Abstract
Asthma is a chronic inflammatory disease that is characterized by increased expression of multiple inflammatory genes. Chromatin modification plays a critical role in the regulation of these genes. Acetyaltion of histones by histone acetyltransferases (HATs) is associated with increased gene transcription, whereas hypocetylation induced by histone deacetylases (HDACs) is associated with suppression of gene expression. We have examined the expression and activity of HATs and HDACs in bronchial biopsies from normal subjects and subjects with asthma. There was no difference in the site of HDAC1–HDAC6 expression between normal subjects and subjects with asthma, but subjects with asthma had reduced HDAC enzymatic activity and reduced HDAC1 and HDAC2 protein expression, as measured by Western blotting. In contrast, subjects with asthma treated with inhaled steroids were found to have greater HDAC activity than untreated subjects with asthma, although still lower than control subjects. In contrast, although there was no change in the site of HAT (CREB binding protein and p300/CREB binding protein–associated factor) expression, HAT activity was increased in subjects with asthma. HAT activity was reduced to control levels in subjects with asthma treated with inhaled steroids. The increase in HAT activity and reduced HDAC activity in asthma may underlie the increased expression of multiple inflammatory genes, and this is reversed, at least in part, by treatment with inhaled steroids.
Inflammation is a central feature of many lung diseases, including asthma (1). The inflammatory response involves the recruitment to and activation of inflammatory cells and changes in the structural cells of the airway (2, 3). This involves a complex cascade of inflammatory mediators whose expression is enhanced during the disease process (1). Because many of these inflammatory genes are not expressed in normal cells under resting conditions, the increased expression of these proteins must result from enhanced gene transcription occurring in a cell-specific manner (1).
At a microscopic level, it has long been recognized that chromatin may become dense or translucent because of the winding or unwinding of DNA around core histones (4). Acetylation of histone residues results in unwinding of the DNA that is wrapped around the histone core. This process opens up the chromatin structure, allowing transcription factors and RNA polymerase II to bind more readily to DNA and thereby increase gene transcription (5).
Transcriptional coactivators such as CREB binding protein (CBP) and p300/CBP-associated factor (PCAF) have intrinsic histone acetylation activity, which is activated by the binding of transcription factors, such as activator protein-1 (AP-1) and nuclear factor-κB (5, 6). Repression of genes is associated with reversal of this process by histone deacetylation, a process controlled by histone deacetylases (HDACs) (7). HDACs consist of a growing family of enzymes of which at least 10 mammalian forms now exist (8). HDAC4 and HDAC5 are distinct from other HDACs in that they are able to shuttle between the nucleus and the cytoplasm (8). Deacetylation of histones increases the winding of DNA round histone residues, resulting in a dense chromatin structure and reduced access of transcription factors to their binding sites, thereby leading to repressed transcription of inflammatory genes (7). Increased gene transcription is therefore associated with an increase in histone acetylation, whereas hypoacetylation is correlated with reduced transcription or gene silencing (5).
Histone acetylation is an active process whereby small changes in acetylases or deacetylases can markedly affect the overall histone acetyltransferase (HAT) activity associated with inflammatory genes (5, 7). Thus, hyperacetylation and increased gene expression may result from increased HAT activation or a reduction in HDAC activity or expression. Importantly, these changes in histone acetylation are targeted to regions of DNA associated with specific activator sites within the promoters of induced inflammatory genes (9), although a global loosening of histone structure has also been proposed (10). In addition we have recently described a requirement for recruitment of HDAC activity to activated transcriptional complexes for glucocorticoid suppression of inflammatory gene expression (11).
The objective of this study was to investigate the distribution, expression, and activity of HDACs within the airways of normal subjects and subjects with asthma to determine whether changes in HDAC activity may play a role in the enhanced inflammatory gene transcription seen in asthma. We further wished to determine whether regular treatment with inhaled steroids could enhance the activity and expression of HDACs in vivo in a similar manner to that described in vitro in cell lines (11).
Ten mild, stable patients with asthma (Table 1)
Control Subjects | Mild Intermittent Asthma | Mild/Moderate Persistent Asthma | |
|---|---|---|---|
| n | 16 | 10 | 14 |
| Age, yr | 28 ± 1.4 | 27 ± 1.6 | 29 ± 4.4 |
| Sex, M/F | 10/6 | 5/5 | 8/6 |
| Atopy | 1/16 | 10/10 | 14/14 |
| FEV1, percentage | |||
| predicted | 99 ± 3 | 92 ± 7 | 91 ± 5 |
| PC20 | ND | 0.58 ± 1.5 | 0.8 ± 0.5 |
Subjects attended the bronchoscopy suite at 8:30 a.m. after having fasted from midnight and were pretreated with atropine (0.6 mg intravenously) and midazolam (5–10 mg intravenously). Bronchoscopy was performed as previously described (12). Bronchial mucosal biopsies were washed in Hank's balanced salt solution (HBSS) before immediately being placed in optimal cutting temperature embedding media and then snap frozen in isopentane precooled with liquid nitrogen and stored at −70°C. Sections (6 μm) were cut on a cryostat and placed on poly-l-lysine–coated microscope slides (Sigma, Poole, UK). The slides were air dried for 30 minutes, then wrapped in aluminum foil, and stored at −70°C before immunostaining.
Other biopsies were stored in lysis buffer (10 mM Tris-HCl, pH 6.5, 50 mM sodium bisulphite, 10 mM MgCl2, 8.6% sucrose, 2% Triton X-100, protease cocktail) at −70°C to be used for HDAC assays. After thawing, tissue samples were homogenized with an A-Type Dounce Homogeniser, and the nuclei were collected by microcentrifugation. Nuclei were incubated on ice for 30 minutes in HDAC buffer A (15 mM Tris-HCl, pH 7.9, 450 mM NaCl, 0.25 mM ethylenediaminetetraacetic acid, 10 mM 2-mercaptoethanol, 10% glycerol) before centrifugation, and the NaCl concentration was restored to 150 mM using HDAC buffer B (15 mM Tris-HCl, pH 7.9, 0.25 mM ethylenediaminetetraacetic acid, 10 mM 2-mercaptoethanol, 10% glycerol). The soluble proteins represent the crude HDAC preparations.
Sections were fixed with periodate-lysine-paraformaldheyde and washed repeatedly with phosphate-buffered saline/15% sucrose solution. The sections were permeabilized with 0.1% saponin in phosphate-buffered saline. Nonspecific labeling was blocked by coating with blocking serum (5% horse or goat normal serum) for 20 minutes at room temperature. After washing in phosphate-buffered saline, the sections were incubated for 60 minutes with a mouse monoclonal antihuman HDAC1 antibody (Santa Cruz Biotechnology, Calne, UK). Alternatively, the sections were incubated with a rabbit polyclonal antihuman HDAC2– HDAC6 antibody (Santa Cruz Biotechnology). All antibodies where used at dilutions of 1:25 of a 200 mg/ml solution. These antibodies do not crossreact with other members of the HDAC family.
For the negative control slides, we either did not add the respective primary antibody or normal mouse or rabbit nonspecific immunoglobulins (Santa Cruz) were used at the same protein concentration as the primary antibody. After incubation and repeated washing steps with phosphate-buffered saline, the sections were subsequently incubated with goat antirabbit biotinylated antibody (ABC-AP Kit; Vector Laboratories, Peterborough, UK) for 30 minutes at room temperature. For HDAC1, a horse–antimouse secondary antibody was used (Vector Laboratories). After further washing, the sections were subsequently incubated with ABC reagent (ABC-AP Kit; Vector Laboratories) for 30 minutes at room temperature. Slides were then incubated with chromogen-fast red for 15–30 minutes, after which they were counterstained in hematoxylin and mounted on aqueous mounting medium. The chromogen-fast red has fluorescent properties, which were used for fluorescent microscopy of HDAC1–HDAC6 expression in normal and asthmatic lung.
Total cellular proteins were extracted from biopsy samples by freeze-thawing samples in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, protease inhibitor cocktail; Boehringer Mannheim, Lewes, UK) (13); 30–50 μg of soluble proteins from the cell lysate was resuspended in 4× Laemmli sample buffer. Pellets were size fractionated on 10% polyacrylamide gels and transblotted onto Nitrocellulose-ECL membranes (Amersham-Pharmacia, Amersham, UK). Membranes were blocked for 45 minutes with 5% skimmed milk at 18°C before incubation with 1:1000 rabbit anti-human CBP or 1:800 goat anti-human PCAF, HDAC1, or HDAC2 antibodies (Santa-Cruz) at 18°C for 2 hours. The level of loading was controlled for by determination of actin expression (1:1000; Santa Cruz). After washing (3× 10 minutes in phosphate-buffered saline-Tween), bound antibody was detected using 1:4000 rabbit antigoat or goat antirabbit antibody (Dako, Ely, UK) linked to horseradish peroxidase, and bound complexes were detected using ECL (Amersham-Pharmacia).
HAT activity assays were performed essentially as previously described (11). Total cellular proteins extracted from biopsies were resuspended in 150 μL of HAT buffer (50 mM Tris-HCl, pH 8.0, 10% glycerol, 1 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, complete protease inhibitor cocktail). Typically, 20 μL of free core histone solution extracted from A549 cells (final amount 10 μg) and 30 μL of cell extract were incubated. Reactions were initiated by the addition of 0.25 μCi of [3H] acetyl-CoA (5 Ci/mmol; Amersham) and performed for 45 minutes at 30°C. After incubation, the reaction mixture was spotted onto p81 phosphocellulose filter paper (Whatman, Maidstone, UK) and washed for 30 minutes with 0.2 M sodium carbonate buffer (pH 9.2) at room temperature with two to three changes of the buffer and then washed briefly with acetone. The dried filters were counted in a liquid scintillation counter.
HDAC activity assays were performed essentially as previously described (11). Radiolabeled histones were prepared from A549 cells after incubation with trichostatin A (TSA) (100 ng/ml, 6 hours) in the presence of 0.1 mCi/ml [3H]acetate. Histones extracted by acid and precipitated with cold acetone were dried and resuspended in distilled water. Crude HDAC preparations were extracted from biopsy homogenates with Tris-based buffer (10 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.25 mM ethylenediaminetetraacetic, 10 mM 2-mercaptoethanol) as previously reported (11). The crude HDAC preparations were incubated with [3H]-labeled histone for 1 hour 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 ethylacetate, and the radioactivity of the supernatant was determined by liquid scintillation counting (14).
Results are expressed as 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). All statistical testing was performed using a two-sided 5% level of significance.
Immunohistochemical analysis of HAT and HDAC expression in biopsy samples from normal subjects and subjects with asthma indicated that CBP, PCAF (Figure 1)
Figure 1. Localization of CBP and PCAF in bronchial biopsies. The localization of the expression of the HATs CBO (A and C) and PCAF (B and D) within the airways of asthmatics (C and D) and nonasthmatic control subjects (A and B) was detected by immunohistochemistry. Results are representative of those for eight subjects in each group.
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Figure 2. Localization of HDAC1– HDAC6 in bronchial biopsies. The localization of the expression of HDAC1–HDAC6 within the airways of asthmatics (D–F and J–L) and nonasthmatic control subjects (A–C and G–I) were detected by immunohistochemistry. Results are representative of those for eight subjects in each group.
[More] [Minimize]Using Western blot analysis, we were unable to detect any difference in the level of CBP or PCAF expression in biopsies between subjects with asthma and normal subjects (Figure 3A)
Figure 3. Expression of CBP, PCAF, and HDAC1 and HDAC2 in bronchial biopsies. (A) The relative expression of the HATs CBP and PCAF within the airways of normal (N, lanes 1 and 2) and subjects with asthma (A, lanes 3 and 4) was detected by Western blotting. β-Actin expression was used as a control for protein loading. Results are representative of those seen in six subjects in each group. (B) Expression of HDAC1 in biopsy samples in N (lanes 1 and 2) and A subjects (lanes 3 and 4) was measured by Western blot analysis and compared with β-actin expression. Results are representative of those seen in eight N and eight A subjects. The panel beneath the blot presents these results graphically. Results are shown as mean ± SEM (*p < 0.05). (C) Expression of HDAC2 in biopsy samples in N (lanes 1 and 2) and asthmatic subjects not on inhaled corticosteroids (A, lanes 3 and 4) was measured by Western blot analysis and compared with β-actin expression. Lanes 5 and 6 show the effect of inhaled corticosteroids on HDAC2 expression (A + ICS). Results are representative of those seen in four subjects in each group. These results are presented graphically in the panel on the right. Results are shown as mean ± SEM (*p < 0.05).
[More] [Minimize]HAT activity, as measured by incorporation of 3H-acetic acid into histones, was induced in biopsy samples from asthmatics (5,800 ± 700 disintegrations per minute [dpm], n = 10) as compared with nonasthmatic control subjects (3,000 ± 500 dpm/μg protein, n = 14, p < 0.01) (Figure 4A)
Figure 4. HAT and HDAC activity in bronchial biopsies. (A) Total HAT activity in biopsies obtained from normal subjects (N), subjects with asthma not taking inhaled corticosteroids (A), and subjects with asthma taking inhaled corticosteroids (A + ICS) was measured by incorporation of 3H-acetate into histones. HAT activity was measured as dpm. Results are expressed as means ± SEM (**p < 0.01 versus normals; #p < 0.05 A versus A + ICS, n = 10 for N, 14 for A, and 3 for A + ICS). (B) Total HDAC activity in biopsies obtained from N (n = 10), A (n = 14), and A + ICS (n = 3) subjects was measured by release of 3H-acetic acid from labeled histones. HDAC activity was measured as dpm/μg protein. Results are expressed as mean ± SEM (**p < 0.01 versus normals; #p < 0.05 A versus A + ICS). (C) Correlation between HDAC activity and FEV1 in biopsy samples.
[More] [Minimize]HDAC activity, as measured by release of 3H-acetic acid from labeled histones, was reduced in biopsy samples from asthmatics (83 ± 13 dpm/μg protein, n = 10) as compared with nonasthmatic control subjects (137 ± 14 dpm/μg protein, n = 14, p < 0.01) (Figure 4B). The patients with asthma treated with inhaled steroids showed increased HDAC activity (105 ± 11 dpm/μg protein, p < 0.05, n = 3), although the levels were still reduced compared with normal (p < 0.01) (Figure 4B). There is a good correlation between HDAC activity and FEV1 (r2 = 0.78, p < 0.0001) across the whole group of subjects, mild intermittent subjects with asthma on inhaled β agonists only, and patients with mild/moderate asthma on inhaled steroids (Figure 4C).
These results show that there is reduced HDAC activity and a reduction in the level of expression of HDAC1 and HDAC2 proteins measured by Western blot analysis in bronchial biopsy samples obtained from subjects with mild asthma compared with normal control subjects. Asthma did not appear to affect the distribution of HAT (CBP and PCAF) or HDAC1–HDAC6 expression in the airway. Finally, we observed greater HDAC and less HAT activity in biopsy samples obtained from subjects with asthma taking inhaled corticosteroids, although these were not restored to normal levels. This confirms previous data obtained in cell lines in vitro where we showed elevation of HDAC activity and expression and decreased HAT activity by dexamethasone (11). Although HDAC activity levels were not restored to normal levels, HAT activity was inhibited after steroid treatment and returned to levels seen in normal nonasthmatic control subjects. This may reflect an additional direct effect of steroids on HAT activity (15).
These effects may have implications for the antiinflammatory actions of steroids in the airways. The data suggest that the epithelium is a major source of inflammatory gene expression and a critical target for steroid action (2, 13). The greater effect of steroids on suppression of HAT activity with respect to induction of HDAC activity suggests that repression of HAT activity may be the predominant action of steroids rather than gene induction of HDACs. The enhanced HDAC expression and activity seen in subjects taking inhaled steroids may further increase steroid suppression of HAT activity via GR recruitment of HDACs to gene activation complexes (9).
Modulation of chromatin structure by histone acetylation plays an important role in inflammatory gene transcription (16). Increased gene transcription is associated with enhanced histone acetylation induced by recruitment and/or activation of specific HATs or coactivator proteins (6). This expanding family of HATs includes the proteins investigated here, CBP and PCAF. Increased HAT activity in the airways of patients with asthma may therefore underlie the increased inflammatory gene expression seen in these patients (1). CBP and PCAF are not representative of all HAT activity; there are other proteins (e.g., p300, Gcn5) that have HAT activity. However, we have chosen to analyze them as representative of HAT activity because they are well characterized and previously reported to be important in many in vitro studies examining cytokine-induced gene induction (7).
Histone acetylation is an active process whereby small changes in acetylases or deacetylases can markedly affect the overall HAT activity associated with inflammatory genes (7). The change in whole-cell HAT activity may reflect either a change in HAT activity per se or a decrease in HDAC activity and/or expression. Here we show a reduced expression of HDAC1 and HDAC2 proteins by Western blotting and a corresponding reduction in HDAC activity. This suggests that changes in HDAC may be more important than altered CBP or PCAF expression. We have previously shown that HDACs are important in regulating maximal expression of interleukin-1β–induced granulocyte-macrophage colony-stimulating factor release and that inhibition of HDAC activity by TSA enhances granulocyte-macrophage colony-stimulating factor production (11). Thus, a reduction in HDAC activity could lead to further enhancement of inflammatory gene transcription. In addition, glucocorticoids require HDAC activity for maximal suppression of cytokine induction by interleukin-1β (11). Thus, in subjects with asthma with reduced HDAC activity, the ability of inhaled steroids to control inflammation may be lost. We have recently reported that reductions in HDAC activity in alveolar macrophages of smokers correlated inversely with dexamethasone suppression of interleukin-8 release (17). This may result in ongoing low-level inflammation occurring even during periods of normal lung function (18).
Although this is not evident clinically in subjects with mild asthma, this reduced HDAC activity may become more important in patients with increasing severity of asthma (19). These changes in histone acetylation are targeted to specific response areas in the promoter regions of inflammatory genes, although a global loosening of histone structure cannot be excluded as a model for the effects of changes in gene transcription (20).
Modulation of HAT and HDAC activity may prove to be important new targets for drug development (21). For example, compounds that inhibit specific HATs may prove to be able to reduce nuclear factor-κB–mediated inflammatory gene expression. Nuclear factor-κB–mediated induction of HAT activity is mediated through specific HATs (11), and inhibition of these activities may therefore be possible. Alternatively, it may be possible to enhance HDAC activity and thereby suppress in a more global fashion the expression of multiple inflammatory genes.
In summary, we have determined the pattern of expression of several members of the HAT and HDAC families in asthmatic airways and report a significant reduction in HDAC activity. This activity could be induced by inhaled corticosteroids that may, in part, account for the repression of inflammation seen with these drugs. These results further suggest that variations in HDAC activity may be important in the regulation of inflammatory gene expression in asthmatic airways.
Supported by research grants from the Clinical Research Committee (Royal Brompton Hospital, London, UK) and GlaxoSmithKline (UK).
| 1. | Barnes PJ, Chung KF, Page CP. Inflammatory mediators of asthma: an update. Pharmacol Rev 1998;50:515–596. |
| 2. | Holgate ST, Lackie P, Wilson S, Roche W, Davies D. Bronchial epithelium as a key regulator of airway allergen sensitization and remodeling in asthma. Am J Respir Crit Care Med 2000;162:S113–S117. |
| 3. | D'Ambrosio D, Mariani M, Panina-Bordignon P, Sinigaglia F. Chemokines and their receptors guiding T lymphocyte recruitment in lung inflammation. Am J Respir Crit Care Med 2001;164:1266–1275. |
| 4. | Allfrey VG, Faulkner R, Mirsky AE. Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc Natl Acad Sci USA 1964;51:786–794. |
| 5. | Imhof A, Wolffe AP. Transcription: gene control by targeted histone acetylation. Curr Biol 1998;8:R422–R424. |
| 6. | Janknecht R, Hunter T. Versatile molecular glue: transcriptional control. Curr Biol 1996;6:951–954. |
| 7. | Pazin MJ, Kadonaga JT. What's up and down with histone deacetylation and transcription? Cell 1997;89:325–328. |
| 8. | Bertos NR, Wang AH, Yang XJ. Class II histone deacetylases: structure, function, and regulation. Biochem Cell Biol 2001;79:243–252. |
| 9. | Urnov FD, Wolffe AP. Chromatin remodeling and transcriptional activation: the cast (in order of appearance). Oncogene 2001;20:2991–3006. |
| 10. | Waterborg JH. Steady-state levels of histone acetylation in Saccharomyces cerevisiae. J Biol Chem 2000;275:13007–13011. |
| 11. | Ito K, Barnes PJ, Adcock IM. Glucocorticoid receptor recruitment of histone deacetylase 2 inhibits interleukin-1β-induced histone H4 acetylation on lysines 8 and 12. Mol Cell Biol 2000;20:6891–6903. |
| 12. | Lim S, Groneberg D, Fischer A, Oates T, Caramori G, Mattos W, Adcock I, Barnes PJ, Chung KF. Expression of heme oxygenase isoenzymes 1 and 2 in normal and asthmatic airways: effect of inhaled corticosteroids. Am J Respir Crit Care Med 2000;162:1912–1918. |
| 13. | Adcock IM, Gilbey T, Gelder CM, Chung KF, Barnes PJ. Glucocorticoid receptor localization in normal and asthmatic lung. Am J Respir Crit Care Med 1996;154:771–782. |
| 14. | Kolle D, Brosch G, Lechner T, Lusser A, Loidl P. Biochemical methods for analysis of histone deacetylases. Methods 1998;15:323–331. |
| 15. | Ito K, Jazrawi E, Cosio B, Barnes PJ, Adcock IM. p65-activated histone acetyltransferase activity is repressed by glucocorticoids: mifepristone fails to recruit HDAC2 to the p65-HAT complex. J Biol Chem 2001;276:30208–30215. |
| 16. | Adcock IM. Molecular mechanisms of glucocorticosteroid actions. Pulm Pharmacol Ther 2000;13:115–126. |
| 17. | Ito K, Lim S, Caramori G, Chung KF, Barnes PJ, Adcock IM. Cigarette smoking reduces histone deacetylase 2 expression, enhances cytokine expression, and inhibits glucocorticoid actions in alveolar macrophages. FASEB J 2001;15:1110–1112. |
| 18. | van den Toorn LM, Overbeek SE, de Jongste JC, Leman K, Hoogsteden HC, Prins JB. Airway inflammation is present during clinical remission of atopic asthma. Am J Respir Crit Care Med 2001;164:2107–2113. |
| 19. | Matthews JG, Ito K, Barnes PJ, Adcock IM. Corticosteroid-resistant and corticosteroid-dependent asthma: two clinical phenotypes can be associated with the same in vitro defects in GR nuclear translocation and acetylation of histone H4 [abstract]. Am J Respir Crit Care Med 2000;161:A189. |
| 20. | Gregory PD, Horz W. Chromatin and transcription—how transcription factors battle with a repressive chromatin environment. Eur J Biochem 1998;251:9–18. |
| 21. | Thompson PR, Kurooka H, Nakatani Y, Cole PA. Transcriptional coactivator protein p300: kinetic characterization of its histone acetyltransferase activity. J Biol Chem 2001;276:33721–33729. |
