Fig. 7.

Antagonism of HSP90 has less impact on the activity of NSG ligands. (A,D) HeLa cells transfected with a TAT3-Luc reporter plasmid were treated with 100 nM Dex, 3 nM GSK47867A (67A) or 3 nM GSK47869A (69A) for 24 hours. Subsequently cells were either co-treated with 10 mM geldanamycin (GA) (D) or washed (WO) and placed in serum-free recording medium (A) for a further 24 hours. The production of luciferase was tracked by measuring the relative light units (RLU) emitted from each sample. Graphs tracks RLU production for 24 hours following GA addition or ligand removal. Graphs are representative of three separate experiments. (B,C) HeLa cells were treated with DMSO vehicle (not shown), 100 nM Dex, 3 nM FP, 3 nM 67A or 3 nM 69A for 24 hours or 1 hour followed by washes (WO) and then cultured in ligand-free medium for 24 hours. Subsequently cells were lysed and RNA was extracted using an RNeasy kit. RNA was reverse transcribed and subjected to qPCR of GILZ (B) and FKBP5 (C) using Sybr Green detection in an ABI q-PCR machine and data analysed by the ΔΔCT method. Graphs (mean±s.e.m.) combine data from three separate experiments and display percentage induction compared with the equivalent constant treatment for 24 hours. HeLa cells were treated with 100 nM Dex, 3 nM 67A or 69A for 2 hours and then co-treated with 10 mM GA for a further 2 hours (E) or 22 hours (F), and a constant 4-hour or 24-hour treatment was used as a comparison. Following treatment, cells were lysed in RIPA buffer containing phosphatase and protease inhibitors and analysed by immunoblotting for GR abundance and GR Ser211 phosphorylation. α-Tubulin was used as a loading control. Statistical significance was evaluated by one-way ANOVA followed by Tukey post-test. *P<0.01 compared with both Dex and FP. (G) Mechanism of GR action. Upon binding glucocorticoids (Gc) (1) the GR interacts with the translocation machinery enabling nuclear import (2). In the nucleus, GR binds to cis-elements to activate or repress target gene expression (3). The GR undergoes dynamic cycles of dissociation, and re-binding of ligand, which occurs in an HSP90-dependent manner (4). Interaction with PP5 facilitates nuclear export of the GR (5) enabling it to be recycled or targeted for degradation by the proteasome (6).