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Estrogen Receptor Β Sustains Epithelial Differentiation by Regulating Prolyl Hydroxylase 2 Transcription

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Estrogen Receptor Β Sustains Epithelial Differentiation by Regulating Prolyl Hydroxylase 2 Transcription Estrogen receptor β sustains epithelial differentiation by regulating prolyl hydroxylase 2 transcription Paul Mak, Cheng Chang, Bryan Pursell, and Arthur M. Mercurio1 Department of Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605 Edited* by Jan-Åke Gustafsson, University of Houston, Houston, TX, and approved February 5, 2013 (received for review December 12, 2012) Estrogen receptor β (ERβ) promotes the degradation of hypoxia target genes, including VEGF, lysyl oxidase, and TWIST, have the inducible factor 1α (HIF-1α), which contributes to the ability of this ability to promote epithelial dedifferentiation (9, 18–20). A chal- hormone receptor to sustain the differentiation of epithelial and lenging problem that emerges from these findings is how a nuclear carcinoma cells. Although the loss of ERβ and consequent HIF-1 hormone receptor induces the degradation of HIF-1α (21). HIF- activation occur in prostate cancer with profound consequences, 1α is degraded in normoxia by a well-established mechanism that the mechanism by which ERβ promotes the degradation of HIF-1α involves its hydroxylation on specific prolines by prolyl hydrox- is unknown. We report that ERβ regulates the ligand (3β-adiol)- ylases (PHDs), which target HIF-1α for recognition by the E3 li- dependent transcription of prolyl hydroxylase 2 (PHD2) also gase von Hippel-Lindau (VHL) and consequent degradation in – fi α known as Egl nine homolog 1 (EGLN1), a 2-oxoglutarate-depen- the proteosome (22 24). More speci cally, HIF-1 is hydroxylated dent dioxygenase that hydroxylates HIF-1α and targets it for rec- on two conserved proline residues (p402 and p564), which allows ognition by the von Hippel-Lindau tumor suppressor and consequent for its interaction with VHL E3 ubiquitin ligase for subsequent β PHD2 polyubiquitination and proteasomal degradation (22). The pri- degradation. ER promotes transcription by interacting with α a unique estrogen response element in the 5′ UTR of the PHD2 gene mary PHD that targets HIF-1 under normal conditions is PHD2, that functions as an enhancer. PHD2 itself is critical for maintaining also called Egl nine homolog 1 (EGLN1) (22, 25). In our quest to understand how ERβ destabilizes HIF-α,we epithelial differentiation. Loss of PHD2 expression or inhibition of its β fi function results in dedifferentiation with characteristics of an epithe- pursued the hypothesis that ER regulates speci cprolylhydrox- ylases. The results obtained demonstrate that ERβ regulates the lial–mesenchymal transition, and exogenous PHD2 expression in transcription of PHD2 but not other PHD genes and that this dedifferentiated cells can restore an epithelial phenotype. Moreover, regulation provides a mechanism for how a nuclear hormone re- expression of HIF-1α in cells that express PHD2 does not induce de- α α ceptor controls HIF-1 stability. They also reveal an unexpected differentiation but expression of HIF-1 containing mutations in the role for PHD2 in regulating epithelial differentiation. proline residues that are hydroxylated by PHD2 induces dedifferen- tiation. These data describe a unique mechanism for the regulation Results of HIF-1α stability that involves ERβ-mediated transcriptional regu- PHD2 Expression Is Regulated by Ligand-Dependent Activation of ERβ lation of PHD2 and they highlight an unexpected role for PHD2 in in Epithelial Cells. PNT1a cells are immortalized, normal prostate maintaining epithelial differentiation. epithelial cells (26) that express ERβ but lack ERα (Fig. 1A). Depletion of ERβ in these cells using shRNAs disrupts their he role of estrogen receptors (ERs), which are transcription cobblestone, epithelial appearance in 2D culture and promotes a Tfactors belonging to the steroid/thyroid nuclear receptor su- mesenchymal phenotype as evidenced by their morphology, loss perfamily (1–3), in regulating epithelial differentiation is an of E-cadherin and increased HIF-1α, vimentin, and N-cadherin emerging area of considerable biological interest and pathological expression (Fig. 1A). PNT1a cells form compact spheroids in 3D relevance. In the prostate, the discovery of ERβ (4, 5) has gen- culture that are characteristic of other epithelial cells in 3D (27), erated intense interest in the roles played by this ER in several butlossofERβ induces invasive outgrowths (Fig. 1A), similar to tissues including prostate and breast epithelia (6–11). Increasing that observed for other EMT cells in 3D (28) (29). These data evidence supports the hypothesis that ERβ functions to maintain support the hypothesis that ERβ contributes to the maintenance epithelial differentiation in the prostate and breast (9, 10, 12, 13). of an epithelial phenotype in normal prostate epithelial cells. In the normal prostate, ERβ contributes to epithelial differentia- To examine the putative relationship between ERβ and PHDs, tion as evidenced by the observation that ERβ knockout mice we quantified the expression of PHD1, PHD2, and PHD3 mRNAs exhibit altered differentiation in the ventral prostate, whereas the in control and ERβ-depleted PNT1a cells. As shown in Fig. 1B, glands of ERα knockout mice lack these lesions and appear to be ERβ contributes to the expression of PHD2 but not to either normal (12). ERβ in human prostate cancer is of substantial rel- PHD1 or PHD3. This observation is consistent with the loss of evance because there is an inverse relationship between the ex- PHD2 protein expression observed in ERβ-depleted cells (Fig. 1B). pression of ERβ and highly invasive prostate cancer (9, 14). In These data were confirmed using LNCaP cells, a differentiated pursuit of a functional basis for this relationship, we demonstrated prostate carcinoma cell line derived from a lymph node metastasis that ERβ sustains an epithelial phenotype and impedes a mesen- (Fig. 1B). LNCaP cells express ERβ but lack ERα (8). Loss of ERβ chymal transition in prostate cancer and we identified a metabolite also promotes the expression of HIF-1α in LNCaP cells (Fig. 1B). of dihydrotestosterone, 5α-androstane-3β,17β4-diol (3β-adiol) as The ligand dependency of ERβ-mediated regulation of PHD2 the specificERβ ligand that mediates this function (9). This ob- expression was evaluated by treating both PNT1a and LNCaP servation is in agreement with the recent findings that 3β-adiol is cells with 3β-adiol, a natural ligand for ERβ in the prostate (9, a natural ligand for ERβ in prostate (13, 15–17). 13, 15–17). Indeed, 3β-adiol treatment caused a significant in- A key issue that arises from the above observations is how crease in PHD2 mRNA and protein expression in both cell types loss of ERβ promotes a de-differentiated, epithelial mesenchymal transition (EMT) phenotype. We reported that ERβ has a causal role in the genesis of this phenotype because it impedes the ex- Author contributions: P.M., C.C., and A.M.M. designed research; P.M., C.C., and B.P. per- pression and activation of hypoxia inducible factor 1 (HIF-1). formed research; P.M. contributed new reagents/analytic tools; P.M., C.C., B.P., and A.M.M. More specifically, we made the seminal finding that 3β-adiol/ERβ analyzed data; and P.M. and A.M.M. wrote the paper. destabilize HIF-1α by promoting its proteasomal degradation (9). The authors declare no conflict of interest. Consequently, HIF-1α is stabilized upon loss of ERβ expression or *This Direct Submission article had a prearranged editor. function, enabling HIF-1–mediated transcription. Several HIF- 1To whom correspondence should be addressed. E-mail: [email protected]. 4708–4713 | PNAS | March 19, 2013 | vol. 110 | no. 12 www.pnas.org/cgi/doi/10.1073/pnas.1221654110 Downloaded by guest on September 27, 2021 A shGFP shER #1 shER #2 kDa ER 50 kDa 2D E-cad 100 50 ER HIF-1 50 100 -actin vimentin 50 N-cad 100 T-47DPNT1a 50 -actin 3D #1 #2 Fig. 1. PHD2 expression is regulated by ligand-dependent shGFP shER shER activation of ERβ in epithelial cells. (A)ERβ ablation induces dedifferentiaton in PNT1a epithelial cells. ERb expression was #1 #2 #1 #2 ablated in PNT1a cells using two independent shRNAs B PNT1a LNCaP kDa kDa kDa (shERb#1 and shERb#2). These cells and control cells (shGFP) shGFPshER shER shGFPshER shER shGFPshER 50 50 were maintained in either 2D or 3D culture. Control cells ex- PHD2 PHD2 100 HIF-1 -tubulin -tubulin 50 -actin hibit a distinct epithelial phenotype in 2D and a spheroid 2 50 1.6 50 PHD1 shape with a distinct polarity in 3D. In contrast, loss of ERβ PHD2 PHD1 1.6 PHD3 PHD2 expression promotes a mesenchymal phenotype in 2D and 1.2 PHD3 invasive outgrowths in 3D. (Scale bars, 100 μm.) Morphological 1.2 changes observed in response to loss of ERβ are accompanied 0.8 by diminished E-cadherin expression and an increase in mes- 0.8 * enchymal markers (N-cadherin, vimentin, and HIF-1α). Immu- * 0.4 noblot on the Far Right demonstrates that PNT1a cells lack Fold Change (qPCR) * Fold Change (qPCR) 0.4 α * expression of ER in comparison with T47D breast carcinoma cells. (B)ERβ ablation suppresses PHD2 expression. ERβ-ablated 0 0 shGFP shER #1 shER #2 shGFP shER #1 shER #2 PNT1a cells were assessed for PHD2 expression by qPCR and C immunoblotting. Loss of ERβ expression is accompanied by PNT1a PNT1a (shER LNCaP a marked reduction in PHD2 mRNA and protein compared with kDa − + 3 -adiol kDa − + 3 -adiol kDa − + 3 -adiol 50 50 50 the control cells. The reduction of PHD2 expression as a con- PHD2 PHD2 PHD2 50 sequence of ERβ ablation is specific because there is no effect -actin -actin -actin 37 37 on PHD1 and PHD3 mRNA (*P < 0.05). Immunoblot on the Far * β PHD2 3 PHD2 * Right demonstrates that loss of ER in LNCaP cells induces HIF- 2 α β β 2 1 .(C) PHD2 expression is induced by an ER ligand, 3 -adiol.
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