Endocrine Abstracts

Glucocorticoids (GCs) are highly effective anti-inflammatory drugs, however their use is limited by serious side effects. We have previously shown that 5αTHB binds GC receptor (GR) and suppresses inflammation in vitro and in vivo, without affecting metabolism. Here the underlying molecular mechanisms were explored in cell models of ligand-induced GR phosphorylation, nuclear localisation and gene transcription. Data are mean±S.E.M. (three experiments); *P<0.05 vs vehicle.

Phosphorylation of Ser211GR, influencing nuclear localisation and gene transcription, was assessed by western blot in A549 cells treated (1h) with vehicle, corticosterone (B) (1 μM) or 5αTHB (1–30 μM). Phosphorylation was not induced by 5αTHB alone, in contrast to B (fold induction: 1.4±0.4 (5αTHB, 1 μM), 6.6±1.6* (B).

To determine mobility of ligand-bound GR, localisation of green fluorescent protein tagged-rat GR (GR-GFP) transfected in HEK293 cells was monitored by fluorescence microscopy. Nuclear translocation of GR-GFP by 5αTHB (1 μM) was incomplete (82.7±1.5% reaching the nucleus within 5 h). Conversely, translocation with B (1 μM) was complete within 30 min. A greater proportion (~3×) of GR-GFP translocated to the nucleus following a sub-maximal dose of B (3 nM; 45 min)) when 5αTHB (1 μM) was present. Nuclear export after steroid washout was observed only with 5αTHB (32.1±6.1% remaining after 24 h). The rate of recovery from nuclear photobleaching (FRAP) suggested that 5αTHB-bound GR-GFP was more mobile in the nucleus than with B (half-life: 5α-THB 3.12±0.38 vs B 4.40±0.42 s; P<0.05).

Ligand ability to induce transcription of GR dimer- and multimer-dependent reporter genes (MMTV and PNMT respectively) was tested. Compared to B, 5αTHB (0.1–3 μM; 24 h) was unable to activate either reporter plasmid (fold induction, 5αTHB and B: MMTV-Luc, 1.2±0.04; 16.9±1.1*; PNMT-Luc, 1.6±0.6; 3.1±0.5*).

GR nuclear translocation is slower in presence of 5αTHB, and activation of classical homodimer- and multimer-dependent gene transcription is absent. This may be linked, to an inability to 5αTHB-bound GR to be phosphorylated on key residue Ser211.

Declaration of interest

The co-authors hold a relevant patent for the use of 5aTHB as an anti-inflammatory drug.

Declaration of funding: This work was supported by the British Heart Foundation.