Stuart Handwerger

A Placenta-Specific Enhancer of the Human Syncytin Gene

Abstract 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 β-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.

New insights into the regulation of human cytotrophoblast cell differentiation.

This monograph presents a broad overview of human trophoblast differentiation, emphasizing the hormonal factors and transcription factors involved in the differentiation process. Particular focus is given to the role for syncytin in the fusion of mononuclear cytotrophoblast (CTB) cells and the roles for the transcription factor TFAP2A in terminal syncytiotrophoblast (STB) differentiation. Also presented are some studies relating to villous trophoblast differentiation in pre-eclampsia, choriocarcinoma and other pathologic conditions of pregnancy associated with abnormal placentation.

Isolation and Characterization of the Human Syncytin Gene Promoter

Abstract Syncytin, a protein encoded by an envelope gene of a human endogenous retrovirus-W (HERV-W), plays a critical role in trophoblast differentiation. We isolated the 5′-flanking region of the syncytin gene from human genomic DNA by PCR and identified cis-acting elements on the promoter that are important for transcription. The major transcription initiation site identified by mung bean nuclease protection assays is 56 base pairs (bp) downstream from a putative CCAAT box. Deletion analysis of the 5′-flanking region of the syncytin gene indicated that the proximal 148 bp are essential for minimal promoter activity and that regions of the promoter from nt −1519 to −984 and nt −294 to −148 are required for maximal expression in normal trophoblast cells. DNase I footprint analysis of the region between nt −252 and +110 revealed three protected regions, FP1–FP3. Mutagenesis of a hepatocyte-specific nuclear protein-1 (HAPF1) binding site in FP1 and a TATA box in FP3 had no effects on basal promoter activity. However, mutation of the CCAAT motif and the octamer protein (Oct) binding site in FP2 decreased promoter activity by 88% and 76%, respectively. Mutation of the ecdysone receptor (EcR) response element in FP2, which may bind a nuclear hormone receptor, increased basal promoter activity by 2-fold. Gel shift and supershift assays indicated that CCAAT-binding factor (CBF) binds to the CCAAT motif and that Oct binds to the Oct binding site. Taken together, these findings indicate that the syncytin promoter is located in the 5′ long terminal repeat (LTR) of the HERV-W gene and that binding sites for CBF and Oct in the proximal promoter are critical for transcriptional regulation of the gene in trophoblast cells.

Autocrine Prolactin Inhibits Human Uterine Decidualization: A Novel Role for Prolactin

Abstract 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 μM), estradiol (10 nM), and prostaglandin E2 (1 μM) 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 μg/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.

Cannabinoid receptor I activation markedly inhibits human decidualization

The role of cannabinoid receptor I (CBR-1) in the induction of decidualization was examined using decidual fibroblasts and human endometrial stromal cells as model systems. Decidual fibroblasts decidualized in vitro for 3 and 6 days in the presence of the CBR-1 agonist R(+)-WIN 55,212-2 mesylate (WIN, 0.1-10 microM) expressed less of the decidualization-specific markers prolactin, CBR-1, forkhead (FKHR), TIMP-3, laminin, endometrial bleeding associated factor (EBAF), decorin and insulin-like growth factor binding protein-1 (IGFBP-1) mRNA levels compared to control cells. The maximal decrease for each transcript was in the range of 50-99%. In contrast, cells exposed to the CBR-1 inhibitor AM-251 (1 microM) expressed about two-fold higher levels of the decidualization-specific marker gene mRNAs. The WIN-exposed cells showed a marked decrease in intracellular cAMP levels and a progressive, concentration-dependent increase in DNA fragmentation (TUNEL assay) and caspase 3 levels during decidualization compared to control cells. These studies strongly suggest that activation of CBR-1 inhibits human decidualization and stimulates apoptosis by a cAMP-dependent mechanism.

3. Regulation of the Synthesis and Release of Decidual Prolactin by Placental and Autocrine/Paracrine-Factors"

Human decidual tissue synthesizes and secretes a protein that is identical to pituitary prolactin in its chemical, biological and immunological properties. Nevertheless, the factors that regulate the synthesis and release of prolactin from the decidual tissues appear to be different to those regulating the synthesis and release of pituitary prolactin. Studies from our laboratory over the past few years indicate that the synthesis and release of decidual prolactin are regulated, at least in part, by factors released by placenta, fetal membranes and decidua. The placenta releases a 23.5 KMr protein [decidual prolactin-releasing factor (PRL-RF)] that stimulates a rapid release of prolactin within the first few minutes of exposure and a sustained, prolonged, increase in the synthesis and release of prolactin beginning 6-8 h after exposure. The acute release of prolactin in response to PRL-RF is inhibited by decidual prolactin release-inhibitory factor (PRL-IF), a 35-45 K Mr protein that is released by the decidua. The secondary increase in the synthesis and release of prolactin in response to PRF-RF is blocked by lipocortin I, which is synthesized by both the placenta and decidua. IGF-I, insulin and relaxin also stimulate the synthesis and release of prolactin. However, the stimulation in response to these factors does not occur until 24-48 h after exposure. The cellular mechanisms involved in the release of decidual prolactin are as yet unknown. However, recent studies implicate activation of adenylate cyclase, phospholipase C-mediated phosphoinositide hydrolysis and phospholipase A2-mediated arachidonic acid release in the regulation of prolactin release. The finding that the synthesis and release of decidual prolactin are regulated, at least in part, by PRL-RF, IGF-I, insulin, relaxin and lipocortin I strongly suggests that there is novel feedback regulation between the placenta, fetal membranes, and decidua in the regulation of the synthesis and release of decidual prolactin.

Silencing or Amplification of Endocannabinoid Signaling in Blastocysts via CB1 Compromises Trophoblast Cell Migration

Background: Mouse preimplantation embryo growth is retarded under elevated or silenced endocannabinoid signaling involving CB1. Results: Developing preimplantation embryos under these conditions have abnormal expression of migration-related genes and compromised trophoblast stem cell migration. Conclusion: Embryo development and trophoblast migration require appropriate endocannabinoid signaling. Significance: A tightly regulated endocannabinoid signaling threshold is critical for female reproductive success. 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 CB1 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 CB1. 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 CB1 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.

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 104 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.

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-2α), 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.

N5 endometrial stromal cell line: A model system to study decidual prolactin gene expression

SummaryProlactin gene expression in extrapituitary tissues, such as decidua and lymphocytes, is regulated by a distinct promoter approximately 6 kb upstream of the pituitary prolactin gene transcription start site. Here we describe studies in a human endometrial stromal cell line, N5, that was immortalized by transfection with an SV40 mutant and which expresses the prolactin gene driven by the extrapituitary promoter. The N5 cells have phenotypic features of primary cultures of decidualized human endometrial stromal cells and secrete low levels of prolactin and insulin-like growth factor binding protein-1 (IGFBP-1), both of which are markers of decidualized endometrial stromal cells. As in primary cultures of endometrial stromal cells, treatment of N5 cells with progesterone and estradiol alone or in combination with prostaglandin E2 stimulated the synthesis and release of prolactin. Transient transfection of the N5 cells with an expression vector containing −2927/+66 bp of the decidual prolactin promoter coupled to a luciferase reporter gene resulted in a 20 to 25-fold increase in luciferase activity, a magnitude similar to that which occurs in primary cultures of endometrial stromal cells decidualized in vitro by treatment with progesterone and estradiol. Luciferase expression levels were similar in untreated N5 cells and N5 cells treated with progesterone and estradiol. Taken together, these results indicate that the N5 human endometrial stromal cell line has phenotypic characteristics of normal decidualized stromal cells and is a useful model to study regulation of decidual prolactin gene expression.