Heparin-binding EGF-like growth factor regulates human extravillous cytotrophoblast development during conversion to the invasive phenotype

Cytotrophoblasts of the anchoring villi convert during human placentation from a transporting epithelium to an invasive, extravillous phenotype that expresses a distinct repertoire of adhesion molecules. Developing extravillous trophoblasts accumulate heparin-binding EGF-like growth factor (HB-EGF), a multifunctional cytokine, which binds HER1 and HER4 of the human EGF receptor (HER/ErbB) family. HB-EGF is downregulated in placentae of women with preeclampsia, a disorder associated with deficient trophoblast invasion, raising important questions about its physiological impact on cytotrophoblasts. Addition of HB-EGF during explant culture of first-trimester chorionic villi enhanced extravillous trophoblast differentiation and invasive activity. Using a first-trimester human cytotrophoblast line, the potential for autocrine and paracrine regulation of the developing trophoblast was established based on the expression of all four HER isoforms, as well as HB-EGF and related growth factors. HB-EGF did not alter proliferation, but initiated extravillous differentiation, with decreased alpha6 integrin expression, increased alpha1, and elevated cell migration. Function-blocking antibodies against EGF family members reduced basal cell motility and antibody inhibition of either HER1 or HER4 ligation prevented HB-EGF-induced integrin switching. We conclude that HER-mediated autocrine and paracrine signaling by HB-EGF or other EGF family members induces cytotrophoblast differentiation to an invasive phenotype.     

Epidermal growth factor inhibits cytokine-induced apoptosis of primary human trophoblasts

In the placenta, as in other organs, the development and maintenance of the differentiated phenotype depend on a balance between cell proliferation, maturation, and death. We are interested in the mechanisms that regulate the survival and differentiation of placental trophoblasts and have recently demonstrated that the inflammatory cytokines tumor necrosis factor alpha (TNFα) and gamma interferon (IFNγ) act in concert to induce apoptotic cell death in normal cytotrophoblasts in culture. In this report we show that exposure to epidermal growth factor (EGF), a 6,700 dalton polypeptide that is abundantly expressed in maternal and fetal tissues, blocks the in vitro TNF/IFN-induced cytotoxicity of human cytotrophoblasts and syncytiotrophoblasts from normal term placentas. This antagonistic effect is dose-related (10⁻¹⁰ M EGF, half-maximal) and proceeds via the interruption of an early step in the cytokine-induced apoptotic response. These observations suggest a novel role for EGF in normal placental development and indicate that the interplay between EGF, TNFα, and IFNγ may determine the rate of trophoblast growth and renewal during gestation. © 1996 Wiley-Liss, Inc.     

Epidermal growth factor rescues trophoblast apoptosis induced by reactive oxygen species

Pre-eclampsia and intrauterine growth restriction are associated with increased apoptosis of placental villous trophoblast. This may result from placental hypoperfusion, leading to the generation of reactive oxygen species (ROS). Apoptosis can be induced in villous trophoblast following exposure to oxidative stress. Epidermal growth factor (EGF) reduces trophoblast apoptosis resulting from exposure to hypoxia. We hypothesised that exposure to hydrogen peroxide, a potent generator of ROS, would induce apoptosis in term placental villous explants and that this could be reduced by treatment with EGF. Placental explants were taken from normal term pregnancies and exposed to increasing doses of hydrogen peroxide (0–1,000 μM) or to a combination of increasing doses of hydrogen peroxide and EGF (0–100 ng/ml) for either 6 or 48 h. Apoptosis was assessed by TUNEL, proliferation by Ki-67 immunostaining, necrosis by lactate dehydrogenase activity and trophoblast differentiation by human chorionic gonadotrophin (hCG) secretion in conditioned culture media. Immunoperoxidase staining was performed to identify phosphorylated-AKT (p-AKT) and phosphorylated-PI3 kinase (p-PI3k). Exposure to 1,000 μM hydrogen peroxide for 48 h induced apoptosis in placental explants. The increase in TUNEL positive nuclei predominantly localised to syncytiotrophoblast. The amount of apoptosis was reduced to control levels by treatment with 10 and 100 ng/ml EGF. Proliferation of cytotrophoblasts within villous explants was significantly reduced following exposure to 1,000 μM hydrogen peroxide, this was restored to control levels by simultaneous treatment with 10 or 100 ng/ml EGF. Neither exposure to hydrogen peroxide or EGF altered the amount of necrosis. There was increased immunostaining for pPI3K following treatment with EGF. This study shows that apoptosis may be induced in villous trophoblast following exposure to ROS, and demonstrates the anti-apoptotic effect of EGF in trophoblast, the maintenance of which is essential for normal pregnancy.     

Cripto-1 induces apoptosis in HC-11 mouse mammary epithelial cells

Cripto-1 (CR-1) is an epidermal growth factor (EGF)-related protein. CR-1 can inhibit beta-casein and whey acidic protein expression in mouse mammary epithelial cells. The present study demonstrates that CR-1 can induce apoptosis in HC-11 mouse mammary epithelial cells, as measured by bis-benzimide stained nuclei, TUNEL assay and cell death ELISA. Apoptosis could be observed after 2 days of exposure of confluent HC-11 cells to CR-1 in the absence of the survival factors EGF and insulin, with maximum apoptosis occurring at 3 days. A reduction in poly(ADP-ribose) polymerase (PARP) expression and an increase in beta-catenin cleavage was found after 18 h of exposure to CR-1 suggesting that apoptosis was preceded by the induction of a caspase activity since the caspase inhibitor ZFAD.FMK could block the CR-1-induced reduction in PARP expression and CR-1-induced apoptosis. CR-1 was found to increase the expression of caspase-3-like protease. Although, the levels of p27kip1 and p21Bax did not change after exposure to CR-1 for 18 h, the levels of Bcl-xL became undetectable. These studies suggest that CR-1 promotes apoptosis by mediating the induction of caspase-3-like protease and downregulating the expression of Bcl-xL.     

Death receptor Fas/Apo-1/CD95 expressed by human placental cytotrophoblasts does not mediate apoptosis

Trophoblasts, the fetal cells that line the villous placenta and separate maternal blood from fetal tissue, express both Fas antigen and the tumor necrosis factor (TNF) receptor p55 (TNFRp55), two members of the TNF receptor family that contain a cytoplasmic "death domain" that mediates apoptotic signals. We show that Fas mRNA expressed by cultured villous cytotrophoblasts isolated from term placentas encodes transmembrane sequences and that the protein is full-length (approximately 45 kDa), suggesting that the product is an active plasma membrane-anchored receptor. Its location on the cell surface was confirmed by cellular ELISA analysis of live cells. Although cytotrophoblast apoptosis was induced by TNFalpha, and both anti-Fas antibody (CH11) and FasL-expressing T lymphocyte hybridoma (activated A1.1) cells induced HeLa cell apoptosis, neither CH11 antibody nor activated A1.1 cells stimulated apoptosis in term or first-trimester cytotrophoblasts or in term syncytiotrophoblasts. We conclude that Fas- but not TNFRp55-mediated apoptosis is blocked in primary villous trophoblasts. These data suggest that the Fas response is specifically inactivated by unknown mechanisms to avoid autocrine or paracrine killing by Fas ligand constitutively expressed on neighboring cyto- or syncytiotrophoblasts.     

Cell death in fetal oocytes: many players for multiple pathways

We devised a short-term culture system allowing us to define novel characteristics of programmed cell death (PCD) of fetal oocytes and to underscore new aspects of this process. Mouse fetal oocytes cultured in conditions allowing meiotic progression underwent apoptotic degeneration as revealed by TUNEL staining, DNA ladder, Annexin V binding, PARP cleavage and, usually, caspase activation. TEM observations show, however, recurrent atypical apoptotic morphologies characterized by the absence of chromatin margination and nuclear fragmentation; oocytes with autophagic and necrotic features are also observed. Moreover, under the fluorescence microscope a subpopulation of TUNEL(+) oocytes appear morphologically healthy and do not show detectable caspase activity. Finally, caspase inhibitors are able to slow down, but not to abolish, oocyte cell death, whereas calpain inhibitor I significantly reduces the number of TUNEL(+) oocytes after 4 days of culture, and rapamycin (mTOR inhibitor) increases such numbers both at day 3 and 4. These observations together with results showing expression in cultured oocytes undergoing cell death of apoptosis inducing factor and Beclin 1, two important players of caspase independent and autophagic cell death, respectively, demonstrate that fetal oocytes possess and are able to activate several players of various forms of cell death. However, causal correlation among different cell death pathways in such oocytes remains to be determined and stimuli causing the activation of these pathways in vitro and in vivo also clarified.     

Epidermal growth factor stimulation of trophoblast differentiation requires MAPK11/14 (p38 MAP kinase) activation

Cultured human term villous cytotrophoblasts (CT) have been reported to be nonproliferating but differentiate when exposed to epidermal growth factor (EGF). Here we show that CT differentiate into chorionic gonadotropin (beta-hCG/CGB)-expressing cells when cultured with medium alone. The addition of EGF decreases CGB secretion and prolongs production for up to 13 days. EGF stimulates the phosphorylation (activation) of the signaling intermediate p38 (MAPK11/14), and blocking phosphorylation pharmacologically with either SB203580 or SB202190 strongly inhibited spontaneous and EGF-stimulated secretion of CGB. In addition, EGF-stimulated fusion of cytotrophoblasts into syncytial units was strongly inhibited by SB203580. EGF upregulated trophoblast proliferation (measured by bromodeoxyuridine uptake) and SB203580 increased this proliferation after 5 days. In agreement with these observations, EGF and SB203580 increased expression of the G1-phase-specific gene cyclin-D1 (CCND1) and SB203580 downmodulated its inhibitor p21 (CDKN1A). When added to villous explant cultures, EGF did nothing to the pattern of CGB secretion, but addition of SB203580 prevented the normal surge in secretion during syncytial regeneration over Days 3-7. These data support the hypothesis that EGF-stimulated cytotrophoblast differentiation to syncytium requires MAPK11/14 activation, and that cytotrophoblast proliferation can be stimulated in culture by EGF and enhanced by MAPK11/14 inhibition with a consequent reduction of differentiation.     

Reactive oxygen species (ROS) mediates the mitochondrial-dependent apoptosis induced by transforming growth factor (beta) in fetal hepatocytes.

Treatment of fetal rat hepatocytes with transforming growth factor beta (TGF-beta) is followed by apoptotic cell death. Analysis of radical oxygen species (ROS) content and mitochondrial transmembrane potential (Deltapsim), using specific fluorescent probes in FACScan and confocal microscopy, showed that TGF-beta mediates ROS production that precedes the loss of Deltapsim, the release of cytochrome c, and the activation of caspase 3. TGF-beta induces a decrease in the protein and mRNA levels of bcl-xL, an antiapoptotic member of the Bcl-2 family. In contrast, there is no change in the expression and/or translocation of Bax, a proapoptotic member of the same family. EGF maintains Bcl-xL, preventing Deltapsim collapse and release of cytochrome c. The presence of radical scavengers blocks the decrease in bcl-xL levels, Deltapsim collapse, cytochrome c release, and activation of caspase 3; in contrast, the presence of glutathione synthesis inhibitors such as BSO accentuated the effect. The incubation of fetal hepatocytes in the presence of ter-butyl-hydroperoxide alone produces a decrease in bcl-xL. These results indicate that during the apoptosis mediated by TGF-beta in fetal hepatocytes, ROS may be responsible for the decrease in bcl-xL mRNA levels that precedes the loss of Deltapsim, the release of cytochrome c, and the activation of caspase 3, culminating in cell death.     

Insulin-like growth factor I and II regulate the life cycle of trophoblast in the developing human placenta

The main disorders of human pregnancy are rooted in defective placentation. Normal placental development depends on proliferation, differentiation, and fusion of cytotrophoblasts to form and maintain an overlying syncytiotrophoblast. There is indirect evidence that the insulin-like growth factors (IGFs), which are aberrant in pregnancy disorders, are involved in regulating trophoblast turnover, but the processes that control human placental growth are poorly understood. Using an explant model of human first-trimester placental villus in which the spatial and ontological relationships between cell populations are maintained, we demonstrate that cytotrophoblast proliferation is enhanced by IGF-I/IGF-II and that both factors can rescue cytotrophoblast from apoptosis. Baseline cytotrophoblast proliferation ceases in the absence of syncytiotrophoblast, although denuded cytotrophoblasts can proliferate when exposed to IGF and the rate of cytotrophoblast differentiation/fusion and, consequently, syncytial regeneration, increases. Use of signaling inhibitors suggests that IGFs mediate their effect on cytotrophoblast proliferation/syncytial formation through the MAPK pathway, whereas effects on survival are regulated by the phosphoinositide 3-kinase pathway. These results show that directional contact between cytotrophoblast and syncytium is important in regulating the relative amounts of the two cell populations. However, IGFs can exert an exogenous regulatory influence on placental growth/development, suggesting that manipulation of the placental IGF axis may offer a potential therapeutic route to the correction of inadequate placental growth.     

Epidermal growth factor protects epithelial cells against Fas-induced apoptosis. Requirement for Akt activation.

Chemotherapeutic drugs that damage DNA kill tumor cells, in part, by inducing the expression of a death receptor such as Fas or its ligand, FasL. Here, we demonstrate that epidermal growth factor (EGF) stimulation of T47D breast adenocarcinoma and embryonic kidney epithelial (HEK293) cells protects these cells from Fas-induced apoptosis. EGF stimulation of epithelial cells also inhibited Fas-induced caspase activation and the proteolysis of signaling proteins downstream of the EGF receptor, Cbl and Akt/protein kinase B (Akt). EGF stimulation of Akt kinase activity blocked Fas-induced apoptosis. Expression of activated Akt in MCF-7 breast adenocarcinoma cells was sufficient to block Fas-mediated apoptosis. Inhibition of EGF-stimulated extracellular signal-regulated kinase (ERK) activity did not affect EGF protection from Fas-mediated apoptosis. The findings indicate that EGF receptor stimulation of epithelial cells has a significant survival function against death receptor-induced apoptosis mediated by Akt.     

ETOH inhibits embryonic neural stem/precursor cell proliferation via PLD signaling

While a mother’s excessive alcohol consumption during pregnancy is known to have adverse effects on fetal neural development, little is known about the underlying mechanism of these effects. In order to investigate these mechanisms, we investigated the toxic effect of ethanol (ETOH) on neural stem/precursor cell (NSC) proliferation. In cultures of NSCs, phospholipase D (PLD) is activated following stimulation with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). Exposure of NSCs to ETOH suppresses cell proliferation, while it has no effect on cell death. Phosphatidic acid (PA), which is a signaling messenger produced by PLD, reverses ETOH inhibition of NSC proliferation. Blocking the PLD signal by 1-butanol suppresses the proliferation. ETOH-induced suppression of NSC proliferation and the protective effect of PA for ETOH-induced suppression are mediated through extracellular signal-regulated kinase signaling. These results indicate that exposure to ETOH impairs NSC proliferation by altering the PLD signaling pathway.     

Neuroprotective peptide ADNF-9 in fetal brain of C57BL/6 mice exposed prenatally to alcohol

Background  

A derived peptide from activity-dependent neurotrophic factor (ADNF-9) has been shown to be neuroprotective in the fetal alcohol exposure model. We investigated the neuroprotective effects of ADNF-9 against alcohol-induced apoptosis using TUNEL staining. We further characterize in this study the proteomic architecture underlying the role of ADNF-9 against ethanol teratogenesis during early fetal brain development using liquid chromatography in conjunction with tandem mass spectrometry (LC-MS/MS).     

Human cytomegalovirus interleukin-10 downregulates metalloproteinase activity and impairs endothelial cell migration and placental cytotrophoblast invasiveness in vitro

At the uterine-placental interface, fetal cytotrophoblasts invade the decidua, breach maternal blood vessels, and form heterotypic contacts with uterine microvascular endothelial cells. In early gestation, differentiating- invading cytotrophoblasts produce high levels of matrix metalloproteinase 9 (MMP-9), which degrades the extracellular matrix and increases the invasion depth. By midgestation, when invasion is complete, MMP levels are reduced. Cytotrophoblasts also produce human interleukin-10 (hIL-10), a pleiotropic cytokine that modulates immune responses, helping to protect the fetal hemiallograft from rejection. Human cytomegalovirus (CMV) is often detected at the uterine-placental interface. CMV infection impairs cytotrophoblast differentiation and invasion, altering the expression of the cell adhesion and immune molecules. Here we report that infection with a clinical CMV strain, VR1814, but not a laboratory strain, AD169, downregulates MMP activity in uterine microvascular endothelial cells and differentiating-invading cytotrophoblasts. Infected cytotrophoblasts expressed CMV IL-10 (cmvIL-10) mRNA and secreted the viral cytokine, which upregulated hIL-10. Functional analyses showed that cmvIL-10 treatment impaired migration in endothelial cell wounding assays and cytotrophoblast invasion of Matrigel in vitro. Comparable changes occurred in cells that were exposed to recombinant hIL-10 or cmvIL-10. Our results show that cmvIL-10 decreases MMP activity and dysregulates the cell-cell and/or cell-matrix interactions of infected cytotrophoblasts and endothelial cells. Reduced MMP activity early in placental development could impair cytotrophoblast remodeling of the uterine vasculature and eventually restrict fetal growth in affected pregnancies.     

Recombinant Hep G2 cells that express alcohol dehydrogenase and cytochrome P450 2E1 as a model of ethanol-elicited cytotoxicity

HepG2 cells were transfected with recombinant plasmids, one carrying the murine alcohol dehydrogenase (ADH) gene and the other containing the gene encoding human cytochrome P450 2E1 (CYP2E1). One of recombinant clones called VL-17A exhibited ADH and CYP2E1 specific activities comparable to those in isolated rat hepatocytes. VL-17A cells oxidized ethanol and generated acetaldehyde, the levels of which depended upon the initial ethanol concentration. Compared with unexposed VL-17A cells, ethanol exposure increased the cellular redox (lactate:pyruvate ratio) and caused cell toxicity, indicated by increased leakage of lactate dehydrogenase into the medium,. Exposure of VL-17A cells to 100mM ethanol significantly elevated caspase 3 activity, an indicator of apoptosis, but this ethanol concentration did not affect caspase 3 activity in parental HepG2 cells. Because ethanol consumption causes a decline in hepatic protein catabolism, we examined the influence of ethanol exposure on proteasome activity in HepG2, VL-17A, E-47 (CYP2E1(+)) and VA-13 (ADH(+)) cells. Exposure to 100mM ethanol caused a 25% decline in the chymotrypsin-like activity of the proteasome in VL-17A cells, but the enzyme was unaffected in the other cell types. This inhibitory effect on the proteasome was blocked when ethanol metabolism was blocked by 4-methyl pyrazole. We conclude that recombinant VL-17A cells, which express both ADH and CYP2E1 exhibit hepatocyte-like characteristics in response to ethanol. Furthermore, the metabolism of ethanol by these cells via ADH and CYP2E1 is sufficient to bring about an inhibition of proteasome activity that may lead to apoptotic cell death.     

EPHB4 regulates chemokine-evoked trophoblast responses: a mechanism for incorporating the human placenta into the maternal circulation

In humans, fetal cytotrophoblasts leave the placenta and enter the uterine wall, where they preferentially remodel arterioles. The fundamental mechanisms that govern these processes are largely unknown. Previously, we have shown that invasive cytotrophoblasts express several chemokines, as well as the receptors with which they interact. Here, we report that these ligand-receptor interactions stimulate cytotrophoblast migration to approximately the same level as a growth factor cocktail that includes serum. Additionally, cytotrophoblast commitment to uterine invasion was accompanied by rapid downregulation of EPHB4, a transmembrane receptor associated with venous identity, and upregulation of ephrin B1. Within the uterine wall, the cells also upregulated expression of ephrin B2, an EPH transmembrane ligand that is associated with arterial identity. In vitro cytotrophoblasts avoided EPHB4-coated substrates; upon co-culture with 3T3 cells expressing this molecule, their migration was significantly inhibited. As to the mechanisms involved, cytotrophoblast interactions with EPHB4 downregulated chemokine-induced but not growth factor-stimulated migration. We propose that EPHB4/ephrin B1 interactions generate repulsive signals that direct cytotrophoblast invasion toward the uterus, where chemokines stimulate cytotrophoblast migration through the decidua. When cytotrophoblasts encounter EPHB4 expressed by venous endothelium, ephrin B-generated repulsive signals and a reduction in chemokine-mediated responses limit their interaction with veins. When they encounter ephrin B2 ligands expressed in uterine arterioles, migration is permitted. The net effect is preferential cytotrophoblast remodeling of arterioles, a hallmark of human placentation.     

TNFalpha induces and insulin inhibits caspase 3-dependent adipocyte apoptosis.

Regulation of fat cell number by apoptosis is proposed to be part of a normal physiological cycle in adipose growth and development. To investigate this process, cultured rat adipocytes were treated with various concentrations of tumor necrosis factor alpha (TNFalpha) and/or insulin to determine the roles of these factors in adipocyte apoptosis. The cells were analyzed by flow cytometry using a TUNEL assay. TNFalpha increased adipocyte apoptosis in a dose-dependent fashion. TNFalpha-mediated apoptosis was detectable within 6 h of treatment and continued to increase with time. Decreasing media insulin concentration from 8.5 to 0.85 nM resulted in increased adipocyte apoptosis, whereas high doses of insulin protected adipocytes from TNFalpha-induced apoptosis. TNFalpha-activated apoptosis was accompanied by an increase in caspase 3 activity and could be inhibited by a caspase 3-specific inhibitor. These data suggest that adipose tissue cell number is regulated, in part, by an apoptotic signaling pathway that involves TNFalpha, insulin, and caspase 3.     

Apoptosis in human cultured trophoblasts is enhanced by hypoxia and diminished by epidermal growth factor

Preeclampsia and fetal growthrestriction are associated with placental hypoperfusion and villoushypoxia. The villous response to this environment includes diminishedtrophoblast differentiation and enhanced apoptosis. We tested thehypothesis that hypoxia induces apoptosis in cultured trophoblasts, andthat epidermal growth factor (EGF), an enhancer of trophoblastdifferentiation, diminishes hypoxia-induced apoptosis. Trophoblastsisolated from placentas of term-uncomplicated human pregnancies werecultured up to 72 h in standard (PO₂ = 120 mmHg) or hypoxic (PO₂ < 15 mmHg) conditions. Exposure to hypoxia for 24 h markedly enhanced trophoblast apoptosis as determined by DNA laddering, internucleosomal in situ DNA fragmentation, and histomorphology, as well as by thereversibility of the apoptotic process with a caspase inhibitor. Apoptosis was accompanied by increased expression of p53 and Bax anddecreased expression of Bcl-2. Addition of EGF to cultured trophoblastsor exposure of more differentiated trophoblasts to hypoxiasignificantly lowered the level of apoptosis. We conclude that hypoxiaenhances apoptosis in cultured trophoblasts by a mechanism thatinvolves an increase in p53 and Bax expression. EGF and enhancement ofcell differentiation protect against hypoxic-induced apoptosis.

     

Bcl-2 inhibits cell death of serum-free mouse embryo cells caused by epidermal growth factor deprivation

SFME cells are brain-derived neural precursor cells that are acutely dependent on epidermal growth factor (EGF) for survival, undergoing apoptosis within 24 h after EGF withdrawal. Because the expression of the protooncogene bcl-2 inhibits apoptosis induced by the withdrawal of interleukins or nerve growth factor in some growth factor-dependent haematopoietic or neuronal cell cultures, we examined the effect of Bcl-2 expression on cell death of SFME cells in the absence of EGF. SFME cells expressing human Bcl-2 showed prolonged survival when deprived of EGF compared to control cells not expressing Bcl-2. A significant fraction of Bcl-2-expressing cells remained viable for 4 days in the absence of EGF and resumed proliferation upon readdition of EGF to the cultures. These results suggest that apoptosis induced by EGF withdrawal in SFME cells may share common mechanisms with other growth factor-related apoptotic systems.