of July 27, 2018.This information is current as

Pathways-Monophosphate-Mediated Signaling

′Calcium- and Cyclic Adenosine 5Subtypes, E-Prostanoid (EP) 3 and EP4, Via

Receptor2Cooperation of Two PGEProlactin Expression in Human T Cells:

-Induced2Mechanism of Prostaglandin (PG)E

Hooghe-Peters and Ron KooijmanSarah Gerlo, Peggy Verdood, Birgit Gellersen, Elisabeth L.

http://www.jimmunol.org/content/173/10/5952doi: 10.4049/jimmunol.173.10.5952

2004; 173:5952-5962; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/173/10/5952.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2004 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Mechanism of Prostaglandin (PG)E2-Induced ProlactinExpression in Human T Cells: Cooperation of Two PGE2

Receptor Subtypes, E-Prostanoid (EP) 3 and EP4, ViaCalcium- and Cyclic Adenosine 5�-Monophosphate-MediatedSignaling Pathways1

Sarah Gerlo,2* Peggy Verdood,* Birgit Gellersen,† Elisabeth L. Hooghe-Peters,* andRon Kooijman*

We previously reported that prolactin gene expression in the T-leukemic cell line Jurkat is stimulated by PGE2 and that cAMPacts synergistically with Ca2� or protein kinase C on the activation of the upstream prolactin promoter. Using the transcriptioninhibitor actinomycin D, we now show that PGE2-induced prolactin expression requires de novo prolactin mRNA synthesis andthat PGE2 does not influence prolactin mRNA stability. Furthermore, PGE2-induced prolactin expression was inhibited by proteinkinase inhibitor fragment 14–22 and BAPTA-AM, which respectively, inhibit protein kinase A- and Ca2�-mediated signalingcascades. Using specific PGE2 receptor agonists and antagonists, we show that PGE2 induces prolactin expression through en-gagement of E-prostanoid (EP) 3 and EP4 receptors. We also found that PGE2 induces an increase in intracellular cAMPconcentration as well as intracellular calcium concentration via EP4 and EP3 receptors, respectively. In transient transfections,3000 bp flanking the leukocyte prolactin promoter conferred a weak induction of the luciferase reporter gene by PGE2 and cAMP,whereas cAMP in synergy with ionomycin strongly activated the promoter. Mutation of a C/EBP responsive element at �214partially abolished the response of the leukocyte prolactin promoter to PGE2, cAMP, and ionomycin plus cAMP. The Journalof Immunology, 2004, 173: 5952–5962.

T he polypeptide hormone, prolactin (PRL),3 is of majorimportance for reproduction. Whereas the pituitary is themain source of circulating PRL, the hormone is also syn-

thesized at extrapituitary sites such as the decidua, the brain, themammary gland, and the immune system (1, 2). PRL expressionhas been demonstrated in thymocytes, T cells, B cells, and mono-cytes (3, 4), and the PRLR, which belongs to the cytokine-receptorfamily, is expressed on both lymphocytes and monocytes (4), sug-gesting that the hormone may act in an auto- or paracrine, cyto-kine-like fashion in the immune system. Whereas no immunedeficiencies could be detected in steady-state conditions in bothPRL�/� and PRLR�/� mice (5, 6), there is compelling evidence

for in vitro effects of PRL on immune cell function. For instance,PRL stimulates inducible NO synthase production, Ig release, andcytokine expression in human leukocytes (7–10). Furthermore,PRL has anti-apoptotic properties that have been demonstrated inthe Nb2 rat lymphoma (11) and in dexamethasone-treated thymo-cytes (12). The apparently conflicting results from the in vivo stud-ies using knockout mice and in vitro data have led to the hypoth-esis that the immunomodulatory effects of PRL come into playonly under conditions in which the organism is subjected to stress(13). Indeed, PRL administration following hemorrhagic shock inmice restored the decreased ability of macrophages to release cy-tokines, and thus decreased mortality from subsequent sepsis (14).Also, in PRL�/� mice, the lack of PRL enhanced the negativeeffects of thermal injury on myelopoiesis and T lymphocyte pro-liferation (15).

Hyperprolactinemia, correlating with disease activity, has beendescribed in autoimmune conditions such as systemic lupus ery-thematosus (SLE; Ref. 16) and rheumatoid arthritis (17, 18), sug-gesting PRL is involved in the pathophysiology of these diseases.Peeva et al. (19) recently reported that PRL augments the expansionof anti-DNA B cells in mice in a CD4� T cell-dependent manner, thuscausing a lupus-like phenotype. The importance of leukocyte-derivedPRL in SLE is suggested by enhanced PRL production in T cells frompatients as compared with normal controls (20, 21). Interestingly, asingle nucleotide polymorphism in the PRL promoter influences PRLexpression in lymphocytes and is associated with SLE in a cohort ofpatients from the U.K. (22). Furthermore, in patients with rheumatoidarthritis, PRL produced by synovium infiltrating T cells causes aber-rant synovial cell function and might thus influence disease progres-sion (23). These findings, implicating a role for leukocyte-derived

*Laboratory of Neuroendocrine Immunology, Department of Pharmacology, FreeUniversity of Brussels, Brussels, Belgium; and †Endokrinologikum Hamburg, Ham-burg, Germany

Received for publication March 17, 2004. Accepted for publication August 26, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This research was supported by the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (G.0126.02) and by institutional grants from the Free University of Brus-sels (Onderzoeksraad, Geoconcerteerde Onderzoeksactie 97-02-04).2 Address correspondence and reprint requests to Dr. Sarah Gerlo, Laboratory ofNeuroendocrine Immunology, Department of Pharmacology, Free University of Brussels,Laarbeeklaan 103, B-1090 Brussels, Belgium. E-mail address: Sarah.Gerlo@vub.ac.be3 Abbreviations used in this paper: PRL, prolactin; SLE, systemic lupus erythemato-sus; EP, E-prostanoid; [Ca2�]i, intracellular calcium concentration; [cAMP]i, intra-cellular cAMP concentration; cptcAMP, 8-(4-chloro-phenyl-thio)-cAMP; RpcAMPs,8-(4-chloro-phenyl-thio)-adenosine-3�,5�-cyclic monopsporothioate, RP-isomer; PKI,protein kinase inhibitor fragment 14–22; 17-P-T-PGE2, 17-phenyl-trinor-PGE2;MSK-1, mitogen- and stress-activated protein kinase-1; PKA, protein kinase A; RT,room temperature; EPAC, exchange protein directly activated by cAMP; IP3, inositol1,4,5-triphosphate; PLC, phospholipase C.

The Journal of Immunology

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00

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PRL in immune responses, prompted us to investigate the regulationof PRL expression in T cells.

Extrapituitary PRL expression is mostly (e.g., in the decidua andthe immune system) regulated by an alternative promoter, located5.8 kb upstream to the pituitary PRL promoter (24, 25). Whereasregulation of pituitary PRL expression has been extensively stud-ied, little is known about the factors that regulate PRL expressionin leukocytes and what DNA sequences in the upstream PRL pro-moter, hereafter called the leukocyte promoter, are targeted bythese regulators. It has been shown that PRL expression in PBMCis stimulated by Con A and phytohemagglutinin (26), and we re-cently reported that PRL expression in Jurkat leukemic T cells andin PBMC is regulated by cAMP and physiological stimuli thatsignal through cAMP such as PGE2 (27). PGE2 modulates immuneresponses by stimulating and inhibiting the functions of many dif-ferent types of immune cells both in vivo and in vitro (28). Mostreports describe inhibitory effects of PGE2 on T cell function:PGE2 inhibits T cell proliferation and expression of both IL-2 andthe IL-2R (29–31). Several reports indicate PGE2 has differenteffects on Th1 vs Th2 cells, resulting in a shift from a Th1 cellularimmune response toward a Th2-driven humoral response (32, 33).However, the overall impact of PGE2 in an inflammatory responsecan be either positive or negative, depending on the level of im-mune activation, the presence of other mediators, and the physio-logical state of the organism (34). PGE2 exerts its effects throughbinding to at least four different receptors, termed E-prostanoid(EP) 1 to EP4, which activate different second messengers, ac-counting for the specificity and diversity of PGE2 effects. The EP1receptor induces a rise in intracellular calcium concentration([Ca2�]i). The EP2 and EP4 receptors activate Gs and thus increaseintracellular cAMP concentration ([cAMP]i). Multiple EP3 iso-forms have been described, which couple to different signalingpathways: binding of PGE2 to the EP3R can decrease [cAMP]i,which was elevated by other agonists, via Gi or increase [Ca2�]i

via Gq. In nonhuman species, EP3 isoforms have been describedthat activate Gs and transduce increases in [cAMP]i (35).

In this study, we have shown that PGE2 up-regulates PRL ex-pression in T cells through transcriptional activation and has noeffect on PRL mRNA stability. Both Ca2� and cAMP appear to beindispensable second messengers for PGE2-induced PRL expres-sion. Using EPR-specific agonists and antagonists, we establishedthe involvement of both EP3 and EP4 receptors, through elevationof Ca2� and cAMP, respectively, in the effect of PGE2 on PRLexpression. We also showed that cAMP and Ca2� exert their syn-ergistic action through a CAAT/enhancer-binding protein (C/EBP)binding site at �214 in the leukocyte PRL promoter.

Materials and MethodsReagents

Actinomycin D, EGTA, H89, KT5720, BAPTA-AM, thapsigargin, Fluo-3-AM, PGE2, 8-(4-chloro-phenyl-thio)-cAMP (cptcAMP), 8-(4-chloro-phenyl-thio)-adenosine-3�,5�-cyclic monopsporothioate, RP-isomer (Rp-cAMPs), myristoylated protein kinase inhibitor fragment 14–22 (PKI),PMA, ionomycin, and wortmannin were purchased from Sigma-Aldrich(Bornem, Belgium). Butaprost, 17-phenyl-trinor-PGE2 (17-P-T-PGE2),sulprostone, SC-19220, and PGE1-OH were from Cayman Chemical (AnnArbor, MI). L-161982 was a gift from Merck Frost (Kirkland, Quebec,Canada). Recombinant human PRL was produced in Escherichia coli inour laboratory, using a cDNA obtained from J. Martial (University ofLiege, Liege, Belgium). The PRL-3000-Luc, PRL-332-Luc, PRL-32-Luc,PRL-332/D-Bmut/Luc, PRL-332/Fmut/Luc, and PRL-332/Gmut/Luc weredescribed earlier (36). The mitogen-stimulated kinase 1 (MSK-1) expres-sion vectors were obtained from Dr. D. Alessi (University of Scotland,Dundee, U.K.).

Cell culture

Jurkat cells, obtained from the European Collection of Cell Cultures (Salis-bury, U.K.), were maintained in RPMI 1640 with Glutamax-I, supple-mented with 10% FCS, 100 U/ml penicillin, and 100 �g/ml streptomycin(all from Invitrogen Life Technologies, Merelbeke, Belgium). For all stim-ulation experiments, cells were washed and resuspended in RPMI 1640with Glutamax-I at a concentration of 2 � 106/ml. In stimulation experi-ments for ELISA, real-time PCR, and reporter gene assays, cells werecultured in Falcon polystyrene 96-well plates (BD Biosciences Labware,Erembodegem, Belgium) in a humidified 5% CO2 atmosphere at 37°C.Stimulations for cAMP and protein kinase A (PKA) assays and Westernblotting experiments were performed in 2 ml reaction tubes (BrinkmanInstruments, Hamburg, Germany) in a waterbath at 37°C.

PRL ELISA

PRL concentrations were determined by sandwich ELISA. A 96-wellMaxisorp-Nunc immunoplates (VWR, Leuven, Belgium) were coatedovernight at 4°C with a 1/500 dilution of a monoclonal anti-hPRL Ab(BioGenex, San Ramon, CA) in bicarbonate buffer (40 mM Na2CO3, 60mM NaHCO3, pH 9.6). Supernatants were incubated in the Ab-coatedplates for 2 h at room temperature. Next, a rabbit anti-hPRL antiserum,NIDDK-IC-5, donated by Dr. Parlow (National Hormone and Peptide Pro-gram, Harbor-University of California Los Angeles Medical Center, Tor-rance, CA) was added to the plates for 2 h (1/500 dilution in PBS, 0.1%Tween 20, and 1% nonfat dry milk), followed by a 2-h incubation with aperoxidase-conjugated anti-rabbit Ig (Amersham Biosciences, Roosendaal,The Netherlands) diluted 1/5000 in PBS, 0.1% Tween 20, and 1% nonfatdry milk. Every Ab incubation was followed by three washing steps inPBS, 0.1% Tween 20. In the final step, the peroxidase substrate (87 �g/ml3, 3�,5,5�-tetramethylbenzidine dihydrochloride hydrate in 109 mM citricacid, 0.05% H2O2) was added. Absorbance of the samples was measured at450 nm with a reference wavelength at 540 nm using a microtiter platereader (Labsystems iEMS Reader MF; Labsystems, Helsinki, Finland). Thedetection limit of this assay was 10 pg/ml, using recombinant human PRLas a standard.

Real-time PCR

Total RNA was isolated using TRIzol reagent (Invitrogen LifeTechnologies) according to the manufacturer’s instructions. Reversetranscription was performed using the TaqMan reverse transcription kitfrom Applied Biosystems (Nieuwerkerk a/d IJssel, The Netherlands). Forreal-time cDNA amplification, we used the Applied Biosystems SYBRGreen Mastermix and the following primers for PRL: sense, 5�-ACC AAGAAG AAT CGG AAC ATA C-3�; antisense, 5�-ACA GGA GCA GGTTTG ACA-C-3�. Fluorescence was monitored using the TaqMan 7700Sequence Detector (Applied Biosystems). The results of a series ofstandards prepared by successive dilution (0.02–200 ng) of total RNA andplotted against the logarithm of the concentration were used to estimate therelative amounts of PRL mRNA initially present in each sample. Therelative PRL mRNA levels are shown as compared with �-Actin controls.�-actin cDNA was amplified using the following primers: sense, 5�-GGATGC AGA AGG AGA TCA CCT G-3�; antisense, 5�-CGA TCC ACACGG AGT ACT TG-3�.

cAMP assays

Jurkat cells were preincubated in RPMI 1640 for 1 h at 37°C, before stim-ulation with PGE2 or EP agonists, for 10 min. Pellets were collected andsnap frozen in liquid nitrogen. Extracts were prepared by boiling the frozencell pellets for 10 min in assay buffer (delivered with the Amersham cAMPRIA). cAMP concentrations in cellular extracts were determined by RIA(Biotrak cAMP[125I] assay system; Amersham Biosciences) according tothe manufacturer’s instructions.

PKA assays

Jurkat cells were preincubated for 1 h with or without PKA inhibitors at37°C, before stimulation with cptcAMP. Extracts were prepared by soni-cation of cells in buffer A (20 mM Tris, pH 7.5, 0.25 M sucrose, 10 mMEGTA, 2 mM EDTA, 1 mM AEBSF, 10 �g/ml leupeptin, and 2 mM DTT).PKA activity was determined using the Peptag assay kit (Promega, Madison,WI). Briefly, the Peptag assay uses a fluorescent PKA-specific peptide sub-strate (kemptide). Phosphorylation of the substrate by PKA alters the peptidesnet charge from �1 to �1. This change in net charge allows the phosphory-lated and nonphosphorylated forms of the substrate to be separated on agarosegel. The bands were visualized using a UV-transilluminator.

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Western blotting

For phospho-MSK-1 detection, cellular extracts were prepared in TritonX-100 extraction buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA,1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% TritonX-100, 10% glycerol, 0.1% SDS, 0.5% sodium deoxycholate, 1 mMPMSF, 10 �g/ml aprotinin, 50 �g/ml leupeptin, 50 �g/ml pepstatin A, and500 �g/ml soybean trypsin inhibitor; all from Sigma-Aldrich) and sub-jected to PAGE. Proteins were transferred to polyvinylidene difluoridemembranes. After blocking for 1 h at room temperature in blocking buffer(PBS, 5% nonfat dry milk, and 0.1% Tween 20), blots were probed withanti-phospho-MSK-1 (Cell Signaling, Beverly, MA) at a 1/1000 dilution inPBS, 5% BSA, and 0.1% Tween 20 for 18 h at 4°C. Subsequently, blotswere washed three times in PBS and 0.1% Tween 20 and incubated for 60 minat room temperature (RT) with a 1/5000 dilution (in PBS, 5% nonfat dry milk,and 0.1% Tween 20) of a peroxidase-labeled anti-rabbit Ig (Amersham Bio-sciences). Following three washes in PBS and 0.1% Tween 20, immunoreac-tive bands were detected using ECL (PerkinElmer, Boston, MA).

Ca2� measurements

Jurkat cells (10 � 106/ml) were loaded with the Ca2�-sensitive fluorescentdye Fluo-3-AM (8.8 �M) at 37°C for 60 min in RPMI 1640. Next, cellswere washed three times in RPMI 1640 and resuspended in RPMI 1640at 1 � 106 cells/ml. Two milliliters of cell suspension was transferredto a 1-cm light path cuvette, and fluorescence intensity was measuredusing a spectrofluorophotometer (Shimadzu RF-5301 PC; ShimadzuScientific Instruments, Kyoto, Japan), at an excitation wavelength of506 nm and an emission wavelength of 526 nm. Fluorescence measure-ments were acquired at 0.5-s intervals for 60 s. Fmax and Fmin weredetermined by incubating the cells with 0.1% Triton X-100 or 25 mMEGTA, respectively. The fluorescence intensities were converted tonanomolar concentrations by the equation [Ca2�]i � Kd(F � Fmin)/(Fmax � F), where Kd denotes the apparent dissociation constant (390nM) of the Fluo-3-Ca2� complex (37).

Transient transfections

For reporter gene assays, Jurkat cells were transfected by electroporation.Cells growing in the log phase were resuspended in RPMI 1640 at aconcentration of 15 � 106/ml. Plasmids were added to a concentrationof 30 �g/ml and electroporation was performed using the followingsettings: 330 V and 1800 �F (Electropore 2000; Eurogentec, Liege,Belgium). Cell lysates were prepared and assayed for luciferase activityusing the Promega Luciferase Assay kit (Promega). For MSK-1 over-expression experiments, cells were transfected by nucleofection usingsolution V and program S18 (Amaxa, Cologne, Germany) according tothe manufacturer’s instructions.

Statistical analysis

Statistical differences between group means were determined by ANOVAwith Tukey’s post-test. Differences were considered significant when p �0.05. Data represented are means � SD of quadruplicate incubations. Thepresented experiments are representative of at least three independentexperiments.

ResultsPGE2 enhances PRL expression by activating PRL transcriptionand does not affect PRL mRNA stability

We previously showed by conventional RT-PCR and ELISA thatPRL mRNA levels and PRL secretion are up-regulated by PGE2 inJurkat cells (27). To address the level(s) at which regulation ofPRL expression by PGE2 takes place, we studied the kinetics ofPRL expression in the presence and absence of the transcriptionalinhibitor actinomycin D. As depicted in Fig. 1A, an increase inPRL mRNA level was already detected after 4 h of stimulationwith PGE2 (100 nM), whereas an increase in PRL secretion wasdetected after 8 h (Fig. 1B). Preincubation of Jurkat cells withactinomycin D completely abrogated the effect of PGE2 on bothPRL mRNA levels and secretion (Fig. 1, A and B). These resultsindicate that PRL expression is regulated at the transcriptionallevel and that PGE2 does not stimulate PRL translation or secre-tion. To exclude modulation of mRNA stability by PGE2, we com-

pared the decrease in absolute levels of PRL mRNA in the pres-ence and absence of actinomycin D. As shown in Fig. 1C, PGE2

does not interfere with PRL mRNA decay, indicating that it doesnot affect mRNA stability.

FIGURE 1. PGE2 regulates PRL expression at the level of transcription.Jurkat cells were preincubated for 1 h with vehicle or the transcriptionalinhibitor, actinomycin D (5 �g/ml), and next stimulated with PGE2 (100nM) for 4, 8, or 18 h. A, PRL mRNA levels were quantified by real-timePCR and normalized to �-actin mRNA expression. Normalized PRLmRNA levels are represented as a percentage of control (no actinomycin Dor PGE2 at time 4 h). B, PRL secretion as assessed by ELISA. C, Decay ofPRL mRNA levels as measured by real-time PCR. PRL mRNA levels arerepresented as percentage of control (5 �g/ml actinomycin D, no PGE2 attime 4 h).

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Both cAMP/PKA and Ca2� are involved in PGE2-inducedsignaling leading to PRL expression

Because we showed that the two principal second messengers in-volved in PGE2 receptor signaling (cAMP and Ca2�) synergisti-cally activate PRL expression in Jurkat cells (27), we further ad-dressed the roles of these messengers in PGE2-induced PRLexpression. PKA is the best known effector of cAMP signal-ing. Therefore, we investigated the effects of four structurally un-related PKA inhibitors (H89, KT5720, RpcAMPs, and PKI) onPGE2-induced PRL mRNA levels and protein secretion. As shownin Fig. 2, A and B, only H89 and PKI blocked PGE2-induced PRLexpression at the mRNA level. Morever, H89 and PKI completelyblocked PGE2-induced PRL secretion, whereas KT5720 andRpcAMPs only exerted small inhibitory effects (Fig 2, C and D).Furthermore, H89, but not KT5720 or RpcAMPs, blocked PRLexpression induced by the long-acting cAMP analog, cptcAMP, atthe level of transcription as well as at the level of secretion. There-fore, to find out whether KT5720 and RpcAMPs were effective inblocking PKA activation in our system, we investigated their effecton cptcAMP-induced activation of PKA using the Peptag PKAassay system. As shown in Fig. 3A, H89, but not KT5720 orRpcAMPs, effectively blocked PKA activation. Although H89 isoften considered as a selective inhibitor for PKA, several studiesindicate that H89 inhibits other protein kinases with a potencygreater than or equal to that for PKA (37). One of these other H89targets is MSK-1. MSK-1 is directly activated by ERK2 and p38and can mediate activation of CREB (39). To address the possi-bility that H89 blocks induction of PRL expression through inhi-bition of MSK-1, we investigated whether PGE2 or cptcAMPphosphorylate overexpressed MSK-1 in Jurkat cells. As shown inFig. 3B, neither PGE2 nor cptcAMP induced phosphorylation ofMSK-1.

It recently became clear that cAMP exerts some of its effects ina PKA-independent manner through direct activation of a Rap-1guanine-nucleotide-exchange factor, an exchange protein directly

activated by cAMP (EPAC; Ref. 40). We investigated the effect ofa methylated cptcAMP analog, which specifically activates EPAC,on PRL expression in Jurkat cells. As shown in Fig. 4, whereascptcAMP stimulates PRL expression in Jurkat cells, the effect ofMe-cptcAMP is inhibitory, suggesting EPAC is not involved instimulating PRL expression.

To address the role of Ca2� in PGE2-induced PRL expression,we preincubated Jurkat cells with the cell-permeable Ca2� chelatorBAPTA-AM, or the cell-impermeable Ca2� chelator, EGTA. Arole for Ca2� is indicated by the inhibitory effects of bothBAPTA-AM and EGTA on the stimulation by PGE2 of PRLmRNA expression and secretion (Fig. 5, A and B). We next ad-dressed the role of Ca2� from intracellular stores. Preincubation ofJurkat cells with thapsigargin, which depletes the intracellularCa2� stores, did not affect either PGE2-induced PRL mRNA ex-pression or PRL secretion (Fig. 5, A and B).

It has recently been shown that PGE2 induces T cell factor/lymphoid enhancer factor-mediated transcriptional activation via aPI3K-dependent pathway (41). Therefore, we investigated the ef-fect of the PI3K inhibitor wortmannin on PGE2-induced PRL ex-pression. As shown in Fig. 6, wortmannin did neither affect PGE2-induced PRL mRNA expression nor PRL secretion.

EP receptors involved in the generation of second messengersand PRL expression

PGE2-induced PRL expression. To address the role of EP re-ceptors in the stimulating effect of PGE2 on PRL expression, wefirst incubated cells with different doses of PGE2, the EP1/EP3agonist 17-P-T-PGE2, the EP2 agonist, butaprost, or the EP3 ag-onist, sulprostone, and measured PRL expression by ELISA. Asshown in Fig. 7A, 10 nM PGE2 and 1 �M 17-P-T-PGE2 enhancedPRL expression, whereas butaprost and sulprostone were ineffec-tive. Next, we investigated the effect of specific antagonists forEP1 and EP4 on PGE2- and 17-P-T-PGE2-induced PRL expres-sion. Whereas the EP1 antagonist SC-19220 had no effect on

FIGURE 2. Effect of PKA inhibi-tors on PGE2-induced PRL expression.Jurkat cells were pretreated for 2 h withvehicle, H89 (10 �M), KT5720 (0.2�M), RpcAMPs (50 �M; A and C), orincreasing doses of PKI (B and D) be-fore stimulation with 100 nM PGE2 or250 �M cptcAMP for 18 h. A and B,PRL mRNA levels were quantified byreal-time PCR and normalized to �-ac-tin mRNA expression. NormalizedPRL mRNA levels are represented aspercentage of control (no inhibitor, nostimulus). C and D, PRL secretion asmeasured by ELISA. The figure legendis shared for A–D. #, Significantly dif-ferent (p � 0.05).

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PGE2- or 17-P-T-PGE2-induced PRL expression, the EP4 antag-onist L-161982 nearly completely blocked PGE2- and 17-P-T-PGE2-induced PRL expression (Fig. 7, B and C), suggesting theinvolvement of EP4 in PGE2- and 17-P-T-PGE2-induced PRLexpression.

Augmentation of [cAMP]i by PGE2 via the EP4 receptor. MostPGE2 effects on immune cells have been attributed to rises in[cAMP]i, as a result of binding of PGE2 to EP2 or EP4 receptors(42–44). Using a cell permeable cAMP analog, we showed earlierthat augmenting [cAMP]i stimulates PRL expression in Jurkat

cells (27). To investigate whether PGE2 does indeed increase[cAMP]i levels in Jurkat cells and if so, which EP receptor medi-ates this rise in [cAMP]i, we measured the effects of increasingdoses of PGE2, 17-P-T-PGE2, or butaprost on [cAMP]i. We foundthat 100 nM PGE2 induced a rise in [cAMP]i (Fig. 8A). Butaprostdid not alter [cAMP]i, suggesting that functional EP2 receptors arenot present on Jurkat cells. As observed for PRL expression, 17-P-T-PGE2 enhanced [cAMP]i at a dose of 1 �M, which is higherthan the dose required for activating EP1 or EP3 receptors. Thehypothesis that PGE2 increases [cAMP]i via the EP4 receptor wasconfirmed by the finding that the EP4 antagonist, L-161982, at adose of 10 nM completely blocked PGE2-induced [cAMP]i accu-mulation (Fig. 8B).

PGE2 raises [Ca2�]i through the EP3 receptor. Our finding that,in addition to cAMP, Ca2� is also involved in PRL induction byPGE2, prompted us to measure the effects of PGE2 and EP agonistsand antagonists on [Ca2�]i. Using the Grynkiewicz equation (37),the [Ca2�]i was calculated, and results were represented as a per-centage of the [Ca2�]i at 0.5 s before stimulation. Indeed, 100 nMPGE2 did increase [Ca2�]i in Jurkat cells (Fig. 9, A, B, and D).

FIGURE 4. The EPAC activator Me-cptcAMP does not stimulate PRLexpression. Jurkat cells were stimulated for 18 h with increasing doses ofcptcAMP or the methylated analog Me-cptcAMP, which specifically acti-vates EPAC. PRL secretion was measured by ELISA.

FIGURE 5. Effect of Ca2� chelators on PGE2-induced PRL expressionin Jurkat cells. Jurkat cells were pretreated for 2 h with vehicle,BAPTA-AM (10 �M), EGTA (2 mM), or thapsigargin (0.1 �M) beforestimulation with PGE2 for 18 h. A, PRL mRNA levels were quantified byreal-time PCR and normalized to �-actin mRNA expression. NormalizedPRL mRNA levels are represented as a percentage of 100% control (noinhibitor, no stimulus). B, PRL secretion as measured by ELISA. The fig-ure legend is shared for A and B. #, Significantly different (p � 0.05).

FIGURE 3. Inhibition of cptcAMP-induced PKA activation. PGE2 orcptcAMP do not phosphorylate overexpressed MSK-1. A, Jurkat cells werepreincubated with or without PKA inhibitors for 1 h in a 37°C waterbathbefore stimulation with cptcAMP for 20 min at 37°C. PKA activity in cellpellets was quantified using the Peptag assay with kemptide as a substrate.NP, nonphosphorylated kemptide; P, phosphorylated kemptide. Lane 1,control; lane 2, 250 �M cptcAMP. Cells in lanes 3–8 were stimulated with250 �M cptcAMP plus the following inhibitors: lane 3, 1 �M H89; lane4, 10 �M H89; lane 5, 0.2 �M KT5720; lane 6, 2 �M KT5720; lane 7, 10�M RpcAMPs; and lane 8, 50 �M RpcAMPs. Lane 9, negative control (noPKA); lane 10, positive control (0.5 ng PKA). B, Jurkat cells were prein-cubated for 1 h in a 37°C waterbath, before stimulation with TPA (10ng/ml), cptcAMP (250 �M), or PGE2 (100 nM) for 10 or 30 min at 37°C.Phosphorylated MSK-1 was detected by Western blotting.

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When the cells were stimulated with PGE2 in the presence of theEP4 antagonist L-161982, a further increase in [Ca2�]i was ob-served (Fig. 9D). At a concentration of 100 nM, both sulprostoneand 17-P-T-PGE2 enhanced [Ca2�]i to an extent that exceeds thatof PGE2 (Fig. 9, A and B). Preincubation of Jurkat cells with theEP1 antagonist SC-19220 had no effect on the 17-P-T-PGE2-in-duced rise in [Ca2�]i (Fig. 9C), suggesting its effect on [Ca2�]i isnot via the EP1 receptor. In conclusion, these results suggest thatPGE2 augments [Ca2�]i via the EP3 receptor.

We previously reported synergistic effects of cAMP and iono-mycin on PRL expression in Jurkat cells, whereas ionomycin byitself was ineffective (27). To obtain further evidence for the ideathat EP3 receptors synergize with EP4 receptors that induce[cAMP]i, we incubated Jurkat cells with a combination of the EP3agonist sulprostone and cptcAMP. As shown in Fig. 10, sulpros-tone and cAMP indeed synergistically stimulated PRL expression.

Localization of cAMP- and PGE2-responsive elements in theleukocyte PRL promotor

We previously reported that the effect of PGE2 on endogenousPRL expression in Jurkat cells exceeded the effect of PGE2 onactivation of an 1842 bp-carrying promoter construct (27). To ad-dress the role of more distant promoter sequences in PRL tran-scription, we transfected Jurkat cells with a promotor constructcarrying 3000 bp of the leukocyte PRL promoter (PRL-3000-Luc).However, using this larger promoter construct, we failed to mimic

the PGE2 effect on endogenous PRL expression, suggesting thatnot all PGE2 responsive elements are contained within these 3000bp of the leukocyte PRL promoter. PRL-3000-Luc was verystrongly activated by a synergistic action of cAMP and ionomycin,

FIGURE 6. Effect of inhibition of PI3K on PGE2-induced PRL expres-sion. Jurkat cells were pretreated for 2 h with increasing doses of wort-mannin before stimulation with PGE2 (100 nM) for 18 h. A, PRL mRNAlevels were quantified by real-time PCR and normalized to �-actin mRNAexpression. Normalized PRL mRNA levels are represented as a percentageof 100% control (no inhibitor, no stimulus). B, PRL secretion as measuredby ELISA. The figure legend is shared for A and B. #, Significantly dif-ferent (p � 0.05).

FIGURE 7. Effect of EP agonists and antagonists on PRL expression inJurkat cells. A, Jurkat cells were incubated for 18 h with increasing dosesof PGE2, the EP2 agonist butaprost, the EP3 agonist sulprostone, or theEP1/EP3 agonist 17-P-T-PGE2. B, Jurkat cells were preincubated for 2 hwith increasing doses of the EP1 antagonist SC-19220 or the EP4 antag-onist L-161982 before stimulation for 18 h with PGE2 (100 nM). C, Jurkatcells were preincubated for 2 h with increasing doses of the EP1 antagonistSC-19220 or the EP4 antagonist L-161982 before stimulation for 18 h with17-P-T-PGE2 (10 �M). PRL levels in conditioned medium were quantifiedby ELISA.

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whereas the effects of cAMP and PGE2 were much weaker (Fig.11A). An identical response was observed with only 332 bp of theupstream promoter. In contrast, a 32-bp promoter construct did notrespond to PGE2, cAMP, or cAMP in combination with ionomycin(Fig. 11A). Importantly, deletion of the �332 and �32 regionsignificantly inhibited the elevated promoter activity in the pres-ence of PGE2, cAMP, or cAMP plus ionomycin. The region be-tween �332 and �32 contains three C/EBP consensus sequences(two overlapping sites, D-B, between �310 and �285; one site, F,between �214 and �201) and one Ets consensus sequence (G,�248 and �239; Ref. 35). Mutation of either the DB or the G sitedid not affect promoter activation by PGE2, cAMP, or cAMP plusionomycin (Fig. 11B). In contrast, mutation of site F significantlyreduced the responsiveness of the wild-type PRL-332-Luc con-struct to cAMP and PGE2 by 52 and 36%, respectively. In addi-tion, mutation of this site also reduced the synergistic response tocAMP and ionomycin by 61% (Fig. 11B).

DiscussionIn this report, we show that, in Jurkat cells, induction of PRLexpression and secretion by PGE2 is regulated at the transcrip-tional level. Both induction of PRL mRNA and PRL secretionwere abrogated in the presence of the transcriptional inhibitor, ac-tinomycin D. In the human B-lymphoblastoid cell line IM-9-P3

PRL mRNA half-life is reduced by dexamethasone (45), whereasit is increased by retinoic acid (46). These findings suggest that theleukocyte PRL mRNA is susceptible to posttranscriptional regu-lation by modification of its clearance rate. Because we were un-able to detect any changes in the rate of mRNA degradation in thepresence of PGE2, we conclude that, in Jurkat cells, PGE2 does notregulate PRL expression beyond the level of transcription.

The specificity and diversity of PGE2 effects can be in part ex-plained by the use of four PGE2 receptor subtypes (EP1, EP2, EP3,and EP4), which transduce their signals predominantly via changesin [cAMP]i, [Ca2�]i, or both (35). To investigate the signalingpathways involved in PGE2-induced PRL expression, we tested theeffects of inhibitors of several signaling cascades. A role for PKAwas indicated by the inhibition of PRL expression by H89 andPKI. Another target of H89, MSK-1, which can activate CREB(39) was not phosphorylated by PGE2 or cAMP in Jurkat cells. Inaddition, PGE2 and cAMP did not phosphorylate kinases upstreamto MSK-1 (p38 and ERK), and inhibitors of these pathways wereunable to block PGE2-induced PRL expression (data not shown).Furthermore, overexpression of MSK-1 or dominant-negative C-terminal kinase-dead MSK-1 did not affect cptcAMP- or PGE2-induced activation of the upstream PRL promoter (data notshown). At nontoxic doses, two PKA inhibitors that are structur-ally unrelated to H89 (KT5720 and RpcAMPs), failed to block theeffects of PGE2. However, unlike H89, KT5720 and RpcAMPswere ineffective in inhibiting cptcAMP-induced PKA activation inJurkat cells. Finally, we found that activation of an alternativeroute for cAMP signaling, through EPAC, did not stimulate PRLexpression but instead resulted in the inhibition of PRL expression.Indeed, opposing effects of PKA and EPAC have recently alsobeen shown on protein kinase B activation in Hek-293 cells (47).In conclusion, these data support the importance of the classicalcAMP effector, PKA, in mediating the effect of PGE2 on PRLexpression. Results from experiments using Ca2� chelatorsBAPTA-AM and EGTA furthermore suggest that, besides cAMP,Ca2� signals are also involved in PGE2-regulated PRL expression.It has recently been shown that PGE2 can activate PI3K in T cellsvia the EP4 receptor (41). The effects of PGE2 on PRL expressionwere, however, unaffected by the PI3K inhibitor wortmannin. Insummary, our results indicate that cAMP, via PKA, as well asCa2� are involved in the regulation of PRL expression at the tran-scriptional level, as inhibition of these pathways blocked PGE2-induced PRL mRNA levels (Figs. 2 and 5). The roles of Ca2� andcAMP in PGE2-induced PRL expression are furthermore sup-ported by the stimulating effects of PGE2 on [cAMP]i and [Ca2�]i

(Figs. 8 and 9), and the synergy of these effectors on PRL promoteractivation (27).

Using EP-specific agonists and antagonists, we have addressedthe involvement of EP receptors in PGE2-induced signaling andPRL expression in Jurkat cells. Our observation that the EP4 an-tagonist L-161982 blocked PGE2-induced PRL expression, indi-cates a role for the EP4 receptor. Furthermore, we found that theEP1/EP3 agonist 17-P-T-PGE2 enhanced PRL expression,whereas the EP2 agonist, butaprost, and the EP3 agonist, sulpros-tone, were ineffective. Although 17-P-T-PGE2 is described as aspecific EP1/EP3 (EP1�EP3) agonist, at high doses this agonistcan also activate the EP4 receptor (Ki � 1 �M; Ref. 35). The factthat a dose of 1 �M is required to augment PRL expression inJurkat cells, argues for an action of 17-P-T-PGE2 through the EP4receptor. Our observation that the EP1 antagonist SC-19220 hadno effect on PGE2- or 17-P-T-PGE2-induced PRL expression,whereas the EP4 antagonist L-161982 nearly completely blocked17-P-T-PGE2-induced PRL expression (Fig. 7, B and C), confirms

FIGURE 8. Effects of EP agonists and antagonists on [cAMP]i. A, Cellswere preincubated for 1 h in a 37°C waterbath, before stimulation for 10min with increasing doses of PGE2, the EP2 agonist butaprost or the EP1/EP3 agonist 17-P-T-PGE2. B, Cells were preincubated for 1 h withL-161982 before stimulation with PGE2 (100 nM) for 10 min. [cAMP]i incell pellets was determined by RIA. Results are represented as a fold in-duction relative to unstimulated controls.

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the involvement of EP4 in PGE2-induced PRL expression. An ac-tion of PGE2 through the EP4 receptor was furthermore supportedby the finding that PGE2 stimulated [cAMP]i in Jurkat cells andthat this effect was blocked by L-161982. Butaprost had no effecton [cAMP]i, suggesting EP2 receptors are not present on Jurkatcells. This is in accordance with earlier findings in which the EP2receptor was undetectable on Jurkat cells by RT-PCR (48). Thefact that the EP1/EP3 agonist 17-P-T-PGE2 raised [cAMP]i at adose of 1 �M confirms the hypothesis that at high doses, thisagonist activates the EP4 receptor, because in human cells EP1 andEP3 receptors do not classically couple to increases in [cAMP]i

(49, 50). Although Ca2� signaling is required for the induction ofPRL expression by PGE2, it appeared that the EP4 antagonistL-161982 did not block the increase in [Ca2�]i. This implies thatanother EP receptor is involved, that accounts for raising [Ca2�]i.We showed that PGE2, sulprostone and 100 nM 17-P-T-PGE2,which does not activate EP4, did indeed raise [Ca2�]i. Further-more, the effects of 17-P-T-PGE2 on [Ca2�]i were not blocked bythe EP1 antagonist SC-19220, suggesting PGE2 exerts its effect on[Ca2�]i via EP3 receptors.

Multiple isoforms of the EP3 receptor have been cloned, in sev-eral species, with variations only in their C-termini, resulting in theactivation of different signaling pathways through different G pro-

tein. The activation of second messengers is best known for thebovine EP3 receptor, of which four isoforms (A, B, C, and D) havebeen described. EP3A, -B, or -C isoforms can either inhibit oractivate adenylyl cyclase through Gi or Gs, respectively. The EP3Dreceptor can also be coupled to the activation of phospholipase C(PLC) via Gq, in addition to Gi and Gs (35, 51). PLC cleaves PIP2to generate diacylglycerol and inositol 1,4,5-triphosphate (IP3)which is pivotal to the entry of Ca2� to the cytosol from the en-doplasmic reticulum and the extracellular space. Our data suggestthe EP3 isoform present on Jurkat cells is coupled to Ca2� sig-naling. An elevation of [Ca2�]i is essential in T cell activationthrough the TCR complex. In T cells, IP3-induced release of Ca2�

from the endoplasmic reticulum serves as a trigger for controllinga large influx of extracellular Ca2�, which plays a pivotal role ingene regulation (52). Although the EP3D receptor has been shownto activate PLC, leading to the generation of IP3, our results sug-gest that the release of [Ca2�]i from the endoplasmic reticulum isnot involved in the stimulation of PRL expression by PGE2. In-cubation of Jurkat cells with thapsigargin, an inhibitor of the sarco-endoplasmic reticulum Ca2� ATPase, leads to a rapid depletion ofintracellular Ca2� stores. Our observation that pretreatment of Jur-kat cells with thapsigargin did not alter the effect of PGE2 on PRLexpression, whereas BAPTA and EGTA were inhibitory, suggeststhat PGE2 generates a store-independent influx of extracellularCa2�. Interestingly, in Jurkat cells, receptors for the Ca2�-releas-ing messenger IP3 were detected on the plasma membrane and itwas shown that these receptors are responsible for the entry ofextracellular Ca2� during proliferative responses (53). A possiblemechanism for the PGE2-induced rise in [Ca2�]i in Jurkat cellscould thus be via the activation of membrane-bound IP3 receptors.

We previously showed that raising intracellular cAMP levelsusing cptcAMP induces PRL expression in Jurkat cells. Ionomycintreatment did not affect PRL expression, suggesting the Ca2� sig-nal by itself is insufficient to trigger PRL expression. This is in linewith our finding that sulprostone alone does not induce PRL ex-pression. However, in combination, cAMP and ionomycin syner-gized to induce PRL expression to an extent comparable to theinduction by PGE2 (27). This is in accordance with the presentobservation that PGE2 not only stimulates cAMP generation, butalso Ca2� influx, and that both these signals are required for fullPRL expression. Furthermore, the fact that we could mimic theeffect of PGE2 on PRL expression using a combination of the EP3agonist sulprostone, which increases [Ca2�]i, and cptcAMP argues

FIGURE 10. The EP3 agonist, sulprostone, induces PRL expression insynergy with cAMP. Jurkat cells were stimulated for 18 h with cptcAMP(250 �M) in combination with increasing doses of sulprostone. PRL levelsin conditioned medium were quantified by ELISA.

FIGURE 9. Effects of EP agonistsand antagonists on [Ca2�]i. Fluo-3-loaded Jurkat cells were stimulatedwith EP agonists or EP antagonists and[Ca2�]i was determined by spectroflu-orometry. Results are represented aspercentage of [Ca2�]i at 0.5 s beforestimulation. The addition of the stimu-lus is indicated by an arrow. A, Cellswere stimulated with 100 nM PGE2 or17-P-T-PGE2. B, Cells were stimulatedwith 100 nM PGE2 or sulprostone. C,Cells were preincubated with or with-out SC-19220 (100 nM) for 1 h at RTbefore stimulation with 17-P-T-PGE2

(100 nM). D, Cells were preincubatedwith or without L-161982 (100 nM) for1 h at RT before stimulation with PGE2

(100 nM).

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for a combined action of PGE2 through EP3 and EP4. These resultsfurthermore indicate that the cAMP signal is the sine qua non forPRL expression, whereas the Ca2� signal can probably enhancethe activation of factors that are induced by cAMP and thus syn-ergize with cAMP to induce PRL expression. The molecular basisfor this synergism remains to be elucidated, yet a possible targetfor the synergistic effect of the Ca2� signal could be the transcrip-tion factor CREB. Indeed, coordinate activation of different ki-nases, among which are the calcium/calmodulin-dependent ki-nases, leading to optimal activation of CREB has been shown in Tcells (54, 55).

The EP4 receptor has been previously described on Jurkat cellsby RT-PCR (48) and flow cytometric analysis (56). To our knowl-edge, we are the first to describe PGE2 effects through EP3 onJurkat cells. Indeed, effects of PGE2 on T lymphocytes are mostoften attributed to rises in [cAMP]i through occupation of EP2and/or EP4 receptors (42–44). However, it was shown that in thehuman leukemic T cell line, HSB.2, expression of matrix metal-loproteinase-9 is regulated by PGE2 binding to the EP3 receptor(57). Furthermore, PGE2 and dexamethasone act synergistically toinhibit TCR signaling, and this effect was mimicked in primary Tcells by a specific agonist for the EP3 receptor (58).

Using a promoter construct carrying 1842 bp of the leukocytePRL promoter, we previously found that the effect of PGE2 onpromoter activation was markedly smaller than the effect onmRNA levels (27). Our observation that PRL mRNA stability wasnot affected by PGE2, indicates that sites beyond �1842 may berequired for full promoter activation. To address this possibility,we transfected Jurkat cells with a construct carrying 3000 bp of theleukocyte PRL promoter. However, the activation of this promoterconstruct was not different from that of the 1842 bp construct,suggesting even more distant or perhaps intronic sites would beinvolved in regulating PRL expression. Indeed, a binding site fora lymphoid-specific factor has been located downstream of thelymphoid-specific transcription start site in intron 1A, which is infact the superdistal region of the pituitary PRL promoter. Whentransfected into Jurkat cells, this footprinted region activated tran-scription of a heterologous promoter, suggesting it might be afunctional enhancer (59). Also, we cannot exclude the possibilitythat full promoter activation requires additional signals, besidescAMP and Ca2�, converging to responsive elements that are notcontained in the 3000-bp promoter construct. Yet another possi-bility is that the transfection of Jurkat cells through electroporation

caused a loss of EP receptors on the cell membrane, as observedfor fMLP receptors in COS cells (60). This idea is in accordancewith our observation that direct activation of second messengers,via ionomycin in combination with cptcAMP, accomplishes fullpromoter activation, comparable to the effect on PRL mRNAlevels.

The effects of cptcAMP, cptcAMP in combination with iono-mycin, and PGE2 on PRL-3000-Luc were preserved using a pro-moter construct carrying 332 bp of the upstream PRL promoter.The promoter harbors an imperfect CRE immediately upstream ofthe transcriptional start site (61). Although Reem et al. (62)showed that mutation of this CRE partially abolished cAMP re-sponsiveness of the upstream PRL promoter in Jurkat cells, wewere unable to activate a construct carrying 32 bp of the upstreamPRL promoter (and thus, the CRE), suggesting this site is notsolely responsible for the cAMP induction. Our results further in-dicate the involvement of a C/EBP site at �214 in the effects ofcptcAMP, cptcAMP plus ionomycin, and PGE2 on the leukocytePRL promoter. Earlier studies in human endometrial stromal cells,showed the requirement of two overlapping C/EBP sites in theregion �332/�270 for PKA inducibility, whereas the C/EBP siteat �214 was not involved (36). The C/EBP family of basic region/leucine zipper DNA-binding proteins consists of six members:C/EBP�, -�, -�, -�, -�, and -�. Specificity of gene control byC/EBPs is ensured by their cell-specific and temporal expressionpattern and through their ability to homo- and heterodimerize andinteract with other transcription factors (63). Recently, it wasshown that PGE2 activates the HIV-1 long terminal repeat in Ju-rkat cells by a cooperative interaction of C/EBP� and CREB, bind-ing to two proximal C/EBP sites (64). Additional experiments willbe required to identify the transcription factors binding to theC/EBP site in the PRL promoter in Jurkat cells.

PBMC from SLE patients produce higher amounts of PRL thanthose from control donors (20, 21). Also, in patients with rheuma-toid arthritis, synovium infiltrating T cells produce PRL whichstimulates the release of proinflammatory factors from synovialcells (23). Taken together, these observations are compatible witha deleterious role of leukocyte PRL in the progression of autoim-mune disease. PGE2 levels are increased in synovial fluids frompatients with rheumatoid arthritis, ranging from 127 to 886 nM(65–68). Furthermore, several lines of evidence suggest that atleast some of the proinflammatory aspects of the disease are me-diated by PGE2 (69–71). Because PRL produced by infiltrating T

FIGURE 11. Promoter sites involved in PGE2 and cptcAMP-induced PRL expression. Jurkat cells were transiently transfected with equimolar amountsof PRL promoter constructs and stimulated for 18 h with cptcAMP (250 �M), ionomycin (200 ng/ml), PGE2 (100 nM), or a combination of cptcAMP andionomycin. Data are shown as a fold induction relative to unstimulated controls. A, Effect of stimuli on 5� deletions of the leukocyte PRL promoter:PRL-3000-Luc, PRL-332-Luc, and PRL- 32-Luc carrying respectively 3000, 332, and 32 bp of the leukocyte PRL promoter. The figure legend is sharedfor A and B. #, Different from the induction of PRL-3000-Luc (p � 0.05). B, Effect of stimuli on wild-type PRL-332-Luc or site directed mutants DB(mutation of two overlapping C/EBP sites at �310 and �298), F (mutation of a C/EBP site at �214), or G (mutation of an Ets site at �248). #, Differentfrom the induction of wild-type PRL-332-Luc (p � 0.05).

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cells could transduce some of the proinflammatory aspects attrib-uted to PGE2, we have investigated the mechanisms at the basis ofthe stimulation of PRL expression by PGE2 in Jurkat cells. Wehave described a novel mechanism, based on the cooperation ofEP3 and EP4 receptors, by which physiologically relevant PGE2

concentrations can induce gene expression in T cells. Our datamight furthermore contribute to a better understanding of the roleof PRL in the pathophysiology of autoimmune diseases such asrheumatoid arthritis and perhaps to the development of new ther-apeutic strategies. Whereas the Jurkat T cell line has been suc-cessfully used as a model system to study various aspects of T cellfunctioning (72), the physiological relevance of our findings re-mains to be confirmed in primary T cells.

AcknowledgmentsWe thank Dr. Dario Alessi for the MSK-1 expression vectors, Issam Harfifor his advice concerning the Ca2� measurements, and Robert Hooghe forrevision of the manuscript.

References1. Ben-Jonathan, N., J. L. Mershon, D. L. Allen, and R. W. Steinmetz. 1996. Ex-

trapituitary prolactin: distribution, regulation, functions, and clinical aspects. En-docr. Rev. 17:639.

2. Kooijman, R., and S. Gerlo. 2002. Prolactin expression in the immune system. InNeuroimmune Biology, Vol. 2. R. Rapaport and L. Matera, eds. Elsevier Science,New York, p. 147.

3. Montgomery, D. W., G. K. Shen, E. D. Ulrich, L. L. Steiner, P. R. Parrish, andC. F. Zukoski. 1992. Human thymocytes express a prolactin-like messenger ri-bonucleic acid and synthesize bioactive prolactin-like proteins. Endocrinology131:3019.

4. Pellegrini, I., J. J. Lebrun, S. Ali, and P. A. Kelly. 1992. Expression of prolactinand its receptor in human lymphoid cells. Mol. Endocrinol. 6:1023.

5. Horseman, N. D., W. Zhao, E. Montecino-Rodriguez, M. Tanaka, K. Nakashima,S. J. Engle, F. Smith, E. Markoff, and K. Dorshkind. 1997. Defective mammo-poiesis, but normal hematopoiesis, in mice with a targeted disruption of the pro-lactin gene. EMBO J. 16:6296.

6. Bouchard, B., C. J. Ormandy, J. P. Di Santo, and P. A. Kelly. Immune systemdevelopment and function in prolactin receptor deficient mice. J. Immunol. 163:576.

7. Dogusan, Z., R. Hooghe, P. Verdood, and E. L. Hooghe-Peters. 2001. Cytokine-like effects of prolactin in human mononuclear and polymorphonuclear cells.J. Neuroimmunol. 120:58.

8. Lahat, N., A. Miller, R. Shtiller, and E. Touby. 1993. Differential effects ofprolactin upon activation and differentiation of human B lymphocytes. J. Neu-roimmunol. 47:35.

9. Matalka, K. Z. 2003. Prolactin enhances production of interferon-�, interleukin-12, and interleukin-10, but not of tumor necrosis factor-� in a stimulus-specificmanner. Cytokine 21:187.

10. Matera, L., M. Contarini, G. Bellone, B. Forno, and A. Biglino. 1999. Upmodu-lation of interferon-� mediates the enhancement of spontaneous cytotoxicity inprolactin-activated natural killer cells. Immunology 98:386.

11. Leff, M. A., D. J. Buckley, J. S. Krumenacker, J. C. Reed, T. Miyashita, andA. R. Buckley. 1996. Rapid modulation of the apoptosis regulatory genes, bcl-2and bax by prolactin in rat Nb2 lymphoma cells. Endocrinology 137:5456.

12. Krishnan, N., O. Thellin, D. J. Buckley, N. D. Horseman, and A. R. Buckley.2003. Prolactin suppresses glucocorticoid-induced thymocyte apoptosis in vivo.Endocrinology 144:2102.

13. Dorshkind, K., and N. D. Horseman. 2000. The roles of prolactin, growth hor-mone, insulin-like growth factor-1, and thyroid hormones in lymphocyte devel-opment and function: insights from genetic models of hormone and hormonereceptor deficiency. Endocr. Rev. 21:292.

14. Zellweger, R., X. H. Zhu, M. W. Wichmann, A. Ayala, C. M. DeMaso, andI. H. Chaudry. 1996. Prolactin administration following hemorrhagic shock im-proves macrophage cytokine release capacity and decreases mortality from sub-sequent sepsis. J. Immunol. 157:5748.

15. Dugan, A. L., O. Thellin, D. J. Buckley, A. R. Buckley, C. K. Ogle, andN. D. Horseman. 2002. Effects of prolactin deficiency on myelopoiesis andsplenic T-lymphocyte proliferation in thermally injured mice. Endocrinology143:4147.

16. Miranda, J. M., R. E. Prieto, R. Paniagua, G. Garcia, D. Amato, L. Barile, andL. J. Jara. 1998. Clinical significance of serum and urine prolactin levels in lupusglomerulonephritis. Lupus 7:387.

17. Kullich, W. C., and G. Klein. 1998. High levels of macrophage inflammatoryprotein-1� correlate with prolactin in female patients with active rheumatoidarthritis. Clin. Rheumatol. 17:263.

18. Seriolo, B., V. Ferretti, A. Sulli, D. Fasciolo, and M. Cutolo. 2002. Serum pro-lactin levels in male patients with rheumatoid arthritis. Ann. NY Acad. Sci. 966:258.

19. Peeva, E., D. Michael, J. Cleary, J. Rice, X. Chen, and B. Diamond. 2003. Pro-lactin modulates the naive B cell repertoire. J. Clin. Invest. 111:275.

20. Larrea, F., A. Martinez-Castillo, V. Cabrera, J. Alcocer-Varela, G. Queipo,C. Carino, and D. Alarcon-Segovia. 1997. A bioactive 60-kilodalton prolactinspecies is preferentially secreted in cultures of mitogen-stimulated and nonstimu-lated peripheral blood mononuclear cells from subjects with systemic lupus er-ythematosus. J. Clin. Endocrinol. Metab. 82:3664.

21. Gutierrez, M. A., J. F. Molina, L. J. Jara, M. L. Cuellar, C. Garcia,S. Gutierrez-Urena, A. Gharavi, and L. R. Espinoza. 1995. Prolactin and systemiclupus erythematosus: prolactin secretion by SLE lymphocytes and proliferative(autocrine) activity. Lupus 4:348.

22. Stevens, A., D. Ray, A. Alansari, A. Hajeer, W. Thomson, R. Donn, W. E. Ollier,J. Worthington, and J. R. Davis. 2001. Characterization of a prolactin gene poly-morphism and its associations with systemic lupus erythematosus. ArthritisRheum. 44:2358.

23. Nagafuchi, H., N. Suzuki, A. Kaneko, T. Asai, and T. Sakane. 1999. Prolactinlocally produced by synovium infiltrating T lymphocytes induces excessive sy-novial cell functions in patients with rheumatoid arthritis. J. Rheumatol. 26:1890.

24. Berwaer, M., J. A. Martial, and J. R. Davis. 1994. Characterization of an up-stream promoter directing extrapituitary expression of the human prolactin gene.Mol. Endocrinol. 8:635.

25. Gellersen, B., R. Kempf, R. Telgmann, and G. E. DiMattia. 1994. Nonpituitaryhuman prolactin gene transcription is independent of Pit-1 and differentially con-trolled in lymphocytes and in endometrial stroma. Mol. Endocrinol. 8:356.

26. Sabharwal, P., R. Glaser, W. Lafuse, S. Varma, Q. Liu, S. Arkins, R. Kooijman,L. Kutz, K. W. Kelley, and W. B. Malarkey. 1992. Prolactin, synthesized byhuman peripheral blood mononuclear cells: an autocrine growth factor for lym-phoproliferation. Proc. Natl. Acad. Sci. USA 89:7713.

27. Gerlo, S., W. Vanden Berghe, P. Verdood, E. L. Hooghe-Peters, andR. Kooijman. 2003. Regulation of prolactin expression in leukemic cell lines andperipheral blood mononuclear cells. J. Neuroimmunol. 135:107.

28. Goetzl, E. J., S. An, and W. L. Smith. 1995. Specificity of expression and effectsof eicosanoid mediators in normal physiology and human diseases. FASEB J.9:1051.

29. Goodwin, J. S., A. D. Bankhurst, and R. P. Messner. 1977. Suppression of humanT-cell mitogenesis by prostaglandin: existence of a prostaglandin-producing sup-pressor cell. J. Exp. Med. 146:1719.

30. Walker, C., F. Krsitensen, F. Bettens, and A. L. deWeck. 1983. Lymphokineregulation of activated (G1) lymphocytes. I. Prostaglandin E2-induced inhibitionof IL-2 production. J. Immunol. 130:1770.

31. Rincon, M., A. Tugores, A. Lopez-Rivas, A. Silva, M. Alonso, M. O. De Landaz,and M. Lopez-Botet. 1988. Prostaglandin E2 and the increase of intracellularcAMP inhibits the expression of interleukin 2 receptors in human T cells. Eur.J. Immunol. 18:1791.

32. Betz, M., and B. S. Fox. 1991. Prostaglandin E2 inhibits production of Th1lymphokines but not of Th2 lymphokines. J. Immunol. 146:108.

33. Roper, R. L., D. M. Brown, and R. P. Phipps. 1995. Prostaglandin E2 promotesB lymphocyte Ig isotype switching to IgE. J. Immunol. 154:162.

34. Tilley, S. L., T. M. Coffman, and B. H. Koller. 2001. Mixed messages: modu-lation of inflammation and immune responses by prostaglandins and trombox-anes. J. Clin. Invest. 108:15.

35. Narumyia, S., Y. Sugimoto, and F. Ushikubi. 1999. Prostanoid receptors: struc-tures, properties and functions. Physiol. Rev. 79:1193.

36. Pohnke, Y., R. Kempf, and B. Gellersen. 1999. CCAAT/enhancer-binding pro-teins are mediators in the protein kinase A-dependent activation of the decidualprolactin promoter. J. Biol. Chem. 274:24808.

37. Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. A new generation of Ca2�

indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440.

38. Davies, S. P., H. Reddy, M. Caivano, and P. Cohen. 2000. Specificity and actionof some commonly used protein kinase inhibitors. Biochem. J. 351:95.

39. Deak, M., A. D. Clifton, J. M. Lucocq, and D. R. Alessi. 1998. Mitogen- andstress-activated protein kinase-1 is directly activated by MAPK and SAPK2/p38,and may mediate activation of CREB. EMBO J. 17:4426.

40. de Rooij, J., F. J. Zwartkruis, M. H. Verheyen, R. H. Cool, S. M. Nijman,A. Wittinghofer, and J. Bos. 1998. Epac is a Rap1 guanine-nucleotide-exchangefactor directly activated by cyclic AMP. Nature 396:474.

41. Fujino, H., K. A. West, and J. W. Regan. 2002. Phosphorylation of glycogensynthase kinase-3 and stimulation of T-cell factor signaling following activationof EP2 and EP4 prostanoid receptors by PGE2. J. Biol. Chem. 277:2614.

42. Kabashima, K., T. Saji, T. Murata, M. Nagamachi, T. Matsuoka, E. Segi,K. Tsuboi, Y. Sugimoto, T. Kobayashi, Y. Miyachi, et al. 2002. The prostaglan-din receptor EP4 suppresses colitis, mucosal damage, and CD4 cell activation inthe gut. J. Clin. Invest. 109:883.

43. Nataraj, C., D. W. Thomas, S. L. Tilley, M. Nguyen, R. Mannon, B. H. Koller,and T. M. Coffman. 2001. Receptors for prostaglandin E2 that regulate cellularimmune responses in the mouse. J. Clin. Invest. 108:1229.

44. Takahashi, H. K., H. Iwagaki, T. Yoshino, S. Mori, T. Morishika, H. Itoh,M. Yokoyama, E. Kondo, T. Akagi, N. Tanaka, and M. Nishibori. 2002. Pros-taglandin E2 inhibits IL-18-induced ICAM-1 and B7.2 expression through EP2/EP4 receptors in human peripheral blood mononuclear cells. J. Immunol. 168:4446.

45. Gellersen, B., G. E. DiMattia, H. G. Friesen, and H. G. Bohnet. 1989. Regulationof prolactin secretion in the human B-lymphoblastoid cell line IM-9-P3 by dexa-methasone but not other regulators of pituitary prolactin secretion. Endocrinology125:2853.

46. Gellersen, B., R. Kempf, S. Hartung, A. Bonhoff, and G. E. DiMattia. 1992.Posttranscriptional regulation of the human prolactin gene in IM-9-P3 cells byretinoic acid. Endocrinology 131:1017.

5961The Journal of Immunology

by guest on July 27, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

47. Mei, F. C., J. Qiao, O. M. Tsygankova, J. L. Meinkoth, L. A. Quilliam, andX. Deng. 2002. Differential signaling of cyclic AMP. J. Biol. Chem. 277:11497.

48. Blaschke, V., K. Jungermann, and G. P. Puschel. 1996. Exclusive expression ofthe Gs-linked prostaglandin E2 receptor subtype 4 mRNA in human mononuclearJurkat and KM-3 cells and coexpression of subtype 4 and 2 mRNA in U-937cells. FEBS Lett. 394:39.

49. An, S., J. Yang, S. W. So, L. Zeng, and E. G. Goetzl. 1994. Isoforms of the EP3subtype of the human prostaglandin E2 receptor that transduce both intracellularcAMP and cAMP signals. Biochemistry 33:14496.

50. Schmid, A., K. Thierauch, W. Schleuning, and H. Dinter. 1995. Splice variantsof the human EP3 receptor for prostaglandin E2. Eur. J. Biochem. 228:23.

51. Hatae, N., Y. Sugimoto, and A. Ichikawa. 2002. Prostaglandin receptors: ad-vances in the study of EP3 receptor signaling. J. Biochem. 131:781.

52. Lewis, R. S. 2001. Calcium signaling mechanisms in T lymphocytes. Annu. Rev.Immunol. 19:497.

53. Khan, A. A., J. P. Steiner, M. G. Klein, M. F. Schneider, and S. H. Snyder. 1992.IP3 receptor: localization to plasma membrane of T cells and cocapping with theT cell receptor. Science 257:815.

54. Shaywitz, A. J., and M. E. Greenberg. 1999. CREB: a stimulus-induced tran-scription factor activated by a diverse array of extracellular signals. Annu. Rev.Biochem. 68:821.

55. Yu, C.-T., H. Shih, and M.-Z. Lai. 2001. Multiple signals required for cyclicAMP-responsive element binding protein (CREB) binding protein interaction in-duced by CD3/CD28 costimulation. J. Immunol. 166:284.

56. Dumais, N., B. Barbeau, M. Olivier, and M. J. Tremblay. 1998. Prostaglandin E2

upregulates HIV-1 long terminal repeat driven gene activity in T cells via NF-�B-dependent and -independent signaling pathways. J. Biol. Chem. 273:27306.

57. Zeng, L., S. An, and E. J. Goetzl. 1996. Regulation of expression of matrixmetalloproteinase-9 in early human T-cells of the HSB.2 cultured line by the EP3subtype of prostaglandin E2 receptor. J. Biol. Chem. 271:27744.

58. Elliot, L. H., A. K. Levay, B. Sparks, M. Miller, and T. L. Roszman. 1996.Dexamethasone and prostaglandin E2 modulate T-cell receptor signaling througha cAMP-independent mechanism. Cell Immunol. 169:117.

59. Van de Weerdt, C., B. Peers, A. Belayew, J. A. Martial, and M. Muller. 2000. Farupstream sequences regulate the human prolactin promoter transcription. Neu-roendocrinology 71:124.

60. Baker, E. A., M. W. Vaughn, and D. L. Haviland. 2000. Choices in transfectionmethodologies: transfection efficiency should not be the sole criterion. Focus22:31.

61. Telgmann, R., E. Maronde, K. Tasken, and B. Gellersen. 1997. Activated proteinkinase A is required for differentiation-dependent transcription of the decidualprolactin gene in human endometrial stromal cells. Endocrinology 138:929.

62. Reem, G. H., D. W. Ray, and J. R. Davis. 1999. The human prolactin geneupstream promoter is regulated in lymphoid cells by activators of T-cells and bycAMP. J. Mol. Endocrinol. 22:285.

63. Lekstrom-Himes, J., and K. G. Xanthopoulos. 1998. Biological role of theCCAAT/enhancer-binding protein family of transcription factors. J. Biol. Chem.273:28545.

64. Dumais, N., S. Bounou, M. Olivier, and M. J. Tremblay. 2002. ProstaglandinE2-mediated activation of HIV-1 long terminal repeat transcription in human Tcells necessitates CCAAT/enhancer binding protein (C/EBP) binding sites in ad-dition to cooperative interactions between C/EBP� and cyclic adenosine 5�-monophosphate response element binding protein. J. Immunol. 168:274.

65. Egg, D., R. Gunther, M. Herold, and F. Kerschbaumer. 1980. Prostaglandins E2

and F2� concentrations in the synovial fluid in rheumatoid and traumatic kneejoint diseases. Z. Rheumatol. 39:170.

66. Hishinuma, T., H. Nakamura, T. Sawai, M. Uzuki, Y. Itabash, and M. Mizugaki.1999. Microdetermination of prostaglandin E2 in joint fluid in rheumatoid arthri-tis patients using gas chromatography/selected ion monitoring. ProstaglandinsOther Lipid Mediat. 58:179.

67. Duffy, T., O. Belton, B. Bresnihan, O. Fitzgerald, and D. Fitzgerald. 2003. In-hibition of PGE2 production by nimesulide compared with diclofenac in theacutely inflamed joint of patients with arthritis. Drugs 63:31.

68. Punzi, L., A. Pozzuoli, M. Pianon, N. Bertazzolo, F. Oliviero, and R. Scapinelli.2001. Pro-inflammatory interleukins in the synovial fluid of rheumatoid arthritisassociated with joint hypermobility. Rheumatology 40:202.

69. Robinson, D. R., A. H. Tashijian, Jr., and L. Levine. 1975. Prostaglandin-stim-ulated bone resorption by rheumatoid synovia: a possible mechanism for bonedestruction in rheumatoid arthritis. J. Clin. Invest. 56:1181.

70. Portanova, J. P., Y. Zang, G. D. Anderson, S. D. Hauser, J.L. Masferrer,K. Seibert, S. A. Gregory, and P. C. Isakson. 1996. Selective neutralization ofprostaglandin E2 blocks inflammation, hyperalgesia, and IL-6 production in vivo.J. Exp. Med. 184:883.

71. McCoy, J. M., J. R. Wicks, and L. P. Audoly. 2002. The role of prostaglandin E2

receptors in the pathogenesis of rheumatoid arthritis. J. Clin. Invest. 110:651.72. Abraham, R. T., and A. Weiss. 2004. Jurkat cells and the development of the

T-cell receptor paradigm. Nat. Rev. Immunol. 4:301.

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