Figure 7.

Rapid activation of HMTs in prostaspheres by short-term E2 exposure. (A) Day 7 prostaspheres were harvested, treated with vehicle, 10 nM E2, and E2 plus pathway inhibitors for 30 min and analyzed by western blots. MLL activation was monitored by immunoblotting for MLL-C protein, which increased by E2 exposure. MLL activation was attenuated by pretreatment with ICI and LY294002 (LY), which reduced p-AKT, but not by U0126 (UO), indicating that ER-mediated p-AKT activates MLL. EZH2 was phosphorylated at S21 by 30-min E2 exposure. This was blocked when spheres were pretreated with ICI and U0126, which blocked p-ERK1/2, but not by LY294002, indicating that ER-mediated p-ERK1/2 phosphorylates EZH2. AKT and ERK phosphorylation were monitored as controls for the inhibitors on a duplicate blot. The GAPDH loading control is shown for both blots. Representative images are from three independent trials. (B) Model depicting possible mechanism of epigenetic control of gene transcription via MLL activation and EZH2 phosphorylation via short-term E2 action.