Differentiation of human endometrial stromal cells (HESCs) into decidual cells is associated with induction of the forkhead transcription factor forkhead box O1A (FOXO1). We performed a genomic screen to identify decidua-specific genes under FOXO1 control. Primary HESCs were transfected with small interfering RNA targeting FOXO1 or with nontargeting control small interfering RNA before treatment with a cAMP analogue and the progestin, medroxyprogesterone acetate for 72 h. Total RNA was processed for whole genome analysis using high-density oligonucleotide arrays. We identified 3405 significantly regulated genes upon decidualization of HESCs, 507 (15.3%) of which were aberrantly expressed upon FOXO1 knockdown. Among the most up-regulated FOXO1-dependent transcriptional targets were WNT signaling-related genes (WNT4, WNT16 ), the insulin receptor (INSR), differentiation markers (PRL, IGFBP1, and LEFTY2), and the cyclin-dependent kinase inhibitor p57(Kip2) (CDKN1C). Analysis of FOXO1-dependent down-regulated genes uncovered several factors involved in cell cycle regulation, including CCNB1, CCNB2, MCM5, CDC2 and NEK2. Cell viability assay and cell cycle analysis demonstrated that FOXO1 silencing promotes proliferation of differentiating HESCs. Using a glutathione-S-transferase pull-down assay, we confirmed that FOXO1 interacts with progesterone receptor, irrespectively of the presence of ligand. In agreement, knockdown of PR disrupted the regulation of FOXO1 target genes involved in differentiation (IGFBP1, PRL, and WNT4) and cell cycle regulation (CDKN1, CCNB2 and CDC2) in HESCs treated with either cAMP plus medroxyprogesterone acetate or with cAMP alone. Together, the data demonstrate that FOXO1 engages in transcriptional cross talk with progesterone receptor to coordinate cell cycle regulation and differentiation of HESCs.

Cell cycle arrest by FoxO transcription factors involves transcriptional repression of cyclin D, although the exact mechanism remains unclear. In this study, we used the BCR-ABL-expressing cell line BV173 as a model system to investigate the mechanisms whereby FoxO3a regulates cyclin D2 expression. Inhibition of BCR-ABL by STI571 results in down-regulation of cyclin D2 expression, activation of FoxO3a activity, and up-regulation of BCL6 expression. Using reporter gene assays, we demonstrate that STI571, FoxO3a, and BCL6 can repress cyclin D2 transcription through a STAT5/BCL6 site located within the cyclin D2 promoter. We propose that BCR-ABL inhibition leads to FoxO3a activation, which in turn induces the expression of BCL6, culminating in the repression of cyclin D2 transcription through this STAT5/BCL6 site. This process was verified by mobility shift and chromatin immunoprecipitation analyses. We find that conditional activation of FoxO3a leads to accumulation of BCL6 and down-regulation of cyclin D2 at protein and mRNA levels. Furthermore, silencing of FoxO3a and BCL6 in BCR-ABL-expressing cells abolishes STI571-mediated effects on cyclin D2. This report establishes the signaling events whereby BCR-ABL signals are relayed to cyclin D2 to mediate cell cycle progression and defines a potential mechanism by which FoxO proteins regulate cyclin D2 expression.In mammalian cells, the commitment to divide is made in the G 1 phase of the cell cycle in response to various stimuli, including growth factors. After passing the restriction point at mid-to late G 1 , cells become refractory to growth inhibition signals or do not require growth factors to progress into S phase (37). Progression of eukaryotic cells through the cell cycle is controlled by the two families of G 1 cyclins: (i) D-type cyclins (cyclins D1, D2, and D3) and cyclin E (cyclins E1 and E2) (29, 44) and (ii) the cyclin-dependent kinases (cdk's), their catalytic counterparts. The primary targets of the G 1 cyclin-cdk complexes are the retinoblastoma protein (pRb) family of pocket proteins, consisting of pRb, p107, and p130 (20,27,34). The phosphorylation state of pRb regulates the activity of the E2F family of transcription factors; in their hypophosphorylated forms, the pRb-related pocket proteins associate with members of the E2F family, negatively regulating transcription activity of E2F-regulated genes that are required for entry into the S phase of the cell cycle (15,35,40).In mammals, the phosphatidylinositol 3-kinase/protein kinase B (PI3-K/PKB) pathway is stimulated by a variety of growth factors and cytokines and by cell-matrix interactions, and it controls many biological functions, including cell proliferation, cell survival, and insulin responses (30). Importantly, constitutive activation of the PI3-K pathway facilitates tumor formation by two different mechanisms: it supports S-phase entry, and it confers resistance to apoptotic signals which normally restrict uncontrolled cell growth (49). Recently, it has been demonstrated that the member...

Menstruation, or cyclic shedding of nonpregnant endometrial tissue with associated bleeding, occurs only in humans and a few other species. This breakdown of the endometrium in response to falling ovarian progesterone levels is a complex process, characterized by local leukocyte infiltration, expression and activation of matrix metalloproteinases, and apoptosis. Spontaneous decidualization (differentiation) of the stromal compartment precedes the cyclic shedding of the endometrium in various menstruating species but the mechanisms that link these processes are not understood. In this study, we identified FOXO1 as a key transcription factor responsible for mediating apoptosis of decidualized human endometrial stromal cells (HESCs) in response to progesterone withdrawal. We demonstrate that medroxyprogesterone acetate (MPA, a synthetic progestin) enhances the expression of FOXO1 in differentiating HESCs while simultaneously inducing cytoplasmic retention and inactivation of FOXO1. Withdrawal of MPA from decidualized HESCs results in rapid nuclear accumulation of FOXO1, increased BIM expression, a proapoptotic FOXO1 target gene, and cell death. Conversely, silencing of FOXO1 expression completely abolishes cell death induced by MPA withdrawal. In summary, the observation that differentiating HESCs become dependent on progesterone signaling for survival through induction and reversible inactivation of FOXO1 suggests a novel mechanism that links decidualization of the endometrium to menstruation.

Heparin is used clinically for the prevention of pregnancy complications associated with prothrombotic disorders, especially antiphospholipid antibody syndrome. Recent studies have suggested that heparin may exert direct effects on placental trophoblast, independently of its anticoagulant activity. We now demonstrate that heparin abrogates apoptosis of primary first trimester villous trophoblast in response to treatment with the pro-inflammatory cytokines interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha. This multifunctional glycosaminoglycan also inhibited apoptosis induced by other agents, including staurosporin, broad-spectrum kinase inhibitor and thrombin. Furthermore, heparin attenuated caspase-3 activity, a hallmark of apoptosis, in human first trimester villous and extravillous trophoblast cell lines treated with peptidoglycan, a Toll-like receptor-2 agonist isolated from Staphylococcus aureus. The ability of heparin to antagonize cell death induced by such diverse apoptotic signals suggested that it acts as a survival factor for human trophoblast. We demonstrate that heparin, like epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), elicits phosphorylation of the EGF receptor and activation of the phosphatidyl inositol 3-kinase (PI3K)-, the extracellular signal-related kinase 1/2 (ERK1/2)- and the c-Jun NH2 terminal kinase (JNK)-signal transduction pathways in primary villous trophoblast. In summary, we have demonstrated that heparin activates multiple anti-apoptotic pathways in human trophoblast. Our results suggest that heparin may be useful in the management of at-risk patients, even in the absence of an identifiable thrombophilic disorder.

Antiphospholipid syndrome (APS), characterized by circulating antiphospholipid (aPL) antibodies, is a major cause of early pregnancy failure and placental insufficiency. In this study, we examined whether impaired endometrial differentiation before conception contributes to the high incidence of pregnancy complications in APS. Timed secretory endometrial biopsies were obtained from a cohort of women with recurrent pregnancy loss (RPL). Real-time quantitative (RTQ)-PCR was used to determine the expression levels of transcripts that encode for decidual markers, proinflammatory cytokines and complement regulatory proteins. Expression of decidual markers such as prolactin (PRL), tissue factor (TF) and signal transducer and activator of transcription 5 (Stat5), but not insulin-like growth factor-binding protein 1 (IGFBP-1), was significantly lower in samples obtained from aPL(+) patients (n = 24) when compared with aPL(-) group (n = 58) (P < 0.05). The abundance of transcripts encoding for interferon gamma (IFNgamma), tumour necrosis factor alpha (TNFalpha) or Stat1 did not differ significantly between both groups (P >/= 0.05). However, analysis of transcripts that encode for complement regulatory proteins showed a marked decrease in decay-accelerating factor (DAF/CD55) levels in aPL(+) patients (P = 0.005), which was mimicked at protein level as demonstrated by immunohistochemistry. In summary, patients with RPL have distinct endometrial gene expression profiles depending on the presence or absence of circulating aPL antibodies. In APS, impaired endometrial differentiation and lower DAF/CD55 expression before conception may compromise implantation and predispose to complement-mediated pregnancy failure.

Chronic myeloid leukaemia is a haemopoietic stem cell disorder, the hallmark of which is the expression of the Bcr-Abl Protein Tyrosine Kinase (PTK). We have previously reported that activation of a temperature sensitive Bcr-Abl PTK in the multipotent haemopoietic cell line FDCP-Mix for short periods resulted in subtle changes including, a transient suppression of apoptosis and no inhibition of di erentiation. In contrast, activation of the Bcr-Abl PTK for 12 weeks results in cells that display a delay in di erentiation at the early granulocyte stage. Flow cytometric analysis also indicates that the expression of cell surface di erentiation markers and nuclear morphology are uncoupled. Furthermore, a signi®cant number of the mature neutrophils display abnormal morphological features. Prolonged exposure to Bcr-Abl PTK results in interleukin-3 independent growth and decreased p53 protein levels. FDCP-Mix cells expressing a dominant negative p53 and p53 null FDCP-Mix cells demonstrate that the reduction in p53 is causally related to the delay in development. Returning the cells to the restrictive temperature restores the p53 protein levels, the growth factor dependence and largely relieves the e ects on development. We conclude that prolonged Bcr-Abl PTK activity within multipotent cells results in a reduction of p53 that drives a delayed and abnormal di erentiation. Oncogene (2000) 19, 5487 ± 5497.

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All cardinal events during the reproductive cycle, including ovulation, implantation, and menstruation, are characterized by a profound tissue remodeling and an associated local inflammatory response. The ovarian hormone progesterone is a key modulator of inflammatory signals in reproductive tissues, but the underlying mechanisms are not well understood. In this study, we report that differentiating human endometrial stromal cells (ESCs) acquire resistance to interferon-gamma (IFNgamma)-dependent signal transducers and activators of transcription (STAT) 1 signaling, although phosphorylation, nuclear translocation, and binding of STAT1 to DNA, are unaffected. These observations prompted an investigation into the role of nuclear repressors of STAT1 signaling. We demonstrate that protein inhibitor of activated STAT-y is complexed to the progesterone receptor (PR) in human ESCs and that its ability to repress STAT1 signaling is dependent upon activation of PR in response to hormone binding. Conversely, IFNgamma and protein inhibitor of activated STAT-y synergistically inhibited PR-dependent transcription, demonstrating that the progesterone and IFNgamma signaling pathways engage in reciprocal transcriptional antagonism in human endometrium.