Session: OR03-Glucocorticoids & Glucocorticoid Actions
Room 130 (Moscone Center)
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
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