Evidence for coordinated interaction of cyclin D3 with p21 and cdk6 in directing the development of uterine stromal cell decidualization and polyploidy during implantation.

Uterine decidualization, characterized by stromal cell proliferation, and differentiation into specialized type of cells (decidual cells) with polyploidy, during implantation is critical to the pregnancy establishment in mice. The mechanisms by which the cell cycle events govern these processes are poorly understood. The cell cycle is tightly regulated at two particular checkpoints, G1-S and G2-M phases. Normal operation of these phases involves a complex interplay of cyclins, cyclin-dependent kinases (cdks) and cdk inhibitors (CKIs). We previously observed that upregulation of uterine cyclin D3 at the implantation site is tightly associated with decidualization in mice. To better understand the role of cyclin D3 in this process, we examined cell-specific expression and associated interactions of several cell cycle regulators (cyclins, cdks and CKIs) specific to different phases of the cell cycle during decidualization in mice. Among the various cell cycle molecules examined, coordinate expression and functional association of cyclin D3 with cdk4 suggest a role for proliferation and, that of cyclin D3 with p21 and cdk6 is consistent with the development of polyploidy during stromal cell decidualization.     

Maternal Smad3 deficiency compromises decidualization in mice

Transforming growth factor (TGF)‐β and activin, members of TGF‐β superfamily, are abundantly expressed in the endometrium and regulate decidualization of endometrial stroma. Smad2 and Smad3 are receptor‐regulated Smads (R‐Smads) that transduce extracellular TGF‐β/activin/Nodal signaling. In situ hybridization results showed that Smad3 was highly expressed in the decidual zone during the peri‐implantation period in mice. By using artificial decidualization, we found that Smad3 null mice showed partially compromised decidualization. We therefore hypothesized that Smad2 might compensate for the function of Smad3 during the process of decidualization. Smad2 was also highly expressed in the decidual zone and phosphorylated Smad2 was much more abundantly increased in the deciduoma of Smad3 null mice than for wild‐type (WT) mice. We further employed an in vitro uterine stromal cell decidualization model, and found that decidual prolactin‐related protein (dPRP) and cyclin D3, which are well‐known markers for decidual cells, were significantly down‐regulated in Smad3 null decidual cells, and were much more significantly reduced when the expression of Smad2 was simultaneously silenced by its siRNA (P < 0.05). However, the expression levels of dPRP and cyclin D3 remained the same when Smad2 was silenced in WT decidual cells. Collectively, these findings provide evidence for an important role of Smad3 in decidualization and suggest that Smad2 and Smad3 may have redundant roles in decidualization. J. Cell. Biochem. 113: 3266–3275, 2012. © 2012 Wiley Periodicals, Inc.     

Adam12 plays a role during uterine decidualization in mice

In mouse, decidualization is characterized by the proliferation of stromal cells and their differentiation into specialized type of cells (decidual cells) with polyploidy, surrounding the implanting blastocyst. However, the mechanisms involved in these processes remain poorly understood. Using multiple approaches, we have examined the role of Adam12 in decidualization during early pregnancy in mice. Adam12 is spatiotemporally expressed in decidualizing stromal cells in intact pregnant females and in pseudopregnant mice undergoing artificially induced decidualization. In the ovariectomized mouse uterus, the expression of Adam12 is upregulated after progesterone treatment, which is primarily mediated by nuclear progesterone receptor. In a stromal cell culture model, the expression of Adam12 gradually rises with the progression of stromal decidualization, whereas the attenuated expression of Adam12 after siRNA knockdown significantly blocks the progression of decidualization. Our study suggests that Adam12 is involved in promoting uterine decidualization during pregnancy.     

Progesterone and DNA damage encourage uterine cell proliferation and decidualization through up-regulating ribonucleotide reductase 2 expression during early pregnancy in mice

Embryo implantation into the maternal uterus is a crucial step for the successful establishment of mammalian pregnancy. Following the attachment of embryo to the uterine luminal epithelium, uterine stromal cells undergo steroid hormone-dependent decidualization, which is characterized by stromal cell proliferation and differentiation. The mechanisms underlying steroid hormone-induced stromal cell proliferation and differentiation during decidualization are still poorly understood. Ribonucleotide reductase, consisting of two subunits (RRM1 and RRM2), is a rate-limiting enzyme in deoxynucleotide production for DNA synthesis and plays an important role in cell proliferation and tumorgenicity. Based on our microarray analysis, Rrm2 expression was significantly higher at implantation sites compared with interimplantation sites in mouse uterus. However, the expression, regulation, and function of RRM2 in mouse uterus during embryo implantation and decidualization are still unknown. Here we show that although both RRM1 and RRM2 expression are markedly induced in mouse uterine stromal cells undergoing decidualization, only RRM2 is regulated by progesterone, a key regulator of decidualization. Further studies showed that the induction of progesterone on RRM2 expression in stromal cells is mediated by the AKT/c-MYC pathway. RRM2 can also be induced by replication stress and DNA damage during decidualization through the ATR/ATM-CHK1-E2F1 pathway. The weight of implantation sites and deciduoma was effectively reduced by specific inhibitors for RRM2. The expression of decidual/trophoblast prolactin-related protein (Dtprp), a reliable marker for decidualization in mice, was significantly reduced in deciduoma and steroid-induced decidual cells after HU treatment. Therefore, RRM2 may be an important effector of progesterone signaling to induce cell proliferation and decidualization in mouse uterus.     

Lefty inhibits in vitro decidualization by regulating P57 and cyclin D1 expressions

Endometrial decidualization is highly important for successful construction and maintenance of embryo implantation and pregnancy. Lefty gene at different menstrual cycle phases has different expressions, indicating its regulatory significance. To study the mechanism of Lefty in decidualization, human endometrial stromal cells (hESCs) were cultured and induced with medroxyprogesterone acetate (MPA) and 8‐bromoadenosine‐cAMP (8‐Br‐cAMP) in vitro as a research model. Our results showed that Lefty1 overexpression inhibited MPA‐ and 8‐Br‐cAMP‐induced hESC decidualization and significantly reduced the secretion of prolactin (PRL) and insulin‐like growth factor‐binding protein 1 (IGFBP‐1). With the inhibition of Lefty1 expression, hESC decidualization induced by MPA and 8‐Br‐cAMP became more remarkable, and the secretions of PRL and IGFBP‐1 were higher too. Further tests indicated that during the process of decidualization, P57 expression increased, whereas cyclin D1 expression decreased. Although Lefty1 overexpression did not significantly change the expressions of P57 and cyclin D1, inhibition of Lefty1 expression resulted in more evident changes in P57 and cyclin D1 expressions. Meanwhile, cell cycle examination showed that Lefty1 overexpression reduced the cell cycle arrest at G1/S phase in the in vitro hESC decidualization model. Therefore, Lefty1 could regulate the cell cycle via modulating the expressions of P57 and cyclin D1 and then inhibit the decidualization in vitro. Copyright © 2014 John Wiley & Sons, Ltd.     

Ovarian steroid receptors and activated MAPK in the regional decidualization in rats

Though the decidua serves a critical function in implantation, the hormonal regulated pathway in decidualization is still elusive. Here we describe in detail the regional distribution and the effects of progesterone receptors (PGR), estrogen receptors (ESR), and MAPK activation on decidualization. We showed an increase in PGR A, PGR B, ESR1, and phosphorylated MAPK3-1 proteins (p-MAPK3-1), but not in ESR2, in the decidual tissue up to Day 8 of pregnancy. PGR was predominantly found in the nuclei of mesometrial decidual cells and of undifferentiated stromal cells where it colocalizes with ESR2 and ESR1. In the antimesometrial decidua, all the receptors showed cytoplasmic localization. MAPK was activated exclusively in undifferentiated stromal cells of the junctional zone between the antimesometrial and mesometrial decidua and at the border of the antimesometrial decidua. Treatment with the progesterone antagonist onapristone and/or the estrogen antagonist faslodex reduced the extent of decidual tissue and downregulated the levels of PGR and ESR1. The expression level of ESR2 was affected only by the progesterone receptor antagonist, while neither the antiprogestin nor the antiestrogen significantly modified the p-MAPK3-1 level. The inhibition of MAPK3-1 phosphorylation by PD98059 impaired the extent of decidualization and the closure reaction of the implantation chamber, and significantly downregulated ESR1. These results confirm a role of both steroid receptors in the growth and differentiation of the different decidual regions and suggest a new function for p-MAPK3-1 in regulating expression levels of ESR1, thereby maintaining the proliferation capacity of stromal cells and limiting the differentiation process in specified regions of decidual tissues.     

Anandamide targets aromatase: A breakthrough on human decidualization

In each menstrual cycle endometrial stromal cells (hESC) proliferate and differentiate into specialized decidual cells, a process termed decidualization, which regulates endometrial receptivity. Decidualization is mainly controlled by sex ovarian hormones, estradiol (E₂) and progesterone. E₂ plays an important role in the expression of the progesterone receptor and promotes the endometrial stromal cells differentiation. Our group previously reported that anandamide (AEA) impairs decidualization through cannabinoid receptor 1 (CB1). In this study, we hypothesized whether AEA inhibitory effect on cell decidualization could be mediated through interaction with aromatase and consequent interference in estradiol production/signaling. We used an immortalized human endometrial stromal cell line (St-T1b) and human decidual fibroblasts (HdF) derived from human term placenta. In cells exposed to a differentiation stimulus, AEA-treatment prevents the increase of the expression of CYP19A1 gene encoding aromatase, E₂ levels and of estradiol receptor expression, that are observed in differentiating cells. Regarding CYP19A1 mRNA levels, the effect was partially reverted by a CB1 receptor antagonist and by a COX2 inhibitor. In addition, we report that AEA presents anti-aromatase activity in placental microsomes, the nature of the inhibition being the uncommon mixed type as revealed by the kinetic studies. Structural analysis of the AEA-Aromatase complexes determined that AEA may bind to the active site pocket of the enzyme. In overall we report that AEA inhibits aromatase activity and may affect E₂ signaling crucial for the decidualization process, indicating that a deregulation of the endocannabinoid system may be implicated in endometrial dysfunction and in fertility/infertility disorders.     

Cyclin D1 Gene Silencing Promotes IL‐1β‐Induced Apoptosis in Rat Chondrocytes

This study investigated the effects of cyclin D1 gene silencing on cell proliferation and apoptosis of interleukin‐1β (IL‐1β)‐induced osteoarthritis (OA) chondrocytes. Chondrocytes from healthy sprague‐dawley rats were divided into blank, OA model (chondrocytes underwent IL‐1β inducement), OA trial (chondrocytes underwent IL‐1β inducement with cyclin D1‐shRNA treatment), and negative control (NC; chondrocytes underwent IL‐1β inducement and control‐shRNA treatment) groups. Cell proliferation was assessed by CCK‐8 assay, and cell cycle and apoptosis by flow cytometry. qRT‐PCR and Western blotting were performed to detect cyclin D1 and apoptosis‐related factors expression levels. Chondrocyte proliferation increased after 72–96 h after incubation. The OA trial group exhibited reduced cell proliferation at 48, 72, and 96 h after treatment. The OA model, OA trial, and NC groups all contained more cells arrested in G1 phase and had higher apoptosis rates than the blank group. Additionally, the OA trial group contained more cells arrested in G1 phase, with increased apoptosis rates compared to the OA model and NC groups. The OA model group had lowest expression of cyclin D1 whereas the blank group contained the highest among the four groups. qRT‐PCR also showed that the OA model, OA trial, and NC groups all had increased expression levels of Bax and reduced expression levels of Bcl‐2 and P53 compared to the blank group, whereby by the OA group had the most significant change. The combined evidence in our study shows that cyclin D1 gene silencing suppresses proliferation and induces apoptosis of rat chondrocytes in IL‐1β‐induced OA. J. Cell. Biochem. 119: 290–299, 2018. © 2017 Wiley Periodicals, Inc.     

Notch1 mediates uterine stromal differentiation and is critical for complete decidualization in the mouse

Uterine receptivity implies a dialogue between the hormonally primed maternal endometrium and the free-floating blastocyst. Endometrial stromal cells proliferate, avert apoptosis, and undergo decidualization in preparation for implantation; however, the molecular mechanisms that underlie differentiation into the decidual phenotype remain largely undefined. The Notch family of transmembrane receptors transduce extracellular signals responsible for cell survival, cell-to-cell communication, and differentiation, all fundamental processes for decidualization and pregnancy. Using a murine artificial decidualization model, pharmacological inhibition of Notch signaling by γ-secretase inhibition resulted in a significantly decreased deciduoma. Furthermore, a progesterone receptor (PR)-Cre Notch1 bigenic (Notch1(d/d)) confirmed a Notch1-dependent hypomorphic decidual phenotype. Microarray and pathway analysis, following Notch1 ablation, demonstrated significantly altered signaling repertoire. Concomitantly, hierarchical clustering demonstrated Notch1-dependent differences in gene expression. Uteri deprived of Notch1 signaling demonstrated decreased cellular proliferation; namely, reduced proliferation-specific antigen, Ki67, altered p21, cdk6, and cyclinD activity and an increased apoptotic-profile, cleaved caspase-3, Bad, and attenuated Bcl2. The results demonstrate that the preimplantation uterus relies on Notch signaling to inhibit apoptosis of stromal fibroblasts and regulate cell cycle progression, which together promotes successful decidualization. In summary, Notch1 signaling modulates multiple signaling mechanisms crucial for decidualization and these studies provide additional perspectives to the coordination of multiple signaling modalities required during decidualization.     

Dysregulated glycolysis underpins high-fat-associated endometrial decidualization impairment during early pregnancy in mice

Pregnancy complications are more likely to occur in obese women because of defective decidualization. However, the specific mechanism of glycolysis in decidual modulation associated with obesity remains unknown. Therefore, we explored the role of glycolysis in the endometrium of obese pregnant mice during decidualization. C57BL/6J mice were fed a high-fat diet (HFD) to induce obesity. All obesity related parameters were significantly higher in the HFD mice than control. Furthermore, the HFD mice had fewer implantation sites, a smaller decidual area growth, and decreased decidualization marker protein expression than control. The HFD mice also had significantly decreased lactate production and glycolytic enzyme expression. To confirm the functional role of glycolysis during the decidual period in obese pregnant mice, we extracted endometrial stromal cells (ESCs) and treated them with oleic acid (OA) and palmitic acid (PA) to mimic a high-fat environment. Decidualization and glycolysis were significantly restricted in the OA-and PA-treated groups. Moreover, we administered a glycolytic inhibitor, 2-DG, and an agonist, pioglitazone. 2-DG treatment considerably decreased the cells' glycolysis and decidualization. However, pioglitazone treatment improved glycolysis and alleviated defective decidualization. In conclusion, obesity-induced endometrial glycolysis modifications and key glycolytic enzyme downregulation during early pregnancy might cause abnormal decidualization, leading to an unsustainable pregnancy.     

Involvement of cyclin D3, CDKN1A (p21), and BIRC5 (Survivin) in interleukin 11 stimulation of decidualization in mice

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.     

Bone morphogenetic protein 2 functions via a conserved signaling pathway involving Wnt4 to regulate uterine decidualization in the mouse and the human

A critical role of progesterone (P) during early pregnancy is to induce differentiation of the endometrial stromal cells into specialized decidual cells that support the development of the implanting embryo. The P-induced signaling pathways that participate in the formation and function of the decidual cells remain poorly understood. We report here that the expression of the bone morphogenetic protein 2 (BMP2), a morphogen belonging to the TGFbeta superfamily, is induced downstream of P action in the mouse uterine stroma during decidualization. To determine the function of BMP2 during this differentiation process, we employed a primary culture system in which undifferentiated stromal cells isolated from pregnant mouse uterus undergo decidualization. When recombinant BMP2 was added to these stromal cultures, it markedly advanced the differentiation program. We also found that siRNA-mediated silencing of BMP2 expression in these cells efficiently blocked the differentiation process. Gene expression profiling experiments identified Wnt4 as a downstream target of BMP2 regulation in stromal cells undergoing decidualization. Attenuation of Wnt4 expression by siRNAs greatly reduced stromal differentiation in vitro, indicating that it is a key mediator of BMP2-induced decidualization. We also observed a remarkable induction in the expression of BMP2 in human endometrial stromal cells during decidualization in vitro in response to steroids and cAMP. Addition of BMP2 to these cultures led to a robust enhancement of Wnt4 expression and stimulated the differentiation process. Collectively, our studies uncovered a unique conserved pathway involving BMP2 and Wnt4 that mediates P-induced stromal decidualization in the mouse and the human.     

Expression of stathmin family genes in the murine uterus during early pregnancy

Stathmin, a cytosolic phosphoprotein that regulates microtubule dynamics during cell-cycle progression, is abundantly expressed at embryo implantation sites in rats. Here, we characterized the expression of stathmin and its family genes in the murine uterus during the peri-implantation period. Stathmin protein was expressed in the glandular and luminal epithelium, blood vessels, and stromal cells on day 3 of pregnancy. On the day of implantation (day 5), stathmin was mainly localized in blood vessels in the endometrium. On day 7, intense stathmin expression was limited to capillary vessels and secondary decidual cells. Stathmin expression was higher at implantation sites than at uterine segments between implantation sites and increased during oil-induced decidualization. Although the artificially-induced deciduoma weights and number of implantation sites were similar between stathmin-knockout (KO) and wild-type (WT) mice, the stathmin-KO mice had fewer newborn pups (reduced by 30%). The expression of alkaline phosphatase, desmin, and cyclin D3 was attenuated in decidual zones of stathmin-KO mice. Messenger RNA level of the stathmin family gene, SCG10, was high at the time of decidualization in WT and stathmin-KO mice. In contrast, the others of stathmin family members, SCLIP and RB3 were highly expressed in stathmin-KO mice compared to WT mice. These results suggest that stathmin and stathmin family genes are expressed in the murine endometrium with enhanced expression in the implantation or the decidualization process.