Figure 4

20β-Dihydrocortisol binds, translocates and activates glucocorticoid receptor inducing gene transcription and suppressing inflammatory gene transcription. (A) Docking of cortisol and 20β-DHF into the ligand binding site of GR. The automatically created pharmacophore indicates the essential structural features for ligand binding (red and green arrows with spheres display hydrogen-bond (H-bond) interactions and yellow spheres hydrophobic interactions). Amino acid residues crucial for ligand binding are shown as sticks. Compared to the binding interactions of cortisol 20β-DHF differs only in the hydroxyl group at the position 20, representing a H-bond donor instead of the carbonyl group of cortisol serving as H-bond acceptor. (B) Unlabelled 20β-DHF displaced ³[H]-dexamethasone from GR in the lysate of SF9 cells expressing GR. (C) 1uM 20β-DHF induced translocation of cytoplasmic GR to the nucleus of HEK293 cells within 30 minutes visualised by fluroescence imaging at 20x magnification. (D) 2.5uM 20β-DHF induced luciferase activation in A549 cells transfected with glucocorticoid responsive plasmid MMTV-luc. (E) 20β-DHF induced transcription of GR-responsive genes IGFBP1 (EC₅₀ 0.51 µM), DUSP1 (EC₅₀ 0.32 µM), FKBP51 (EC₅₀ 0.44 µM) and GILZ (EC₅₀ 1.25 µM) in A549 cells. (F) TNFα induced transcription of IL-1β in A549 cells, this was inhibited by cortisol and by 20β-DHF at 0.15 µM. Transcription was not reduced by co-incubation of cortisol or 20β-DHF with the GR antagonist RU486. Experiments were performed in triplicate on three occasions. Data are mean ± SEM (N = 3). Data were compared by two-way ANOVA and Bonferroni correction test: *P < 0.05, **P < 0.01 compared to vehicle.