Increased glucocorticoid receptor beta alters steroid response in glucocorticoid-insensitive asthma

Abstract

Rationale: Glucocorticoids (GCs) are highly effective in the treatment of asthma. However, some individuals have GC-insensitive asthma.

Objectives: To evaluate the functional response to steroids of bronchoalveolar lavage (BAL) cells from sites of airway inflammation from patients with GC-insensitive versus GC-sensitive asthma. As well, to attempt to define the functional role of glucocorticoid receptor (GCR)beta (a splicing variant, and dominant negative inhibitor of, the classic GCRalpha) in controlling GCRalpha nuclear translocation and transactivation at a molecular level.

Methods and measurements: Fiberoptic bronchoscopy with collection of BAL fluid was performed on seven patients with GC-sensitive asthma and eight patients with GC-insensitive asthma. GCRalpha cellular shuttling in response to 10(-6) M dexamethasone treatment and GCRbeta expression were analyzed in BAL cells by immunofluorescence staining. The effects of overexpression and silencing of GCRbeta mRNA on GCRalpha function were assessed.

Main results: Significantly reduced nuclear translocation of GCRalpha in response to steroids was found in BAL cells from patients with GC-insensitive asthma. BAL macrophages from patients with GC-insensitive asthma had significantly increased levels of cytoplasmic and nuclear GCRbeta. It was demonstrated that GCRalpha nuclear translocation and its transactivation properties were proportionately reduced by level of viral transduction of the GCRbeta gene into the DO-11.10 cell line. RNA silencing of GCRbeta mRNA in human BAL macrophages from patients with GC-insensitive asthma resulted in enhanced dexamethasone-induced GCRalpha transactivation.

Conclusions: GC insensitivity is associated with loss of GCRalpha nuclear translocation in BAL cells and elevated GCRbeta, which may inhibit GCRalpha transactivation in response to steroids.

Figure 1.

Suppression of bronchoalveolar lavage (BAL) macrophage production of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) by dexamethasone (DEX) in patients with glucocorticoid (GC)-insensitive asthma (n = 8; solid triangles) and patients with GC-sensitive asthma (n = 7; solid squares) 24 h after LPS (A and B) or zymosan (C and D) stimulation. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

Impaired GCRα nuclear translocation in BAL macrophages from GC-insensitive patients (original magnification, ×400; blue [DAPI], nuclear staining; red [Cy3], GCRα). Representative images of GCRα cellular shuttling in BAL macrophages from patients with GC-insensitive asthma (A) and patients with GC-sensitive asthma (B) in response to 3 h of 10⁻⁶ M DEX treatment in vitro are shown. Note the cytoplasmic localization of GCRα in the cells after DEX treatment of the patient with GC-insensitive asthma versus nuclear localization of GCRα in the cells from the patient with GC-sensitive asthma, respectively (arrows). The mean fluorescence intensity (MFI) of Cy3 staining (GCRα) of BAL cells from GC-insensitive patients (C) and GC-sensitive patients (D) was assessed by analysis software within the computer-generated masks for nuclear and cytoplasmic regions of the cells and is expressed as the GCRα (Cy3) nuclear:cytoplasmic ratio. Fifty to 100 cells were analyzed for each patient studied.

Figure 3.

Increased expression of GCRβ in BAL macrophages of patients with GC-insensitive asthma. GCRβ expression in BAL macrophages from patients with asthma was analyzed by immunofluorescence staining (A and B) (original magnification, ×400; blue [DAPI], nuclear staining; red [Cy3], GCRβ) and revealed significantly higher GCRβ in BAL macrophages from GC-insensitive patients. The MFI of Cy3 staining (GCRβ) in BAL cells from patients with GC-insensitive asthma and from patients with GC-sensitive asthma was assessed by analysis software within the computer-generated masks for nuclear and cytoplasmic regions of the cells. Forty to 60 cells were analyzed for each patient studied. Note the nuclear and cytoplasmic expression of GCRβ in human macrophages (A). The nuclear and cytoplasmic distribution of GCRβ in BAL macrophages was confirmed by Western blot (C). Compared with the GC-sensitive group, increased GCRβ mRNA expression (D), but not GCRα mRNA expression (E), was observed by real-time polymerase chain reaction (PCR) in freshly isolated BAL cells from patients with GC-insensitive asthma.

Figure 4.

Overexpression of GCRβ controls GCRα nuclear translocation and its transactivation properties. (A) Distribution of green fluorescent protein (GFP) expression in GFP-GCRβ retrovirally transduced DO-11.10 cell lines. As shown previously (27), the level of GFP expression in transgenic DO-11.10 cells correlates with GCRβ expression, because GCRβ cDNA was subcloned into the viral vector as a bicistronic coding unit containing GFP. (B) GCRα has cytoplasmic localization in wild-type (WT) and GCRβ-expressing DO-11.10 cells. GFP GCRβ^dim transgenic cells have nuclear and cytoplasmic localization of GCRβ, with the majority of protein residing in the cell cytoplasm. To control the quality of nuclear and cytoplasmic protein preparations, the membranes were reprobed with anti–NF-1 and anti–β-tubulin antibodies as nuclear and cytoplasmic proteins, respectively. (C) GCRα nuclear shuttling in response to DEX is impaired in the cells expressing GCRβ. (D) GCRα transactivation properties are proportionately reduced by GCRβ expression. DO-11.10 wild-type, GFP GCRβ^dim, and GFP GCRβ^bright cells were collected at various time points after DEX treatment. MKP-1 mRNA induction by DEX in the cell isolates as compared with medium-treated cells was analyzed by real-time PCR.

Figure 5.

Specific GCRβ siRNA enhances GCRα transactivation in BAL macrophages from patients with GC-insensitive asthma. Introduction of GCRβ siRNA into the cells resulted in specific inhibition of GCRβ (A) but not GCRα (B) mRNA expression by the cells as shown by real-time PCR. Data are expressed as the percentage of GCR mRNA expression as compared with such after introduction of nonsilencing control siRNA. (C) Silencing of GCRβ significantly increased DEX-induced MKP-1 mRNA production by BAL macrophages. The cells were transfected with siRNA or remained untreated, and were cultured for 20 h and treated with 10⁻⁶ M DEX for 4 h. MKP-1 mRNA induction by DEX as compared with medium-treated cells was analyzed by real-time PCR.