Journal of Steroid Biochemistry & Molecular Biology 102 (2006) 2–10

Progesterone signaling in breast and endometrium

Cecilia Ballare a,!, Griselda Vallejo b, Guillermo P. Vicent a,Patricia Saragueta b, Miguel Beato a

a Centre de Regulacio Genomica (CRG), Universitat Pompeu Fabra (UPF), PRBB, Dr. Aiguader 88, E-08003 Barcelona, Spainb Universidad de Buenos Aires, Instituto de Biologıa y Medicina Experimental (IBYME-CONICET), Buenos Aires, Argentina

Abstract

In addition to transcriptional effects, steroid hormones rapidly activate cytoplasmic signaling cascades. The ultimate targets of these cascadesare not well-defined and likely include transcription factors and coactivators. To better understand the role of the rapid “non-transcriptional”effects of progestins, we investigated the mechanisms leading to activation of these pathways and their relevance in the biological response,using two model systems: breast cancer and endometrial stromal cells. Our results demonstrated that progestins rapidly activate the Src/Erk1/2and PI3K/Akt pathways in both cellular types via crosstalk between PR and ER! or ER". This activation is essential for triggering proliferativeresponse. However, even when the activation of kinase cascades is similar in both cellular types, the biological outcome of progestin treatmentis different. A different ability of PR to mediate transcriptional effects might account for this discrepancy. Also differences in amount andsubcellular location of PR, presence of ER! or ER" and alternative receptors could be also important for determining the cellular response.

We also explored the connection between rapid activation of kinase cascades and transcriptional induction by progestins. Our results uncovera novel function of the rapid Erk activation by progestins, namely its direct involvement in transcriptional induction of MMTV promoter andother progesterone-target genes.© 2006 Elsevier Ltd. All rights reserved.

Keywords: Progesterone; Breast cancer; Endometrium; Steroid receptors; Signal transduction; MAPK; PI3K/Akt; Proliferation; Chromatin; Mouse mammarytumor virus

1. Introduction

Estrogen and progesterone influence a variety of cellu-lar functions, depending on the nature of the target cell andthe multiple signals impinging on the cell at a given time.During many years attention has been mainly focused on thetranscriptional effects of these hormones. Steroid hormonereceptors (SHR) are ligand-dependent transcription factors,which upon activation with the specific hormone can interactwith hormone responsive elements (HREs) in the promoterof target genes. SHRs can also activate genes lacking HREsby interaction with other sequence-specific transcription fac-tors bound to their target sequences [3]. In addition to theseso-called “genomic effects”, steroid hormones are known toproduce rapid responses similar to those induced by pep-tide growth factors. Estrogens activate the Src/p21ras/Erk and

! Corresponding author. Tel.: +34 93 224 09 14; fax: +34 93 224 08 99.E-mail address: cecilia.ballare@crg.es (C. Ballare).

the PI3K/Akt pathways via direct interaction of the estrogenreceptor (ER) with c-Src and the regulatory subunit of PI3K,respectively [18,6]. Activation of these kinase pathways isessential for estrogen induction of cell proliferation in breastcancer cells. Progestins can also activate these signal cas-cades, either via an interaction of the progesterone receptor(PR) with ER, which itself activates c-Src, or by direct inter-action of PR with c-Src [19,5,1,27]. The ultimate targets ofthe activated kinase cascades are not well-defined and likelyinclude transcription factors and co-factors involved in cellcycle control [4]. Whether this action on kinases and their tar-gets relates to the transcriptional effects of steroid hormonesis an interesting open question.

It has been reported that during activation of immediateearly genes by diverse stimuli the chromatin over the c-fos andc-jun genes becomes rapidly and transiently hyperacetylatedand phosphorylated at histone H3 [16,2,7]. The modifica-tions of histone H3 seem to be required for transcriptionalactivation of these genes [25]. These findings suggest a link

0960-0760/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.jsbmb.2006.09.030

C. Ballare et al. / Journal of Steroid Biochemistry & Molecular Biology 102 (2006) 2–10 3

between signaling-mediated chromatin phosphorylation andgene regulation.

To better understand the role of these “non-genomic”effects activated by progestins we have investigated the mech-anisms leading to the activation of these signal pathwaysand their relevance on progestin-induced cell proliferation.We have also explored the connection between rapid kinaseactivation and transcriptional induction by progestins. Herewe summarise some of our recent work published elsewhere[1,27] as well as new results showing that the rapid acti-vation of Erk1/2 and PI3K/Akt pathways by progestins inbreast cancer and endometrial cells is mediated by crosstalkbetween PR and ER! or ER" and is essential for the pro-liferative response to progestins. We also uncover a novelfunction of the rapid activation of Erk by progestins in breastcancer cells, that is, its relevance for transcriptional inductionof hormone-responsive genes. Our results establish a noveland direct connection between rapid kinase activation and“genomic effects” of progestins.

2. Materials and methods

2.1. Cell culture and hormone treatments

T47D human breast cancer cells were routinely grownin Dulbecco’s modified Eagle medium (DMEM) sup-plemented with 10% FBS, 2 mM l-glutamine, 100 U/mlpenicillin and 100 #g/ml streptomycin. UIII rat normaluterine stromal cells were maintained in M199 mediumsupplemented with 10% FBS, 2 mM l-glutamine, penicillin(100 U/ml) and streptomycin (100 #g/ml). COS-7 cells weregrown routinely in DMEM supplemented with 5% FBS,2 mM l-glutamine, penicillin (100 U/ml) and streptomycin(100 #g/ml).

For hormone treatment experiments, cells were plated inmedium without phenol red plus 10% dextran-coated char-coal treated FBS (DCC/FBS) and 48 h later medium wasreplaced by fresh medium without serum. After 1 day (T47D)or 2 days (UIII) in serum-free conditions, cells were incu-bated with R5020 or vehicle (ethanol) for different times at37 "C.

When indicated, RU486 (1 #M), ICI182780 (10 #M),PD98059 (50 #M) or Wortmannin (WM) (0.1 #M) wereadded 1 h (T47D) or 30 min (UIII) before hormoneinduction.

2.2. Cell proliferation experiments

For DNA synthesis analysis, cells were treated as indi-cated above and incubated with 10 nM R5020 or vehicle(ethanol) for 15 h. BrdU (10 #M) was added during the last2 h of hormone induction. After treatment, cells were fixed,permeabilised and BrdU incorporation evaluated with anti-BrdU antibodies by indirect immunofluorescence followedby FACs analysis. For cell proliferation experiments in UIII,

cells were cultured as indicated above and treated with 10 pMof R5020 or vehicle (ethanol). The number of cells was deter-mined at time zero and after 5 days of hormone treatment.

2.3. Activation of Erk1/2 and Akt pathways

Cells treated as indicated above were incubated for 0, 5or 10 min at 37 "C with R5020 or vehicle (ethanol: –). Celllysates were prepared in 1% SDS, 25 mM Tris–HCl pH 7.6,1 mM EDTA, 1 mM EGTA plus protease and phosphataseinhibitors and analysed by Western blots with antibodies rec-ognizing the phosphorylated forms of Erk, Msk, Akt andGSK3 (Cell Signaling). Total Erk, Msk-1, Akt, PR and tubu-lin were detected with the corresponding antibodies (SantaCruz).

2.4. Analysis of PR and ER content in T47D and UIIIcells

It was performed by Western blot using specific antibod-ies against PR (Santa Cruz), ER! (Santa Cruz) and ER"(anti-DBD, gift of Jan-Ake Gustafsson). PR content wasalso determined by immunofluorescence as was previouslydescribed [27].

2.5. Coimmunoprecipitation experiments

Cells were lysed in 50 mM Tris–HCl pH 7.4, 150 mMNaCl, 1 mM EDTA, 1 mM EGTA, 5 mM MgCl2, 0.5% tritonX-100 plus protease and phosphatase inhibitors. Coimmuno-precipitation experiments of PR and ER were performed aspreviously described [1,27]. For coimmunoprecipitation PR-Erk, cell extracts (2 mg/ml for each immunoprecipitation)were precleared 2 h with 40 #l of 50% slurry protein G/Aagarose beads (Oncogene) and incubated overnight at 4 "Cwith 60 #l protein G/A agarose beads previously coupledwith 4 #g of anti-Erk2 antibody (Santa Cruz) or an unspe-cific control antibody. The immunoprecipitated proteins (IP)were eluted by boiling in SDS sample buffer. Inputs and IPswere analysed for PR, Erk by Western blot using specificantibodies.

2.6. Transient transfections

Cells were transiently transfected with the LipofectaminePlus reagent (Invitrogene) according to the manufacturer’sinstructions.

2.7. Luciferase assay

Cell lysates were prepared and luciferase and "-galactosidase activities were determined as was previouslydescribed [27] with assay kits from Promega, according tomanufacturer’s instructions. To correct for differences intransfection efficiencies, luciferase units were normalized for"-galactosidase activities.

4 C. Ballare et al. / Journal of Steroid Biochemistry & Molecular Biology 102 (2006) 2–10

2.8. Erk immunoprecipitation and activity assay

Cos7 cells were transfected with expression vectorsfor ER! (pSG5-ER!) and PRB (pSG5-PR). Alternatively,pSG5-PR was replaced for PR mutants as indicated in theexperiment. Erk immunoprecipitation and activity assay wereperformed as was previously reported [1]. Briefly, cell lysateswere immunoprecipitated with anti-Erk antibodies (SantaCruz) and the immunoprecipitates were assayed for MAPkinase activity using myelin basic protein (MBP) as a sub-strate and [$-32P] ATP.

3. Results and discussion

3.1. Progestins activate Erk and PI3K pathways inbreast cancer and endometrial cells

In addition to their transcriptional effects, progestins areknown to crosstalk to kinase cascades activated by signalsimpinging on membrane receptors. In T47D breast can-cer cells, which express high levels of PR and ER!, wehave observed that treatment of 5 or 10 min with 10 nMof the PR agonist R5020 enhanced Erk 1/2 kinase activity(Fig. 1A, upper panel) and this activation was inhibited byPD98059 (PD), an specific inhibitor of mitogen-activatedprotein kinase kinase 1, as well as the specific PR antag-onist RU486 (RU) and ER antagonist ICI182.780 (ICI).Thus, progestin activation of the Erk1/2 pathway requireda crosstalk between PR and ligand-free ER. Msk kinases aretargets of Erk1/2 that have been shown to participate in therapid activation of immediate early genes via phosphoryla-tion of histone H3 following mitogen stimulation [23]. After5 or 10 min of progestin treatment we have also observedan increase in the activated-phosphorylated form of Msk1,which paralleled the increase in phosphorylated Erk and thatwas inhibited by PD (Fig. 1A, lower panel). Msk activa-tion by R5020 was also abrogated by RU and ICI (data notshown).

It has been reported that the PI3K/Akt pathway is involvedin estrogen- as well as medroxyprogesterone acetate (MPA)-induced cell proliferation in breast cancer cells [6,22]. Wehave then analysed phosphorylation of Akt in response toR5020 in T47D cells. Five minutes after progestin treat-ment there was an increase in activated-phosphorylated Akt(Fig. 1B, upper panel). We have also evaluated the phospho-rylation state of the glycogen synthase kinase 3!/" (GSK-3!/"), a critical downstream element of the PI3K/Akt cellproliferation and survival pathways. GSK-3 is a ubiquitouslyexpressed serine/threonine protein kinase that phosphory-lates and inactivates glycogen synthase. It can also phospho-rylates cyclin D1 in threonine-286, triggering proteosomaldegradation of cyclin D1 [12,10]. GSK-3 is constitutivelyactive in arrested cells and can be inhibited by Akt-mediatedphosphorylation at Serine-21 of GSK-3! and Serine-9 ofGSK-3" [11]. As shown in Fig. 1B (lower panel), there was

a rapid increase in phosphorylation of GSK3 !/" concomi-tantly with the activation of Akt by R5020.

Progesterone is an essential stimulus in the uterus, neededfor in vivo proliferation of stromal cells [8,30]. The molecularmechanism underlying physiological action of progesteroneon uterine function is unclear; although it has been assumedthat it involves transcriptional activation of progesterone tar-get genes mediated by its nuclear PR. To better understand themechanism of action of progesterone in uterine stromal cells,we have explored the involvement of signaling pathways inthe proliferative response of normal endometrial stromal cellsto progestins. We have used as a model the established ratuterine stromal UIII cell line [9]. Treatment of UIII cells for 5or 10 min with progestin R5020 induced a rapid activation ofErk (Fig. 1C, upper panel) and the PI3K/Akt (Fig. 1D) path-ways. Even picomolar concentrations of R5020 were enoughto activate both kinase cascades (Fig. 1E). As in breast cancercells, RU and ICI abrogated these activations (Fig. 1C, lowerpanel and Fig. 1D), showing that the classical PR and ER arerequired for activation of signaling cascades by progesterone.

Conversely to T47D cells, UIII have very low levels of PR(Fig. 2A, left panel), no ER! (Fig. 2B) and low amounts ofER" (Fig. 2C) and these receptors are located mainly in thecytoplasm (Fig. 2A, right panel; [27]). The lack of ER! inthese cells suggests that it is ER" which crosstalk with PRfor the activation of Erk and PI3K pathways in UIII cells.

3.2. Crosstalk ER–PR

It was previously demonstrated that progestins can acti-vate kinase cascades through an interaction between a prolinecluster in PR and the SH3 domain of c-Src [5,1]. How-ever, in breast cancer cells containing ER!, the progestineffect on Src/Erk pathway is mediated by an interaction ofPR B with the unliganded ER!, which itself binds to theSH2 domain of c-Src and stimulates the c-Src kinase activity[19,1]. Using yeast two-hybrid assays and pull-down exper-iments and analysing several PR and ER mutants we haveidentified two domains in the N-terminal half of PR B, whichdirectly interact with the ligand-binding domain of ER! [1].Fig. 3A resumes the molecular interaction between PR, ER!and c-Src. Coimmunoprecipitation experiments showed thata small fraction of PR is forming a complex with ER! invivo in untreated T47D cells (Fig. 3B, left panel). BothER-interacting domains (ERID-I and ERID-II) in PR B areneeded for the interaction with ER! in vivo, since the deletionof ERID-I or -II avoided the coimmunoprecipitation of PRand ER! (Fig. 3B, right panel). Moreover, neither the indi-vidual PR deletion mutants (PR !ERID-I or PR !ERID-II),nor the double mutant (PR !ERID I + II), were able to medi-ate the activation of Erk in response to progestins in a MAPkinase activity assay (Fig. 3C).

Coimmunoprecipitation experiments performed in uterinestromal cells UIII lacking ER! showed that in the absence ofhormone a fraction of PR form a complex with ER" (Fig. 3D),suggesting that in these cells the activation of signal cascades

C. Ballare et al. / Journal of Steroid Biochemistry & Molecular Biology 102 (2006) 2–10 5

Fig. 1. R5020 activates the MAPK Erk1/2 and PI3K/Akt pathways in breast cancer and endometrial cells. (A) R5020 activates the Erk1/2 pathway in T47Dcells: T47D cells were cultured in medium deprived of steroids for 48 h, followed by 24 h in serum-free conditions, prior to the addition of 10#8 M R5020 (R)for 5 or 10 min. When indicated, 1 h before hormonal induction cells were pretreated with 50 #M PD 98059 (PD), 10 #M ICI 182.780 (ICI) or 1 #M RU486(RU). Cell lysates were analysed by Western blotting with antibodies against phosphorylated Erk1/2 (pErk1/2) and total Erk2 (upper panel) or phosphorylatedMsk1 (pMsk1) and total Msk (lower panel). T0: time zero. (B) R5020 activates the PI3K/Akt pathway in T47D cells: Cells were treated as described in (A).Cell lysates were analysed by Western blotting with antibodies against phosphorylated Akt (pAkt) and total Akt (upper panel) or phosphorylated GSK3 !/"(pGSK3 !/") and tubulin (lower panel). (C) R5020 activates Erk1/2 in UIII cells: UIII cells were cultured in medium deprived of steroids for 48 h, followedby 48 h in serum-free conditions, prior to the addition of 10#8 M R5020 (R) for 5 or 10 min. When indicated (lower panel), 30 min before hormonal inductioncells were pretreated with 50 #M PD or 10 #M ICI or 1 #M RU. Cell lysates were analysed by Western blotting with antibodies against pErk1/2 and total Erk2.(D) R5020 activates Akt in UIII cells: UIII cells were treated as described in (C). R5020 was added at concentration 10#11 M. Cell lysates were analysed byWestern blotting with antibodies against pAkt and total Akt. (E) Dose dependence: UIII cells were cultured as described in (C) and incubated 5 min with eithervehicle (0) or different concentrations of R5020 (10#13 to 10#7 M), followed by Western blot analysis using antibodies against pErk1/2 and total Erk2 (upperpanel) or against pAkt and total Akt (lower panel).

by progestins is mediated by the interaction PR and ER". Theexistence of a complex PR-ER" is consistent with immuno-cytochemistry results of intracellular location of PR and ER",showing that both receptors co-localize mainly in cytoplasmof UIII cells [27].

3.3. Activation of the Erk and the PI3K/Akt pathways isrequired for progestin-induced cell proliferation

Estrogen and progesterone are critical hormones for mam-mary gland growth and are implicated in the development andprogression of breast cancer [20]. Treatment of T47D cells

with 10 nM R5020 induced a consistent increase in DNA syn-thesis measured by BrdU incorporation (Fig. 4A) and thiseffect was completely blocked by the specific antagonists ofPR and ER, as well as inhibitors of the Erk pathway, PD andthe PI3K/Akt pathway, Wortmannin. Thus, Erk and PI3K/Aktactivation via crosstalk between PR and ER! is essential forthe stimulatory effect of progestin on cell proliferation inbreast cancer cells.

We also explored the participation of signaling cascadesin the proliferative response of the endometrial stromal cellsto progesterone. Our results showed that even picomolar con-centrations of R5020 induced cell proliferation measured by

6 C. Ballare et al. / Journal of Steroid Biochemistry & Molecular Biology 102 (2006) 2–10

Fig. 2. PR and ER expression in T47D and UIII cells. Western blot analysis of cell extracts were performed using specific antibodies against PR (A, leftpanel), ER! (B) and ER" (C). The amount of protein extract used is indicated under each lane. (A) Right panel: The expression of PR was determined byimmunofluorescence in UIII and T47D cells with an antibody against PR (Santa Cruz). Fluorescence images were registered by confocal laser microscopy system.

increase in number of cells (Fig. 4B and [27]) or in DNAsynthesis (data not shown), This proliferative response wasErk and PI3K/Akt dependent and mediated by the classicalPR and ER, since it was completely abrogated by PD, WM,RU and ICI (Fig. 4B).

We have then tested the transcriptional competence ofthe endogenous low levels of PR in UIII cells. For this,we performed transient transfection studies with a reporterplasmid carrying the mouse mammary tumor virus (MMTV)promoter fused to the luciferase gene. R5020 did not trans-activate the MMTV promoter at concentrations going from10#11 to 10#8 M (Fig. 5A, white bars). Under the same condi-tions, luciferase was strongly induced in T47D cells (Fig. 5B).The lack of response in UIII was not due to limiting factorsother than PR, since the reporter gene was induced by R5020when an expression vector for PR B was co-transfected(Fig. 5A, grey bars). Similar experiments with three othertransfected reporters gave the same results, showing that thelack of transcriptional activation by the endogenous PR wasnot promoter specific [27]. Whereas the induction was opti-mal at 10#8 M of R5020, there was no response at 10#11 M(Fig. 5A), a concentration sufficient for optimal activation ofkinase cascades (Fig. 1E) and for triggering cell proliferation(Fig. 4B). We conclude that in UIII cells the proliferativeeffects of progestins required activation of kinase cascadesand are independent of direct genomic effects of PR.

Together, our data illustrated that in both model systems,breast cancer and endometrial stromal cells, progesteroneinduces rapid activation of Erk and PI3K/Akt signaling cas-cades, and these pathways are essential for triggering pro-liferative response. However, the biological outcome of pro-gestin treatment in both cellular types is different. In UIIIcells, progesterone regulates selected target genes involvedin cell cycle control and proliferation (Vallejo, manuscript inpreparation). Treatment of these cells with progestins inducessustained proliferation with a consistent increase in the num-ber of cells. Conversely, in breast cancer cells the response

is more complex and still controversial. Studies using humanbreast cancer cell lines have shown a biphasic effect of pro-gestins on cell growth, with an initial proliferation burst,followed by an arrest of the cells in late G1 phase of the sec-ond cycle. During the early phase several genes associatedwith cell cycle progression are induced, as cyclin D1, D3, E,A and B, myc and fos, and there is an acceleration of cellsthrough one mitotic cycle. The later arrest is accompaniedby decreased levels of cyclins D1, D3 and E, disappear-ance of cyclins A and B, and sequential induction of thecyclin-dependent kinase (cdk) inhibitors p21 and p27Kip1[14,15].

Several reasons could account for the different outcomesto progesterone treatment in breast cancer and uterine stro-mal cells. The different ability of PR to mediate “genomiceffects” might be one of the main explanations. Direct tran-scriptional action of PR could be required for the inductionof several genes in response to progestins. Moreover, resultsfrom our group and others [4,31] show that an importantproportion of the hormone responsive genes need the par-ticipation of both, “genomic” and “non-genomic pathwaysfor their induction. On the other hand, differences in amountand subcellular location of PR and ER could be important todetermine which pathways are activated in response to thehormone. The presence of ER! or ER" and the participationof other alternative membrane receptors also involved in acti-vation of signaling pathways [13] might also be significantfor the cellular response. Further studies will be necessaryto clarify the relevance of each one of these factors in thebiological response to progestins.

3.4. Activated Erk1/2 forms a complex with PR, isrecruited to the promoter of a reporter gene and isrequired for hormonal induction

We next explored the connection between rapid kinaseactivation and the transcriptional effects of steroid hormones

C. Ballare et al. / Journal of Steroid Biochemistry & Molecular Biology 102 (2006) 2–10 7

Fig. 3. Interaction between PR and ER. (A) Schematic representation of the molecular interactions between PR, ER" and c-Src: The domain structure of humanPR B and ER! is indicated. The activation functions (AF1, AF2 and AF3), the inhibitory domain (IF), the DNA binding domain (DBD) and the ligand-bindingdomain (LBD) are indicated. The numbers refer to the aminoacid residues. The ER-interacting domains ERID-I (aminoacids 165–345) and ERID-II (aminoacids456–546) in PR, the proline cluster (pro cluster) of PR responsible to the interaction with the SH3 domain of Src, as well as the phospho-Tyr537 in ER! whichmediates the interaction with the SH2 domain of Src [17] are also indicated. (B) Interaction between PR and ER" in vivo: left panel: cell lysates of T47D wereimmunoprecipitated with antibodies against ER!, PR or non-specific IgG (control: ctr). The immunoprecipitates (IPs) and input were analysed by Westernblotting with antibodies against ER! and PR. Right panel: COS-7 cells were transiently cotransfected with expression vectors for ER! and PR B wild type orthe deletion mutants PR !ERID I, PR !ERID II and PR !ERID I + II. The expression of ER! and the PR B constructs was verified by immunoblotting of totalcell lysates with specific antibodies (lanes 1–4). Cell lysates were immunoprecipitated with anti-ER! antibody and the IPs were analysed by Western blottingwith antibodies against ER! and PR (lanes 5–8). (C) Effect of progestin R5020 on Erk 1/2 activity: Cos-7 cells transfected as indicated in (B) were treated for10 min with 10#8 M R5020 or with vehicle (ethanol: –). Cell lysates were prepared, immunoprecipitated with anti-Erk antibodies and assayed for MAP kinaseactivity with MBP as a substrate and [$-32P] ATP. (D) Interaction between PR and ER# in UIII cells: Lysates of UIII cells were immunoprecipitated withantibodies against PR or non-specific IgG (control: ctr). The immunoprecipitates (IP) and inputs were analysed by Western blotting with antibodies against PRand ER".

in breast cancer. For this, we studied progestin transacti-vation of the MMTV promoter in the T47D-MTVL breastcancer cell line, which carries an integrated single copy ofthe MMTV-LTR promoter (Fig. 6A) driving the luciferasegene [26]. The MMTV promoter is transcriptionally activatedby progestins via an hormone responsive region includinga cluster of degenerated HREs and an adjacent site for theubiquitous transcription factor nuclear factor 1 (NF1) [3].

This region of the MMTV promoter is wrapped around apositioned nucleosome [21], which upon hormone induc-tion becomes hypersensitive to nucleases [26] due to theselective displacement of histone H2A and H2B dimers[28].

After progestin treatment of T47D-MTVL cells weobserved a strong induction of luciferase activity (Fig. 6B).Treatment of the cells with PD or ICI did not influence the

8 C. Ballare et al. / Journal of Steroid Biochemistry & Molecular Biology 102 (2006) 2–10

Fig. 4. Activation of the Erk1/2 and PI3K/Akt pathways is essential forprogestin-induced cell proliferation. (A) Inhibition of Erk or PI3K/Akt path-ways prevents the progesterone-induced increase in DNA synthesis in T47Dcells: Serum starved T47D cells were treated with 10#8 M R5020 (R) orvehicle (ethanol: –) for 15 h. When indicated, 1 h before hormonal inductioncells were pretreated with 10 #M ICI or 1 #M RU or 50 #M PD or 0.1 #MWortmannin. BrdU 10 #M was added during the last 2 h of hormone induc-tion. Cells were fixed, permeabilised and BrdU incorporation was evaluatedby indirect immunofluorescence, followed by FACs analysis. The valuesrepresent the mean and standard deviation from two experiments performedin duplicate. (B) Progestin induction of UIII cell proliferation is mediated byErk and PI3K/Akt pathways and requires the classical PR and unligandedER: Serum starved UIII cells were incubated with 10#11 M R5020 or vehicle(ethanol: –) as indicated in Section 2. Cell number was determined after 5days of culture. When indicated 50 #M PD, 10#9 M WM, 10 #M ICI or 1 #MRU were added 30 min before hormonal induction. The values represent themean and standard deviation from at least three independent experiments.***P < 0.001 vs. control (black column).

basal MMTV transcription but strongly decreased hormoneinduction. Moreover, the induction of a significant fractionof endogenous progestin-target genes was also compromisedby blocking the Erk pathway [31]. None of the inhibitors hada significant effect on transcription from the "-actin promoterused as control (data not shown). We conclude that activationof the Erk1/2 pathway, likely via an interaction of ligand-activated PR with ligand-free ER, is required for hormoneinduction of the MMTV promoter and other progesterone-target genes.

We next investigated whether PR and Erk1/2 form a com-plex and how this is affected by hormone treatment. Coim-munoprecipitation experiments in T47D cells showed that

Fig. 5. Transcriptional activity of PR in UIII and T47D cells. (A) The PRof UIII is transcriptional incompetent: An MMTV-Luc reporter plasmidcarrying the MMTV promoter linked to the luciferase gene was transientlycotransfected with the PRB expression plasmid pSG5:PR (grey columns) orthe empty plasmid pSG5 (white columns). Cells were incubated for 36 h withvehicle (0) or with the indicated concentrations of R5020 (10#11 to 10#8 M).When indicated 10#8M RU was added. Luciferase activity was determinedin whole extracts. The mean and standard deviation of two independentexperiments performed in duplicate are shown. (B) Transcriptional activityof PR in T47D cells: T47D cells cultured in medium depleted of steroids wereincubated for 36 h with different concentrations of R5020 (10#11 to 10#7 M).Luciferase activity was determined in whole extracts. The values representmean and standard deviation of two independent experiments performed induplicate.

PR did not bind to Erk in untreated cells but already after5–10 min of hormone treatment both proteins co-precipitatedas a complex (Fig. 6C). Formation of this complex requiredErk activation and ligand-free ER, since PD and ICI inhibitedPR-Erk interaction.

Using chromatin immunoprecipitation (ChIP) experi-ments we have analysed changes taking place at nucleosomeB of the MMTV promoter during the initial phase of hormoneinduction. We observed that already 5 min after progestinaddition PR and the activated form of Erk and Msk1 wererecruited to the MMTV promoter [31]. Inhibition of Erkactivation by PD hinders hormone-induced recruitment ofthe kinases, as well as recruitment of chromatin remodelingcomplexes and RNA polymerase II. Using nucleosome res-olution ChIP assays we found that the recruitment of PR,

C. Ballare et al. / Journal of Steroid Biochemistry & Molecular Biology 102 (2006) 2–10 9

Fig. 6. Activated Erk1/2 forms a complex with PR and is required for hormonal induction of the MMTV promoter. (A) Schematic representation of nucleosomepositioning of the MMTV-LTR in the T47D-MTVL cell line. The HREs, the binding site for NF1 and the position of nucleosome B are indicate. (B) ActivatedErk pathway is required for transactivation of the MMTV promoter by progestins: T47D cells cultured as indicated in Fig. 1A were incubated for 24 h with10#8 M R5020 (R) or vehicle (–). When indicated, 1 h before hormonal induction cells were pretreated with 50 #M PD or 10 #M ICI. Luciferase activity wasdetermined in total extracts. The values represent the mean and standard deviation from two experiments performed in duplicate. (C) R5020 induces interactionbetween Erk and PR: T47D cells cultured as indicated in Fig. 1A were incubated for 10 min with 10#8 M R5020 (R) or vehicle (–). When indicated, 1 h beforehormonal induction cells were pretreated with 50 #M PD (R + PD) or 10 #M ICI (R + ICI). Left panel: Cell lysates were immunoprecipitated with an antibodyagainst Erk2, the immunoprecipitates (IP) were analysed by Western blotting with antibodies against Erk-2 and against PR. Right panel: Western blot analysisof the inputs (2.5% of cell lysate used for immunoprecipitation) performed with !-PR antibody.

Erk and Msk was limited to nucleosome containing HREs,supporting a PR-mediated targeting. A targeted recruitmentof activated kinases to the MMTV promoter could play anessential role in preparing the promoter chromatin for geneexpression. Msk mediated phosphorylation of chromatin pro-teins, as histone H3, has been associated with gene inductionof early genes by mitogenic stimuli [24]. Phosphorylation ofhistone H3 could promote recruitment of chromatin remod-eling complexes that mediates removal of H2A/H2B dimers,thus allowing binding of further PR molecules and NF1, co-activators and the basal transcriptional machinery includingthe RNA polymerase [29,31]. Our results establish a noveland direct connection between rapid signaling by steroid hor-mones to kinases cascades ant their transcriptional effects inthe nucleus.

Acknowledgments

We wish to thank Nora Spinedi and A. Silvina Nacht,CRG, for technical assistance. The experimental work wassupported by grants from the Departament d’UniversitatsRecerca i Societat de la Informacio (DURSI), Ministerio deEducacion y Ciencia (MEC) BMC 2003, 02902, Fondo deInvestigacion Sanitaria (FIS) PI0411605 and CP04/00087,Universidad de Buenos Aires (UBACYT 2004-2007 X318),Agencia Nacional de Promocion Cientıfica y Tecnologica(PICT 0005-09044) Argentina, and Consejo Nacional deInvestigaciones Cientificas y Tecnicas (PIP 2005-2006 Nro5348). P.S. is an established investigator from the CONICETand had a fellowship from the Departament d’UniversitatsRecerca i Societat de la Informacio (DURSI). G.P.V. was a

10 C. Ballare et al. / Journal of Steroid Biochemistry & Molecular Biology 102 (2006) 2–10

recipient of a fellowship of the Ramon y Cajal Programme.G.V. was the recipient of a CONICET predoctoral fellowship.

References

[1] C. Ballare, M. Uhrig, T. Bechtold, E. Sancho, M. Di Domenico, A.Migliaccio, F. Auricchio, M. Beato, Two domains of the progesteronereceptor interact with the estrogen receptor and are required for proges-terone activation of the c-Src/Erk pathway in mammalian cells, Mol.Cell. Biol. 23 (2003) 1994–2008.

[2] M.J. Barratt, C.A. Hazzalin, E. Cano, L.C. Mahadevan, Mitogen-stimulated phosphorylation of histone H3 is targeted to a smallhyperacetylation-sensitive fraction, Proc. Natl. Acad. Sci. U.S.A. 91(1994) 4781–4785.

[3] M. Beato, P. Herrlich, G. Schutz, Steroid hormone receptors: manyactors in search of a plot, Cell 83 (1995) 851–857.

[4] L. Bjornstrom, M. Sjoberg, Mechanisms of estrogen receptor signaling:convergence of genomic and nongenomic actions on target genes, Mol.Endocrinol. 19 (2005) 833–842.

[5] V. Boonyaratanakornkit, M.P. Scott, V. Ribon, L. Sherman, S.M. Ander-son, J.L. Maller, W.T. Miller, D.P. Edwards, Progesterone receptorcontains a proline-rich motif that directly interacts with SH3 domainsand activates c-Src family tyrosine kinases, Mol. Cell 8 (2001) 269–280.

[6] G. Castoria, A. Migliaccio, A. Bilancio, M. Di Domenico, A. de Falco,M. Lombardi, R. Fiorentino, L. Varricchio, M.V. Barone, F. Auricchio,PI3-kinase in concert with Src promotes the S-phase entry of oestradiol-stimulated MCF-7 cells, EMBO J. 20 (2001) 6050–6059.

[7] P. Cheung, K.G. Tanner, W.L. Cheung, P. Sassone-Corsi, J.M. Denu,C.D. Allis, Synergistic coupling of histone H3 phosphorylation andacetylation in response to epidermal growth factor stimulation, Mol.Cell 5 (2000) 905–915.

[8] C.L. Clarke, R.L. Sutherland, Progestin regulation of cellular prolifer-ation, Endocr. Rev. 11 (1990) 266–301.

[9] H. Cohen, J.F. Pageaux, C. Melinand, J.M. Fayard, C. Laugier, Normalrat uterine stromal cells in continuous culture: characterization andprogestin regulation of growth, Eur. J. Cell Biol. 61 (1993) 116–125.

[10] P. Cohen, S. Frame, The renaissance of GSK3, Nat. Rev. Mol. CellBiol. 2 (2001) 769–776.

[11] D.A. Cross, D.R. Alessi, P. Cohen, M. Andjelkovich, B.A. Hemmings,Inhibition of glycogen synthase kinase-3 by insulin mediated by proteinkinase B, Nature 378 (1995) 785–789.

[12] J.A. Diehl, M. Cheng, M.F. Roussel, C.J. Sherr, Glycogen synthasekinase-3beta regulates cyclin D1 proteolysis and subcellular localiza-tion, Genes Dev. 12 (1998) 3499–3511.

[13] D.P. Edwards, Regulation of signal transduction pathways by estrogenand progesterone, Annu. Rev. Physiol. 67 (2005) 335–376.

[14] S.D. Groshong, G.I. Owen, B. Grimison, I.E. Schauer, M.C. Todd,T.A. Langan, R.A. Sclafani, C.A. Lange, K.B. Horwitz, Biphasic regu-lation of breast cancer cell growth by progesterone: role of the cyclin-dependent kinase inhibitors, p21 and p27(Kip1), Mol. Endocrinol. 11(1997) 1593–1607.

[15] C.A. Lange, J.K. Richer, K.B. Horwitz, Hypothesis: progesteroneprimes breast cancer cells for cross-talk with proliferative or antipro-liferative signals, Mol. Endocrinol. 13 (1999) 829–836.

[16] L.C. Mahadevan, A.C. Willis, M.J. Barratt, Rapid histone H3 phospho-rylation in response to growth factors, phorbol esters, okadaic acid, andprotein synthesis inhibitors, Cell 65 (1991) 775–783.

[17] A. Migliaccio, G. Castoria, M. Di Domenico, A. de Falco, A. Bilancio,M. Lombardi, M.V. Barone, D. Ametrano, M.S. Zannini, C. Abbon-

danza, F. Auricchio, Steroid-induced androgen receptor-oestradiolreceptor beta-Src complex triggers prostate cancer cell proliferation,EMBO J. 19 (2000) 5406–5417.

[18] A. Migliaccio, M. Di Domenico, G. Castoria, A. de Falco, P. Bontempo,E. Nola, F. Auricchio, Tyrosine kinase/p21ras/MAP-kinase pathwayactivation by estradiol-receptor complex in MCF-7 cells, EMBO J. 15(1996) 1292–1300.

[19] A. Migliaccio, D. Piccolo, G. Castoria, M. Di Domenico, A. Bilan-cio, M. Lombardi, W. Gong, M. Beato, F. Auricchio, Activation of theSrc/p21ras/Erk pathway by progesterone receptor via cross-talk withestrogen receptor, EMBO J. 17 (1998) 2008–2018.

[20] A.M. Raafat, S. Li, J.M. Bennett, L.J. Hofseth, S.Z. Haslam, Estro-gen and estrogen plus progestin act directly on the mammary gland toincrease proliferation in a postmenopausal mouse model, J. Cell Phys-iol. 187 (2001) 81–89.

[21] H. Richard-Foy, G.L. Hager, Sequence-specific positioning of nucleo-somes over the steroid-inducible MMTV promoter, EMBO J. 6 (1987)2321–2328.

[22] M. Saitoh, M. Ohmichi, K. Takahashi, J. Kawagoe, T. Ohta, M.Doshida, T. Takahashi, H. Igarashi, A. Mori-Abe, B. Du, S. Tsutsumi,H. Kurachi, Medroxyprogesterone acetate induces cell proliferationthrough up-regulation of cyclin D1 expression via phosphatidylinosi-tol 3-kinase/Akt/nuclear factor-kappaB cascade in human breast cancercells, Endocrinology 146 (2005) 4917–4925.

[23] A. Soloaga, S. Thomson, G.R. Wiggin, N. Rampersaud, M.H. Dyson,C.A. Hazzalin, L.C. Mahadevan, J.S. Arthur, MSK2 and MSK1 mediatethe mitogen- and stress-induced phosphorylation of histone H3 andHMG-14, EMBO J. 22 (2003) 2788–2797.

[24] A. Soloaga, S. Thomson, G.R. Wiggin, N. Rampersaud, M.H. Dyson,C.A. Hazzalin, L.C. Mahadevan, J.S. Arthur, MSK2 and MSK1 mediatethe mitogen- and stress-induced phosphorylation of histone H3 andHMG-14, EMBO J. 22 (2003) 2788–2797.

[25] S. Thomson, A.L. Clayton, C.A. Hazzalin, S. Rose, M.J. Barratt, L.C.Mahadevan, The nucleosomal response associated with immediate-early gene induction is mediated via alternative MAP kinase cascades:MSK1 as a potential histone H3/HMG-14 kinase, EMBO J. 18 (1999)4779–4793.

[26] M. Truss, J. Bartsch, A. Schelbert, R.J.G. Hache, M. Beato, Hormoneinduces binding of receptors and transcription factors to a rearrangednucleosome on the MMTV promoter in vivo, EMBO J. 14 (1995)1737–1751.

[27] G. Vallejo, C. Ballare, J.L. Baranao, M. Beato, P. Saragueta, Pro-gestin activation of nongenomic pathways via cross talk of pro-gesterone receptor with estrogen receptor beta induces proliferationof endometrial stromal cells, Mol. Endocrinol. 19 (2005) 3023–3037.

[28] G.P. Vicent, A.S. Nacht, C.L. Smith, C.L. Peterson, S. Dimitrov, M.Beato, DNA instructed displacement of H2A and H2B at an induciblepromoter, Mol. Cell 16 (2004) 439–452.

[29] G.P. Vicent, A.S. Nacht, C.L. Smith, C.L. Peterson, S. Dimitrov, M.Beato, DNA instructed displacement of histones H2A and H2B at aninducible promoter, Mol. Cell 16 (2004) 439–452.

[30] Z. Zhang, C. Funk, S.R. Glasser, J. Mulholland, Progesterone regula-tion of heparin-binding epidermal growth factor-like growth factor geneexpression during sensitization and decidualization in the rat uterus:effects of the antiprogestin, ZK 98.299, Endocrinology 135 (1994)1256–1263.

[31] G.P. Vicent, C. Ballare, A.S. Nacht, J. Clausell, A. Subtil-Rodrigez, I.Quiles, A. Jordan, M. Beato, Induction of progesterone target genesrequires activation of Erk and Msk kinases and phosphorylation ofhistone H3, Mol. Cell, in press.