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Molecular Endocrinology, doi:10.1210/me.2006-0296
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Molecular Endocrinology 20 (12): 3240-3250
Copyright © 2006 by The Endocrine Society

The Role of Interleukin-11 in Pregnancy Involves Up-Regulation of {alpha}2-Macroglobulin Gene through Janus Kinase 2-Signal Transducer and Activator of Transcription 3 Pathway in the Decidua

Lei Bao, Sangeeta Devi, Jennifer Bowen-Shauver, Susan Ferguson-Gottschall, Lorraine Robb and Geula Gibori

Department of Physiology and Biophysics (L.B., S.D., J.B.-S., S.F.-G., G.G), University of Illinois College of Medicine, Chicago, Illinois 60612; and The Walter and Eliza Hall Institute of Medical Research (L.R.), Parkville, 3050 Victoria, Australia

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
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IL-11 expressed by endometrial stromal cells is crucial for normal pregnancy. IL-11 receptor {alpha} (IL-11R{alpha}) null mice are infertile due to abnormal development of the placenta. In these mice, the mesometrial decidual tissue, which is the site of trophoblast invasion, thins and disappears at mid-pregnancy. Degeneration of the decidua is accompanied by uncontrolled trophoblast invasion. In this report, we show, using IL-11R{alpha} null mice, that a defect in IL-11 signaling in the decidua leads to severe down-regulation of {alpha}2-macroglobulin ({alpha}2-MG), a metalloproteinase inhibitor crucial for limiting trophoblast invasion. We also present evidence, using uterine stromal cells that decidualize in culture, that IL-11 robustly stimulates the endogenous {alpha}2-MG expression and enhances {alpha}2-MG promoter activity. Serial 5' deletion and internal deletion of the promoter reveal two important signal transducer and activator of transcription (Stat) binding sites. Mutation of either one of these motifs decreases IL-11 stimulation, whereas double mutation prevents IL-11 action. We also found that IL-11 activates Janus kinase 2 (Jak2) and induces rapid phosphorylation, nuclear translocation, and promoter binding activity of Stat3 in decidual cells, whereas Jak1, Tyk2, and Stat5 activities are not affected. In addition, Jak2 inhibitor totally prevents {alpha}2-MG expression in decidual cells. Taken together, results of this investigation provide, at least in part, an explanation for the overinvasiveness of the trophoblast in IL-11R{alpha} null mice and reveal, for the first time, that IL-11 signals through the Jak2/Stat3 pathway in decidual cells to stimulate the expression of {alpha}2-MG, a protease inhibitor essential for normal placentation in pregnancy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE SUCCESSFUL DEVELOPMENT of the embryo during pregnancy, in both humans and rodents, requires a severe readjustment of the uterus that involves proliferation and differentiation of endometrial stromal cells into decidua. In rodents, decidualization initially occurs in the antimesometrial endometrium and results in the formation of the antimesometrial decidua or decidua capsularis. Subsequent decidualization of the mesometrial stromal cells gives rise to the mesometrial decidua or decidua basalis. The decidua capsularis is composed of large-sized polyploid and closely packed cells, and it has all the characteristics of an endocrine organ (1). On the other hand, the decidua basalis, formed by small loosely packed fibroblast-like cells, and with no reported endocrine function, is the site of trophoblast invasion (1).

The decidua is known to express several cytokines (2), and gene deletion studies have revealed key roles for two of these cytokines, leukemia inhibitory factor (LIF) and IL-11, in implantation and decidualization. Whereas LIF is expressed in uterine epithelial cells around the day of implantation (3, 4), IL-11 is expressed uniquely in decidual cells and its expression peaks between d 6 and 9 of pregnancy (5, 6). IL-11 is not detected in uteri of cycling mice or in early pregnancy (5, 6). Deletion of LIF and IL-11 receptor {alpha} (IL-11R{alpha}) provides evidence for the different roles of these cytokines. Whereas implantation is defective in LIF null mice (7), implantation and early stromal cell proliferation are normal in IL-11R{alpha} null mice. However, in IL-11R{alpha} null mice, decidual development becomes defective, leading to uncontrolled trophoblast invasion, small decidua, and fetal death (5, 6). Two different laboratories (5, 6) have established that the inadequate formation of the placenta and fetal death are due to the inability of IL-11 to signal in decidual cells of IL-11R{alpha} null mice.

IL-11, originally cloned from a primate bone marrow stromal cell line (8), is known to be a multifunctional hematopoietic growth factor (8, 9, 10) with a wide spectrum of biological activities (11). This cytokine was reported to stimulate myelopoiesis (9), erythropoiesis (10), and megakaryopoiesis (12). There are two loci for the murine IL-11R{alpha} chain, IL-11R{alpha} and IL-11R{alpha}2 (13). Whereas IL-11R{alpha} is widely expressed, Il-11R{alpha}2 is expressed only in the testis, lymph node, and thymus (13). When IL-11R{alpha} null mice were generated, they surprisingly displayed normal hematopoiesis (14). The mice had normal peripheral blood cells, platelets, and normal hematocrit. Bone marrow examination revealed normal cells for all hematopoietic lineages. Most surprisingly, it was found that female IL-11R{alpha} null mice were infertile (5, 6). Most embryos in IL-11R{alpha} null mice appear phenotypically normal up to d 8 of pregnancy (5). However, the mesometrial decidua, which is the site of trophoblast invasion, thins with the progress of pregnancy and begins to disappear from d 9. The degeneration of the maternal decidua is accompanied by an invasion of trophoblast giant cells that fill the space usually occupied by the mesometrial decidua. The invasion of trophoblast is similar to that seen when embryos are grafted to ectopic sites such as kidney, spleen, or nonpregnant uterus, suggesting that the decidua of mice with IL-11R{alpha} deletion is unable to prevent trophoblast invasion and that the invading trophoblast causes decidual degradation and ultimately death of the embryo. In pseudopregnant IL-11R{alpha} null mice, although the mesometrial decidua is smaller than that of wild-type mice, it never disappears completely, which suggests that the total loss of mesometrial decidual cells is due in large part to uncontrolled trophoblast invasion (5, 6).

It is well established that embryo implantation in humans and rodents is a highly invasive yet tightly controlled process. The trophoblast cells invade the uterus by secreting matrix metalloproteinases (MMPs) that are capable of degrading extracellular matrix. On the other hand, the decidua can limit this invasion by producing tissue inhibitors of metalloproteinase (TIMPs) and {alpha}2-macroglobulin ({alpha}2-MG) (15, 16). To date, four distinct TIMPs have been identified: TIMP-1, -2, and -4 are secreted proteins, and TIMP-3 is bound to extracellular matrix. Only TIMP-3 is expressed in the mesometrial decidual cells adjacent to trophoblast invasion (17) and has been implicated in the regulation of trophoblast invasion (18). We found that TIMP-3 was down-regulated in IL-11R{alpha} null mice and can be stimulated in decidual cell culture by IL-11 (19). In addition to TIMP-3, we have previously found that {alpha}2-MG, produced specifically by the mesometrial decidua adjacent to the trophoblast, is by far the most abundant protein synthesized by rat mesometrial decidual cells (20, 21, 22). {alpha}2-MG is a potent proteinase inhibitor that binds and deactivates a broad range of proteinases including the MMPs (23). {alpha}2-MG was shown to limit trophoblast invasion (15, 16, 24). The fact that trophoblast invasion in IL-11R{alpha} null mice is not limited by the decidua suggests that a lack of protease inhibitors may cause the trophoblast cells to invade and destroy the decidual cells, leading to fetal death.

In this study, we have established that disruption in IL-11 signaling leads to total disappearance of {alpha}2-MG from the mouse decidua. We have also established that IL-11 is a potent stimulator of {alpha}2-MG in a uterine cell line that decidualizes in culture and have shown that this stimulation is at the transcriptional level and involves the Janus kinase (Jak)2/signal transducer and activator of transcription (Stat)3 pathway.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Either IL-11R{alpha} null mice or wild-type mice were mated with fertile males to induce pregnancy. As shown in Fig. 1Go, uteri of d 9 pregnant IL-11R{alpha} null mice are much smaller than the ones in wild-type mice.


Figure 1
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  		Fig. 1. A Dramatic Difference in Size of the Uterus Is Observed between Wild-Type and IL-11R{alpha} Null Mice during Pregnancy

IL-11R{alpha} null mice and wild type-mice were mated with fertile males to induce pregnancy. On d 9 of pregnancy, mice were killed and their uteri were compared.

 
To examine whether {alpha}2-MG was defectively expressed in IL-11R{alpha} null mice and to eliminate any possible fetal tissue contamination, pseudopregnancy was induced by mating females with vasectomized males. On d 4 of pseudopregnancy, decidualization was induced with intrauterine administration of oil. Decidual tissue was collected on d 9 and was used to examine the expression of {alpha}2-MG. Deletion of IL-11R{alpha} dramatically down-regulated {alpha}2-MG expression in the decidua at the mRNA (Fig. 2AGo) and protein levels (Fig. 2BGo), suggesting that {alpha}2-MG expression in the decidua depends on IL-11 signaling.


Figure 2
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  		Fig. 2. {alpha}2-MG Is Not Expressed in the Decidua of IL-11R{alpha} Null Mice

IL-11R{alpha} null mice or wild-type mice were mated with vasectomized males to induce pseudopregnancy. On d 4 of pseudopregnancy, decidualization was induced with intrauterine administration of oil. Decidua tissues were collected on d 9. {alpha}2-MG expression was compared between wild-type mice and IL-11R{alpha} null mice by RT-PCR (A) and Western analysis (B). Values are expressed as the mean ± SEM (n = 3). *, P < 0.05 vs. +/+.

 
To examine whether IL-11 indeed regulates {alpha}2-MG expression in decidual cells, we used a nontransformed rat uterine stromal cell line (UIII), which decidualizes in the presence of arachidonic acid. This cell line was previously characterized by our laboratory (25). Cells were treated with different doses of IL-11 for 24 h, and {alpha}2-MG mRNA expression was examined by RT-PCR and by immunocytochemistry. The results indicate that IL-11 robustly stimulates the expression of {alpha}2-MG. At doses ranging from 25–100 ng/ml, a 4-fold stimulation was observed (Fig. 3AGo). Higher doses of IL-11 had no stimulatory effect. Immunocytochemical analysis of cells treated with IL-11 (100 ng/ml) or vehicle also established that this cytokine markedly stimulated {alpha}2-MG protein levels in UIII cells (Fig. 3BGo).


Figure 3
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  		Fig. 3. IL-11 Stimulates {alpha}2-MG Expression in UIII Cells

A, UIII cells were treated with different doses of IL-11 for 24 h, and total RNA was subjected to RT-PCR analysis using specific primers for rat {alpha}2-MG and L19 as internal control. Values are expressed as the mean ± SEM (n = 3). *, P < 0.05 vs. 0 ng/ml. B, UIII cells were treated with IL-11(100 ng/ml) or vehicle for 24 h and were subjected to immunocytochemistry as described in Materials and Methods. {alpha}2-MG, Red; nucleus, blue. Experiments were repeated three times.

 
To investigate further the effect of IL-11 on the expression of {alpha}2-MG gene, we transfected decidualized UIII cells with a 2.6-kb rat {alpha}2-MG promoter linked to a luciferase reporter gene ({alpha}2-MG-Luc). Cells were cotransfected with a plasmid expressing renilla luciferase, allowing for normalization of transfection efficiency. IL-11 treatment induced a 6.5-fold increase in the activity of the 2.6-kb {alpha}2-MG-Luc construct (Fig. 4Go). Serial 5' deletion of the {alpha}2-MG promoter revealed that the region between –371 and –159 bp upstream of the transcription start site conferred regulation of {alpha}2-MG by IL-11 gene. Internal deletion study further demonstrated that the minimal and necessary elements for IL-11 stimulation were located between the –209 and –159 bp region. Deletion of this region completely abolished the stimulatory effect of IL-11. Analysis of this region revealed one proximal Stat site (pStat: TTCTGGGAA, –166 to – 158 bp) and one distal Stat homology site (dStat: TAACTGGAA, –187 to –179 bp).


Figure 4
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  		Fig. 4. IL-11 Activates {alpha}2-MG Promoter in UIII Cells

Using Effectene reagent, UIII cells were transfected with equal amounts of different rat {alpha}2-MG promoter reporter constructs obtained by either serial 5' deletion or by internal deletion. Control cells were transfected with empty vector. Sixteen to 19 h after transfection, cells were treated with IL-11 (100 ng/ml) for another 24 h. Transient expression of the reporter gene was quantified by a standard luciferase assay and normalized against renilla luciferase. The experiment was repeated three times with triplicate wells for each group. Values are expressed as the mean ± SEM. *, P < 0.05 vs. vehicle. Two putative Stat response elements were found between –209 and –159 bp.

 
To examine whether these two Stat sites are necessary for IL-11 stimulation of {alpha}2-MG, we mutated the proximal, the distal, or both of these Stat sites in the context of a 300-bp {alpha}2-MG-Luc. As shown in Fig. 5Go, mutation of either the distal or proximal Stat site decreased IL-11 stimulation, whereas double mutation obliterated IL-11 action, suggesting that both of these response elements may be important for IL-11 stimulation of {alpha}2-MG promoter activity.


Figure 5
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  		Fig. 5. Mutation of the Stat-Binding Sites Abolished the IL-11 Stimulation of {alpha}2-MG Promoter Activity

A 371-bp rat {alpha}2-MG promoter was mutated either at the pStat site (mpStat), dStat site (mdStat), or at both sites (dm). UIII cells were transfected with equal amounts of the {alpha}2-MG promoter reporter constructs using Effectene reagent. Sixteen to 19 h after transfection, cells were treated with IL-11 (100 ng/ml) for another 24 h. The experiment was repeated three times with triplicate wells for each group. Values are expressed as the mean ± SEM. *, P < 0.05 vs. vehicle.

 
Because the two Stat sites are important for IL-11 stimulation of {alpha}2-MG promoter activity in UIII cells, we examined which Stat is involved in IL-11 action. IL-11 (100 ng/ml) treatment for different time points showed that IL-11 induced Stat3 phosphorylation in UIII cells within 10–20 min with no apparent effect on Stat5 phosphorylation (Fig. 6AGo). Immunocytochemical analysis of IL-11 action revealed clearly that this cytokine induced total translocation of Stat3 to the nucleus (Fig. 6BGo).


Figure 6
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  		Fig. 6. Stat3 Phosphorylation and Nuclear Translocation Is Stimulated by IL-11 Treatment in UIII Cells

A, UIII cells were treated with IL-11 (100 ng/ml) for different lengths of time. Phosphorylation of Stat3 and Stat5 was determined by Western analysis using specific antibodies. B, UIII cells were cultured on chamber slides for 24 h in M199 medium supplemented with 2% charcoal-dextran-treated FBS. The cells were serum starved for 4 h and then treated for 30 min with IL-11 (100 ng/ml) or vehicle. Cells were prepared for immunocytochemistry as described in Materials and Methods. Cells treated with vehicle (a–c) or IL-11 (d–f) were stained with total Stat3 antibody (1:100, final dilution). Nuclei were stained with 4',6-diamino-2-phenylindole. Stat3, Red; nucleus, blue. Experiments were repeated three times.

 
To further examine Stat3 involvement in IL-11 stimulation of {alpha}2-MG gene expression, UIII cells were cotransfected with {alpha}2-MG promoter and with either dominant negative (DN)-Stat5a, DN-Stat5b, DN-Stat3, or empty vector. After 16–19 h, cells were treated with IL-11 for another 24 h. As shown in Fig. 7Go, DN-Stat3 completely obliterated IL-11 stimulation of {alpha}2-MG promoter activity. Interestingly, although IL-11 had no stimulatory effect on Stat5 phosphorylation, either DN-Stat5a or DN-Stat5b inhibited IL-11 stimulation of {alpha}2-MG promoter activity to a significant level. Stat5 and Stat3 are known to dimerize (26). Because these dimers are found only in the cytosol and not in the nucleus, it was concluded that the dimerization of Stat5 with Stat3 serves as a negative modulator for Stat3 activity, preventing translocation of a fraction of Stat3 to the nucleus (26). The DN-Stat5 overexpressed in this investigation were mutated in the DNA binding and activation domain while their protein-protein interaction remained intact.


Figure 7
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  		Fig. 7. DN-Stat3 Suppresses the Stimulation of {alpha}2-MG Promoter by IL-11 in UIII Cells

UIII cells were cotransfected with {alpha}2-MG promoter reporter construct and with empty vector, DN-Stat5a, DN-Stat5b, or DN-Stat3 for 16–19 h. Cells were then treated with either IL-11 or vehicle for another 24 h. Transient expression of the reporter gene was quantified by the standard luciferase assay and normalized against renilla luciferase. The experiment was repeated three times with triplicate wells for each group. Values are expressed as the mean ± SEM. *, P < 0.05 vs. vehicle.

 
We next examined whether IL-11 affected binding of Stat3 to the {alpha}2-MG promoter (Fig. 8Go). EMSA showed that IL-11 treatment increased Stat3 binding (lane 2 vs. 3). This binding was competed with unlabeled wild-type probe (lane 5), the unlabeled dStat site mutated probe (lane 4), and to a much lesser degree by the cold pStat site mutated probe (lane 6). Binding was not competed by cold probe with double mutation (land 7). This suggests that the pStat site has a higher affinity for Stat3 than the distal site, and binding of Stat3 to both the proximal and distal sites (interaction of two Stat3 dimers) may form the most stable DNA-transcription factor complex. Addition of Stat3 antibody to the probe/nuclear-extract mix (lane 10) led to the formation of a supershift band, whereas an antibody to Stat5a/b failed to supershift the DNA/protein band (lane 8).


Figure 8
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  		Fig. 8. IL-11 Treatment Increases Stat3 Binding to Its Cognate Binding Sites in the {alpha}2-MG Promoter in UIII Cells

UIII cells were treated with IL-11 (100 ng/ml) or vehicle for 20 min. Nuclear extracts were subjected to gel shift assay. Lane 1, Negative control; lane 2, nuclear extract from cells treated with vehicle; lane 3, nuclear extract from cells treated with IL-11; lane 4, 50-fold molar excess of unlabeled dStat site mutated probe (mdStat); lane 5, 50-fold molar excess of unlabeled wild-type (wt) probe; lane 6, 50-fold molar excess of unlabeled pStat mutated probe (mpStat); lane 7, 50-fold molar excess of unlabeled double mutation probe (dm); lane 8, Stat5 antibody for supershift; lane 9, no antibody; lane 10, Stat3 antibody for supershift. The experiment was repeated three times.

 
Next we examined which Jak was involved in IL-11 signaling in the decidua. UIII cells were treated with IL-11 for different time points as shown in Fig. 9Go. It was found that IL-11 increased Jak2 phosphorylation but had no apparent effect on either Jak1 or Tyk2 phosphorylation. To further examine the role of Jak2 on {alpha}2-MG expression, we chose one member of the tyrphostin family of tyrosine kinase inhibitors, AG490 (27). 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 (27). This inhibitor has also been shown to inhibit Jak3 (28). The ability of AG490 to selectively block Jak2 and -3 may be explained by the high level homology shared between these two most closely related members of the Jak family. Indeed, AG490 displays similar micromolar inhibitory concentration requirements to block Jak2 and -3 activity. AG490 is considered to be a Jak2-specific inhibitor in UIII cells that do not express Jak3 (25). As shown in Fig. 9BGo, AG490 suppressed totally {alpha}2-MG expression. This suppression was both concentration and time dependent. Maximal effect (down-regulation >70%) was achieved in less than 3 h. This effect could not be explained by general toxicity because no loss in cell viability was observed within the range of the concentrations used and appeared to be solely due to inhibition of Jak2 activity.


Figure 9
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  		Fig. 9. Jak2 But Not Jak1 or Tyk2 Is Phosphorylated by IL-11 in UIII Cells

A, UIII cells were kept in serum free medium for 24 h before they were treated with IL-11 (100 ng/ml) for different lengths of time. Phosphorylation of Tyk2, Jak1, and Jak2 was studied by Western analysis. B, Total RNA obtained from UIII cells treated with different doses of AG490 and for different times was subjected to RT-PCR analysis, as described in Materials and Methods. The autoradiograms are representative of three separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Proper decidualization and trophoblast invasion are two events crucial for the formation of a functional placenta and for establishment and maintenance of pregnancy. Defects in IL-11 signaling in mice result in trophoblast overinvasion and fetal loss (5, 6). Similar results were shown in women with tubal pregnancy where inadequate IL-11 signaling and dysregulation of trophoblast invasion take place (29). The trophoblast cells invade the uterus by secreting MMPs that are capable of degrading extracellular matrix. Although trophoblast cells can be highly invasive and destructive when the embryo implants in other sites rather than the uterus, the process is tightly controlled in the decidua, which limits this invasion by producing TIMPs and {alpha}2-MG (15, 16).

It is clear from the results obtained in this investigation that IL-11 plays a crucial role in the expression of decidual {alpha}2-MG, a potent protease inhibitor known to limit trophoblast invasion (24) and to be abundantly produced by the mesometrial decidual cells, which are at the site of trophoblast invasion (1).

IL-11R{alpha} null mice are deficient in natural killer (NK) cells in the decidua (30) and have defective placental vascularization. Both NK cells and {alpha}2-MG are found in the mesometrial decidua where the trophoblast invades. NK precursor cells are recruited to the mesometrial decidua from secondary lymphoid tissues after embryo implantation where they differentiate into mature NK cells with a pregnancy-specific phenotype and appear to play an important role in the normal vascularization during trophoblast invasion. Disruptions in NK cell function have been postulated to cause early pregnancy failure (31, 32). Croy et al. (33) have provided evidence to suggest that {alpha}2-MG not only limits trophoblast invasion but also plays a role in NK cell proliferation and normal vascularization of the placenta. This therefore implies that IL-11 stimulation of {alpha}2-MG is a crucial upstream step necessary for all these events that take place during pregnancy to allow for normal placentation.

During pregnancy, the liver and the decidua are the major sites of {alpha}2-MG production in rodents (34), and {alpha}2-MG represents by far the most abundant protein produced by mesometrial decidual cells (20, 21, 22). In the liver, {alpha}2-MG is stimulated by IL-6 (35, 36). However, in the decidua, IL-6 expression is suppressed (37, 38, 39), and its expression in the uterus has been implicated in the termination of pregnancy. IL-6 production by decidual tissue in response to inflammation and in conjunction with other inflammatory mediators is considered to play a role in the pathophysiology of preterm labor due to infection (40, 41, 42). High levels of IL-6 are detected in the amniotic fluid of women delivering prematurely due to uterine infection (40, 43). Because production of IL-6 during pregnancy may compromise fetal survival by triggering an inflammatory response, the decidua instead produces IL-11.

IL-11 was shown to activate gene expression in different tissues through different mechanisms including the inhibition of nuclear factor-{kappa}b and the activation of MAPK, phosphatidylinositol 3-kinase, and Jak2 (44, 45, 46). It is clear from this investigation that IL-11 stimulation of {alpha}2-MG in decidual cells involves the Jak2/Stat3 pathway.

Although stimulation of {alpha}2-MG by IL-11 appears to be of great importance for placentation and fetal survival, IL-11 is not the only cytokine able to stimulate this gene in the decidua. IL-11 expression peaks only for a short period of time (6–9 d) early in pregnancy (2, 6), whereas levels of {alpha}2-MG expression in the mesometrial decidua increase rather than decrease with the advance of pregnancy. We have previously shown that the rodent decidua expresses the prolactin (PRL) gene starting from d 8–9 of pregnancy (47), produces PRL, and expresses both forms of the PRL receptors (48). We have also shown that PRL can stimulate {alpha}2-MG gene expression (1) and activate Stat5 (49) in rodent primary decidual cells and that the decidua of PRL-null mice does not express {alpha}2-MG (data not shown). Administration of PRL to PRL null mice stimulated {alpha}2-MG expression. It appears therefore that IL-11 and PRL act consecutively in vivo to maintain high levels of this important protease inhibitor. Interestingly, whereas deletion of IL-11 gene leads to abortion early in pregnancy, knockout of the PRL gene causes abortion later in pregnancy despite progesterone treatment (Binart, N., S. Elizur, and G. Gibori, unpublished observations). This suggests that both these cytokines expressed sequentially during development act to limit trophoblast invasion and assure proper formation and maintenance of the placenta.

In addition to down-regulation of {alpha}2-MG, we have also found that several cell cycle factors are dysregulated in the decidua of IL-1R{alpha} null mice (50). This suggests that the infertility of these mice may be due to multiple reproductive defects.

Taken together, results of this investigation provide, at least in part, an explanation for the overinvasiveness of the trophoblast in IL-11R{alpha} null mice and reveal, for the first time, that IL-11 signals through the Jak2/Stat3 pathway in decidual cells to stimulate the expression of {alpha}2-MG, a protease inhibitor essential for normal placentation in pregnancy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
[{alpha}-32P]dCTP, [{gamma}-32P]dATP, poly(dI-dC) and AutoSeq G-50 columns were purchased from Amersham Biosciences, Inc. (Piscataway, NJ); dNTP, ExTaq DNA polymerase, and ExTaq buffer were purchased from Takara Biomedicals (Shiga, Japan); medium M199 without phenol red, the reverse transcriptase (RT) kit, the T4 kinase kit, Trizol reagent, the nucleotides used as primers for RT reaction were obtained from Invitrogen (Carlsbad, CA); Western blotting Luminol Reagent was obtained from Santa Cruz Biotechnology (Santa Cruz, CA); medium M199 with phenol red, antibiotic-antimycotic solution, 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); arachidonic acid, IL-11, aprotinin, leupeptin, and phenylmethylsulfonyl fluoride were purchased from Sigma (St. Louis, MO); protease inhibitor cocktail was from Roche Applied Science (Indianapolis, IN); 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); Passive lysis 5X buffer, Dual-Luciferase Reporter Assay was purchased from Promega (Madison WI); Effectene Transfection Reagent was obtained from QIAGEN Inc. (Valencia, CA); QuikChange II site-directed mutagenesis kit was from Stratagene (La Jolla, CA); AG490 was purchased from Calbiochem (La Jolla, CA). The DN-Stat3 was kindly given to us by Dr. Toshio Hirano (Osaka University, Osaka, Japan). Tyr705 was substituted by phenylalanine so that it could not be phosphorylated (51). The DN-Stat5a was a gift from Dr. Alice Mui (University of British Columbia, British Columbia, Canada). It contains a C-terminal deletion in the transactivation domain and is transcriptionally inactive (52). The DN-Stat5b, a gift from Dr. Li-yuan Yu-Lee (Baylor College of Medicine, Houston, TX), contains a four-amino-acid substitution in the DNA binding domain, which prevents it from binding to DNA (53, 54).

Animals
IL-11R{alpha} 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 Southern blotting using tail DNA. IL-11R{alpha} null mice or wild-type mice were mated with vasectomized males to induce pseudopregnancy. Decidualization was induced with intrauterine administration of oil on d 4. The decidual tissues were collected on d 9. 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.

RNA Isolation and RT-PCR Analysis
Total RNA from the decidual tissue of mice or from UIII cells was isolated using Trizol reagent according to the manufacturer’s instructions. The RT and PCRs were conducted as previously described (55). 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. 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 were washed three times. The resulting gels were photographed using UV transilluminator and a digital camera (Electrophoresis Documentation and Analysis System 120; Eastman Kodak Co., New Haven, CT). For PCR with [{alpha}-32P]dCTP, reaction products were electrophoresed on 8% polyacrylamide nondenaturing gel and were subjected to autoradiography.

For the mouse, the {alpha}2-MG primers used were 5'-CAA TGC GAT GAA GGG TGG TGT-3' and 5'-GTG GGC CGG GAA GAC TCA GA-3'. The 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 {alpha}2-MG primers used were 5'-GTA ATC CTT CTA ACT GCT TCG GCG-3' and 5'-CCA ATG AAG ATC GTT TCA TAC GGA-3'. The L19 primers used were 5'-CTG AAG GTC AAA GGG AAT GTG-3' and 5'-GGA CAG AGT CTT GAT GAT CTC-3'.

Western Blot Analysis
Western blots were performed as described previously (56). Antibodies to phospho-Jak2, phospho-Tyk2 (Cell Signaling, Danvers, MA), Jak2, phopho-Stat5a/b, phospho-Stat3, Stat3 (Upstate Biotechnology, Lake Placid, NY), Stat5a/b, Jak1, phospho-Jak1 (BioSource International, Inc., Camarillo, CA), and Tyk2 (BD Biosciences, San Jose, CA) were used. {alpha}2-MG antibody was kindly given by Dr. Jack Gauldie (McMaster University, Ontario, Canada). Briefly, 40 µg of proteins were 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 or 5% BSA (when studying phosphorylated proteins) in Tris-buffered saline (TBS) 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 being extensively washed, blots were analyzed using an enhanced chemiluminescence detection system and exposed to x-ray film.

Cell Culture
The rat uterine stromal cells, UIII, established from rat uterine stromal cells (57) and characterized in our laboratory (25), were grown in M199 medium supplemented with FBS (10%), nonessential amino acids, and antibiotic-antimycotic solution. Arachidonic acid (30 µM) is essential for decidualization of UIII cells (58). They were incubated in a humidified atmosphere of 5% CO2 at 37 C. Culture media were replaced every 48 h. Cells were treated with various concentrations of IL-11 in M199 medium supplemented with 1% charcoal-dextran-treated FBS. After 24 h, cells were washed twice with ice cold PBS and were frozen at –80 C until RNA extraction.

Transfection of UIII Cells
Transfection was done by using Effectene transfection reagent according to the manufacturer’s instructions. Briefly, UIII cells were seeded in 12-well plates and cultured until 60–80% confluency. A total of 0.4–0.6 µg DNA was transfected per well. Sixteen to 19 h after transfection, cells were treated with IL-11 (100 ng/ml) for another 24 h. Transient expression of the reporter gene was quantified by a standard luciferase assay and was normalized against renilla luciferase according to the manufacturer’s instructions. Luciferase activities were measured using a luminometer (Lumat LB 9507 luminometer; EG&G Berthold, Oak Ridge, TN).

Immunocytochemistry
UIII cells were grown for 24 h in M199 medium supplemented with 2% charcoal-dextran-treated FBS on Lab-Tek chamber slides (Nalge Nunc International, Rochester, NY). Cells were then cultured with IL-11 (100 ng/ml) or vehicle for 30 min or 24 h. At the end of the treatment, cells were washed twice in PBS and were fixed for 10 min in 4% paraformaldehyde solution in PBS at room temperature. After being rinsed in TBS buffer, the cells were permeabilized for 15 min at room temperature in TBS containing 10% BSA, 0.1% Triton X-100, and 0.2% Tween 20 solution. After blocking, the cells were then incubated overnight at 4 C with either a polyclonal antibody to total Stat3 (Santa Cruz Biotechnology) or a polyclonal antibody to {alpha}2-MG (a gift from Dr. Jack Gauldie) at 1:100. The cells were then exposed for 4 h at room temperature to Cy3-conjugated donkey antirabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at 1:800 dilution. The slides were mounted in Vectashield medium (Vector Laboratories, Inc., Burlingame, CA) containing a counterstain for DNA 4',6-diamino-2-phenylindole and were observed with a Carl Zeiss (Oberkochen, Germany) LSM 510 laser confocal microscope equipped with a X63 water immersion objective lens (NA 1.2).

Mutation of {alpha}2-MG Promoter
Mutations to the promoter were made via PCR using the QuikChange II site-directed mutagenesis kits (Stratagene) according to the manufacturer’s protocol. An {alpha}2-MG promoter (–371 to +54 bp) in pSVOA{Delta}5' was used as the template. Mutation was confirmed through DNA sequencing. pStat site mutation was created by using primers 5'-AAAAAGTGAGCAGTAACTGGAAAGTCCTTAATCCggaTGGGAATTCTGGC-3' and 5'-GCCAGAATTCCCAtccGGATTAAGGACTTTCCAGTTACTGCTCACTTTTT-3'. dStat site mutation was created by using primers 5'-AAAAAGTGAGCAGTAACTttccAGTCCTTAATCCTTCTGGGAATTCTGGC-3' and 5'-GCCAGAATTCCCAGAAGGATTAAGGACTggaaA GTTACTGCTCACTTTTT-3'. Double mutation was generated by using primers 5'-AAAAAGTGAGCAGTAACTttccAGTCCTTAATCCggaTGGGAATTCTGGC-3' and 5'- GCCAGAATTCCCAtccGGATTAAGGACTggaaAGTTACTGCTCACTTTTT-3'. The underlined portions represent the pStat and dStat sites.

EMSA
UIII cells were treated with IL-11 (100 ng/ml) or vehicle for 20 min. Cells were washed with cold PBS twice and were frozen at –80 C until use. Four hundred microliters of hypotonic buffer [10 mM NaCl; 3 mM MgCl2; 10 mM Tris, pH 7.4; 0.5% Nonidet P-40; 0.5 mM dithiothreitol (DTT); 0.5 mM phenylmethylsulfonyl fluoride; 1 mM Na3VO4; and 1x protease inhibitor cocktail] was added to each 10 cm dish. Cells were scratched and vortexed vigorously for 30 sec. Tubes were centrifuged for 1 min at maximal speed, and the pellet was resuspended with 50 µl of extraction buffer C (420 mM KCl; 20 mM HEPES, pH 7.9; 1.5 mM MgCl2; 0.2 mM EDTA; 20% glycerol; 0.5 mM DTT; 0.5 mM phenylmethylsulfonyl fluoride; 1 mM Na3VO4; and 1x protease inhibitor cocktail). The solution was vortexed vigorously for 25 min and centrifuged for 5 min at maximal speed at 4 C. The supernatant containing the nuclear extract was aliquoted and stored at –80 C. Annealed wild-type probes (5 pmol) were labeled using 10 U of T4 kinase and 25 µCi of [{gamma}-32P]dATP. Five micrograms of nuclear extracts were incubated with 1 µg of poly(dI-dC) and different cold competitor probes (2.5 pmol) in 1x binding buffer (10 mM Tris-HCl, pH 7.4; 150 mM KCl; 0.1 mM EDTA; 12.5% glycerol; 0.1 mM DTT) on ice for 30 min. Hot wild-type probe (50 fmol) was added. Samples were run on a 4% nondenaturing polyacrylamide gel in 0.5x Tris borate EDTA buffer at 200 V for 2–3 h. The gels were then dried and analyzed by autoradiography. For supershift assays, nuclear extract, poly(dI-dC), and hot wild-type probe were incubated on ice for 20 min, and antibodies were added for another 30 min at room temperature. Stat3 antibody was purchased from Santa Cruz Biotechnology, and Stat5a/b antibody was from BioSource International, Inc.

Statistical Analysis
Data were examined by t test (Fig. 2Go), one-way ANOVA followed by the Tukey test (Fig. 3Go), or two-way ANOVA followed by Bonferroni post-tests (Figs. 4Go, 5Go, and 7Go) using Prism software (GraphPad Software, Inc., San Diego, CA). Values were considered statistically significant at P < 0.05.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Georg H. Fey (University of Erlangen-Nuremberg, Erlangen, Germany) for the rat {alpha}2-MG promoters, Dr. Toshio Hirano for the DN-Stat3, Dr. Li-yuan Yu-Lee for the DN-Stat5b, Dr. Alice Mui for the DN-Stat5a, and Dr. Jack Gauldie for the {alpha}2-MG antibody.


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

Disclosure statement: The authors have nothing to disclose.

First Published Online September 7, 2006

Abbreviations: DN, Dominant negative; DTT, dithiothreitol; dStat, distal Stat; FBS, fetal bovine serum; IL-11R{alpha}, IL-11 receptor {alpha}; Jak, Janus kinase; LIF, leukemia inhibitory factor; {alpha}2-MG, {alpha}2-macroglobulin; MMP, matrix metalloproteinase; NK, natural killer; PRL, prolactin; pStat, proximal Stat; TIMP, tissue inhibitor of metalloproteinase; Stat, signal transducer and activator of transcription; TBS, Tris-buffered saline.

Received for publication July 19, 2006. Accepted for publication August 28, 2006.


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