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Endocrinology Vol. 142, No. 3 1242-1250
Copyright © 2001 by The Endocrine Society

ARTICLES

Characterization of a Rat Uterine Cell Line, UIII Cells: Prolactin (PRL) Expression and Endogenous Regulation of PRL-Dependent Genes; Estrogen Receptor {beta}, {alpha}2-Macroglobulin, and Decidual PRL Involving the Jak2 and Stat5 Pathway1

Anne Prigent-Tessier2, Uriel Barkai2, Christian Tessier, Hélène Cohen and Geula Gibori

Department of Physiology and Biophysics (A.P.-T., U.B., C.T., G.G.), College of Medicine, University of Illinois, Chicago, Illinois 60612; Institut National des Sciences Appliquées de Lyon-INSERM U352 (H.C.), Laboratoire de Biochimie et Pharmacologie, 69100 Villeurbanne Cedex, France

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Decidualization of endometrial stroma in the rat induces the expression and secretion of rat decidual PRL (rdPRL). Recently, we have generated a nontransformed rat uterine stromal cell line (UIII) that decidualizes spontaneously in culture. In this report, we have established by immunocytochemistry, RT-PCR, Western blot analysis, labeled amino acid incorporation and RIA that these cells express the rat PRL messenger RNA as well as synthesize and secrete PRL. We have also cloned by RT-PCR a 403-bp complementary DNA fragment whose sequence is identical with that of rat pituitary PRL. In addition, UIII cells express the PRL receptor (PRL-R) long form, all the components involved in the PRL signal transduction pathway, estrogen receptor {beta} (ER{beta}) and {alpha}2-macroglobulin ({alpha}2-MG), which are known to be PRL-regulated genes. However, when UIII cells were treated with PRL, no regulation of these genes was observed. Moreover, in these cells, the PRL signaling components: the tyrosine kinase Jak2 and the transcription factor Stat5 were endogenously phosphorylated and their phosphorylation states were not enhanced in the presence of exogenous PRL. To examine whether the endogenously secreted PRL affects the expression of PRL-regulated genes, UIII cells were treated with either an anti-PRL receptor antibody or a Jak2 inhibitor, AG490. The anti-PRL receptor antibody decreased {alpha}2-MG expression. AG490 inhibited Jak2 and Stat5 phosphorylation, prevented Stat5 binding to its DNA consensus sequence, and also caused a dose-dependent down-regulation of {alpha}2-MG and ER{beta} expression. In contrast, AG490 enhanced PRL mRNA levels. In summary, we have established that the UIII stromal cells of uterine origin produce PRL. Furthermore, we have shown for the first time that decidual PRL may act locally to activate the Jak2/Stat5 pathway and up-regulate important genes involved in decidual growth and placentation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE RAT, the uterine stromal cells undergo a marked cell proliferation and differentiation to form the decidua. Decidualization leads to numerous cell modifications including morphological and biological changes (1, 2, 3). We have recently established that decidualization causes a defined cell population located in the antimesometrial site of the uterus to express PRL mRNA as well as synthesize and secrete this hormone and have named it rat decidual PRL (rdPRL) (4). We have also shown that decidualization induces the expression of the PRL receptor (5) and a potent protease inhibitor, {alpha}2-MG, which is implicated in limiting trophoblast invasion (6, 7, 8, 9, 10, 11). The expression of {alpha}2-MG is regulated by PRL either in the ovaries or the decidua (12, 13). Another PRL-regulated gene, the estrogen receptor {beta} (ER{beta}) is also expressed in decidual cells (14). However, it is still not known whether the decidual-derived PRL regulates the expression of these genes locally.

Because primary decidual cells are difficult to obtain and rapidly lose the PRL-R in culture (5), making the investigation of the role and regulation of decidual PRL difficult, we have used a previously generated SV-40-transformed and temperature-sensitive decidual cell line (GG-AD) (15). However, similar to many transformed cell lines, these cells do not express several of the genes found in the decidual cells in vivo. In this investigation, we have characterized a nontransformed rat uterine stromal cell line (UIII), which differentiates spontaneously in culture, giving rise to large cells presenting several characteristics of the cells forming the rat decidua (16, 17). We have shown that these cells express the PRL-R, ER{beta} and the components implicated in PRL signaling pathway. Most importantly, our results revealed that the UIII cells produce PRL, which may act by an autocrine mechanism to regulate the level of {alpha}2-MG, ER{beta}, and PRL mRNA. To our knowledge the UIII cells are the only nontransformed rat cell line that has the characteristic of rat decidual cells. These cells provide a powerful tool to study the role and regulation of rdPRL and other genes induced by decidualization.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Tissue culture medium M199, antibiotic-antimycotic solution, nonessential amino acids were obtained from Mediatech (Washington, DC). FBS was purchased from HyClone Laboratories, Inc. (Logan, UT). Phenylmethyl sulfonyl fluoride (PMSF), leupeptin, pepstatin-A, aprotinin, fluorescein isothiocyanate (FITC), horse radish peroxidase-conjugated second antibodies and all other reagent grade chemicals were purchased from Sigma (St. Louis, MO). Acrylamide and bis-acrylamide were obtained from Accurate Chemical Inc. (Westbury, NY) and Eastman Kodak Co. (Rochester, NY) respectively. ExTaq DNA polymerase was purchased from Panvera (Madison, WI). The oligonucleotides used as primers for sequencing and RT-PCR analysis were obtained from Life Technologies, Inc. (Grand Island, NY). [{alpha}-32P] deoxycytidine triphosphate (dCTP) was from Amersham Pharmacia Biotech (Arlington Heights, IL). Trans 35S-label was purchased from ICN Radiochemicals (Costa Mesa, CA). AG490 was purchased from Calbiochem (La Jolla, CA). Ovine PRL (APF 10677 C) rPRL I6, iodination grade (AFP 10505 B), rPRL RP3 (AFP 4459 B) and anti-rPRL antibody S9 (AFP 131581570) were kindly supplied by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK, Bethesda, MD). The PRL receptor antibody was generously providing by Jean Djiane (18). The monoclonal anti-Stat5 (G-2) and anti-p-Tyr (PY99) antibodies and the polyclonal anti-Jak2 (HR-758) antibody were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Animals and surgical procedures
Pseudopregnant Holtzman Sprague Dawley-derived females rats were obtained from Harlan (Madison, WI), and decidualization of pseudopregnant uterus endometrium was induced as described previously (13). Uteri from day 12 of pseudopregnancy were isolated and trimmed of adherent tissues, washed thoroughly with ice cold PBS and the antimesometrial decidual tissue was separated from the mesometrial tissue as described by Martel et al. (19). Tissues were stored at -80 C until used for nucleic acid and protein isolation.

Cell culture
The rat uterine stromal cells, UIII, established from adult, untreated uteri (16) were grown in M199 medium supplemented with FBS (10%), nonessential amino acids (1x), antibiotic-antimycotic solution (2x). They were incubated in a humidified atmosphere of 5% CO2 at 37 C. Culture media were replaced every 48 h and cells harvested at 70–90% confluence. Cells were treated with various concentrations of AG490 and PRL in M199 medium supplemented with 1% FBS containing arachidonic acid which is essential for the in vitro differentiation of UIII cells (17). At the end of the treatment, the cells were washed twice with ice cold PBS and frozen at -80 C until RNA extraction.

Isolation of total RNA and RT-PCR analysis
Total RNA from UIII cells and decidual tissues was isolated by one-step guanidinium- thiocyanate-phenol extraction procedure according to the manufacturer’s protocol (RNA-NOW; Biogentex, Houston, TX). The RT-PCR was performed as previously described (13). Oligonucleotide primer pairs were constructed for different rat specific genes based on their published DNA sequence. An additional pair of oligonucleotides specific to the rat ribosomal protein L19 mRNA was included as internal control in each PCR. The PCR conditions were such that the amplification of the products was in the exponential phase, and the assay was linear with respect to the amount of input RNA. PCR products were electrophoresed on 2.5% Metaphore agarose gel (FMC Bioproducts, Rockland, ME) containing 0.5 µg/ml ethidium bromide. The resulting gels were photographed using a UV transilluminator and a digital camera (Electrophoresis Documentation and Analysis System 120, Kodak, New Haven, CT). For PCR with [{alpha}-32P] deoxy-CTP (2 µCi of 3000 Ci/mmol), reaction products were electrophoresed on 8% polyacrylamide nondenaturing gel. After autoradiography, data were analyzed using a Molecular Dynamics, Inc. PhosphorImager and ImageQuant version 3 software (Molecular Dynamics, Inc., Sunnyvale, CA).

For the detection of Stat5a and Stat5b, a common sense primer-5'-GGGCATCACCATTGCTTGGAAG-3' was combined with a specific Stat5a antisense-5'-GGAGCTTCTGGCAGAAGTGAAG-3' or with a specific Stat5b antisense-5'-CACGACTAGTATTAACACTTCAC-3'. The sizes of the coamplified complementary DNA (cDNA) products were 542 and 610 bp for Stat5a and Stat5b, respectively. The other sets of primers were as followed: PRL receptor long form (PRL-RL), 5'-AAAGTATCTTGTCCAGACTCGCTG-3' and 5'-AGCAGTTCTTCAGACTTGCCCTT-3' (279-bp cDNA fragment); {alpha}2-MG-5'-GTAATCCTTCTAACTGCTTCGGCG-3' and 5'-CCAATGAAGATCGTTTCATACGGA-3' (343-bp cDNA fragment); Stat3–5'-ACTTCTTCACTAAGCCTCCGTTTG-3' and 5'-GGGATACCAGGATGTTGGTAGCG-3' (535bp cDNA fragment); Jak1–5'-CTATGAGCCAGCTGAGTTTCGATC-3' and 5'-CATCTCGGACACAGACGCCGTA-3' (275-bp cDNA fragment); Jak2–5'-GTTCTTACCGAAGTGCGTGCGA-3' and 5'-GGTAATGGTGTGCATCCGCAGTT-3' (523-bp cDNA fragment); Jak3–5'-CCAGGAAGCTGGAACGCTCAAC-3' and 5'-CGAACAGCAGTAGGCGGTGGTT-3' (700-bp cDNA fragment); SHP-2–5'-CGGGAGTTAAGCAAGCTAGCCG-3' and 5'-CCTCACACGCATGACGCCATAC-3' (465-bp cDNA fragment); rat PRL-5'-ATGAACAGCCAAGTGTCAGC-3' and 5'-CTTCATGGATTCCACCTAGTC-3' (403-bp cDNA fragment); L19–5'-CTGAAGGTCAAAGGGAATGTGC-3' and 5'-GGACAGAGTCTTGATGATCTCG-3' (198-bp cDNA fragment).

Molecular cloning of the rat PRL cDNA by PCR
Total RNA from UIII cells was isolated and RT-PCR was performed using specific oligonucleotide primers based on the sequence of the rat pituitary PRL gene. A sense oligonucleotide corresponding to the first 23 nucleotides of the coding region (5'-ATGAACAGCCAGGTGTCAGCCCG-3') was combined with an antisense oligonucleotide corresponding to the 383–403 nucleotides of the coding region (5'CTTCATGGATTCCACCTAGTC-3'). The predicted size of the PCR-amplified product was 403 bp. The PCR products from three independent experiments were electrophoresed on a 0.7% agarose gel. Only one band was detected by ethidium bromide at 403 bp. The cDNA fragments were extracted from the agarose gel, purified, and reamplified by PCR using the specific oligonucleotide primers for rat pituitary PRL containing four CUA repeats for subcloning into the CLONEAMP pAMP10 vector (Life Technologies, Inc., Gaithersburg, MD). DH5{alpha} competent cells were then transformed with the vector. Clones were selected for DNA sequencing from both strands by the dideoxy-chain-termination method (Perkin-Elmer Corp., Foster City, CA). Sequencing was performed by the DNA Sequencing Facility of the University of Chicago.

Immunocytochemistry
PRL expression in UIII cells was performed by immunocytochemistry as previously described for decidual cells in primary culture (4) using an antirabbit FITC conjugated antibody (Sigma) as secondary antibody.

Western blot and immunoprecipitation analyses
For Western blot analysis, equal amounts (30 to 100 µg) of total cellular proteins prepared as already described previously (13) were dissolved in sample buffer (62.5 mM Tris-HCL, pH 6.7, 2% SDS, 10% glycerol, 0.1% bromophenol blue and 5% {beta}-mercapto ethanol). For immunoprecipitation analyses, 1 µg of mouse IgG was added to 800 µg protein extract samples and incubated overnight at 4 C, with either a monoclonal anti-Stat5 antibody (G-2) or a polyclonal anti-Jak2 (HR-758) antibody. Complexes were then precipitated with Protein A/G Sepharose and boiled for 5 min in sample buffer (62.5 mM Tris-HCl, pH 6.7, 2% SDS, 20% glycerol, 0.1% bromophenol blue and 5% {beta}-mercapto ethanol). Proteins were resolved on 10 or 12% denaturing polyacrylamide gels according to the Laemmli method (20). After gel electrophoresis, the proteins were electrophoretically transferred to nitrocellulose filters (Protran, Schleicher & Schuell, Inc., Keene, NH). The blots were incubated overnight at 4 C in 5% nonfat dry milk to block nonspecific binding. Blots were washed, incubated for 4 h at room temperature with the primary antibody (anti-p-Tyr PY99 or anti-rPRL antibody S9), washed again, and then incubated with a horseradish peroxidase-conjugated antirabbit or antimouse IgGs (1:5000) for 1 h. Protein-antibody complexes were visualized using the enhanced chemiluminescence Western blotting detection system (ECL, Amersham Pharmacia Biotech).

De novo synthesis and secretion
Cells were depleted from endogenous methionine following 1-h incubation in methionine-free medium. They were then grown overnight in methionine-free medium containing 100 µCi/ml trans-35S-label. Labeled media were collected, dialyzed against 500-fold excess of 10 mM NaHCO3 (pH 8.0), and concentrated using a microcon filter (Amicon, Inc., Beverly, MA). Labeled cells were washed in PBS and lysed in 50 mM Tris pH 6.7, 2 mM EDTA, 150 mM NaCl, and the protease inhibitors cocktail. Cells were sonicated and cleared by brief centrifugation (10,000 x g for 10 min). Samples were precleared with 50 µg/ml Pansorbin (Calbiochem, La Jolla, CA) and incubated overnight at room temperature with the S9 anti-rPRL antibody (1:1000) in the presence or absence of rat pituitary PRL (2 µg/ml). Pansorbin was again added for 1 h at room temperature and complexes precipitated by centrifugation. Protein gel electrophoresis was performed as described previously. Gels were dried and exposed to x-ray film for 3–5 days.

RIA of rat PRL
Samples of culture media and cell homogenates were assayed for rat PRL using a RIA supplied by the National and Pituitary Program of the NIDDK. Briefly, iodination of PRL was performed by the lactoperoxidase method (21). 125I labeled rat PRL was separated from the free 125I and damaged hormone by chromatography on Sephadex G-100. The limit of detection when a 400 µl sample was assayed, was 156 pg/ml. Intra and interassay variabilities were less than 5%.

Electromobility gel shift assay (EMSA)
To prepare nuclear extracts, UIII cells were scraped from 75 cm2 flasks and pelleted. The pellet was resuspended in 400 µl buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, and 1 µg/ml aprotinin). Cells were allowed to swell for 15 min and 10% (vol/vol) of Nonidet NP-40 (Fluka Chemical Co., Buchs, Sweden) was then added. The tubes were rigorously vortexed for 30 sec, then centrifuged at 10,000 x g for 1 min. The resulting pellet was resuspended in 50 µl Buffer B (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, and 1 µg/ml aprotinin), rocked at 4 C for 20 min and then centrifuged at 10,000 x g for an additional 10 min. The supernatants were frozen at -80 C. For EMSA, Gel Shift Assay System (Promega Corp.; Madison, WI) was applied. Stat5 consensus oligonucleotide (5'AGATTTCTAGGAATTCAATCC-3') was end-labeled with 32P-{gamma}ATP. Nuclear extracts (5 µg) were incubated for 30 min in binding buffer together with 1 x 105 cpm of labeled Stat5 oligonucleotide. Samples were electrophoresed for 3 h on 4.5% non denaturing polyacrylamide gels. Gels were dried and exposed to x-ray film for 3–5 days.

Statistics
Data were examined by one-way ANOVA, followed by Duncan’s multiple-range test. A level of P < 0.05 was accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of PRL cDNA and characterization of PRL expression by UIII cells
To examine whether UIII cells maintained in culture express the PRL gene, we isolated RNA from these cells and amplified by RT-PCR a 403-bp cDNA fragment using specific sense and antisense oligonucleotide primers based on the sequence of the rat pituitary PRL gene. The PCR-amplified products from three independent experiments were directly cloned into the pAMP10 vector. Four clones were selected for each experiment and sequenced on both strands using the dideoxy-chain-termination method. The nucleotide and amino acid deduced sequence from all clones revealed 100% homology with the rat pituitary PRL (Fig. 1AGo). To examine PRL expression in UIII cells, total RNA was isolated and subjected to RT-PCR analysis using specific oligonucleotide primers for PRL. RNA obtained from either antimesometrial decidua or from pituitary was used as positive control. As shown in Fig. 1BGo, the UIII cells express PRL mRNA, although at lower levels than the pituitary and the antimesometrial decidua obtained from day 12 of pseudopregnancy, a time when PRL is maximally expressed in this tissue (4). To further characterize PRL expression in UIII cells, immunocytochemistry analysis was performed using a polyclonal anti-PRL antibody. PRL was detected in the cytoplasm of UIII cells and was concentrated around the nucleus (Fig. 2AGo). Western blot analysis shown in Fig. 2BGo revealed that PRL expressed by UIII cells migrates with an apparent molecular mass of 23 kDa, the same as pituitary PRL. Finally, to study the de novo synthesis and secretion of PRL, UIII cells were cultured in the presence of labeled 35S methionine. Cell extracts and conditioned medium (Fig. 3AGo) were immunoprecipitated with rat polyclonal anti-PRL antibody in the presence (non specific binding) or absence (specific binding) of excess unlabeled rat pituitary PRL. Proteins were separated by SDS-PAGE and gels were exposed to x-ray film. Iodinated pituitary PRL was used as control. The results revealed that UIII cells not only synthesize but also secrete PRL.



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  		Figure 1. Isolation of PRL cDNA and expression of PRL mRNA in UIII cells. A, A 403-bp fragment was amplified by RT-PCR using RNA from UIII cells and specific sense and antisense oligonucleotide primers, based on the sequence of the rat pituitary PRL. This fragment was cloned into the pAMP10 vector. The nucleotides and amino acids are numbered from the initiation codon, ATG (nucleotides1–3). The specific primers used to determine the complete sequence of the 403bp PCR-amplified fragment are underlined. The nucleotide and deduced amino acid sequences of the four clones revealed 100% homology with the rat pituitary PRL. B, Total RNA (1 µg) from antimesometrial decidua of day 12 pseudopregnant rats and UIII cells or total RNA (0.1 µg) from rat pituitary, depicted as Pit, were isolated and analyzed by RT-PCR using specific primers for rat PRL as described in Materials and Methods. The experiment was repeated several times and one representative autoradiogram is shown.

 


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  		Figure 2. Expression of immunoreactive PRL protein in UIII cells. A, UIII cells were grown on sterile cover glass and processed by immunocytochemistry with an antirat PRL antibody as indicated in Materials and Methods. Negative control was obtained by omitting the primary antibody. A positive signal is indicated by the green staining. B, Total proteins (30 µg) obtained from UIII cell extracts were separated by SDS-PAGE, transferred to nitrocellulose and analyzed by Western blotting using a polyclonal antirat PRL antibody. A standard of rat pituitary PRL (30 ng) was used as positive control.

 


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  		Figure 3. Synthesis and secretion of PRL. A, 35S-methionine labeled proteins from either UIII cell extracts (Cells) or conditioned medium (Medium) were immunoprecipitated with the antirat PRL antibody in the presence (nonspecific binding, NS) or absence (specific binding, S) of added unlabeled rat pituitary PRL. Iodinated rat pituitary PRL (Pit PRL, 3 ng:2105 dpm) was used as molecular weight control. Samples were electrophoresed on 12% denaturing polyacrylamide gels. Gels were dried and exposed to x-ray film for 3–5 days. The autoradiogram from one representative experiment is shown (n = 3). B, PRL secretion in UIII cells cultured for 24 h in 1% FBS, were examined using rat PRL RIA as described in the Materials and Methods. Results are the average of six independent experiments.

 
To estimate the amount of PRL secreted by UIII cells, an RIA for rat PRL was performed. As shown in Fig. 3BGo, PRL was significantly secreted by these cells. Interestingly, the cellular content of this hormone was almost 3-fold higher than its release in the medium, indicating that UIII cells have storage capacity.

Expression of PRL signaling components in UIII cells
To examine whether UIII cells express all the components involved in the PRL signaling through the Jak/Stat pathway, cells were isolated and subjected to RT-PCR analysis using specific primers for the genes studied. Previous results (13) and those shown in Fig. 4Go establish that UIII cells express the PRL receptor long form, Jak1 and 2, but not Jak3, as well as Stat3 and 5 (a and b). The tyrosine phosphatase SHP-2, which is activated by Jak2 and acts as a positive regulator of PRL signaling (22), is also expressed in these cells.



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  		Figure 4. PRL signaling components in UIII cells. Total RNA was isolated from UIII cells and subjected to RT-PCR analysis using specific primer sets for the long form of the PRL-R (PRL-RL), the three Jak members (Jak1, 2, 3), the three Stat members (Stat3 and the two Stat5 isoforms) and the tyrosine phosphatase, SHP-2, as described in Materials and Methods.

 
Stat5 and Jak2 protein expression and phosphorylation in UIII cells: effect of AG490
Because mRNA expression does not necessarily imply that Jak2 and Stat5 proteins are phosphorylated by PRL in UIII cells, total proteins were extracted from cells treated with or without PRL and immunoprecipitated with either anti-Stat5 or anti-Jak2 antibodies (Fig. 5Go). They were then analyzed by Western blot using an anti-phosphotyrosine antibody. As shown in Fig. 5AGo, Stat5 was detectable in UIII cells and was phosphorylated on tyrosine before the addition of exogenous PRL. Moreover, addition of exogenous PRL for up to 20 min did not promote further Stat5 phosphorylation. However, pretreatment of UIII cells for 8 h with the Jak2 inhibitor, AG490, effectively decreased the state of Stat5 phosphorylation. As shown for Stat5, Jak2 was also phosphorylated in the absence of PRL (Fig. 5BGo). The state of Jak2 phosphorylation was not enhanced by PRL but was markedly reduced by treatment with AG490.



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  		Figure 5. Stat5 and Jak2 phosphorylation in UIII cells treated with either PRL or AG490. UIII cells were treated up to 20 min with 1 µg/ml PRL or for 8 h with 50 µM AG490. Cellular extracts were precipitated either with the anti-Stat5 or Jak2 antibody, separated on 10% denaturing polyacrylamide gel, transferred to nitrocellulose and analyzed by Western blotting using the monoclonal anti-phosphotyrosine antibody ({alpha}Py) to examine for phosphorylated Stat5 (A) or Jak2 (B).

 
Effect of AG490 on DNA binding activity in UIII cells
Further downstream in the PRL signaling pathway, phosphorylation of Stat5 leads to its translocation to the nucleus and subsequent binding to specific promoter sequences. Therefore, we looked at the DNA binding activity of the immunoreactive Stat5 protein found in UIII cells. Nuclear proteins from these cells were isolated and incubated in the presence of a labeled Stat5 oligonucleotide probe. Results in Fig. 6Go show one DNA-protein complex that disappeared after 10 min of AG490 treatment and remained undetectable throughout the 60 min of treatment.



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  		Figure 6. Binding activity of UIII cells nuclear extracts to consensus Stat5 oligonucleotide. EMSA was performed using a radiolabeled Stat5 consensus oligonucleotide as described in Materials and Methods. Nuclear extracts from cells treated with AG490 (50 µM) for 10 to 60 min were incubated with Stat5 response element. The autoradiograms are representative of three separate experiments.

 
Expression of {alpha}2-MG and ER{beta} in UIII cells: effect of PRL, PRL receptor antibody, and AG490
Because previous investigations in our laboratory have shown that PRL stimulates {alpha}2-MG gene expression in the rat decidual tissue (6), we looked at the expression of {alpha}2-MG in UIII cells treated with different doses of PRL for 24 h. The results show that the UIII cells express this protease inhibitor and that addition of exogenous PRL had no stimulatory effect on {alpha}2-MG mRNA levels (Fig. 7Go). Because PRL is produced and secreted by UIII cells, and because Jak2 and Stat5 are phosphorylated in these cells before the addition of exogenous PRL, we treated the UIII cells with either an anti-PRL receptor antibody or the Jak2 inhibitor, AG490 to prevent PRL signaling. Results shown in Fig. 8Go indicate that treatment of UIII cells with a high concentration of anti-PRL receptor antibody (5% final concentration), which has been shown to block PRL action on casein synthesis (18), induced a significant decrease in {alpha}2-MG mRNA expression. Inhibition of Jak2 by AG490 in UIII cells also led to a down-regulation of {alpha}2-MG mRNA expression (Fig. 9Go). This effect was both dose (Fig. 9AGo) and time (Fig. 9BGo) dependent. Interestingly, the AG490 effect was rapid and caused more than 70% decrease in mRNA levels in less than 3 h. The range of effective inhibitory levels of AG490 was not toxic to the cells and has no effect on cell viability.



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  		Figure 7. {alpha}2-MG gene expression in UIII cells and lack of effect of exogenous PRL. Total RNA obtained from UIII cells treated for 24 h with different doses of PRL was subjected to RT-PCR using specific primers for {alpha}2-MG. The upper panel depicts one representative radiogram and the lower panel represents the normalized mRNA levels as the mean ± SEM (n = 3).

 


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  		Figure 8. Effect of PRL-receptor antibody on {alpha}2-MG mRNA expression in UIII cells. UIII cells were cultured for 48 h in M199–1% FBS medium with or without 5% final concentration of the anti-PRL receptor antiserum (18 ). Total RNA was prepared and subjected to RT-PCR analysis, as described in Materials and Methods. RT-PCR products were visualized by autoradiography (upper panel) and normalized to the amount of the L19 mRNA internal control (lower panel, mean ± SEM, n = 3).

 


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  		Figure 9. Inhibition of {alpha}2-MG mRNA expression in UIII cells by AG490. Total RNA obtained from UIII cells treated with different doses (A) and for different times (B) of AG490 was subjected to RT-PCR analysis using specific primers for {alpha}2-MG, as described in Materials and Methods. The right panels depict representative radiograms and the left panels represent the normalized mRNA levels as the mean ± SEM (n = 3).

 
To examine the effect of AG490 on another decidual PRL target gene (14), we looked whether UIII cells express ER{beta} and whether this gene is regulated through a Jak2-dependent event. Results shown in Fig. 10Go indicate that UIII cells express ER{beta} and that the expression of this gene is down-regulated in a dose-related manner within 8 h of AG490 treatment. The inactive tyrphostin A1 showed no effect.



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  		Figure 10. Estrogen receptor {beta} mRNA expression in UIII cells and its inhibition by AG490. UIII cells were incubated in the presence of different doses of AG490 for 8 h. The inactive tyrphostin A1 (50 µM) was used as control. Total RNA was prepared and subjected to RT-PCR analysis as described in Materials and Methods using specific oligonucleotide pairs for estrogen receptor {beta}. The upper panel depicts one representative radiogram and the lower panel represents the normalized mRNA levels as the mean ± SEM (n = 3).

 
Effect of AG490 on PRL, Jak2 and Stat5 mRNA expression in UIII cells
In contrast to its inhibitory effect on {alpha}2-MG and ER{beta}, AG490 caused an increase in UIII PRL mRNA levels (Fig. 11Go), suggesting that PRL may reduce the expression of its own gene in this cell line. Indeed in the rat decidua, PRL limits rdPRL expression and secretion by the decidua (4). We further analyzed the effect of AG490 on both Jak2 and Stat5 mRNA levels and found that the concentration range of AG490 that caused a marked change in the expression of {alpha}2MG, ER{beta} and PRL had no effect whatsoever on either Jak2 or Stat5 mRNA expression in UIII cells (Fig. 12Go).



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  		Figure 11. Effect of AG490 on PRL expression. UIII cells were cultured for 8 h in M199 medium supplemented with 1% FBS and with various concentrations of AG490. PRL mRNA expression was analyzed by RT-PCR as described in Materials and Methods. One representative radiogram is shown (n = 3).

 


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  		Figure 12. Jak2 and Stat5 mRNA expression in UIII cells treated with AG490. UIII cells were treated with different doses of AG490. Total RNA (1 µg) was reverse transcribed and 40 ng of the cDNA was amplified by RT-PCR using specific primers for Jak2 (A) and Stat5 (a and b isoforms) (B). The experiments were repeated several times and one representative radiogram is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have characterized a rat uterine stroma-derived nontransformed cell line, UIII that has the characteristics of rat decidual cells, and we have provided evidence that several PRL-responsive genes may be regulated by an autocrine mechanism in these cells. Decidualization of rat uterine stromal cells induces the expression of PRL (4) and PRL-R (5) as well as {alpha}2-MG (6) and ER{beta} (14). Using cloning strategy, immunocytochemistry, Western analysis, amino acid incorporation, immunoprecipitation, and RIA, we have demonstrated that UIII cells express the rat PRL gene as well as synthesize and secrete PRL. The recent discovery that the rat decidua expresses, as does the human decidua, the PRL gene and the development of a rat decidual nontransformed cell line that produces PRL provide a powerful tool to investigate and understand the role of rdPRL in reproduction. Indeed, a recent work from Reese et al. (23) suggests a key role for PRL as luteotropic factor during implantation, whereas the work of Kelly’s group (24) suggests strongly that uterine PRL receptor is necessary to fully maintain pregnancy.

To activate target genes, PRL signal transduction requires the presence of the effector inducing membranal receptor dimerization that leads downstream to the activation of several specific mediators (25, 26, 27, 28, 29, 30, 31). Thus, we investigated the expression of PRL signaling core elements involved in the Jak/Stat pathway in UIII cells. All of the essential components, the PRL receptor long form, Jak2, Stat5 (a and b isoforms) were found to be expressed in UIII cells. SHP-2, a cytoplasmic tyrosine phosphatase acting as a positive regulator of PRL signaling (22), is also present in this cell line.

To propagate the signal further downstream, Jak2, associated with the PRL-R, needs to be phosphorylated (27). It then phosphorylates Stat5, which translocates into the nucleus to activate specific gene promoters (27, 32, 33). In this report, we have documented that phosphorylated Jak2 and Stat5 can be immunoprecipitated from whole cell extracts of cultured UIII cells and that activated Stat5 exists in nuclear extracts of these cells. Moreover, because these cells produce PRL, exogenous PRL could not further enhance the phosphorylation state of either Jak2 or Stat5, indicating that these modifications in UIII cells are initially carried to their maximal extent.

We also did not observe any induction above basal levels of {alpha}2-MG mRNA expression when UIII cells were supplemented with exogenous PRL, suggesting that PRL is sufficiently expressed in these cells to induce Jak2/Stat5 phosphorylation and that the expression of {alpha}2-MG may be controlled in an autocrine manner. Because up-regulation of {alpha}2-MG mRNA expression by PRL could not be established in UIII cells, and because PRL-mediated up-regulation of {alpha}2MG in the ovary is through the Jak/Stat pathway, we analyzed different ways to down-regulate its expression by interfering with the Jak/Stat signaling. For this purpose, we used an anti-PRL receptor antibody and chose one member of the tyrphostin family of tyrosine kinase inhibitors, AG490 (34). AG490 is a well known inhibitor of Jak2 activation, unique in that it does not inhibit other tyrosine kinases such as Jak1, Src, Tyk2, or lyn (34). This tyrphostin has also been involved in Jak3-dependent signals implicating Jak3, Stat1, 3 and 5 (a and b) inhibition (35). The ability of AG490 to selectively block Jak2 and 3 may be explained by the high level homology shared between these two closest related members of the Jak family. Indeed, AG490 displays similar micromolar inhibitory concentration requirements to block Jak2 and 3 activity. However, AG490 is not a general kinase inhibitor (34) and is considered to be a Jak2 specific inhibitor in cells that lack detectable levels of Jak3. In UIII cells, as in decidual cells (data not shown), Jak3 is not expressed. In a series of experiments, we demonstrated that Jak2 and Stat5 phosphorylation as well as the expression of the PRL target gene, {alpha}2-MG, were down-regulated by AG490. The inhibition of PRL-induced {alpha}2-MG expression by AG490, in the micromolar range, is both concentration and time dependent. Maximal effect (down-regulation greater than 70%) was achieved in less than 3 h. This effect could not be explained by general toxicity as no loss in cell viability was recorded within the range of the concentrations used. Thus, it is likely that the inhibition of Jak2 activity is responsible for the following decline in the phosphorylation state of Stat5. This, in turn, brings about the loss of its DNA binding activity and attenuates {alpha}2-MG mRNA expression. Similar inhibition of {alpha}2-MG expression was seen with the PRL-R antibody.

We have recently shown that the rat decidua expresses ER{beta} (14), which appears to transduce estradiol signaling to IL-6 (36). Estradiol inhibits the production of IL-6 in decidual cells and prevents the expression of this cytokine, which has detrimental effects on pregnancy (37). We have also shown that PRL up-regulates the expression of ER{beta} in decidual cells (14). Our present finding that ER{beta} expression in UIII cells can be severely inhibited by the Jak2 inhibitor led to the conclusion that this receptor may be up-regulated as is {alpha}2-MG through the Jak/Stat system in an autocrine manner.

Whereas AG490 caused a down-regulation of both {alpha}2-MG and ER{beta} in UIII cells, it enhanced the expression of decidual PRL mRNA and had no effect on the mRNA levels of Jak2 and Stat5 in UIII cells. Previous reports (4, 38) have shown that PRL down-regulates its own expression in the decidua. Results of the present investigation further demonstrate that rdPRL may have a direct effect on the regulation of its own secretion and that this negative feed back involves the Jak/Stat pathway.

Pituitary PRL is known to affect several physiological processes such as regulation of mammary gland development, stimulation of lactation, immuno-modulation, osmo-regulation (for review see Ref. 39). However, Nagy and Berczi (40) reported that immunoneutralization of PRL in hypophysectomized rats resulted in increasing mortality compared with hypophysectomized control rats. This result strongly suggests that extrapituitary PRL compensates at least in part for a deficiency in pituitary PRL and underlines the crucial role of local PRL production (41). Several sites of PRL production have been discovered in rodents specifically in reproductive organs (4, 42), immune system (43), brain (44), and exocrine glands (45). However, the putative autocrine/paracrine function of PRL is not known. Thus, the decidua, which is a specialized endometrial stromal tissue, produces PRL that can be transported to the amniotic fluid where this hormone could access to the fetal circulation and regulate fetal functions as suggested in humans (46). Effect on implantation, immunological rejection of the blastocyst, and inhibition of uterine contractility before labor has also been proposed for human decidua PRL (47). In breast tissues, the role of locally produced PRL seems also very important. Indeed, in rodents, PRL was shown to act as a local growth factor that stimulates the proliferation of mammary tumors (48). Moreover, PRL produced by immune cells was implicated in the development and maturation of these cells (for review see Ref. 49). Altogether, these results suggest that extrapituitary PRLs play essential role as does pituitary PRL.

In summary, results of this investigation have revealed that decidual PRL may act in an autocrine fashion to regulate target genes involved in normal placentation. Our results further the hypothesis that locally produced PRL plays a crucial role in the maintenance of pregnancy.


   Acknowledgments
 
We thank Dr. Jean Djiane for generously providing us with the PRL receptor antibody. We are grateful to the NIDDK and the National Hormone and Pituitary Program (NIH) for the oPRL and to Rosemary Clepper for animal care. The editing and preparation of the manuscript by Vivian Rogala and Gil Gibori is highly appreciated.


   Footnotes
 
1 This work was supported by NIH Grants HD-12356 and HD-11119 (to G.G.) and Ernst Schering AG Research Foundation (to C.T.). Back

2 These authors contributed equally to this work. Back

Received August 3, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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