The effect of leukemia inhibitory factor (LIF) on trophoblast differentiation: a potential role in human implantation

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein strongly associated with normal implantation in the mouse. We have recently determined that LIF is expressed in the human endometrium in a menstrual cycle dependent manner. Maximal expression is observed between days 19 and 25 of the menstrual cycle, coinciding with the time of human implantation. In this study we have utilized purified cultures of human cytotrophoblasts to examine the effects of LIF on several morphologic and biochemical markers of the trophoblastic differentiation. We purified human cytotrophoblasts from term placentae and cultured them with and without LIF (10 ng/mL). The secretion of human CG, oncofetal fibronectin, and progesterone were measured at 24, 48, 72, and 96 h. Northern blot analysis was used to assess messenger RNA (mRNA) expression of beta hCG and oncofetal fibronectin. We found that LIF markedly decreased trophoblast production of hCG protein at 72 and 96 h, as well as expression of beta hCG mRNA. LIF also significantly increased the expression of oncofetal fibronectin mRNA and secretion of the protein. LIF did not affect steroidogenic activity of cultured trophoblasts, as determined by progesterone production. These biochemical changes are characteristic of cytotrophoblast differentiation toward an anchoring extravillous phenotype. Thus, LIF appears to be an important regulator of human embryonic implantation by directly modulating trophoblast differentiation.     

The rhesus monkey analogue of human lymphocyte antigen-G is expressed primarily in villous syncytiotrophoblasts

The human placenta expresses the nonclassical major histo-compatibility complex (MHC) class I molecule, human lymphocyte antigen (HLA)-G, which may contribute to the establishment of maternal-fetal immune tolerance. Although the HLA-G ortholog of the rhesus monkey, Mamu-G, is a pseudogene, another nonclassical MHC class I locus, Mamu-AG, is expressed in the rhesus monkey placenta. Mamu-AG encodes MHC class I A locus-related molecules that exhibit all the characteristics of human HLA-G, including limited polymorphism and a truncated cytoplasmic domain. We have examined MHC class I glycoprotein and Mamu-AG mRNA expression in the rhesus placenta and in cultured trophoblasts. Immunocytochemical analysis of rhesus placental tissues with the W6/32 monoclonal antibody demonstrated a high level of MHC class I expression in villous syncytiotrophoblasts, whereas villous cytotrophoblasts were largely MHC class I negative. Only low levels of MHC class I expression were seen in extravillous cytotrophoblasts of cell columns and the trophoblastic shell. In situ hybridization demonstrated that Mamu-AG mRNAs were expressed at a high level in first-trimester villous syncytiotrophoblasts. MHC class I and Mamu-AG expression was significantly up-regulated during in vitro culture and differentiation of freshly isolated villous cytotrophoblasts into syncytiotrophoblasts. Preferential Mamu-AG expression in syncytiotrophoblasts suggests that rhesus monkey MHC class I-bearing trophoblasts could potentially interact with maternal peripheral blood lymphocytes rather than with uterine decidual lymphocytes as has been proposed for human trophoblasts.     

Analysis of WT1 in granulosa cell and other sex cord-stromal tumors

The molecular genetic events involved in the etiology of granulosa cell, Sertoli cell, and Leydig cell tumors are unknown. The expression of the Wilms' tumor suppressor gene WT1 in granulosa and Sertoli cells prompted us to analyze this gene for mutations in 11 granulosa cell tumors, three Leydig cell tumors, and one Sertoli/Leydig cell tumor. Although most of these tumors express WT1 mRNA, none harbors a WT1 mutation in the zinc finger domains where > 90% of WT1 mutations in sporadic Wilms' tumors have been found. In addition we were able to exclude tumor-specific loss of heterozygosity in 13 of 15 cases. Taken together these results suggest that the WT1 gene is unlikely to play an important role in the development of sex cord-stromal tumors.     

Midkine as a novel target gene for the Wilms' tumor suppressor gene (WT1)

Midkine (MK) is a heparin-binding growth factor which is strongly expressed during the midgestation period of mouse embryogenesis. Wilms' tumor is an embryonal kidney malignancy in infants, and WT1 has been identified as its tumor suppressor gene. The high expression level of MK in all Wilms' tumor specimens so far examined and the presence of two WT1 elements (5'-GCGGGGGCG-3') in the human MK promoter region led us to examine the possible role of the WT1 gene product in the regulation of MK gene expression. A gel shift assay verified the complex formation between the WT1 gene product and WT1 consensus sequence of MK gene. DNase1 footprint analysis also demonstrated that the downstream WT1 element was protected from DNase1 cleavage by the addition of the WT1 protein. The human MK promoter fused with the chloramphenicol acetyltransferase gene (phMK2.3kCAT) was co-transfected with an effector plasmid containing the WT1 gene into several cell lines. Transient transfection assays showed suppression of the MK promoter by WT1 co-transfection in recipient cells; deletion of the WT1 binding site abolished the suppression. The evidence reported in this study indicates that MK gene is a newly identified WT1 target gene.     

Regulation of ornithine decarboxylase gene expression by the Wilms' tumor suppressor WT1

The importance of ornithine decarboxylase (ODC) to cell proliferation is underscored by the complex array of cell-specific mechanisms invoked to regulate its synthesis and activity. Misregulation of ODC has severe negative consequences on normal cell function, including the acquisition of tumorigenic growth properties by cells overexpressing ODC. We hypothesize that ODC gene expression is a candidate target for the anti-proliferative function of certain tumor suppressors. Here we show that the Wilms' tumor suppressor WT1 binds to multiple sites within the human ODC promoter, as determined by DNase I protection and methylation interference assays. The expression of WT1 in transfected HCT 116, NIH/3T3 and HepG2 cells represses activity of the ODC promoter controlling expression of a luciferase reporter gene. In contrast WT1 expression enhances ODC promoter activity in SV40-transfected HepG2 cells. Both the extent of modulation of ODC gene expression and the mediating WT1 binding elements are cell specific. Constructs expressing WT1 deletion mutants implicate two regions required for repressor function, as well as an intrinsic activation domain. Understanding the regulation of ODC gene expression by WT1 may provide valuable insights into the roles of both WT1 and ODC in development and tumorigenesis.     

p53, Bax and Bcl-2 expression, and apoptosis in gestational trophoblast of complete hydatidiform mole

This study investigates the extent of apoptosis in complete hydatidiform mole (CHM), using an in situ 3'-end DNA labelling (TUNEL) technique on formalin-fixed and paraffin-embedded sections. The sections were also immunostained with antibodies to p53, Bax and Bcl-2 proteins. In 10 normal placenta cases and 15 CHM cases, the apoptotic index was <1 and 2-4 per cent, respectively. The labelled trophoblastic cells possessed pyknotic nuclei and densely eosinophilic cytoplasm which corresponded well to the so-called apoptotic bodies by light and electron microscopy. The p53 positive reaction was restricted to the nuclei of cytotrophoblasts and intermediate trophoblasts, while the syncytiotrophoblasts showed only rare immunolocalization. Strong p53 expression was seen most often in cytotrophoblasts of CHM (>30 per cent of nuclei) which also showed a higher apoptosis index, while cytotrophoblasts in normal placentae were weakly and focally labelled (<10 per cent of nuclei). There were statistical differences between normal and CHM cases (P<0.05). Bcl-2 accumulation, on the other hand, was observed predominantly in syncytiotrophoblasts of normal placentae, and cytotrophoblasts and intermediate trophoblasts did not express Bcl-2 in all cases. Interestingly, syncytiotrophoblasts were found to be negative for Bax protein and positive in cytotrophoblast, which is consistent with the function of the protein in conveying increased apoptosis susceptibility to this cell population. The results show that the level of apoptosis correlates with the histological type of the gestational trophoblasts, and apoptosis index is higher in cytotrophoblasts in CHM. The fact that p53 quantitative expression and an increase in the Bax/Bcl-2 ratio were also observed in CHM suggested that they may contribute partly to the high level of apoptosis.     

Human cytotrophoblast invasion is up-regulated by epidermal growth factor: evidence that paracrine factors modify this process

Formation of the human placenta requires a subset of cytotrophoblast stem cells to acquire an invasive phenotype. We examined the effect on cytotrophoblast invasiveness of growth factors that control the differentiation of other cells. Exogenous TGF-beta 1, PDGF-AA, PDGF-BB, and TNF-alpha affected neither cell morphology nor the rate of cytotrophoblast invasion in vitro. In contrast, addition of EGF to first trimester cytotrophoblast cultures produced dramatic changes in morphology and a severalfold increase in invasive capacity. The effects of EGF on later gestation cytotrophoblasts, whose invasive capacity is diminished, were much less pronounced. Next we investigated whether cytotrophoblasts themselves produce ligands that interact with the EGF receptor. A radioimmunoassay and a radioreceptor assay failed to detect EGF receptor ligands in cytotrophoblast-conditioned medium. Likewise, by RT-PCR cytotrophoblasts expressed neither EGF nor TGF-alpha mRNA. In contrast, EGF receptor mRNA was expressed and its protein levels remained constant during the experiment. Immunolocalization using F(ab') fragments of an anti-human EGF antibody failed to detect this growth factor in the chorionic villus. We conclude that maternal ligands that interact with the EGF receptor could play an important role by up-regulating trophoblast invasion, particularly during the early stages of pregnancy.     

Infrequent mutation of the WT1 gene in 77 Wilms' Tumors

Homozygous deletions in Wilms' tumor DNA have been a key step in the identification and isolation of the WT1 gene. Several additional loci are also postulated to contribute to Wilms' tumor formation. To assess the frequency of WT1 alterations we have analyzed the WT1 locus in a panel of 77 Wilms' tumors. Eight tumors showed evidence for large deletions of several hundred or thousand kilobasepairs of DNA, some of which were also cytogenetically detected. Additional intragenic mutations were detected using more sensitive SSCP analyses to scan all 10 WT1 exons. Most of these result in premature stop codons or missense mutations that inactivate the remaining WT1 allele. The overall frequency of WT1 alterations detected with these methods is less than 15%. While some mutations may not be detectable with the methods employed, our results suggest that direct alterations of the WT1 gene are present in only a small fraction of Wilms' tumors. Thus, mutations at other Wilms' tumor loci or disturbance of interactions between these genes likely play an important role in Wilms' tumor development.     

A new family of heparin-binding growth/differentiation factors: increased midkine expression in Wilms' tumor and other human carcinomas

Midkine (MK) and heparin-binding growth-associated molecule/pleiotrophin form a new family of heparin-binding growth/differentiation factors. We studied MK gene expression in human tumors. In normal human reference tissues, MK was highly expressed in the mucosal tissue of the small intestine, moderately in the thyroid, weakly in the tissues of the lung, colon, stomach, kidney, and spleen, and not at all in the liver. All of 6 surgically removed specimens of Wilms' tumor highly expressed MK. Also, a moderate to intense level of MK expression was noted in the majority of surgically removed hepatocellular carcinomas. The MK mRNA level was analyzed in a number of cultured and nude mice-transplanted lines of human tumors. In stomach, colon, pancreatic, lung, and esophageal carcinomas, a moderate to high level of MK expression was found in the majority of them. These results suggest an important role of MK in the development and/or biological behavior of tumors and raised a possibility to use MK as a diagnostic marker. Heparin-binding growth associated molecule/pleiotrophin mRNA was low or scarcely detectable in samples analyzed thus far except for significant levels of the expression that were observed in PA-1 teratocarcinoma cells and in some surgical specimens of Wilms' tumor.     

Human placental cytotrophoblasts produce the immunosuppressive cytokine interleukin 10

The mechanism by which the mammalian mother accepts the implanting fetus as an allograft remains unexplained, but is likely to be the result of a combination of factors. Mononuclear cytotrophoblasts, the specialized fetal cells of the placenta that invade the uterus, play an important role. These cells express HLA-G, an unusual major histocompatibility complex class I-B molecule, and secrete cytokines and pregnancy-specific proteins that can regulate immune function. We investigated whether cytotrophoblasts secrete interleukin 10 (IL-10), a cytokine that potently inhibits alloresponses in mixed lymphocyte reactions. Cytotrophoblasts from all stages of pregnancy produced IL-10 in vitro, but neither placental fibroblasts nor choriocarcinoma (malignant trophoblast) cell lines did so. Spontaneous IL-10 production averaged 650, 853, and 992 pg/10(6) cells in the first, second, and third trimesters of pregnancy, respectively. IL-10 secretion dropped approximately 10-fold after the first 24 h of culture, and was paralleled by a decrease in messenger RNA. IL-10 messenger RNA was detected in biopsies of the placenta and the portion of the uterus that contains invasive cytotrophoblasts, suggesting that this cytokine is also produced in vivo. IL-10 secreted by cytotrophoblasts in vitro is bioactive, as determined by its ability to suppress interferon gamma production in an allogeneic mixed lymphocyte reaction. We conclude that human cytotrophoblast IL-10 may be an important factor that contributes to maternal tolerance of the allogeneic fetus.