ETS1 induces human trophoblast differentiation.

A possible role for the transcription factor v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS1) in human trophoblast cell differentiation was examined using a highly enriched fraction of human mononuclear cytotrophoblast cells (CTBs) that differentiate spontaneously in vitro to a multinucleated syncytiotrophoblast cell (STB) phenotype. ETS1 mRNA and protein levels were abundant in freshly isolated CTBs and decreased as the cells differentiated. Silencing of ETS1 expression in freshly prepared CTBs markedly attenuated syncytialization, as demonstrated by desmoplakin staining, and blocked the induction of syncytin, the transcription factor activator protein-2α, placental lactogen, and other STB-specific genes. Conversely, overexpression of ETS1 in primary trophoblast cells induced STB marker gene mRNAs and transactivated each of the gene proximal promoters. Taken together, these findings strongly suggest a critical role for ETS1 in the induction of human villus CTB differentiation. The effect of ETS1 on syncytialization likely results, at least in part, from inhibition of syncytin expression, whereas the induction of STB marker genes likely results in part from transactivation by activator protein-2α.

FOXO1 expression in villous trophoblast of preeclampsia and fetal growth restriction placentas.

Oxidative stress and increased apoptosis are implicated in the pathogenesis of many disorders of pregnancy, including preeclampsia (PE) and fetal growth restriction (FGR). Since the transcription factor FOXO1 (forkhead box protein O1) is implicated in the regulation of a variety of cellular processes, including resistance to oxidative stress, apoptosis and morphogenesis of the placenta, we examined whether FOXO1 expression is abnormal in placentas from patients with PE or FGR. Paracentral sections from grossly unremarkable areas of 9 or 10 placentas each from early third trimester patients (31.7±5.0 weeks) with mild PE, severe PE, FGR and a gestational age-matched comparison group (GA controls) were double immunostained for FOXO1 and E-cadherin, the latter distinguishing villous cytotrophoblast cells (CTB) from syncytiotrophoblast (STB). The numbers of FOXO1-positive and FOXO1 negative STB and CTB nuclei were determined on ten 20x objective fields of each placenta section by three observers who were blinded to the clinical outcome. The results were evaluated by a generalized linear mixed model. In mild PE, FOXO1-positive STB nuclei were significantly decreased in number and FOXO1-negative STB nuclei were increased as compared to GA controls. However, the number of FOXO1-positive and FOXO1-negative CTB nuclei were not significantly changes as compared to GA controls. In severe PE and FGR, the numbers of FOXO-positive and FOXO1-negative STB and CTB were not statistically different from GA controls. Since FOXO1 is critical for placental cellular morphogenesis, abnormal FOXO1 expression may contribute in part to the abnormal trophoblast differentiation in mild PE. The differences in FOXO1 expression in mild and severe PE are consistent with other studies suggesting that the two forms of PE are different disease processes.

Silencing or amplification of endocannabinoid signaling in blastocysts via CB1 compromises trophoblast cell migration.

Endocannabinoid signaling plays key roles in multiple female reproductive events. Previous studies have shown an interesting phenomenon, that mice with either silenced or elevated endocannabinoid signaling via Cnr1 encoding CB(1) show similar defects in several pregnancy events, including preimplantation embryo development. To unravel the downstream signaling of this phenomenon, microarray studies were performed using RNAs collected from WT, Cnr1(-/-), and Faah(-/-) mouse blastocysts on day 4 of pregnancy. The results indicate that about 100 genes show unidirectional changes under either silenced or elevated anandamide signaling via CB(1). Functional enrichment analysis of the microarray data predicted that multiple biological functions and pathways are affected under aberrant endocannabinoid signaling. Among them, genes enriched in cell migration are suppressed in Cnr1(-/-) or Faah(-/-) blastocysts. Cell migration assays validated the prediction of functional enrichment analysis that cell mobility and spreading of either Cnr1(-/-) or Faah(-/-) trophoblast stem cells are compromised. Either silenced or elevated endocannabinoid signaling via CB(1) causes similar changes in downstream targets in preimplantation embryos and trophoblast stem cells. This study provides evidence that a tightly regulated endocannabinoid signaling is critical to normal preimplantation embryo development and migration of trophoblast stem cells.

Abnormal expression of transcription factor activator protein-2α in pathologic placentas.

Recent studies from our laboratory have indicated that the transcription factor activator protein-2α plays a critical role in the differentiation of human villous cytotrophoblast cells to syncytiotrophoblast cells. However, little is known about the expression of activator protein-2α in placentas from pathologic pregnancies. This study compares the expression of activator protein-2α in placentas from high-risk pregnancies to gestational age-matched controls. Paracentral sections from grossly unremarkable areas of 10 placentas from each group of pregnancies complicated by mild preeclampsia, severe preeclampsia, diabetes mellitus, chronic hypertension, and fetal growth restriction and 10 control cases of placentas from normal pregnancies matched for gestational age were double immunostained for activator protein-2α and E-cadherin. The total numbers of cytotrophoblast cells and syncytiotrophoblast nuclei and the numbers of activator protein-2α-positive and activator protein-2α-negative nuclei in both of these cell types were counted by 2 pathologists blinded to disease status, in 10 representative×40 high-power fields for each placenta. Abnormal placental maturation in most of pathologic pregnancies was evidenced by a 1.5- to 1.7-fold lower expression ratio of syncytiotrophoblast cell to cytotrophoblast cell. Activator protein-2α in syncytiotrophoblast cells was lower in mild preeclampsia, diabetes mellitus, hypertension, and fetal growth restriction (P<.0001 in each instance) and was higher by 2-fold in severe preeclampsia, although this increase was not statistically significant (P=.3). Because activator protein-2α has been shown to be critical for villous cytotrophoblast cell differentiation, our findings suggest that abnormalities in the activator protein-2α cascade of transcription factors and/or signaling molecules may contribute to the pathogenesis of the abnormal maturation in placentas in certain types of high-risk pregnancies. The different pattern of activator protein-2α expression in mild and severe preeclampsia clearly suggests that these conditions may have 2 independent pathogenic mechanisms.

Critical role for TWIST1 in the induction of human uterine decidualization.

The importance of the transcription factor TWIST1 for uterine decidualization was examined in human uterine fibroblast (HUF) cells decidualized in vitro with medroxyprogesterone, estradiol (E2), and prostaglandin E2. TWIST1 mRNA levels increased by 6.0- to 6.8-fold during the first 1-2 d of decidualization and remained above predecidualization levels for up to 15 d. Pretreatment of HUF cells with a TWIST1 small interfering RNA (siRNA) for 3 d before the induction of decidualization resulted in less morphologic differentiation than HUF cells pretreated with a nonsilencing control RNA. In addition, the cells pretreated with TWIST1 siRNA expressed 75-95% less IGF binding protein 1, LEFTY2, fibromodulin, laminin, and several other mRNA during decidualization, including the mRNA for the transcription factors forkhead box protein O1 and v-ets-erythroblastosis virus E26, both of which were previously shown to be critical for the induction of decidualization. The HUF cells pretreated with the TWIST1 siRNA also underwent less apoptosis during decidualization than the control cells, as evidenced by a 20% decrease in DNA fragmentation (terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling assay) and a 43-48% decrease in caspase 3, BCL2-associated X protein, and TNF receptor superfamily member 6 mRNA levels. Although the knockdown of TWIST1 expression markedly attenuated the induction of decidualization, overexpression of TWIST1 alone was insufficient to induce the decidualization of HUF cells. Taken together, these findings strongly implicate an essential role for TWIST1 in the initiation of human decidualization and uterine stromal cell apoptosis that occurs upstream of the induction of forkhead box protein O1 and v-ets-erythroblastosis virus E26 mRNA.

Parathyroid hormone-like hormone (PTHLH) represses decidualization of human uterine fibroblast cells by an autocrine/paracrine mechanism.

CONTEXT: Parathyroid hormone-like hormone (PTHLH) is abundantly expressed by human endometrial stromal cells during decidualization. However, the role for PTHLH in the decidualization process is unknown. OBJECTIVE: To examine the effects of PTHLH on the induction and maintenance of decidualization of human uterine fibroblast (HUF) cells in vitro. DESIGN: HUF cells were treated with a PTHLH siRNA or a PTHLH receptor antagonist (bPTH(7-34)) before or after decidualization with medroxyprogesterone acetate (MPA), estradiol (E(2)), and prostaglandin E(2) (PGE(2)). Decidualization was monitored by immunocytochemistry and the induction of decidualization-specific marker genes, including IGFBP-1, prolactin, lefty, and transcription factor FOXO1. RESULTS: HUF cells decidualized after pretreatment with a PTHLH siRNA showed greater morphologic changes of decidualization, greater IGFBP-1 protein, and two- to threefold more IGFBP-1, prolactin, lefty, and FOXO1 mRNAs than cells pretreated with a nonsilencing RNA. The PTHLH siRNA pretreated cells also had 31% less DNA fragmentation (TUNEL assay) and 30-35% less caspase 3 levels during decidualization than cells pretreated treated with nonsilencing RNA. Treatment of HUF cells with PTHLH siRNA or bPTH(7-34) at 9 d after the induction of decidualization also resulted in 2.1- to 3.2-fold greater IGFBP-1, prolactin, lefty, and FOXO1 mRNA levels than that noted in control cells treated with nonsilencing RNA. CONCLUSIONS: These finding strongly suggest that PTHLH represses the induction of human decidualization, stimulates stromal cell apoptosis, and limits the extent of uterine stromal cell differentiation. Because PTHLH and its receptor are expressed by HUF cells and placental cells, the inhibitory effect of PTHLH on decidualization appears to be due, at least in part, to an autocrine/paracrine mechanism.

Involvement of transcription factor NR2F2 in human trophoblast differentiation.

BACKGROUND: During the in vitro differentiation of human villous cytotrophoblast (CTB) cells to a syncytiotrophoblast (STB) phenotype, mRNA levels for the nuclear hormone receptor NR2F2 (ARP-1, COUP-TFII) increase rapidly, reaching a peak at day 1 of differentiation that is 8.8-fold greater than that in undifferentiated CTB cells. To examine whether NR2F2 is involved in the regulation of villous CTB cell differentiation, studies were performed to determine whether NR2F2 regulates the expression of TFAP2A (AP-2alpha), a transcription factor that is critical for the terminal differentiation of these cells to a STB phenotype. METHODOLOGY/PRIMARY FINDINGS: Overexpression of NR2F2 in primary cultures of human CTB cells and JEG-3 human choriocarcinoma cells induced dose-dependent increases in TFAP2A promoter activity. Conversely, siRNA mediated silencing of the NR2F2 gene in villous CTB undergoing spontaneous differentiation blocked the induction of the mRNAs for TFAP2A and several STB cell specific marker genes, including human placental lactogen (hPL), pregnancy specific glycoprotein 1 (PSG1) and corticotropin releasing hormone (CRH) by 51-59%. The induction of TFAP2A promoter activity by NR2F2 was potentiated by the nuclear hormone receptors retinoic acid receptor alpha (RARA) and retinoid X receptor alpha (RXRA). CONCLUSIONS/SIGNIFICANCE: Taken together, these results strongly suggest that NR2F2 is involved in villous CTB cell differentiation and that NR2F2 acts, at least in part, by directly activating TFAP2A gene expression and by potentiating the transactivation of TFAP2A by RARA and RXRA.

LEFTY, a member of the transforming growth factor-beta superfamily, inhibits uterine stromal cell differentiation: a novel autocrine role.

LEFTY is expressed in normal endometrium in cells that decidualize. To understand the importance of this expression, we have studied the effect of LEFTY on decidualization in vitro and in vivo. Exposure of human uterine fibroblast (HuF) cells to recombinant LEFTY blocked the induction of the decidual differentiation-specific marker genes, IGFBP1 (IGF-binding protein 1) and PRL (prolactin) in response to medroxyprogesterone acetate, estradiol, and prostaglandin E2. The inhibitory effect was associated with decreased induction of the transcription factors ETS1 and FOXO1, both of which are essential for decidualization. Overexpression of LEFTY in decidualized HuF cells with an adenovirus that transduced LEFTY caused a marked decrease in IGFBP1 secretion, and withdrawal of medroxyprogesterone acetate from decidualized cells resulted in a decrease in IGFBP1 secretion and an increase in LEFTY expression. Moreover, overexpression of LEFTY in decidualized cells reprogrammed the cells to a less differentiated state and attenuated expression of decidual markers. Uterine decidualization was markedly attenuated and litter size was significantly reduced by retroviral transduction of LEFTY in the uterine horns of pregnant mice or by induction of LEFTY expression by doxycycline treatment in Tet-On conditional LEFTY transgenic pregnant mice. In addition, administration of the contraceptive agent drospirenone to ovariectomized mice induced a marked increase in LEFTY expression and inhibited decidualization. Taken together, these finding indicate that LEFTY acts as a molecular switch that modulates both the induction of decidual differentiation and the maintenance of a decidualized state. Because decidual cells express abundant amounts of LEFTY, the action of LEFTY on decidualization occurs by an autocrine mechanism.

TEAD1 inhibits prolactin gene expression in cultured human uterine decidual cells.

Forced overexpression of TEAD1 in human uterine fibroblast (HUF) and human endometrial stromal cells markedly inhibited prolactin promoter activity in both cell types in a dose-dependent manner, with maximal inhibition of greater than 90%. Conversely, the knockdown of TEAD1 expression in HUF cells with a TEAD1 siRNA resulted in a 75-80% increase in prolactin mRNA levels (p<0.01) compared to control cells exposed to a scrambled nonsense RNA. Mutagenesis of the putative TEAD site inhibited basal promoter activity by about 80%. However, mutagenesis of the TEAD site did not prevent TEAD1-induced inhibition of promoter activity; and the transcription activity of a minimal promoter fragment lacking a putative TEAD binding site was repressed by overexpression of TEAD1. Taken together, these findings suggest that the TEAD binding site on the prolactin promoter is important for the maintenance of basal prolactin promoter activity and that overexpression of TEAD1 has a dominant-negative effect on prolactin promoter activity, probably by interacting directly with other transcription factors.

Epigenetic activation of the human growth hormone gene cluster during placental cytotrophoblast differentiation.

The hGH cluster contains a single human pituitary growth hormone gene (hGH-N) and four placenta-specific paralogs. Activation of the cluster in both tissues depends on 5' remote regulatory elements. The pituitary-specific locus control elements DNase I-hypersensitive site I (HSI) and HSII, located 14.5 kb 5' of the cluster (position -14.5), establish a continuous domain of histone acetylation that extends to and activates hGH-N in the pituitary gland. In contrast, histone modifications in placental chromatin are restricted to the more 5'-remote HSV-HSIII region (kb -28 to -32) and to the placentally expressed genes in the cluster, with minimal modification between these two regions. These data predict distinct modes of hGH cluster gene activation in the pituitary and placenta. Here we used cell culture models to track structural changes at the hGH locus through placental-gene activation. The data revealed that this process was initiated in primary cytotrophoblasts by histone H3K4 di- and trimethylation and H4 acetylation restricted to HSV and to the individual placental-gene repeat (PGR) units within the cluster. Later stages of transcriptional induction were accompanied by enhancement and extension of these modifications and by robust H3 acetylation at HSV, at HSIII, and throughout the placental-gene regions. These data suggested that elements restricted to HSIII-HSV regions and each individual PGR might be sufficient for activation of the hCS genes. This model was tested by comparing hCS transgene expression in the placentas of mouse embryos carrying a full hGH cluster to that in placentas in which the HSIII-HSV region was directly linked to the individual hCS-A PGR unit. The findings indicate that the HSIII-HSV region and the PGR units, although targeted for initial chromatin structural modifications, are insufficient to activate gene expression and that this process is dependent on additional, as-yet-unidentified chromatin determinants.

Cytomegalovirus infection of human syncytiotrophoblast cells strongly interferes with expression of genes involved in placental differentiation and tissue integrity.

The principle route of acquisition of cytomegalovirus (CMV) for the fetus is believed to be via the placenta. We subjected purified cytotrophoblast cells obtained from full-term placentas to CMV infection and examined placental gene expression using microarray analyses. Cytotrophoblast cells purified from term placentas differentiated in vitro into a multinucleated syncytium that could be productively infected with CMV, with peak virus titers of approximately 10 plaque-forming units (PFU)/mL identified in supernatants at late time points postinoculation. Infected syncytiotrophoblast cells expressed CMV-specific transcripts and proteins, as demonstrated by Northern blot and immunofluorescence assays. Microarray analyses revealed that CMV infection strongly and reproducibly altered trophoblast gene expression, elevating expression of mitotic cell cycle genes, and repressing expression of genes associated with trophoblast differentiation, particularly those associated with formation and stabilization of the extracellular matrix. We conclude that purified, differentiated syncytiotrophoblasts are permissive for CMV replication. Infection of these cells induces significant perturbations in trophoblast transcription. An improved understanding of the molecular events that occur during CMV infection of trophoblasts could provide insights into interventions that might prevent or minimize congenital transmission.

Autocrine prolactin inhibits human uterine decidualization: a novel role for prolactin.

Human prolactin (PRL) and its receptor (PRLR) are markedly induced during human uterine decidualization, and large amounts of PRL are released by decidual cells as differentiation progresses. However, the role of PRL in decidualization is unknown. In order to determine whether PRL plays an autocrine role in decidualization, human uterine fibroblast cells that were decidualized in vitro with medroxyprogestrerone acetate (1 microM), estradiol (10 nM), and prostaglandin E(2) (1 microM) were exposed to exogenous PRL and/or the pure PRLR antagonist delta1-9-G129R-PRL. As measured by quantitative PCR, cells that were decidualized in the presence of exogenous PRL (0.25-2 microg/ml) expressed significantly lower levels of mRNA for the genes that encode insulin-like growth factor binding protein 1 (IGFBP1), left-right determination factor 2 (LEFTY2), PRL, decorin (DCN), and laminin alpha 1 (LAMA1), all of which are known to be induced during decidualization. These effects were blocked when the cells were exposed simultaneously to PRL and the PRLR antagonist, which confirms the specific inhibitory action of PRL on the expression of decidualization markers. In addition, cells exposed to the PRLR antagonist alone expressed higher levels of the marker gene mRNAs than cells that were decidualized in control media. Taken together, these results strongly suggest that PRL acts via an autocrine mechanism to regulate negatively the extent of differentiation (decidualization) of human uterine cells.

Transcription factor FOXF1 regulates growth hormone variant gene expression.

Deletion analysis of the human growth hormone variant (GHV) promoter in transient transfection studies in BeWo choriocarcinoma and HepG2 cells indicated that the region extending from nt -158/+57 retained full transcriptional activity. DNase I footprint analysis of the fragment revealed a protected region at nt -82/-77, which is in a putative FOXF1/FOXF2 binding site. Supershift assays using an antiserum to human FOXF1 demonstrated that the protected region binds FOXF1. Overexpression of FOXF1 in BeWo and HepG2 cells induced the GHV promoter, whereas overexpression of FOXF2 was without effect. Mutagenesis of the FOXF1/FOXF2 site reduced basal promoter activity by 50-60% and markedly attenuated transactivation of the promoter by FOXF1. These studies indicate that FOXF1 induces GHV expression by interaction with a FOXF1/FOXF2 cis-element in the proximal promoter.

Transcription factor ETS1 is critical for human uterine decidualization.

The aim of this study was to examine whether the transcription factor ETS1 plays a critical role in the regulation of human decidualization. Decidual fibroblast cells were decidualized in vitro by treatment with medroxyprogesterone, estradiol (E(2)) and dibutyryl cyclic AMP or prostaglandin E(2) in the absence or presence of an ETS1 antisense oligonucleotide (oligo) that blocks the translation of ETS1 mRNA. Control experiments were performed using a control oligo that did not affect ETS1 expression and the induction of specific marker genes for decidualization. The ETS1 antisense oligo markedly inhibited ETS1 protein expression and significantly inhibited downstream targets of ETS1 action. On day 6 of culture, the decidualized fibroblast cells that had been exposed to the ETS1 antisense oligo contained 40-90% less mRNAs for prolactin, insulin growth factor binding protein 1 (IGFBP-1) and other decidualization-specific markers (laminin, tissue inhibitor of metalloproteinase-3 [TIMP3], endometrial bleeding associated factor [EBAF] and decorin) than those of control cells that had not been exposed to the ETS1 antisense oligo. GAPDH mRNA levels, which do not change during decidualization, were unaffected by either the ETS1 antisense or the control oligo. The cells decidualized in the presence of the ETS1 antisense oligo also released significantly less prolactin, EBAF and IGFBP-1 protein, determined by western blot analyses, than the control cells. Taken together, these findings strongly suggest that ETS1 plays a critical role in the induction of human decidualization.

Gene expression microarray data analysis of decidual and placental cell differentiation.

Gene expression analysis using DNA microarray approaches have provided new insights into the physiology and pathophysiology of many biological processes. These include identification of genetic programs and pathways that underlie cell and tissue differentiation and gene expression programs responsive to genetic perturbations, drugs, toxins, and infectious agents. In this chapter, we present methods for the analysis of microarray data using earlier investigations from our laboratory as examples of how gene expression patterns for cellular differentiation may be detected and analyzed for biological significance and how regulated genes may be classified into functional categories and pathways.

Mitogen-activated protein kinase activation induces corticotrophin-releasing hormone gene expression in human placenta.

Corticotropin-releasing hormone (CRH) gene expression in human placental cells is induced by activation of the cyclic AMP and protein kinase C signal transduction pathways, but the role of the mitogen-activated kinase (MAPK) pathway is unknown. In this study, we showed that the MAPK inhibitor, PD098059, causes a dose-dependent inhibition of placental CRH gene expression. In contrast, overexpression of RAF in human choriocarcinoma JEG cells stimulates CRH promoter activity by 15-fold, and the stimulation is inhibited by 65% by co-transfection of the cells with a plasmid expressing a RAF dominant/negative protein. The stimulation by RAF was completely abolished by mutation of the cyclic AMP response element (CRE) in the proximal region of the CRH promoter. Taken together, these results strongly suggest that the MAPK signal transduction pathway plays a pivotal role in the regulation of CRH gene expression in human placenta, and that the CRE binding site in the proximal CRH promoter acts as a point of convergence for different signal transduction pathways in the regulation of CRH gene expression in placenta cells.

A placenta-specific enhancer of the human syncytin gene.

The cis- and trans-acting factors that are critical for placenta-specific expression of the human syncytin gene are unknown. We identified a 146-base pair (bp) region of the 5'-flanking region of the human syncytin gene from nt-294 to -148 that is essential for basal gene expression in human BeWo and JEG3 choriocarcinoma cell lines but not in hepatoblastoma and kidney cell lines. Ligation of the 146-bp fragment to a SV40 promoter or a human beta-globin minimal promoter markedly enhanced promoter activity in the placenta cells but not in the liver and kidney cells. DNase I footprint assays indicated that nuclear extracts from BeWo cells but not HepG2 cells protected four regions (FP1-FP4) of the 146-bp fragment. Site-directed mutagenesis of an SP1-binding site in FP3 and a GATA-binding site in FP4 significantly repressed promoter activity in the placenta cells. Overexpression of SP1 (Sp1 transcription factor) and GATA2 (GATA binding protein 2) and GATA3 induced syncytin promoter activity but had little or no effect on the activities of syncytin promoter fragments containing mutations in the SP1- and GATA-binding sites. GATA2 and -3 mRNA levels increased markedly during spontaneous in vitro differentiation of human cytotrophoblast cells when the cytotrophoblast cells fused to form a syncytium. These findings strongly suggest that the 146-bp region of the 5'-flanking region (nt-294/-148) of the human syncytin gene acts as a placenta-specific enhancer. Binding of SP1 and GATA family members to this enhancer is critical for cell-specific expression of the syncytin gene.