Glucocorticoids (GC) are potent anti-inflammatory compounds, acting mainly through the glucocorticoid receptor (GR). GC therapy, however, has debilitating side-effects, necessitating safer new alternative therapies. The natural GC metabolite 5α-Tetrahydrocorticosterone (5αTHB) is anti-inflammatory in vivo in mice, but with fewer side-effects. Its mechanism of action is unknown, and here we test signalling via GR and the mineralocorticoid receptor (MR). 5αTHB displaced dexamethasone (Dex) from primary rat hepatocytes (n=6, Kd (nM): Dex 37±8, corticosterone 153±59, 5αTHB 268±78) but only negligibly from isolated human GR (n=3, EC50 (μM): Dex 0.004, cortisol 0.019, 5αTHB 480). GR nuclear translocation was quantified by western blot after nuclear (N) and cytoplasmic (C) separation of steroid-treated A549 cells. Dex (100 nM) increased the N/C GR ratio, after 30 minutes (0.59±0.26 to 2.97±0.50, P<0.0001) and 24 h (0.47±0.29 to 2.23±0.72, P<0.05). In contrast, GR translocation was not observed with 5αTHB (1, 3 or 10 μM) at either time (n=6). Similarly, GR Ser211 phosphorylation was increased by (1 μM) corticosterone (B; 5.00±1.05 fold vs vehicle, P<0.0001) but not by (10 μM) 5αTHB (2.74±0.35 fold vs vehicle, not significant). MARCoNI (Pamgene) peptide array analysis (n=3) demonstrated that Dex altered the interaction of GR ligand binding domain (LBD) with 75 co-regulator peptides, whereas 5αTHB induced weak changes with only 3. Interestingly, interactions between the MR LBD and its co-regulator peptides were altered to a comparable extent by F and 5αTHB treatment (76 vs 41 interactions altered, respectively). In addition, both aldosterone (10 nM) and 5αTHB (5 μM) increased transcriptional activity of a luciferase tagged MR reporter construct in HEK293 cells (13.50±1.09 fold and 11.20±1.24 fold vs vehicle, respectively, both P<0.0001, n=4). Both effects were antagonised by spironolactone. In conclusion, the mechanisms underlying the action of 5αTHB differ from those of classical GCs, consistent with its reduced side-effect profile, and may involve MR as much as GR.
20 May 2017 - 23 May 2017