p65-activated histone acetyltransferase activity is repressed by glucocorticoids: Mifepristone fails to recruit HDAC2 to the p65/HAT complex

Glucocorticoids acting through their specific receptor can either enhance or repress gene transcription. Dexamethasone represses IL-1 b -stimulated histone acetylation and GM-CSF 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 IL-1 b -stimulated histone acetylation and GM-CSF 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. This data suggests that the maximal repressive actions of glucocorticoids require recruitment of HDAC2 to a p65/HAT complex. This data also suggests that pharmacological manipulation of specific histone acetylation status is a potentially useful approach for the treatment of inflammatory diseases. Cag CACC-3’ switch on our model acetylation/deacetylation may prove useful examination of glucocorticoids.


Introduction
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 organised and dynamic protein-DNA complex. The fundamental subunit of chromatin, the nucleosome, is composed of an octamer of 4 core histones; an H3/H4 tetramer and two H2A/H2B dimers, surrounded by 146 bp 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 ubiquitinisation of specific amino acid residue (6).
Increased gene transcription is associated with an increase in histone acetylation, whereas hypo-acetylation 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)(9)(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 pro-inflammatory transcription factors such as AP-1 (Fos:Jun heterodimers) and NF-κB (p65:p50 heterodimers) (15)(16)(17). We have recently shown that GR represses NF-κB-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-κB 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 over-expression 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 non-transfected 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 4 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); 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). PCR reactions 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 U 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 45s.

Direct histone extraction
Histones were extracted from nuclei overnight using HCl and H2SO4 at 4°C as previously described (18). Cells were microfuged for 5 min and the cell pellets extracted with ice-cold lysis buffer (10mM Tris-HCl, 50mM sodium bisulphite, 1% Triton X-100, 10mM MgCl2, 8.6% sucrose, complete protease inhibitor cocktail (Boehringer-Mannheim, Lewes, UK) for 20 min at 4°C. The pellet was repeatedly washed in buffer until the supernatant was clear (centrifuge at 8000rpm, 5min after each wash) and the nuclear pellet washed in nuclear wash buffer (10mM Tris-HCl, 13mM EDTA) and resuspended in 50µl of 0.2N HCl and 0.4N H2SO4 in distilled water. The nuclei were extracted overnight at 4°C and the residue microfuged for 10 min. The supernatant was mixed with 1ml ice-cold acetone and left overnight at -20°C. The sample was microfuged 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 (BioRad, Hemel Hempstead, UK).

Western blotting
Immunoprecipitates, whole cell extractions or isolated histones were measured by SDS-PAGE and Western blot analysis using ECL (Amersham, Amersham, UK). Proteins were size-fractionated by SDS-PAGE and transferred to Hybond-ECL membranes.
Immunoreactive bands were detected by ECL.

Histone acetylation activity
Cells were plated at a density of 0.25 x 10 6 cells/ml and exposed to 5µCi/ml of [ 3 H] acetate (NEN life science). After incubation for 10 min at 37°C cells were stimulated for 6 hr. Histones were isolated and separated by electrophoresis on SDS-16% polyacrylamide gel. Gels were stained with Coommasie brilliant blue and the core histones (H2A, H2B, H3 and H4) excised.
The radioactivity in extracted core histones was determined by liquid scintillation counting and normalised to protein level.

Histone deacetylation assay
Radiolabelled histones were prepared from A549 cells following incubation with TSA

Immunoprecipitation
Extracts were prepared using 100 µl of mild IP buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% NP-40, complete protease inhibitor cocktail (Boehringer-Mannheim). The lysis mixture was incubated on ice for 15 min and microfuged for 10 min at 4°C. Extracts were precleared with 20 µl of A/G agarose (a 50:50 mix; Santa Cruz Biotech Inc.) and 2 µg of normal rabbit IgG. After microcentrifugation, 20 µl of A/G agarose conjugated with 5µg of p65 antibody were incubated for 6 hours at 4°C with rotation. The immune complexes were pelleted by gentle centrifugation and washed 3 times with 1ml of IP buffer. For the HAT assay, immunoprecipitates were washed twice with IP-HAT buffer, and for Western blotting, after final wash with IP buffer, the buffer was aspirated completely and resuspended in Laemmli buffer.

Statistics
Results are expressed as means ± standard error of the mean (SEM). 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, USA). 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 11 by guest on July 8, 2020 http://www.jbc.org/ Downloaded from seen (EC 50 ) were calculated from concentration-response curves by linear regression.

Effect of anti-glucocorticoid mifepristone on cytokine production and histone acetylation
We

Mifepristone fails to induce histone acetylation but inhibits IL-1ß-stimulated acetylation of bulk histone
In order to determine whether dexamethasone or mifepristone could affect bulk IL-1βstimulated histone acetylation, experiments were performed in whole cell extracts from cells treated with IL-1β and/or mifepristone or dexamethasone. IL-1β induced a 4-fold increase in histone acetylation ( Figure 2). Mifepristone alone had no effect on basal histone acetylation but inhibited IL-1β-induced histone acetylation with maximal inhibition of 53% (Figure 2 upper panel). In contrast, dexamethasone had a biphasic effect on IL-1β-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.

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-1β had no effect on GR translocation (Figure 3 a-d).
In contrast to this result, IL-1β, but not dexamethasone and mifepristone, enhanced NF-κB (p65 subunit) nuclear translocation (Figure 3e-h). We examined histone H4 lysine (K) acetylation in order to confirm the role of histone acetylation in IL-1β, dexamethasone or mifepristone mediated effects. IL-1β caused acetylation of K8 ( Figure 3p) and K12 residues (data not shown) whilst dexamethasone targeted acetylation on K5 (Figure 3k) and K16 (data not shown) as previously reported (18). Mifepristone failed to stimulate acetylation of any lysine residue (Figure 3 j, n).

H4-K5 acetylation
Western analysis of specific acetylated lysines showed that dexamethasone, but not mifepristone, induced acetylation of K5 residues at the concentration of 10 -6 M (Figure 4a).
In addition, dexamethasone significantly inhibited IL-1ß-stimulated K8 acetylation ( Figure   4b). Mifepristone also reduced IL-1β-stimulated K8 acetylation but to a lesser extent than dexamethasone. This data suggests that mifepristone can slightly inhibit histone acetylation induced by IL-1β and, in contrast to dexamethasone, is unable itself to induce histone acetylation.
The above data examines bulk histone acetylation status. In order to be functionally relevant these events must occur at the correct promoter sites.

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-1β and/or mifepristone or dexamethasone stimulated cells. We have previously shown that IL-1βstimulated HAT activity induced acetylation of K5 and K12 residues only after mild, but not stringent, CBP-immunoprecipitation conditions suggesting that CBP itself does not play a role in NF-κB-induced histone acetylation (18). Histone acetylation was increased 3-fold following IL-1β stimulation (p<0.05, Figure 5a). Dexamethasone and mifepristone both inhibited p65- In the same immunoprecipitates, mifepristone was found to have little or no effect on histone deacetylation except at the highest concentrations studied ( (Figure 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

Discussion
It has been postulated that mifepristone and related compounds may dissociate transrepression from transactivation at AP-1 and NF-κB 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-1β caused a concentrationdependent increase in GM-CSF expression, which was inhibited by 50% maximally by mifepristone. A similar effect of mifepristone was seen on IL-1β-stimulated histone acetylation and on p65-associated HAT activity. We have previously demonstrated that dexamethasone was able to inhibit IL-1β-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-κB activity and or its interactions with GR (31-37). All of these studies involve over-expression of one or more of the factors thought to be involved in the interactions or micro-injection of antibodies. As such these must be considered as contrived systems which must be confirmed in the natural cell.
In this study, in the absence of over-expression, we were unable to show any role for CBP, or PCAF, in mediating the IL-1β-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  (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 dimerisation-deficient mutant mouse in which GR is unable to dimerise and therefore 20 by guest on July 8, 2020 http://www.jbc.org/ Downloaded from 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 were 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 are waiting to be determined.
The results presented in this study differ from those of Heck and colleagues (20) in regards to the ability of mifepristone to cause transrepression and others with regards to NF-κB requirement for CBP (31). The most obvious reason for this is the use of reporter gene assays and GR over-expression in the study of Heck whilst they were not employed here. This has important ramifications for the use of reporter genes and over-expression 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-1β-stimulated histone acetylation and GM-CSF release by 50% maximally. IL-1β-stimulated NF-κB activated distinct p65associated HATs (35 and 55kDa) but did not activate CBP or PCAF HAT activity.
Mifepristone was able to inhibit p65-associated HAT activity to the same extent as