Glucocorticoid receptor recruitment of histone deacetylase 2 inhibits interleukin-1beta-induced histone H4 acetylation on lysines 8 and 12

Abstract

We have investigated the ability of dexamethasone to regulate interleukin-1beta (IL-1beta)-induced gene expression, histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity. Low concentrations of dexamethasone (10(-10) M) repress IL-1beta-stimulated granulocyte-macrophage colony-stimulating factor (GM-CSF) expression and fail to stimulate secretory leukocyte proteinase inhibitor expression. Dexamethasone (10(-7) M) and IL-1beta (1 ng/ml) both stimulated HAT activity but showed a different pattern of histone H4 acetylation. Dexamethasone targeted lysines K5 and K16, whereas IL-1beta targeted K8 and K12. Low concentrations of dexamethasone (10(-10) M), which do not transactivate, repressed IL-1beta-stimulated K8 and K12 acetylation. Using chromatin immunoprecipitation assays, we show that dexamethasone inhibits IL-1beta-enhanced acetylated K8-associated GM-CSF promoter enrichment in a concentration-dependent manner. Neither IL-1beta nor dexamethasone elicited any GM-CSF promoter association at acetylated K5 residues. Furthermore, we show that GR acts both as a direct inhibitor of CREB binding protein (CBP)-associated HAT activity and also by recruiting HDAC2 to the p65-CBP HAT complex. This action does not involve de novo synthesis of HDAC protein or altered expression of CBP or p300/CBP-associated factor. This mechanism for glucocorticoid repression is novel and establishes that inhibition of histone acetylation is an additional level of control of inflammatory gene expression. This further suggests that pharmacological manipulation of of specific histone acetylation status is a potentially useful approach for the treatment of inflammatory diseases.

Figures

FIG. 1

Histone acetylation is associated with IL-1β- and dexamethasone (Dex)-induced gene expression. (A) Effect of TSA (10 ng/ml) on IL-1β-stimulated GM-CSF release (upper panel). Cells were stimulated with IL-1β (1 ng/ml) for 6 h. Supernatants were collected and assayed for GM-CSF by ELISA. ∗, P < 0.05; ∗∗, P < 0.01 versus nontreatment. (Lower panel) Effect of 30 min of preincubation with dexamethasone on IL-1β stimulated GM-CSF release. The effect of TSA (10 ng/ml) on dexamethasone inhibition of IL-1β stimulated GM-CSF release was also measured. Results are expressed as mean ± the SEM (n = at least three independent experiments; ∗, P < 0.05, or ∗∗, P < 0.01, versus IL-1β alone). (B) Effects of dexamethasone and IL-1β on SLPI production. Cells were treated with dexamethasone alone (upper panel) or preincubated with dexamethasone for 30 min before incubation with IL-1β (1 ng/ml) for 6 h (lower panel). Supernatants were assayed by ELISA. The effects of TSA (10 ng/ml) on IL-1β-stimulated SLPI release were also measured. Results are expressed as mean ± the SEM (n = at least three independent experiments; ∗, P < 0.05, and ∗∗, P < 0.01, versus IL-1β alone).

FIG. 2

IL-1β and dexamethasone (Dex) acetylate specific and distinct lysine residues. Immunocytochemical staining for specific histone H4 acetylated lysine residues. Cells were incubated with dexamethasone (10−7 M) (b, f, j, and n), IL-1β (1 ng/ml) (c, g, k, and o), or TSA (100 ng/ml) (d, h, l, and p) for 6 h before staining for acetylated forms of histone H4 lysine residues K5 (a to d), K8 (e to h), K12 (i to l), and K16 (m to p). Results are representative of four independent experiments.

FIG. 3

Effects of dexamethasone (Dex) on IL-1β-induced histone acetylation. (A) PCAF acetylates specific histone residues. Cells were treated with IL-1β (1 ng/ml) for 1 h before total cellular proteins were extracted. PCAF was immunoprecipitated under stringent IP conditions and incubated with histones and acetyl-CoA (see Materials and Methods). Using antibodies against specific acetylated lysine residues the level of histone acetylation in the IP sample was measured by immunoassay. Histone acetylation at each lysine residue is expressed in units (1 U is equivalent to the absorbance produced by 50 ng of TSA-treated hyperacetylated histone). Results are expressed as mean ± the SEM (n = at least three independent experiments). (B) CBP acetylation of histone H4 lysine residues. Cells were treated with IL-1β (1 ng/ml) for 1 h before total cellular proteins were extracted. CBP was immunoprecipitated under stringent IP conditions and incubated with histones and acetyl-CoA (see Materials and Methods). Using antibodies against specific acetylated lysine residues the level of histone acetylation in the IP sample was measured by immunoassay. (C) CBP-associated proteins acetylate specific lysine residues. Cells were treated with IL-1β (1 ng/ml) for 1 h before total cellular proteins were extracted. CBP was immunoprecipitated under mild IP conditions and incubated with histones and acetyl-CoA (see Materials and Methods). Using antibodies against specific acetylated lysine residues the level of histone acetylation in the IP sample was measured by immunoassay. (D) Dexamethasone inhibits IL-1β-induced histone acetylation in total cell extracts. Cells were pretreated with dexamethasone for 30 min before incubation with IL-1β (1 ng/ml) for 1 h in the presence of 0.05 mCi of [3H]acetate. Histones were isolated and separated by SDS-PAGE, and [3H]acetate incorporated histones were counted and normalized to the protein level. The data represent the means ± the SEM of three independent experiments. ∗∗, P < 0.01. (E) Western blot analysis of dexamethasone actions on IL-1β-stimulated histone acetylation. Cells were incubated with IL-1β (1 ng/ml) for 6 h in the presence of increasing concentrations of dexamethasone. Protein extracts were obtained and examined for pan-acetylated histone H4 lysine residues and for specific K5, K8, K12, and K16 acetylation by Western blotting. Lanes: control (lane 1); IL-1β stimulation (lane 2); IL-1β stimulation in the presence of dexamethasone at 10−12 M (lane 3), 10−10 M (lane 4), 10−8 M (lane 5), and 10−6 M (lane 6); and dexamethasone, 10−6 M alone (lane 7). The results are representative of three independent experiments.

FIG. 4

Association of specific acetylated lysine residues with GM-CSF and SLPI gene promoters. (A) GM-CSF and SLPI promoter regions. The sequence of the GM-CSF (−191 to +10) and SLPI (−170 to +32) promoter regions amplified by PCR primer pairs. Primers are indicated by overlined sequences. The NF-κB response element in the GM-CSF promoter is underlined. The coding region (CR) of each gene is indicated by an arrow. An enrichment of the GM-CSF promoter DNA is shown following PCR amplification of IP of p65-associated DNA from cells treated with IL-1β (1 ng/ml) for 1 h. (B) Specific lysine residue acetylation at the GM-CSF and SLPI promoters. Cells were incubated with IL-1β (1 ng/ml) in the presence of dexamethasone. Proteins and DNA were cross-linked by formaldehyde treatment, and chromatin pellets were extracted. Following sonication, acetylated histone H4 lysine residues (AcK5, AcK8, and AcK12) were immunoprecipitated, and the associated DNA was amplified by PCR. The results are representative of three independent experiments.

FIG. 5

Dexamethasone (Dex) inhibits p65-associated histone acetylation: a role for HDAC. (A) Dexamethasone inhibits IL-1β-induced p65-immunoprecipitated histone acetylation. Cells were preincubated with dexamethasone for 30 min before IL-1β (1 ng/ml) treatment for a further 1 h. Total cellular proteins were isolated, and p65 was immunoprecipitated under stringent conditions. The associated histone acetylation activity was measured following incubation of the p65-immunoprecipitated extract with 10 μg of free core histones and 0.25 mCi of [3H]acetyl-CoA for 45 min. Radiolabeled histones were counted, and the results are presented as the mean ± the SEM of at least three independent experiments. ∗, P < 0.05; ∗∗, P < 0.01. (B) TSA represses dexamethasone inhibition of p65-associated histone acetylation. Histone acetylation experiments were performed as in panel A in the presence of TSA (100 ng/ml). This resulted in a reduced ability of dexamethasone to suppress p65-associated histone acetylation. Results are presented as the mean ± the SEM of at least three independent experiments. ∗∗, P < 0.01. (C) Effect of IL-1β and dexamethasone on p65-associated histone deacetylation. Using the same immunoprecipitates as in panel A, HDAC activity was measured by incubation of extracts with 3H-labeled histones for 30 min. Free 3H-labeled acetic acid was extracted and counted. The results are presented as the mean ± the SEM of at least three independent experiments. ∗∗, P < 0.01. (D) Specific lysine acetylation by p65. Cells were treated with IL-1β, and total cellular proteins were extracted. p65 was immunoprecipitated under stringent IP conditions and incubated with histones and acetyl-CoA. Using antibodies against specific acetylated lysine residues, the level of histone acetylation in the IP sample was measured by immunoassay. Histone acetylation at each lysine residue is expressed in units (1 U is equivalent to the absorbance produced by 50 ng of TSA-treated hyperacetylated histone). The results are expressed as the mean ± the SEM (n = at least three independent experiments).

FIG. 6

Effect of dexamethasone (Dex) on p65-associated coactivators and GR recruitment. (A) Effect of dexamethasone on CBP and PCAF expression. Cells were incubated with vehicle (control) or increasing concentrations of dexamethasone for 6 h. The results are representative of three independent experiments. (B) Effect of dexamethasone on CBP-p65 interaction and PCAF-p65 interaction. Cells were preincubated with vehicle (control), IL-1β (1 ng/ml), IL-1β and dexamethasone (10−6 M), or dexamethasone (10−6 M) alone for 1 h before total cellular or nuclear proteins were extracted. p65 IP was performed in mild IP buffer. Immunoprecipitates were separated by SDS-PAGE and detected by Western blotting. The results are representative of three independent experiments. (C) Effect of dexamethasone on CBP-PCAF interaction. Cells were preincubated with vehicle (control), IL-1β, or IL-1β and dexamethasone (10−6 M) before protein extraction and PCAF immunoprecipitation under mild IP conditions. The results are representative of three independent experiments. (D) Effect of dexamethasone on CBP phosphorylation. Cells were incubated with [32P]orthophosphate for 30 min before stimulation with IL-1β for 6 h in the presence of dexamethasone. Radioactive bands were excised and counted. The results are expressed as the mean ± the SEM (n = 3; ∗∗, P < 0.01).

FIG. 7

Effect of dexamethasone (Dex) on IL-1β-stimulated CBP-associated histone acetylation and deacetylation activity. (A) No effect of IL-1β and dexamethasone on PCAF IP histone acetylation. Cells were preincubated with dexamethasone (30 min) before IL-1β treatment for 6 h. Total cellular proteins were extracted and PCAF was immunoprecipitated under stringent IP conditions. The associated histone acetylation activity was measured following incubation of the PCAF IP extract with 10 μg of free core histones and 0.25 mCi of [3H]acetyl-CoA for 45 min. Radiolabeled histones were counted, and the results are presented as the mean ± the SEM of at least three independent experiments. (B) Effect of dexamethasone on IL-1β-stimulated CBP immunoprecipitated histone acetylation. Cells were treated as in panel A, and CBP was immunoprecipitated under stringent IP conditions. CBP-associated HAT activity is presented as the mean ± the SEM of at least three independent experiments (∗, P < 0.05). (C) Effect of dexamethasone on IL-1β-stimulated CBP-associated histone acetylation. Cells were treated as in panel A, and CBP was immunoprecipitated under mild IP conditions. CBP-HAT activity is presented as the mean ± the SEM of at least three independent experiments (∗, P < 0.05). (D) Dexamethasone suppression of IL-1β-induced CBP-associated HAT activity requires GR. Cells were treated with IL-1β for 6 h, cellular proteins were extracted, and CBP was immunoprecipitated under stringent IP conditions. CBP IP was incubated with dexamethasone alone or with dexamethasone and GR together with [3H]acetyl-CoA for 45 min in the presence of TSA (100 ng/ml). Results are presented as the mean ± the SEM of at least three independent experiments (∗, P < 0.05). (E) Effect of IL-1β and dexamethasone on histone deacetylation. Using the same immunoprecipitates as in panel C, HDAC activity was measured by incubation of CBP IP extracts with 3H-labeled histones for 30 min. Free 3H-labeled acetic acid was extracted and measured by liquid scintillation counting. The results are presented as the mean ± the SEM of at least three independent experiments (∗∗, P < 0.01). (F) Effect of IL-1β and dexamethasone on GR-mediated histone deacetylation. Cells were treated as in panel A, and total cellular proteins were immunoprecipitated using an anti-GR antibody under stringent IP conditions. The results are presented as the mean ± the SEM of at least three independent experiments (∗∗, P < 0.01).

FIG. 8

Effect of dexamethasone (Dex) on HDAC protein expression, HDAC activity, and HDAC recruitment to the p65 complex. (A) Relative expression of HDAC1 and HDAC2 in A549 cells. A 30-μg portion of protein was size fractionated by SDS–10% PAGE, and Western blot analysis was performed. The results are representative of three independent observations. (B) Effect of dexamethasone on HDAC2 protein expression and HDAC activity. Cells were incubated increasing concentrations of dexamethasone (10−10 to 10−6 M) for 6 h. Western blot analysis of HDAC2 expression is shown in the upper panel, and total cellular HDAC activity is shown in the lower panel. The results are expressed as the mean ± the SEM of three separate experiments (∗, P < 0.05). (C) Recruitment of HDAC2 to p65, CBP, and GR IP complexes. Cells were incubated with IL-1β in the presence of dexamethasone (10−10 M) for 6 h. Total cellular proteins were isolated and immunoprecipitated with anti-p65, anti-CBP, or anti-GR antibodies using mild IP conditions. HDAC2 expression in the IP complexes was measured by Western blotting. p65, CBP, and GR expression in the same samples is shown as a control for protein loading. The results are representative of three separate experiments.

FIG. 9

Proposed model for dexamethasone-GR complex inhibition of IL-1β-stimulated histone acetylation. DNA bound p65 induces histone acetylation via activation of CBP and a CBP-associated HAT complex. This results in local unwinding of DNA and increased gene transcription. GR, possibly acting as a monomer, interacts with CBP, causing an inhibition of CBP-associated HAT activity. In addition, GR also recruits HDAC2 to the activated p65-CBP complex, further reducing local HAT activity and leading to enhanced nucleosome compaction and repression of transcription.