Biol Reprod Lalor Postdoctoral Fellowships -- Application Deadline January 15, 2009
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print September 19, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.063313
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
78/1/127    most recent
biolreprod.107.063313v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, F.
Right arrow Articles by Gibori, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, F.
Right arrow Articles by Gibori, G.
Agricola
Right arrow Articles by Li, F.
Right arrow Articles by Gibori, G.
BIOLOGY OF REPRODUCTION 78, 127–133 (2008)
DOI: 10.1095/biolreprod.107.063313
© 2008 by the Society for the Study of Reproduction, Inc.

Involvement of Cyclin D3, CDKN1A (p21), and BIRC5 (Survivin) in Interleukin 11 Stimulation of Decidualization in Mice1

Feixue Li 3 4, Y. Sangeeta Devi 3, Lei Bao , Jifang Mao , and Geula Gibori 2

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

ABSTRACT

Interleukin 11 receptor alpha (Il11ra) null mice are infertile due to defective decidualization and abnormal trophoblast invasion. We have previously shown in these mice that downregulation of decidual proteinase inhibitors plays a role in uncontrolled trophoblast invasion. However, the decidua is abnormally smaller in pseudopregnant Il11ra null mice, where trophoblast invasion is not a factor. Here, we examined whether defective decidualization is due to dysregulation of key molecules involved in decidual cell growth and differentiation. We found a dramatic downregulation of cyclin D3 in Il11ra null mice. We also found that IL11 robustly stimulates the expression of cyclin D3 in cell culture. CDK4 and CDK6, known partners of cyclin D3, are not affected. Immunolocalization studies show absence of cyclin D3 in the mesometrial site and absence of differentiated polyploid cells in the antimesometrial site of Il11ra null mice. We also examined the expression of cell differentiation factors CDKN1A (p21) and CDKN1B (p27), and found that in both in vivo and cell culture the expression of CDKN1A (p21) but not CDKN1B (p27) is under the control of IL11. Another clear target of IL11 in the decidua is BIRC5 (Survivin), whose expression is repressed in the decidua of Il11ra null mice and stimulated by IL11 in cell culture. Taken together, these results provide, at least in part, an explanation for the defective small decidua of mice lacking the Il11ra gene, and reveal for the first time that cyclin D3, CDKN1A (p21), and BIRC5 (Survivin) are targets of IL11 in the decidua.

CDKN1A, cyclin D3, deciduas, IL11, implantation

INTRODUCTION

Implantation and development of the embryo require extensive reorganization of the different tissues forming the uterus. Rapid growth and differentiation of the endometrial stroma are the earliest and most striking events that take place in the uterine milieu. This leads to the formation of special cells, termed decidual cells, that differ greatly from the original endometrial stromal cells. In rodents, the attachment of the embryo to the antimesometrial uterine epithelium elicits a local and extensive response that results in the formation of the antimesometrial decidua. Decidualization then extends mesometrially to form the mesometrial decidua comprised of cells much smaller and less differentiated than the antimesometrial decidual cells. The mechanisms involved in the differential growth and transformation of endometrial cells located in different sites of the uterus are not completely understood; however, the involvement of hormones is well known. For decidualization to occur, the uterus must be primed with progesterone alone or progesterone and estradiol [1–3]. Decidualization can be also induced artificially by various stimuli, such as trauma or injection of oil into the lumen of the hormonally primed pseudopregnant uterus. Artificial decidualization shares many of the features of natural decidualization. While the local molecular mechanisms driving decidualization are still largely unknown, a number of decidual-derived factors have been identified as differentiation factors in this process. These include extracellular matrix remodeling factors, lipid mediators, and homeobox and C/EBPβ transcription factors as well as cytokine leukemia inhibitory factor (LIF) and interleukin 11 (IL11) [4, 5].

IL11 is a member of the IL6 family of cytokines that is active on a range of different types of cells. Its effects include stimulation of hematopoietic progenitors, anti-inflammatory actions, effects on bone remodeling, and induction of acute-phase protein secretion by the liver [6, 7]. The biological effects of IL11 are mediated by association of the ligand with both its receptors (IL11R) and the signal transducer, gp130 [8]. IL11 is not detected in cycling or in early pregnant mice uterus; however, after decidualization, this cytokine becomes expressed in decidual cells, and its expression peaks between Days 6 and 9 of pregnancy [9, 10]. Gene deletion studies have revealed a key role for this cytokine in the process of decidualization. Female mice with either a null mutation of the gene encoding Il11ra [9] or a hypomorphic Il11ra allele [10] were found to be infertile due to defective decidualization. The decidua of Il11ra null mice is much smaller than that of wild-type mice and does not support successful pregnancy, causing fetal death between Days 8 and 9 [9]. This suggests that IL11 plays a key role in decidual growth and/or differentiation. During differentiation, decidual cells, located principally in the antimesometrial region of the uterus, acquire polyploidization, a hallmark for this event. The decidual cell polyploidy is characterized by formation of large mononucleated or binucleated cells, a characteristic of nuclear endoreduplication, consisting of DNA with four, eight, and even higher multiples of the haploid complement [11–13]. It is believed that endoreduplicated cells exhibit an array of cell cycle activities that direct continuous DNA synthesis without cell division. It has been reported previously that coordinate expression of cyclin D3 with CDKN1A (p21) and CDK6 drives development of polyploidy during stromal cell decidualization [14, 15].

In this report, using both in vivo and cell culture approaches, we examined whether IL11 regulates the expression of key molecules involved in decidual cell growth and endoreplication. We also examined whether deletion of Il11ra causes dysregulation in the expression of any of these cell cycle-regulating factors.

MATERIALS AND METHODS

Chemicals

ExTaq DNA polymerase, ExTaq buffer, and deoxyribonucleotide triphosphate (dNTP) were purchased from Takara Biomedicals (Shiga, Japan); medium M199 without Phenol red, reverse transcrptase kit, T4 kinase kit, TRizol reagent, and the nucleotides used as primers in the RT-PCR analysis 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); and fetal bovine serum (FBS) was purchased from HyClone Laboratories (Logan, UT). Arachidonic acid, IL11, aprotinin, leupeptin, and phenylmethylsulfonyl fluoride were purchased from Sigma (St. Louis, MO); RIPA buffer was purchased from Boston Bioproducts (Ashland, MA).

Tissue Collection

Mice with germline transmission of the Il11ra null mutation were kept at 25°C with a 14L:10D cycle and were fed a commercial pelleted diet ad libitum. Heterozygous mutants in the C57BL/6 x 129/sv background were intercrossed to generate +/+, +/–, and –/– (null) mice, which were genotyped by Southern blot using tail DNA. Il11ra null and wild-type mice were mated with either vasectomized males or wild-type males to induce either pseudopregnancy or pregnancy. Decidualization was induced in pseudopregnant mice with intrauterine administration of oil on Day 4. The decidual tissues were collected on Days 7, 8, and 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.

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, and antibiotic-antimycotic solution. 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 IL11 in medium M199 supplemented with 1% charcoal dextran-stripped FBS containing arachidonic acid (30 µM), which is essential for the in vitro differentiation of UIII cells [17]. At the end of the treatment, cells were washed twice with ice-cold PBS and frozen at –80°C until RNA extraction.

RNA Isolation and RT-PCR Analysis

Total RNA was isolated from the decidual tissue of mice using Trizol reagent (Life Technologies Inc., Gaithersburg, MD) according to the manufacturer's instructions. The RT and PCR reactions were conducted as previously described [18]. For the PCR reaction, 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 2% agarose gel. Gels were stained with Tris-Borate-EDTA (TBE) containing 0.5 µg/ml ethidium bromide and washed three times. The resulting gels were photographed using a UV transilluminator and a digital camera (Electrophoresis Documentation and Analysis System 120; Eastman Kodak Co., New Haven, CT).

Primers for RT-PCR are described as follows. For mice, cyclin D3 primers used were 5'-CGTGCAAAAGGAGATCAAGC-3' and 5'-AATCAAGGCCAGGAAGTCG-3'; Cdkn1a (p21) primers used were 5'-CGGTGGAACTTTGACTTCGT-3' and 5'-CAGGGCAGAGGAAGTACTGG-3'; Cdkn1b (p27) primers used were 5'-CAGAATCATAAGCCCCTGGA-3' and 5'-TCTGACGAGTCAGGCATTTG-3'; and Birc5 (Survivin) primers used were 5'-TACCTCAAGAACTACCGCATCG-3' and 5'-AAGGCTCAGCATTAGGCAGC-3'. For rat uterine cell line, Cdkn1a (p21) primers used were 5'-TGGACAGTGAGCAGTTGAGC-3' and 5'-ACACGCTCCCAGACGTAGTT-3'; and Cdkn1b (p27) primers used were 5'-AGGAGAGCTTGGATGTCAGC-3' and 5'-TCTGACGAGTCAGGCATTTG-3'.

Western Blot Analysis

Western blots were performed as described previously [19]. Briefly, 40 µg proteins were separated on SDS-PAGE gel and transferred to a nitrocellulose membrane. Western blotting was performed by blocking nonspecific binding with 5% dry milk in Tris-buffered saline buffer containing 1% Tween 20 for 1 h. Blots were then incubated with the primary antibody at a final dilution of 1:1000, cyclin D3 (Santa Cruz, sc-182; Santa Cruz Biotechnology), CDKN1A/p21 (Santa cruz, sc-397), CDK4 (Santa cruz, sc-601), CDK6 (Santa cruz, sc-7180), BIRC5/Survivin (Santa cruz, sc-10811), and CDKN1B/p27 (2552; Cell Signaling Technology), and 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 h. After extensive washing, blots were analyzed using an enhanced chemiluminescence detection system and exposed to x-ray film.

Immunohistochemical Staining

Paraffin-embedded sections were subjected to the avidin-biotin-peroxidase complex (ABC) method using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Briefly, formalin-fixed, paraffin embedded sections (5 µm) were deparaffinized, hydrated, and irradiated in a microwave oven for the antigen retrieval and then with 0.3% (w/v) hydrogen peroxidase for 30 min to block endogenous peroxidase activity. After washing in PBS, sections were treated with normal goat serum for 30 min to eliminate any nonspecific binding of conjugated secondary antibodies. Then, the slides were incubated overnight at 4°C with a polyclonal antibody to cyclin D3 (Santa Cruz Biotechnology) at a final dilution of 1:400. After rinsing with PBS, sections were incubated with a biotinylated second antibody for 30 min at room temperature, and then incubated for another 30 min with the ABC reagent. Finally, they were incubated with diaminobenzidine solution containing 0.03% (w/v) hydrogen peroxidase for 5 min. Sections were washed in PBS for 5 min three times between each step. Negative controls consisted of sections incubated with normal goat serum instead of primary antibody.

Statistical Analysis

Data were examined by t-test, one-way ANOVA followed by the Tukey test using Prism software (GraphPad Software Inc., San Diego, CA). Values were considered statistically significant at P < 0.05.

RESULTS

Regulation of Cyclin D3 by IL11

We first examined whether there is any defect in the expression of cell cyclin D3 in decidualization in Il11ra null mice, since this cell cycle regulator was shown to be essential for decidual cell growth [14]. Either wild-type or Il11ra null mice were mated with vasectomized males to induce pseudopregnancy. On Day 4 of pseudopregnancy, decidualization was induced with intrauterine administration of oil. Decidual tissue was collected on Days 8 and 9, and cyclin D3 expression was examined by RT-PCR and Western blot analysis. Cyclin D3 expression was dramatically downregulated at both mRNA (Fig. 1A) and protein (Fig. 1B) levels in the decidua of Il11ra null mice, suggesting that cyclin D3 expression in the decidua depends on IL11 signaling.


Figure 01
View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 1 The expression of cyclin D3 is inhibited in the decidua of Il11ra null mice (–/–) and is stimulated by IL11 in UIII cells. A, B) Il11ra null and wild-type mice (+/+) were mated with vasectomized males to induce pseudopregnancy. On Day 4 of pseudopregnancy, decidualization was induced with intrauterine administration of oil. Decidual tissues were collected on Days 8 (D8) and 9 (D9), and mRNA and total protein were extracted. Cyclin D3 expression was examined by RT-PCR and Western blot analysis. L19 and β-actin were used as loading controls. The relative intensities of the cyclin D3 were quantified by densitometry and normalized to corresponding L19 or β-actin intensities. Values are expressed as mean ± SEM (n = 3). *P < 0.05. C) UIII cells were treated with different doses of IL11 for 24 h and were processed for analysis of cyclin D3 expression by Western blot. β-Actin was used as loading control. Comparative intensities were shown at the right panel. Values are expressed as mean ± SEM (n = 3). *P < 0.05 versus 0 ng/ml.

To further examine whether IL11 indeed regulates cyclin D3 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 [20] and found to be responsive to IL11 [21]. As shown in Figure 1C, IL11 markedly stimulates the expression of cyclin D3 in cultured decidual cells.

Immunohistochemical analysis (Fig. 2, A and B) indicates that expression of cyclin D3 protein is almost absent from the mesometrial decidual cells of Il11ra null mice. This lack of expression correlates with severe defect in the normal growth of the mesometrial decidua, as reported previously [9], and as shown in Figure 2A. In contrast, cyclin D3 is localized in the antimesometrial decidual cells of both Il11ra–/– (Fig. 2D) and Il11ra+/+ (Fig. 2E) mice. Interestingly, however, large nuclei, characteristic of polyploidy of antimesometrial cells, are barely seen in Il11ra–/– mice.


Figure 02
View larger version (117K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 2 Localization of cyclin D3 in the decidua of Il11ra null mice. Il11ra null and wild-type mice were mated with males to induce pregnancy. Decidual tissues were collected on Day 8 and processed for immunohistochemistry as described in Materials and Methods. A) Cyclin D3 was detected in the mesometrial (M) and antimesometrial (AM) decidua of Il11ra null mice using a specific antibody to cyclin D3 (x200). Higher-magnification (x400) image of mesometrial (B) and antimesotrial (D) decidua. Cyclin D3 expression in mesometrial (C) and antimesometrial (E) decidua of wild-type mice (x400). The arrowheads indicate large nuclei of polyploid cells. Only a few representative polylpoid cells are indicated in the wild-type antimesometrium decidua (E).

Lack of Effect of IL11 on CDK4 and CDK6 Expression

We examined the expression of CDK4 and CDK6, the known partners of cyclin D3, in the Il11ra null mouse decidua. As shown in Figure 3, A and B, there are no differences in the expression of either CDK in the decidua of wild-type and Il11ra null mice. In addition, IL11 has no effect on the expression of either CDK4 or CDK6 in UIII cells at any doses used (Fig. 3, C and D).


Figure 03
View larger version (26K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 3 The expression of CDK4 and CDK6 is normal in the decidua of Il11ra null mice and is not stimulated by IL11 in UIII cells. A, B) Il11ra null and wild-type mice were mated with vasectomized males to induce pseudopregnancy. On Day 4 of pseudopregnancy, decidualization was induced with intrauterine administration of oil. Decidual tissues were collected on Days 8 and 9. Protein was isolated and subjected to Western blot analysis for CDK4 and CDK6. The relative intensities of CDK4 and CDK6 were quantified by densitometry and normalized to corresponding β-actin intensities. Values are expressed as mean ± SEM (n = 3). C, D) UIII cells were treated with different doses of IL11 for 24 h. Total protein was extracted, and CDK4 and CDK6 expression was analyzed by Western blot. β-Actin was used as loading control. The relative intensities were quantified by densitometry and normalized to corresponding β-actin intensities. Values are expressed as mean ± SEM (n = 3).

Expression and Regulation of Cell Differentiation Factors CDKN1A (p21) and CDKN1B (p27)

Initiation of cell cycle arrest and cell differentiation involves the induction of endogenous CDK inhibitors (CDKN1A and CDKN1B), which bind to cyclin/CDK complexes to inhibit their activity [22, 23]. A role for CDKN1A (p21) in the development of polyploidy during stromal cell decidualization has been shown [12]. This led us to investigate whether dysregulation in CDKN1A (p21) and/or CDKN1B (p27) expression contributes to the decidual defect of Il11ra null mice. As shown in Figure 4, CDKN1A (p21) is dramatically reduced in the decidua of Il11ra null mice at both mRNA (Fig. 4A) and protein (Fig. 4B) levels compared with that in wild-type mice. Furthermore, IL11 treatment of UIII cells upregulates exquisitely Cdkn1a (p21) expression (Fig. 4C). In contrast to Cdkn1a (p21), Cdkn1b (p27) mRNA and protein expression is not effected at all by deletion of Il11ra in vivo (Fig. 5, A and B). IL11 treatment of UIII cells also has no detectable effect (Fig. 5C). These findings indicate that CDKN1A (p21) is clearly a target of IL11 and that decidual CDKN1A (p21) is highly sensitive to IL11 stimulation.


Figure 04
View larger version (31K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 4 The expression of CDKN1A (p21) is dramatically reduced in the decidua of Il11ra null mice and is stimulated by IL11 in UIII cells. Decidual tissues were collected from Day 8 and Day 9 pseudopregnant mice. CDKN1A (p21) expression was compared between wild-type mice and Il11ra null mice by RT-PCR (A) and Western blot analysis (B). The relative intensities of CDKN1A (p21) were quantified by densitometry and normalized to corresponding L19 or β-actin intensities. Values are expressed as mean ± SEM (n = 3). *P < 0.05. UIII cells were treated with different doses of IL11 for 24 h, and total RNA was subjected to RT-PCR analysis using specific primers for mouse Cdkn1a (p21). L19 was used as internal control (C). The relative intensities of Cdkn1a (p21) were quantified by densitometry and normalized to corresponding L19 intensities. Values are expressed as mean ± SEM (n = 3). *P < 0.05.


Figure 05
View larger version (38K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 5 The expression of CDKN1B (p27) is normal in the decidua of Il11ra null mice and is not stimulated by IL11 in UIII cells. Decidual tissues were collected from Day 8 and Day 9 pseudopregnant mice. CDKN1B (p27) expression was compared between wild-type mice and Il11ra null mice by RT-PCR (A) and Western blot analysis (B). L19 and β-actin were used as loading controls. The relative intensities of CDKN1B (p27) were quantified by densitometry and normalized to corresponding L19 or β-actin intensities. Values are expressed as mean ± SEM (n = 3). C) UIII cells were treated with different doses of IL11 for 24 h, and total RNA was subjected to RT-PCR analysis using specific primers for mouse Cdkn1b (p27). L19 was used as internal control. The relative intensities of Cdkn1b (p27) were quantified by densitometry and normalized to corresponding L19 intensities. Values are expressed as mean ± SEM (n = 3).

IL11 Effect on BIRC5 (Survivin) Expression

Because BIRC5 (Survivin), a protein shown to play an important role in cell survival, is stimulated by IL11 in other cell types [24, 25], we examined its expression in the Il11ra null mouse decidua and examined whether this protein is a target of IL11. We found Birc5 (Survivin) to be barely detectable in the Il11ra null mice (Fig. 6A). We also found that 50 and 100 ng/ml IL11 stimulates significantly BIRC5 (Survivin) expression in cell culture.


Figure 06
View larger version (31K):
[in this window]
[in a new window]
[Download PPT slide]
  		FIG. 6 BIRC5 (Survivin) expression is stimulated by IL11. A) Decidual tissues were isolated from Day 8 and Day 9 pseudopregnant mice; mRNA was extracted and subjected to RT-PCR using L19 as loading control. B) UIII cells were treated with different doses of IL11 for 24 h, and total protein was subjected to Western blot analysis. β-Actin was used as loading control. The relative intensities of BIRC5 (Survivin) were quantified by densitometry and normalized to corresponding β-actin intensities. Values are expressed as mean ± SEM (n = 3). *P < 0.05 versus 0 ng/ml.

DISCUSSION

Uterine decidualization, characterized by stromal cell proliferation and differentiation with polyploidy, is critical for pregnancy in mice. Il11ra null mice are infertile due to defects in decidual formation. Growth and differentiation of endometrial stromal cells do take place in the uterus of Il11ra null mice after implantation, but the deciduas that form are much smaller than normal and are unable to limit trophoblast invasion [9, 10, 21]. We have previously shown that downregulation of decidual proteinase inhibitors, such as {alpha}2-macroglobulin and tissue inhibitor of metalloproteinases 3, could lead to uncontrolled trophoblast invasion and failure of functional placenta formation in these mice [21]. However, the finding that the decidua of Il11ra null mice is abnormally small not only in pregnant but also in pseudopregnant mice where trophoblast invasion is not a factor indicates that other defects intrinsic to decidual cells contribute to the infertility of these mice. In this report, we show that IL11 plays an important role in the expression of specific decidual proteins involved in growth, differentiation, and survival.

The best known regulators of mammalian cell proliferation are the D-type cyclins, which are considered to be key regulators of cell proliferation and transition of G1/S phase in the cell cycle [26–29]. These proteins mediate their action by binding to catalytic subunits of specific cyclin-dependent kinases [22, 27, 29]. A role for cyclin D3 in both growth and polyploidy of decidual cells was previously shown [12–15]. In this report, we found that IL11 stimulates cyclin D3 expression at both mRNA and protein levels in decidual cells. We also found a profound downregulation of this cell cycle protein in the decidua of Il11ra null mice. Cyclin D3 generally associates with CDK4 or CDK6 for cell proliferation. We found no change in the expression of either CDK in the Il11ra null mouse decidua. Nevertheless, the low expression of cyclin D3 indicates low complex formation between cyclin D3/CDKs 6 and 4 and decreased CDK activity. Interestingly, the defect in cyclin D3 is localized principally in the mesometrial decidual cells. This protein, normally expressed highly in Day 8 mesometrial decidual cells that are actively dividing at this stage [12], is absent from the mesometrial decidual cells of Il11ra null mice. This lack of expression correlates with and may provide an explanation for the severe defect in the normal growth of the mesometrial deciduas, as shown in this report and as previously reported [9, 10].

In contrast to its defective expression in the mesometrial decidua, cyclin D3 expression appears normal in the antimesometrial decidua of the Il11ra null mice. However, cell polyploidization is profoundly compromised, and the large nuclei specific to polyploid cells in the antimesometrial zone of wild-type mice are rarely observed in the Il11ra null decidua. This suggests a possible defect in the differentiation and polyploidization of stromal cells in the absence of IL11 signaling. This possibility is further strengthened by our findings that CDKNIA (p21) is a target of IL11 and is exquisitely upregulated by this cytokine in decidual cells. CDKNIA (p21) and CDKNIB (p27) are CDK inhibitors involved in cell cycle arrest and cell differentiation. They bind to cyclin/CDK complexes to inhibit their activity [22, 23]. CDKNIA (p21) was shown to be related to terminal differentiation of decidual cells in a region-specific manner, whereas functional association of cyclin D3 with CDKNIA (p21) and CDK6 has been implicated in the development of polyploidy during stromal cell decidualization [12, 14, 15]. Our finding that, similarly to cyclin D3, CDKNIA (p21) is barely detectable in the decidua of Il11ra null mice and is a sensitive target to IL11 provides a likely explanation for the lack of differentiation and polyploidization in absence of IL11 signaling.

A third decidual target of IL11 found in this investigation is BIRC5 (Survivin), which is normally expressed in human endometrium and decidua [30, 31] and is stimulated by IL11 in other cell types [24, 25]. BIRC5 (Survivin) is a cell survival gene discovered by Ambrosini et al. in 1997 [32] and cloned in the mouse in 1999 [33]. BIRC5 (Survivin) is proposed to function as a mitotic regulator and as cell death inhibitor during development and pathogenesis. As such, BIRC5 (Survivin) has attracted great interest in both basic and translational research. BIRC5 (Survivin) acts as a subunit of the chromosomal passenger complex and is essential for chromosome segregation and cytokinesis. Recent findings indicate that interaction of BIRC5 (Survivin) with several proteins, such as Crm1 [34, 35] and HBXIP [36], seems to be required for its cytoprotective activity. BIRC5 (Survivin) is expressed and regulated in normal tissues characterized by self-renewal and proliferation, such as the decidua. The signaling pathways that activate BIRC5 (Survivin) expression are not yet totally characterized. Activation of STAT3 was, however, shown to upregulate BIRC5 (Survivin) expression [25, 37–39]. Our recent finding that IL11 does activate Jak2 and STAT3 in decidual cells [21] suggests strongly that IL11 stimulation of BIRC5 (Survivin) may involve this pathway. Interestingly, repression of cyclin D3 expression was reported to reduce BIRC5 (Survivin) levels profoundly [40]. It is therefore possible that the very low levels of BIRC5 (Survivin) in Il11ra null mice are due to lack of IL11 signaling and the low cyclin D3 expression.

Taken together, results of this investigation provide one explanation for the defect in the normal formation of the decidua and infertility of the Il11ra null mice and demonstrate for the first time that cyclin D3, CDKN1A (p21), and BIRC5 (Survivin) are downstream targets of IL11. In the absence of Il11ra, IL11 becomes unable to stimulate these proteins, which appears to be crucial for the normal growth, differentiation, and survival of the decidua and for maintenance of normal pregnancy.

ACKNOWLEDGMENTS

We are grateful to Dr. L. Robb (The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia) for providing us with Il11ra null mice.

FOOTNOTES

3These authors contributed equally to this work. Back

4Current address: Departments of Obstetrics and Gynecology, University of Kentucky, Lexington, KY 40536. Back

1Supported by National Institutes of Health grants HD12356, U54 HD40093, and HD 11119. Back

Correspondence: 2Geula Gibori, Department of Physiology and Biophysics (M/C 901), University of Illinois at Chicago, 835 South Wolcott Ave., Chicago, IL 60612. FAX: 312 413 0159; e-mail: ggibori{at}uic.edu

Received: 5 June 2007.

First decision: 29 June 2007.

Accepted: 14 September 2007.

REFERENCES

  1. Psychoyos A. Hormonal control of ovoimplantation Vitam Horm 1973 31201–256[Medline]
  2. J Reprod Fertil Suppl 1976 Psychoyos A. Hormonal control of uterine receptivity for nidation 17–28
  3. Bagchi IC, Li Q, Cheon YP. Role of steroid hormone-regulated genes in implantation Ann N Y Acad Sci 2001 94368–76[Medline]
  4. Wang H and Dey SK. Roadmap to embryo implantation: clues from mouse models Nat Rev Genet 2006 7185–199[CrossRef][Medline]
  5. Mantena SR, Kannan A, Cheon YP, Li Q, Johnson PF, Bagchi IC, Bagchi MK. C/EBPbeta is a critical mediator of steroid hormone-regulated cell proliferation and differentiation in the uterine epithelium and stroma Proc Natl Acad Sci U S A 2006 1031870–1875[Abstract/Free Full Text]
  6. Du X and Williams DA. Interleukin-11: review of molecular, cell biology, and clinical use Blood 1997 893897–3908[Free Full Text]
  7. Schwertschlag US, Trepicchio WL, Dykstra KH, Keith JC, Turner KJ, Dorner AJ. Hematopoietic, immunomodulatory and epithelial effects of interleukin-11 Leukemia 1999 131307–1315[CrossRef][Medline]
  8. Jenkins BJ, Roberts AW, Greenhill CJ, Najdovska M, Lundgren-May T, Robb L, Grail D, Ernst M. Pathologic consequences of STAT3 hyperactivation by IL-6 and IL-11 during hematopoiesis and lymphopoiesis Blood 2007 1092380–2388[Abstract/Free Full Text]
  9. Robb L, Li R, Hartley L, Nandurkar HH, Koentgen F, Begley CG. Infertility in female mice lacking the receptor for interleukin 11 is due to a defective uterine response to implantation Nat Med 1998 4303–308[CrossRef][Medline]
  10. Bilinski P, Roopenian D, Gossler A. Maternal IL-11Ralpha function is required for normal decidua and fetoplacental development in mice Genes Dev 1998 122234–2243[Abstract/Free Full Text]
  11. O'Shea JD, Kleinfeld RG, Morrow HA. Ultrastructure of decidualization in the pseudopregnant rat Am J Anat 1983 166271–298[CrossRef][Medline]
  12. Tan J, Raja S, Davis MK, Tawfik O, Dey SK, Das SK. Evidence for coordinated interaction of cyclin D3 with p21 and cdk6 in directing the development of uterine stromal cell decidualization and polyploidy during implantation Mech Dev 2002 11199–113[CrossRef][Medline]
  13. Tan Y, Li M, Cox S, Davis MK, Tawfik O, Paria BC, Das SK. HB-EGF directs stromal cell polyploidy and decidualization via cyclin D3 during implantation Dev Biol 2004 265181–195[CrossRef][Medline]
  14. Das SK, Lim H, Paria BC, Dey SK. Cyclin D3 in the mouse uterus is associated with the decidualization process during early pregnancy J Mol Endocrinol 1999 2291–101[Abstract]
  15. Dey SK, Lim H, Das SK, Reese J, Paria BC, Daikoku T, Wang H. Molecular cues to implantation Endocr Rev 2004 25341–373[Abstract/Free Full Text]
  16. Cohen H, Pageaux JF, Melinand C, Fayard JM, Laugier C. Normal rat uterine stromal cells in continuous culture: characterization and progestin regulation of growth Eur J Cell Biol 1993 61116–125[Medline]
  17. Tessier-Prigent A, Willems R, Lagarde M, Garrone R, Cohen H. Arachidonic acid induces differentiation of uterine stromal to decidual cells Eur J Cell Biol 1999 78398–406[Medline]
  18. Tessier C, Deb S, Prigent-Tessier A, Ferguson-Gottschall S, Gibori GB, Shiu RP, Gibori G. Estrogen receptors alpha and beta in rat decidua cells: cell-specific expression and differential regulation by steroid hormones and prolactin Endocrinology 2000 1413842–3851[Abstract/Free Full Text]
  19. Frasor J, Barkai U, Zhong L, Fazleabas AT, Gibori G. PRL-induced ERalpha gene expression is mediated by Janus kinase 2 (Jak2) while signal transducer and activator of transcription 5b (Stat5b) phosphorylation involves Jak2 and a second tyrosine kinase Mol Endocrinol 2001 151941–1952[Abstract/Free Full Text]
  20. Prigent-Tessier A, Barkai U, Tessier C, Cohen H, Gibori G. Characterization of a rat uterine cell line, U(III) 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 pathway Endocrinology 2001 1421242–1250[Abstract/Free Full Text]
  21. Bao L, Devi S, Bowen-Shauver J, Ferguson-Gottschall S, Robb L, Gibori G. The role of interleukin-11 in pregnancy involves up-regulation of alpha2-macroglobulin gene through janus kinase 2-signal transducer and activator of transcription 3 pathway in the decidua Mol Endocrinol 2006 203240–3250[Abstract/Free Full Text]
  22. Sherr CJ and Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression Genes Dev 1999 131501–1512[Free Full Text]
  23. Nguyen L, Besson A, Heng JI, Schuurmans C, Teboul L, Parras C, Philpott A, Roberts JM, Guillemot F. p27kip1 independently promotes neuronal differentiation and migration in the cerebral cortex Genes Dev 2006 201511–1524[Abstract/Free Full Text]
  24. Kirkiles-Smith NC, Mahboubi K, Plescia J, McNiff JM, Karras J, Schechner JS, Altieri DC, Pober JS. IL-11 protects human microvascular endothelium from alloinjury in vivo by induction of survivin expression J Immunol 2004 1721391–1396[Abstract/Free Full Text]
  25. Mahboubi K, Li F, Plescia J, Kirkiles-Smith NC, Mesri M, Du Y, Carroll JM, Elias JA, Altieri DC, Pober JS. Interleukin-11 up-regulates survivin expression in endothelial cells through a signal transducer and activator of transcription-3 pathway Lab Invest 2001 81327–334[Medline]
  26. Hunter T and Pines J. Cyclins and cancer. II: Cyclin D and CDK inhibitors come of age Cell 1994 79573–582[CrossRef][Medline]
  27. Grana X and Reddy EP. Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs) Oncogene 1995 11211–219[Medline]
  28. Herzinger T and Reed SI. Cyclin D3 is rate-limiting for the G1/S phase transition in fibroblasts J Biol Chem 1998 27314958–14961[Abstract/Free Full Text]
  29. Grossel MJ and Hinds PW. Beyond the cell cycle: a new role for Cdk6 in differentiation J Cell Biochem 2006 97485–493[CrossRef][Medline]
  30. Konno R, Yamakawa H, Utsunomiya H, Ito K, Sato S, Yajima A. Expression of survivin and Bcl-2 in the normal human endometrium Mol Hum Reprod 2000 6529–534[Abstract/Free Full Text]
  31. Li H, Yang J, Sun Y. Expression of survivin in early villus and decidua and its implication J Huazhong Univ Sci Technolog Med Sci 2002 22170–120
  32. Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma Nat Med 1997 3917–921[CrossRef][Medline]
  33. Li F and Altieri DC. The cancer antiapoptosis mouse survivin gene: characterization of locus and transcriptional requirements of basal and cell cycle-dependent expression Cancer Res 1999 593143–3151[Abstract/Free Full Text]
  34. Knauer SK, Mann W, Stauber RH. Survivin's dual role: an export's view Cell Cycle 2007 6518–521[Medline]
  35. Song Z, Yao X, Wu M. Direct interaction between survivin and Smac/DIABLO is essential for the anti-apoptotic activity of survivin during taxol-induced apoptosis J Biol Chem 2003 27823130–23140[Abstract/Free Full Text]
  36. Marusawa H, Matsuzawa S, Welsh K, Zou H, Armstrong R, Tamm I, Reed JC. HBXIP functions as a cofactor of survivin in apoptosis suppression EMBO J 2003 222729–2740[CrossRef][Medline]
  37. Fulda S and Debatin KM. Sensitization for anticancer drug-induced apoptosis by the chemopreventive agent resveratrol Oncogene 2004 236702–6711[CrossRef][Medline]
  38. Abu-El-Asrar AM, Dralands L, Missotten L, Al-Jadaan IA, Geboes K. Expression of apoptosis markers in the retinas of human subjects with diabetes Invest Ophthalmol Vis Sci 2004 452760–2766[Abstract/Free Full Text]
  39. Aoki Y, Feldman GM, Tosato G. Inhibition of STAT3 signaling induces apoptosis and decreases survivin expression in primary effusion lymphoma Blood 2003 1011535–1542[Abstract/Free Full Text]
  40. Lu M, Kwan T, Yu C, Chen F, Freedman B, Schafer JM, Lee EJ, Jameson JL, Jordan VC, Cryns VL. Peroxisome proliferator-activated receptor gamma agonists promote TRAIL-induced apoptosis by reducing survivin levels via cyclin D3 repression and cell cycle arrest J Biol Chem 2005 2806742–6751[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
78/1/127    most recent
biolreprod.107.063313v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, F.
Right arrow Articles by Gibori, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, F.
Right arrow Articles by Gibori, G.
Agricola
Right arrow Articles by Li, F.
Right arrow Articles by Gibori, G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS