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The Journal of Immunology, 2004, 173: 5952-5962.
Copyright © 2004 by The American Association of Immunologists

Mechanism of Prostaglandin (PG)E2-Induced Prolactin Expression in Human T Cells: Cooperation of Two PGE2 Receptor Subtypes, E-Prostanoid (EP) 3 and EP4, Via Calcium- and Cyclic Adenosine 5'-Monophosphate-Mediated Signaling Pathways1

Sarah Gerlo2,*, Peggy Verdood*, Birgit Gellersen{dagger}, Elisabeth L. Hooghe-Peters* and Ron Kooijman*

* Laboratory of Neuroendocrine Immunology, Department of Pharmacology, Free University of Brussels, Brussels, Belgium; and {dagger} Endokrinologikum Hamburg, Hamburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously reported that prolactin gene expression in the T-leukemic cell line Jurkat is stimulated by PGE2 and that cAMP acts synergistically with Ca2+ or protein kinase C on the activation of the upstream prolactin promoter. Using the transcription inhibitor actinomycin D, we now show that PGE2-induced prolactin expression requires de novo prolactin mRNA synthesis and that PGE2 does not influence prolactin mRNA stability. Furthermore, PGE2-induced prolactin expression was inhibited by protein kinase inhibitor fragment 14–22 and BAPTA-AM, which respectively, inhibit protein kinase A- and Ca2+-mediated signaling cascades. Using specific PGE2 receptor agonists and antagonists, we show that PGE2 induces prolactin expression through engagement of E-prostanoid (EP) 3 and EP4 receptors. We also found that PGE2 induces an increase in intracellular cAMP concentration 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 –214 partially abolished the response of the leukocyte prolactin promoter to PGE2, cAMP, and ionomycin plus cAMP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The polypeptide hormone, prolactin (PRL),3 is of major importance for reproduction. Whereas the pituitary is the main source of circulating PRL, the hormone is also synthesized at extrapituitary sites such as the decidua, the brain, the mammary gland, and the immune system (1, 2). PRL expression has been demonstrated in thymocytes, T cells, B cells, and monocytes (3, 4), and the PRLR, which belongs to the cytokine-receptor family, is expressed on both lymphocytes and monocytes (4), suggesting that the hormone may act in an auto- or paracrine, cytokine-like fashion in the immune system. Whereas no immune deficiencies could be detected in steady-state conditions in both PRL–/– 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, and cytokine expression in human leukocytes (7, 8, 9, 10). Furthermore, PRL has anti-apoptotic properties that have been demonstrated in the Nb2 rat lymphoma (11) and in dexamethasone-treated thymocytes (12). The apparently conflicting results from the in vivo studies using knockout mice and in vitro data have led to the hypothesis that the immunomodulatory effects of PRL come into play only under conditions in which the organism is subjected to stress (13). Indeed, PRL administration following hemorrhagic shock in mice restored the decreased ability of macrophages to release cytokines, and thus decreased mortality from subsequent sepsis (14). Also, in PRL–/– mice, the lack of PRL enhanced the negative effects of thermal injury on myelopoiesis and T lymphocyte proliferation (15).

Hyperprolactinemia, correlating with disease activity, has been described in autoimmune conditions such as systemic lupus erythematosus (SLE; Ref.16) and rheumatoid arthritis (17, 18), suggesting PRL is involved in the pathophysiology of these diseases. Peeva et al. (19) recently reported that PRL augments the expansion of anti-DNA B cells in mice in a CD4+ T cell-dependent manner, thus causing a lupus-like phenotype. The importance of leukocyte-derived PRL in SLE is suggested by enhanced PRL production in T cells from patients as compared with normal controls (20, 21). Interestingly, a single nucleotide polymorphism in the PRL promoter influences PRL expression in lymphocytes and is associated with SLE in a cohort of patients from the U.K. (22). Furthermore, in patients with rheumatoid arthritis, PRL produced by synovium infiltrating T cells causes aberrant synovial cell function and might thus influence disease progression (23). These findings, implicating a role for leukocyte-derived PRL in immune responses, prompted us to investigate the regulation of PRL expression in T cells.

Extrapituitary PRL expression is mostly (e.g., in the decidua and the immune system) regulated by an alternative promoter, located 5.8 kb upstream to the pituitary PRL promoter (24, 25). Whereas regulation of pituitary PRL expression has been extensively studied, little is known about the factors that regulate PRL expression in leukocytes and what DNA sequences in the upstream PRL promoter, hereafter called the leukocyte promoter, are targeted by these regulators. It has been shown that PRL expression in PBMC is stimulated by Con A and phytohemagglutinin (26), and we recently reported that PRL expression in Jurkat leukemic T cells and in PBMC is regulated by cAMP and physiological stimuli that signal through cAMP such as PGE2 (27). PGE2 modulates immune responses by stimulating and inhibiting the functions of many different types of immune cells both in vivo and in vitro (28). Most reports describe inhibitory effects of PGE2 on T cell function: PGE2 inhibits T cell proliferation and expression of both IL-2 and the IL-2R (29, 30, 31). Several reports indicate PGE2 has different effects on Th1 vs Th2 cells, resulting in a shift from a Th1 cellular immune response toward a Th2-driven humoral response (32, 33). However, the overall impact of PGE2 in an inflammatory response can be either positive or negative, depending on the level of immune activation, the presence of other mediators, and the physiological state of the organism (34). PGE2 exerts its effects through binding to at least four different receptors, termed E-prostanoid (EP) 1 to EP4, which activate different second messengers, accounting for the specificity and diversity of PGE2 effects. The EP1 receptor induces a rise in intracellular calcium concentration ([Ca2+]i). The EP2 and EP4 receptors activate Gs and thus increase intracellular cAMP concentration ([cAMP]i). Multiple EP3 isoforms have been described, which couple to different signaling pathways: 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 described that activate Gs and transduce increases in [cAMP]i (35).

In this study, we have shown that PGE2 up-regulates PRL expression in T cells through transcriptional activation and has no effect on PRL mRNA stability. Both Ca2+ and cAMP appear to be indispensable second messengers for PGE2-induced PRL expression. Using EPR-specific agonists and antagonists, we established the involvement of both EP3 and EP4 receptors, through elevation of Ca2+ and cAMP, respectively, in the effect of PGE2 on PRL expression. We also showed that cAMP and Ca2+ exert their synergistic action through a CAAT/enhancer-binding protein (C/EBP) binding site at –214 in the leukocyte PRL promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

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 (RpcAMPs), 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 (Ann Arbor, MI). L-161982 was a gift from Merck Frost (Kirkland, Quebec, Canada). Recombinant human PRL was produced in Escherichia coli in our laboratory, using a cDNA obtained from J. Martial (University of Liège, Liège, 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 were described earlier (36). The mitogen-stimulated kinase 1 (MSK-1) expression 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 (Salisbury, U.K.), were maintained in RPMI 1640 with Glutamax-I, supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Invitrogen Life Technologies, Merelbeke, Belgium). For all stimulation experiments, cells were washed and resuspended in RPMI 1640 with Glutamax-I at a concentration of 2 x 106/ml. In stimulation experiments for ELISA, real-time PCR, and reporter gene assays, cells were cultured 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 Western blotting experiments were performed in 2 ml reaction tubes (Brinkman Instruments, Hamburg, Germany) in a waterbath at 37°C.

PRL ELISA

PRL concentrations were determined by sandwich ELISA. A 96-well Maxisorp-Nunc immunoplates (VWR, Leuven, Belgium) were coated overnight at 4°C with a 1/500 dilution of a monoclonal anti-hPRL Ab (BioGenex, San Ramon, CA) in bicarbonate buffer (40 mM Na2CO3, 60 mM NaHCO3, pH 9.6). Supernatants were incubated in the Ab-coated plates for 2 h at room temperature. Next, a rabbit anti-hPRL antiserum, NIDDK-IC-5, donated by Dr. Parlow (National Hormone and Peptide Program, Harbor-University of California Los Angeles Medical Center, Torrance, 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 a peroxidase-conjugated anti-rabbit Ig (Amersham Biosciences, Roosendaal, The Netherlands) diluted 1/5000 in PBS, 0.1% Tween 20, and 1% nonfat dry milk. Every Ab incubation was followed by three washing steps in PBS, 0.1% Tween 20. In the final step, the peroxidase substrate (87 µg/ml 3, 3',5,5'-tetramethylbenzidine dihydrochloride hydrate in 109 mM citric acid, 0.05% H2O2) was added. Absorbance of the samples was measured at 450 nm with a reference wavelength at 540 nm using a microtiter plate reader (Labsystems iEMS Reader MF; Labsystems, Helsinki, Finland). The detection limit of this assay was 10 pg/ml, using recombinant human PRL as a standard.

Real-time PCR

Total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. Reverse transcription was performed using the TaqMan reverse transcription kit from Applied Biosystems (Nieuwerkerk a/d IJssel, The Netherlands). For real-time cDNA amplification, we used the Applied Biosystems SYBR Green Mastermix and the following primers for PRL: sense, 5'-ACC AAG AAG AAT CGG AAC ATA C-3'; antisense, 5'-ACA GGA GCA GGT TTG ACA-C-3'. Fluorescence was monitored using the TaqMan 7700 Sequence Detector (Applied Biosystems). The results of a series of standards prepared by successive dilution (0.02–200 ng) of total RNA and plotted against the logarithm of the concentration were used to estimate the relative amounts of PRL mRNA initially present in each sample. The relative PRL mRNA levels are shown as compared with {beta}-Actin controls. {beta}-actin cDNA was amplified using the following primers: sense, 5'-GGA TGC AGA AGG AGA TCA CCT G-3'; antisense, 5'-CGA TCC ACA CGG AGT ACT TG-3'.

cAMP assays

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

PKA assays

Jurkat cells were preincubated for 1 h with or without PKA inhibitors at 37°C, before stimulation with cptcAMP. Extracts were prepared by sonication of cells in buffer A (20 mM Tris, pH 7.5, 0.25 M sucrose, 10 mM EGTA, 2 mM EDTA, 1 mM AEBSF, 10 µg/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 substrate (kemptide). Phosphorylation of the substrate by PKA alters the peptides net charge from +1 to –1. This change in net charge allows the phosphorylated and nonphosphorylated forms of the substrate to be separated on agarose gel. The bands were visualized using a UV-transilluminator.

Western blotting

For phospho-MSK-1 detection, cellular extracts were prepared in Triton X-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% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM PMSF, 10 µg/ml aprotinin, 50 µg/ml leupeptin, 50 µg/ml pepstatin A, and 500 µg/ml soybean trypsin inhibitor; all from Sigma-Aldrich) and subjected to PAGE. Proteins were transferred to polyvinylidene difluoride membranes. After blocking for 1 h at room temperature in blocking buffer (PBS, 5% nonfat dry milk, and 0.1% Tween 20), blots were probed with anti-phospho-MSK-1 (Cell Signaling, Beverly, MA) at a 1/1000 dilution in PBS, 5% BSA, and 0.1% Tween 20 for 18 h at 4°C. Subsequently, blots were washed three times in PBS and 0.1% Tween 20 and incubated for 60 min at 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 Biosciences). Following three washes in PBS and 0.1% Tween 20, immunoreactive bands were detected using ECL (PerkinElmer, Boston, MA).

Ca2+ measurements

Jurkat cells (10 x 106/ml) were loaded with the Ca2+-sensitive fluorescent dye Fluo-3-AM (8.8 µM) at 37°C for 60 min in RPMI 1640. Next, cells were washed three times in RPMI 1640 and resuspended in RPMI 1640 at 1 x 106 cells/ml. Two milliliters of cell suspension was transferred to a 1-cm light path cuvette, and fluorescence intensity was measured using a spectrofluorophotometer (Shimadzu RF-5301 PC; Shimadzu Scientific Instruments, Kyoto, Japan), at an excitation wavelength of 506 nm and an emission wavelength of 526 nm. Fluorescence measurements were acquired at 0.5-s intervals for 60 s. Fmax and Fmin were determined by incubating the cells with 0.1% Triton X-100 or 25 mM EGTA, respectively. The fluorescence intensities were converted to nanomolar concentrations by the equation [Ca2+]i = Kd(F – Fmin)/(Fmax – F), where Kd denotes the apparent dissociation constant (390 nM) 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 a concentration of 15 x 106/ml. Plasmids were added to a concentration of 30 µg/ml and electroporation was performed using the following settings: 330 V and 1800 µF (Electropore 2000; Eurogentec, Liége, Belgium). Cell lysates were prepared and assayed for luciferase activity using the Promega Luciferase Assay kit (Promega). For MSK-1 overexpression experiments, cells were transfected by nucleofection using solution V and program S18 (Amaxa, Cologne, Germany) according to the manufacturer’s instructions.

Statistical analysis

Statistical differences between group means were determined by ANOVA with Tukey’s post-test. Differences were considered significant when p < 0.05. Data represented are means ± SD of quadruplicate incubations. The presented experiments are representative of at least three independent experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PGE2 enhances PRL expression by activating PRL transcription and does not affect PRL mRNA stability

We previously showed by conventional RT-PCR and ELISA that PRL mRNA levels and PRL secretion are up-regulated by PGE2 in Jurkat cells (27). To address the level(s) at which regulation of PRL expression by PGE2 takes place, we studied the kinetics of PRL expression in the presence and absence of the transcriptional inhibitor actinomycin D. As depicted in Fig. 1A, an increase in PRL mRNA level was already detected after 4 h of stimulation with PGE2 (100 nM), whereas an increase in PRL secretion was detected after 8 h (Fig. 1B). Preincubation of Jurkat cells with actinomycin D completely abrogated the effect of PGE2 on both PRL mRNA levels and secretion (Fig. 1, A and B). These results indicate that PRL expression is regulated at the transcriptional level and that PGE2 does not stimulate PRL translation or secretion. To exclude modulation of mRNA stability by PGE2, we compared the decrease in absolute levels of PRL mRNA in the presence and absence of actinomycin D. As shown in Fig. 1C, PGE2 does not interfere with PRL mRNA decay, indicating that it does not affect mRNA stability.



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

 
Both cAMP/PKA and Ca2+ are involved in PGE2-induced signaling leading to PRL expression

Because we showed that the two principal second messengers involved in PGE2 receptor signaling (cAMP and Ca2+) synergistically activate PRL expression in Jurkat cells (27), we further addressed the roles of these messengers in PGE2-induced PRL expression. PKA is the best known effector of cAMP signaling. Therefore, we investigated the effects of four structurally unrelated PKA inhibitors (H89, KT5720, RpcAMPs, and PKI) on PGE2-induced PRL mRNA levels and protein secretion. As shown in Fig. 2, A and B, only H89 and PKI blocked PGE2-induced PRL expression at the mRNA level. Morever, H89 and PKI completely blocked PGE2-induced PRL secretion, whereas KT5720 and RpcAMPs only exerted small inhibitory effects (Fig 2, C and D). Furthermore, H89, but not KT5720 or RpcAMPs, blocked PRL expression induced by the long-acting cAMP analog, cptcAMP, at the level of transcription as well as at the level of secretion. Therefore, to find out whether KT5720 and RpcAMPs were effective in blocking PKA activation in our system, we investigated their effect on cptcAMP-induced activation of PKA using the Peptag PKA assay system. As shown in Fig. 3A, H89, but not KT5720 or RpcAMPs, effectively blocked PKA activation. Although H89 is often considered as a selective inhibitor for PKA, several studies indicate that H89 inhibits other protein kinases with a potency greater than or equal to that for PKA (37). One of these other H89 targets is MSK-1. MSK-1 is directly activated by ERK2 and p38 and can mediate activation of CREB (39). To address the possibility that H89 blocks induction of PRL expression through inhibition of MSK-1, we investigated whether PGE2 or cptcAMP phosphorylate overexpressed MSK-1 in Jurkat cells. As shown in Fig. 3B, neither PGE2 nor cptcAMP induced phosphorylation of MSK-1.



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  		FIGURE 2. Effect of PKA inhibitors on PGE2-induced PRL expression. Jurkat cells were pretreated for 2 h with vehicle, H89 (10 µM), KT5720 (0.2 µM), RpcAMPs (50 µM; A and C), or increasing doses of PKI (B and D) before stimulation with 100 nM PGE2 or 250 µM cptcAMP for 18 h. A and B, PRL mRNA levels were quantified by real-time PCR and normalized to {beta}-actin mRNA expression. Normalized PRL mRNA levels are represented as percentage of control (no inhibitor, no stimulus). C and D, PRL secretion as measured by ELISA. The figure legend is shared for A–D. #, Significantly different (p < 0.05).

 


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  		FIGURE 3. Inhibition of cptcAMP-induced PKA activation. PGE2 or cptcAMP do not phosphorylate overexpressed MSK-1. A, Jurkat cells were preincubated with or without PKA inhibitors for 1 h in a 37°C waterbath before stimulation with cptcAMP for 20 min at 37°C. PKA activity in cell pellets was quantified using the Peptag assay with kemptide as a substrate. NP, nonphosphorylated kemptide; P, phosphorylated kemptide. Lane 1, control; lane 2, 250 µM cptcAMP. Cells in lanes 3–8 were stimulated with 250 µM cptcAMP plus the following inhibitors: lane 3, 1 µM H89; lane 4, 10 µM H89; lane 5, 0.2 µM KT5720; lane 6, 2 µM KT5720; lane 7, 10 µM RpcAMPs; and lane 8, 50 µM RpcAMPs. Lane 9, negative control (no PKA); lane 10, positive control (0.5 ng PKA). B, Jurkat cells were preincubated for 1 h in a 37°C waterbath, before stimulation with TPA (10 ng/ml), cptcAMP (250 µM), or PGE2 (100 nM) for 10 or 30 min at 37°C. Phosphorylated MSK-1 was detected by Western blotting.

 
It recently became clear that cAMP exerts some of its effects in a PKA-independent manner through direct activation of a Rap-1 guanine-nucleotide-exchange factor, an exchange protein directly activated by cAMP (EPAC; Ref.40). We investigated the effect of a methylated cptcAMP analog, which specifically activates EPAC, on PRL expression in Jurkat cells. As shown in Fig. 4, whereas cptcAMP stimulates PRL expression in Jurkat cells, the effect of Me-cptcAMP is inhibitory, suggesting EPAC is not involved in stimulating PRL expression.



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

 
To address the role of Ca2+ in PGE2-induced PRL expression, we preincubated Jurkat cells with the cell-permeable Ca2+ chelator BAPTA-AM, or the cell-impermeable Ca2+ chelator, EGTA. A role for Ca2+ is indicated by the inhibitory effects of both BAPTA-AM and EGTA on the stimulation by PGE2 of PRL mRNA expression and secretion (Fig. 5, A and B). We next addressed the role of Ca2+ from intracellular stores. Preincubation of Jurkat cells with thapsigargin, which depletes the intracellular Ca2+ stores, did not affect either PGE2-induced PRL mRNA expression or PRL secretion (Fig. 5, A and B).



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  		FIGURE 5. Effect of Ca2+ chelators on PGE2-induced PRL expression in Jurkat cells. Jurkat cells were pretreated for 2 h with vehicle, BAPTA-AM (10 µM), EGTA (2 mM), or thapsigargin (0.1 µM) before stimulation with PGE2 for 18 h. A, PRL mRNA levels were quantified by real-time PCR and normalized to {beta}-actin mRNA expression. Normalized PRL mRNA levels are represented as a percentage of 100% control (no inhibitor, no stimulus). B, PRL secretion as measured by ELISA. The figure legend is shared for A and B. #, Significantly different (p < 0.05).

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



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  		FIGURE 6. Effect of inhibition of PI3K on PGE2-induced PRL expression. Jurkat cells were pretreated for 2 h with increasing doses of wortmannin before stimulation with PGE2 (100 nM) for 18 h. A, PRL mRNA levels were quantified by real-time PCR and normalized to {beta}-actin mRNA expression. Normalized PRL mRNA levels are represented as a percentage of 100% control (no inhibitor, no stimulus). B, PRL secretion as measured by ELISA. The figure legend is shared for A and B. #, Significantly different (p < 0.05).

 
EP receptors involved in the generation of second messengers and PRL expression

PGE2-induced PRL expression. To address the role of EP receptors in the stimulating effect of PGE2 on PRL expression, we first incubated cells with different doses of PGE2, the EP1/EP3 agonist 17-P-T-PGE2, the EP2 agonist, butaprost, or the EP3 agonist, sulprostone, and measured PRL expression by ELISA. As shown in Fig. 7A, 10 nM PGE2 and 1 µM 17-P-T-PGE2 enhanced PRL expression, whereas butaprost and sulprostone were ineffective. Next, we investigated the effect of specific antagonists for EP1 and EP4 on PGE2- and 17-P-T-PGE2-induced PRL expression. Whereas the EP1 antagonist SC-19220 had no effect on PGE2- or 17-P-T-PGE2-induced PRL expression, the EP4 antagonist L-161982 nearly completely blocked PGE2- and 17-P-T-PGE2-induced PRL expression (Fig. 7, B and C), suggesting the involvement of EP4 in PGE2- and 17-P-T-PGE2-induced PRL expression.



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

 
Augmentation of [cAMP]i by PGE2 via the EP4 receptor. Most PGE2 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, 43, 44). Using a cell permeable cAMP analog, we showed earlier that 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 mediates this rise in [cAMP]i, we measured the effects of increasing doses of PGE2, 17-P-T-PGE2, or butaprost on [cAMP]i. We found that 100 nM PGE2 induced a rise in [cAMP]i (Fig. 8A). Butaprost did not alter [cAMP]i, suggesting that functional EP2 receptors are not present on Jurkat cells. As observed for PRL expression, 17-P-T-PGE2 enhanced [cAMP]i at a dose of 1 µM, which is higher than the dose required for activating EP1 or EP3 receptors. The hypothesis that PGE2 increases [cAMP]i via the EP4 receptor was confirmed by the finding that the EP4 antagonist, L-161982, at a dose of 10 nM completely blocked PGE2-induced [cAMP]i accumulation (Fig. 8B).



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  		FIGURE 8. Effects of EP agonists and antagonists on [cAMP]i. A, Cells were preincubated for 1 h in a 37°C waterbath, before stimulation for 10 min 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 with L-161982 before stimulation with PGE2 (100 nM) for 10 min. [cAMP]i in cell pellets was determined by RIA. Results are represented as a fold induction relative to unstimulated controls.

 
PGE2 raises [Ca2+]i through the EP3 receptor. Our finding that, in addition to cAMP, Ca2+ is also involved in PRL induction by PGE2, prompted us to measure the effects of PGE2 and EP agonists and antagonists on [Ca2+]i. Using the Grynkiewicz equation (37), the [Ca2+]i was calculated, and results were represented as a percentage of the [Ca2+]i at 0.5 s before stimulation. Indeed, 100 nM PGE2 did increase [Ca2+]i in Jurkat cells (Fig. 9, A, B, and D). When the cells were stimulated with PGE2 in the presence of the EP4 antagonist L-161982, a further increase in [Ca2+]i was observed (Fig. 9D). At a concentration of 100 nM, both sulprostone and 17-P-T-PGE2 enhanced [Ca2+]i to an extent that exceeds that of PGE2 (Fig. 9, A and B). Preincubation of Jurkat cells with the EP1 antagonist SC-19220 had no effect on the 17-P-T-PGE2-induced rise in [Ca2+]i (Fig. 9C), suggesting its effect on [Ca2+]i is not via the EP1 receptor. In conclusion, these results suggest that PGE2 augments [Ca2+]i via the EP3 receptor.



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  		FIGURE 9. Effects of EP agonists and antagonists on [Ca2+]i. Fluo-3-loaded Jurkat cells were stimulated with EP agonists or EP antagonists and [Ca2+]i was determined by spectrofluorometry. Results are represented as percentage of [Ca2+]i at 0.5 s before stimulation. The addition of the stimulus is indicated by an arrow. A, Cells were stimulated with 100 nM PGE2 or 17-P-T-PGE2. B, Cells were stimulated with 100 nM PGE2 or sulprostone. C, Cells were preincubated with or without SC-19220 (100 nM) for 1 h at RT before stimulation with 17-P-T-PGE2 (100 nM). D, Cells were preincubated with or without L-161982 (100 nM) for 1 h at RT before stimulation with PGE2 (100 nM).

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



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

 
Localization of cAMP- and PGE2-responsive elements in the leukocyte PRL promotor

We previously reported that the effect of PGE2 on endogenous PRL expression in Jurkat cells exceeded the effect of PGE2 on activation of an 1842 bp-carrying promoter construct (27). To address the role of more distant promoter sequences in PRL transcription, we transfected Jurkat cells with a promotor construct carrying 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 that not all PGE2 responsive elements are contained within these 3000 bp of the leukocyte PRL promoter. PRL-3000-Luc was very strongly activated by a synergistic action of cAMP and ionomycin, whereas the effects of cAMP and PGE2 were much weaker (Fig. 11A). An identical response was observed with only 332 bp of the upstream promoter. In contrast, a 32-bp promoter construct did not respond to PGE2, cAMP, or cAMP in combination with ionomycin (Fig. 11A). Importantly, deletion of the –332 and –32 region significantly inhibited the elevated promoter activity in the presence of PGE2, cAMP, or cAMP plus ionomycin. The region between –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 site did not affect promoter activation by PGE2, cAMP, or cAMP plus ionomycin (Fig. 11B). In contrast, mutation of site F significantly reduced the responsiveness of the wild-type PRL-332-Luc construct to cAMP and PGE2 by 52 and 36%, respectively. In addition, mutation of this site also reduced the synergistic response to cAMP and ionomycin by 61% (Fig. 11B).



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  		FIGURE 11. Promoter sites involved in PGE2 and cptcAMP-induced PRL expression. Jurkat cells were transiently transfected with equimolar amounts of PRL promoter constructs and stimulated for 18 h with cptcAMP (250 µM), ionomycin (200 ng/ml), PGE2 (100 nM), or a combination of cptcAMP and ionomycin. 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 shared for 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). #, Different from the induction of wild-type PRL-332-Luc (p < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we show that, in Jurkat cells, induction of PRL expression and secretion by PGE2 is regulated at the transcriptional level. Both induction of PRL mRNA and PRL secretion were abrogated in the presence of the transcriptional inhibitor, actinomycin D. In the human B-lymphoblastoid cell line IM-9-P3 PRL mRNA half-life is reduced by dexamethasone (45), whereas it is increased by retinoic acid (46). These findings suggest that the leukocyte PRL mRNA is susceptible to posttranscriptional regulation by modification of its clearance rate. Because we were unable to detect any changes in the rate of mRNA degradation in the presence of PGE2, we conclude that, in Jurkat cells, PGE2 does not regulate PRL expression beyond the level of transcription.

The specificity and diversity of PGE2 effects can be in part explained by the use of four PGE2 receptor subtypes (EP1, EP2, EP3, and EP4), which transduce their signals predominantly via changes in [cAMP]i, [Ca2+]i, or both (35). To investigate the signaling pathways involved in PGE2-induced PRL expression, we tested the effects of inhibitors of several signaling cascades. A role for PKA was indicated by the inhibition of PRL expression by H89 and PKI. Another target of H89, MSK-1, which can activate CREB (39) was not phosphorylated by PGE2 or cAMP in Jurkat cells. In addition, PGE2 and cAMP did not phosphorylate kinases upstream to MSK-1 (p38 and ERK), and inhibitors of these pathways were unable 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 not shown). At nontoxic doses, two PKA inhibitors that are structurally unrelated to H89 (KT5720 and RpcAMPs), failed to block the effects of PGE2. However, unlike H89, KT5720 and RpcAMPs were ineffective in inhibiting cptcAMP-induced PKA activation in Jurkat cells. Finally, we found that activation of an alternative route for cAMP signaling, through EPAC, did not stimulate PRL expression but instead resulted in the inhibition of PRL expression. Indeed, opposing effects of PKA and EPAC have recently also been shown on protein kinase B activation in Hek-293 cells (47). In conclusion, these data support the importance of the classical cAMP effector, PKA, in mediating the effect of PGE2 on PRL expression. Results from experiments using Ca2+ chelators BAPTA-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 cells via the EP4 receptor (41). The effects of PGE2 on PRL expression were, however, unaffected by the PI3K inhibitor wortmannin. In summary, our results indicate that cAMP, via PKA, as well as Ca2+ are involved in the regulation of PRL expression at the transcriptional level, as inhibition of these pathways blocked PGE2-induced PRL mRNA levels (Figs. 2 and 5). The roles of Ca2+ and cAMP in PGE2-induced PRL expression are furthermore supported by the stimulating effects of PGE2 on [cAMP]i and [Ca2+]i (Figs. 8 and 9), and the synergy of these effectors on PRL promoter activation (27).

Using EP-specific agonists and antagonists, we have addressed the involvement of EP receptors in PGE2-induced signaling and PRL expression in Jurkat cells. Our observation that the EP4 antagonist L-161982 blocked PGE2-induced PRL expression, indicates a role for the EP4 receptor. Furthermore, we found that the EP1/EP3 agonist 17-P-T-PGE2 enhanced PRL expression, whereas the EP2 agonist, butaprost, and the EP3 agonist, sulprostone, were ineffective. Although 17-P-T-PGE2 is described as a specific EP1/EP3 (EP1>EP3) agonist, at high doses this agonist can also activate the EP4 receptor (Ki = 1 µM; Ref.35). The fact that a dose of 1 µM is required to augment PRL expression in Jurkat cells, argues for an action of 17-P-T-PGE2 through the EP4 receptor. Our observation that the EP1 antagonist SC-19220 had no effect on PGE2- or 17-P-T-PGE2-induced PRL expression, whereas the EP4 antagonist L-161982 nearly completely blocked 17-P-T-PGE2-induced PRL expression (Fig. 7, B and C), confirms the involvement of EP4 in PGE2-induced PRL expression. An action of PGE2 through the EP4 receptor was furthermore supported by the finding that PGE2 stimulated [cAMP]i in Jurkat cells and that this effect was blocked by L-161982. Butaprost had no effect on [cAMP]i, suggesting EP2 receptors are not present on Jurkat cells. This is in accordance with earlier findings in which the EP2 receptor was undetectable on Jurkat cells by RT-PCR (48). The fact that the EP1/EP3 agonist 17-P-T-PGE2 raised [cAMP]i at a dose of 1 µM confirms the hypothesis that at high doses, this agonist activates the EP4 receptor, because in human cells EP1 and EP3 receptors do not classically couple to increases in [cAMP]i (49, 50). Although Ca2+ signaling is required for the induction of PRL expression by PGE2, it appeared that the EP4 antagonist L-161982 did not block the increase in [Ca2+]i. This implies that another 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. Furthermore, the effects of 17-P-T-PGE2 on [Ca2+]i were not blocked by the 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 several species, with variations only in their C-termini, resulting in the activation of different signaling pathways through different G protein. The activation of second messengers is best known for the bovine EP3 receptor, of which four isoforms (A, B, C, and D) have been described. EP3A, -B, or -C isoforms can either inhibit or activate adenylyl cyclase through Gi or Gs, respectively. The EP3D receptor can also be coupled to the activation of phospholipase C (PLC) via Gq, in addition to Gi and Gs (35, 51). PLC cleaves PIP2 to generate diacylglycerol and inositol 1,4,5-triphosphate (IP3) which is pivotal to the entry of Ca2+ to the cytosol from the endoplasmic reticulum and the extracellular space. Our data suggest the EP3 isoform present on Jurkat cells is coupled to Ca2+ signaling. An elevation of [Ca2+]i is essential in T cell activation through the TCR complex. In T cells, IP3-induced release of Ca2+ from the endoplasmic reticulum serves as a trigger for controlling a large influx of extracellular Ca2+, which plays a pivotal role in gene regulation (52). Although the EP3D receptor has been shown to activate PLC, leading to the generation of IP3, our results suggest that the release of [Ca2+]i from the endoplasmic reticulum is not involved in the stimulation of PRL expression by PGE2. Incubation of Jurkat cells with thapsigargin, an inhibitor of the sarco-endoplasmic reticulum Ca2+ ATPase, leads to a rapid depletion of intracellular Ca2+ stores. Our observation that pretreatment of Jurkat cells with thapsigargin did not alter the effect of PGE2 on PRL expression, whereas BAPTA and EGTA were inhibitory, suggests that PGE2 generates a store-independent influx of extracellular Ca2+. Interestingly, in Jurkat cells, receptors for the Ca2+-releasing messenger IP3 were detected on the plasma membrane and it was shown that these receptors are responsible for the entry of extracellular Ca2+ during proliferative responses (53). A possible mechanism for the PGE2-induced rise in [Ca2+]i in Jurkat cells could thus be via the activation of membrane-bound IP3 receptors.

We previously showed that raising intracellular cAMP levels using cptcAMP induces PRL expression in Jurkat cells. Ionomycin treatment did not affect PRL expression, suggesting the Ca2+ signal by itself is insufficient to trigger PRL expression. This is in line with our finding that sulprostone alone does not induce PRL expression. However, in combination, cAMP and ionomycin synergized to induce PRL expression to an extent comparable to the induction by PGE2 (27). This is in accordance with the present observation that PGE2 not only stimulates cAMP generation, but also Ca2+ influx, and that both these signals are required for full PRL expression. Furthermore, the fact that we could mimic the effect of PGE2 on PRL expression using a combination of the EP3 agonist sulprostone, which increases [Ca2+]i, and cptcAMP argues for a combined action of PGE2 through EP3 and EP4. These results furthermore indicate that the cAMP signal is the sine qua non for PRL expression, whereas the Ca2+ signal can probably enhance the activation of factors that are induced by cAMP and thus synergize with cAMP to induce PRL expression. The molecular basis for this synergism remains to be elucidated, yet a possible target for the synergistic effect of the Ca2+ signal could be the transcription factor CREB. Indeed, coordinate activation of different kinases, among which are the calcium/calmodulin-dependent kinases, leading to optimal activation of CREB has been shown in T cells (54, 55).

The EP4 receptor has been previously described on Jurkat cells by RT-PCR (48) and flow cytometric analysis (56). To our knowledge, we are the first to describe PGE2 effects through EP3 on Jurkat cells. Indeed, effects of PGE2 on T lymphocytes are most often attributed to rises in [cAMP]i through occupation of EP2 and/or EP4 receptors (42, 43, 44). However, it was shown that in the human leukemic T cell line, HSB.2, expression of matrix metalloproteinase-9 is regulated by PGE2 binding to the EP3 receptor (57). Furthermore, PGE2 and dexamethasone act synergistically to inhibit TCR signaling, and this effect was mimicked in primary T cells by a specific agonist for the EP3 receptor (58).

Using a promoter construct carrying 1842 bp of the leukocyte PRL promoter, we previously found that the effect of PGE2 on promoter activation was markedly smaller than the effect on mRNA levels (27). Our observation that PRL mRNA stability was not affected by PGE2, indicates that sites beyond –1842 may be required for full promoter activation. To address this possibility, we transfected Jurkat cells with a construct carrying 3000 bp of the leukocyte PRL promoter. However, the activation of this promoter construct was not different from that of the 1842 bp construct, suggesting even more distant or perhaps intronic sites would be involved in regulating PRL expression. Indeed, a binding site for a lymphoid-specific factor has been located downstream of the lymphoid-specific transcription start site in intron 1A, which is in fact the superdistal region of the pituitary PRL promoter. When transfected into Jurkat cells, this footprinted region activated transcription of a heterologous promoter, suggesting it might be a functional enhancer (59). Also, we cannot exclude the possibility that full promoter activation requires additional signals, besides cAMP and Ca2+, converging to responsive elements that are not contained in the 3000-bp promoter construct. Yet another possibility is that the transfection of Jurkat cells through electroporation caused a loss of EP receptors on the cell membrane, as observed for fMLP receptors in COS cells (60). This idea is in accordance with our observation that direct activation of second messengers, via ionomycin in combination with cptcAMP, accomplishes full promoter activation, comparable to the effect on PRL mRNA levels.

The effects of cptcAMP, cptcAMP in combination with ionomycin, and PGE2 on PRL-3000-Luc were preserved using a promoter construct carrying 332 bp of the upstream PRL promoter. The promoter harbors an imperfect CRE immediately upstream of the transcriptional start site (61). Although Reem et al. (62) showed that mutation of this CRE partially abolished cAMP responsiveness of the upstream PRL promoter in Jurkat cells, we were unable to activate a construct carrying 32 bp of the upstream PRL promoter (and thus, the CRE), suggesting this site is not solely responsible for the cAMP induction. Our results further indicate the involvement of a C/EBP site at –214 in the effects of cptcAMP, cptcAMP plus ionomycin, and PGE2 on the leukocyte PRL promoter. Earlier studies in human endometrial stromal cells, showed the requirement of two overlapping C/EBP sites in the region –332/–270 for PKA inducibility, whereas the C/EBP site at –214 was not involved (36). The C/EBP family of basic region/leucine zipper DNA-binding proteins consists of six members: C/EBP{alpha}, -{beta}, -{delta}, -{epsilon}, -{gamma}, and -{zeta}. Specificity of gene control by C/EBPs is ensured by their cell-specific and temporal expression pattern and through their ability to homo- and heterodimerize and interact with other transcription factors (63). Recently, it was shown that PGE2 activates the HIV-1 long terminal repeat in Jurkat cells by a cooperative interaction of C/EBP{beta} and CREB, binding to two proximal C/EBP sites (64). Additional experiments will be required to identify the transcription factors binding to the C/EBP site in the PRL promoter in Jurkat cells.

PBMC from SLE patients produce higher amounts of PRL than those from control donors (20, 21). Also, in patients with rheumatoid arthritis, synovium infiltrating T cells produce PRL which stimulates the release of proinflammatory factors from synovial cells (23). Taken together, these observations are compatible with a deleterious role of leukocyte PRL in the progression of autoimmune disease. PGE2 levels are increased in synovial fluids from patients with rheumatoid arthritis, ranging from 127 to 886 nM (65, 66, 67, 68). Furthermore, several lines of evidence suggest that at least some of the proinflammatory aspects of the disease are mediated by PGE2 (69, 70, 71). Because PRL produced by infiltrating T cells could transduce some of the proinflammatory aspects attributed to PGE2, we have investigated the mechanisms at the basis of the stimulation of PRL expression by PGE2 in Jurkat cells. We have described a novel mechanism, based on the cooperation of EP3 and EP4 receptors, by which physiologically relevant PGE2 concentrations can induce gene expression in T cells. Our data might furthermore contribute to a better understanding of the role of PRL in the pathophysiology of autoimmune diseases such as rheumatoid arthritis and perhaps to the development of new therapeutic strategies. Whereas the Jurkat T cell line has been successfully used as a model system to study various aspects of T cell functioning (72), the physiological relevance of our findings remains to be confirmed in primary T cells.


   Acknowledgments
 
We thank Dr. Dario Alessi for the MSK-1 expression vectors, Issam Harfi for his advice concerning the Ca2+ measurements, and Robert Hooghe for revision of the manuscript.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 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 Brussels (Onderzoeksraad, Geoconcerteerde Onderzoeksactie 97-02-04). Back

2 Address correspondence and reprint requests to Dr. Sarah Gerlo, Laboratory of Neuroendocrine Immunology, Department of Pharmacology, Free University of Brussels, Laarbeeklaan 103, B-1090 Brussels, Belgium. E-mail address: Sarah.Gerlo{at}vub.ac.be Back

3 Abbreviations used in this paper: PRL, prolactin; SLE, systemic lupus erythematosus; EP, E-prostanoid; [Ca2+]i, intracellular calcium concentration; [cAMP]i, intracellular 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, inositol 1,4,5-triphosphate; PLC, phospholipase C. Back

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


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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