gCA ggA ATC AAg CTT TC-3' (54 °C, 408 bp); and GAPDH sense 5'-CCC TgA ATT TgA Cag TCT CACC-3' and antisense 5'-CAC AAT AAA ACT TgC CCA gAA AAA-3' (62 °C, 175 bp). Extraction of RNA from A549 cells was performed using an RNeasy Mini Kit according to the manufacturer's instructions (Qiagen, Crawley, UK). Sample RNA was quantified by spectrophotometry, and 1 µg was reverse transcribed to cDNA as previously described (23). PCRs were performed on 5 µl of the cDNA with a Hybaid Omnigene thermal cycler (Hybaid, Ashford, Middlesex, UK) in a final reaction volume of 25 µl in the presence of 0.4 units of Taq DNA polymerase. 35 cycles were used, with a denaturing step at 94 °C for 45 s, followed by the specific primer annealing temperature for 45 s and an extension step at 72 °C for 45 s.

Direct Histone Extraction-- Histones were extracted from nuclei overnight using HCl and H₂SO₄ at 4 °C as previously described (18). Cells were microcentrifuged for 5 min, and the cell pellets were extracted with ice-cold lysis buffer (10 mM Tris-HCl, 50 mM sodium bisulfite, 1% Triton X-100, 10 mM MgCl₂, 8.6% sucrose, complete protease inhibitor mixture (Roche Molecular Biochemicals) for 20 min at 4 °C. The pellet was repeatedly washed in buffer until the supernatant was clear (centrifuge at 8000 rpm, 5 min after each wash), and the nuclear pellet was washed in nuclear wash buffer (10 mM Tris-HCl, 13 mM EDTA) and resuspended in 50 µl of 0.2 N HCl and 0.4 N H₂SO ₄ in distilled water. The nuclei were extracted overnight at 4 °C, and the residue was microcentrifuged for 10 min. The supernatant was mixed with 1 ml of ice-cold acetone and left overnight at 20 °C. The sample was microcentrifuged for 10 min, washed with acetone, dried, and diluted in distilled water. Protein concentrations of the histone-containing supernatant were determined by Bradford protein assay kit (Bio-Rad).

Western Blotting-- Immunoprecipitates, whole cell extractions, or isolated histones were measured by SDS-polyacrylamide gel electrophoresis and Western blot analysis using ECL (Amersham Pharmacia Biotech). Proteins were size-fractionated by SDS-polyacrylamide gel electrophoresis and transferred to Hybond-ECL membranes. Immunoreactive bands were detected by ECL.

Immunocytochemistry-- A549 cells (0.5 × 10 ⁶) were cultured in eight-well slide chambers with mifepristone (10⁶ M), dexamethasone (10⁶ M), or IL-1 (1 ng/ml). Cells were washed with Hanks' solution and air-dried for 30 min at room temperature. Cells were then fixed in ice-cold acetone-methanol (50/50, w/w) (20 °C) for 10 min. Slides were air-dried and incubated with blocking buffer (20% normal swine serum in phosphate-buffered saline, 0.1% saponin) (Dako) for 20 min, followed by 1-h incubation with primary antibody solution (phosphate-buffered saline, 0.1% saponin, 1% bovine serum albumin). Antibodies against acetylated H4 Lys⁵, H4 Lys⁸ (Serotec) (24), GR (Serotec), and NF-B/p65 (Santa Cruz Biotechnology. Inc., Santa Cruz, CA) were used at 1:100 to 1:300 dilution. Slides were washed twice and incubated with biotinylated swine anti-rabbit IgG (Dako) (1:200) for 45 min. Slides were washed again before incubation with fluorescein isothiocyante-conjugated streptavidin (1:100) for 45 min. The slides were washed twice more before counterstaining with 20% hematoxylin, and mounting. Stained cells were observed by fluorescent microscopy.

Histone Acetylation Activity-- Cells were plated at a density of 0.25 × 10 ⁶ cells/ml and exposed to 5 µCi/ml of [³H]acetate (PerkinElmer Life Sciences). After incubation for 10 min at 37 °C cells were stimulated for 6 h. Histones were isolated and separated by electrophoresis on an SDS-16% polyacrylamide gel. Gels were stained with Coomassie Brilliant Blue, and the core histones (H2A, H2B, H3, and H4) were excised. The radioactivity in extracted core histones was determined by liquid scintillation counting and normalized to protein level.

Histone Deacetylation Assay-- Radiolabeled histones were prepared from A549 cells following incubation with TSA (100 ng/ml, 6 h) in the presence of 0.1 mCi/ml [³H]acetate. Histones were dried and resuspended in distilled water. Crude HDAC preparations were extracted from total cellular homogenates with Tris-based buffer (10 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.25 mM EDTA, 10 mM 2-mercaptoethanol) as previously reported (25). The crude HDAC preparation or immunoprecipitates were incubated with ³H-labeled histone for 30 min at 30 °C before the reaction was stopped by the addition of 1 N HCl, 0.4 N acetic acid. Released ³H-labeled acetic acid was extracted by ethyl acetate, and the radioactivity of the supernatant was determined by liquid scintillation counting.

Immunoprecipitation-- Extracts were prepared using 100 µl of mild IP buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, complete protease inhibitor mixture (Roche Molecular Biochemicals). The lysis mixture was incubated on ice for 15 min and microcentrifuged for 10 min at 4 °C. Extracts were precleared with 20 µl of A/G agarose (a 50/50 mix; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and 2 µg of normal rabbit IgG. After microcentrifugation, 20 µl of A/G agarose conjugated with 5 µg of p65 antibody were incubated for 6 h at 4 °C with rotation. The immune complexes were pelleted by gentle centrifugation and washed three times with 1 ml of IP buffer. For the HAT assay, immunoprecipitates were washed twice with IP-HAT buffer, and for Western blotting, after a final wash with IP buffer, the buffer was aspirated completely and resuspended in Laemmli buffer.

IP-HAT Assays-- IP-HAT assays were performed using a modified method of Ogryzko (26). Immune complexes with resin were resuspended in 150 µl of HAT buffer (50 mM Tris-HCl, pH 8.0, 10% glycerol, 1 mM dithiothreitol, 0.1 mM EDTA, complete protease inhibitor mixture). Typically, 20 µl of free core histone solution extracted from A549 cells (final concentration 10 µg) and 30 µl of immunoprecipitate were incubated. Reactions were initiated by the addition of 0.25 µCi of [³H]acetyl-CoA (5 Ci/mmol) (Amersham Pharmacia Biotech) and performed for 45 min at 30 °C. After incubation, the reaction mixture was spotted onto Whatman p81 phosphocellulose filter paper (Whatman) and washed for 30 min with 0.2 M sodium carbonate buffer (pH 9.2) at room temperature with 2-3 changes of the buffer and then washed briefly with acetone. The dried filters were counted in a liquid scintillation counter.

Chromatin Immunoprecipitation Assay-- A-549 cells pretreated for 30 min with dexamethasone or mifepristone were treated with IL-1 (1 ng/ml) for 4 h. Protein-DNA complexes were fixed by formaldehyde (1% final concentration) and treated as previously described (27). Cells were resuspended in 200 µl of SDS lysis buffer (50 mM Tris, pH 8.1, 1% SDS, 5 mM EDTA, complete proteinase inhibitor mixture) and subjected to three 10-s pulses of sonication on ice. Sonicated samples were centrifuged to spin down cell debris, and the soluble chromatin solution was immunoprecipitated using sonicated salmon sperm DNA-agarose A slurry (Upstate Biotechnology, Buckingham, UK) as described by Chen et al. (28). Protein-bound immunoprecipitated DNA was washed with LiCl wash buffer and Tris-EDTA buffer, and immune complexes were eluted by adding elution buffer (1% SDS, 0.1 M NaHCO₃). The elution was treated successively for 4 h at 65 °C in 200 mM NaCl, 1% SDS to reverse cross-links and incubated for 1 h at 45 °C with 70 µg/ml proteinase K (Sigma). DNA was extracted with phenol/chloroform; precipitated with ethanol, 0.3 M NaHCOOH, 20 µg glycogen; and resuspended in 50 µl of Tris-EDTA buffer. Quantitative PCR was performed with 10 µl of DNA sample and 30 cycles. Primer pairs of GM-CSF were as follows: forward (5-CTGACCACCTAGGGAAAAGGC-3) and reverse (5-CAGCCACATCCTCCTCCAGAGAAC-3). PCR products were resolved by 3% agarose gel and visualized with ethidium bromide.

Statistics-- Results are expressed as means ± S.E. A multiple comparison was made between the mean of the control and the means from each individual treatment group by Dunnett's test using SAS/STAT software (SAS Institute Inc., Cary, NC). All statistical testing was performed using a two-sided 5% level of significance test. The concentrations of dexamethasone or mifepristone producing 50% of the maximal inhibition seen (EC₅₀) were calculated from concentration-response curves by linear regression.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Effect of Anti-glucocorticoid Mifepristone on Cytokine Production and Histone Acetylation-- We used the partial glucocorticoid agonist mifepristone (RU-486) to examine the different roles of GR transactivation and transrepression in control of GM-CSF and SLPI expression. Using reporter gene assays and overexpression of GR, it has been postulated that mifepristone and related compounds may discriminate between transrepression and transactivation at GRE and AP-1- and NF-B-driven genes (19, 20). Mifepristone inhibited IL-1-stimulated GM-CSF release with a maximal inhibition of 50.4% (EC₅₀ = 0.7 × 10⁹ M; Fig. 1A) and failed to induce SLPI expression (Fig. 1B). This was in contrast to dexamethasone, which totally suppressed IL-1-stimulated GM-CSF release (EC₅₀ = 1.4 × 10⁹ M) and caused a marked induction of SLPI release (7.6 ± 0.5 versus 1.5 ± 0.7 ng/ml at 10⁶ M). Thus, at the level of functional mediator release, there is a clear discrimination between the activation and repressive roles of mifepristone.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Mifepristone inhibits IL-1-stimulated gene expression. A, inhibitory effects of mifepristone and dexamethasone on IL-1-induced GM-CSF production. Cells were preincubated with mifepristone (1012 to 105 M) or dexamethasone (1012 to 10-6 M) for 30 min before incubation with IL-1 (1 ng/ml) for 24 h. Supernatants were collected and assayed for GM-CSF by ELISA. Results are expressed as mean ± S.E., n = 3 independent experiments; *, p < 0.05; **, p < 0.01. NS, nonstimulated. B, effects of mifepristone and dexamethasone on SLPI production. Cells were incubated for 24 h before supernatants were collected and assayed for SLPI by ELISA. Results are expressed as mean ± S.E. Each experiment was performed in duplicate on three separate occasions. **, p < 0.01. C, effect of mifepristone and dexamethasone on IL-1-stimulated SLPI production. Cells were preincubated with concentrations of mifepristone or dexamethasone for 30 min before incubation with IL-1 (1 ng/ml) for 24 h. Supernatants were collected and assayed for SLPI release by ELISA. Results are expressed as mean ± S.E., n = 3 independent experiments; *, p < 0.05; **, p < 0.01. D, effect of TSA on the inhibitory effect of mifepristone (Mif) and dexamethasone (Dex) on IL-1-induced GM-CSF production. Cells were preincubated with concentrations of mifepristone (106 M) or dexamethasone (106 M) for 30 min before incubation with IL-1 (1 ng/ml) for 24 h. Supernatants were collected and assayed for GM-CSF by ELISA. TSA (10 ng/ml) was added 10 min before IL-1 stimulation. Results are expressed as mean ± S.E., n = 3 independent experiments; *, p < 0.05; **, p < 0.01 versus IL-1 alone control; #, p < 0.05 versus TSA treatment.

In addition, mifepristone inhibited IL-1-induced SLPI production with maximal inhibition of 65% at 10⁵ M (Fig. 1C). Dexamethasone strongly inhibited IL-1-stimulated SLPI release (73%) at a concentration of 10^-10 M before stimulating further SLPI release to levels seen with dexamethasone (10⁶ M) alone. Many reports have suggested that control of gene expression is dependent upon changes in chromatin structure resulting from alterations in the acetylation status of core histones (29). We used the HDAC inhibitor TSA (10 ng/ml) to determine the role of HDACs in mediating the effects of dexamethasone and mifepristone on inhibition of IL-1-stimulated GM-CSF release. TSA further stimulated IL-1-induced GM-CSF release, suggesting a role for HDACs in modulating gene induction, possibly reflecting a feedback mechanism to enable switching off of GM-CSF after an initial inflammatory pulse. In addition, TSA attenuated both the maximal inhibition and the EC₅₀ for dexamethasone (maximum inhibition, 61 versus 91% at 10⁶ M; EC₅₀, 7.8 × 10⁸ versus 1.4 × 10⁹ M) while having no effect on mifepristone actions (maximum inhibition, 50 versus 45% at 10⁶ M; EC₅₀, 0.7 × 10⁵ versus 1.2 × 10⁵ M) (Fig. 1D). These data suggest that mifepristone actions in repressing IL-1-stimulated GM-CSF release are independent of HDAC activity, whereas the full repressive effects of dexamethasone require HDAC activity.

Mifepristone Fails to Induce Histone Acetylation but Inhibits IL-1-stimulated Acetylation of Bulk Histone-- In order to determine whether dexamethasone or mifepristone could affect bulk IL-1-stimulated histone acetylation, experiments were performed in whole cell extracts from cells treated with IL-1 and/or mifepristone or dexamethasone. IL-1 induced a 4-fold increase in histone acetylation (Fig. 2). Mifepristone alone had no effect on basal histone acetylation but inhibited IL-1-induced histone acetylation with maximal inhibition of 53% (Fig. 2, upper panel). In contrast, dexamethasone had a biphasic effect on IL-1-stimulated histone acetylation (Fig. 2, lower panel), similar to the effects seen on SLPI production. Dexamethasone alone also induced histone acetylation in a concentration-dependent manner.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effect of mifepristone on IL-1-induced histone acetylation. Mifepristone inhibits IL-1-induced histone acetylation in total cell extracts. Cells were pretreated with mifepristone or dexamethasone for 30 min before incubation with IL-1 (1 ng/ml) for 6 h in the presence of 0.05 mCi of [3H]acetate. Histones were isolated and separated by SDS-polyacrylamide gel electrophoresis, and [3H]acetate incorporated histones were counted and normalized to protein level. Data represent mean ± S.E. of three independent experiments. **, p < 0.01; *, p < 0.05.

Mifepristone Induces GR Nuclear Translocation but Fails to Induce Chromatin Acetylation-- Immunofluorescence and confocal microscopy showed that dexamethasone and mifepristone enhanced GR nuclear translocation. IL-1 had no effect on GR translocation (Fig. 3, a-d). In contrast to this result, IL-1, but not dexamethasone and mifepristone, enhanced NF-B (p65 subunit) nuclear translocation (Fig. 3, e-h). We examined histone H4 lysine acetylation in order to confirm the role of histone acetylation in IL-1-, dexamethasone-, or mifepristone-mediated effects. IL-1 caused acetylation of Lys⁸ (AcH4K8; Fig. 3p) and Lys¹² residues (data not shown), while dexamethasone targeted acetylation on Lys⁵ (AcH4K5; Fig. 3k) and Lys¹⁶ (data not shown) as previously reported (18). Mifepristone failed to stimulate acetylation of any lysine residue (Fig. 3, j and n).

View larger version (46K): [in this window] [in a new window]   Fig. 3.   Immunocytochemistry of GR, NF-B (p65 subunit), and acetylated histone 4. Cells were incubated with mifepristone (10-6 M) (b, f, j, and n), dexamethasone (10-6 M) (c, g, k, and o), or IL-1 (1 ng/ml) (d, h, l, and p) for 6 h. Cells were analyzed for nuclear localization of GR (a-d), NF-B (p65 subunit) (e-h), and acetylated lysine residues Lys5 (AcH4K5; i-l) and Lys8 (AcH4K8; m-p) by immunocytochemistry. Results are representative of four independent experiments.

Mifepristone Inhibits IL-1-stimulated Acetylation of Histone H4 Lys⁸ but Does Not Enhance H4 Lys⁵ Acetylation-- Western analysis of specific acetylated lysines showed that dexamethasone, but not mifepristone, induced acetylation of Lys⁵ residues at the concentration of 10⁶ M (Fig. 4A). In addition, dexamethasone significantly inhibited IL-1-stimulated Lys⁸ acetylation (Fig. 4B). Mifepristone also reduced IL-1-stimulated Lys⁸ acetylation but to a lesser extent than dexamethasone. These data suggest that mifepristone can slightly inhibit histone acetylation induced by IL-1 and, in contrast to dexamethasone, is unable itself to induce histone acetylation.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effect of mifepristone on IL-1-induced specific histone acetylation and promoter activation. A, cells were incubated with IL-1 (1 ng/ml) for 6 h in the presence of mifepristone (Mif, 106 M) or dexamethasone (Dex, 106 M). Protein extracts were obtained and examined for acetylated histone H4 lysine residue Lys5 (Ac-H4K5) by Western blot analysis. Results are representative of three independent experiments. Densitometric analysis was also done, and results are presented as mean ± S.E. of at least three independent experiments. *, p < 0.05; **, p < 0.01. B, Western blot analysis of acetylated histone H4 lysine residue Lys8 (Ac-H4K8) in the same samples as above. Results are representative of three independent experiments. Densitometric analysis was also done, and results are presented as mean ± S.E. of at least three independent experiments. *, p < 0.05. C, association of acetylated histone 4 Lys5 and Lys8 with the GM-CSF promoter. A549 cells pretreated with mifepristone (Mif, 106 M) or dexamethasone (Dex, 106 M) for 30 min were incubated with IL-1 (1 ng/ml) for 4 h. Proteins and DNA were cross-linked by formaldehyde treatment, and chromatin pellets were extracted. Following sonication, acetylated histone H4 Lys5 and Lys8 were immunoprecipitated, and the associated DNA was amplified by PCR. Results are representative of three independent experiments. D, GM-CSF and SLPI are regulated at the level of gene expression. IL-1 (1 ng/ml, lane 2) stimulates the expression of both GM-CSF and SLPI mRNA compared with unstimulated cells (lane 1) at 6 h as measured by reverse transcription-PCR. The effects of increasing concentrations of dexamethasone (Dex, lanes 3-5) and mifepristone (Mif, lanes 6-8) on IL-1-stimulated GM-CSF and SLPI steady-state mRNA expression are shown. GAPDH expression is used as a control for mRNA extraction. Results are representative of three independent experiments.

The above data examine bulk histone acetylation status. In order to be functionally relevant, these events must occur at the correct promoter sites. Using chromatin immunoprecipitation assays, we showed that H4 Lys⁸ acetylation, but not H4 Lys⁵ acetylation, was involved in IL-1-stimulated GM-CSF promoter activation. Both dexamethasone and mifepristone inhibited the IL-1-stimulated increase of GM-CSF promoter associated with acetylated H4 Lys⁸ (Fig. 4C). The effect of dexamethasone was greater (64% reduction) than that seen with mifepristone (45% reduction). This data confirms the earlier results showing that dexamethasone was more effective than mifepristone in inhibiting GM-CSF release and also suggests that the effect occurs at the level of gene expression. This was confirmed using reverse transcription-PCR. This showed that IL-1 induced expression of both GM-CSF and SLPI steady-state mRNA (Fig. 4D, compare lanes 1 and 2). GM-CSF steady state mRNA levels were reduced to a greater extent with dexamethasone (1 µM, 95%) than by mifepristone (1 µM, 51%)(Fig. 4D, compare lanes 5 and 8), mimicking the effect seen at the protein level. SLPI steady-state mRNA expression also followed its release patterns with a reduction at low concentrations of dexamethasone (10⁹ M, lane 3) and subsequent gene induction at higher concentrations (10⁶ M, lane 5). In contrast, mifepristone reduced SLPI steady-state mRNA levels at low concentrations (10⁹ M, lane 7) without subsequent induction of gene expression at higher concentrations (Fig. 4D, lane 8). These data indicate that release is controlled at the level of gene expression. Although not formally demonstrated here, previous data have suggested that dexamethasone regulation of GM-CSF release in lung epithelial cells is regulated at the level of gene transcription (30).

Effect of Mifepristone on p65-induced Histone Acetylation and Deacetylation-- In order to clarify the inhibitory mechanism of mifepristone on histone acetylation, we investigated p65-associated histone acetylation and deacetylation in IL-1 and/or mifepristone- or dexamethasone-stimulated cells. We have previously shown that IL-1-stimulated HAT activity induced acetylation of Lys⁵ and Lys¹² residues only after mild, but not stringent, CBP immunoprecipitation conditions, suggesting that CBP itself does not play a role in NF-B-induced histone acetylation (18). Histone acetylation was increased 3-fold following IL-1 stimulation (p < 0.05, Fig. 5A). Dexamethasone and mifepristone both inhibited p65-associated IL-1-induced histone acetylation in a concentration-dependent manner (IC₅₀ = 3.7 × 10¹⁰ and 3.1 × 10^-7 M, p < 0.05) (Fig. 5A, open bars). Dexamethasone and mifepristone, in the absence of IL-1, produced no change in p65-associated histone acetylation from that seen in control untreated samples (Fig. 5A, open bars). In order to confirm that dexamethasone and mifepristone targeted p65-associated HAT activity specifically, control experiments with a blocking peptide were performed, which showed that no histone acetylation activity was pulled down in these assays (184 ± 41 versus >2500 dpm/10⁶ cells). To confirm the earlier data on GM-CSF release (Fig. 1D), we examined the effect of HDAC inhibition on dexamethasone and mifepristone actions on IL-1-induced p65-associated HAT activity. TSA (10 ng/ml) reduced dexamethasone suppression of p65-associated HAT activity (IC₅₀ = 1.9 × 10^-8 versus 3.7 × 10^-10 M, p < 0.05) but had no effect on the mifepristone concentration-response curve (IC₅₀ = 5.5 × 10^-8 M versus 3.1 × 10^-7 M) (Fig. 5A, closed bar). In the presence of TSA, there was no significant difference in IC₅₀ for IL-1-stimulated p65-associated HAT activity between dexamethasone and mifepristone (IC₅₀ = 1.9 × 10^-8 M versus 5.5 × 10^-8 M).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   p65-associated histone acetylation and deacetylation. A, effects of mifepristone (Mif) and dexamethasone (Dex) on IL-1-induced p65-immunoprecipitated histone acetylation in the absence (open bar) or presence (closed bar) of TSA (10 ng/ml). Cells were preincubated with increasing concentrations of mifepristone or dexamethasone for 30 min before IL-1 (1 ng/ml) treatment for a further 1 h. Total cellular proteins were isolated, and p65 was immunoprecipitated. The associated histone acetylation activity was measured following incubation of the p65-IP extract with 10 µg of free core histones and 0.25 µCi of [3H]acetyl-CoA for 45 min. Radiolabeled histones were counted, and results are presented as mean ± S.E. of at least three independent experiments. #, p < 0.05 compared with nonstimulated cells. , p < 0.05 compared with IL-1-stimulated cells. *, p < 0.05 compared with non-TSA-treated cells. B, effects of mifepristone and dexamethasone on p65-associated histone deacetylation. Using the same immunoprecipitates as in A, histone deacetylase activity was measured by incubation of extracts with 3H-labeled histones for 30 min. Free 3H-labeled acetic acid was extracted by ethyl acetate and measured by liquid scintillation counting. Results are presented as mean ± S.E. of at least three independent experiments. **, p < 0.01.

In the same immunoprecipitates, mifepristone was found to have little or no effect on histone deacetylation except at the highest concentrations studied (10⁶ M; 103.2 ± 15.0 versus 15.3 ± 3.9 dpm/10⁶ cells, p < 0.05). In comparison, dexamethasone caused a concentration-dependent increase in p65-associated HDAC activity (10⁶ M; 939.4 ± 58.3 versus 15.3 ± 3.9 dpm/10⁶ cells, p < 0.01) (Fig. 5B).

Effect of Mifepristone on HDAC Expression, Activity, and Recruitment-- We have previously shown that dexamethasone induced HDAC2 recruitment to the p65-associated HAT complex. We therefore examined whether this was the case for mifepristone. We determined the effect of mifepristone on HDAC2 expression, histone deacetylase activity, and p65/HDAC association. In marked contrast to dexamethasone, mifepristone failed to induce either HDAC2 expression or histone deacetylation activity (Fig. 6A). Western blot analysis of p65 immunoprecipitates showed a recruitment of HDAC2 to the p65-immunoprecipitated complex following treatment of cells with IL-1 and low concentrations (10^-8 M) of dexamethasone (Fig. 6B), suggesting a role for HDAC2 in the suppressive actions of dexamethasone. In contrast, mifepristone failed to mediate recruitment of HDAC2 to the p65-associated HAT complex.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Effect of mifepristone and dexamethasone on HDAC protein expression, HDAC activity, and HDAC recruitment to the p65 complex. A, effect of mifepristone and dexamethasone on HDAC2 protein expression and HDAC activity. Cells were incubated with increasing concentrations of mifepristone (10-8, 106 M) for 24 h. Western blot analysis of HDAC2 expression is shown (upper panel), and total cellular HDAC activity is shown in the lower panel. Results are expressed as mean ± S.E. of three separate experiments. *, p < 0.05. B, recruitment of HDAC2 to p65-immunoprecipitated complexes. Cells were incubated with IL-1 in the presence of mifepristone (Mif; 10-8, 106 M) or dexamethasone (Dex; 10-8, 106 M) for 4 h. Total cellular proteins were isolated and immunoprecipitated with anti-p65 antibodies. HDAC2 content in the immunoprecipitated complexes was measured by Western blotting. The level of p65 within the same samples is shown as a control for protein loading. The result is representative of four separate experiments. NS, nonstimulated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

It has been postulated that mifepristone and related compounds may dissociate transrepression from transactivation at AP-1- and NF-B-driven promoters (19, 20). Therefore, we used mifepristone to examine the roles of GR transactivation and transrepression in the control of GM-CSF and SLPI expression and histone acetylation status. Mifepristone was unable to stimulate histone H4 acetylation and SLPI release. IL-1 caused a concentration-dependent increase in GM-CSF expression, which was inhibited by 50% maximally by mifepristone. A similar effect of mifepristone was seen on IL-1-stimulated histone acetylation and on p65-associated HAT activity. We have previously demonstrated that dexamethasone was able to inhibit IL-1-stimulated histone acetylation by a combination of direct inhibition of p65-activated HAT activity and by recruitment of HDACs to the activated p65-HAT complex. Here we show that mifepristone has a similar ability to dexamethasone in repressing p65-associated HAT activity but, in contrast, is unable to recruit HDAC2 to the activated p65-HAT complex. We have previously shown that this p65-induced HAT activity is associated with, but not due to, CBP and PCAF (18). We show that the p65-associated HAT activity was directly inhibited by both dexamethasone and mifepristone in vitro, suggesting that this is the target for glucocorticoid action rather than CBP itself. It is possible that CBP may play a scaffolding role in this process.

Many previous studies have reported a role for CBP in mediating NF-B activity and/or its interactions with GR (31-37). All of these studies involve overexpression of one or more of the factors thought to be involved in the interactions or microinjection of antibodies. As such, these must be considered as contrived systems that must be confirmed in the natural cell. In this study, in the absence of overexpression, we were unable to show any role for CBP or PCAF in mediating the IL-1-stimulated p65-associated increase in histone acetyltransferase and GM-CSF gene expression. An alternative scenario is that dexamethasone and mifepristone inhibit p65 association with co-activators such as CBP and PCAF. Although not measured directly here, we have previously shown that this is not the case for dexamethasone (18).

Many of the anti-inflammatory effects of corticosteroids may be mediated by repression of transcription factors (transrepression), whereas the endocrine and metabolic effects of corticosteroids are mediated via GRE binding (transactivation) (38). This has led to a search for novel corticosteroids that selectively repress inflammatory gene transcription and would thus reduce the risk of systemic side effects. Transactivation via GR/GRE binding involves a GR homodimer, while transrepression of transcription factor (AP-1 and NF-B) activity involves a GR monomer. A separation of transactivation and transrepression has been demonstrated using reporter gene constructs in transfected cells using selective mutations of GR (19). Furthermore, some corticosteroids, such as RU24858, mifepristone, and ZK98299, have a greater transrepression than transactivation effect (19, 20). Indeed, the topical corticosteroids used in asthma therapy today, such as fluticasone propionate and budesonide, appear to have more potent transrepression than transactivation effects, which may account for their selection as potent anti-inflammatory agents (39). Recently, a novel class of corticosteroids has been described in which there is potent transrepression with relatively little transactivation. These "dissociated" corticosteroids, including RU24858 and RU40066 have anti-inflammatory effects in vivo (21).

The clinical relevance of these effects of GR mutants is indicated by the construction of a GR dimerization-deficient mutant mouse in which GR is unable to dimerize and therefore bind to DNA, thus separating the transactivation and transrepression activities of corticosteroids (40). These animals, in contrast to GR knockout animals, survive to adulthood. In these animals, dexamethasone was able to inhibit AP-1-driven gene transcription, but the ability to facilitate GRE-mediated effects such as cortisol suppression and T-cell apoptosis was markedly inhibited. This suggests that the development of corticosteroids with a greater margin of safety is possible and may predict the development of oral corticosteroids that may be safe to use in asthma and other inflammatory diseases. The results of glucocorticoid actions on airway hyperresponsiveness and airway inflammation in these animals await determination.

The results presented in this study differ from those of Heck and colleagues (20) in regard to the ability of mifepristone to cause transrepression and others with regard to NF-B requirement for CBP (31). The most obvious reason for this is the use of reporter gene assays and GR overexpression in the study of Heck, while they were not employed here. This has important ramifications for the use of reporter genes and overexpression assays in understanding the role of specific proteins in complex systems.

In summary, we have shown that the glucocorticoid receptor agonist mifepristone has no ability to induce gene transcription but represses IL-1-stimulated histone acetylation and GM-CSF release by 50% maximally. IL-1-stimulated NF-B activated distinct p65-associated HATs (35 and 55 kDa) but did not activate CBP or PCAF HAT activity. Mifepristone was able to inhibit p65-associated HAT activity to the same extent as dexamethasone but failed to recruit HDAC2 to the p65-HAT complex. These data suggest that the maximal transcriptional repressive action of glucocorticoids requires recruitment of HDAC2 to the p65-HAT complex. In addition, our results suggest dissociation between the ability of mifepristone to repress histone acetylation and gene expression and of its ability to activate histone acetylation and to switch on gene expression. This suggests that our model of histone acetylation/deacetylation may prove to be useful for the examination of dissociated glucocorticoids.

	FOOTNOTES

^* This work was funded by the Clinical Research Committee (Brompton Hospital), the British Lung Foundation, and GlaxoSmithKline.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A European Respiratory Society Clinical Fellow.

^§ To whom correspondence should be addressed: Thoracic Medicine, Imperial College School of Medicine at the National Heart and Lung Institute, Dovehouse St., London SW3 6LY, United Kingdom. Tel.: 44 171 352 8121; Fax: 44 171 351 8126; E-mail: ian.adcock@ic.ac.uk.

Published, JBC Papers in Press, June 6, 2001, DOI 10.1074/jbc.M103604200

	ABBREVIATIONS

The abbreviations used are: bp, base pairs; SLPI, secretory leukocyte proteinase inhibitor; IL, interleukin; GR, glucocorticoid receptor; GM-CSF, granulocyte-macrophage colony-stimulating factor; TSA, trichostatin A; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IP, immunoprecipitation; HDAC, histone deacetylase; CBP, CREB-binding protein; PCAF, P300/CBP associated factor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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