r e p r o d u c t i v e b i o l o g y 1 3 ( 2 0 1 3 ) 1 1 3 – 1 2 1

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Original Research Article

Constitutive activity of Erk1/2 and NF-kB protects humanendometrial stromal cells from death receptor-mediatedapoptosis

Herbert Fluhr *, Julia Spratte, Marike Bredow, Stephanie Heidrich, Marek Zygmunt

Department of Obstetrics and Gynecology, University of Greifswald, Sauerbruchstr., 17475 Greifswald, Germany

a r t i c l e i n f o

Article history:

Received 17 December 2012

Accepted 4 March 2013

Keywords:

Apoptosis

Death receptor

Endometrium

Erk1/2

NF-kB

a b s t r a c t

Apoptosis in the human endometrium plays an essential role for endometrial receptivity

and early implantation. A dysbalance of pro- and anti-apoptotic events in the secretory

endometrium seems to be involved in implantation disorders and consecutive pregnancy

complications. However, little is known about the mechanisms regulating apoptosis-sensi-

tivity in the human endometrium. Therefore this studywas performed to identifymolecular

mechanisms underlying the resistance toward apoptosis in human endometrial stromal

cells (ESCs). Human ESCs were isolated from hysterectomy specimens and used as undif-

ferentiated cells or after decidualization in vitro. Cells were incubated with an activating

anti-Fas antibody, tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL),

TNF-a and inhibitors of protein- and RNA-syntheses, a caspase-inhibitor and inhibitors of

extracellular signal regulated kinase (Erk)1/2, nuclear factor (NF)-kB and Akt. Apoptosis was

measured by flow cytometric detection of hypodiploid nuclei. Caspase-activity was detected

by luminescencent assays. Several pro- and anti-apoptotic molecules and the activation of

Erk1/2, NF-kB and Akt were analyzed by in-cell Western assays or flow cytometry. Inhibition

of protein- and RNA-syntheses differentially sensitized human ESCs for death receptor-

mediated apoptosis in a caspase-dependent manner, based on the up-regulation of the

death receptors Fas and TRAIL-R2. The constitutive activity of Erk1/2 and NF-kB could be

identified as a reason for the apoptosis-resistance of human ESCs. These results suggest the

pro-survival signaling pathways Erk1/2 and NF-kB as key regulators of the sensitivity of

human ESCs for death receptor-mediated apoptosis. The modulation of these pathways

might play an important role in the physiology of implantation.

# 2013 Society for Biology of Reproduction & the Institute of Animal Reproduction and

Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban &

Partner Sp. z o.o. All rights reserved.

* Corresponding author. Tel.: +49 3834 866500; fax: +49 3834 866501.E-mail addresses: fluhr@uni-greifswald.de, herbert.fluhr@uni-greifswald.de (H. Fluhr).Abbreviations: Bcl, B-cell lymphoma/leukemia; FLIP, FLICE-inhibitory protein; TNF, tumor necrosis factor; TRAIL, TNF-related apopto-

sis-inducing ligand; XIAP, X-inked inhibitor of apoptosis protein.1642-431X/$ – see front matter # 2013 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research ofPolish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.http://dx.doi.org/10.1016/j.repbio.2013.03.001

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1. Introduction

The regulation of endometrial apoptosis plays an importantrole for tissue homeostasis as well as tissue remodeling duringthemenstrual cycle, therebypreparing theendometriumfor theimplantation of an embryo [1]. Apoptosis-inducing molecules,especially the death receptor-stimuli Fas-ligand (FasL), tumornecrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)and TNF-a, are present at the feto-maternal interface and seemto be involved in the process of early implantation [1–6]. Theinteraction of these stimuli with their respective deathreceptors (Fas, TRAIL-R1, -R2, TNF-aR1) typically leads to thesequential activation of caspases and finally results in apopto-sis. Moreover, depending on the type of cell and tissue,stimulation of death receptors can also activate non-apoptoticsignaling pathways and transcription factors, such as extracel-lular signal regulated kinase (Erk)1/2, nuclear factor (NF)-kB orphosphatidylinositol-3 kinase (PI3K)/Akt-signaling [7–10].

Several intracellular anti-apoptotic molecules are known toregulate the process of programmed cell death on differentlevels of the apoptotic signaling cascade. For example, XIAP (X-linked inhibitor of apoptosis protein) and survivin inhibit theactivity of caspases, including the effector-caspase-3 [11]. Bcl-2(B-cell lymphoma/leukemia-2) and Bcl-xL are inhibiting apo-ptosis on the mitochondrial level and FLIP (FLICE-inhibitoryprotein) interacts with caspase-8-activation at the deathreceptor-complex [11]. Insomecell types resistance toapoptosishasbeenshowntobeanactivemetabolicprocess,dependingonde novo RNA- and protein-syntheses [12]. Based on theseobservations, several signaling pathways could be identified aspro-survival factors, such asmitogen-activated protein kinases(MAPKs), NF-kB or PI3K/Akt [13]. We have previously observed,that human endometrial stromal cells (ESCs) are resistant toFas- and TRAIL-mediated apoptosis, although the respectivereceptors aswell as essential apoptosis-molecules like caspasesare expressed and functional in these cells [14–16]. In addition,stimulation of death-receptors in human ESCs regulates thesecretion of cytokines and chemokines in a caspase-dependentnon-apoptotic manner [16]. The primary resistance to Fas--mediated apoptosis in ESCs can be overcome by the combinedtreatment with the inflammatory molecules TNF-a andinterferon (IFN)-g due to an up-regulation of Fas [14].

Based on these observations we investigated themolecularmechanisms responsible for the primary resistance of humanESCs to death receptor-mediated apoptosis. Therefore, wetested the influence of general metabolic inhibitors as well asthe inhibition of single signaling pathways on the sensitivity ofthe cells for Fas, TRAIL- and TNF-a-mediated apoptosis. Inparallel we characterized the role of caspases as well as pro-- and anti-apoptotic molecules in human ESCs.

2. Materials and methods

2.1. Tissue collection and cell culture

Endometrial tissue sampleswere obtained frompremenopaus-al women undergoing hysterectomy for benign reasons afterwritten informed consent. The study protocol was approved by

the institutional ethical board of the University of Greifswald,Germany. ESCs were isolated, cultured and characterizedfollowing a standard protocol [16]. Briefly, minced endometrialtissue was digested by incubation with collagenase (Biochrom,Berlin, Germany) and the dispersed endometrial cells wereseparatedbyfiltration. ESCsweremaintained inDMEM/F-12cellculture medium without phenol red (Gibco/Invitrogen, Karls-ruhe, Germany) containing 10% charcoal-stripped fetal bovineserum (Biochrom) and 50 mg/ml gentamycin (Ratiopharm, Ulm,Germany). The purity of ESC cultures was proven by standardimmunofluorescent staining of vimentin.

2.2. Decidualization in vitro and experimental conditions

ESCsweredecidualized invitroby incubating thecellswith 1 mMprogesteroneand30 nM17b-estradiol(bothfromSigma–Aldrich,Taufkirchen, Germany) for 9 days. Decidualization was provenby measuring a significantly increased secretion of insulin-likegrowth factor binding protein (IGFBP)-1 and prolactin [17].Decidualized as well as undifferentiated ESCs were incubatedwith the following agents: cycloheximide (CHX; Calbiochem/Merck, Nottingham, UK), actinomycin D (ActD; Biomol, Ham-burg, Germany), activating anti-Fas antibody (cloneCH-11; MBL,Woburn, MA, USA), recombinant human TRAIL (Alexis Bio-chemicals, Lörrach, Germany), recombinant human TNF-a(Biosource, Camarillo, CA, USA), Z-VAD-FMK or Z-FA-FMK (bothfrom BD Biosciences, Heidelberg, Germany), etoposide (Sigma–Aldrich), staurosporine (Sigma–Aldrich), PD98059 (Cell SignalingTechnology, Danvers, MA, USA), parthenolide (Calbiochem/Merck), wortmannin (Calbiochem/Merck) recombinant humanepidermal growth factor (EGF; Biomol) and insulin (Actrapid®,Novo Nordisk Pharma, Mainz, Germany). The different combi-nations, dosages and incubation times of thementioned agentsare indicated in detail in the corresponding figures.

2.3. Measurement of apoptosis

The rate of apoptotic nuclei was measured by the method ofNicoletti et al. [18]. Apoptotic nuclei were prepared by lysingthe cells in a hypotonic buffer (0.1% sodium citrate, 0.1% TritonX-100) containing 50 mg/ml propidiumiodide (Sigma–Aldrich)and subsequently analyzed by flow cytometry. Nuclei to theleft of the 2N peak containing hypodiploid DNA wereconsidered apoptotic. Measurements were performed on aFACSCantoTM flow cytometer using FACSDiva software foranalysis (both from BD Biosciences).

2.4. Caspase activity assays

The activity of caspases in ESCs was detected using theCaspase-Glo® assay system (Promega, Madison, WI, USA).Caspase-Glo®-substrates LEHD or DEVDwere added to the cellcultures followed by incubation for 60 min. Luminescence wasrecorded using the FLUOstar OPTIMA system (BMG Labtech,Offenburg, Germany).

2.5. In-cell Western assays

To quantify the levels of XIAP, survivin, Bcl-2, Bcl-xL, FLIP,phosphorylated (Thr202/Tyr204) Erk1/2, phosphorylated

[(Fig._1)TD$FIG]

Fig. 1 – Cycloheximide (CHX) and actinomycin D (ActD)sensitize human endometrial stromal cells (ESCs) to deathreceptor-mediated apoptosis. Undifferentiated (A) anddecidualized (B) ESCs were incubated with CHX (1 mg/ml),ActD (0.01 mg/ml), an activating anti-Fas antibody,recombinant human tumor-necrosis factor-a (TNF-a) orrecombinant human TNF-related apoptosis-inducingligand (TRAIL; 100 ng/ml in each case) alone and in theindicated combinations for 48 h. Thereafter, the rate of

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(Ser536) NF-kB p65 and phosphorylated (Ser473) Akt in ESCsweused an in-cell Western assay, based on immunofluorescentstaining performed in a 96-well microplate format. The cellswere fixed with 3.7% formaldehyde and permeabilized withice-coldmethanol. Blockingwith Odyssey® Blocking Buffer (LI--COR®, Lincoln, NE, USA) was followed by the incubation withthe respective primary antibodies (all from Cell SignalingTechnology) and IRDye®800CW conjugated secondary anti-bodies (LI-COR®). Normalization of each sample was per-formed by parallel DNA-staining using DRAQ5TM (BiostatusLimited, Shepshed, UK). The assays were visualized andanalyzed using the LI-COR® Odyssey imaging system (LI--COR®).

2.6. Flow cytometry

The expression of Fas, TRAIL-R1, -R2 and TNF-aR1 on the cellsurface of ESCs was determined by extracellular staining withspecific monoclonal antibodies (from eBioscience, Frankfurt,Germany and Alexis Biochemicals, Lörrach, Germany) and asecondary antibody conjugated with Alexa Fluor® 647 (Molec-ular Probes, Eugene, OR, USA). Staining was performedaccording to a standard protocol [14]. Each staining samplewas accompanied by a parallel staining with an unspecificisotype control (mouse IgG1, BD). A FACSCantoTM flowcytometer and FACSDiva software were used for measure-ments and analysis.

2.7. Statistics

Each experiment was performed in triplicate or quadruplicateon cell cultures derived from three to five different patients.Statistical analysis was carried out with one-way ANOVA,followed by Dunnett's and Bonferroni multiple comparisontests or unpaired Mann–Whitney t-tests using GraphPadPRISM version 5 software (GraphPad, San Diego, CA, USA).The results are expressed as mean � standard error of themean (SEM). Differences were considered to be significant ifp < 0.05.

apoptosis was measured by flow-cytometric detection ofhypodiploid nuclei. Bars represent meanW SEM; *p < 0.05compared to untreated ESCs.

3. Results

3.1. Metabolic inhibitors differentially sensitize ESCs fordeath receptor-mediated apoptosis

First of all, we investigated whether the resistance of humanESCs to death receptor-mediated apoptosis is an activemetabolic process requiring de novo protein or RNAsynthesis. Therefore, undifferentiated as well as decidua-lized human ESCs were incubated with the protein synthesisinhibitor CHX or the RNA synthesis inhibitor ActD incombination with death receptor-stimulation by an activat-ing anti-Fas antibody, TRAIL and TNF-a, followed by themeasurement of apoptosis. The concentrations of CHX andActD used in these and the following experiments have beentested in our cell culture model in preparation of this studyto have low toxicity but strong inhibitory effects on proteinand RNA synthesis. As shown in Fig. 1, undifferentiated(Fig. 1A) and decidualized (Fig. 1B) ESCs were sensitized to

Fas-mediated apoptosis by CHX and to Fas- and TRAIL--mediated apoptosis by ActD. However, there was nosensitization to TNF-a-induced apoptosis by none of thetwo inhibitors.

3.2. Caspases are mediating apoptosis of ESCs induced bymetabolic inhibition and death receptor-stimulation

We also tested whether the observed induction of apoptosisin ESCs under the influence of metabolic inhibitors anddeath receptor-stimuli is mediated by the activation ofcaspases. The cells were incubated with CHX or ActD incombination with an activating anti-Fas antibody, TRAILand TNF-a and the enzymatic activity of caspases wasmeasured using the caspase-substrates LEHD (Fig. 2A) and

[(Fig._2)TD$FIG]

Fig. 2 – Caspases are activated upon death-receptorstimulation in human endometrial stromal cells (ESCs)being sensitized by cycloheximide (CHX) and actinomycinD (ActD). Decidualized ESCs were incubated with CHX(1 mg/ml), ActD (0.01 mg/ml), an activating anti-Fasantibody, recombinant human tumor-necrosis factor-a(TNF-a) or recombinant human TNF-related apoptosis-inducing ligand (TRAIL; 100 ng/ml in each case) alone andin the indicated combinations for 24 h. Thereafter, theenzymatic activity of caspases was measured byluminescent activity assays using two different substratesLEHD (A) and DEVD (B). Results are standardized relative tothe values of untreated ESCs, which are set to 1 arbitraryunit. Bars represent meanW SEM; *p < 0.05 compared tountreated ESCs, #p < 0.05 compared to ESCs treated withanti-Fas only.

[(Fig._3)TD$FIG]

Fig. 3 – Caspases mediate death receptor-induced apoptosisin human endometrial stromal cells (ESCs) being sensitizedby cycloheximide (CHX) and actinomycin D (ActD).Decidualized ESCs were incubated for 30 min with thecaspase-inhibitor Z-VAD-FMK (20 mM) or the control Z-FA-FMK (20 mM) followed by the addition of CHX (1 mg/ml),ActD (0.01 mg/ml), an activating anti-Fas antibody orrecombinant human tumor-necrosis factor-relatedapoptosis-inducing ligand (TRAIL; 100 ng/ml in each case)and in the indicated combinations for another 48 h.Thereafter the rate of apoptosis was measured by flow-cytometric detection of hypodiploid nuclei. Bars representmeanW SEM; *p < 0.05 compared to untreated ESCs,#p < 0.05 compared to the respective conditions markedwith asterisks.

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DEVD (Fig. 2B). Fas-stimulation alone led to a (non-apoptotic)induction of caspase-activity in decidualized ESCs,which was significantly enhanced by the addition ofCHX or ActD (Fig. 2A and B). Under the influence of TRAILthere was an increase in caspase-activity only in thepresence of ActD, whereas TNF-a alone or in the combina-tion with the inhibitors did not stimulate caspase-activity(Fig. 2A and B).

To find out whether caspase activation is necessary for theinduction of apoptosis in this setting, the cells were incubatedwith the death-inducing agents plus/minus the generalcaspase-inhibitor Z-VAD-FMK as well as its unspecific controlZ-FA-FMK, followed by the measurement of apoptosis. Asshown in Fig. 3, inhibition of caspases completely abolishedthe apoptosis-inducing effects of Fas (plus CHX or ActD)- andTRAIL (plus ActD)-stimulation. Similar results regarding theactivation of caspases and their necessity for apoptosis wereseen in undifferentiated ESCs (data not shown).

3.3. Metabolic inhibitors modulate the apoptotic signaling-cascade in ESCs on the level of death receptors

Based on the results described above we wanted to furtherclarify the mechanism behind the observed apoptosis-sensitization of ESCs. In a first orientating step we wantedto see, whether metabolic inhibitors also cause an increasedsensitivity to stimulation of the intrinsic pathway of apopto-sis to see whether the sensitizing effects are up- ordownstream of the mitochondrium. Thus, ESCs were incu-bated with the topoisomerase-inhibitor etoposide – anactivator of the intrisic apoptotic pathway – alone and incombination with CHX or ActD. As shown in Fig. 4, etoposideinduces apoptosis in ESCs to a low extent, but this effect is notfurther enhanced by the addition of CHX or ActD. Similarresults were obtained in undifferentiated as well as decid-ualized ESCs.

[(Fig._4)TD$FIG]

Fig. 4 – Cycloheximide (CHX) and actinomycin D (ActD) donot further sensitize human endometrial stromal cells(ESCs) to etoposide-induced apoptosis. UndifferentiatedESCs were incubated with CHX (1 mg/ml), ActD (0.01 mg/ml),etoposide (100 mg/ml) alone and in the indicatedcombinations as well as staurosporine (2.5 mM) as positivecontrol for 48 h. Thereafter, the rate of apoptosis wasmeasured by flow-cytometric detection of hypodiploidnuclei. Bars represent meanW SEM; *p < 0.05 compared tountreated ESCs.

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In the next stepwe analyzed changes of relevantmoleculesin the apoptotic signaling-cascade under the influence ofmetabolic inhibitors. Hence, undifferentiated and decidua-lized ESCs were incubated with CHX and ActD, followed by themeasurement of the proteins listed in Table 1. In accordancewith the results of the previous experiment, testing etoposide,CHX and ActD had no impact on the anti-apoptotic moleculesXIAP, survivin, Bcl-2 and Bcl-xL, but upregulated the pro--apoptotic death receptors Fas and TRAIL-R2 (Table 1). Theseresultswere similar in undifferentiated anddecidualized ESCs.

3.4. Basal activity of Erk1/2 and NF-kB is involved in theresistance of ESCs to death receptor-mediated apoptosis

To provide further evidence that the primary resistance ofESCs to death receptor-mediated apoptosis is an active process

Table 1 – Impact of cycloheximide (CHX) or actinomycin D(ActD) on apoptosis-molecules.

CHX ActD

XIAP No effect No effectSurvivin No effect No effectBcl-2 No effect No effectBcl-xL No effect No effectFLIP No effect No effectFas Enhancement EnhancementTRAIL-R1 a a

TRAIL-R2 No effect EnhancementTNF-aR1 a a

a Barely detectable.

we tested whether typical pro-survival signaling pathways areinvolved. First of all we tested the functionality of thefollowing inhibitory substances in ESCs: PD98059 to inhibitErk1/2-signaling, parthenolide to inhibit NF-kB-signaling andwortmannin to inhibit Akt-signaling. The cells were incubatedwith these inhibitors alone or in combination with strongstimuli of the respective signaling pathways andmeasured theactivation (phosphorylation) of Erk1/2, NF-kB and Akt. Asshown in Fig. 5, the chosen substances effectively inhibited thebasal as well as stimulated activity of Erk1/2, NF-kB and Akt inESCs. Similar results were obtained in undifferentiated as wellas decidualized ESCs.

Based on these results we tested the impact of the abovementioned signaling-inhibitors on the sensitivity of ESCs fordeath receptor-stimulation. Therefore, the cells were incubat-ed with PD98059, parthenolide or wortmannin followed byFas-, TRAIL- and TNF-a-stimulation and the measurement ofapoptosis. As shown in Fig. 6, PD98059 sensitized ESCs for Fas--mediated apoptosis, whereas parthenolide additionally sen-sitized for TRAIL- and (only in undifferentiated ESCs) TNF-a--induced apoptosis. In addition, the incubation of theundifferentiated ESCs with parthenolide alone and decidua-lized ESCs with PD98059 plus parthenolide alone caused asignificant increase of apoptosis (Fig. 6A and B). Wortmanninalone or in combination with death receptor-stimulation hadno significant impact on the rate of apoptosis in undifferenti-ated and decidualized ESCs (data not shown).

To further clarify whether stimulation of death receptorsitself causes an activation of the signaling pathways involvedin the resistance of ESCs to apoptosis, we incubated the cellswith activating anti-Fas, TRAIL and TNF-a and measured theactivation of Erk1/2 and NF-kB. Neither Fas- nor TRAIL-stimulation activated Erk1/2 or NF-kB in undifferentiated(Fig. 7A and C) and decidualized (Fig. 7B and D) ESCs. Incontrast, TNF-a caused a weak activation of Erk1/2 (Fig. 7A andB) and a strong activation of NF-kB (Fig. 7C and D) in ESCs.

4. Discussion

In the present study, we observed constitutive activity of thesignaling pathways Erk1/2 and NF-kB as a underlyingmechanism of the previously described primary resistanceof human ESCs to death receptor-mediated apoptosis [14,15].Metabolic inhibitors such as CHX or ActD have been shown tosensitize different types of tumor cells for death receptor--mediated apoptosis [12,19,20]. As molecular mechanismsinvolved in this sensitization, the down-regulation of theanti-apoptoticmolecules FLIP and XIAP by CHX and ActDwasseen in neuroblastoma cells [12]. In accordance with thesestudies we also observed a sensitization to Fas- as well asTRAIL-mediated apoptosis in human ESCs, but we saw nochanges of the anti-apoptotic molecules XIAP and FLIP.However, inhibition of de novo RNA- and protein-synthesisin human ESCs caused an up-regulation of the deathreceptors Fas and TRAIL-R2. This observation is in line witha study reporting anup-regulationof Fas in the rat liver in vivoafter the applicationof CHX [21]. The lack of effects of CHXandActD on anti-apoptotic molecules interacting with theapoptotic cascade at the level of the mitochondrium (e.g.

[(Fig._6)TD$FIG]

Fig. 6 – Inhibition of extracellular signal regulated kinase 1/2(Erk1/2) and nuclear factor NF-kB sensitizes humanendometrial stromal cells (ESCs) to death receptor-mediated apoptosis. Undifferentiated (A) and decidualized(B) ESCs were incubated with the MEK-inhibitor, PD98059(50 mM) and/or the NF-kB-inhibitor (20 mM), parthenolidefor 1 h, followed by the addition of an activating anti-Fasantibody, recombinant human tumor-necrosis factor-a(TNF-a) or recombinant human TNF-related apoptosis-inducing ligand (TRAIL; 100 ng/ml in each case) in theindicated combinations for another 48 h. Thereafter, therate of apoptosis was measured by flow-cytometricdetection of hypodiploid nuclei. Bars representmeanW SEM; *p < 0.05 compared to untreated ESCs,#p < 0.05 compared to ESCs incubated with parthenolideonly, §p < 0.05 compared to ESCs treated with bothinhibitors only.

[(Fig._5)TD$FIG]

Fig. 5 – The constitutive and stimulated activity ofextracellular signal regulated kinase 1/2 (Erk1/2), nuclearfactor NF-kB and kinase Akt in human endometrial stromalcells (ESCs) can be strongly reduced by specific inhibitors.Undifferentiated ESCs were incubated with the mitogen-activated protein kinase kinase (MEK)-inhibitor, PD98059(50 mM), the NF-kB-inhibitor, parthenolide (20 mM) or thephosphatidyinositol 3-kinase (PI3K)-inhibitor, wortmannin(500 nM) for one hour followed by the addition of epidermalgrowthfactor (EGF;100 ng/ml), tumor-necrosis factor-a (TNF-a;100 ng/ml) or insulin (1 mM) alone and in the indicatedcombinations for 10 min. Thereafter, the induction of Erk1/2(A), NF-kB (B) and Akt (D) was measured by phopho-specificantibodies using in-cell Western assays. Bars representmeanW SEM; *p < 0.05 compared to untreated ESCs, #p < 0.05compared to stimulated (EGF, TNF-a, insulin) ESCs.

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Bcl-2 and Bcl-xL) or even downstream (e.g. XIAP and survivin)was further underlined by the observation that metabolicinhibitors did not sensitize human ESCs for etoposide as astimulus of the intrinsic apoptotic pathway. Similar resultshave been shown by others for different T and B cell lines [22].Caspase activation is often mentioned synonymously withapoptotic cell death. However, in addition to their initiallyreported role as executors of apoptosis these cysteineproteases are also able to mediate non-apoptotic signals[23,24]. For example, the initiator-caspase-8 is involved insyncytiotrophoblast formation and the differentiation ofmacrophages during early pregnancy and the effector--caspase-3 have been shown to be involved in osteogenic

[(Fig._7)TD$FIG]

Fig. 7 – Extracellular signal regulated kinase 1/2 (Erk1/2) and nuclear factor NF-kB are activated by tumor-necrosis factor-a(TNF-a) in human endometrial stromal cells (ESCs), but not by Fas- or TRAIL stimulation. Undifferentiated (A and C) anddecidualized (B and D) ESCs were incubated with an activating anti-Fas antibody, recombinant human TNF-relatedapoptosis-inducing ligand (TRAIL; 100 ng/ml) or recombinant human TNF-a (100 ng/ml in each case) for 10 min. Thereafter,the induction of Erk1/2 (A and B) and NF-kB (C and D) was measured by phopho-specific antibodies using in-cell Westernassays. Bars represent meanW SEM; *p < 0.05 compared to untreated ESCs.

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differentiation and the differentiation of keratinocytes [25].Additionally, we have previously shown that caspases areactivated upon stimulation of Fas andmediate non-apoptoticeffects on cytokines in human ESCs [16]. In the presentstudy we again detected non-apoptotic activation ofcaspases under the influence of Fas-stimulation, beingsignificantly enhanced and switched to an apoptotic effectin the presence of CHX or ActD. This observation suggeststhat the extent of caspase-activation in human ESCsdetermines the fate of the cells and therefore the regulatedsusceptibility to caspase-stimulation seems to be an impor-tant property of the human endometrium in the context ofphysiology versus pathophysiology.

According to this hypothesis, a significantly increasedactivity of caspase-3 and apoptotic cell death is seen in the latesecretory endometrium, being involved in the tissue remodel-ing in the transition to the proliferative phase [26,27]. Anincrease of apoptosis in the endometrial stroma is alsoreported under the influence of intrauterine levonorgestrel--releasing systems used as contraceptive device or to treatmenorrhagia [28]. High levels of apoptosis have been describedin the luteal out-of-phase endometrium from infertile patients

[29]. In addition, human chorionic gonadotropin (hCG) as wellas progesterone as physiologically essential hormones duringimplantation and early pregnancy and also used as lutealsupport in infertile patients have been shown to reduceendometrial apoptosis in vivo and in vitro [30,31]. In contrast, areduced sensitivity to apoptotic stimulation has been de-scribed in the ectopic endometrium from women sufferingfrom endometriosis [1].

Anti-apoptotic signaling plays an important role forcontinued growth and/or differentiation of cells and conse-quently the homeostasis of specialized tissues. Several pro-survival signaling pathways have been described to inhibitapoptosis, including the MAPK Erk1/2, NF-kB or PI3K/Akt [13].The resistance of HeLa-cells to Fas-mediated apoptosis is aconsequence of self-induced MAPK-activation [32]. BlockingNF-kB sensitizes primary glomerular mesangial cells to TNF--induced apoptosis, but not to other mediators including FasL[33]. Similar results have been seen in keratinocytes, beingsensitized to TNF- but not TRAIL-mediated apoptosis by theinhibition of NF-kB [34]. In first trimester trophoblast cells, thebasal activity of Akt is responsible for the resistance to Fas--mediated apoptosis and a similar mechanism is seen in

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murine hepatocytes [35,36]. By the use of selected specificinhibitors [37] we could identify Erk1/2- andNF-kB-signaling tobe involved in the resistance of human ESCs to Fas-, TRAIL-- and TNF-a-mediated apoptosis. In contrast to HeLa-cells [32]the observed active resistance-mechanism of human ESCswas not induced by a stimulation of the respective signalingpathways by Fas or TRAIL itself, but relies on the constitutiveactivity of Erk1/2 and NF-kB. However, regarding TNF-a, such aself-induced protection-mechanism seems to play a role, asTNF-a stimulated the pro-survival pathways Erk1/2 andNF-kB.Taking our results and the mentioned observations in othercell types into account, apoptosis-resistance caused by activesignaling seems to be cell type- and tissue-specific, showing aspecific pattern in human ESCs.

Taken together, we demonstrate that the resistance ofhuman ESCs to death receptor-mediated apoptosis is an activemetabolic process involving the constitutive activity of Erk1/2-- and NF-kB-signaling. These observations further underlinethe concept of death receptor-mediated apoptosis as a verytightly regulated event in thehuman endometrium. The subtlebalance between apoptosis-resistance and -sensitivity seemsto be essential for the successful implantation of the embryo. Abetter understanding of the physiological as well as patho-physiological regulation of apoptosis in the human endome-trium can reveal targets for the development of newprophylactic and therapeutic concepts for women sufferingfrom repeated implantation failure or habitual miscarriages.

Funding

Thisworkwas supported by a grant from theGermanResearchFoundation, Bonn, Germany to H.F. (grant FL 667/2-1).

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