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Regular Article |
a Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912-3000
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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decidua, mechanisms of hormone action, pregnancy, progesterone receptor, signal transduction, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The STAT (signal transducer and activator of transcription) family of proteins represents another group of transcription factors that may also act as coregulatory proteins. STAT molecules are stored in the cytoplasm in an inactive state until they are phosphorylated by tyrosine kinases associated with growth factor and cytokine transmembrane receptors [10, 11]. Ligand activation of the epidermal growth factor receptor (EGF-R) leads to specific activation of several STAT family members, including STAT1, STAT3, and STAT5. Once these molecules are activated, they leave the sites of phosphorylation, form homodimers or heterodimers, and become rapidly translocated to the nucleus [10, 11]. Activated STATs can also form multiprotein complexes with unrelated proteins, including steroid hormone receptors, to modulate gene expression [12–16]. In fact, STAT3 and STAT5 have been shown to modulate transcription by direct interaction with the glucocorticoid receptor and the PR [13, 16]. The transcriptional complexes thus formed can trigger transcription through STAT and non-STAT DNA binding elements [13–17].
The essential nature of these relationships is illustrated by the mouse mammary gland model, wherein the phenotypic changes observed in PR-null mice are similar to those observed in mice lacking the STAT5 gene, and in mice lacking functional EGF-Rs (reviewed in [2]). These observations suggest that full expression of progestin action depends not only on its cognate receptor but also on functional EGF/EGF-R and STAT systems. It is interesting that these same elements are also present in the decidualized mesometrium of the pregnant rat [1]. In fact, a landmark event in the decidualization process is progesterone-induced expression of EGF-R [18–21]. However, little is known about STAT proteins or their role in decidual cell function.
In this paper we report a potential role for this class of proteins in decidual cell function. We recently screened decidual homogenates for STAT expression by Western blot analysis and found that only STAT3 and STAT6 were expressed at appreciable levels [1]. Because STAT3 is known to be an important substrate of EGF-R, whereas STAT6 is dependent on interleukin-4 and interleukin-13 receptors for activation, we chose to evaluate the interrelationship between STAT3 expression and progesterone action in rat decidualized mesometrium [11]. We show that STAT3 is a progesterone-dependent protein and that STAT3 interacts with the PR by direct protein-protein association.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult female Holtzman rats weighing 225–250 g were purchased from Harlan Sprague-Dawley, Inc. (Indianapolis, IN) and were bred in the laboratory animal care facility of our institution. All animal care and use was conducted in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and with a protocol approved by the Medical College of Georgia Committee on Animal Use in Research and Education. Pregnancy (Day 1) was identified by the presence of vaginal sperm after overnight exposure to a fertile male. Rats were killed at 0800–0900 h between Days 8 and 17 of pregnancy (term is on Day 22) or at other times as mentioned for specific experiments.
Antiprogestin Treatment
Normal pregnant rats received a single injection of 0.5 mg RU486 (mifepristone; 11ß-[4-dimethylaminophenyl]-17ß-hydroxy-17
-[prop-1-ynyl]-estra-9,9-dien-3-one; Sigma Chemical Company, St. Louis, MO) dissolved in 0.5 ml of vehicle solution (10% ethanol in propylene glycol). The i.p. injection was administered either 24, 12, 6, or 3 h before the rats were killed at 1400 h on Day 10. This regimen of RU486 treatment does not alter serum concentrations of progesterone [22]. Although RU486 is most potent as an antiprogestin, it can exhibit appreciable antiglucocorticoid activity [23]. Blockade of progesterone action most likely accounts for the in vivo effects of RU486 on the decidua basalis, because these effects were similar to those induced by ovariectomy and were reversed by treatment with progesterone [19, 24, 25]. Furthermore, glucocorticoids do not appear to influence the process of stromal cell decidualization [26]. The use of RU486 allows direct study of progestin action on the decidua in the presence of functional ovaries with normal production of estrogen and progesterone.
Tissue Preparation for Western Blot Analysis
The decidualized mesometrium (also termed the decidua basalis) was isolated as previously described [27]. Briefly, tissues were separated from the myometrium, chorioallantoic placental tissues, and antimesometrial decidua and snap-frozen in liquid nitrogen and stored at -80°C until homogenized. The decidua basalis begins to regress on Day 14 and becomes too thin to dissect reliably after Day 17. Tissues from a single litter were pooled to yield 8–12 mg protein/ml of the cytosol fraction and 18–25 mg/ml of particulate fraction. To achieve sufficient protein when the amount of decidua basalis tissue was very small, it was necessary to combine tissues from more than 1 litter. For example, as many as 30 decidua basalis tissues were required per pool during early pregnancy and for some treatment groups (e.g., RU486 for 24 h). Four to six tissue pools were analyzed per treatment group.
Each pool of decidual tissues was homogenized in 1 ml of TEGS buffer (20 mM Tris base pH 7.5, 7.5 mM EDTA, 1.0 mM EGTA, and 0.32 M sucrose) with 200 mM sodium vanadate, 0.05 mM leupeptin, 1 mM PMSF, 50 mM mercaptoethanol, 100 µg/ml aprotinin, and 1 mg/ml pepstatin A added immediately before use. The homogenate was divided into particulate and cytosolic fractions by centrifugation at 185 000 x g for 50 min at 4°C. The supernatant (cytosol fraction) was decanted to a 1.5-ml minivial, and an equal volume of Laemmli buffer [28] was added after an aliquot was taken for protein determination [29]. The pellet was resuspended in 0.4 ml of 2% SDS in Tris buffer (20 mM pH 7.5) and dissolved by sonication. An aliquot was taken for protein determination before an equal volume of Laemmli buffer was added. The homogenates were then boiled for 10 min and stored at -20°C until analysis. Aliquots containing 100 µg of protein were withdrawn from the minivials mixed with a volume of Laemmli buffer to provide a volume of 80 µl, and boiled as before. Each sample preparation was applied to a 10% polyacrylamide gel under denaturing conditions and subsequently electroblotted to polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia Biotech UK Limited, Buckinghamshire, U.K.) according to procedures described elsewhere [19, 30]. Blots were incubated for 18 h at 4°C in the presence of a monoclonal antibody to rat STAT3 diluted 1:2500 (BD Transduction Laboratories, Lexington, KY). After washing and incubating with secondary antibody, the blots were exposed to chemiluminescent detection substrates (ECLPlus, Amersham Pharmacia) and analyzed as previously described [25, 27, 30]. Specificity of the reaction was checked by incubating PVDF membranes in the same solution but without addition of primary antibody. The STAT3 band detected in decidual homogenates corresponded in molecular size (i.e., gel mobility) to the band detected in the positive control sample provided by the manufacturer (88–90 kDa). Progesterone receptor antibody (Mab-h928) was kindly provided by Dean Edwards (Denver Colorado Health Sciences University, Denver, CO).
Antiprogesterone Receptor and Anti-STAT3 Immunoprecipitations
Decidua basalis tissue pooled from Day 10 pregnant rats was homogenized in lysate buffer (20 mM Tris base pH 7.5, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) with 1 mM phenylmethylsulfonyl fluoride and aprotinin (20 µg/ml) added just before use. The cell lysates were precleared by adding 0.25 µg of normal mouse immunoglobulin (Ig) G together with 20 µl of resuspended Protein A-Agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After a 30-min incubation at 4°C, the beads were pelleted by centrifugation in a microcentrifuge at the high-speed setting (approximately 1000 x g) for 6 min at 4°C. The supernatant was then divided into equal aliquots (about 2 mg/ml each). One aliquot was immunoprecipitated with 10 µg/ml of an antibody to PR (Mab-h928) and the other with 10 µg/ml of STAT3 antibody (as above) by constant rotation at 4°C for 1 h. Immunocomplexes were captured after overnight exposure to 20 µl/ml of protein-A-agarose beads by centrifugation in a microcentrifuge at the high-speed setting for 6 min at 4°C. The supernatant was decanted, and the pellet was resuspended and washed four times in 1 ml of Tris-buffered saline. The washed pellets were then resuspended in 80 µl of Laemmli buffer, boiled for 5 min, and stored at -20°C until applied to a gel. Control groups were immunoprecipitated with PR antibody or STAT3 antibody and treated exactly as the experimental aliquots except that blotted proteins were exposed only to the secondary antibody.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1 shows changes in STAT3 expression over the course of pregnancy in particulate and cytosol cellular fractions of decidual homogenates. STAT3 expression in the cytosolic fraction was maintained at a relatively abundant level through the first 17 days of pregnancy. On the other hand, STAT3 accumulation in the particulate fraction (presumably most reflective of activation and translocation to nuclei) was highest on Days 8 and 10, and fell to much lower levels on Days 12–17 (P < 0.02).
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Regulation of STAT3 Protein Expression by the Antiprogestin RU486
The next experiment was designed to determine whether STAT3 was dependent on progesterone action for expression. Pregnant rats were treated with antiprogestin at several intervals during the 24-h period between Days 9 and 10 of pregnancy and all were killed at 1400 h on Day 10. Figure 2 shows that the level of STAT3 expression in the cytosolic fraction was severely curtailed after 12 and 24 h of exposure to the antiprogestin (P < 0.001), whereas expression of the STAT3 protein in the particulate fraction was at least as abundant at these time periods as in the untreated control group (Day 10).
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Protein-Protein Interaction Between PR and STAT3
Because the glucocorticoid and progesterone receptors have both been shown to associate with STAT5, STAT3, and STAT1 in breast epithelial cells [12–15], we speculated that PR and STAT3 may also form protein complexes in rat decidua. To test this notion, decidual lysates were divided into two aliquots; one was treated with anti-PR and the other with anti-STAT3 to immunoprecipitate the cognate antigen. The immunoprecipitates were then blotted and probed with antibodies specific for STAT3 and PR, respectively (Fig. 3). Lane 2 in Figure 3A shows that STAT3 was present in the fraction that immunoprecipitated with the PR antibody. The immunoprecipitate from a different decidual lysate gave identical results (not shown). Lane 1 in Figure 3B shows that two PR bands, corresponding to the molecular weights of PR-B (
110 kDa) and PR-A (90 kDa), were present in the anti-STAT3 immunoprecipitate, whereas in another experiment using a different decidual lysate (lane 2), only PR-A was detected. In a third experiment, only PR-B was found (results not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The regulation of STAT3 during pregnancy as revealed in the present study appeared to deviate somewhat from this model. We expected cytosolic expression to decline markedly on Days 14 and 17 (representing the late pregnancy progesterone "resistant" state) compared with Days 8 and 10. Instead, we found STAT3 to be abundantly expressed at all stages of pregnancy (Fig. 1). Two explanations may account for these findings. First, we know that progesterone is still essential for maintenance of pregnancy during this period; thus, even though decidual cells may be relatively insensitive to progesterone, some actions of progesterone are maintained [22, 25]. Second, other factors in addition to progesterone may regulate STAT3 expression.
These two hypotheses are not mutually exclusive, although it seems clear that at least in early pregnancy, basal expression is very dependent on progesterone (Fig. 2). In contrast, abundance of STAT3 in the particulate fraction did decline markedly in late pregnancy from the levels found on Days 8 and 10, which corresponds to the pattern of EGF-R abundance noted in a previous study [19]. Treatment of pregnant rats with antiprogestin resulted in an 80% decline in cytosolic abundance of STAT3 within 12 h (Fig. 2), but did not influence the amount of the protein expressed in the particulate fraction. Although the relatively high level of expression in the particulate fraction in late pregnancy may seem inconsistent, it is in keeping with findings from a previous study in which we found that more than 12 h of RU486 exposure was required to alter the abundance of activated EGF-R [19]. Thus, accumulation in the particulate fraction could be expected to continue unaltered late into the treatment period. In addition, RU486-PR signaling itself may have enhanced STAT3 accumulation in the particulate fraction because it is known that the antiprogestin has the ability to promote efficient binding of PR to progesterone-responsive elements [33]. Perhaps RU486-PR complexes are able to associate directly with STAT3 molecules and increase nuclear accumulation of STATs as progestin-PR complexes are able to do [13]. The findings from the present study are consistent with those reported for a breast cancer cell line by Horwitz and coworkers [12, 13]. In their studies, progestin was shown to increase STAT message and protein expression, whereas enhanced EGF/EGF-R signaling influenced nuclear accumulation primarily.
It would be of interest to know whether the three isoforms of PR (A, B, and C) in decidua interacted differentially with STAT3, because they would be expected to differ in promoter specificity [34]. However, our experiments could not evaluate this subtlety. In three experiments using anti-STAT3 immunoprecipitates of decidual basalis lysates, PR-A and PR-B were detected together in one preparation, and either A or B in two other preparations. Our immunoprecipitate experiments were not able to reveal PR-C (60 kDa) because proteins from the protein-A agarose preparation reacted with the secondary antibody over a range of 40–70 kDa.
STAT3 is known to be involved in cell proliferation and prevention of apoptosis, and in fact may be essential for some types of oncogenesis [35–37]. A similar role for STAT3 in the decidua may be inferred from its pattern of expression wherein localization to the particulate cell fraction was most abundant during the active period of stromal cell proliferation and decidualization (Days 8–10), falling thereafter by 75%. This pattern fits closely with that of EGF-R expression and the onset of apoptosis in late pregnancy [19, 27], suggesting that suppression of cell death signals may be relieved in part by waning levels of "activated" STAT3.
Our results indicate that progesterone action potentiates growth factor signaling in decidual tissue by at least three mechanisms: 1) up-regulation of the EGF-R protein [19]; 2) enhancing cytoplasmic stores of STAT3, thereby providing phosphorylation substrates for activated EGF-R; and 3) formation of PR and STAT3 complexes. Such multilevel cross-talk indicates the intricate nature of decidual cell communication. The complexities of this network go far beyond altering sensitivity to various hormonal influences and extend to direct interaction between the transcription factors. Indeed, PR and STAT3 may act as coactivators for each other in modulating gene expression and, in this way, regulate differentiated function of decidualized stromal cells during pregnancy.
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FOOTNOTES |
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1 Supported by National Institutes of Health grant HD 38724. 
2 Correspondence: Thomas F. Ogle, Department of Physiology, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912. FAX: 706 721 7299; togle{at}hargray.com
Accepted: January 29, 2002.
Received: September 19, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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