Compound A, a dissociated glucocorticoid receptor modulator, inhibits T-bet (Th1) and induces GATA-3 (Th2) activity in immune cells

Abstract

Background: Compound A (CpdA) is a dissociating non-steroidal glucocorticoid receptor (GR) ligand which has anti-inflammatory properties exerted by down-modulating proinflammatory gene expression. By favouring GR monomer formation, CpdA does not enhance glucocorticoid (GC) response element-driven gene expression, resulting in a reduced side effect profile as compared to GCs. Considering the importance of Th1/Th2 balance in the final outcome of immune and inflammatory responses, we analyzed how selective GR modulation differentially regulates the activity of T-bet and GATA-3, master drivers of Th1 and Th2 differentiation, respectively.

Results: Using Western analysis and reporter gene assays, we show in murine T cells that, similar to GCs, CpdA inhibits T-bet activity via a transrepressive mechanism. Different from GCs, CpdA induces GATA-3 activity by p38 MAPK-induction of GATA-3 phosphorylation and nuclear translocation. CpdA effects are reversed by the GR antagonist RU38486, proving the involvement of GR in these actions. ELISA assays demonstrate that modulation of T-bet and GATA-3 impacts on cytokine production shown by a decrease in IFN-γ and an increase in IL-5 production, respectively.

Conclusions: Taken together, through their effect favoring Th2 over Th1 responses, particular dissociated GR ligands, for which CpdA represents a paradigm, hold potential for the application in Th1-mediated immune disorders.

Figure 1. Compound A inhibits T-bet transcriptional activity.

A, EL4 cells were transfected with 9 µg of a reporter plasmid which contains T-bet response elements upstream of the luciferase gene (T-bet-RE-Luc) with or without 9 µg of T-bet and GR expression vectors. After 16 h in culture, cells were stimulated for 5 h with or without Compound A (CpdA, 0.1, 1, 5 and 10 µM), Dexamethasone (Dex, 10 nM) or the GR specific antagonist RU38486 (1 µM). Results, as folds, normalized to β-galactosidase activity, are expressed as mean ± SEM (n  =  6, * p<0.001 vs. basal without T-bet, ** p<0.05 vs. T-bet without CpdA), averaged from three independent experiments. B, Lysates obtained from transfection experiments performed under similar conditions as mentioned above, were prepared for T-bet analysis by Western Blot. Single bands corresponding to T-bet were obtained. GAPDH signal was used as loading control; one of three independent experiments with similar results is shown.

Figure 2. Compound A inhibits T-bet transcriptional activity by transrepression.

A, EL4 cells were cotransfected with 9 µg of κB-Luc reporter plasmid and 9 µg of Rel A plus 9 µg of phGR-SB (wt) wild-type GR expression vectors or the GR mutants A458T or S425G. After 16 h, cells were stimulated for 8 h with IL-1β (10 ng/ml), which induces κB-Luc activity, and with CpdA (10 µM) or Dex (100 nM). Results, as % of inhibition of κB-Luc, normalized to β-galactosidase activity, are expressed as mean ± SEM (n  =  3, * p<0.001 vs. Rel A plus IL-1β without Dex or CpdA), of one representative experiment of three independent experiments with similar results. B, EL4 cells were transfected with 9 µg of a reporter plasmid containing two palindromic GR-binding sites coupled to the TK promoter reporter plasmid (TK-GRE2-Luc) plus 9 µg of phGR-SB (wt) wild-type GR expression vector or the GR mutants A458T or S425G. After 16 h, cells were stimulated for 5 h with CpdA or Dex. Results, as % of activation of TK-GRE2-Luc, normalized to β-galactosidase activity, are expressed as mean ± SEM (n  =  3, * p<0.05 vs. basal without Dex or CpdA, ** p<0.05 vs. Dex treated wild-type or S425G mutant GR), of one representative experiment of three independent experiments with similar results. C, EL4 cells were transfected with 9 µg of T-bet-RE-Luc reporter plasmid and with 9 µg of T-bet and wild-type GR expression vectors (ph-GR-SB) or the GR mutants A458T or S425G. After 16 h, cells were stimulated for 5 h with CpdA. Results, as % of inhibition of T-bet-RE-Luc, normalized to β-galactosidase activity, are expressed as mean ± SEM (n  =  3, * p<0.001 vs. ph-GR-SB or A458T-mediated T-bet-RE-Luc relative inhibition) of one representative experiment of three independent experiments with similar results.

Figure 3. Compound A inhibits IFN-γ promoter activity and cytokine production.

A, EL4 cells were cotransfected with 9 µg of IFN-γ promoter-driven luciferase plasmid (IFN-γ-Luc) plus 9 µg of GR expression vector and with 9 µg of T-bet expression vector. After 16 h in culture, cells were stimulated for 5 h with Compound A (CpdA, 10 µM). Results, as folds, normalized to β-galactosidase activity, are expressed as mean ± SEM (n  =  6, * p<0.001 vs. basal without T-bet, ** p<0.001 vs. T-bet without CpdA), averaged from three independent experiments. B, Purification of T cells was achieved by a conventional technique involving cell adhesion to plastic and then to a nylon wool column and alternatevely by FACS sorting. Purified T cells were activated with PMA (P) and Ionomycin (I) during 24 h and then incubated with CpdA for 5 h. Supernatants were used to measure mouse IFN-γ according to the manufacturer’s instructions by ELISA. Results are expressed as mean ± SEM (n  =  4, * p<0.001 vs. basal without CpdA, ** p<0.001 vs. P+I without CpdA) of one representative experiment of three independent experiments with similar results.

Figure 4. Compound A induces GATA-3 transcriptional activity by signaling through p38 MAPK.

A, EL4 cells were transfected with 9 µg of a reporter plasmid, which contains GATA-3 response elements upstream of the luciferase gene (GATA-RE-Luc) and with 9 µg of GATA-3 and GR expression vectors. After 16 h, cells were stimulated for 5 h with Compound A (CpdA, 1, 5 and 10 µM), Dexamethasone (Dex, 10 nM), cAMP (0.3 mM) and with the GR specific antagonist RU38486 (1 µM). n  =  6, * p<0.001 vs. GATA-3 without CpdA and cAMP, ** p<0.001 vs. GATA-3 with cAMP and without CpdA. B, Lysates obtained from transfection experiments performed under similar conditions as mentioned above, were analysed by Western Blot. GAPDH was used as loading control. C, EL4 cells were transfected with 9 µg of GATA-RE-Luc reporter and with 9 µg of GATA-3 and GR expression vectors. After 16 h, cells were stimulated for 5 h with CpdA (10 µM) and cAMP. Also, EL4 cells were pretreated during 1 h with the p38 MAPK inhibitor, SB203580 (10 µM). n  =  6, * p<0.001 vs. GATA-3 without cAMP and CpdA, ** p<0.001 vs. GATA-3 with cAMP and without CpdA, *** p<0.001 vs. GATA-3 with cAMP and CpdA. D, EL4 cells were transfected with 9 µg of GATA-RE-Luc reporter and 9 µg of GATA-3, GR and p38 MAPK expression vectors. After 16 h, cells were stimulated for 5 h with CpdA and cAMP. n  =  6, * p<0.001 vs. GATA-3 without cAMP and CpdA, ** p<0.001 vs. GATA-3 with cAMP and without CpdA, *** p<0.001 vs. GATA-3 with cAMP and p38. E, EL4 cells were transfected with 20 µg of the GR expression vector. After 16 h, EL4 cells were pretreated during 30 minutes with CpdA and then with cAMP during 25 minutes. Cell lysates were prepared for Western Blot analysis against phospho-p38 (p-p38) MAPK. GAPDH and total p38 signals were used as loading controls. Lower panel: NIH Image semiquantification. F, EL4 cells were transfected with 9 µg of pFA-ATF2 and 9 µg of pG5-Luc reporter plasmid, and the GR expression vector. After 16 h, cells were stimulated for 5 h with CpdA and cAMP. Also, EL4 cells were pretreated during 1 h with the p38 MAPK inhibitor, SB203580. n  =  6, * p<0.001 vs. basal without cAMP and CpdA, ** p<0.001 vs. cAMP without CpdA, *** p<0.001 vs. cAMP and CpdA. For all the transfections experiments, results, as folds, normalized to β-galactosidase activity, are expressed as mean ± SEM, averaged from three independent experiments.

Figure 5. Compound A induces GATA-3 phosphorylation and nuclear translocation.

A, EL4 cells were transfected with 15 µg of the GR and 15 µg of GATA-3 expression vectors. After 16 h in culture, EL4 cells were pretreated during 30 minutes with or without Compound A (CpdA, 10 µM) and then treated under basal or activated conditions using cAMP (0.3 mM) during 25 minutes. Cell lysates were prepared for Western Blot analysis against phospho-GATA-3 (p-GATA-3) (bands of 55 kDa). Total GATA-3 signal was used as loading control. One out of three independent experiments with similar results are shown. B, EL4 cells were transfected with 15 µg of the GR and 15 µg of GATA-3 expression vectors. After 16 h in culture, EL4 cells were pretreated during 30 minutes with or without CpdA (10 µM) and then treated under basal or activated conditions using cAMP (0.3 mM) during 25 minutes. Cell nuclear and cytoplamic extracts were prepared for GATA-3 analysis by Western Blot. Histone H3 and GAPDH signals were used as nuclear and cytoplasmic extracts control respectively. One out of three independent experiments with similar results are shown.

Figure 6. Compound A inhibits IL-5 promoter activity and cytokine production.

A, EL4 cells were cotransfected with 9 µg of IL-5-Luc reporter plasmid plus 9 µg of GR and GATA-3 expression vectors. After 16 h, cells were stimulated for 5 h with CpdA (10 µM) and cAMP. Also, EL4 cells were pretreated during 1 h with p38 inhibitor, SB203580 (10 µM). n  =  6, * p<0.001 vs. GATA-3 without cAMP and CpdA, ** p<0.001 vs. GATA-3 with cAMP and without CpdA, *** p<0.001 vs. GATA-3 with cAMP and CpdA, # p<0.001 vs. GATA-3 with cAMP and SB203580 and without CpdA. B, EL4 cells were cotransfected with 9 µg of the wild-type or GATA-3 mutated-binding site on the IL-5 promoter (IL-5-Luc-WT and IL-5-Gm-Luc) plus 9 µg of GR and GATA-3 expression vectors. After 16 h, cells were stimulated for 5 h with CpdA and cAMP. n  =  6, * p<0.001 vs. GATA-3 without cAMP and CpdA on IL-5-Luc WT, ** p<0.001 vs. GATA-3 with cAMP and without CpdA on IL-5-Luc WT, *** p<0.001 vs. GATA-3 with cAMP and CpdA on IL-5-Luc WT, # p<0.05 vs. GATA-3 with cAMP and without CpdA on IL-5-Gm-Luc. C, A20 cells were cotransfected with 9 µg of IL-5-Luc reporter plus 9 µg of GR and GATA-3 expression vectors. After 16 h, cells were stimulated for 5 h with CpdA and cAMP. n  =  6, * p<0.001 vs. basal, ** p<0.001 vs. GATA-3, *** p<0.001 vs GATA-3 with cAMP. D, Purification of T cells was achieved as described in Materials and Methods. Cells were activated with PMA (P), Ionomycin (I) and cAMP during 24 h and then with CpdA for 5 h. Supernatants were used to measure mouse IL-5 by ELISA. n  =  4, * p<0.05 vs. basal without P+I, ** p<0.001 vs. P+I without cAMP, *** p<0.001 vs. P+I and cAMP, of one representative experiment of three independent experiments with similar results. For the transfections experiments, results, as folds, normalized to β-galactosidase activity, are expressed as mean ± SEM, averaged from three independent experiments.