OR03-5 Investigation into the Molecular Mechanisms Underlying the Anti-inflammatory Properties of 5α-Tetrahydrocorticosterone

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: OR03-Glucocorticoids & Glucocorticoid Actions
Saturday, June 15, 2013: 11:30 AM-1:00 PM
Presentation Start Time: 12:30 PM
Room 130 (Moscone Center)
Annalisa Gastaldello*, Mark Nixon, Chenjing Yang, Philippa T Saunders, Brian R Walker, Karen Elizabeth Chapman and Ruth Andrew
University of Edinburgh, Edinburgh, United Kingdom
Glucocorticoids (GCs) are highly effective anti-inflammatory drugs, however their chronic use is limited by serious metabolic side effects. The 5α-reduced metabolite of corticosterone (B), 5α-tetrahydrocorticosterone (5αTHB), binds the GC receptor (GR) and suppresses inflammation in vitro and in vivo. Here the underlying molecular mechanisms of the anti-inflammatory action of 5αTHB were explored in cell models of ligand-induced GR phosphorylation, nuclear localisation and gene transcription. Data are mean±SEM (of 4 experiments, each in triplicates; * p<0.05).

Phosphorylation of Ser211GR, known to influence GR nuclear localisation and gene transcription, was assessed by Western blot in A549 cells treated (1h) with vehicle, corticosterone (B) (1μM) or 5αTHB (1-30μM). Phosphorylation was not induced by 5αTHB alone, in contrast to B (fold induction over vehicle: 1.4±0.4 (5αTHB, 1μM), 6.6±1.6* (B)). Co-incubation of B and 5μTHB did not significantly alter GR phosphorylation compared to B alone.

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 (1μM) was incomplete (82.7±1.5% reaching the nucleus within 5h). Translocation with B (1μM) was complete within 30mins. Compared to a sub maximal dose of B alone (3nM, 45mins), co-incubation with 5αTHB (1μM) resulted in a greater proportion (3x) of GR-GFP nuclear localisation. Nuclear export after steroid washout (24h) was observed only with 5αTHB (32.1±6.1% remaining). The rate of recovery from nuclear photobleaching suggested that 5αTHB-bound GR-GFP was more mobile in the nucleus than B-bound GR-GFP (half-life: 5αTHB 3.12±0.38* vs B 4.40±0.42s).

Ligand ability to induce transcription of GR dimer- and multimer-dependent reporter genes (MMTV and PNMT respectively) was tested in A549 cells. In contrast to B, 5αTHB (0.1-3μM; 24h) was unable to activate either reporter plasmid (fold induction over vehicle MMTV-Luc: 5αTHB 1.2±0.04; B 16.9±1.1*; PNMT-Luc: 5αTHB 1.6±0.6; B 3.1±0.5*).

Effects of B and 5α-THB on mRNA levels of metabolic genes were investigated in BWTG3 mouse hepatoma cells naturally expressing GR. While B (1μM, 16h) significantly increased the levels of mRNA encoding the GC-induced genes tyrosine aminotransferase (Tat) and gamma-glutamyltransferase 1 (Ggt1) (fold over vehicle: x23±3, x1.4±0.2 respectively), 5α-THB (1-30μM) did not. However, 5α-THB (1-3μM) suppressed the induction of Tat (by 67±4%) and Ggt1(by 33±5%) by B alone.

In conclusion, 5αTHB exhibits the properties of a partial agonist. In its presence, GR translocates slowly, does not become phosphorylated at Ser211 and fails to increase the abundance of metabolic genes. However, 5αTHB restrains the ability of B to activate metabolic genes, protecting tissues from excess glucocorticoid action.

Nothing to Disclose: AG, MN, CY, PTS, BRW, KEC, RA

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: British Heart Foundation (BHF)