{"paper_id":"86ee9867-c8ec-4024-8d48-7efc70b15f17","body_text":"Castro et al. Biol Res  (2015) 48:56 \nDOI 10.1186/s40659-015-0047-2\nRESEARCH ARTICLE\nDifferential expression of upstream \nstimulatory factor (USF) 2 variants in eutopic \nendometria from women with endometriosis: \nestradiol regulation\nJazmin Castro1, Germán Araya1, Pamela Inostroza1, Paulina Hidalgo1, Reinaldo González‑Ramos1,2, \nHugo Sovino1,2, M. Angélica Boric1, Ariel Fuentes1,2 and M. Cecilia Johnson1*\nAbstract \nBackground: Endometriosis, pro‑inflammatory and invasive benign disease estrogen dependent, abnormally \nexpress in endometria the enzyme P450Arom, positively regulated by steroid factor‑1 (SF‑1). Our objective was to \nstudy the nuclear protein contents of upstream stimulating factor 2 (USF2a and USF2b), a positive regulator of SF‑1, \nthroughout the menstrual cycle in eutopic endometria from women with and without (control) endometriosis and \nthe involvement of nuclear estrogen receptors (ER) and G‑coupled protein estrogen receptor (GPER)‑1.\nResults: Upstream stimulating factor 2 protein contents were higher in mid (USF2b) and late (USF2a and USF2b) \nsecretory phase in eutopic endometria from endometriosis than control (p < 0.05). In isolated control epithelial cells \nincubated with E2 and PGE2, to resemble the endometriosis condition, the data showed: (a) significant increase of \nUSF2a and USF2b nuclear protein contents when treated with E2, PPT (specific agonist for ERα) or G1 (specific agonist \nfor GPER1); (b) no increase in USF2 binding to SF‑1 E‑Box/DNA consensus sequence in E2‑treated cells; (c) USF2 vari‑\nants protein contents were not modified by PGE2; (d) SF‑1 nuclear protein content was significantly higher than basal \nwhen treated with PGE2, E2 or G1, stimulation unaffected by ICI (nuclear ER antagonist); and (e) increased (p < 0.05) \ncytosolic protein contents of P450Arom when treated with PGE2, E2, PPT or G1 compared to basal, effect that was addi‑\ntive with E2 + PGE2 together. Nevertheless, in endometriosis cells, the high USF2, SF‑1 and P450Arom protein contents \nin basal condition were unmodified.\nConclusion: These data strongly suggest that USF2 variants and P450Arom are regulated by E2 through ERα and \nGPER1, whereas SF‑1 through GPER1, visualized by the response of the cells obtained from control endometria, being \nunaffected the endogenously stimulated cells from endometriosis origin. The lack of E2 stimulation on USF2/SF‑1 \nE‑Box/DNA‑sequence binding and the absence of PGE2 effect on USF2 variants opposite to the strong induction \nthat they exert on SF1 and P450 proteins suggest different mechanisms and indirect regulations. The sustained USF2 \nvariants protein expression during the secretory phase in eutopic endometria from women with endometriosis may \nparticipate in the pathophysiology of this disease strongly associated with infertility and its characteristic endometrial \ninvasion to ectopic sites in the pelvic cavity.\nKeywords: USF2, GPER1, Endometriosis, Eutopic endometrium, Estrogen receptor, Estrogen receptor specific \nagonists, SF‑1, P450Arom\n© 2015 Castro et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License \n(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, \nprovided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, \nand indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/\npublicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.\nOpen Access\n*Correspondence:  cjohnson@med.uchile.cl \n1 Faculty of Medicine, Institute of Maternal and Child Research, University \nof Chile, P .O. Box 226‑3, Santiago, Chile\nFull list of author information is available at the end of the article\n\nPage 2 of 11Castro et al. Biol Res  (2015) 48:56 \nBackground\nEndometriosis is an estrogen-dependent gynecologic dis -\nease, characterized by the presence and growth of endo -\nmetrium outside the uterine cavity. This pathology affects \nabout 10  % of reproductive-age women and is associated \nwith infertility, chronic pelvic pain, dysmenorrhea, and \ndyspareunia [1– 4]. The etiology of this disease remains \nincompletely understood [5 ]. Retrograde menstruation \nwith viable endometrial fragments has been advocated \nas one of the mechanisms by which the endometrium \nreaches the peritoneal cavity [6]. However, this theory fails \nto explain why only a select group of women experiencing \nretrograde menstruation develops endometriosis [7].\nEndometrial estrogenic microenvironment has been \nshown to be an important factor in the pathophysiology \nof endometriosis by abnormal expression of enzymes \ninvolved in estrogen synthesis and degradation [8, 9]. The \nactivation of CYP19A1 gene induces P\n450Arom expres -\nsion, the rate-limiting enzyme in conversion of androgens \nto estrogens. Normally, steroid factor-1 (SF-1) positively \nregulates the CYP19A1 gene in the ovary, though not in \nthe normal endometrium. Nevertheless, SF-1, expressed \nin eutopic and ectopic endometria from women with \nendometriosis, aberrantly activates CYP19A1 and the \nexpression of P\n450Arom in stroma [10–12] or gland [13, \n14] in these tissues as has been extensively described \nfavoring this estrogenic microenvironment in this \ndisease.\nSF-1 gene is recognized in a region called E-box by \nupstream stimulatory factor (USF), the ubiquitous tran\n-\nscription factor involved on embryonic development, \nfertility, stress, growth and lipid and carbohydrate metab\n-\nolisms [15, 16]. Although two types of USF, USF1 and \nUSF2, have been reported, it is USF2 that shows the high-\nest binding activity on SF-1 promoter and its knockdown \nresults in down-regulation of SF-1 and also of its target \ngene CYP19A1 in ectopic endometrium from endome\n-\ntriosis women [17]. Two variants of USF2, produced by \nalternative splicing, have been reported, the bigger USF2a \n(44 kDa) and the smaller USF2b (38 kDa) by the loss of 67 \ninternal amino acid in the N-terminal domain [15, 18].\nEstradiol (E\n2) acts through the classic nuclear estrogen \nreceptors (ER), ERα and ERβ, both strictly regulated by \nthe ovarian steroid hormones during the menstrual cycle, \nwith a predominance of ERα over ERβ in the normal \nendometrium and reducing their expression during the \nsecretory phase [19–22]. In eutopic endometrium from \nwomen with endometriosis, although each ER isoforms \nare increased, the ERα/ERβ ratio is decreased affecting \nthe normal actions of E\n2 in this tissue [23, 24]. In addi -\ntion, a membrane receptor called G-protein estrogen \nreceptor 1 (GPER1) presents high affinity for E\n2 in vitro \n[25, 26] and has been described as potentially responsible \nof early and non genomic responses of estrogen in several \ncell lines and tissues including the endometrium [27–29].\nThe aberrant expression of CYP19A1 and SF-1 genes in \neutopic and ectopic endometria of women with endome\n-\ntriosis led us to study USF2 variants protein contents in \nhuman endometrium throughout the menstrual cycle and \nthe effect of estrogenic and proinflammatory environments \nin epithelial cells of eutopic endometrium from women with \nand without endometriosis. The involvement of nuclear \nERα, ERβ or GPER1 in the E\n2 action on USF2 variants, SF-1 \nand P450Arom protein expression was also evaluated.\nResults\nUSF2 protein contents in endometrium throughout the \nmenstrual cycle\nThe protein content of both USF2a and USF2b variants \nwere detected by immunoblot (Fig.  1) in endometrium \nfrom women with endometriosis and controls. No inter -\nactions between USF2 protein studies and subject age \nwas found by ANCOVA.\nTwo protein bands (44 and 38 kDa), corresponding to \nUSF2a and USF2b variants, respectively, were found in \nthe nuclear compartment of control and endometrio\n-\nsis endometria (Fig.  1). In control endometria, nuclear \nUSF2a protein content decreased in early and late \nsecretory phases (37 and 57 %, respectively, p < 0.05) as \ncompared to the proliferative phase. On the other hand, \nin endometriosis, USF2a contents were lower during the \nlate secretory as compared to the mid secretory phase, \nalthough higher than late control endometria (Fig. 1 a).\nDuring mid and late secretory phases, USF2b (38 kDa) \nnuclear protein content significantly decreased (39 and \n34 %, respectively) as compared to proliferative phase in \ncontrol endometria, instead it was observed an increase \nin endometriosis endometria during the same stages of \nthe menstrual cycle, being higher 100 and 155 %, respec\n-\ntively, than control (Fig. 1b).\nStimulatory effect of E2 and PGE2 on USF2, SF‑1 \nand P450Arom protein contents\nIn endometriosis epithelial cells, nuclear protein contents \nof USF2a and USF2b were significantly higher in basal con\n-\ndition than control cells. Nevertheless, only in control cells, \nE\n2 increased USF2a (103 %) and USF2b (91 %) nuclear pro-\ntein contents, effect also partially blocked by the presence \nof ICI (Fig.  2a, b). Nuclear protein homogenate obtained \nfrom control and endometriosis epithelial cells bound to \ntarget E-Box motif, complexes displaced by cold probe. \nThe previous incubation of the nuclear protein homogen\n-\nates with USF2 antibody shifted partially the protein/E-Box \ncomplex in basal or E2-treated conditions (Fig. 2c).\nWe observed a null effect on USF2a and USF2b nuclear \nprotein contents of 10\n−8 mol/L PGE 2, in the presence or \n\nPage 3 of 11\nCastro et al. Biol Res  (2015) 48:56 \nabsence of 10−8 mol/L E2 in isolated epithelial cells from \nboth control and endometriosis endometria (Fig. 2a, b).\nEpithelial cells obtained from endometriosis endome -\ntria had high SF-1 protein expression in basal condition, \nwhich were resistant to E 2 and PGE 2 (Fig.  3a). On the \ncontrary, in control epithelial cells, the SF-1 protein con -\ntent was strongly increased by E 2 (126  %) as compared \nto basal, effect not modified by the presence of ICI. The \npresence of PGE\n2 also increased the content of SF-1 pro -\ntein (154 %) as compared to basal, although no additive \nor synergistic effects were observed when E 2 and PGE 2 \nwere added together (Fig. 3a).\nSimilarly to SF-1 protein results, the cytosolic protein \ncontent of P 450Arom was strongly high in epithelial cells \nfrom endometriosis endometria in basal condition, and \nalso resistant to E\n2 and PGE2 presences (Fig.  3b). In con-\ntrol epithelial cells, P450Arom protein content was signifi-\ncantly increased by E 2 (292 %), effect partially blocked by \nICI pre-treatment. PGE 2 increased (258  %) the protein \ncontent of P 450Arom and the presence of both, E 2 and \nFig. 1 USF2a (a) and USF2b (b) nuclear protein contents in eutopic endometria throughout the menstrual cycle. Endometria were obtained \nfrom seven women without (control) and seven women with endometriosis in each stage of the menstrual cycle. Representative immunoblots \nare shown. Data were normalized with TFIIB protein contents. Results are the mean ± SEM. *p < 0.05 vs. control; #p < 0.05 vs. proliferative phase; \nºp < 0.05 vs. late secretory phase\n\nPage 4 of 11Castro et al. Biol Res  (2015) 48:56 \nFig. 2 E 2 and PGE2 effect on USF2 variants protein contents and SF‑1 E‑Box DNA binding. USF2a (a) and USF2b (b) nuclear protein contents of \nendometrial epithelial cells (EEC) from women with and without (control) endometriosis treated with E2 (10−8  mol/L) and/or PGE2 (10−8  mol/L) in \nthe presence or absence of ICI (10−6  mol/L; 30 min previously added); all data were normalized with TFIIB. Representative immunoblot is shown. \nResults are the mean ± SEM of EEC obtained from 7 control women and 7 women with endometriosis. c Representative SF‑1 E‑Box DNA shift assay \nfrom 3 gels. Lane 1 free probe; lanes 2–5 nuclear protein from control EEC (2 basal condition; 3 E2 treated; 4 basal + anti USF2 antibody; 5 E2 + anti \nUSF2 antibody); lanes 6 and 7: nuclear protein from endometriosis endometrial epithelial cells (6 basal condition; 7 basal + anti USF2 antibody), \nand lane 8 basal condition + cold competitor. Protein procurements and assays are described in “Methods” . *p < 0.05 control; \n#p < 0.05 vs. baseline; \nºp < 0.05 vs. E2\n\nPage 5 of 11\nCastro et al. Biol Res  (2015) 48:56 \nPGE2, shows an additive effect (525  %) on the protein \ncontent (Fig. 3b).\nSpecific agonists of estrogen receptors involved on USF2, \nSF‑1 and P450Arom protein content by E2 stimulation\nTaking into account that cells obtained from endometrio-\nsis women are highly endogenously stimulated, control \nepithelial cells were used for the following experiments to \nassess the ER isoform involvement. For that, control cells \nwere incubated with specific agonists for ERα (PPT), ERβ \n(DPN) and GPER1 (G1).\nThese cells responded to PPT at 10\n−7 mol/L and G1 at \n10−6  mol/L, increasing the protein content of USF2a by \n153 and 164 % and USF2b by 169 and 109 %, respectively \nFig. 3 E 2 and PGE2 effect on SF‑1 and P450Arom protein levels in endometrial epithelial cells. Isolated endometrial epithelial cells (EEC) obtained \nfrom 4 to 6 women with or without (control) endometriosis were treated for 24 h with E2 (10−8  mol/L) and/or PGE2 (10−8  mol/L) in the presence and \nabsence of ICI (10−6  mol/L; 30 min previously added). Representative immunoblots are shown. Data for SF‑1 (nuclear homogenates) were normal‑\nized with TFIIB (a) and for P450Arom (cytosol homogenates) with GAPDH (b). Results are the mean ± SEM. *p < 0.05 vs.control; #p < 0.05 vs. basal, \nºp < 0.05 vs. one treatment\n\nPage 6 of 11Castro et al. Biol Res  (2015) 48:56 \n(p < 0.05). The stimulatory effect of PPT was blocked by \nthe presence of ICI. Paradoxically, ICI alone increased \nboth USF2 variants. No significant effect was observed \nwith DPN (Fig. 4a, b).\nOnly G1 increased SF-1 nuclear protein content by \n250 % at 10\n−6 mol/L in the control cells (Fig. 5a). Similarly \nto SF-1, P450Arom cytosolic protein content was increased \nin a dose-dependent manner by G1 (242  %) and also by \nPPT (232 %) as compared to basal condition (Fig. 5b).\nDiscussion\nTo our knowledge, this is the first report on human \nendometrial USF2a and USF2b protein co-expression \nthroughout the menstrual cycle, and their positive regu\n-\nlation by E2 through ERα and GPER1.\nThe reduced nuclear protein content of USF2 vari -\nants during the late secretory phase in control endo -\nmetria is consistent with the decreased plasma E 2 and \nprogesterone levels during this period of the menstrual \ncycle. In contrast, eutopic endometria from endometrio\n-\nsis patients exhibited high USF2 variants protein con -\ntents during this stage coincidently with the estrogenic \nmicroenvironment described in the eutopic and ectopic \nendometria of these patients [10, 13, 14, 30–32]. On the \nother hand, the opposite expression of USF2a and USF2b \nfound in control endometria during the mid secretory \nphase, when the embryo implantation occurs, was not \nobserved in eutopic endometria from endometriosis \nwomen, which may contribute to the infertility associ\n-\nated to this pathology. The different expression patterns \nFig. 4 Dose‑response curves of specific agonists on USF2a (a) and USF2b (b) nuclear protein content. Endometrial epithelial cells obtained from 4 \ncontrol women were treated for 24 h with E2 (10−8  mol/L), PPT (10−9  to 10−7  mol/L), DPN (10−9  to 10−7  mol/L), and G1 (10−8  to 10−6  mol/L) in the \npresence or absence of ICI (10−6  mol/L; 40 min previously added). Representative immunoblot is shown. Data were normalized with TFIIB. Results \nare the mean ± SEM of EEC obtained from at least 4 control women. *p < 0.05 vs. basal; #p < 0.05 vs. agonist\n\nPage 7 of 11\nCastro et al. Biol Res  (2015) 48:56 \nof endometrial USF2 variants between women with and \nwithout endometriosis throughout the menstrual cycle \nadd new molecules to those abnormally expressed in this \ntissue as has been widely reported [33–36]\nWe observed a strong E\n2-stimulatory effect on USF2 \nvariants nuclear protein contents in epithelial cells from \ncontrol endometria. These findings are supported by the \nepithelial cells response to ICI, an antagonist of ERα and \nERβ, which partially blocked those effects induced by \nE\n2, but completely blocked those induced by PPT (spe -\ncific agonist of ERα) and acting as agonist for GPER1, \nunaffected those effects induced by G1 (specific agonist \nFig. 5 Dose‑response curves of specific agonists on SF‑1 (a) and P450Arom (b) protein content. Endometrial epithelial cells obtained from 4 control \nwomen were treated with E2 (10−8  mol/L), PPT (10−9  to 10−7  mol/L), DPN (10−9  to 10−7  mol/L), and G1 (10−8  to 10−6  mol/L). Representative immu‑\nnoblots are shown. Data for SF‑1 (nuclear homogenates) were normalized with TFIIB and for P450Arom (cytosol homogenates) with GAPDH. Results \nare the mean ± SEM. *p < 0.05 vs. basal\n\nPage 8 of 11Castro et al. Biol Res  (2015) 48:56 \nof GPER1), confirming the dual action of ICI on estro -\ngen receptors [37]. These data suggest that this process \nis under ovarian steroid regulation through the classic \nnuclear ERα and also GPER1. Interestingly, positive regu\n-\nlation of the ERα expression by USF2 has been reported \nin sheep uterine arteries [32, 38] showing a complex rela\n-\ntionship between both transcription factors. The action \nof E\n2 through GPER1 not only may be involved on the \nUSF2 protein synthesis or viability, but also on the USF2 \nactivation through several pathways described for GPER1 \n[25, 39–41]. This aspect is of high relevance consider\n-\ning the important role, beside the cell-specificity, that \nspecific phosphorylation plays on the activation of USF \nprotein that modifies its function from tumor suppressor \nin prostate cancer to tumor promoter in lung cancer and \nthyroid cancer as recently Horbach et  al. reported [15, \n42].\nOur first hypothesis was that the strong increase of \nUSF2 induced by E\n2 might stimulate the SF-1 transcrip -\ntion activating the E-box motif, which in turn may induce \nCyp19A1 gene increasing the key enzyme P\n450Arom. \nHowever, the discordance between the high USF2 pro -\ntein expression and the weak binding observed on SF-1 \nE-Box DNA consensus studies suggests a partial effect \nof USF2 on SF-1 gene promoter in cell treated with E\n2. \nNevertheless, more studies are needed to confirm or to \ndiscard this pathway. Furthermore, the proinflammatory \nenvironment, generated by PGE\n2, was unable to modify \nUSF2 variants protein, although induced a strong stimu -\nlation on SF-1 and P450Arom protein contents, indicating \ndifferent regulations.\nThe up-regulation of P 450Arom by PGE 2 through \ncAMP/CREB signaling pathway was previously reported \n[1, 11, 12, 43]. The additive effect of E\n2 and PGE 2 on \nP450Arom protein contents indicates different activation \nmechanisms. Similar additive effect we reported pre -\nviously in isolated control epithelial cells treated with \nperitoneal fluid from endometriosis women (PF-E) and \nBu\n2cAMP [44] mimicking the conditions of the endo -\nmetriotic lesions. In our control epithelial cell model, \nP\n450Arom stimulation by E2 was through ERα and GPER1, \nbut not through ERβ as it was previously proposed [1, 45] \nprobably by the use of isolated control epithelial cells and \nnot endometriotic stromal cells. It is known the impor\n-\ntant role of SF-1 on steroid hormone biosynthesis, and \nalso on development, differentiation, and function of the \nendocrine tissues [46]. The non-classic receptor GPER1 \nmediating the E\n2 stimulatory action on SF-1 protein con -\ntent as shown by our G1 data, is in agreement with SF-1 \nactivation and endometrial cell proliferation through the \nPI3K and MAPK pathways activated in several cell lines \ntransfected with GPER1 [39, 40]. However, cAMP path\n-\nway cannot be ruled out according to similar response to \n(Bu)2cAMP of control or SF-1-transfected endometrial \nepithelial cells as we previously reported [44].\nIn the present study, control epithelial cells were sen -\nsitive to E 2 and/or PGE 2 treatments, mimicking the \nestrogenic and pro-inflammatory microenvironment \ndescribed in endometriosis, inducing abnormal mol\n-\necule expression similarly to endometria from women \nwith endometriosis as has been widely reported by sev\n-\neral authors including our own group [14, 33–36, 47]. \nVery little information are regarding USF2, and even less \nabout USF2 variants. Our data of sustained USF2 protein \nexpression during the secretory phase in eutopic endo\n-\nmetria of women with endometriosis, an invasive estro -\ngen-dependent disease, and the fact that the USF2 action \nis cell specific and may change its function from tumor \nsuppressor to tumor promoter with invasive character\n-\nistics [15, 42], suggest that USF2 may be involved in the \npathophysiology of the endometriosis.\nConclusions\nTo our knowledge, this is the first report that shows USF2 \nvariants protein expression patterns in human normal \nand pathologic endometria during the menstrual cycle \nand its E\n2 stimulation mediated by ERα and GPER1 vis -\nualized by the response of cells obtained from control \nendometria, being unaffected the endogenously stimu\n-\nlated cells from endometriosis origin. The lack of E2 stim-\nulation on USF2/SF-1 E-Box/DNA-sequence binding and \nthe absence of PGE\n2 effect on USF2 variants opposite to \nthe strong induction that they exert on SF1 and P450 pro-\nteins suggest different mechanisms and regulations. The \nsustained USF2 protein expression during the secretory \nphase in eutopic endometria of women with endometrio\n-\nsis may participate in the pathophysiology of this disease \nstrongly associated with infertility and its characteristic \nendometrial invasion to ectopic sites in the pelvic cavity.\nMethods\nSubjects\nEutopic endometrium was obtained from 37 women \nundergoing diagnostic laparoscopy for endometriosis \nassociated with pain and/or infertility (endometriosis \ngroup), and 49 women without endometriosis undergo\n-\ning laparoscopy for tubal ligation or hysterectomy for a \nbenign non-endometrial gynecologic condition (control \ngroup) in the Clinical Hospital San Borja-Arriarán. The \nage of these women was 33.9 ± 5.6 years for the endome\n-\ntriosis group and 36.7  ± 6.5 years for the control group \n(p < 0.05). Both groups of women had abstained from any \nhormonal treatment for at least 3  months prior to sur\n-\ngery. Endometrial biopsies were obtained during surgery \nwith Cornier pipelle suction curettage from the corpus \nof the uterus, kept in cold sterile phosphate buffer saline \n\nPage 9 of 11\nCastro et al. Biol Res  (2015) 48:56 \n(PBS), and transported to the laboratory at 4  °C. One \npiece of the tissue was fixed in formalin for histological \nevaluation, others pieces were frozen for protein studies \nor used for endometrial epithelial cells isolation.\nThe endometriosis grade was 49 % minimal-mild (score \n1–15 points) and 51  % moderate-severe (score ≥16 \npoints) according to American Society of Reproductive \nMedicine criteria [48]. Endometriosis was diagnosed \nduring surgery by visual evaluation by an experimented \nsurgeon in each patient. This study was approved by the \nethical committees of Faculty of Medicine of University \nof Chile and Metropolitan Central Health Service of \nChile; each patient signed a written informed consent \nbefore surgery.\nEndometrial samples were dated according to Noyes \ncriteria [49] and classified as proliferative (days 6–14; \n12 control and 9 endometriosis samples) phase or early \n(days 15–18; 12 control and 10 endometriosis), mid (days \n19–23; 12 control and 10 endometriosis), and late secre\n-\ntory phase (days 24–28; 13 control and 8 endometriosis).\nCell culture\nSecretory endometrium was washed in PBS, minced, and \ndigested according to previous indication [50]. The glands \nwere separated and cultured according to previous indi\n-\ncation [44, 50] and after the first or second passage, the \ncells were reseeded in duplicate protein studies until sub-\nconfluence. Then, the cells were incubated in fetal bovine \nserum-free medium (defined-medium) for 24  h, and \ntreated for another 24  h in fresh defined-medium with\n-\nout (basal) or with prostaglandin E 2 (PGE 2, 10−8  mol/L; \nSigma), or E 2 (10−8  mol/L; Sigma). Increasing concentra-\ntions of Propylpyrazole-triol (PPT, 10 −9 to 10 −7  mol/L, \nTocris  Bioscience, Bristol, UK) and Diarylpropionitrile \n(DPN, 10\n−9 to 10−7 mol/L, Tocris), specific agonists of ERα \nand ERβ, respectively, or G1 (10 −8 to 10−6  mol/L, Merck \nKGaA, Darmstadt, Germany) specific agonist of GPER1, \nwere also added to cell cultures for 24 h in the presence or \nabsence of ICI-182,780 (10\n−6 mol/L, ERα and ERβ antago-\nnist; Tocris) added 40 min before of ER agonists.\nProtein homogenate preparation\nCytosolic and nuclear proteins from endometrial pieces \nand epithelial cells were obtained as previously reported \n[47]. The protein concentration was determined using \nthe Bradford Assay reagent (BioRad, Hercules, CA, USA).\nThirty μg of cytosolic and nuclear proteins were dena\n-\ntured, resolved in 10 % PAGE-SDS, and electrotransferred \ninto nitrocellulose membranes (BioRad) as previously \nindicated [44, 50]. After blocking with 5  % BSA, the \nmembranes were incubated overnight at 4  °C with pri\n-\nmary antibodies against USF2 (polyclonal, 1:800; Abcam \nInc, Cambridge, MA, USA), SF-1 (polyclonal, 1:800; ABR \nAffinity BioReagents, Golden, CO., USA), P\n450Arom \n(monoclonal; 1:600; Serotec, Oxford, UK), TFIIB (mon -\noclonal, 1:500; BD Biosciences, MD, USA), or GAPDH \n(polyclonal; 1:5000; Abcam). The images were captured \nwith Discovery10gD (Ultralum, Claremont, CA, USA) \nusing UltraQuant 6.0.0.344 software, analyzed with \nCarestreamMI5.0.6.20 software (Carestream Health, \nInc., Rochester, NY, USA). The results were normalized \nwith GAPDH or TFIIB analysis for cytosolic or nuclear \nextracts, respectively.\nSF‑1 E‑Box DNA shift assay\nThe assay was performed using LightShift Chemilu -\nminescent EMSA kit (Thermo Scientific, Rockford, IL, \nUSA). Briefly, 5 µg nuclear proteins obtained as described \nabove were incubated during 20  min at room tempera\n-\nture in a reaction mix which included 20 fmol biotin end-\nlabeled oligonucleotides that represented the SF-1 gene \npromoter containing the E-box (Integrated DNA Tech\n-\nnologies, Inc., Coralville, IO, USA) following the manu -\nfacturer’s indications and as described Utsunomiya et al. \n[17] For supershift study, nuclear proteins were previ\n-\nously incubated with 1 µg USF2 antibody (Abcam) during \n2 h at 4 °C. The samples were resolved in non denaturing \n4  % polyacrylamide gel, electrotransferred to biodyne-\nB membrane (Pall Corporation, Port Washington, NY, \nUSA), which was UV-light crosslinked (UVP HL-2000 \nHybriLinker, Cambridge, UK), blocked and the label \ndetected following the manufacturer’s indications (Ther\n-\nmos). The images were captured with Discovery10gD \nusing UltraQuant 6.0.0.344 software.\nStatistical analysis\nResults are expressed as mean  ±  SEM. Kolmogorov–\nSmirnov test was used to evaluate normal distribution. \nWhen non-parametric distribution was present Mann–\nWhitney or Kruskal–Wallis tests were used, followed by a \nDunn test. Means were expressed as percent of increase. \nAnalysis of covariance (ANCOVA) was employed to \ntest statistical interaction with co-variables like age and \nphases of the menstrual cycle.\nAbbreviations\nUSF2: upstream stimulatory factor; SF‑1: steroid factor‑1; GPER1: G‑coupled \nprotein estrogen receptor 1; P450Arom: aromatase enzyme; PPT: 4,4′4″‑(4‑Pro‑\npyl‑[1H]‑pyrazole‑1,3,5‑triyl)trisphenol; DPN: 2.3‑bis(4‑Hydroxyphenyl)‑\npropionitrile; ICI 182,780: 7α,17β‑[9[4,4,5,5,5‑Pentafluoropentyl]sulfinyl]nonyl]\nestra‑1,3,5(10)‑triene‑3,17‑diol; G1: 1‑(4‑(6‑Bromobenzo[1,3]dioxol‑5‑yl)‑\n3a,4,5,9b‑tetrahydro‑3H‑cyclopenta[c]quinolin‑8‑yl)‑ethanone; E\n2: estradiol; \nPGE2: prostaglandin E2; ER: estrogen receptor.\nAuthors’ contributions\nJC participated in experimental protocols and drafted the manuscript. GA \ncarried out cell cultures and EMSA/SS studies and in the analysis of data. PI \nand PH carried out cell cultures and immunoblots studies and the analysis \nof data. RG‑R diagnosed the endometriosis during surgery, participated in \nthe interpretation of data and helped reviewing the manuscript critically. \n\nPage 10 of 11Castro et al. Biol Res  (2015) 48:56 \nHS diagnosed the endometriosis during surgery and participated in the \ninterpretation of data. MAB participated in the interpretation of data, helped \nto draft the article and reviewed the manuscript critically. AF carried out the \nstatistical analysis and helped in the interpretation of data and reviewing the \nmanuscript critically. MCJ conceived and design the study as principal investi‑\ngator; participated in the analysis and interpretation of data and drafting the \nmanuscript. All authors read and approved the final manuscript.\nAuthor details\n1 Faculty of Medicine, Institute of Maternal and Child Research, University \nof Chile, P .O. 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