Estrogen Receptor ESR1 Mediates Activation of ERK1/2, CREB, and ELK1 in the Corpus of the Epididymis

F N CAVALCANTI and others ESR1 activates ERK1/2, CREB, and 54:3 339–349 Research ELK1

Estrogen receptor ESR1 mediates activation of ERK1/2, CREB, and ELK1 in the corpus of the epididymis

Correspondence Fernanda N Cavalcanti, Thais F G Lucas, Maria Fatima M Lazari and Catarina S Porto should be addressed to C S Porto Section of Experimental Endocrinology, Department of Pharmacology, Escola Paulista de Medicina, Email Universidade Federal de Sa˜ o Paulo, Rua Treˆ s de maio 100, INFAR, Vila Clementino, Sa˜ o Paulo, [email protected] or Sa˜ o Paulo 04044-020, Brazil [email protected]

Abstract

Expression of the estrogen receptor ESR1 is higher in the corpus than it is in the initial Key Words segment/caput and cauda of the epididymis. ESR1 immunostaining in the corpus has been " ESR1 localized not only in the nuclei but also in the cytoplasm and apical membrane, which " ERK1/2 indicates that ESR1 plays a role in membrane-initiated signaling. The present study " CREB investigated whether ESR1 mediates the activation of rapid signaling pathways by estradiol " ELK1

(E2) in the epididymis. We investigated the effect of E2 and the ESR1-selective agonist " epididymis (4,40,400-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol (PPT) on the activation of extracellular signal-regulated protein kinases (ERK1/2), CREB protein, and ETS oncogene-related protein (ELK1). Treatment with PPT did not affect ERK1/2 phosphorylation in the cauda, but it rapidly

Journal of Molecular Endocrinology increased ERK1/2 phosphorylation in the initial segment/caput and corpus of the epididymis. PPT also activated CREB and ELK1 in the corpus of the epididymis. The PPT-induced phosphorylation of ERK1/2, CREB, and ELK1 was blocked by the ESR1-selective antagonist MPP and by pretreatment with a non-receptor tyrosine kinase SRC inhibitor, an EGFR kinase inhibitor, an MEK1/2 inhibitor, and a phosphatidylinositol-3-kinase inhibitor. In conclusion, these results indicate that the corpus, which is a region with high expression of the estrogen

receptor ESR1, is a major target in the epididymis for the activation of rapid signaling by E2.

The sequence of events that follow E2 interaction with ESR1 includes the SRC-mediated transactivation of EGFR and the phosphorylation of ERK1/2, CREB, and ELK1. This rapid estrogen signaling may modulate gene expression in the corpus of the epididymis, and it Journal of Molecular may play a role in the dynamic microenvironment of the epididymal lumen. Endocrinology (2015) 54, 339–349

Introduction

The epididymis plays an important role in sperm of these segments possesses a distinct pattern of gene transport, maturation, protection, and storage. In expression related to the physiological functions of rodents, this organ can be functionally divided into the epididymis (Johnston et al. 2005, Henderson et al. four regions: the initial segment, the caput, the corpus, 2006, Guyonnet et al. 2009; reviewed by Belleanne´e and the cauda (Turner 1995, Turner et al. 2003). Each et al. 2012).

http://jme.endocrinology-journals.org Ñ 2015 Society for Endocrinology Published by Bioscientiﬁca Ltd. DOI: 10.1530/JME-15-0086 Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 01:26:57AM via free access Research F N CAVALCANTI and others ESR1 activates ERK1/2, CREB, and 54:3 340 ELK1

Epididymal development and physiology are regu- & Gustafsson 2011), this steroid also mediates rapid effects lated by a complex interplay of hormones and testicular that occur within seconds or minutes. These rapid effects factors (lumicrine factors) (Robaire & Viger 1995, may be mediated by: i) ESR1 and ESR2 localized at or near Hinton et al. 1998, Hess et al. 2011). Androgens and the plasma membrane after exposure to ligand (reviewed lumicrine factors have been extensively studied, and their by Song & Santen 2006, Acconcia & Marino 2011 and Levin contribution to the regulation of epididymal function is 2014); ii) a variant of ESR1 called ERa-36 (Wang et al. 2005, indubitable (Robaire & Viger 1995, Hinton et al. 1998). 2006); and/or iii) GPER (reviewed by Lucas et al. 2011 and Androgens and lumicrine factors mainly influence the Prossnitz & Barton 2014). These rapid responses include the expression of genes specific to the initial segment and activation of different downstream signaling pathways, for caput, and they are less efficient in the more distal corpus example, the MAPK (ERK1/2) and phosphatidylinositol-3- and cauda regions of the epididymis (Lan et al. 1998, Sipila¨ kinase (PI3K) pathways, which in turn modulate nuclear et al. 2006). Therefore, other factors (i.e., estrogens, transcriptional events in a variety of cells (reviewed by temperature, pressure, and small RNAs) may contribute to Lucas et al. 2011, Levin 2014 and Prossnitz & Barton 2014). the regulatory mechanisms found in the epididymis These protein kinases play a key role in cell growth, (reviewed by Belleanneé et al. 2012). differentiation, and function at both the transcriptional Estrogen biosynthesis may occur not only in the and the post-transcriptional levels by phosphorylating a luminal sperm (Hess et al. 2011) but also in the epididymal range of proteins, including nuclear transcription factors, epithelium (Pereyra-Martinez et al. 2001, Carpino et al. such as CREB protein and ETS-oncogene-related protein 2004, Shayu & Rao 2006, Joseph et al. 2011). Cytochrome (ELK1), cytoskeletal proteins, other protein kinases, and P450 aromatase has been shown to be present in the caput receptors for hormones and growth factors. However, it is epithelium and interstitium of the epididymis of the mouse not known whether the ERK1/2 (MAPK3/1) and PI3K

(Joseph et al. 2011), rat, human, and monkey (Pereyra- pathways are activated by the rapid action of E2 in the Martinez et al.2001, Carpino et al.2004). Estrogen epididymis and whether they play a role in the activation of sulfotransferase has also been shown to be present in the transcription factors, such as CREB and ELK1. epithelium and intraluminal ﬂuid of the epididymis (Tong In the present study we therefore investigated whether

& Song 2002, Frenette et al. 2009, Hoffmann et al. 2010), ESR1 mediates the E2-induced activation of rapid signaling and it may control the pool of bioactive luminal estrogens pathways in the epididymis. in this organ (Frenette et al. 2009). The classical estrogen receptors ESR1 and ESR2 (also

Journal of Molecular Endocrinology known as ERa and ERb respectively) and the G-protein- Materials and methods coupled estrogen receptor (GPER, GPR30) are present in Animals and tissue preparation the epididymis (Hess et al. 2011). Knockout of ESR1 K K (Esr1 / ) in the male mouse leads to the misregulation Ninety-day-old male Wistar rats were born and housed in of acid/base transporters, failure of epididymal acidifica- the Animal Facility at the Instituto Nacional de tion, and infertility (Joseph et al. 2010a). Spermatozoa Farmacologia e Biologia Molecular (INFAR), Escola recovered from the epididymides of these animals exhibit Paulista de Medicina, Universidade Federal de Saõ Paulo abnormal flagellar coiling and increased incidence of (EPM-UNIFESP), and they were maintained on a 12 h spontaneous acrosome reactions (Joseph et al. 2010b). light:12 h darkness schedule at 23 8C and were allowed Whereas ESR2 expression does not change among the to feed and drink water ad libitum. The experimental epididymal regions in adult rats, the expression of ESR1 is procedures were approved by the Research Ethical higher in the corpus as compared with the initial segment/ Committee at EPM-UNIFESP (no. 2043/11). Epididymides caput and cauda, which indicates that the effects of were dissected and sectioned into three regions: initial estrogen may vary among epididymal regions (Hess et al. segment/caput, corpus, and cauda (Turner et al. 1990, 2011) and may contribute to regional differences in gene Turner 1995). For the removal of sperm and luminal fluid, expression. Furthermore, cytoplasmic and apical mem- each region was cut into pieces (approximately 1 mm3), brane immunostaining for ESR1 have been observed in the which were placed in six-well dishes and washed three corpus region (Hess et al. 2011), which indicates that ESR1 times with nutrient solution (Vreeburg et al. 1992, Siu et al. plays a role in membrane-initiated signaling. 2006, Gomes et al. 2011) with the following composition:

In addition to the well-established transcriptional 136.89 mM NaCl, 5.63 mM KCl, 1.80 mM CaCl2, 0.36 mM

effects of E2 (reviewed by Heldring et al. 2007, and Nilsson NaH2PO4, 14.88 mM NaHCO3, and 5.55 mM glucose (pH

7.6–7.8; Sigma Chemical Co.), bubbled with air at 30 8C. centrifuged at 1610 g for 30 min at 4 8C. The supernatant Each region of the left epididymis was incubated in was separated, and the protein concentration was nutrient solution and used as a control (incubation with determined with the Bio-Rad protein assay using BSA as vehicle), and each region of the right epididymis was the standard (Bio-Rad Laboratories, Inc.). Total proteins

incubated with E2 (Sigma Chemical Co., 10 nM) or the (5 mg/lane) were incubated with sample buffer containing ESR1-selective agonist PPT (4,40,400-(4-propyl-(1H)-pyra- b-mercaptoethanol and subjected to 10% SDS–PAGE. zole-1,3,5-triyl)trisphenol, Tocris Bioscience (Ellisville, Proteins were electrotransferred onto PVDF membranes MI, USA), 100 nM) for 5–30 min. In another series of (pore size 0.45 mm, ImmobilonP, Millipore, Bedford, experiments, the effect of PPT (100 nM, 5 min) was MA, USA) using 20 V overnight at 4 8C. Membranes were analyzed after pretreatment or no pretreatment with the blocked in Tris-buffered saline (TBS; 10 mM Tris, 150 mM ESR1-selective antagonist MPP (1,3-bis[4-hydroxyphenyl]- NaCl) containing 0.1% Tween 20 (TBST) and 5% nonfat dry 4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole milk (pH 7.6) for 2 h at room temperature. After washes in dihydrochloride, Tocris Bioscience, 100 nM, 60 min), the TBST, membranes were incubated overnight at 4 8C with a non-receptor tyrosine kinase SRC inhibitor PP2 (AG 1879, rabbit polyclonal antibody raised against a synthetic 4 amino-5-[4-chlorophenyl]-7-(t-buthyl)pyrazolo[3,4- peptide (KLH-coupled) derived from the sequence of the d]pyrimidine, Calbiochem, Merck Biosciences (Darmstadt, Germany), 2.5 mM, 30 min), the EGFR kinase inhibitor 8.0 A C EGF AG1478 ([4-(3-chloroanilino)-6,7-dimethoxyquinazo- p-ERK1 44 kDa 6.0 * p-ERK2 42 kDa line], Calbiochem, 25 mM, 30 min), the MEK1/2 inhibitor 4.0 * ERK1 44 kDa U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophe- ERK2 42 kDa 2.0 0.0 EGF

nylthio] butadiene, Cell Signaling Technology (Beverly, Phosphorylation of ERK1/2 B E2 E2 MA, USA), 1 mM, 30 min), or the PI3K inhibitor Wortman- C 10 nM 0.1 nM C C 10 nM 0.1 nM C nin (Cell Signaling Technology, 1 mM, 30 min). Epidermal growth factor (EGF, Sigma, 10 ng/ml, 5 min) was used as a

3.0 positive control. At these concentrations, the agonists and * * p-ERK1 ERK1 44 kDa 2.0 ,# antagonists are highly selective, as was previously reported ,# * p-ERK2 ERK2 42 kDa * (Lucas et al. 2008, 2010, Royer et al. 2012). In preliminary 1.0

experiments, we tested different concentrations of the 0.0 E2 10 nM E2 0.1 nM inhibitors (PP2, AG1478, U0126, and Wortmannin), and Phosphorylation of ERK1/2

Journal of Molecular Endocrinology the best concentrations were similar to those previously used in rat Sertoli cells (Lucas et al. 2008, 2010, Royer et al. C E 2 3.0 2012) and mouse proximal caput epididymis-1 cells C 5′ 10′ 15′ 30′ p-ERK1 44 kDa (Hamzeh & Robaire 2011). All of the controls and treated p-ERK2 42 kDa 2.0

ERK1 44 kDa 1.0 tissues were processed in exactly the same way, and they ERK2 42 kDa

remained in the nutrient solution for the same total 0.0 5′ 10′ 15′ 30′ Phosphorylation of ERK1/2 incubation time. After this incubation period, the tissues E2 10 nM were frozen in liquid nitrogen and pulverized in a tissue grinder, and western blotting was immediately performed. Figure 1 Effects of E2 and EGF on the expression and phosphorylation of ERK1 and ERK2 in the corpus of the epididymis. Tissues were incubated at 30 8Cinthe Western blot to detect total and phospho-ERK1/2 absence (control, C) and presence of epidermal growth factor (EGF,10 ng/ml, 5min)(A),E2 (10 and 0.1 nM, 5 min) (B), or E2 (10 nM, different periods of Western blotting was performed as previously described by time) (C). Total tissue lysates (5 mg of protein/lane) were resolved in 10% SDS–PAGE, transferred to PVDF membrane, and probed with antibody Lucas et al. (2008). Briefly, 25 mg of pulverized tissue were specific to phosphorylated ERK1/2 (p-ERK1/2) or with antibody that lysed in ice-cold lysis buffer (20 mM HEPES (pH 7.5), recognizes total (phosphorylation state-independent) ERK1/2. The relative 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM positions of p-ERK1/2 and total ERK1/2 proteins are shown on the right. The data shown are representative of three (A and B) and two (C) independent MgCl2, 1 mM EGTA, 10 ml/ml aprotinin, 10 ml/ml leupep- experiments. Bars represent the densitometric analysis of the western blot. tin, 1 mM phenylmethylsulphonyl fluoride, 2 mM White barsZERK1; black barsZERK2. Results were normalized to total ERK1/2 expression in each sample and plotted (meanGS.E.M.) in relation to the Na3VO4, 50 mM NaF, and 10 mM Na4P2O7) and homogen- control (CZ1). *ERK1/2 activation was significantly greater than that of the ized in Ultra Turrax once at 8200 g for 30 s at 4 8C. The control (P!0.05, ANOVA and Newman–Keuls). #Significantly different from ! homogenate was incubated for 30 min at 4 8C and then E2 10 nM (P 0.05, ANOVA and Newman–Keuls).

C-terminus of rat p44 MAPK (no. 9102, Cell Signaling electrotransferred onto PVDF membranes using 20 V over- Technology, 1:2000 dilution) or a polyclonal antibody night at 4 8C. Membranes were blocked in TBST containing raised against a synthetic phosphopeptide that corre- 10% nonfat dry milk (pH 7.6) for 2 h at room temperature. sponded to the residues that surround Thr202/Tyr204 of After washes in TBST, membranes were incubated over- human p44 MAPK (no. 9101, Cell Signaling Technology, night at 4 8C with a rabbit MAB against phospho-CREB 1:1000 dilution), which cross-reacts with rat p44 MAPK. Ser133 (phospho-CREB (Ser 133), no. 9198, Cell Signaling Proteins were visualized using ECL reagent (GE Healthcare Technology) diluted in blocking solution (1:1000) or a UK Ltd, Buckinghamshire, UK) after being incubated with rabbit polyclonal antibody against the synthetic phospho- donkey anti-rabbit HRP-conjugated secondary antibody peptide derived from the amino acids that contain the (GE Healthcare UK Ltd) at 1:3000 dilution for 1 h at room Ser383 from human ELK1 (p-ELK1, Ser383, no. 9181S, Cell temperature. Actin levels were monitored to ensure equal Signaling Technology) diluted in blocking solution protein loading by using a rabbit antibody raised against a (1:4000). The membranes used for CREB determination synthetic peptide derived from actin residues 20–33 with N- were stripped as previously described (Gomes et al. 2011) terminus-added lysine (1:4000; A5060, Sigma) overnight at and reused for ELK1 and lamin A determinations. Lamin A, 4 8C. Apparent molecular masses were determined from which was used as a nuclear protein loading control molecular mass standards (no. 26634, Thermo Fisher (Prawan et al. 2009, Royer et al. 2012), was detected with a Scientific, Inc., Suwanee, GA, USA). rabbit polyclonal antibody against the sequence of amino Band intensities of total ERK1/2 and phosphorylated acids 563–664 of human lamin A (sc-20680, Santa Cruz ERK1/2 from individual experiments were quantified by Biotechnology, Inc.) diluted in blocking solution (1:250). densitometric analysis of linear-range autoradiograms Proteins were visualized using ECL reagent after being using an Epson Expression 1680 scanner (Epson America, incubated with donkey anti-rabbit HRP-conjugated sec- Long Beach, CA, USA) and Quick Scan 2000 WIN Software ondary antibody (GE Healthcare UK Ltd) at 1:3000 dilution (Helena Laboratories, Beaumont, TX, USA). Results were for 1 h at room temperature. normalized based on total ERK1/2 expression in each Band intensities of phospho-CREB, phospho-ELK1, sample and plotted (meanGS.E.M.) in relation to control and lamin A from individual experiments were quantified (CZ1) (Lucas et al. 2008). as described in the previous section for ERK1/2. Results were normalized to the respective lamin A expression and plotted (meanGS.E.M.) in relation to control (CZ1). Western blot to detect phospho-CREB (Ser133) and phospho-ELK1 (Ser 383) Journal of Molecular Endocrinology Statistical analysis Nuclear fractions were obtained as previously described by Royer et al. (2012). Nuclear extracts (40 mg/lane) were Data were expressed as meanGS.E.M. Statistical analysis incubated with sample buffer containing b-mercapto- was carried out by ANOVA followed by the Newman–Keuls ethanol and subjected to 10% SDS–PAGE. Proteins were test. P values of !0.05 were accepted as significant.

A B 3.0 * 3.0 MPP C 5′ C 10′ C 10′ C 15′ C 15′ C 30′ * * * * * * C PPT PPT 2.0 p-ERK1 44 kDa 2.0 * p-ERK2 42 kDa * p-ERK1 44 kDa * p-ERK2 42 kDa # # ERK1 44 kDa ERK1/2 1.0 1.0 ERK2 42 kDa ERK1 44 kDa ERK2 42 kDa

Phosphorylation of 0.0 5′ 10′ 15′ 30′

Phosphorylation of ERK1/2 0.0 PPT PPT MPP/PPT MPP

Figure 2 Effects of ESR1-selective agonist PPT on the expression and phosphorylation state-independent) ERK1/2 proteins (bottom panels). The relative positions of ERK1 and ERK2 in the corpus of the epididymis. Tissues were incubated of p-ERK1/2 and total ERK1/2 proteins are shown on the right. The data in the absence (control, C) and presence of PPT (100 nM) for 5–30 min at shown are representative of three or four independent experiments. Bars 30 8C. (A) Tissues were pretreated or not pretreated with ESR1-selective represent the densitometric analysis of the western blot. White barsZ antagonist MPP (100 nM, 60 min). Afterward, tissues were stimulated with ERK1; black barsZERK2. Results were normalized to total ERK1/2 PPT (100 nM) for 5 min at 30 8C. (B) Total tissue lysates (5 mg of protein/lane) expression in each sample and plotted (meanGS.E.M.) in relation to the were resolved using 10% SDS–PAGE, transferred to PVDF membrane, and control. *ERK1/2 activation was significantly greater than that of the probed with antibody specific to phosphorylated ERK1/2 (p-ERK1/2, top control (P!0.05, ANOVA and Newman–Keuls). #Significantly different from panels) or with antibody that recognizes total (phosphorylation PPT (P!0.05, ANOVA and Newman–Keuls).

Results Figure S1). The ESR1-selective agonist PPT (100 nM, 5 min) increased the phosphorylation state of ERK1/2 in the Effects of the E and ESR1-selective agonist PPT on the 2 initial segment/caput (1.8-fold for both ERK1 and ERK2) expression and phosphorylation of ERK1 and ERK2 in the and in the corpus of the epididymis (2.5- and 2.8-fold corpus of the epididymis respectively for ERK1 and ERK2), but it did not change the EGF receptor is present in the epididymis (Hamzeh & ERK1/2 activity in the cauda of the epididymis, which Robaire 2011). Therefore, EGF (10 ng/ml, 5 min) was used indicates that the effects of PPT are stronger in the corpus as a positive control for the phosphorylation of ERK1/2 of the epididymis and could be related to the higher in the corpus of the epididymis (Fig. 1A). expression of ESR1 in this region. Activation of ERK1/2 induced by a 5 min treatment A 2.5 * with E2 was concentration-dependent, and it was greater PP2 * 2.0 with 10 nM of E2 (2.3- and 2.5-fold respectively for ERK1 CPPT PPT 1.5 p-ERK1 44 kDa # p-ERK2 42 kDa # and ERK2) than it was with 0.1 nM (Fig. 1B). A whole 1.0

representative gel is shown to demonstrate that no other ERK1 44 kDa 0.5 ERK2 42 kDa 0.0 protein bands were detected in control or E2-treated tissues. Phosphorylation of ERK1/2 PPT PP2/PPT

E2 (10 nM) induced a rapid and transient increase in the B AG1478 2.5 * phosphorylation state of ERK1/2, with a peak at 5 min C PPT PPT 2.0 * p-ERK1 44 kDa 1.5 # # (Fig. 1C). No differences were observed in total ERK1/2 p-ERK2 42 kDa 1.0 protein expression under any of these conditions (Fig. 1B ERK1 44 kDa 0.5 ERK2 42 kDa and C). The expression of total ERK1 protein was eightfold 0.0

Phosphorylation of ERK1/2 PPT AG1478/PPT higher than that of ERK2 in the corpus of the epididymis.

C 2.5 The ESR1-selective agonist PPT (100 nM) also induced a * U0126 rapid increase of ERK1/2 phosphorylation that was already 2.0 * C PPT PPT 1.5 # detected 5 min after treatment with PPT. The activity of p-ERK1 44 kDa # p-ERK2 42 kDa 1.0 ERK1/2 almost returned to control levels by 30 min (Fig. 2A 0.5 ERK1 44 kDa ERK2 top panels). After 5 min of stimulation with PPT, ERK1 and 42 kDa 0.0 Phosphorylation of ERK1/2 PPT U0126/PPT ERK2 activation were 2.2- and 2.4-fold higher respectively D 2.5 as compared with the control. No differences were observed Wortmannin 2.0 in total ERK1/2 protein expression under any of these C PPT PPT Journal of Molecular Endocrinology * * p-ERK1 44 kDa 1.5 # # p-ERK2 conditions (Fig. 2A, lower panel). No signiﬁcant differences 42 kDa 1.0 among different controls were observed in the expression ERK1 44 kDa 0.5 ERK2 42 kDa O 0.0 or phosphorylation state of ERK1/2 (P 0.05) (Fig. 2A). Phosphorylation of ERK1/2 PPT Wort/PPT Thus, the results were normalized based on the total

ERK1/2 expression in each sample and plotted in relation Figure 3 to the average value for controls (CZ1) (Fig. 2A). Signaling pathway involved in the phosphorylation of ERK1 and ERK2 Activation of ERK1/2 induced by a 5 min treatment induced by PPT in the corpus of the epididymis. Tissues were incubated in the absence (control, C) and presence of PPT (100 nM) for 5 min at 30 8C. with PPT (100 nM) was blocked by pretreatment with an Tissues were pretreated or not pretreated with a non-receptor tyrosine ESR1-selective antagonist (MPP, 100 nM, 60 min) (Fig. 2B), kinase SRC inhibitor (PP2, 2.5 mM) (A), an EGFR kinase inhibitor (AG1478, which indicates that ESR1 is the upstream component that 25 mM) (B), a MEK1/2 inhibitor (U0126, 1 mM) (C), or a PI3K inhibitor (Wortmannin, 1 mM) (D) for 30 min. Afterwards, tissues were stimulated regulates ERK1/2 activity in this rapid action. Pretreat- with PPT (100 nM) for 5 min at 30 8C. Total tissue lysates (5 mgof ment with MPP in the absence of PPT did not have any protein/lane) were resolved in 10% SDS–PAGE, transferred to PVDF membrane, and probed with antibody specific to phosphorylated ERK1/2 effect on basal ERK1/2 phosphorylation (Fig. 2B). (p-ERK1/2, top panels) or with antibody that recognizes total (phos- The effects of PPT were also examined using the phorylation state-independent) ERK1/2 proteins (bottom panels). The three epididymal regions: the initial segment/caput, the relative positions of p-ERK1/2 and total ERK1/2 proteins are shown on the right. The data shown are representative of three or four independent corpus, and the cauda (Supplementary Figure S1, see experiments. Bars represent the densitometric analysis of the western blot. section on supplementary data given at the end of this White barsZERK1; black barsZERK2. Results were normalized to total article). The total ERK1/2 protein expression, which was ERK1/2 expression in each sample and plotted (meanGS.E.M.) in relation to the control. *ERK1/2 activation was significantly greater than that of the normalized to the endogenous control actin, was similar control (P!0.05, ANOVA and Newman–Keuls). #Significantly different from among the distinct epididymal regions (Supplementary PPT (P!0.05, ANOVA and Newman–Keuls).

A MPP

C PPT PPT 5.0 4.0

4.0 p-CREB 43 kDa 3.0 3.0 2.0 p-ELK1 47 kDa 2.0 1.0 1.0 Lamin A 69 kDa Phosphorylation of ELK1 Phosphorylation of CREB 0.0 0.0 PPT MPP/PPT MPP PPT MPP/PPT MPP

B PP2 C PPT PPT 5.0 4.0 * 4.0 p-CREB 43 kDa 3.0 3.0 * 2.0 p-ELK1 47 kDa 2.0 # # 1.0 1.0 Phosphorylation of ELK1 Lamin A 69 kDa Phosphorylation of CREB 0.0 0.0 PPT PP2/PPT PP2 PPT PP2/PPT PP2

C AG1478 C PPT PPT 5.0 4.0

4.0 p-CREB 43 kDa 3.0 * * 3.0 2.0 p-ELK1 47 kDa 2.0 # # 1.0 1.0 Phosphorylation of ELK1 Lamin A 69 kDa Phosphorylation of CREB 0.0 0.0 PPT AG1478/PPT AG1478 PPT AG1478/PPT AG1478

D U0126 C PPT PPT 8.0 4.0 p-CREB 43 kDa * 6.0 3.0 *

p-ELK1 47 kDa 4.0 2.0 # 2.0 1.0 # Journal of Molecular Endocrinology Lamin A 69 kDa Phosphorylation of ELK1 Phosphorylation of CREB 0.0 0.0 PPT U0126/PPT U0126 PPT U0126/PPT U0126

E Wortmannin C PPT PPT 5.0 4.0 * 4.0 * p-CREB 43 kDa 3.0 3.0 2.0 p-ELK1 47 kDa 2.0 # # 1.0 1.0 Phosphorylation of ELK1 Lamin A 69 kDa Phosphorylation of CREB 0.0 0.0 PPT Wort/PPT Wort PPT Wort/PPT Wort

Figure 4 Effects of PPT and signaling pathways involved in the phosphorylation of antibody specific to phosphorylated ELK1 Ser383 (p-ELK1[Ser383], middle CREB (Ser133) and ELK1 (Ser383) in the corpus of the epididymis. Tissues panels) or with antibody that recognizes the nuclear protein lamin A were incubated in the absence (control, C) and presence of PPT (100 nM) (bottom panels). The relative positions of p-CREB, p-ELK1, and lamin A for 5 min at 30 8C. Tissues were pretreated or not pretreated with the ESR1- proteins are shown on the right. The data shown are representative of two selective antagonist MPP (100 nM, 60 min) (A), a non-receptor tyrosine (A) or four (B, C, D, and E) independent experiments. Bars represent the kinase SRC inhibitor (PP2, 2.5 mM) (B), an EGFR kinase inhibitor (AG1478, densitometric analysis of the western blot. Black barsZp-CREB; hatched 25 mM) (C), a MEK1/2 inhibitor (U0126, 1 mM) (D), or a PI3K inhibitor barsZp-ELK1. Results were normalized to lamin A expression in each (Wortmannin, 1 mM) (E) for 30 min. Afterward, tissues were stimulated with sample and plotted (meanGS.E.M.) in relation to the control (CZ1). *CREB PPT (100 nM) for 5 min at 30 8C. Nuclear extracts (40 mg of protein/lane) or ELK1 activation was significantly greater than that of the control were resolved in 10% SDS–PAGE, transferred to PVDF membrane, and (P!0.05, ANOVA and Newman–Keuls). #Significantly different from PPT probed with antibody specific to phosphorylated CREB Ser133 (P!0.05, ANOVA and Newman–Keuls). (p-CREB[Ser133], top panels). The membrane was stripped and reused with

Signaling pathways involved in the phosphorylation of with the selective antagonist of ESR1 (MPP) blocked this ERK1/2 induced by PPT in the corpus of the epididymis effect, which indicates that ESR1 is the upstream component that regulates ELK1 activity (Fig. 4A). Activation of ERK1/2 induced by a 5 min treatment with The activation of ELK1 induced by PPT was blocked PPT (100 nM) was tested in the presence of the following by pretreatment with a non-receptor tyrosine kinase SRC inhibitors: a non-receptor tyrosine kinase SRC inhibitor inhibitor (PP2, Fig. 4B), the EGFR kinase inhibitor (AG (PP2, 2.5 mM), an EGFR kinase inhibitor AG 1478 (25 mM), a 1478, Fig. 4C), the MEK1/2 inhibitor (U0126, Fig. 4D), and MEK1/2 inhibitor (U0126, 1 mM), and a PI3K inhibitor the PI3K inhibitor (Wortmannin, Fig. 4E). (Wortmannin, 1 mM) for 30 min (Fig. 3). The effect of each Pretreatment with MPP, PP2, AG 1478, U0126, or inhibitor (PP2, AG1478, U0126, and Wortmannin) on the Wortmannin in the absence of PPT did not affect the basal phosphorylation of ERK1 and ERK2 was tested and phosphorylation of ELK1 (Fig. 4A, B, C, and E). compared with the control (absence of an inhibitor). Any change produced by the inhibitor on the basal phosphorylation of ERK1/2 was subtracted from the effect of the Discussion inhibitor on phosphorylation induced by PPT. Only U0126 Several pieces of evidence are indicative of a role for produced a statistically signiﬁcant inhibition of the basal estrogen in the control of epidydimal ﬂuid absorption and G G phosphorylation of ERK1/2 (78 5% of ERK1 and 79 7% secretion (Hess et al. 2011). Whereas fewer genes are Z of ERK2 basal phosphorylation; n 4 independent experi- regulated by estrogens as compared with androgens in the ments). The results shown in the graphs (Fig. 3) show this mouse epididymis (Hamzeh & Robaire 2010), estrogen result. All of the inhibitors completely blocked the regulates the expression of genes involved in solute and phosphorylation of ERK1/2 that was induced by PPT (Fig. 3). water transport (Hess et al. 1997, 2001, Snyder et al. 2009), which is a function that is essential for normal reproduc- Activation of ESR1 induces phosphorylation of CREB tive performance (Hess et al. 1997). Among these genes, (Ser 133) in the corpus of the epididymis aquaporin 9 contains both AREs and EREs, and its expression is regulated by both estrogen and androgen The ESR1-selective agonist PPT (100 nM, 5 min) increased (Pastor-Soler et al. 2010, Joseph et al. 2011). Furthermore, phosphorylation of CREB about 3.5-fold. Pretreatment we have also previously reported that treatment with the with MPP, a selective antagonist of ESR1, blocked this anti-estrogen fulvestrant affected the expression of effect, which indicates that ESR1 is the upstream com- proteins that are important for epithelial organization

Journal of Molecular Endocrinology ponent that regulates CREB activity (Fig. 4A). No and absorption/secretion in the rat epididymis (Pereira differences were observed in lamin A expression under et al. 2014). This indicates that the balance of estrogen and any of these conditions (Fig. 4, lower panels). androgen actions may be important to ensure proper The involvement of SRC, EGFR, ERK1/2, and PI3K in epididymal function. the phosphorylation of CREB induced by PPT (100 nM, The higher expression of the estrogen receptor ESR1 in 5 min) was tested in the presence of the non-receptor the corpus of the rat epididymis and its localization not tyrosine kinase SRC inhibitor (PP2), the EGFR kinase only in the nucleus but also in the cytoplasm and inhibitor (AG 1478), the MEK1/2 inhibitor (U0126), and membrane regions of the epithelial cells led us to

the PI3K inhibitor (Wortmannin). The activation of CREB investigate the role of this receptor in E2-mediated rapid induced by PPT was blocked by pretreatment with PP2 signaling pathways. (Fig. 4B), AG 1478 (Fig. 4C), U0126 (Fig. 4D), and Regulation of the ERK and AKT pathways is important Wortmannin (Fig. 4E). for cell survival, proliferation, metabolism, and motility Pretreatment with MPP, PP2, AG 1478, U0126, or (reviewed by Mendoza et al. 2011). The high level of Wortmannin in the absence of PPT did not affect the activity of the ERK pathway components seems to be phosphorylation of CREB (Fig. 4A, B, C, and E). essential for differentiation of the epithelium from the initial segment of the mouse epididymis (Xu et al. 2010, 2011). Similar to the results of previous studies using mice Activation of ESR1 induces phosphorylation of ELK1 (Xu et al. 2010), the relatively high basal activity was (Ser383) in the corpus of the epididymis detected in the present study of ERK1/2 in all of the The ESR1-selective agonist PPT (100 nM, 5 min) increased epididymal regions in adult rats. The role of the ERK1/2 the phosphorylation of ELK1 about 2.5-fold. Pretreatment pathway in the adult epididymis is still not clear, but it

may be important for cell survival and secretion. The high between the ERK1/2 and the PI3K/AKT pathways, either activity of the ERK pathway in the epididymis of mice was positively by cross-activation or negatively by cross- abolished by efferent duct ligation (Xu et al. 2010, 2011), inhibition, depending on the cellular environment which indicates that the testicular luminal ﬂuid may (reviewed by Mendoza et al. 2011). PI3K-mediated contain factors that activate the ERK pathway. In fact, the activation of the ERK1/2 pathway induced by relaxin in

results of the present study indicated that E2 is a possible rat Sertoli cells (Nascimento et al. 2012) and by DHT in the component of the testicular luminal ﬂuid that is involved mouse proximal caput epididymis PC-1 cell line (Hamzeh in the regulation of the ERK1/2 pathway in the & Robaire 2011) has been shown.

epididymis. Overlapping and interconnected actions of E2 (in the ERK1/2 activation by the ESR1-selective agonist PPT present study), androgens (Hamzeh & Robaire 2011), and was stronger in the corpus of the epididymis, which growth factors (reviewed by Cotton et al. 2008) on ERK1/2 correlates with the higher concentration of ESR1 in this activation may occur in the epididymis. An imbalance of region. This rapid action of PPT was blocked by the ESR1- these actions may result in infertility, and much remains selective antagonist MPP, which indicates that ESR1 is the to be learned about the complex interplay of hormonal upstream estrogen receptor that regulates ERK1/2 activity. actions in the epididymis. Further experimental

The intracellular mechanism by which the E2-ESR1 approaches using a combination of these hormones and complex initiates rapid signaling in epididymal cells is not growth factors are necessary to address these issues of known. The non-receptor tyrosine kinase SRC has been epididymis biology.

implicated in the activation of ERK1/2 by E2 and androgen. It is now well known that rapid, membrane-initiated

Both steroids induce the interaction of ESR1 and androgen signaling mechanisms stimulated by E2 may lead to the receptor (AR) with SRC as well as a conformational change activation of gene transcription (reviewed by Lucas et al. that activates this kinase in breast cancer MCF-7 and 2011, Levin 2014 and Prossnitz & Barton 2014). prostate cancer LNCaP cells (Migliaccio et al. 2000; The phosphorylation of CREB protein at Ser133 induced

reviewed by Migliaccio et al. 2005). An SRC-dependent by E2 through the MAPK and PI3K pathways plays a role ERK activation by 5a-dihydrotestosterone (DHT) has also in gene transcription in several cells (Aronica et al. 1994,

been described in the mouse proximal caput epididymis Genua et al. 2009, Royer et al. 2012). E2-mediated CREB PC-1 cell line (Hamzeh & Robaire 2011). The phosphory- phosphorylation has not, to our knowledge, been lation of ERK1/2 induced by the activation of ESR1 was analyzed in the epididymis so far. The phosphorylation

blocked by pretreatment with a non-receptor tyrosine of CREB induced by E2-ESR1 in the corpus of the

Journal of Molecular Endocrinology kinase SRC inhibitor (PP2) in the corpus of the epididymis, epididymis was blocked by PP2, AG 1478, U0126, and which indicates that SRC is involved in this signal Wortmannin, which indicates that the SRC-EGFR-MEK- transduction. ERK1/2 and PI3K pathways are involved in the phos-

The complex E2-ESRs (Razandi et al. 2003, Lucas et al. phorylation of CREB.

2008) and E2-GPER (Filardo et al. 2000, Lucas et al. 2010), The phosphorylation of CREB may be involved in the via the activation of SRC, induce transactivation of EGFR regulation of gene expression related to epidydimal with a consequent phosphorylation of ERK1/2 in breast secretion. For instance, CREB plays a role in the cancer cells and rat Sertoli cells. Another model of this transcriptional regulation of pannexin1, a channel-form- cross-talk has been proposed in which the ligand-activated ing protein that has been implicated in cellular communi- EGFR phosphorylates ESR1 on tyrosine 537, thereby cation through the secretion of biomolecules, such as ATP inducing ESR1-SRC association and kinase activation. and glutamate, in the epididymis (Dufresne & Cyr 2014). SRC phosphorylates EGFR, which ampliﬁes receptor Another transcription factor that may be activated

activity and induces signal transduction activation by the E2-ERK1/2 pathway in different cells is ELK1 (Song (reviewed by Migliaccio et al. 2010). PPT-induced ERK1/2 et al. 2002, Kim et al. 2011). The presence of ELK1 in the phosphorylation in the corpus of the epididymis was epididymis has not, to our knowledge, been analyzed so decreased by the EGFR kinase inhibitor, which indicates far. The pathway responsible for the activation of ELK1 is the involvement of ESR1-SRC-EGFR in the activation of triggered by the relocation of the GRB2/SOS complex from ERK1/2. the cytoplasm to the cell membrane, where GRB2 is PPT-induced activation of ERK1/2 in the corpus of the recruited to phosphorylated residues of EGFR. Transloca- epididymis was inhibited by the PI3K inhibitor Wortman- tion of the GRB2/SOS complex facilitates the interaction nin. In fact, there is substantial evidence of cross-talk of membrane-associated RAS with SOS and RAS-RAF-1-

MEK1/2-ERK1/2 activation. Activation of the ERK1/2 Aronica SM, Kraus WL & Katzenellenbogen BS 1994 Estrogen action via the pathway causes de-SUMOylation and phosphorylation of cAMP signaling pathway: stimulation of adenylate cyclase and cAMP regulated gene transcription. PNAS 91 8517–8521. (doi:10.1073/pnas. ELK1, which permits ELK1 to transition from a transcrip- 91.18.8517) tionally repressive to a transcriptionally active form Belleanneé C, Thimon V & Sullivan R 2012 Region-specific gene expression (reviewed by Jorissen et al. 2003 and Yang & Sharrocks in the epididymis. Cell and Tissue Research 349 717–731. (doi:10.1007/ s00441-012-1381-0) 2006). The activation of ELK1 induced by PPT was seen in Carpino A, Romeo F & Rago V 2004 Aromatase immunolocalization in the corpus of the epididymis. human ductuli efferentes and proximal ductus epididymis. Journal of ELK1 modulates the expression of genes that encode Anatomy 204 217–220. (doi:10.1111/j.0021-8782.2004.00272.x) Cotton LM, O’Bryan MK & Hinton BT 2008 Cellular signaling by fibroblast proteins that control proteolytic activity, such as the growth factors (FGFs) and their receptors (FGFRs) in male reproduction. inhibitors of plasminogen activator 1 and metalloprotei- Endocrine Reviews 29 193–216. (doi:10.1210/er.2007-0028) nase 2 and 9 (MMP2 and MMP9) (reviewed by Kasza 2013). Dufresne J & Cyr DG 2014 Regulation of the pannexin-1 promoter in the rat epididymis. Biology of Reproduction 91 143. (doi:10.1095/ MMP2 and MMP9 are present in the epididymis (Warinrak biolreprod.114.122168) et al. 2015). Whether the phosphorylation of ELK1 Filardo EJ, Quinn JA, Bland KI & Frackelton AR Jr 2000 Estrogen-induced activation of ERK1 and ERK2 requires the G protein-coupled receptor induced by E2 plays a role in the regulation of the trans metalloproteinases remains to be explored. homolog, GPR30, and occurs via -activation of the epidermal growth factor receptor through release of HB-EGF. Molecular Endo- In conclusion, the present results indicate that crinology 14 1649–1660. (doi:10.1210/mend.14.10.0532) estrogen, when it interacts with the classical estrogen Frenette G, Leclerc P, D’Amours O & Sullivan R 2009 Estrogen receptor ESR1, induces transactivation of EGFR via the sulfotransferase is highly expressed along the bovine epididymis and is secreted into the intraluminal environment. Journal of Andrology 30 activation of SRC, with the consequent phosphorylation 580–589. (doi:10.2164/jandrol.108.006668) of ERK1/2, which in turn triggers an increase in CREB and Genua M, Pandini G, Sisci D, Castoria G, Maggiolini M, Vigneri R & Belfiore ELK1 phosphorylation in the corpus of the epididymis. A 2009 Role of cyclic AMP response element-binding protein in insulin-like growth factor-1 receptor up-regulation by sex steroids in This rapid action of estrogen may ultimately modulate prostate cancer cells. Cancer Research 69 7270–7277. (doi:10.1158/ the transcription of genes, and it may play a role in the 0008-5472.CAN-09-0088) dynamic microenvironment of the epididymal lumen. Gomes GRO, Yasuhara F, Siu ER, Fernandes SAF, Avellar MCW, Lazari MFM & Porto CS 2011 In vivo treatments with Fulvestrant and Anastrozole differentially affect gene expression in the rat efferent ductules. Biology of Reproduction 84 52–61. (doi:10.1095/biolreprod.110.085340) Supplementary data Guyonnet B, Marot G, Dacheux JL, Mercat MJ, Schwob S, Jaffre´zic F & Gatti JL This is linked to the online version of the paper at http://dx.doi.org/10.1530/ 2009 The adult boar testicular and epididymal transcriptomes. BMC JME-15-0086. Genomics 10 369. (doi:10.1186/1471-2164-10-369) Hamzeh M & Robaire B 2010 Identification of early response genes and

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Received in ﬁnal form 25 April 2015 Accepted 29 April 2015 Journal of Molecular Endocrinology