Compound A, a Dissociated Glucocorticoid Receptor Modulator, Inhibits T-bet (Th1) and Induces GATA-3 (Th2) Activity in Immune Cells
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.