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Endocrinology Vol. 140, No. 10 4442-4450
Copyright © 1999 by The Endocrine Society

ARTICLES

The Expression of Interleukin-6 (IL-6), IL-6 Receptor, and gp130-Kilodalton Glycoprotein in the Rat Decidua and a Decidual Cell Line: Regulation by 17ß-Estradiol and Prolactin1

S. Deb, C. Tessier, A. Prigent-Tessier, U. Barkai, S. Ferguson-Gottschall, R. K. Srivastava, J. Faliszek and G. Gibori2

Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, Illinois 60612

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
 
The cytokine interleukin 6 (IL-6), a major mediator of immune and acute phase responses of the liver, has been implicated in the termination of pregnancy once expressed in the uterus. This study was undertaken to investigate the expression and regulation of genes encoding IL-6 and IL-6 receptor (IL-6R) in rat decidual tissue. Total RNA obtained from rat decidual tissue on different days of pseudopregnancy was analyzed by RT-PCR using specific primers for IL-6, IL-6R, and 130-kDa glycoprotein (gp130). Ribosomal L19 primers served as an internal control. IL-6R and gp130 were found to be expressed in the decidua throughout development, while no messenger RNA (mRNA) for IL-6 was detected. Interestingly, within several hours of culture, decidual explants acquired the ability to express IL-6. The apparent ability of decidual cells to express IL-6 and its lack of expression in vivo led us to examine whether the IL-6 gene is actively inhibited. Primary decidual cells were cultured in the presence of estradiol, progesterone, or PRL. Progesterone showed no effect, whereas estradiol and PRL reduced the level of IL-6 mRNA expression. To examine the mechanism by which these hormones inhibit IL-6 expression, we used a simian virus 40-transformed decidual cell line (GG-AD), which expresses only estrogen receptor-ß (ERß). Like primary decidual cells in culture, GG-AD cells express IL-6, IL-6R, and gp130 mRNA. When cultured in the presence of estradiol (0–100 ng/ml), mRNA for IL-6 and its receptor components were down-regulated in a dose-dependent manner. Estradiol also caused a dose-dependent decrease in IL-6 protein secretion into the culture medium. The inhibitory effect of estradiol on IL-6 mRNA expression was reversed by the antiestrogen ICI-164,384. Similar inhibition of IL-6 and gp130 mRNA expression was observed with PRL treatment. However, PRL had no effect on IL-6R mRNA levels. PRL inhibition of IL-6 expression was totally reversed by tyrphostin AG490, a JAK2 inhibitor. In summary, the results of this investigation indicate that IL-6 expression, which is detrimental to the maintenance of pregnancy, is inhibited in the rat decidual tissue. This inhibition is induced by PRL and estradiol, which down-regulate not only IL-6 expression, but also the expression of IL-6 receptor and signaling proteins. The results also suggest that PRL signaling to the IL-6 gene is mediated through the long form of PRL receptor and involves JAK2 activation, whereas that of estradiol can be transduced by estrogen receptor-ß.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MULTIFUNCTIONAL cytokine, interleukin-6 (IL-6), is produced by a variety of cells, including fibroblast, epithelial, and endothelial cells (1), in response to infection, trauma, and injury (2, 3, 4). Besides the acute phase response, IL-6 is also known to play important roles in normal physiology. It participates in the growth of endometrial tissue, formation of bone, and synthesis of hormones from various glands, including the pituitary (5, 6). Although IL-6 can directly affect steroid synthesis in the adrenal glands (7), testis (8), and ovary (9, 10, 11), its expression in various tissues is regulated by steroid hormones. Progesterone and glucocorticoid were found to be very potent inhibitors of IL-6 synthesis in granulosa and luteal cells in culture (12). Estrogen is the primary regulator of the IL-6 gene in bone (13, 14). In postmenopausal women, lower estrogen levels cause bone loss, which appears to be mediated by an elevated IL-6 concentration (15, 16, 17). However, estrogen was also shown to have no effect on the expression of IL-6 in normal human osteoblasts (18). Besides steroid hormones, PRL also regulates IL-6 expression (19).

IL-6 actions are mediated by a specific cell surface receptor [IL-6 receptor (IL-6R)], which is an 80-kDa glycoprotein (gp80), considered to be the {alpha}-subunit of the IL-6R system (20). Binding of IL-6 to IL-6R causes homodimerization of gp130, which is also known as the ß-subunit of the IL-6R. gp130 is a common signaling molecule for various cytokines, and its homodimerization induced by the IL-6-IL-6R complex is crucial for IL-6 action. Dimerization of gp130 is followed by the activation of tyrosine kinases leading to the phosphorylation of various transcriptional factors, including Stats (signal transducer and activator of transcription).

Examination of mouse uterine tissue revealed little or no IL-6 messenger RNA (mRNA). However, treatment of primary decidual cells with LPS caused synthesis of IL-6 (21, 22). Very little is known about the expression of IL-6 in rat and human decidua. Evidence suggests that IL-6 is produced in human decidua in response to inflammation (23). IL-1{alpha} and tumor necrosis factor (TNF) have been shown to induce IL-6 production in cultured human decidual cells, and this induction can be prevented by actinomycin treatment (23). High levels of IL-6 are detected in the amniotic fluid of women delivering prematurely due to uterine infection (24). Several studies have shown that IL-6 production by decidual tissue in response to inflammation and in conjunction with other inflammatory mediators may play a role in the pathophysiology of preterm labor due to infection (25, 26, 27). Moreover, IL-6 mRNA is not only expressed in gestational tissues after preterm labor, but is also present in normal labor with or without associated intrauterine infection (28, 29). How IL-6 participates in the termination of pregnancy is not yet clear. Evidence obtained with primary cell cultures (30) indicates that IL-6 may induce the synthesis of PG, which is a known factor in normal as well as infection-induced parturition. Because excessive production of IL-6 may compromise pregnancy by triggering an inflammatory response, its expression in the decidua and uterine tissue ought to be tightly regulated. Indeed, the results of this investigation indicate that IL-6 expression is silenced in the decidua and that estradiol and PRL act to prevent the expression of both IL-6 and its receptor signaling proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Acrylamide and bis-acrylamide were obtained from Accurate Chemical, Inc. (Westbury, NY), and Eastman Kodak Co. (Rochester, NY) respectively; Taq DNA polymerase was purchased from Pan Vera Corp. (Madison, WI); [32P]deoxy-CTP was obtained from Amersham Pharmacia Biotech (Arlington Heights, IL); the oligonucleotides used as primers in the RT-PCR analysis were obtained from Life Technologies, Inc. (Grand Island, NY); RPMI 1640 medium, antibiotic-antimycotic solution, nonessential amino acids, and sodium pyruvate were purchased from Mediatech (Washington DC); FBS was obtained from HyClone Laboratories, Inc. (Logan, UT); trypsin-EDTA was obtained from Life Technologies, Inc.; 17ß-estradiol was purchased from Steraloids, Inc. (Wilton, NH); progesterone, phenylmethylsulfonylfluoride, leupeptin, pepstatin A, aprotinin, and all other reagent grade chemicals were purchased from Sigma Chemical Co. (St. Louis, MO); ovine PRL (oPRL; PRL-18; 30 IU/mg) was a gift from the NIDDK (NIH, Bethesda, MD); the IL-6 enzyme-linked immunoassay (ELISA) kit was obtained from R&D Systems (Minneapolis, MN); tyrphostin AG490 was obtained from Calbiochem (La Jolla, CA); ICI-164,384 was a gift from Zeneca Pharmaceuticals Pharmaceutical (Cheshire, UK).

Animal model and tissue preparation
Pseudopregnancy was induced by mating Holtzman Sprague Dawley female rats with vasectomized male rats at the Harlan facilities (Madison, WI). The day a vaginal plug was found was designated day 1 of pseudopregnancy. Pseudopregnant rats were kept under controlled conditions of light (lights on, 0500–1900 h) and temperature (22–24 C) with free access to standard rat chow and water. Animals were handled according to the procedures approved by the institutional animal care and use committee. Decidualization of the uterine endometrium was induced by scratching the antimesometrial surface of both the uterine horns with a hooked needle on day 5 of pseudopregnancy under ether anesthesia. Rats were killed on different days of pseudopregnancy by an overdose of ether. Uterine horns were dissected, washed in cold PBS to remove excess blood, and then either used for decidual primary cell culture or frozen in liquid nitrogen and stored at -80 C until processed for RNA or protein extraction.

Decidual tissue culture
Decidual tissue was collected on day 9 of pseudopregnancy. After dissection, total tissue, corresponding to both antimesometrial and mesometrial decidua, was minced into 2- to 3-mm pieces and rinsed extensively with PBS. Approximately 100 mg tissue were placed into a 35-mm culture dish containing RPMI 1640 serum-free medium supplemented with 2% antibiotic-antimycotic solution and incubated for up to 4 h under a 95% air-5% CO2 humidified atmosphere. After incubation, spent medium was collected for IL-6 protein analysis by ELISA, and the tissue was frozen in liquid nitrogen and stored at -80 C until RNA extraction.

Primary decidual cell culture
Primary decidual cell culture was performed according to the procedure previously reported from this laboratory (31). Briefly, decidual tissue obtained from day 9 pseudopregnant rats was washed in cold PBS. The tissue obtained from five rats was pooled and incubated in RPMI 1640 containing 50 U/ml collagenase, 2.4 U/ml dispase, and 200 U/ml deoxyribonuclease for 1 h at 37 C. At the end of the incubation, the supernatant was filtered through nylon mesh to remove undigested tissue and centrifuged at 3000 rpm in a tabletop centrifuge for 10 min. The cell pellet was resuspended in RPMI 1640 supplemented with 1 x L-glutamine, 2 x antibiotic, 1 x sodium pyruvate, 1 x nonessential amino acids, 0.45% D-glucose, and 10% FBS. Viable decidual cells, as determined by trypan blue dye exclusion staining, were seeded into six-well plates (~2 x 106 cells/well) and incubated at 37 C under a 95% air-5% CO2 humidified atmosphere. Cells were allowed to attach for 3–4 h and then treated for 12 h with various concentrations of estradiol (0.1–10 ng/ml), progesterone (0.3–3 µg/ml), and/or oPRL (0.01–1 µg/ml) in RPMI 1640 phenol red-free medium supplemented with 1% dextran-charcoal-treated FBS.

GG-AD cell culture and transfection with the long form of the PRL receptor
The decidual cell line termed GG-AD was developed from antimesometrial decidual cells after infection with simian virus 40 tsA209 mutant virus as reported previously (32). These cells proliferate at 33 C and differentiate at 39 C. We adopted the procedure of Felgner et al. (33) with some modifications to stably transfect GG-AD cells with the long form of PRL receptor (PRL-RL). The complementary DNA (cDNA) of the PRL-RL was subcloned into the pMT2 poly vector. The pSV2 neo vector was used to confer neomycin resistance to the cells. Plasmid DNA was purified after transformation by equilibrium centrifugation in a cesium chloride-ethidium bromide gradient. GG-AD cells were seeded in six-well plates and grown to 33% confluence in RPMI 1640 medium supplemented with 5% FBS. Cells were then washed with serum- and antibiotic-free medium. For each well, 10 µl lipofectin reagent were mixed with 10 µg plasmid DNA (isolated from pMT2 poly and pSV2 neo) and 180 µl RPMI medium (serum and antibiotics free) and incubated for 15 min at room temperature. This mixture of lipofectin and DNA was then added to the cells in 1.8 ml RPMI (serum and antibiotics-free). After 24-h incubation at 37 C, the medium was replaced with a growth medium containing 5% FBS and 2 x antibiotics, and the cells were cultured for an additional 48 h. The medium was then replaced with fresh growth medium supplemented with 100 µg/ml G418 sulfate. G418 sulfate addition was continued every other day until G418 sulfate-resistant colonies were identified. These colonies were picked and cultured in 5% FBS growth medium until cells were confluent. Cells were grown and passaged several times to identify the successful stable transfection with the PRL-RL. As shown in Fig. 1Go, PRL-RL mRNA expression in these cells was confirmed by RT-PCR using PRL-RL-specific primers, as previously described (34).



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  		Figure 1. Expression of PRL-RL in stably transfected GG-AD cells. GG-AD cells were stably transfected with the PRL-R long form as described in Materials and Methods. Total RNA was obtained from these cells and from corpora lutea (CL) of pregnant rats (used as control) and subjected to RT-PCR using specific primers as previously described (30 ).

 
Both the wild-type plus the stably PRL-RL transfected GG-AD cells were cultured overnight at 39 C to allow the cells to differentiate in RPMI 1640 medium without phenol red and supplemented with 1 x L-glutamine, 2 x antibiotic-antimycotic solution, 1 x nonessential amino acids, 1 x sodium pyruvate, 0.45% D-glucose, and 1% charcoal-dextran-treated FBS. The cells were then treated with estradiol (0.01–10 ng/ml), oPRL (0.01–1 µg/ml), or a combination of estradiol and PRL for 24 h at 39 C. The specificity of PRL action through JAK2 was examined using tyrphostin AG490, which has been shown to inhibit JAK2 phosphorylation (35). GG-AD cells transfected with PRL-RL were treated with PRL (0.1–1 µg/ml) plus dimethylsulfoxide (vehicle) or PRL (0.1–1 µg/ml) plus tyrphostin AG490 (30 µM) for 24 h at 39 C. RNA isolated from GG-AD cells was examined for the expression of IL-6, gp80, and gp130.

RNA isolation and RT-PCR analysis
Total RNA from frozen decidual tissue was isolated using Trizol reagent (Life Technologies, Inc., Gaithersberg, MD) according to the manufacturer’s instructions, whereas total RNA from primary cells or GG-AD cells was isolated by one-step guanidinium-thiocyanate-chloroform extraction (36).

Oligonucleotide primer pairs were based on the sequence of the rat IL-6 gene (37) (5'-GACTGATGTTGTTGACAGCCACTGC-3' and 5'-TAGCCACTCCTTCTGTGACTCTAACT-3'), the rat IL-6R gene (38) (5'-TCACAGAGCAGAGAATGGACT-3' and 5'-GTATGGCTGATACCACAAGGT-3'), and the rat gp130 gene (39) (5'-TCAACTTGTGGAACCATGTGG-3' and 5'-TCCAACTGACACAGCATGTTC-3'). In each reaction, an additional pair of oligonucleotides specific to the rat ribosomal protein L19 (40) was included for use as an internal control (L19 primers, 5'-CTGAAGGTCAAAGGGAATGTG-3' and 5'-CTCTAGTAGTTCTAGGACAGG-3'). The predicted sizes of the PCR-amplified products were 509 bp for IL-6, 480 bp for IL-6R, 375 bp for gp130, and 194 bp for L19. One to 3 µg total RNA were reverse transcribed using the Advantage RT-for-PCR Kit (CLONTECH Laboratories, Inc., Palo Alto, CA) according to the manufacturer’s instructions.

Reverse transcribed products were diluted to 100 µl by adding diethylpyrocarbonate water at the end of the RT reaction. The cDNA was either used immediately for PCR or was stored at -20 C. For PCR amplification, a mixture containing specific oligonucleotide primers for the gene studied (50 pmol each), [{alpha}-32P]deoxy-CTP (2 µCi; 3000 Ci/mmol), deoxy-NTP, and Taq polymerase (0.8 U) was added to 5 µl cDNA. The total volume was increased to 40 µl with 1 x PCR buffer, and the samples were overlaid with 50 µl mineral oil. Touch-down PCR was used for amplification of the gene products. Briefly, cDNA was specifically amplified for 5 cycles with high annealing temperature (annealing temperature of the primer, +4 C), and then amplified for 20–30 cycles using 94 C for denaturing, 62–65 C for annealing depending on the primer, and 71 C for extension in a Robocycler Gradient 40 (Stratagene, La Jolla, CA). The 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 RNA. Each PCR reaction included L19 ribosomal protein as an internal control. Reaction products were electrophoresed on a 8% polyacrylamide nondenaturing gel. Gels were exposed to Kodak x-ray film for 6–24 h. After autoradiography, data were analyzed using a Molecular Dynamics, Inc. PhosphorImager and ImageQuant version 3 software (Molecular Dynamics, Inc., Sunnyvale, CA).

IL-6 assay
IL-6 was measured in the conditioned medium of decidual explant or GG-AD cells treated with or without estradiol, using a mouse IL-6 ELISA kit according to the manufacturer’s instruction (R&D Systems). The sensitivity of the system was 3.1 pg/ml, and the intra- and interassay coefficients of variation were 4.5% and 7.1%, respectively.

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


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Developmental expression of IL-6, IL-6R, and gp130 in rat decidual tissue during pseudopregnancy
To determine whether rat decidual tissue could either produce IL-6 or be a target for IL-6 action, we examined the expression of IL-6, IL-6R, and gp130 mRNA in decidual tissue on days 9–14 of pseudopregnancy. As shown in Fig. 2Go, IL-6R and gp130 were expressed constitutively from days 9–14 without any significant change. Whereas both components of IL-6R are expressed in the rat decidua, mRNA for IL-6 was not detected at any stage examined (Fig. 3AGo). However, when decidual tissue was maintained in culture (Fig. 3BGo) a spontaneous expression of IL-6 mRNA was observed within 2 h of culture.



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  		Figure 2. Developmental expression of IL-6R and gp130 in the rat decidual tissue during pseudopregnancy. Decidua were collected from rats at different stages of pseudopregnancy, and total RNA was isolated and analyzed by RT-PCR using specific primers for IL-6R (A) or gp130 (B) as described in Materials and Methods. Data were quantified by densitometry and corrected using L19 as an internal standard. The mRNA levels for each day are graphically represented in the lower panel as the mean ± SEM (n = 3).

 


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  		Figure 3. IL-6 expression in rat decidua. A, Decidua were collected from pseudopregnant rats on different days of pseudopregnancy. Total RNA isolated from decidual tissue or spleen (as positive control) was subjected to RT-PCR analysis using specific primers for IL-6. B, Time course of IL-6 mRNA expression by decidual explants maintained in culture in serum-free conditions. Decidual tissue obtained from D9 pseudopregnant rats was cultured in vitro. IL-6 mRNA was analyzed by RT-PCR immediately after isolation of the decidua (time zero) or after 1, 2, 3, or 4 h of incubation. Data are representative of three different experiments.

 
Effects of estrogen, progesterone, and PRL on the spontaneous expression of IL-6 mRNA in decidual tissue in culture
The lack of IL-6 mRNA expression in vivo and its expression within several hours in culture suggested that the expression of this cytokine is under inhibitory regulation in the rat decidua. We investigated the effect of estradiol and progesterone, which are known to regulate IL-6 expression in other tissue (12, 13, 14), and PRL, which plays a role in immune defense in the rat, on IL-6 mRNA levels. Receptors for these three hormones are expressed in the rat decidua (34, 42, 43). As shown in Fig. 4AGo, estradiol as well as PRL caused a down-regulation of IL-6 expression in primary decidual cells maintained for 12 h in culture. No synergistic effect was observed when cells were cultured in the presence of both estradiol and PRL. In contrast to the effects of estradiol and PRL, progesterone showed no inhibitory effect on IL-6 expression in decidual cells (Fig. 4BGo).



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  		Figure 4. Effects of estradiol, progesterone, and PRL on IL-6 mRNA expression in primary decidual cells. Decidual cells were isolated from day 9 pseudopregnant rats and cultured in RPMI 1640 phenol-free medium supplemented with 1% charcoal-dextran-treated FBS for 12 h in the presence of estradiol, progesterone, or PRL. The effects of these hormones on IL-6 mRNA were determined by RT-PCR analysis as described in Materials and Methods. A, The effect of estradiol (E; 1 ng/ml), PRL (1 µg/ml), or their combination. Values of decidual IL-6 mRNA obtained for the three groups treated (E, PRL, and E PRL) were significantly different (P < 0.05) from those in vehicle-treated controls. B, The effects of progesterone (P; 1 µg/ml), PRL (1 µg/ml), and their combination. Data are representative of three different experiments (mean ± SEM). Values obtained with PRL and P plus PRL treatments were statistically different (P < 0.05) from control values.

 
Effect of estrogen on IL-6 and IL-6R expression in GG-AD cells
To examine whether estradiol-mediated inhibition of IL-6 and IL-6R is transduced by estrogen receptor-ß (ERß), we investigated the effect of estradiol using GG-AD cells. These cells, generated from rat decidual cells, express only ERß and not ER{alpha} (42). As shown in Fig. 4Go, GG-AD cells express IL-6, and consistent with our primary cell culture results, estradiol inhibited the levels of both IL-6 mRNA (Fig. 5AGo) and IL-6 protein secreted into the culture medium (Fig. 6Go) in a dose-dependent manner. The inhibitory action of estradiol on IL-6 was completely reversed by the highest concentration of antiestrogen, ICI-164,384, used (Fig. 5BGo). As with primary cell culture, progesterone had no effect on IL-6 mRNA expression in GG-AD cells (Fig. 5CGo). Interestingly, estradiol also down-regulated the mRNA levels of both IL-6 receptor (Fig. 7AGo) and gp130 (Fig. 7BGo).



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  		Figure 5. Effect of estradiol and progesterone on IL-6 mRNA in GG-AD cells. GG-AD cells were grown at 33 C, and when 70% confluence was reached they were transferred to 39 C and left overnight to differentiate. Differentiated cells were treated for 24 h at 39 C. A, The effects of different doses of estradiol. IL-6 mRNA values for three groups treated with estradiol (0.01–100 ng/ml) were significantly different (P < 0.05) from those for vehicle-treated controls. B, The effect of the antiestrogen ICI-164,384. Values obtained with estradiol alone and estradiol plus ICI in a 30-fold excess were statistically different (P < 0.05) compared with control values. C, The effect of the progesterone dose response. Total RNA was isolated and reversed transcribed into single stranded complementary DNA. Included in the reaction was a pair of oligonucleotide primers for L19 ribosomal mRNA, which was used as an internal standard. Band intensity was measured with a phosphorimager, and the IL-6 signal was normalized to the L19 for each time point (n = 3), as shown in the lower panel of each representative autoradiogram.

 


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  		Figure 6. Effects of estradiol on IL-6 secretion by GG-AD cells. GG-AD cells were treated with estradiol (E; 0.01–10 ng/ml) as described in Materials and Methods. IL-6 accumulated in the conditioned medium after 24 h of culture was measured by ELISA. Results are the mean ± SEM (n = 3) and are significantly different (P < 0.05) from vehicle-treated control values.

 


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  		Figure 7. Regulation of IL-6R by estradiol in GG-AD cells. GG-AD cells were treated with various concentrations of estradiol (E; 0.01–100 ng/ml). Total RNA was isolated from these cells and analyzed by RT-PCR (n = 4) using specific primer for gp80 (A) and gp130 (B). The band intensities were quantified by phosphorimager, as shown under each representative autoradiogram. Values obtained with doses of 0.1–100 ng/ml estradiol were statistically different (P < 0.05) compared with control values.

 
Effect of PRL on IL-6 and IL-6R components in GG-AD cells
Decidual tissue is constantly exposed to PRL and PRL-like proteins that are produced within the tissue or secreted by placental trophoblast cells. As demonstrated previously, decidual tissue expresses both the short and long forms of the PRL receptor and responds to PRL treatment (34). To determine whether the PRL-mediated inhibition of IL-6 involves the PRL-RL and JAK2, we transfected GG-AD cells with PRL-RL and treated them with either PRL alone or PRL plus tyrphostin AG490. As shown in Fig. 8AGo, PRL inhibited IL-6 mRNA expression (A and B). This PRL-mediated down-regulation of IL-6 was reversed by the JAK2 inhibitor (Fig. 8BGo). The inhibitor alone had no effect on IL-6 expression. PRL also caused a remarkable down-regulation of gp130 mRNA (Fig. 9BGo), but had no effect on the level of IL-6R mRNA (Fig. 9AGo).



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  		Figure 8. PRL-mediated regulation of IL-6 expression in GG-AD. A, GG-AD cells stably transfected with the PRL-RL were cultured at 39 C for 24 h in the absence (C) or presence of different doses of oPRL. IL-6 mRNA values obtained with PRL (0.01–1 µg/ml) were significantly different (P < 0.05) from vehicle-treated control values. B, GG-AD cells were treated for 24 h without (C) or with oPRL (0.1 µg/ml), AG490 (30 µM), or oPRL (0.1 µg/ml) plus AG490 (30 µM). Values obtained with PRL alone and with PRL plus AG490 were statistically different (P < 0.05) compared with control values.

 


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  		Figure 9. Regulation of IL-6R components by PRL in GG-AD cells. GG-AD cells expressing PRL-RL were cultured at 39 C in the presence of PRL (0.01–1 µg/ml). Total RNA isolated from these cells was subjected to RT-PCR using specific primer for IL-6R (n = 3; A) and gp 130 (n = 3; B), as described in Materials and Methods. gp130 mRNA values obtained for the three groups treated (PRL, 0.1 and 1 µg/ml) were statistically different (P < 0.05) compared with control values.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ability of the decidual tissue to spontaneously express IL-6 in vitro together with the lack of IL-6 expression in the decidual tissue in vivo suggest that when decidual tissue is cultured in vitro, potential inhibitors of IL-6 expression are removed. Although multiple agents activate IL-6 gene transcription (44), steroid hormones that bind estrogen, progesterone, and glucocorticoid receptors have been described as potent repressors of IL-6 gene expression (45, 46, 47, 48). Our results reveal that estradiol, and not progesterone, causes a strong inhibition of the spontaneous expression of decidual IL-6 mRNA. The same effect of estradiol on IL-6 secretion was observed in freshly explanted human endometrial stromal cells (49). Moreover, several in vitro and in vivo experiments, including those involving IL-6 knockout mice, clearly established that estrogen is a potent inhibitor of IL-6 in bone cells (13, 15, 17). A drop in estrogen levels in postmenopausal women causes increased synthesis of IL-6 in bone, resulting in osteoporosis (50, 51). Progesterone, which was shown to inhibit IL-6 in the corpus luteum (12), had no effect in decidual cells, suggesting that the effect of steroid hormones on IL-6 is tissue specific. The results of our experiments also reveal that estradiol inhibits not only IL-6 mRNA expression but also that of the proteins necessary for IL-6 signaling.

Inhibition of IL-6 by estradiol and its reversal by ICI-164,384 in GG-AD cells that express only ERß (42) suggest that ERß can mediate estradiol signaling to the IL-6 gene. Although ERß shows strong structural homology with ER{alpha} (52) and interacts with almost all estrogenic compounds (53), very little is known about its mechanism of action. It has been shown that steroid receptors do not bind to the 5'-flanking region of the IL-6 gene (46, 47), but instead interact with the transcription factor nuclear factor-{kappa}B (NF-{kappa}B), a potent enhancer of IL-6 transcription (45, 46, 47, 48), and antagonize its function (48). Whether ERß interacts with the IL-6 promoter and/or with NF-{kappa}B remains to be examined in decidual cells.

The rat decidua expresses PRL receptors and secretes PRL-like proteins (34), which have been shown to regulate various functions of the decidual tissue (54, 55). Although PRL is well known for its immunostimulatory action (56), here we present evidence that PRL inhibits IL-6 and gp 130 gene expression in the decidua. A similar inhibitory action of PRL was observed in Kupffer cells, where PRL administration down-regulated the expression of mRNA for IL-6 and other cytokines such as IL-1ß, and TNF (19). Our findings that PRL inhibits IL-6 mRNA expression in GG-AD cells, which express only the long form of the PRL receptor, and that this effect of PRL is blocked by AG490 suggest that PRL signaling to the IL-6 gene is through the long form of the PRL receptor and is transduced through the JAK-Stat pathway. Whereas PRL inhibits IL-6 and gp130, it stimulates ERß expression (12, 43). It is, therefore, possible that the inhibitory effect of PRL on IL-6 gene expression is amplified by its up-regulation of ERß.

The expression of IL-6R and that of its signaling molecule gp130 in the decidua suggest that decidual cells are capable of responding to IL-6. This may explain why IL-6 expression during pregnancy is detrimental. In the human, cytokines such as TNF, IL-1, and IL-6 are detected in the amniotic fluid during pregnancy (25, 57) and labor (57, 58). Infection caused a much greater rise in these cytokines (57). Recently, it has been shown that the concentration of IL-6 in serum increases significantly after uterine contraction (59) and that IL-6 expression is associated with the synthesis of PGE2 and PGF2{alpha} from uterine and extrauterine tissues (8, 12, 13). It is possible that this phenomenon is analogous to the increase in IL-6 observed in the serum of renal transplant recipients just before acute rejection of the graft (60). Whereas IL-6 may play a physiological role during parturition, its expression is powerfully inhibited in the decidua. The results of this investigation indicate that this inhibition is due in large part to estradiol and PRL. These hormones also reduce the level of expression of proteins necessary for IL-6 signaling, thereby allowing pregnancy to continue to term. These results also indicate that estradiol and PRL signaling to the IL-6 gene occurs through ERß and the long form of the PRL receptor, respectively.


   Acknowledgments
 
We are grateful to the NIDDK and the National Hormone and Pituitary Program (NIH) for the oPRL. We thank Linda Alaniz-Avila for photography, and Janice M. Gentry and Vivian Rogala for preparation of the manuscript. The editing of the manuscript by Jonna Frasor is highly appreciated.


   Footnotes
 
1 This work was supported by NIH Grant HD-12356. Back

2 NIH Merit Awardee (HD-11119). Back

Received March 15, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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