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: 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
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ABSTRACT: Recent studies indicated that both estren and Rg1 appear to be able to activate mitogen-activated protein kinase (MAPK) pathway in estrogen responsive cells. Rg1 could lead to MAPK activation through ligand-independent activation of estrogen receptor (ER), while estren could activate the Src-MAPK pathway in an ERE-independent manner. Thus, it is important to understand the mechanistic insights on the difference in transcriptional activation between estren and Rg1. The present study also addressed the differential abilities of Rg1 and estren in terms of the ability to activate ER and the ability to induce ER translocation in MCF-7 cells. Our data indicated that Rg1 could increase pS2 gene expression, and could recruit the co-activator steroid receptor co-activator-1 (SRC-1) to the pS2 promoter. Rg1 could also induce ERα nuclear translocation as well as ERα phosphorylation at Ser18 principally in the cytoplasm in MCF-7 cells. We deduced that estren induced ERE-dependent transcriptional activity and activated ERα at Ser18 occurred in the nucleus of MCF-7 cells. However, it was found to decrease pS2 gene expression and failed to induce the recruitment of SRC-1 to the pS2 promoter in MCF-7 cells. Our results suggest that the abilities of Rg1 and estren to regulate pS2 gene expression, to recruit co-activators as well as to induce sub-cellular distribution of ERα are dramatically different.The Journal of steroid biochemistry and molecular biology 05/2014; DOI:10.1016/j.jsbmb.2014.01.014 · 4.05 Impact Factor
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ABSTRACT: In the present work we investigated the effect of 17β-estradiol (E2) and basic fibroblast growth factor (FGF2) on the lactotroph cell proliferative response and the related membrane-initiated signalling pathway. Anterior pituitary mixed cell cultures of random cycling 3-month-old female rats were treated with 10nM of E2, E2 membrane-impermeable conjugated (E2-BSA), PPT (ERα agonist) and DPN (ERβ agonist) alone or combined with FGF2 (10ng/ml) for 30min or 4h. Although our results showed that the uptake of BrdU into the nucleus of lactotrophs was not modified by E2 or FGF2 alone, a significant increase in the lactotrophs uptake of BrdU was observed after E2/FGF2 co-incubation, with this effect being mimicked by PPT/FGF2. These proliferative effects were blocked by ICI182780 or PD98059. The involvement of membrane ER in the proliferative response of prolactin cells induced by the steroid and FGF2 co-incubation was confirmed using E2-BSA, and the association between ERα and FGF receptor was observed after E2/FGF2 treatment by immunoprecipitation. A significant increase in the ERK1/2 expression was noted after E2, E2-BSA, PPT and FGF2 alone, which was more noticeable after E2-BSA/FGF-2, E2/FGF2 or PPT/FGF2 treatments. This study provides evidence that E2 and FGF2 exert a cooperative effect on the lactotroph proliferation principally by signalling initiated at the plasma membrane triggering a genomic effect mediated by MEK/ERK1/2, a common signalling pathway, which finally regulates the lactotroph population thus contributing to pituitary plasticity.AJP Endocrinology and Metabolism 05/2013; DOI:10.1152/ajpendo.00027.2013 · 4.09 Impact Factor