Repression of TNF-alpha-induced IL-8 expression by the glucocorticoid receptor-beta involves inhibition of histone H4 acetylation

Abstract

Increased expression of a number of proinflammatory genes, including IL-8, is associated with inflammatory conditions such as asthma. Glucocorticoid receptor (GR)beta, one of the GR isoforms, has been suggested to be upregulated in asthma associated with glucocorticoid insensitivity and to work as a dominant negative inhibitor of wild type GRalpha. However, recent data suggest that GRbeta is not a dominant negative inhibitor of GRalpha in the transrepressive process and has its own functional role. We investigated the functional role of GRbeta expression in the suppressive effect of glucocorticoids on tumor necrosis factor (TNF)-alpha-induced IL-8 release in an airway epithelial cell line. GRbeta expression was induced by treatment of epithelial cells with either dexamethasone or TNF-alpha. GRbeta was able to inhibit glucocorticoid-induced transcriptional activation mediated by binding to glucocorticoid response elements (GREs). The suppressive effect of dexamethasone on TNF-alpha-induced IL-8 transcription was not affected by GRbeta overexpression, rather GRbeta had its own weak suppressive activity on TNF-alpha-induced IL-8 expression. Overall histone deacetylase activity and histone acetyltransferase activity were not changed by GRbeta overexpression, but TNF-alpha-induced histone H4 acetylation at the IL-8 promoter was decreased with GRbeta overexpression. This study suggests that GRbeta overexpression does not affect glucocorticoid-induced suppression of IL-8 expression in airway epithelial cells and GRbeta induces its own histone deacetylase activity around IL-8 promoter site.

Figure 1

Expression of GRα and GRβ. (A) A549 cells were transfected with 0.01 µg and 0.1 µg of pCMV.hGRβ and were harvested 18 h after transfection. RT-PCR for GRβ was performed on cDNA derived from these cells. (B) A549 cells were transfected with pCMV.hGRβ and Western blot was carried out with antibodies raised against GRα and GRβ.

Figure 2

Effects of treatment with dexamethasone or TNF-α on expression of GRβ. 10^-9 M of dexamethasone or 1 ng/ml of TNF-α was used to treat cells for the times indicated. Cells were harvested, cDNA prepared and RT-PCR were performed.

Figure 3

Effects of GRβ expression on dexamethasone-induced transcriptional activation in A549 cell lines. Data are shown as the fold activity over control activity and represent the mean ± SD of three independent experiments. ^*P < 0.05 versus control activity. Dex, dexamethasone.

Figure 4

Effects of GRβ expression on dexamethasone-induced repression of IL-8. A549 IgGκ-NF-κB luciferase cells were transfected with control vector or pCMV.hGRβ. Following 6 h of treatment with TNF-α (5 ng/ml) and/or 10^-7 M of dexamethasone, cells were harvested. (A) Luciferase activity. Data are shown as the fold activity over control activity. (B) mRNA of IL-8 expression was measured by RT-PCR. Densitometric data. The results are shown as the relative ratio to TNF-α induced activity. (C) Supernatants were collected and levels of IL-8 were measured by ELISA. Data represent the mean ± SD of three independent experiments. ^*P < 0.05 versus TNF-α-induced activity. N, no treatment; T, TNF-α treatment; D, dexamethasone treatment.

Figure 5

Effect of GRβ overexpression on HAT and HDAC activities in A549 cells. GRβ overexpression did not change the (A) HAT and (B) HDAC activities. Results are shown as the relative ratio to HAT or HDAC activity with control vector transfection and the mean ± SD of three independent experiments.

Figure 6

GRβ inhibit TNF-α-induced acetylation of histone H4 at IL-8 promoter. (A) Results of a representative chromatin immunoprecipitation (ChIP) experiment showing histone acetylation at IL-8 promoter. A549 cells were transfected with control (pcDNA) or pCMV.hGRβ. Cells were incubated with TNF-α (T; 5 ng/ml) in the presence and absence of dexamethasone (D; 10^-7 M). (B) Graphical analysis of the percentage increase in band density of ChIP data. The results are expressed as mean ± SD of three independent experiments. ^*P < 0.05 versus control vector-induced activity. N, no treatment; T, TNF-α treatment; D, dexamethasone treatment.