Skip to main content
. 2013 Mar 4;110(12):4708–4713. doi: 10.1073/pnas.1221654110

Fig. 1.

Fig. 1.

PHD2 expression is regulated by ligand-dependent activation of ERβ in epithelial cells. (A) ERβ ablation induces dedifferentiaton in PNT1a epithelial cells. ERb expression was ablated in PNT1a cells using two independent shRNAs (shERb#1 and shERb#2). These cells and control cells (shGFP) were maintained in either 2D or 3D culture. Control cells exhibit a distinct epithelial phenotype in 2D and a spheroid shape with a distinct polarity in 3D. In contrast, loss of ERβ expression promotes a mesenchymal phenotype in 2D and invasive outgrowths in 3D. (Scale bars, 100 μm.) Morphological changes observed in response to loss of ERβ are accompanied by diminished E-cadherin expression and an increase in mesenchymal markers (N-cadherin, vimentin, and HIF-1α). Immunoblot on the Far Right demonstrates that PNT1a cells lack expression of ERα in comparison with T47D breast carcinoma cells. (B) ERβ ablation suppresses PHD2 expression. ERβ-ablated PNT1a cells were assessed for PHD2 expression by qPCR and immunoblotting. Loss of ERβ expression is accompanied by a marked reduction in PHD2 mRNA and protein compared with the control cells. The reduction of PHD2 expression as a consequence of ERβ ablation is specific because there is no effect on PHD1 and PHD3 mRNA (*P < 0.05). Immunoblot on the Far Right demonstrates that loss of ERβ in LNCaP cells induces HIF-1α. (C) PHD2 expression is induced by an ERβ ligand, 3β-adiol. PNT1a and LNCaP cells were incubated with DMSO (control) or 5 µM 3β-adiol for 2 d. Cells were examined for PHD2 expression by qPCR and immunoblotting. 3β-Adiol enhances PHD2 mRNA and protein expression significantly. In contrast, 3β-adiol had no effect on PHD2 expression in ERβ-ablated PNT1a cells. (D) 3β-Adiol and DPN but not estradiol enhance PHD2 expression. PHTPP attenuates PHD2 expression in either the absence or presence of 3β-adiol. (E) TGF-β–induced EMT suppresses PHD2 expression. PNT1a cells were cultured in the absence or presence of TGF-β (5 ng/mL) for 3 d and harvested for immunoblotting. TGF-β treatment diminishes PHD2 expression significantly with a concomitant decrease in E-cadherin and ERβ.

HHS Vulnerability Disclosure