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BOR - Papers in Press, published online ahead of print January 31, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.053058
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BIOLOGY OF REPRODUCTION 76, 777–783 (2007)
DOI: 10.1095/biolreprod.106.053058
© 2007 by the Society for the Study of Reproduction, Inc.

Autocrine Prolactin Inhibits Human Uterine Decidualization: A Novel Role for Prolactin1

Ori Eyal 3, Jean-Baptiste Jomain 4 5, Cherie Kessler 3, Vincent Goffin 4 5, and Stuart Handwerger 2 3

Division of Endocrinology,3 Cincinnati Children's Hospital Medical Center, University of Cincinnati School of Medicine, Cincinnati, Ohio 45229-3039 Université Paris Descartes,4 Faculté de Médecine René Descartes 5 site Necker, 75015 Paris, France INSERM,5 Unit 808, Laboratory of PRL, GH and Tumors, 75730 Paris Cedex 15, France

ABSTRACT

Human prolactin (PRL) and its receptor (PRLR) are markedly induced during human uterine decidualization, and large amounts of PRL are released by decidual cells as differentiation progresses. However, the role of PRL in decidualization is unknown. In order to determine whether PRL plays an autocrine role in decidualization, human uterine fibroblast cells that were decidualized in vitro with medroxyprogestrerone acetate (1 µM), estradiol (10 nM), and prostaglandin E2 (1 µM) were exposed to exogenous PRL and/or the pure PRLR antagonist delta1–9-G129R-PRL. As measured by quantitative PCR, cells that were decidualized in the presence of exogenous PRL (0.25–2 µg/ml) expressed significantly lower levels of mRNA for the genes that encode insulin-like growth factor binding protein 1 (IGFBP1), left-right determination factor 2 (LEFTY2), PRL, decorin (DCN), and laminin alpha 1 (LAMA1), all of which are known to be induced during decidualization. These effects were blocked when the cells were exposed simultaneously to PRL and the PRLR antagonist, which confirms the specific inhibitory action of PRL on the expression of decidualization markers. In addition, cells exposed to the PRLR antagonist alone expressed higher levels of the marker gene mRNAs than cells that were decidualized in control media. Taken together, these results strongly suggest that PRL acts via an autocrine mechanism to regulate negatively the extent of differentiation (decidualization) of human uterine cells.

decidua, prolactin, prolactin receptor

INTRODUCTION

During the luteal phase of the menstrual cycle, the uterus undergoes morphological and biochemical changes that are essential for embryo implantation and endometrial stromal cells differentiate into decidual cells by the process known as decidualization. If implantation ensues, the decidual cells become the predominant cell type of the uterine lining.

Much of our knowledge of the regulation of human decidualization comes from studies that utilized in vitro model systems. Several models have been developed in which decidualization is induced in primary cultures of human endometrial stromal cells from the follicular phase of the menstrual cycle cultured in the presence of progesterone and estradiol (E2), relaxin or high levels of cAMP [1–4]. In each instance, the cells undergo differentiation to a morphologic phenotype that is characteristic of decidual cells and they express decidual cell-specific markers, such as PRL, IGFBP1, and PRLR [1, 5–7]. While endometrial stromal cells have been useful in studies on the biology of human decidualization, these studies have been limited by the relatively short life span of endometrial stromal cells in culture, the necessity to obtain endometrial samples from the luteal phase of the menstrual cycle, and the fact that treatment of patients with progestins and other hormones affects the endometrium and interferes with the ability of the cells to undergo further decidualization. In order to overcome these problems, Brosens and coworkers [8] have developed immortalized human endometrial fibroblasts from enriched endometrial stromal cells cultures. These cells have the ability to decidualize in response to stimulation with MPA plus 8-bromo-cAMP, with the induction of the decidual cell-specific markers PRL and IGFBP1.

In an earlier study [9], we have demonstrated that decidualization can also be induced in primary cultures of human uterine fibroblast cells, which are isolated from decidua parietalis adherent to the fetal membranes of placentas delivered at term, by treatment of the uterine fibroblast cells with progesterone and E2 in combination with cAMP or prostaglandin E2 [10]. DNA microarray studies have indicated that prior to treatment the cells do not express decidual cell-specific markers, such as PRL, IGFBP1, and PRLR. Following exposure of the cells to progesterone, E2, and cAMP or prostaglandin E2, the cells undergo differentiation with the induction of genes found to be induced during the decidualization of endometrial stromal cells [9]. The levels of PRL and PRLR mRNA increase to detectable levels by 2–3 days. The PRL mRNA levels then increase progressively to reach a peak at Day 12 of in vitro decidualization [9]. The PRLR mRNA levels increase in parallel with the induction of the PRL gene [11]. Moreover, human uterine fibroblast cells decidualized in vitro show expression of other genes that are expressed in endometrial samples obtained from large pools of fertile volunteers during the luteal phase of the menstrual cycle [12] and in endometrial stromal cells obtained in the late proliferative phase of the cycle and decidualized in vitro [13]. Since uterine fibroblast cells are relatively easy to prepare and can be induced to differentiate to a phenotype that expresses the same genes as decidualized endometrial stromal cells, they represent a very useful model to study the molecular events of human decidualization. However, uterine fibroblast cells do not show the characteristic morphological changes observed in endometrial stromal cells undergoing decidualization [10].

Since PRL and its receptor are induced early during decidualization and are expressed in the same cell type, we have examined whether PRL plays an autocrine role in regulating the differentiation process. In an earlier paper, Prigent-Tessier and coworkers [14] have shown that exogenous PRL inhibits rat decidual PRL expression, although the underlying mechanism has not been elucidated. Since the effects of exogenous PRL on other markers of decidualization were not studied, it is not known whether exogenous PRL inhibits only PRL or the process of decidualization. To examine the possibility that exogenous PRL inhibits human decidualization, we studied the effects of exogenous PRL and the PRLR antagonist {Delta}1–9-G129R-PRL on the in vitro decidualization of human uterine fibroblast cells. The second generation PRLR antagonist {Delta}1–9-G129R-PRL acts as a pure antagonist in vivo and in vitro by competing with PRL for binding to its receptor and interfering with functional receptor dimerization, a molecular event that is essential for activation of the receptor [15–17].

MATERIALS AND METHODS

Cell Culture

Human uterine fibroblast cells were prepared as previously described [10]. Briefly, decidua parietalis tissue was dissected from fetal membranes 1–2 h after delivery, treated with collagenase, and the cells were dispersed on plastic plates and cultured in RPMI 1640 medium that was supplemented with 10% FBS. After incubation for 24 h, the nonadherent cells were freed by agitation and removed by medium exchange. Adherent cells were grown to confluence, split by trypsinization, and replated. Permission to obtain term human placentas from uncomplicated pregnancies was approved by the Institutional Review Boards at the University of Cincinnati, College of Medicine and the Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.

Human uterine fibroblast cells were cultured to confluence in 6-well plates using RPMI medium that contained 10% FBS, 25 µg/ml penicillin, 25 µg/ml streptomycin, and 2.5 µg/ml amphotericin B. The plates were incubated in a humidified atmosphere of 5% CO2 at 37°C. The culture media were replaced every 48 h. After reaching 90–95% confluence, the cells were decidualized by the addition of medroxyprogesterone acetate (MPA, 1 µM), E2 (10 nM), and prostaglandin E2 (PGE2, 1 µM) in RPMI medium that contained 2% FBS for up to 9 d. The cells were divided into four groups: Group 1 cells were exposed to exogenous PRL (0.25 to 2 µg/ml) from the beginning of the decidualization process; Group 2 cells were exposed to both exogenous PRL (0.25 to 2 µg/ml) and {Delta}1–9-G129R-PRL (5–15 µg/ml) from the beginning of the decidualization process; Group 3 cells were exposed to {Delta}1–9-G129R-PRL (5–15 µg/ml) alone from the beginning of the decidualization process; and Group 4 cells were cultured in decidualizing medium alone. Each treatment group consisted of 2 or more culture wells. The medium was exchanged daily for fresh PRL and/or {Delta}1–9-G129R-PRL. RNA was extracted from the cells at time-point 0 and the indicated time-points. In selected instances, in which medium was collected for determination of IGFBP1 protein levels, the medium was changed to serum-free medium 24 h before collection.

Isolation of Total RNA and PCR Analysis

Decidualization was monitored by determination of the mRNA levels of several decidualization-specific marker genes by real-time quantitative PCR using the GAPDH mRNA levels for normalization. Total RNA was extracted from the cells using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's specifications. Two micrograms of total RNA were reverse-transcribed using SuperScript II Reverse Transcriptase (Invitrogen). PCR reactions were performed using the Stratagene Mx30000P instrument (Stratagene, La Jolla, CA). Quantitative PCR amplifications were performed using the Eppendorf HotMasterMix (Brinkmann Instruments, Westbury, NY) supplemented with SYBR Green (Molecular Probes, Eugene, OR) and ROX (Stratagene). The mix was used according to the manufacturer's instructions using a 20-µl final volume. The PCR reaction was performed with a 2-min incubation at 95°C, followed by 40 cycles of 95°C for 30 sec, 55°C for 1 min, and 72°C for 30 sec. Dissociation/association curves for each reaction were determined after the 40th cycle. A single dissociation curve was noted for each primer set. Preliminary experiments determined that the conditions used for each primer were optimized for these conditions. The primers used for quantitative PCR are shown in Table 1.


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  		TABLE 1. List of decidualization markers and primers used to determine effects of PRL and PRL antagonist on decidualization.

Western Blot Analysis

For the analysis of the IGFBP1, PRL, and LEFTY2 proteins, 2 ml of medium was concentrated using an Amicon Centricon YM-10 unit (Millipore, Bedford, MA). The LAMA1 and caspase 3 proteins were detected in cellular extracts. The concentrated medium and cellular extract (15 to 25 µg total protein) were then separated by electrophoresis on 12% resolving/5% stacking reducing gels and transferred to Protran nitrocellulose membranes (Schleicher & Schuell Bioscience, Kenne, NH). The membranes were blocked and then incubated overnight at room temperature with polyclonal anti-IGFBP1, anti-PRL, and anti-LAMA1 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-LEFTY2 (R&D Systems, Minneapolis, MN), and caspase 3 (Cell Signaling, Beverly, MA) IgG antibodies at a final dilution of 1:200. After additional washing and blocking, the blots were incubated with donkey anti-goat or anti-rabbit secondary antibody conjugated to horseradish peroxidase (Chemicon International, Temecula, CA) at a 1:5000 dilution at room temperature for 1 h. Chemiluminescence was measured using the Super Signal West Pico Chemiluminescent Substrate Kit (Pierce, Rockford, IL), followed by autoradiography on Hyperfilm (Amersham Biosciences, Buckinghamshire, UK). The autoradiographic signals were quantitated using the Kodak Digital Science ID Image Analysis software (Eastman Kodak, Rochester, NY).

Statistical Analysis

Statistical analysis was performed by ANOVA followed by the Student-Newman-Keuls multiple comparisons test. P values < 0.05 were considered statistically significant.

RESULTS

Decidualization was monitored by examining the amounts of PRL, IGFBP1, LEFTY2, DCN, and LAMA1 mRNAs, all of which have been shown to be significantly induced during decidualization. Figure 1 shows the mRNA levels for each of these genes on different days during decidualization following exposure to MPA, E2, and PGE2. As anticipated, before decidualization, the cells contained undetectable levels of PRL, IGFBP1, and LEFTY2 mRNAs and relatively low levels of LAMA1 and DCN mRNAs. During the first 2 days of exposure to MPA, E2, and PGE2, the mRNA levels of each of the five genes increased significantly.


Figure 01
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  		FIG. 1. Changes in mRNA levels during decidualization. RT-PCR analysis of PRL, IGFBP1, LEFTY2, DCN, and LAMA1 during decidualization of human uterine fibroblast cells. The amounts of mRNA for each gene were measured at time-point 0 and on different days during decidualization. The top figure shows the RT-PCR products for each of the genes. The bottom table shows the relative mRNA values for each of the genes.

To examine the effects of exogenous PRL on decidualization, human uterine fibroblasts were decidualized with MPA, E2, and PGE2 for 2 to 6 days in the presence or absence of exogenous PRL (0.25 to 2 µg/ml). These levels of PRL are comparable to those observed in human amniotic fluid and human decidual tissues [18–20]. Human uterine fibroblast cells decidualized in the presence of exogenous PRL expressed lower levels of PRL, IGFBP1, LEFTY2, DCN, and LAMA1 mRNAs than control cells (Fig. 2). The cells exposed to exogenous PRL (0.25 µg/ml) during the first 2 days of decidualization contained 30–60% less mRNA for each of PRL, IGFBP1, LEFTY2, LAMA1, and DCN (P < 0.05), and cells exposed to 1.5 µg/ml PRL contained 35–70% less mRNA for these genes (P < 0.05). Decreased levels of each mRNA were also observed on Days 4 and 6 (data not shown). Nearly identical results were obtained in three other experiments.


Figure 02
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  		FIG. 2. Human uterine fibroblast cells exposed to exogenous PRL during decidualization express lower levels of decidualization-specific marker mRNAs than control cells. Human uterine fibroblast cells were decidualized in the presence and absence of exogenous PRL. The medium was changed daily and fresh PRL was added each day. Cell extracts were prepared at the end of Day 2. Each group contained triplicate culture wells. Each bar represents the relative change in mRNA levels compared to control cells. The results of a typical experiment in which the cells were exposed to 0.25 and 1.5 µg/ml PRL are shown. The mean ± SEM (n = 3) is shown. *P < 0.05; **P < 0.01 (control vs. treatment group).

The decrease in IGFBP1 mRNA in the cells exposed to exogenous PRL was accompanied by a significant decrease in the amount of IGFBP1 protein released into the medium. As shown in Figure 3, the amounts of PRL, IGFBP1, and LEFTY2 in the medium on Day 6 of decidualization and the cellular content of LAMA1 protein at the end of Day 6 of decidualization in the presence of 2 µg/ml PRL, as measured by Western blotting, was 35–72% less than that in cells decidualized in the absence of exogenous PRL. Similar results were obtained in four experiments.


Figure 03
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  		FIG. 3. Cells exposed to exogenous PRL release significantly lower amounts of PRL, IGFBP1, and LEFTY2 proteins and contain less LAMA1 protein than control cells. Two experiments are shown in which uterine fibroblast cells exposed to 2 µg/ml exogenous PRL release lower amounts of PRL, IGFBP1, and LEFTY2 protein into the medium than control cells. In Experiment 1, the cellular content of LAMAI protein is decreased relative to that of the control cells. Twenty-four hours before collection, the medium was changed to serum-free medium. The PRL, IGFBP1, LEFTY2, and LAMA1 levels were determined by Western blot analysis using the same amount (15–25 µg) of total protein for each sample. The results of Western blot analyses in two separate experiments performed on Day 6 of decidualization are shown. Similar results were obtained in two other experiments.

The PRLR antagonist {Delta}1–9-G129R-PRL reversed the inhibitory effects of exogenous PRL. Figure 4 shows the results of a representative experiment in which human uterine fibroblast cells were decidualized in the presence of exogenous PRL (1.5 µg/ml) alone or in combination with a 6-fold molar excess of {Delta}1–9-G129R-PRL (10 µg/ml) to achieve efficient competition. At the end of Day 2 of culture, cells that were decidualized in the presence of exogenous PRL showed the expected decreases in mRNA marker levels. The levels of mRNA for IGFBP1, LEFTY2, LAMA1, and PRL were decreased by 35% (P < 0.001), 50% (P < 0.001), 70% (P < 0.05), and 53% (P < 0.05), respectively. The addition of {Delta}1–9-G129R-PRL completely reversed the effects of PRL on these mRNA levels (P < 0.05).


Figure 04
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  		FIG. 4. The addition of {Delta}1–9-G129R-PRL reverses the effects of exogenous PRL on decidualization-specific marker mRNAs. Cells were decidualized in the presence of 1.5 µg/ml exogenous PRL alone or in combination with 10 µg/ml {Delta}1–9-G129R-PRL. Each bar represents the relative change in mRNA levels compared to control cells on Day 2 of decidualization. In each instance, {Delta}1–9-G129R-PRL antagonizes the inhibitory effects of PRL on the decidualization-specific marker mRNAs. The mean ± SEM (n = 3) is shown. *P < 0.05; **P < 0.01 (PRL-treated group vs. PRL plus antagonist-treated group).

Cells that were decidualized in the presence of {Delta}1–9-G129R-PRL alone contained higher levels of IGFBP1, LEFTY2, and PRL mRNAs than control cells. Figure 5 shows the results of a representative experiment in which human uterine fibroblast cells were decidualized in the presence of 10 µg/ml {Delta}1–9-G129R-PRL. After 2 days of decidualization, the IGFBP1, LEFTY2, and PRL mRNA levels in the cells exposed to {Delta}1–9-G129R-PRL were 50–100%, 100–160%, and 20–80% higher, respectively, than those in control cells. Increased mRNA levels for each of the above genes were observed also on Days 4 and 6 of decidualization (data not shown).


Figure 05
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  		FIG. 5. Human uterine fibroblast cells exposed to {Delta}1–9-G129R-PRL during decidualization express higher levels of decidualization-specific marker mRNAs than the control. Human uterine fibroblast cells were decidualized in the presence and absence of 10 µg/ml {Delta}1–9-G129R-PRL for 2 days. The medium was exchanged daily for fresh medium with {Delta}1–9-G129R-PRL. Each bar represents the relative change in mRNA levels compared to control cells on Day 2 of decidualization. The mean ± SEM (n = 3) is shown. *P < 0.05; **P < 0.01 (control vs. antagonist-treated group).

In earlier experiments, we have demonstrated that the cannabinoid receptor agonist R(+)-WIN 55,212–2 mesylate (WIN) inhibits in vitro decidualization and stimulates the apoptosis of uterine fibroblast cells [21]. The stimulation of apoptosis was shown by an increase in DNA fragmentation (TUNEL assay) and the induction of caspase 3 protein and FAS mRNA. To determine whether exogenous PRL also stimulates the apoptosis of uterine fibroblast cells during in vitro decidualization, uterine fibroblast cells were decidualized in vitro for 6 days in the presence or absence of exogenous PRL (1.5 µg/ml). As shown in Figure 6, the amounts of caspase 3 protein and FAS mRNA in cells exposed for 6 h (at which time-point WIN(+) 55,212–2 mesylate stimulates both caspase 3 protein and FAS mRNA levels) to exogenous PRL were not different to those of control cells. Furthermore, no changes in caspase 3 protein and FAS mRNA were noted at 0.25, 0.5, and 12 h and at 2, 4, 6, and 9 days of exposure to PRL. The failure of exogenous PRL to stimulate caspase 3 protein and FAS mRNA was observed in a second experiment performed under identical conditions (data not shown). In both experiments, exogenous PRL lowered the levels of PRL and IGFBP1 mRNAs.


Figure 06
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  		FIG. 6. Exogenous PRL has no effects on caspase 3 protein and FAS mRNA levels. Human uterine fibroblast cells were decidualized for up to 9 days in control medium or medium that contained exogenous PRL (1.5 µg/ml). The amounts of caspase 3 protein and the FAS mRNA levels are shown at the end of 6 h of exposure, at which time the cannabinoid receptor WIN has been shown in earlier studies [21] to stimulate caspase 3 and FAS mRNA levels. No differences were noted between the control and exogenous PRL-exposed cells at other time-points during the 9-day exposure (data not shown). No effect of exogenous PRL on caspase 3 or FAS mRNA was noted in a separate experiment performed under identical conditions.

DISCUSSION

PRL is one of the major proteins synthesized and secreted by human decidualized endometrial stromal cells [1, 6]. The hormone is released into the amniotic fluid but does not appear to be released into the maternal circulation [18]. Indeed, the concentration of PRL in amniotic fluid increases steadily during pregnancy, reaching a peak of 4000 to 6000 ng/ml at 20–25 wk of gestation, at which time the PRL concentrations in the maternal and fetal sera are 100 to 300 ng/ml and 50 to 100 ng/ml, respectively [18, 22]. The PRL concentration in amniotic fluid then declines gradually towards term, reaching a concentration of 100 ng/ml. The amount of PRL released by human decidual tissue into the medium during a 24-h incubation can reach 196 ng/ml [19].

Although the physiological roles of decidual PRL are poorly understood at present, locally produced PRL may play a role in the differentiation of decidual cells and in preparing the uterus for possible embryo implantation. During early pregnancy, placental development and the establishment of the maternal-fetal circulation are dependent upon angiogenic and antiangiogenic factors. The intact PRL molecule stimulates angiogenesis, while its proteolytic N-terminal 16-kDa fragment inhibits angiogenesis [23]. The opposing actions of the intact protein and the 16-kDa fragment are mediated by independent receptors rather than by competition for binding to the same receptor. Therefore, it has been proposed that decidual PRL promotes blood vessel development in the placenta [24].

During decidualization, there is a marked increase in the expression levels of many genes other than PRL. In a DNA microarray study of the genes regulated during in vitro decidualization of human uterine fibroblast cells in response to progesterone, E2, and cAMP, 121 out of 6918 genes queried were upregulated, 110 genes were repressed, and 50 genes showed biphasic behavior over a 15-day period. Most of the regulated genes were related to decidualization-associated processes, such as extracellular organization. The most highly induced genes were IGFBP1, LEFTY2, and somatostatin; and the most repressed genes were IGFBP5 and fibronectin 1 [9]. Similar results were noted in DNA microarray studies of human endometrial stromal cells [13].

The results of the present study show that PRL has an inhibitory effect on the differentiation of human uterine fibroblast cells. Human uterine fibroblast cells decidualized in the presence of exogenous PRL contained lower levels of PRL and other decidualization-specific marker mRNAs, and the PRLR antagonist {Delta}1–9-G129R-PRL reversed the inhibitory effects of exogenous PRL. Since this antagonist exhibits slightly lower affinity for the receptor compared to wild-type PRL [16], the efficient competition that we observed using only a 6-fold molar excess of the antagonist clearly indicates that the effects of PRL on decidualization-specific marker gene mRNAs result from specific PRL-mediated activation of the PRLR and further confirms the intrinsic inhibitory action of PRL on decidualization. Furthermore, treatment of human uterine fibroblast cells with {Delta}1–9-G129R-PRL alone (no addition of exogenous PRL) increased the levels of the decidualization-specific marker mRNAs above the control levels. Since we have previously demonstrated that {Delta}1–9-G129R-PRL is completely devoid of agonistic activity and only acts by inhibiting PRL-induced activation of the PRL receptor [16, 17], our findings strongly suggest that PRL mRNA in decidualized cells is translated and secreted as a bioactive hormone that exerts an autocrine inhibitory effect on human decidualization. In an earlier study [21], we demonstrated that cannabinoid receptor 1 (CNR1) agonists inhibit while CNR1 antagonists induce decidualization of human uterine fibroblast cells. Since the uterus produces endocannabinoid and CNR1 mRNA levels increase markedly during decidualization of human uterine fibroblast cells, it appears that the fibroblast cells synthesize multiple factors that control uterine decidualization.

Although many studies have shown that PRL and its receptor are coexpressed in various tissues, the notion that locally produced PRL acts via an autocrine and/or paracrine mechanism has been regarded as speculative. For example, based on the observations that both PRL and PRLR are expressed in lymphocytes and that exogenous PRL promotes lymphocyte proliferation and increases antibody production, it has been suggested that the PRL produced by lymphocytes also plays an autocrine/paracrine role in the immune system [18]. Similarly, bioactive PRL is synthesized and released from the glandular and adipose compartments of the human breast, with significantly higher PRL secretion by adipose tissue [25]. Since the role of PRL in the cellular growth and differentiation of breast tissue is very well documented, locally produced PRL has been proposed to exert the same effects. In addition, both PRL and its receptor are expressed by normal and neoplastic human mammary gland cells, and PRL is considered to have a mitogenic action on breast cancer cells [26–31].

In most, if not all of these systems, direct evidence for an active autocrine/paracrine loop is lacking, partly due to the fact that there is currently no known negative regulator of PRL synthesis/secretion at extrapituitary sites that could be used to downregulate the putative autocrine effects of PRL produced naturally in these experimental systems. To the best of our knowledge, the report by Ginsburg and Vonderhaar [32] is the only one that clearly shows an autocrine proliferative effect of local PRL on breast cancer cell lines, which was performed by inhibiting the effects of endogenously produced PRL using anti-PRL antibodies. In addition, human breast cancer cell clones that express the PRL gene have higher proliferation rates than human breast cancer cell clones that do not express the PRL gene [33]. A previous study using an animal model showed that the addition of exogenous PRL to primary rat decidual cells in culture caused a marked decrease in rat decidual PRL mRNA expression [14]. The same study found that a decrease in PRLR correlated with an increase in rat decidual PRL expression. The present study, which utilizes a recently developed pure PRLR antagonist that inhibits the effects of PRL in various experimental cells and animal systems [16, 17], provides the first evidence for a human, nontumor system in which locally produced PRL exerts a biological effect via an autocrine/paracrine mechanism.

In a recent study, Jasinska and coworkers [34] have observed that disruption of the cytoskeleton of cultured endometrial stromal cells by cytochalasin D markedly increases apoptosis, and that the induction of apoptosis is inhibited by decidualization of the cells with hCG or dibutyryl cyclic AMP in the presence of E2 and progesterone. Treatment of cytochalasin-treated cells with recombinant PRL or IGFBP1 also inhibited the induction of apoptosis, which suggests that the inhibition of apoptosis in response to cytochalasin may be due, at least in part, to the effects of PRL and IGFBP1. In agreement with this and other studies that have shown PRL to be mainly antiapoptotic, Ueda and co-workers [35] have recently shown that S179D PRL, which is a molecular mimic of phosphorylated PRL that acts as a functional antagonist, exerts antiangiogenic effects, partly by induction of endothelial cell apoptosis via the MAPK pathway and the upregulation of p21. However, while PRL has been shown to affect apoptosis in many cell types, the treatment of human uterine fibroblast cells with exogenous PRL in the present study did not appear to inhibit or promote apoptosis.

In summary, the findings of this study suggest that locally produced PRL has a role in the complex process of human decidualization. It is possible that PRL acts in an autocrine manner to limit the extent of differentiation, although the mechanism(s) by which PRL inhibits human decidualization remains to be elucidated. While earlier studies from our laboratory suggest that endocannabinoids limit the extent of decidualization by inducing apoptosis [22], the effects of PRL on decidualization appear to be due to mechanisms different to those of the endocannabinoids. Since exogenous PRL inhibits PRL expression in rat decidual cells [15] and the expression of PRL in decidual cells is a specific marker for decidualization [6], it is possible that PRL also plays an autocrine role in the regulation of decidualization in the rat.

ACKNOWLEDGMENTS

We thank Jennifer Schroeder and Michael Hubert for their invaluable assistance. Jean-Baptiste Jomain was supported by the Ministry of Research and Technology of France, and by the Association pour la Recherche contre le Cancer.

FOOTNOTES

1Supported by grant HD-15201 from the National Institutes of Health. Back

Correspondence: 2Stuart Handwerger, Cincinnati Children's Hospital Medical Center, Division of Endocrinology, MLC 7012, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. FAX: 513 636 7486; e-mail: stuart.handwerger{at}cchmc.org

Received: 10 April 2006.

First decision: 10 May 2006.

Accepted: 16 January 2007.

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