Regulation of growth and function of the human placenta

The human placenta is a tumor-like tissue in which highly proliferative, migratory, and invasive extra-villous trophoblast cells, migrate and invade the uterus and its vasculature, to provide a vital link between the mother and the developing fetus. In the present article, we review our studies on a series of experiments, designed to identify molecular events responsible for the phenotypic changes during placental growth. Our observations illustrate that the human placenta is endowed with the biochemical machinery to proliferate indefinitely throughout gestation, yet, there are intrinsic mechanisms that effectively circumscribe the extent and duration of trophoblast proliferation. The placenta combines in itself the unique ability to produce a wide variety of protein, peptide and steroid hormones, but intricately interwoven in this process, is also the remarkable capacity to simultaneously regulate their synthesis and secretion. The placenta therefore represents an autonomous or a self-sufficient unit capable of modulating its own growth and function, while assisting the developing fetus until it is capable of independent existence.     

Normal nonmetastatic human trophoblast cells share in vitro invasive properties of malignant cells

First-trimester normal human trophoblast cells show some phenotypic similarities to malignant cells, e.g., rapid proliferation and ability to invade neighboring tissue, including basement membrane in situ, but do not have the ability for unlimited growth or metastasis. The present study examined whether the invasive ability of normal trophoblast cells is an intrinsic property of these cells, independent of the microenvironment provided by the pregnant uterus, and if so, whether they share some of the molecular mechanisms of invasion exercized by metastatic malignant cells. The ability of in vitro grown human trophoblast lines to invade an epithelium-free human amniotic membrane was measured from the temporal kinetics of retention of radioactivity within this membrane resulting from a penetration by 125I-iododeoxyuridine-labeled trophoblast cells. The magnitude of this invasion was compared to that of the highly metastatic human JAR-choriocarcinoma cell line and murine B16F10 melanoma line. Trophoblasts were found to share some of the same molecular mechanisms of invasion with the metastatic cell lines. Inhibitors of collagenase, plasmin, plasminogen, and plasminogen activators completely prevented invasion of the amnion by the trophoblast lines as well as by the metastatic JAR and B16F10 lines. Mersalyl, a compound known to activate collagenase, stimulated invasion by all cell lines tested, including under conditions in which plasmin activity was inhibited. In addition, trophoblasts produced significant levels of type IV collagenase and laminin, both of which appear to be important products of metastatic tumor cells required for basement membrane invasion. It may be concluded from these findings that the invasive property of first trimester human trophoblasts is genetically determined; that the magnitude of amnion invasion cannot differentiate between metastatic cell lines and invasive but nonmetastatic cell lines; and that invasiveness is not a sufficient prerequisite for metastatic ability.     

Human cytotrophoblasts acquire aneuploidies as they differentiate to an invasive phenotype

Through an unusual differentiation process, human trophoblast progenitors (cytotrophoblasts) give rise to tumor-like cells that invade the uterus. By an unknown mechanism, invasive cytotrophoblasts exhibit permanent cell cycle withdrawal. Here, we report molecular cytogenetic data showing that approximately 20 to 60% of these interphase cells had acquired aneusomies involving chromosomes X, Y, or 16. The incidence positively correlated with gestational age and differentiation to an invasive phenotype. Scoring 12 chromosomes in flow-sorted cytotrophoblasts showed that more than 95% of the cells were hyperdiploid. Thus, aneuploidy appears to be an important component of normal placentation, perhaps limiting the proliferative and invasive potential of cytotrophoblasts within the uterus.     

The metastasis-associated genes MTA1 and MTA3 are abundantly expressed in human placenta and chorionic carcinoma cells

Normal placenta development relies on the ability of trophoblast cells to invade into the uterus and to build up an extensively vascularized feto-maternal tissue, necessary for the nutrition of the embryo. The ability of cell migration, invasion, and the ability to induce neovascularization are likewise hallmarks of cancer cells. The metastasis-associated genes MTA1 and MTA3 are known to be involved in cancer cell migration by regulation of cell adhesion proteins and to induce the expression of neoangiogenic cytokines, as recently shown by us for ovarian cancer cells. Therefore, we analyzed the expression of MTA1 and MTA3 in normal human placenta tissues and the chorionic cancer cell lines BeWo, JEG, and JAR. Immunohistochemical analysis revealed a rather strong expression of MTA1 and MTA3 in the nuclei of human trophoblast cells. A high expression level of MTA1 and MTA3 was further observed in the nuclei of human chorionic carcinoma cells, as shown by immunofluorescence analysis, and confirmed by Western blot and RT-PCR analysis. We conclude that the high expression level of MTA proteins in human chorionic cells might facilitate trophoblast cell migration and neoangiogenesis, and might further predispose human chorionic cancer cells with properties that are characteristic for this highly aggressive and metastatic carcinoma type.     

Smurf2 Participates in Human Trophoblast Cell Invasion by Inhibiting TGF-�� Type I Receptor

Successful embryo implantation depends on the ability of the trophoblast cells to invade the endometrium and the receptivity of the endometrium. Unlike tumor invasion, trophoblast invasion is spatio-temporaly restricted. Transforming growth factor (TGF)-β is a key inhibitory factor in the invasion of early trophoblast cells. Smad ubiquitination regulatory factor 2 (Smurf2), a HECT type E3 ubiquitin ligase, is an important regulator of the TGF-β signaling pathway, targeting TGF-β receptors and various Smads for proteasome-mediated degradation. In this context, we wished to determine whether Smurf2 has a physiological role during embryo implantation, especially in trophoblast invasion. We examined the spatio-temporal expression of Smurf2 in human placental villi and the function of Smurf2 in trophoblast cell migration and invasion in a model system involving a human extravillous trophoblast cell line, HTR-8/SVneo. Results from RT-PCR and immunohistochemical studies showed that expression of Smurf2 in placental villi was the highest during the first trimester and decreased as the pregnancy progressed. Overexpression of Smurf2 in HTR-8/SVneo cells reduced TGF-β type I receptor levels, and enhanced cell migration and invasion. Conversely, RNA interference–mediated downregulation of Smurf2 resulted in a significant increase in TGF-β type I receptor protein levels. However, the levels of Smad2, another potential target of Smurf2, remained unchanged. In conclusion, the present study suggests that Smurf2 promotes trophoblast cell migration and invasion, and this function may involve downregulation of TGF-β type I receptor. (J Histochem Cytochem 57:605–612, 2009)     

Human placental trophoblast as an in vitro model for tumor progression

The human placenta is a highly invasive tumor-like structure in which a subpopulation of placental trophoblast cells known as the "extravillous trophoblast" (EVT) invades the uterine decidua and its vasculature to establish adequate fetal-maternal exchange of molecules. By utilizing in vitro-propagated short-lived EVT cell lines we found that molecular mechanisms responsible for their invasiveness are identical to those of cancer cells; however, unlike cancer cells, their proliferation, migration, and invasiveness in situ are stringently controlled by decidua-derived transforming growth factor (TGF)-beta. By SV40T antigen transfection of normal EVT cells followed by a forced crisis regimen in culture we produced an immortalized premalignant derivative that is hyperproliferative, hyperinvasive, and deficient in gap-junctional intercellular communication. Both premalignant and malignant EVT (JAR and JEG-3 choriocarcinoma) cell lines were found to be TGF-beta-resistant. Using these cell lines, we investigated genetic changes responsible for transition of the normal EVT cells to premalignant and malignant phenotype. Hyperinvasiveness in both cases resulted from a downregulation of tissue inhibitor of metalloprotease (TIMP)-1 and plasminogen activator inhibitor (PAI)-1 genes. In contrast to normal EVT cells, both cell types failed to upregulate these genes in response to TGF-beta. Loss of TGF-beta response in malignant EVT cells was explained by the loss of expression of Smad3 gene. Differential mRNA display of normal and premalignant EVT cells identified up- and down-regulation of numerous known or novel genes in premalignant EVT cells, with potential oncogenic and (or) tumor-suppressor functions, e.g., loss of fibronectin and insulin-like growth factor binding protein (IGFBP-5). Premalignant EVT cells also lost IGF receptor type 2 (IGFR-II). IGFBP-5 was shown to be a negative regulator of IGF-1-induced proliferation of premalignant EVT cells, so that loss of IGFBP-5 as well as IGFR-II permitted their unrestricted proliferation in an IGF-I-rich microenvironment of the fetal-maternal interface. The present model may be a good prototype for identifying genetic changes underlying epithelial tumor progression.     

A novel host/tumor cell interaction activates matrix metalloproteinase 1 and mediates invasion through type I collagen.

Along with degradation of type IV collagen in basement membrane, destruction of the stromal collagens, types I and III, is an essential step in the invasive/metastatic behavior of tumor cells, and it is mediated, at least in part, by interstitial collagenase 1 (matrix metalloproteinase 1 (MMP-1)). Because A2058 melanoma cells produce substantial quantities of MMP-1, we used these cells as models for studying invasion of type I collagen. With a sensitive and quantitative in vitro invasion assay, we monitored the ability of these cells to invade a matrix of type I collagen and the ability of a serine proteinase inhibitor and all-trans-retinoic acid to block invasion. Although these cells produce copious amounts of MMP-1, they do not invade collagen unless they are co-cultured with fibroblasts or with conditioned medium derived from fibroblasts. Our studies indicate that a proteolytic cascade that depends on stromal/tumor cell interactions facilitates the ability of A2058 melanoma cells to invade a matrix of type I collagen. This cascade activates latent MMP-1 and involves both serine proteinases and MMPs, particularly stromelysin 1 (MMP-3). All-trans-retinoic acid (10(-6) M) suppresses the invasion of tumor cells by several mechanisms that include suppression of MMP synthesis and an increase in levels of tissue inhibitor of metalloproteinases 1 and 2. We conclude that invasion of stromal collagen by A2058 melanoma cells is mediated by a novel host/tumor cell interaction in which a proteolytic cascade culminates in the activation of pro-MMP-1 and tumor cell invasion.     

Mechanism of control of trophoblast invasion in situ.

We have previously shown that first trimester human trophoblast cells share in vitro invasive properties with malignant cells. In this study we show that the in situ control of trophoblast invasion is provided by the uterine microenvironment. Trophoblast cells were labeled with 125I-deoxyuridine and examined for their ability to invade an epithelium-free human amniotic membrane in vitro under various conditions. The degree of invasion was determined as the percentage of the radioactivity retained within the membrane. Conditioned media from first trimester human decidual cells (DCM) suppressed invasion of trophoblast cells in the amnion invasion assay. This suppression was prevented by addition of neutralizing anti-TGF beta antibody or neutralizing antibody to tissue inhibitor of metalloproteinases (TIMP-1) to the DCM, and mimicked by TGF beta 1. These antibodies also augmented invasion beyond control levels, suggesting that trophoblast cells may also produce these factors. A bioassay for TGF beta activity, measured by antiproliferative effect on the mink lung epithelial cell line Mv 1 Lu, revealed that decidual cells produced this factor only in the latent form, whereas the active form was produced by the trophoblast. A decrease in collagenase type IV activity in the conditioned media of trophoblast cultures was observed when TGF beta 1 was added to these cultures. Removal of endogenous TGF beta in trophoblast cultures by addition of anti-TGF beta antibody resulted in down-regulation of TIMP message as determined by Northern analysis. These results indicate that a) decidua-derived (and to a minor extent trophoblast-derived) TGF beta is the prime mediator in the control of invasion by first trimester trophoblast, the latent form of TGF beta likely being activated by trophoblast-derived proteinases; b) induction of TIMP by TGF beta in both trophoblast and decidua is the final pathway in this control.     

Serum-dependent effects of IGF-I and insulin on proliferation and invasion of human first trimester trophoblast cell models

Extravillous cytotrophoblasts are specialised epithelial cells of the placenta that proliferate or invade the maternal decidua. Little is known about the mechanisms that regulate these processes. Here the effects of several insulin and insulin-like growth factor-I (IGF-I) doses, either singly or in synergy with serum, on human chorionic gonadotropin-beta (hCG-beta) secretion (RIA), proliferation (cell counting, cyclin B(1) levels) and invasion [Matrigel invasion assay, secretion of matrix metalloproteinases (MMP) 2 and 9] were investigated. The choriocarcinoma cell lines BeWo, JAR and JEG-3 served as models for first trimester human trophoblasts. Both growth factors altered hCG-beta secretion and proliferation dependent on the cell line. Insulin stimulated proliferation in JAR cells and, to a lesser extent, in JEG-3 cells, and when cultured in serum-free medium, BeWo was not affected. Invasion was not affected although proMMP-2 levels in culture medium were altered under some conditions. A strong synergistic effect with serum was noted. In the presence of serum both growth factors reduced proliferation and invasion in a similar fashion. Since the cell models differ by their degree of differentiation, the data demonstrate that the effects of insulin and IGF-I strongly depend on serum and the degree of differentiation. It can be speculated that IGF-I can take on tasks of insulin in the regulation of trophoblast functions under conditions of insulinopenia.     

Coordinated regulation of human trophoblast invasiveness by macrophages and interleukin 10

Trophoblast invasion and modification of the spiral arterioles are essential for the establishment of adequate uteroplacental blood flow during pregnancy. However, such vascular remodeling is deficient in preeclampsia. This disease is also associated with increased maternal levels of circulating proinflammatory cytokines such as tumor necrosis factor (TNF) and reduced levels of immunoregulatory cytokines such as interleukin 10 (IL10). We have previously shown that activated macrophages inhibit trophoblast invasiveness in vitro. The present study demonstrates that IL10 interferes with the invasion-inhibitory effect that activated macrophages exert on trophoblast cells. Co-culture experiments revealed that human lipopolysaccharide (LPS)-activated macrophages inhibited the ability of immortalized HTR-8/SVneo human trophoblast cells to invade through reconstituted extracellular matrix. This effect of activated macrophages on trophoblast invasiveness was paralleled by decreased expression of urokinase plasminogen activator receptor (PLAUR) on the surface of trophoblast cells, and by increased secretion of plasminogen activator inhibitor type 1 (SERPINE1). Exposure of LPS-treated macrophages to IL10 prior to co-culture prevented their ability to inhibit trophoblast invasion, PLAUR expression, and to stimulate SERPINE1 secretion. Interleukin 10 prevented macrophage activation by LPS as determined by the lack of secretion of TNF in the culture medium, and a neutralizing TNF antibody completely blocked the effect of macrophages on trophoblast invasion. These results indicate that decreased circulating levels of IL10 associated with preeclampsia may contribute to inadequate trophoblast invasion and remodeling of the uterine spiral arterioles.     

Invasiveness of mouse embryos to human ovarian cancer cells HO8910PM and the role of MMP-9

ABSTRACT: Background Our previous work found that mouse embryos could invade malignant cancer cells. In the process of implantation, embryo trophoblast cells express matrix metalloproteinases and the invasive ability of trophoblast cells is proportional to matrix metalloproteinase-9 protein expression. So the purpose of this study is to observe the effects of mouse embryos on human ovarian cancer cells in the co-culture environment in vitro and explore the possible mechanism of matrix metalloproteinase-9. Methods Several groups of human ovarian cancer cells HO8910PM were co-cultured with mouse embryos for different time duration, after which the effects of mouse embryos on morphology and growth behavior of HO8910PM were observed under the light microscope real-time or by H.E staining. Apoptosis was detected under laser confocal microscope by Annexin V-EGFP/PI staining in situ. Invasion ability of tumor cells was studied by transwell experiments. After matrix metalloproteinase 9 (MMP -9) activity was inhibited by MMP-9 Inhibitor I, the interaction between mouse embryos and human ovarian cancer cells HO8910PM was observed. Results Mouse embryos were able to invade co-cultured human ovarian cancer cell layer which extended in the bottom of the culture dish, and gradually pushed away tumor cells to form their own growth space. The number of apoptosis tumor cells surrounding the embryo increased under laser confocal microscope. After co-cultured with mouse embryos, tumor cells invasive ability was lowered compared with the control group. After MMP-9 activity was inhibited, the interaction between mouse embryos and HO8910PM cells had no significant difference compared with the normal MMP-9 activity group. Conclusion Mouse embryos were able to invade human ovarian cancer cells in vitro and form their own growth space, promote apoptosis of human ovarian cancer cells and lower their invasive ability. The mouse embryo was still able to invade human ovarian cancer cells after MMP-9 activity was inhibited.     

Adhesive and degradative properties of human placental cytotrophoblast cells in vitro

Human fetal development depends on the embryo rapidly gaining access to the maternal circulation. The trophoblast cells that form the fetal portion of the human placenta have solved this problem by transiently exhibiting certain tumor-like properties. Thus, during early pregnancy fetal cytotrophoblast cells invade the uterus and its arterial network. This process peaks during the twelfth week of pregnancy and declines rapidly thereafter, suggesting that the highly specialized, invasive behavior of the cytotrophoblast cells is closely regulated. Since little is known about the actual mechanisms involved, we developed an isolation procedure for cytotrophoblasts from placentas of different gestational ages to study their adhesive and invasive properties in vitro. Cytotrophoblasts isolated from first, second, and third trimester human placentas were plated on the basement membrane-like extracellular matrix produced by the PF HR9 teratocarcinoma cell line. Cells from all trimesters expressed the calcium-dependent cell adhesion molecule cell-CAM 120/80 (E-cadherin) which, in the placenta, is specific for cytotrophoblasts. However, only the first trimester cytotrophoblast cells degraded the matrices on which they were cultured, leaving large gaps in the basement membrane substrates and releasing low molecular mass 3H-labeled matrix components into the medium. No similar degradative activity was observed when second or third trimester cytotrophoblast cells, first trimester human placental fibroblasts, or the human choriocarcinoma cell lines BeWo and JAR were cultured on radiolabeled matrices. To begin to understand the biochemical basis of this degradative behavior, the substrate gel technique was used to analyze the cell-associated and secreted proteinase activities expressed by early, mid, and late gestation cytotrophoblasts. Several gelatin-degrading proteinases were uniquely expressed by early gestation, invasive cytotrophoblasts, and all these activities could be abolished by inhibitors of metalloproteinases. By early second trimester, the time when cytotrophoblast invasion rapidly diminishes in vivo, the proteinase pattern of the cytotrophoblasts was identical to that of term, noninvasive cells. These results are the first evidence suggesting that specialized, temporally regulated metalloproteinases are involved in trophoblast invasion of the uterus. Since the cytotrophoblasts from first trimester and later gestation placentas maintain for several days the temporally regulated degradative behavior displayed in vivo, the short-term cytotrophoblast outgrowth culture system described here should be useful in studying some of the early events in human placen     

Mechanisms of placental invasion of the uterus and their control.

Trophoblast cells of the placenta in many species have acquired mechanisms to invade the uterus, inclusive of its blood vessels, to establish efficient fetomaternal exchange of molecules. This invasion is strictly controlled both spatially and temporally and, in humans, usually continues until midgestation. Key mechanisms underlying various steps in trophoblast invasion are: (i) the attachment to the basement membrane, most likely by binding to laminin; (ii) the detachment from the basement membrane matrix, a process requiring the presence of complex-type oligosaccharides on the cell surface; and (iii) the breakdown of basement membrane components, mediated by secretion of metalloproteases (such as type IV collagenases) and serine proteases (plasminogen activator). Activation of trophoblast-derived metalloproteases appears to be plasmin dependent. Trophoblast invasiveness in situ is controlled by the microenvironment, owing to local production of anti-invasive factors by the decidual tissue of the uterus. One of these factors is TIMP (tissue inhibitor of metalloproteases), which neutralizes metalloproteases in an equimolar ratio. Another is TGF-beta (transforming growth factor-beta), which has a dual effect: it induces TIMP-1 secretion by the trophoblast and decidual cells and promotes differentiation of invasive trophoblast cells into multinucleated giant cells, which are presumably noninvasive. Thus, TGF-beta provides the key control of trophoblast invasiveness in situ. This control is lost in certain choriocarcinomas. In contrast to the response shown by the normal trophoblast, JAR and JEG-3 choriocarcinoma cell invasiveness does not seem to be inhibited by TGF-beta. In fact, in preliminary studies, JAR cells responded to TGF-beta by increased invasiveness.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human trophoblast and JEG choriocarcinoma cells are sensitive to lysis by IL-2-stimulated decidual NK cells.

Freshly isolated decidual large granular lymphocytes (LGL) show natural killer (NK) activity against K562 cells but not against normal human trophoblast. We now show that these decidual LGL proliferate in vitro in response to recombinant interleukin-2 (rIL-2) and that these rIL-2-stimulated cells acquire a broad cytolytic potential that is characteristic of lymphokine-activated killer (LAK) cells. Both fetal fibroblasts and JEG-3 choriocarcinoma cells are resistant to lysis by freshly isolated decidual effectors but are readily killed by IL-2-stimulated decidual LGL. The ability to kill these target cells is acquired after only 18 hr exposure to rIL-2. rIL-2-activated decidual LGL also kill cultured normal trophoblast cells but much lower levels of cytolysis were seen even after the effectors had been stimulated with rIL-2 for 4-6 days. The preferential killing of malignant over normal human trophoblast cells raises questions about the potential role of IL-2-activated decidual LGL in the control of unduly invasive or malignant trophoblast populations in vivo.     

Tumor suppressor properties of the ESCRT-II complex component Vps25 in Drosophila

We have found that the Drosophila gene vps25 possesses several properties of a tumor suppressor. First, vps25 mutant cells activate Notch and Dpp receptor signaling, inducing ectopic organizers in developing eyes and limbs and consequent overproliferation of both mutant and nearby wild-type cells. Second, as the mutant cells proliferate, they lose their epithelial organization and undergo apoptosis. Strikingly, when apoptosis of mutant cells is blocked, tumor-like overgrowths are formed that are capable of metastasis. vps25 encodes a component of the ESCRT-II complex, which sorts membrane proteins into multivesicular bodies during endocytic trafficking to the lysosome. Activation of Notch and Dpp receptor signaling in mutant cells results from an endocytic blockage that causes accumulation of these receptors and other signaling components in endosomes. These results highlight the importance of endocytic trafficking in regulating signaling and epithelial organization and suggest a possible role for ESCRT components in human cancer.