Histone deacetylase 2–mediated deacetylation of the glucocorticoid receptor enables NF-κB suppression

Glucocorticoids are the most effective antiinflammatory agents for the treatment of chronic inflammatory diseases even though some diseases, such as chronic obstructive pulmonary disease (COPD), are relatively glucocorticoid insensitive. However, the molecular mechanism of this glucocorticoid insensitivity remains uncertain. We show that a defect of glucocorticoid receptor (GR) deacetylation caused by impaired histone deacetylase (HDAC) 2 induces glucocorticoid insensitivity toward nuclear factor (NF)-κB–mediated gene expression. Specific knockdown of HDAC2 by RNA interference resulted in reduced sensitivity to dexamethasone suppression of interleukin 1β–induced granulocyte/macrophage colony-stimulating factor production. Loss of HDAC2 did not reduce GR nuclear translocation, GR binding to glucocorticoid response element (GRE) on DNA, or GR-induced DNA or gene induction but inhibited the association between GR and NF-κB. GR becomes acetylated after ligand binding, and HDAC2-mediated GR deacetylation enables GR binding to the NF-κB complex. Site-directed mutagenesis of K494 and K495 reduced GR acetylation, and the ability to repress NF-κB–dependent gene expression becomes insensitive to histone deacetylase inhibition. In conclusion, we show that overexpression of HDAC2 in glucocorticoid-insensitive alveolar macrophages from patients with COPD is able to restore glucocorticoid sensitivity. Thus, reduction of HDAC2 plays a critical role in glucocorticoid insensitivity in repressing NF-κB–mediated, but not GRE-mediated, gene expression.

In the resting cell, chromatin is tightly compacted to prevent transcription factor accessibility. During activation of the cell this compact inaccessible chromatin is made available to DNA-binding proteins, thus allowing the induction of gene transcription (1,2). There is compelling evidence that increased gene transcription is associated with an increase in histone acetylation induced by histone acetyltransferase, whereas hypo-acetylation is correlated with reduced transcription or gene silencing (2,3), which is controlled by histone deacetylases (HDACs).
At least 18 HDACs are now recognized in mammalian cells (4,5) and are grouped into 4 classes based on similarity of genes in yeast. In previous studies, we have found that total nuclear HDAC activity and HDAC2 expression are decreased in lung macrophages and peripheral lung tissue obtained from chronic obstructive pulmonary disease (COPD) (6,7) and that the reduction correlates with disease severity.
Glucocorticoids are the most eff ective therapy for the treatment of many chronic infl ammatory diseases such as asthma and infl ammatory bowel disease (8). They act by binding to cytosolic glucocorticoid receptors (GRs), which upon binding become activated and rapidly translocate to the nucleus. Within the nucleus, GR either induces transcription of genes, such as secretary leukocyte proteinase inhibitor (SLPI) (8) and mitogen-activated kinase phosphatase-1 (9), or inhibits expression of genes such as IL-6 (10) by binding to specifi c DNA elements (glucocorticoid response elements [GREs]) at the promoter/enhancer of responsive genes. At lower concentrations, glucocorticoids reduce infl ammatory gene transcription induced by NF-κB or AP-1 via association between these factors and GR (11). In addition, Ogawa et al. also show that the GR represses a large set of functionally related infl ammatory response genes by disrupting p65-interferon regulatory factor complexes (12). Thus, binding  of GR to p65-NF-κB is crucial for transrepression by corticosteroids. However, it is unclear how the GR dissociates its ability to control infl ammation by suppressing NF-κB from its ability to directly transactivate genes via binding to GRE.
COPD is a common and debilitating chronic infl ammatory disease characterized by progressive airfl ow limitation that is poorly reversible (13) and glucocorticoid insensitive (14,15). We previously demonstrated that total HDAC activity negatively correlates with the inhibitory eff ect of dexamethasone

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(Dex) on TNF-α-induced IL-8 production (16) in alveolar macrophages from smokers and nonsmokers. We also reported that theophylline might restore glucocorticoid sensitivity via enhancement of HDAC activity in COPD macrophages (7). However, it has not been elucidated which HDAC regulates glucocorticoid sensitivity nor how it modulates glucocorticoid action.
In this paper, we identifi ed an HDAC enzyme involved in glucocorticoid-dependent repression of NF-κB-induced gene expression and provided evidence that HDAC2 overexpression was able to restore glucocorticoid sensitivity in glucocorticoid insensitive diseases such as COPD.
Furthermore, HDAC2 KD shifted the concentrationdependent inhibition of IL-1β-stimulated GM-CSF release by Dex to the right, indicating a reduction in glucocorticoid action ( Fig. 1, C and D). A similar result was seen after treatment with TSA (19). A similar action was also seen with suppression of IL-1β-induced IL-8 production ( Fig. 1 D). In contrast, HDAC1, -3, and -8 KD had no eff ect on Dex action on either GM-CSF or IL-8 production (Fig. 1 D). Using individual short interference RNA (siRNA), or combinations of up to four duplexes against HDAC2, we were able to obtain diff erent levels of HDAC2 reduction in cells (Fig. 1 E). This HDAC2 study showed that there was a signifi cant negative correlation between HDAC2 expression after RNAi and Dex EC 50 with respect to IL-1β-induced GM-CSF production (r = −0.771; P = 0.0020; Fig. 1 E). This graded reduction in glucocorticoid responsiveness with reducing HDAC2 expression suggests that HDAC2 plays an important role in the antiinfl ammatory actions of glucocorticoids. Intriguingly, KD of HDAC2 does not aff ect the maximal response to Dex seen at very high concentrations (1 μM; Fig. 1 C) and suggests that, at these supraphysiological concentrations, alternative mechanisms of action may be occurring (20).
HDAC2 KD did not aff ect GR expression or nuclear translocation (Fig. 2 A).

Loss of HDAC2 inhibited association of GR and NF-B
In addition, HDAC2 KD did not aff ect NF-κB expression, nuclear translocation (Fig. 3 A), or NF-κB-DNA binding (Fig. 3 B).[ I D ] F I G 3 [ / I D ] We confi rmed previous data showing that Dex increased GR and HDAC2 association with the p65-NF-κB complex (Fig. 3 A) (19). However, GR was not recruited to the p65-NF-κB complex after HDAC2 KD (Fig. 3 A). Chromatin immunoprecipitation (ChIP) analysis showed that Dex failed to inhibit histone 4 acetylation at the p65-NF-κB binding site in the GM-CSF promoter region after HDAC2 KD (Fig. 3 C). Thus, HDAC2 KD did not reduce the ability of GR to induce GR-sensitive gene expression but had a preferential eff ect on the suppression of NF-κB-mediated infl ammatory gene expression.

GR is an acetylated protein and deacetylated by HDAC2
Previous studies have shown that both estrogen receptor α and androgen receptor are acetylated within their hinge/ ligand binding domains and that this can modulate hormoneinduced gene induction (21,22). This nuclear receptor acetylation site is conserved among members of related nuclear receptors and, based on these fi ndings (22), there is a potential acetylation site at aa 492-495 (KTKK) within the DNA binding domain/hinge region of GR. Indeed, we show that GR is acetylated after Dex binding (Fig. 4, A and  B), and the acetylation levels were decreased when K494 and K495 on GR were mutated to alanine (A), asparagine (N), or glutamine (Q) (Fig. 4 C).

[ I D ] F I G 4 [ / I D ]
These mutants did not further induce SLPI expression by Dex even though overexpression of native GR enhanced SLPI expression by Dex (Fig. 4 D). There was no diff erence in ability of native or mutant GR to bind to p65-NF-κB (Fig. 4 E). However, GR-mediated suppression of IL-1β-stimulated GM-CSF release was not aff ected by TSA with the K494 and K495 mutants (Fig. 4 F) even though the repression was attenuated by TSA in native GR overexpression cells. This suggests that GR acetylation negatively regulates Dex-induced repression of NF-κB-dependent gene expression. In addition, we found that p65-NF-κB-associated GR was deacetylated (Fig. 4 A). Because GR is unable to associate with the p65-NF-κB complex in HDAC2 KD cells, this deacetylation must be important for GR-mediated transrepression (Fig. 4, A and B). Importantly, HDAC2 KD does not inhibit GR-GRE binding or SLPI transactivation (Fig. 2, B and D), indicating that the acetylated GR is still able to activate glucocorticoid-responsive genes, which may be involved in some of the deleterious side eff ects that limit the clinical use of these powerful drugs, as well as some minor antiinfl ammatory eff ects via induction of antiinfl ammatory molecules such as SLPI and MKP-1.
To determine whether acetylated GR was a substrate for HDAC2, acetylated GR was immunoprecipitated in the presence of Dex and incubated with immunopurifi ed HDAC1, -2, -3, and -8 (adjusted to show the same degree of HDAC activity) in a nonisotopic in vitro assay. Acetylated GR was clearly a substrate for HDAC2, although it is also partially deacetylated by HDAC3 (Fig. 4 G). Thus, acetylated GR is a substrate of HDAC2, and deacetylation of GR by HDAC2 may be prerequisite for GR association with the

BRIEF DEFINITIVE REPORT
p65-NF-κB-activated complex and subsequent suppression of infl ammatory gene expression. This mechanism provides a molecular explanation for the ability of GR to distinguish between recruitment of coactivator and corepressor proteins, as previously demonstrated for GRIP (23), and the subsequent ability to transactivate or repress gene transcription.
Overexpression of HDAC2 restores glucocorticoid sensitivity in alveolar macrophages from glucocorticoid-insensitive disease (COPD ) We previously reported that HDAC2 expression and activity is decreased in smokers (16) and patients with COPD (6), who are known to be insensitive to the antiinfl ammatory effects of glucocorticoids (14). In addition, there is a negative correlation between the repressive eff ect of Dex on cytokine production and total HDAC activity in alveolar macrophages from smokers and nonsmokers (16). To determine whether HDAC2 is important for Dex actions in primary cells from patients with glucocorticoid-insensitive disease, we obtained alveolar macrophages from healthy nonsmokers, healthy smokers, and patients with COPD.[ I D ] F I G 5 [ / I D ] There was no substantial diff erence in macrophage GR expression between normal and COPD samples (unpublished data). Total HDAC activity, immunoprecipitated HDAC2 activity (Fig. 5 A), and HDAC2 protein expression (Fig. 5 B, NT) were markedly decreased in cells from patients with COPD. The acetylation level in immunoprecipitated nuclear GR after Dex treatment (10 −8 M) was increased in alveolar macrophages obtained from patients with COPD (Fig. 5 C). In these cells obtained from COPD patients, Dex did not inhibit NF-κB-dependent (24) LPS-induced GM-CSF production in vitro (Fig. 5 D,  top). Overexpression of HDAC2 protein by 6.5 ± 1.1-fold in primary macrophages from COPD patients (Fig. 5 B) restored Dex effi cacy toward suppressing LPS-induced GM-CSF release to levels seen in cells from healthy control subjects (Fig. 5 D, middle). In contrast, HDAC2 overexpression by 3.3 ± 0.86-fold and 5.2 ± 1.1-fold in nonsmokers and smokers, re spectively, did not further increase Dex effi cacy toward LPS-induced GM-CSF production in cells from nonsmokers/smokers, presumably as these cells are already sensitive to Dex actions (Fig. 5 D). HDAC1 overexpression did not aff ect glucocorticoid sensitivity (Fig. 5 D, bottom). Furthermore, 40% KD of HDAC 2 in sputum macrophages from healthy nonsmokers by RNAi caused a reduction in the inhibitory eff ect of Dex (10 −8 M) from 62 to 36% (Fig. 5 E).
Collectively, our results show that HDAC2 is a key protein involved in the suppression of p65-NF-κB-mediated infl ammatory gene expression, which occurs at low concentrations of Dex, whereas modulation of HDAC2 expression does not reduce GR-mediated gene induction. HDAC2 acts by deacetylating GR, thereby enabling p65-NF-κB association and subsequent attenuation of proinfl ammatory gene transcription. The importance of this mechanism in COPD, a glucocorticoid-insensitive disease, is emphasized by overexpression of HDAC2, which restores glucocorticoid sensitivity in primary cells from these patients.

Cultured cells and alveolar macrophages.
The human lung adenocarcinoma type II cell line (A549 cells) was purchased from American Type Culture Collection. Healthy nonsmokers, current healthy smokers, and patients with stage 2-3 COPD were recruited. The study was approved by the Brompton Harefi eld and National Heart and Lung Institute Ethics