Proinflammatory cytokines regulate human glucocorticoid receptor gene expression and lead to the accumulation of the dominant negative beta isoform: a mechanism for the generation of glucocorticoid resistance

Abstract

Inflammatory responses in many cell types are coordinately regulated by the opposing actions of NF-kappaB and the glucocorticoid receptor (GR). The human glucocorticoid receptor (hGR) gene encodes two protein isoforms: a cytoplasmic alpha form (GRalpha), which binds hormone, translocates to the nucleus, and regulates gene transcription, and a nuclear localized beta isoform (GRbeta), which does not bind known ligands and attenuates GRalpha action. We report here the identification of a tumor necrosis factor (TNF)-responsive NF-kappaB DNA binding site 5' to the hGR promoter that leads to a 1.5-fold increase in GRalpha mRNA and a 2.0-fold increase in GRbeta mRNA in HeLaS3 cells, which endogenously express both GR isoforms. However, TNF-alpha treatment disproportionately increased the steady-state levels of the GRbeta protein isoform over GRalpha, making GRbeta the predominant endogenous receptor isoform. Similar results were observed following treatment of human CEMC7 lymphoid cells with TNF-alpha or IL-1. The increase in GRbeta protein expression correlated with the development of glucocorticoid resistance.

Figure 1

GRβ-specific mRNA increases after cytokine treatment. Total RNA from HeLaS3 cells was isolated, and RT-PCR was performed on 0.5 μg total RNA per treatment group by using hGRα- and hGRβ-specific primers. Primers specific for actin were used as an internal control.

Figure 2

Immunostaining of the GR isoforms by using isoform-specific epitope-purified polyclonal antibodies. (A) HeLaS3 cells were treated with 1 ng/ml TNF-α or vehicle for 24 h followed by treatment with 100 nM dexamethasone for one hour (TNF) or vehicle (CON). Cells were fixed and processed as described (14, 21, 23, 24). (B) Image analysis of the fluorescent staining was performed by using NIH Image 1.56. Mean peak intensities were calculated for each isoform with and without exposure to TNF.

Figure 3

Western analysis of GR isoform levels after cytokine treatment. (A) HeLaS3 cells were treated with increasing concentrations of TNF-α (0.1 to 100 ng/ml) for 24 h. The alpha-specific AShGR and beta-specific BShGR antibodies were used for Western analysis. (B) Quantitation of the Western blots showing the average (SEM) of three separate experiments. (C) HeLaS3 cells were treated with 1 ng/ml TNF-α for 0, 12, 24, or 48 h. Whole cell extracts were prepared and analyzed as described. (D) Quantitation of the results given as a mean (SEM) of three experiments.

Figure 4

Preferential up-regulation of GRβ occurs in other cell types and with other cytokines. (A) HeLaS3 cells and CEMC7 cells were left untreated or treated with TNF-α (1 ng/ml) or IL-1 (10 ng/ml) for 24 h. Whole cell extracts were prepared and subjected to Western analysis by using AShGR and BShGR antibodies. (B) HeLaS3 cells were treated with TNF-α for 24 or 48 h or vehicle. Whole cell extracts were prepared and analyzed by Western blot with the anti-hGR antibody (no. 57) directed against an epitope common to both GRα and GRβ.

Figure 5

Location of an NF-κB consensus sequence in the human glucocorticoid receptor promoter. (A) Location of the NF-κB consensus sequence relative to the transcription start site (+1) of the hGR promoter (2). The AP-1 site is also designated. Sequence of the NF-κB site is also shown. (B) COS1 cells were transfected with either pBR322 (MOCK) or the NF-κB consensus sequence linked to a heterologous thymidine kinase promoter driving expression of a luciferase reporter (NF-κB-tk-LUC) and p65 or NF-κB-tk-LUC alone or the tk-LUC plus p65 or tk-LUC alone. Luciferase assays represent the mean of three transfections.

Figure 6

Half-life studies of GRα and GRβ mRNA and protein after TNF-α treatment, and down-regulation of receptor protein in response to glucocorticoids. (A) HeLaS3 cells were treated for 24 h with 1 ng/ml TNF-α then treated with actinomycin D for 1 h. mRNA was obtained at various time intervals, and RT-PCR by using alpha-isoform or beta-isoform specific primers was performed. (B) HeLaS3 cells were treated for 24 h with 1 ng/ml TNF-α then treated with cyclohexamide for 1 h. Whole cell extracts were obtained at various time intervals, and Western analysis was performed by using AShGR or BShGR antibodies. (C) COS1 cells were transfected with either hGRα or hGRβ. Sixteen hours posttransfection, the cells were treated with 100 nM DEX or vehicle (CON) for 24 h. Cell extracts were prepared, and GR protein levels were evaluated by Western analysis by using no. 57 antibody.

Figure 7

Hormone-mediated transactivation is abrogated after pretreatment with cytokine. (A) HeLaS3 cells were transiently transfected with the glucocorticoid-inducible reporter plasmid GRE2-TATA-CAT. Cells were treated for 24 h with vehicle alone (CON), 100 nM DEX, 1 ng/ml TNF-α, or 1 ng/ml TNF-α for 24 h plus 100 nM dexamethasone for 1 h (TNF-α + DEX). Cell extracts were prepared and assayed for CAT activity. (B) Endogenous alkaline phosphatase activity was analyzed by using the same treatment scheme as above. (C) Western analysis of nuclear and cytoplasmic levels of p65 after 0, 1, and 24 h of TNF-α treatment.