Inhibition of histone deacetylase 2 expression by elevated glucocorticoid receptor beta in steroid-resistant asthma

Abstract

Rationale: Cross-talk between glucocorticoid receptors and histone deacetylases (HDACs) under steroid-insensitive conditions has not been explored.

Objectives: To evaluate expression and interaction of HDACs with glucocorticoid receptor isoforms in bronchoalveolar lavage and peripheral blood mononuclear cells from steroid-resistant versus steroid-sensitive patients with asthma.

Methods: Expression of HDACs 1 through 11 was measured by real-time polymerase chain reaction in primary cells and in the DO11.10 cell line, designed to overexpress glucocorticoid receptor β. Glucocorticoid receptor β expression was inhibited in bronchoalveolar lavage cells by small interfering RNA. Human HDAC2 promoter fragments were cloned into a luciferase reporter vector, and transiently transfected with glucocorticoid receptor α- and β-encoding plasmids into the cells. Luciferase activity was then assayed in response to glucocorticoids.

Measurements and main results: Levels of HDAC2 mRNA, but not other histone deacetylases, were significantly decreased in bronchoalveolar lavage cells but not in peripheral blood mononuclear cells from steroid-resistant patients with asthma. Overexpression of glucocorticoid receptor β in DO11.10 cells selectively reduced HDAC2 mRNA and protein levels. Silencing of glucocorticoid receptor β in bronchoalveolar lavage cells from patients with asthma significantly increased HDAC2 mRNA. Luciferase activity assays with HDAC2 promoter reporter constructs identified two glucocorticoid-inducible regions in the HDAC2 promoter. Promoter activity was increased more than fourfold in dexamethasone-treated cells cotransfected with glucocorticoid receptor α. Cotransfection of glucocorticoid receptor β abolished this effect in a dose-dependent manner.

Conclusions: Glucocorticoid receptor β controls expression of histone deacetylase 2 by inhibiting glucocorticoid response elements in its promoter. This highlights a novel mechanism by which glucocorticoid receptor β promotes steroid insensitivity (Li et al.: J Allergy Clin Immunol 2009;123:S146; and Li et al.: J Allergy Clin Immunol 2010;125:AB104).

Figure 1.

Changes in histone deacetylase 2 (HDAC2) mRNA expression in bronchoalveolar lavage (BAL) cells but not peripheral blood mononuclear cells (PBMCs) from subjects with steroid-resistant (SR) asthma. (A) HDAC2 gene expression but not (B) HDAC1 gene expression is significantly decreased in BAL macrophages of subjects with SR asthma as compared with subjects with steroid-sensitive (SS) asthma. No difference in (C) HDAC2 and (D) HDAC1 mRNA expression exists in PBMCs of subjects with SS and SR asthma, as shown by real-time polymerase chain reaction.

Figure 2.

Effects of glucocorticoid receptor β (GCRβ) overexpression on histone deacetylase 2 (HDAC2) expression by DO11.10 cells. (A) Distribution of green fluorescent protein (GFP) expression in GFP/GCRβ and GFP-only retrovirally transduced DO11.10 cells. The level of GFP expression in transgenic DO11.10 cells corresponds to GCRβ expression. (B) Reduced HDAC2 mRNA and (C and D) protein expression in GCRβ-overexpressing cells. HDAC2 gene expression was evaluated by real-time polymerase chain reaction in DO11.10 cells expressing GFP/GCRβ versus DO11.10 cells expressing GFP only (n = 7). HDAC2 protein expression was detected in wild-type (WT), GFP/GCRβ, and GFP DO11.10 cells by Western blot. (C) A representative Western blot experiment and (D) densitometry data are shown (of six independent experiments performed).

Figure 3.

Inhibition of glucocorticoid receptor β (GCRβ) gene expression by use of specific small interfering RNA (siRNA) enhances histone deacetylase 2 (HDAC2) expression. Introduction of GCRβ siRNA into human bronchoalveolar lavage cells increases (A) HDAC2 gene expression as compared with the nonspecific siRNA control group, whereas (B) no effect on HDAC1 gene expression was observed.

Figure 4.

Identification of a functional glucocorticoid response element (GRE) in the human histone deacetylase 2 gene (HDAC2) promoter. (A) Sequences of putative GRE-binding elements within the –5000 bp HDAC2 promoter fragments. Underlined base pairs indicate the conserved GRE sequence. The promoter regions of the HDAC2 gene, encompassing bp −990 to 100, −2070 to −950, −1970 to 3050, −4030 to −3020, and –4010 to −4940 relative to the transcriptional start site (TSS), were fused upstream of the luciferase (Luc) reporter gene to generate four pLG3-HDAC2 constructs, which are designated as pGL3-HDAC2.1 (GRE 1 and 2), HDAC2.2 (GRE 3 and 4), HDAC2.4 (GRE 5), and HDAC2.5 (no GRE). (B) A549 cells and (C) 293-GJ cells were transfected with 250 ng of the four pGL3-HDAC2 promoter reporter plasmids, respectively, along with 50 ng of pRSV-β-galactosidase to monitor transfection efficiency. Cells were incubated for 24 hours ± 10 nM dexamethasone (DEX) before luciferase activity measurements. Luc activity was normalized to β-galactosidase activities, and the data are presented as the fold induction of luciferase activity by DEX. Data represent means ± SD obtained from four independent experiments. *Promoter constructs selected for further functional experiments as DEX inducible (P < 0.05).

Figure 5.

Glucocorticoid receptor β (GCRβ)–mediated inhibition of dexamethasone (DEX)-inducible glucocorticoid response element activity in the histone deacetylase 2 gene (HDAC2) promoter. 293-GJ cells were transfected with 250 ng of (A) HDAC2.2 or (B) HDAC2.4 promoter reporter plasmids either alone or with 100 ng of GCRα and various amounts of GCRβ-encoding plasmids. pGL3-basic plasmid cotransfections were used to adjust the total amount of DNA to 1 μg. Luciferase activities were determined in cell lysates as described in Figure 4. Experiments were performed in triplicate, and the reported values denote means ± SD derived from three independent experiments. *P < 0.05 as compared with GCRα-transfected cells. β-GAL = β-galactosidase.