17β-Estradiol stimulates the translocation of endogenous estrogen receptor α at the plasma membrane of normal anterior pituitary cells
ABSTRACT In the present work we aimed at identifying ERα in the plasma membrane of normal anterior pituitary cells and investigated if 17β-estradiol was able to induce their subcellular redistribution. Our results show that about 8% of anterior pituitary cells expressed ERα in the plasma membrane, with the geometrical mean fluorescence intensity being increased after steroid hormone treatment. 17β-Estradiol and the selective ERα agonist PPT induced an increase of ERα expression in the plasma membrane and activated the PKCα/ERK 1/2 pathway in a time-course not compatible with genomic actions, thus supporting the notion of membrane-initiated effects. These findings suggest that 17β-estradiol stimulates the translocation of endogenous ERα to the plasma membrane, consequently modulating this ER pool and leading to cellular biological effects in normal anterior pituitary gland.
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ABSTRACT: Estrogens are key to anterior pituitary function, stimulating hormone release and controlling cell fate to achieve pituitary dynamic adaptation to changing physiological conditions. In addition to their classical mechanism of action through intracellular estrogen receptors (ERs), estrogens exert rapid actions via cell membrane-localized ERs (mERs). We previously showed that E2 exerts a rapid pro-apoptotic action in anterior pituitary cells, especially in lactotropes and somatotropes, through activation of mERs. In the present study, we examined the involvement of mERα in the rapid pro-apoptotic action of estradiol by TUNEL in primary cultures of anterior pituitary cells from ovariectomized rats using a cell-impermeable E2 conjugate (E2-BSA) and an ERα selective antagonist (MPP dihydrochloride). We studied mERα expression during the estrous cycle and its regulation by gonadal steroids in vivo by flow cytometry. We identified ERα variants in the plasma membrane of anterior pituitary cells during the estrous cycle and studied E2 regulation of these mERα variants in vitro by surface biotinylation and Western Blot. E2-BSA-induced apoptosis was abrogated by MPP in total anterior pituitary cells and lactotropes. In cycling rats, we detected a higher number of lactotropes and a lower number of somatotropes expressing mERα at proestrus than at diestrus. Acute E2 treatment increased the percentage of mERα-expressing lactotropes whereas it decreased the percentage of mERα-expressing somatotropes. We detected three mERα isoforms of 66, 39 and 22 kDa. Expression of mERα66 and mERα39 was higher at proestrus than at diestrus, and short-term E2 incubation increased expression of these two mERα variants. Our results indicate that the rapid apoptotic action exerted by E2 in lactotropes depends on mERα, probably full-length ERα and/or a 39 kDa ERα variant. Expression and activation of mERα variants in lactotropes could be one of the mechanisms through which E2 participates in anterior pituitary cell renewal during the estrous cycle.PLoS ONE 07/2012; 7(7):e41299. DOI:10.1371/journal.pone.0041299 · 3.53 Impact Factor
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ABSTRACT: Regulation of sexual reproduction by estradiol involves the activation of estrogen receptors (ERs) in the hypothalamus. Of the two classical ERs involved in reproduction, ERα appears to be the critical isoform. The role of ERα in reproduction has been found to involve a nuclear ERα that induces a genomic mechanism of action. More recent a plasma membrane ERα has been shown to trigger signaling pathways involved in reproduction. Mechanisms underlying membrane-initiated estradiol signaling are emerging, including evidence that activation of plasma membrane ERα involves receptor trafficking. The present study examined the insertion of ERα into the plasma membrane of N-38 neurons, an immortalized murine hypothalamic cell line. We identified, using western blotting and PCR that N-38 neurons express full-length 66 kDa ERα and a 52 kDa ERα spliced variant missing the fourth exon - ERαΔ4. Using surface biotinylation we observed that treatment of N-38 neurons with estradiol or with a membrane impermeant estradiol elevated plasma membrane ERα protein levels, indicating that membrane signaling increased receptor insertion into the cell membrane. Insertion of ERα was blocked by the ER antagonist ICI 182,780 or with the protein kinase C (PKC) pathway inhibitor bisindolylmaleimide (BIS). Downstream membrane-initiated signaling was confirmed by estradiol activation of PKC-theta (PKCθ) and the release of intracellular calcium. These results indicate that membrane ERα levels in N-38 neurons are dynamically autoregulated by estradiol.Steroids 01/2013; DOI:10.1016/j.steroids.2012.12.008 · 2.72 Impact Factor
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ABSTRACT: BACKGROUND: Prior to the introduction of tamoxifen, high dose estradiol was used to treat breast cancer patients with similar efficacy as tamoxifen, albeit with some undesirable side effects. There is renewed interest to utilize estradiol to treat endocrine resistant breast cancers, especially since findings from several preclinical models and clinical trials indicate that estradiol may be a rational second-line therapy in patients exhibiting resistance to tamoxifen and/or aromatase inhibitors. We and others reported that breast cancer patients bearing protein kinase C alpha (PKCalpha)- expressing tumors exhibit endocrine resistance and tumor aggressiveness. Our T47D:A18/PKCalpha preclinical model is tamoxifen resistant, hormone independent, yet is inhibited by 17beta-estradiol (E2) in vivo. We previously reported that E2-induced T47D:A18/PKCalpha tumor regression requires extranuclear ERalpha and interaction with the extracellular matrix. METHODS: T47D:A18/PKCalpha cells were grown in vitro using two-dimensional (2D) cell culture, three dimensional (3D) Matrigel and in vivo by establishing xenografts in athymic mice. Immunofluoresence confocal microscopy and co-localization were applied to determine estrogen receptor alpha (ERalpha) subcellular localization. Co-immunoprecipitation and western blot were used to examine interaction of ERalpha with caveolin-1. RESULTS: We report that although T47D:A18/PKCalpha cells are cross-resistant to raloxifene in cell culture and in Matrigel, raloxifene induces regression of tamoxifen-resistant tumors. ERalpha rapidly translocates to extranuclear sites during T47D:A18/PKCalpha tumor regression in response to both raloxifene and E2, whereas ERalpha is primarily localized in the nucleus in proliferating tumors. E2 treatment induced complete tumor regression whereas cessation of raloxifene treatment resulted in tumor regrowth accompanied by re-localization of ERalpha to the nucleus. T47D:A18/neo tumors that do not overexpress PKCalpha maintain ERalpha in the nucleus during tamoxifen-mediated regression. An association between ERalpha and caveolin-1 increases in tumors regressing in response to E2. CONCLUSIONS: Extranuclear ERalpha plays a role in the regression of PKCalpha overexpressing tamoxifen-resistant tumors. These studies underline the unique role of extranuclear ERalpha in E2- and raloxifene-induced tumor regression that may have implications for treatment of endocrine-resistant PKCalpha expressing tumors encountered in the clinic.Molecular Cancer 05/2013; 12(1):34. DOI:10.1186/1476-4598-12-34 · 5.40 Impact Factor