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Endocrinology, doi:10.1210/en.2006-1643
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Endocrinology Vol. 148, No. 5 2326-2334
Copyright © 2007 by The Endocrine Society

Decidual Prolactin Silences the Expression of Genes Detrimental to Pregnancy

Lei Bao, Christian Tessier, Anne Prigent-Tessier, Feixue Li, Oscar L. Buzzio, Eduardo A. Callegari, Nelson D. Horseman and Geula Gibori

Department of Physiology and Biophysics (L.B., C.T., A.P.-T., F.L., O.L.B., E.A.C., G.G.), University of Illinois, Chicago, Illinois 60612; and Department of Molecular and Cellular Physiology (N.D.H.), University of Cincinnati, Cincinnati, Ohio 45267

Address all correspondence and requests for reprints to: Geula Gibori, Ph.D., Department of Physiology and Biophysics (M/C 901), University of Illinois at Chicago, 835 South Wolcott Avenue, Chicago, Illinois 60612-7342. E-mail: ggibori{at}uic.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although the main role of prolactin (PRL) in pregnant rodents is to sustain progesterone production by the corpus luteum, progesterone treatment of PRL or PRL receptor (PRL-R) null mice is unable to prevent fetal loss. We have previously shown that the rat decidua is a site of PRL production and action. In this report, we examined the hypothesis, using PRL null mice and rat decidual cell culture, that the absence of this hormone leads to the expression in the decidua of genes detrimental to pregnancy. The results show that decidual growth is normal in PRL null mice treated with PRL, progesterone, or their combination. However, the decidua of mice treated with progesterone starts expressing IL-6 and 20{alpha}-hydroxysteroid dehydrogenase (20{alpha}-HSD), two proteins absent from the decidua of wild-type mice and involved, respectively, in inflammation and progesterone catabolism. The expression of both IL-6 and 20{alpha}-HSD is prevented by PRL treatment. Our results further suggest that PRL inhibition of 20{alpha}-HSD expression is at the level of transcription and that decidual PRL (dPRL) inhibits 20{alpha}-HSD promoter activity. Inhibitors of Janus kinase 2 (Jak2) but not other kinases prevent dPRL down-regulation of the 20{alpha}-HSD promoter. Furthermore, cotransfection of the 20{alpha}-HSD promoter with expression vectors of constitutively active PRL-R, Jak2, or signal transducer and activator of transcription 5b (Stat5b) leads to substantial inhibition of promoter activity. Taken together, our investigation provides an explanation for the inability of progesterone to sustain pregnancy in PRL null mice and suggests that dPRL plays an important role in pregnancy by repressing the expression of IL-6 and 20{alpha}-HSD in the decidua. The study also demonstrates that PRL signals through the Jak2/Stat5 pathway to down-regulate 20{alpha}-HSD expression in the decidua.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ENDOMETRIAL STROMAL CELLS proliferate and differentiate to form decidual tissues during pregnancy in mammalian species. In humans, decidualization occurs in every menstrual cycle and depends primarily on levels of progesterone and estradiol (1). However, in rodents, decidualization requires, in addition to adequate levels of these hormones, an exogenous trigger, either the contact of the blastocyst during pregnancy or artificial stimulation of the uterine endometrium during pseudopregnancy (2).

Extensive investigations from our laboratory and others have shown that the decidua is able to produce hormones and cytokines. The first hormone that was shown to be secreted by the rat decidua was a prolactin (PRL)-related hormone. We had advanced the concept of decidual PRL-like luteotropin in rodents (3, 4) before PRL was shown to be produced by the human decidua (5). This luteotropic hormone was shown to bind to the PRL receptor (PRL-R) in both ovarian (6, 7, 8) and decidual cells (9) and was named decidual luteotropin (DLt). Two PRL-related genes were subsequently cloned from the rat decidua by different laboratories. The first gene cloned by the Friesen laboratory (10) was named PRL-like protein B (PLP-B). The second, cloned by the Soares group (11), was named decidual PRL-related protein (dPRP). This prompted both groups (11, 12) to suggest that these proteins could be the previously found DLt. However, further investigation failed to demonstrate that these PRL-related hormones bind to the PRL-R (13, 14), and their biological actions are still under investigation. Because DLt, identified years ago (3, 4, 6, 15), is actually the only decidual hormone that can bind the PRL-R (7, 8, 9), we reexamined this issue and succeeded at demonstrating that similar to humans, the PRL gene is expressed in the rat decidua (16) and that as previously shown for DLt, the PRL is expressed by decidual cells located in the antimesometrial site of the uterus. The cloned sequence of the decidual PRL (dPRL) is the same as pituitary PRL.

Although extensive investigations have clarified the expression and regulation of dPRL in humans (17), little is known about the role of this hormone in the maintenance of pregnancy. Our finding that the PRL gene is expressed in the decidua of rodents (16) provides an experimental animal model to study the role of this hormone.

Results obtained from PRL-R and PRL knockout (18, 19, 20) mice support an important role for dPRL in the normal progress of pregnancy. PRL and PRL-R null mice are infertile. Because the most important role of PRL in fertility is its ability to sustain progesterone production from the corpus luteum (CL) (21), these mice were treated with progesterone. Progesterone rescued implantation (18, 19, 20) and allowed for normal decidualization (22). However, extensive fetal death started from midpregnancy despite steroid treatment (18, 19, 20). This led investigators to conclude that dPRL may play a key role in the normal progress of pregnancy by acting at the level of the decidua (18, 20). Indeed, our investigations on the roles of PRL in rat decidua have revealed that PRL has potent inhibitory and stimulatory roles on some key decidual genes (23, 24, 25, 26, 27, 28).

Two proteins known to be detrimental for the normal progress of pregnancy are the cytokine IL-6 and the steroidogenic enzyme 20{alpha}-hydroxysteroid dehydrogenase (20{alpha}-HSD). We and others have shown that IL-6 normally is not expressed in the decidua (25, 29, 30). IL-6 production by decidual tissue in response to inflammation and in conjunction with other inflammatory mediators is considered to play an important role in the pathophysiology of preterm labor due to infection (31, 32). As for 20{alpha}-HSD, it is an NADPH-dependent enzyme that catabolizes progesterone, converting it to its inactive form, 20{alpha}-hydroxyprogesterone (33, 34). This enzyme plays an important role in terminating pregnancy by inactivating progesterone synthesized in the CL (34, 35, 36). 20{alpha}-HSD is not expressed in the rat CL throughout pregnancy, but its expression significantly increases just before parturition (35, 37). Similar to the ovary, 20{alpha}-HSD activity in the rat placenta was undetectable until d 20 of pregnancy and then increased dramatically on d 21 (38, 39).

Our overall goal is to examine the role of PRL in the decidua. Using the PRL null mice and primary decidual cell culture, we demonstrate in this study that dPRL is a powerful repressor of IL-6 and 20{alpha}-HSD expression in the decidua. Moreover, our results reveal that dPRL acts locally to down-regulate the transcriptional activity of the 20{alpha}-HSD gene through the long form of the PRL-R and the Janus kinase 2 (Jak2)/signal transducer and activator of transcription 5 (Stat5) signal transduction pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
[{alpha}-32P]Deoxy-CTP (dCTP) was purchased from Amersham Biosciences, Inc. (Piscataway, NJ); deoxynucleotide triphosphate, ExTaq DNA polymerase, and ExTaq buffer were purchased from Takara Biomedicals (Shiga, Japan). Reverse transcriptase kit, Trizol reagent, and the nucleotides used as primers in the RT-PCR analysis were obtained from Invitrogen (Carlsbad, CA). SYBR green PCR Master Mix was obtained from Applied Biosystems (Foster City, CA). Western blotting Luminol reagent was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Tissue culture media RPMI 1640, nonessential amino acids, sodium pyruvate, trypsin-EDTA, antibiotics, and antimycotics were purchased from Mediatech (Herndon, VA). Fetal bovine serum (FBS) was purchased from HyClone Laboratories (Logan, UT). Aprotinin, leupeptin, phenylmethylsulfonyl fluoride, and wortmannin were purchased from Sigma Chemical Co. (St. Louis, MO). RIPA buffer was purchased from Boston Bioproducts (Ashland, MA). Protogel, a 30% acrylamide/bis-acrylamide mixture (37.5:1) was from National Diagnostics (Atlanta, GA). AG490, AG18, PD 98059, rapamycin, and PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) were purchased from Calbiochem (La Jolla, CA). Ovine PRL (APF 10677 C) was kindly provided by the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD).

Animal models
PRL null mice were kept at 25 C with a 14-h light, 10-h dark cycle and were fed a commercial pelleted diet ad libitum. Heterozygous mutants were intercrossed to generate +/+, +/–, and –/– (null) mice, which were genotyped by PCR using tail DNA. PRL null mice or wild-type mice were mated with vasectomized males to induce pseudopregnancy, and the day on which a vaginal plug was found was designated d 1 of pseudopregnancy. Decidualization was induced on d 4 at 1000 h of pseudopregnancy by injecting sesame oil (25 µl/each uterine horn) bilaterally under ether anesthesia. Decidualization was sustained in PRL null mice by injections of progesterone (3 mg sc in sesame oil, once daily), PRL (60 µg sc in polyvinyl pyrrolidone, twice daily), or progesterone plus PRL beginning on the day of vaginal plug detection. The decidual tissues were removed from the uterus and collected on d 10. Pseudopregnant Holtzman Sprague Dawley-derived female rats were obtained from Harlan facilities (Madison, WI), and decidualization of pseudopregnant uterine endometrium was induced as described previously (25). All experimental procedures were performed in accordance with the principles of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee.

Rat primary decidual cell culture
Decidual tissue, removed from the uterus of three to five pseudopregnant rats, was incubated under mild agitation in a water-jacketed cell stirrer (Wheaton Scientific, Millville, NJ) containing RPMI 1640 medium supplemented with collagenase (50 U/ml), dispase (2.4 U/ml), and deoxyribonuclease (200 U/ml) for 1 h at 37 C. At the end of the incubation, dispersed cells were filtered through a nylon mesh to remove undigested tissue and centrifuged at 1500 x g for 10 min. The cell pellet was gently resuspended in RPMI 1640 medium supplemented with 10% FBS, antibiotic-antimycotic solution (2x), nonessential amino acids (1x), sodium pyruvate (1x), and D-glucose (0.45%). Viable decidual cells, determined by the trypan blue exclusion method, were seeded in six-well plates at 1.5–2 x 106 cells per well and cultivated in a humidified atmosphere containing 5% CO2 at 37 C. After allowing the cells to attach for 3–4 h, the unattached blood cells were removed with sterile PBS. The decidual cells were then treated for 12 or 48 h with PRL, progesterone, and AG490 in RPMI 1640 phenol-free medium supplemented with 1% dextran charcoal-treated FBS. At the end of the treatment, the cells were washed twice with ice-cold PBS and frozen at –80 C until RNA extraction. In the case where transient transfections needed to be done, the cells were transfected using lipofectin reagent (Life Technologies, Inc., Rockville, MD) according to the manufacturer’s instructions. The cells were incubated with the lipofectin-reagent-DNA complexes diluted in Opti-MEM I reduced serum medium (Life Technologies) for 4 h before an equal volume of RPMI 1640 phenol red-free medium containing 2% dextran charcoal-treated FBS was added to each well with or without treatment. Medium was changed every 24 h. To harvest cells, each well was washed twice with ice-cold PBS.

RNA isolation and RT-PCR analysis
Total RNA from the decidual tissue of mice or from decidual cells was isolated using Trizol reagent according to the manufacturer’s instructions. RNA concentration was measured by photospectrometry. The RT and PCR were conducted as previously described (26). For the PCR, the conditions were such that amplification of the product was in the exponential phase, and the assay was linear with respect to the amount of input cDNA. Each PCR included ribosomal protein L19 used as an internal control. Reaction products were electrophoresed on an 8% polyacrylamide nondenaturing gel. Gels were stained with Tris-borate EDTA containing 0.5 µg/ml ethidium bromide and washed three times. The resulting gels were photographed using a UV transilluminator and a digital camera (Electrophoresis Documentation and Analysis System 120; Eastman Kodak Co., New Haven, CT). For reactions using [{alpha}-32P]dCTP, reaction products were electrophoresed on 8% polyacrylamide nondenaturing gel and were subjected to autoradiography. After autoradiography, data were analyzed using a Molecular Dynamics PhosphorImager and ImageQuant version 3 software (Molecular Dynamics, Sunnyvale, CA). For each experiment, RT-PCR were repeated at least three times.

For the mouse, the IL-6 primers used were 5'-TGG TGA CAA CCA CGG CCT TC-3' and 5'-GAG CAT TGG AAA TTG GGG TAG GA-3'. The 20{alpha}-HSD primers used were 5'-TCT TCG GTA CTT TCC TGC TGAT-3' and 5'-CTG GGG GTG AGT TGC TAAG-3' (40). The TIMP-3 primers used were 5'-CTT GTC GTG CTC CTG AGC TG-3' and 5'-CAG AGG CTT CCG TGT GAA TG-3' (41). The PRL primers used were 5'-AGC CTC TGC CAA TCT GTT CC-3' and 5'-AGG CCT GGC TAA TTA TCT TCT CA-3'. Ribosomal protein L19 primers used were 5'-AGC GCC TCC AGG CCA AGA AGG-3' and 5'-CCA GGC CGC TAT GTA CAG ACA CGA-3'.

For the rat, the 20{alpha}-HSD primers used were 5'-TAG GGC TGC CAT CTT AGT ATT CA-3' and 5'-GAA TGC CAT CTT TAT CTC AAC CA-3' as described previously (40). The L19 primers used were 5'-CTG AAG GTC AAA GGG AAT GTG-3' and 5'-GGA CAG AGT CTT GAT GAT CTC G-3'.

Real-time RT-PCR
For each real-time PCR (25 µl), cDNA was mixed with 2x SYBR Green PCR Master Mix containing primers. The mouse IL-6 primers used were 5'-CCG GAG AGG AGA CTT CAC AG-3' and 5'-TCC ACG ATT TCC CAG AGA AC-3'. The mouse 20{alpha}-HSD primers used were 5'-GGA GGC CAT GGA GAA GTG TA-3' and 5'-ATG GCA TTC TAC CTG GTT GC-3'. The mouse TIMP-3 and L19 primers used were the same as those used in RT-PCR. The PCR were carried out in duplicate or triplicate, and the samples were analyzed on an ABI 7900HT Real-Time PCR machine (Applied Biosystems). L19 was used as the internal reference. Melting curve analyses were done for both target genes and L19 to confirm the product specificity. Standard curves were generated for both the target genes and L19 to make sure that the amplification efficiencies were approximately equal.

Western blot analysis
Western blots were performed as described previously (42). Antibodies to mouse IL-6 (Sigma), TIMP-3 (Santa Cruz Biotechnology), and 20{alpha}-HSD (43) were used. Briefly 40 µg protein was separated on a 7.5% SDS-PAGE gel and transferred to a nitrocellulose membrane. Western blotting was performed by blocking nonspecific binding with 5% dry milk in Tris-buffered saline buffer containing 1% Tween 20 for 1 h at room temperature. Blots were then incubated with the primary antibody overnight at 4 C on a rocking platform. After a series of washes, blots were incubated with a secondary antibody linked to horseradish peroxidase for 1–2 h at room temperature. After extensive washing, blots were analyzed using an enhanced chemiluminescence detection system and exposed to x-ray film.

Luciferase activity measurement
For promoter analysis, we used the 2.5-kb 20{alpha}-HSD promoter cloned by our laboratory and linked to a luciferase reporter gene (20{alpha}-HSD-Luc) (44), and 0.5 µg of 20{alpha}-HSD-Luc constructs and 0.5 µg of a control ß-galactosidase expression vector (Life Technologies) were transfected into primary decidual cells. At the end of the incubation, 100 µl passive lysis buffer (Promega) was added into each well, and 20 µl cell lysate was used to measure firefly luciferase activity or ß-galactosidase activity using Promega’s Luciferase Reporter or ß-Galactosidase Assay System respectively, in a Lumat LB 9507 Luminometer (EG & G Berthold, Oak Ridge, TN). Relative light units were obtained by dividing the luciferase activity by the ß-galactosidase activity.

Statistical analysis
Data were examined by one-way ANOVA followed by the Tukey test using Prism software (GraphPad Software, Inc., San Diego, CA). For Figs. 1AGo, 2Go, and 4CGo, –/– mice were analyzed by one-way ANOVA, whereas +/+ mice were used as a normal reference. Values were considered statistically significant at P < 0.05.


Figure 1
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  		FIG. 1. Uterus weight of wild-type and PRL null mice. A, PRL null mice were mated with vasectomized males to induce pseudopregnancy. From the day the vaginal plug was found, these mice were treated with vehicle (V), progesterone (P), PRL, or their combination. Decidualization was induced with intrauterine administration of oil. On d 10 of pseudopregnancy, uteri were collected and weighed. The –/– mice were analyzed by one-way ANOVA, whereas +/+ mice were used as a normal reference. Values are expressed as the mean ± SEM (n = 3). *, P < 0.05 vs. –/– treated with vehicle. B, dPRL expression was measured by RT-PCR as described in Materials and Methods.

 

Figure 2
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  		FIG. 2. PRL but not progesterone prevents IL-6 expression in the mouse decidua. Decidual tissues were collected from wild-type mice and PRL null mice treated with progesterone (P), PRL, or both on d 10 of pseudopregnancy. A, TIMP-3, a decidualization marker, was measured by RT-PCR, real-time RT-PCR, and Western analysis as described in Materials and Methods. B, IL-6 expression was measured by RT-PCR, real-time RT-PCR, and Western analysis. The –/– mice were analyzed by one-way ANOVA, whereas +/+ mice were used as a normal reference. Values are expressed as the mean ± SEM (n = 3). *, P < 0.05 vs. –/– treated with progesterone.

 

Figure 4
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  		FIG. 4. Effect of PRL and progesterone on 20{alpha}-HSD expression in the decidua. Rat primary decidual cells were isolated from pseudopregnant rats and cultured for 12 h in the presence of different doses of PRL (A) or progesterone (P) (B). Total RNA was prepared and subjected to RT-PCR analysis. C, decidual tissues were collected from wild-type mice and PRL null mice treated with progesterone, PRL, or both on d 10 of pseudopregnancy. 20{alpha}-HSD expression was measured by RT-PCR, real-time RT-PCR, and Western analysis. The –/– mice were analyzed by one-way ANOVA, whereas +/+ mice were used as a normal reference. Values are expressed as the mean ± SEM (n = 3). *, P < 0.05 vs. 0 µg/ml (cell culture) or –/– treated with progesterone (animal study).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Decidualization can be maintained with progesterone, PRL, or their combination in PRL null mice
To examine the role of PRL in the decidua and to eliminate any possible contamination by trophoblast cells, we mated PRL null mice with vasectomized males to induce pseudopregnancy. From the day the vaginal plug was found, these mice were treated with progesterone, PRL, or their combination. Control mice were PRL null mice treated with vehicle. Decidualization was induced with intrauterine administration of oil. Whereas decidualization does not occur in the uterus of PRL null mice treated with vehicle, decidualization takes place in the uterus of PRL null mice treated with PRL, progesterone, or their combination with an increase in uterine weight similar to that seen in wild-type mice used as a reference (Fig. 1AGo). Although we have previously reported that PRL expressed in the decidua is identical to pituitary PRL (16), we examined whether or not dPRL mRNA was found in the decidua of PRL null mice. As shown in Fig. 1BGo, the dPRL expressed in wild-type mice was not detectable in the decidua of null mice treated with either progesterone or PRL.

PRL but not progesterone down-regulates IL-6 expression in the mouse decidua
We and others have previously shown that IL-6 is an inflammatory cytokine not normally expressed in the decidua (25, 29, 30). Using a decidual cell line, we have previously shown that PRL can inhibit the expression of this cytokine. To examine whether PRL can indeed repress the expression of this gene in vivo, we isolated decidua from either wild-type or PRL null mice treated with PRL, progesterone, or with both hormones. As a decidualization marker (45, 46), tissue inhibitor of metalloproteinase (TIMP-3) expression was examined by RT-PCR, real-time RT-PCR, and Western analysis (Fig. 2AGo). Although PRL appears to stimulate TIMP-3 expression, it did not reach statistical difference and the level of decidual TIMP-3 expression was similar among the different groups. In sharp contrast to TIMP-3, the decidua of wild-type mice did not express IL-6 (Fig. 2BGo), and neither mRNA nor protein could be detected in this tissue. However, in PRL null mice treated with progesterone for several days, significant expression of IL-6 was detected. PRL treatment, when given either alone or with progesterone, significantly down-regulated the expression of this gene, demonstrating clearly that the lack of IL-6 expression in the decidua is due to PRL and that PRL is a physiological inhibitor of this cytokine in vivo.

Characterization of 20{alpha}-HSD expression in the rat decidua
To investigate whether 20{alpha}-HSD is expressed in the rat decidua, decidual tissue was collected on d 9–12 of pseudopregnancy. Total RNA and proteins were isolated and subjected to RT-PCR (Fig. 3AGo) and Western blot (Fig. 3BGo) analysis. The results reveal that 20{alpha}-HSD mRNA and proteins are not detectable during this period of time. However, primary decidual cells maintained in culture acquire the ability to express 20{alpha}-HSD (Fig. 3CGo), suggesting that decidual 20{alpha}-HSD expression is under inhibitory regulation in vivo.


Figure 3
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  		FIG. 3. Correlation of developmental expression of 20{alpha}-HSD mRNA (A) and proteins (B) in the rat decidual tissue throughout pseudopregnancy with 20{alpha}-HSD mRNA expression in primary decidual cells (C). Total RNA and proteins were isolated from decidual tissue dissected from d 9–12 pseudopregnant animals. RNA isolation was also performed with primary decidual cells isolated from d 9 pseudopregnant rats that were cultured for 12 h in RPMI 1640 phenol red-free medium supplemented with 1% dextran charcoal-treated FBS. RT-PCR was performed using specific primer sets for 20{alpha}-HSD and L19 as an internal control. Western blot analysis was performed using the rat 20{alpha}-HSD polyclonal antibody produced by our laboratory (43 ). Results are from three independent experiments for each autoradiogram.

 
Effect of PRL on 20{alpha}-HSD expression in the decidua
To determine whether PRL is an inhibitory factor, we used both rat primary decidual cells and the PRL null mice. Primary decidual cells were maintained in short-term culture (12 h) because in this condition the cells remain expressing the PRL-R (47) but produce little if any PRL (16). Decidual cells were treated with increasing concentrations of either PRL or progesterone. Progesterone was used as control because in vivo, PRL stimulates progesterone production from the CL. As shown in Fig. 4AGo, treatment with PRL led to a concentration-dependent decrease in 20{alpha}-HSD expression. Progesterone had no significant inhibitory effect in culture (Fig. 4BGo). When we examined 20{alpha}-HSD expression in vivo, we found low mRNA and no protein detectable in the decidua of wild-type mice (Fig. 4CGo). Interestingly, the expression of this enzyme was substantial in PRL null mice treated with progesterone. PRL administered to PRL null mice, either alone or with progesterone prevented 20{alpha}-HSD expression.

Mechanism of PRL action on 20{alpha}-HSD promoter activity in rat decidual cells
Because the principal kinase known to be activated by PRL is Jak2 (48), we examined the effect of AG490, a classical inhibitor of this kinase, on PRL repression of 20{alpha}-HSD. As shown in Fig. 5Go, AG490 prevented PRL action, indicating that Jak2 is involved in PRL regulation of 20{alpha}-HSD expression. To determine whether PRL represses 20{alpha}-HSD expression at the level of transcription, we transfected rat decidual cells with a 2.5-kb 20{alpha}-HSD promoter, cloned in our laboratory and linked to a luciferase reporter gene (2.5-kb 20{alpha}-HSD-Luc) (44). Decidual cells were cotransfected with a ß-galactosidase construct to control for transfection efficiency and cultured for 48 h. Because decidual cells lose the PRL receptor when maintained in culture for more than 24 h (47), we also transfected a constitutively active PRL receptor (PRL-RCA) into these cells (49). The results shown in Fig. 6Go indicate that transfection of PRL-RCA induced a dramatic decrease in 20{alpha}-HSD promoter activity. Similar inhibition was observed when the cells were transfected with either a constitutively active Jak2 (Jak2-CA) (50) or a constitutively active Stat5b (Stat5b-CA) (51), indicating that PRL acts through the Jak2/Stat5 pathway to inhibit 20{alpha}-HSD promoter activity. Because rat primary decidual cells produce PRL (16) but lose the PRL-R when maintained in culture for several days, we examined whether endogenous dPRL inhibited the transcriptional activity of this promoter. The 20{alpha}-HSD-Luc promoter reporter construct was cotransfected with the long form of the PRL-R into decidual cells. Cells were allowed to recover and were treated with inhibitors for different signaling pathways. As shown in Fig. 7Go, AG18, a general tyrosine kinase inhibitor, and AG490, the more specific Jak2 inhibitor, were able to induce a 5- to 6-fold increase in 20{alpha}-HSD promoter activity. PD 98059, a MAPK inhibitor; wortmannin, a phosphatidylinositol-3-kinase inhibitor; rapamycin, an mTOR inhibitor, and PP2; a Src kinase inhibitor, had no significant effect on 20{alpha}-HSD promoter activity. These results suggest that dPRL represses 20{alpha}-HSD transcriptional activity through the Jak2/Stat5 pathway.


Figure 5
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  		FIG. 5. Effect of AG490 on PRL-mediated inhibition of 20{alpha}-HSD expression in rat decidual cells. Rat decidual cells were cultured for 12 h with 1 µg/ml PRL in the presence of 20 µM AG490 or vehicle. 20{alpha}-HSD mRNA expression was analyzed by RT-PCR. Values are expressed as the mean ± SEM (n ≥ 3). *, P < 0.05 vs. vehicle-treated control.

 

Figure 6
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  		FIG. 6. PRL inhibition of 20{alpha}-HSD promoter activity through the Jak2/Stat5 pathway in rat decidual cells. The 2.5-kb 20{alpha}-HSD-Luc promoter construct (0.5 µg/well) was cotransfected with PRL-RCA, Jak2-CA, or Stat5b-CA. Transient expression of the reporter gene was quantified by a standard luciferase assay and normalized against ß-galactosidase. Values are expressed as the mean ± SEM (n ≥ 3). *, P < 0.05 vs. control.

 

Figure 7
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  		FIG. 7. Effects of several inhibitors of different signaling pathways on 20{alpha}-HSD promoter activity in rat decidual cells. Rat decidual cells were transfected with the 2.5-kb 20-HSD-Luc promoter construct (0.5 µg/well) in the presence of the long form of the PRL-R expression vector. They were then treated with AG490, a specific Jak2 inhibitor; AG18, a general tyrosine kinase inhibitor; PD 98059, a MAPK inhibitor; wortmanin, a phosphatidylinositol-3-kinase inhibitor; rapamycin, an mTOR inhibitor; and PP2, Src kinase inhibitor, for 48 h because at this time the locally produced dPRL is maximally expressed. Transient expression of the reporter gene was quantified by a standard luciferase assay and normalized against ß-galactosidase. Values are expressed as the mean ± SEM (n ≥ 3). *, P < 0.05 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PRL is a single-chain protein hormone closely related to GH. More than 300 different functions have been reported so far (48, 52). In rodents, one of the major functions of PRL in pregnancy is to stimulate and maintain progesterone production from the CL (48, 52). PRL is mainly secreted by lactotrophs in the anterior pituitary. In addition, it is also secreted by several other tissues, such as lymphocytes (53) and the decidua (16). It is generally accepted that pituitary PRL functions as an endocrine hormone by entering the circulation and acting on remote target tissues, whereas PRL expressed in extrapituitary sites acts through autocrine or paracrine mechanisms to regulate local factors (17, 48).

The findings that rodent decidua produces PRL (16), expresses the PRL-R (7, 22), and responds to PRL stimulation (23, 24, 25, 26, 27, 28) together with the inability of progesterone to completely salvage pregnancy in PRL (Binart, N., S. Elizur, and G. Gibori, unpublished observations) and PRL-R null mice (18, 20) strongly suggest that dPRL may play an important role locally to allow the normal progress of pregnancy and fetal survival.

Decidualization in PRL null mice treated with either progesterone or PRL occurs normally, and decidual weight of these mice remains similar as compared with wild-type mice. This and a previous report (22) suggest that decidualization in the rodent is dependent on ovarian progesterone but not on dPRL. However, in this investigation, we present evidence that the decidua of PRL null mice treated with only progesterone expresses two factors, IL-6 and 20{alpha}-HSD, which can negatively affect pregnancy outcome and normally are not expressed in the decidua of wild-type mice. Interestingly, PRL injected either alone or with progesterone totally prevents the expression of these molecules in vivo.

We have previously shown that PRL but not progesterone inhibits the expression of the proinflammatory cytokine IL-6 in cultured decidual cells (25). This led us to examine whether dPRL is a physiological repressor of IL-6 in vivo. Results of the present investigation clearly indicate that in the absence of PRL, the decidua abundantly expresses IL-6 despite progesterone treatment. Because production of IL-6 during pregnancy may compromise fetal survival by triggering an inflammatory response, the inhibition of its expression in the decidua by PRL may be of great physiological importance. It is therefore highly possible that the premature abortion and fetal death seen in the PRL and PRL-R null mice treated with only progesterone is due, at least in part, to high expression of IL-6 in the decidua.

The second gene found to be repressed by PRL is 20{alpha}-HSD, an enzyme that catabolizes progesterone to its inactive form, 20{alpha}-hydroxyprogesterone. 20{alpha}-HSD expression in the ovary is key for the decrease in progesterone production seen just before parturition in rodents (40). However, during pregnancy, it is crucial that 20{alpha}-HSD remain silent both in the ovary (36, 40, 54, 55, 56), which is the source of progesterone production, and in the decidua, a major site of progesterone action. The high concentration of progesterone found in the CL can down-regulate luteal 20{alpha}-HSD (57). However, only a slight but not significant inhibition was observed in decidual cells treated with doses of progesterone (1 µg/ml) 10-fold higher than the one seen in the circulation of pregnant mice. Moreover, 20{alpha}-HSD is expressed at very high levels in the PRL null mice treated with progesterone, whereas no expression is seen in the decidua of the knockout mice treated with PRL. These results strongly suggest that the extensive fetal death seen in the progesterone-treated null mice from midpregnancy may be due, at least in part, to the local catabolism of progesterone by decidual 20{alpha}-HSD, reducing the levels of progesterone seen by the uterus. Therefore, inhibition of 20{alpha}-HSD by dPRL may be essential for the normal progress of pregnancy. Another important finding of the present study is that dPRL down-regulation of 20{alpha}-HSD is at the transcriptional level and involves the long form of the PRL-R and the Jak2/Stat5 signal transduction pathway.

The decidual tissue of pseudopregnancy is a great model to study gene expression in the decidua without any contamination from the embryo. However, recent work done by Bany and Cross (58) has demonstrated that the conceptus has local effects on uterine gene expression. Whether the embryo has any effect on the expression of IL-6 and 20{alpha}-HSD remains a subject for further investigation.

Taken together, our investigation provides an explanation for the inability of progesterone to sustain pregnancy in PRL null mice and indicates that dPRL plays an important role in pregnancy by repressing two genes, IL-6 and 20{alpha}-HSD, whose expression in the decidua is detrimental for the normal progress of pregnancy.


   Acknowledgments
 
We are grateful to the National Institute of Diabetes and Digestive and Kidney Diseases and the National Hormone and Pituitary Program (National Institutes of Health) for the ovine PRL, Dr. Jean Djiane for the PRL-RCA, Olli Silvennoinen for the Jak2-CA, and Dr. Toshio Kitamura for the Stat5b-CA. We thank Carlos Stocco and Julia Halperin for their input, Susan Ferguson-Gottschall and Aurora Shehu for technical support, and Jamie Le for the preparation of the manuscript.


   Footnotes
 
This work was supported by National Institutes of Health Grants HD12356, HD40093, and U54.

None of the authors has anything to disclose.

First Published Online January 25, 2007

Abbreviations: CL, Corpus luteum; dCTP, deoxy-CTP; DLt, decidual luteotropin; dPRL, decidual PRL; FBS, fetal bovine serum; 20{alpha}-HSD, 20{alpha}-hydroxysteroid dehydrogenase; Jak2, Janus kinase 2; Jak2-CA, constitutively active Jak2; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PRL, prolactin; PRL-R, PRL receptor; PRL-RCA, constitutively active PRL-R; Stat5, signal transducer and activator of transcription 5; Stat5b-CA, constitutively active Stat5b; TIMP, tissue inhibitor of metalloproteinase.

Received December 7, 2006.

Accepted for publication January 18, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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