Myeloperoxidase enhances the migration and invasion of human choriocarcinoma JEG-3 cells.

Myeloperoxidase (MPO) is one of the most abundant proteins in neutrophil granules. It catalyzes the production of reactive oxygen species, which are important in inflammation and immune defense. MPO also binds to several proteins, lipids, and DNA to alter their function. MPO is present at the feto-maternal interface during pregnancy, where neutrophils are abundant. In this study, we determined the effect of MPO on JEG-3 human choriocarcinoma cells as a model of extravillous trophoblasts (EVTs) during early pregnancy. We found that MPO was internalized by JEG-3 cells and localized to the cytoplasm and nuclei. MPO internalization and activity enhanced JEG-3 cell migration and invasion, whereas this effect was impaired by pre-treating cells with heparin, to block cellular uptake, and MPO-activity inhibitor 4-ABAH. This study identifies a novel mechanism for the effect of MPO on EVT function during normal pregnancy and suggests a potential role of MPO in abnormal pregnancies.

Decidual stromal cells support tolerance at the human foetal-maternal interface by inducing regulatory M2 macrophages and regulatory T-cells.

During pregnancy, the semi-allogeneic nature of the foetus requires maternal immune adaption and acquisition of tolerance at the foetal-maternal interface. Macrophages with regulatory properties and regulatory T (Treg) cells are central in promoting foetal tolerance and are enriched in the decidua (the uterine endometrium during pregnancy). Although tissue-resident decidual stromal cells (DSC) have been implicated in regulatory functions, it is not known if they are able to induce the regulatory phenotype of macrophages and T-cells. In this study we report that maternally derived DSC are able to induce homeostatic M2 macrophages and Treg cells. CD14+ monocytes and CD4+ T-cells from healthy non-pregnant women were cultured in the presence or absence of conditioned medium (CM) from DSC isolated from 1st trimester and term placentas. DSC-CM alone was able to promote the survival of macrophages and to induce a regulatory CD14brightCD163+CD209+CD86dim phenotype, typical for decidual macrophages and similar to that induced by M-CSF. Interestingly, DSC-CM was also able to overrule the pro-inflammatory effects of GM-CSF by upregulating CD14, CD163 and CD209. Protein-profiling showed that M-CSF was secreted by DSC, and blocking of M-CSF partially reversed the M2 phenotype and reduced viability. DSC-CM also expanded CD25brightFoxp3+ Treg cells, an expansion that was abolished by a SMAD3-inhibitor, indicating the contribution of TGF-ß signaling. In conclusion, our findings collectively emphasize the role of tissue-resident stromal cells in shaping the tolerogenic environment at the foetal-maternal interface.

3-Dimensional JEG-3 choriocarcinoma cell organoids as a model for trophoblast expansion and differentiation.

JEG-3 choriocarcinoma cells have been widely used as a model for placental trophoblast. Herein, 3-dimensional (3D) JEG-3 organoids (JEG-3-ORGs) were established using a protocol that we recently developed for primary cytotrophoblast organoids (CTB-ORGs). 3D JEG-3-ORGs, cultivated in basic culture medium, rapidly divide and spontaneously undergo differentiation. Under stem cell culture conditions (activation of WNT/EGF signalling and inhibition of TGF-ß signalling) smaller organoids with reduced proliferative capacity were generated specifically abolishing expression of extravillous trophoblast (EVT)-specific genes. Similar to CTB-ORGs, removal of the WNT activator CHIR99021 induced re-expression of these genes in JEG-3-ORGs. Hence, JEG-3-ORGs could be used as a model for directed EVT differentiation.

Expression pattern and phosphorylation status of Smad2/3 in different subtypes of human first trimester trophoblast.

INTRODUCTION: TGF-ß superfamily members are thought to play a pivotal role in placental development and differentiation. However, their downstream effectors, the Smad transcription factors, have been poorly investigated in human trophoblasts. METHODS: Expression and localisation of the canonical TGF-ß targets Smad2/3 and their regulators (Smad4 and Smad7) were investigated in first trimester placenta and purified cytotrophoblast (CTB) subtypes using immunofluorescence, western blotting and qPCR. Canonical and non-canonical activation was analysed in nuclear/cytoplasmic extracts of trophoblast subtypes as well as in tissue sections using antibodies against Smad2/3, phosphorylated either at the C-terminus (pSmad2C/3C) or in their linker regions (pSmad2L/3L). Smad phosphorylation was also examined in differentiating extravillous trophoblasts (EVTs) in the absence or presence of decidual stromal cell (DSC)-conditioned medium. RESULTS: Smad2, Smad4 and Smad7 protein were uniformly expressed between 6th and 12th week placentae and the different isolated CTB subtypes. Activated pSmad2L was mainly detected in nuclei and cytoplasm of villous CTBs, whereas pSmad2C was absent from these cells. In contrast, pSmad2C could be detected in the cytoplasm of cell column trophoblasts and in the cytoplasm/nuclei of EVTs. Smad3 and its phosphorylated forms pSmad3C and pSmad3L specifically localised to EVT nuclei. During EVT differentiation autocrine activation of pSmad2C/3C and pSmad3L was observed. DSC-conditioned medium further increased Smad2/3 phosphorylation in EVTs. DISCUSSION: The lack of pSmad2C in villous CTBs suggests that other mitogens than TGF-ß could promote Smad2 linker phosphorylation under homeostatic conditions. Whereas autocrine signalling activates Smad2/3 in differentiating EVTs, paracrine factors contribute to Smad phosphorylation in these cells.

Notch signalling in placental development and gestational diseases.

Activation of Notch signalling upon cell-cell contact of neighbouring cells controls a plethora of cellular processes such as stem cell maintenance, cell lineage determination, cell proliferation, and survival. Accumulating evidence suggests that the pathway also critically regulates these events during placental development and differentiation. Herein, we summarize our present knowledge about Notch signalling in murine and human placentation and discuss its potential role in the pathophysiology of gestational disorders. Studies in mice suggest that Notch controls trophectoderm formation, decidualization, placental branching morphogenesis and endovascular trophoblast invasion. In humans, the particular signalling cascade promotes formation of the extravillous trophoblast lineage and regulates trophoblast proliferation, survival and differentiation. Expression patterns as well as functional analyses indicate distinct roles of Notch receptors in different trophoblast subtypes. Altered effects of Notch signalling have been detected in choriocarcinoma cells, consistent with its role in cancer development and progression. Moreover, deregulation of Notch signalling components were observed in pregnancy disorders such as preeclampsia and fetal growth restriction. In summary, Notch plays fundamental roles in different developmental processes of the placenta. Abnormal signalling through this pathway could contribute to the pathogenesis of gestational diseases with aberrant placentation and trophoblast function.

ERBB2 gene amplification increases during the transition of proximal EGFR(+) to distal HLA-G(+) first trimester cell column trophoblasts.

INTRODUCTION: Although, DNA copy-number alterations (CNAs) have been well documented in a number of adverse phenotypic conditions, accumulating data suggest that CNAs also occur during physiological processes. Interestingly, extravillous trophoblasts induce the expression of the transforming, proto-oncogene ERBB2, which is frequently amplified in human cancer. However, no data are available to address whether trophoblast-related ERBB2 expression might also be linked to genomic amplification. METHODS: Dual color silver as well as fluorescence in situ hybridization analyses were carried out to evaluate frequency and degree of ERBB2 gene and chromosome 17 copy numbers in first trimester placental cell columns and isolated trophoblasts. Proliferative EGFR(+) and differentiated HLA-G(+) trophoblasts were identified or separated by means of in situ immunofluorescence co-stainings and magnetic beads cell isolation, respectively. RESULTS: ERBB2 gene amplification is detected in approximately 40% of isolated HLA-G(+) trophoblasts. Although already detectable in EGFR(+) cells, the percentage and extent of ERBB2 amplification was markedly increased in HLA-G(+) trophoblasts in situ and after isolation. Accordingly, HLA-G(+) trophoblasts highly express ERBB2 on protein level. Finally, ERBB2 copy number variations occur independently of aneuploidy as the majority of ERBB2 amplifying cells were cytogenetically diploid for chromosome 17. DISCUSSION: ERBB2 gene amplification is a frequent event during EVT differentiation. This finding challenges the long standing paradigm, which associates gene amplification with pathological conditions and further supports recent evidences suggesting that CNAs are a normal feature of developmental processes.

Expression pattern and function of Notch2 in different subtypes of first trimester cytotrophoblast.

INTRODUCTION: Notch signalling has been shown to control cytotrophoblast (CTB) proliferation, differentiation and motility suggesting that the conserved signalling pathway could be critical for human placental development. Since individual Notch receptors have not been elucidated, we herein investigated expression pattern and function of Notch2 in different first trimester trophoblast subpopulations. METHODS: Localisation of Notch2 was analysed in first trimester placental and decidual tissues using immunofluorescence. Notch2 transcript and protein levels were studied by qRT-PCR and Western blotting in proliferative EGF receptor (EGFR)(+) and differentiated HLA-G(+) CTBs, respectively, isolated from early placentae by MACS. CTB migration through fibronectin-coated transwells as well as proliferation (EdU labelling) in floating villous explant cultures and primary CTBs were investigated in the presence of Notch2 siRNAs or specific antibodies blocking Notch2 cleavage. RESULTS: In tissue sections Notch2 expression was higher in HLA-G(+) distal cell column trophoblasts (dCCTs) compared to proximal CCTs. Accordingly, expression of Notch2 mRNA and protein were elevated in isolated HLA-G(+) CTBs compared to EGFR(+) CTBs. Notch2 was also detectable in interstitial CTBs as well as in intramural CTBs associated with maternal decidual vessels. Antibody-mediated inhibition of Notch2 signalling did not affect proliferation, but increased migration of SGHPL-5 cells and primary CTBs. Similarly, Notch2 siRNA treatment promoted trophoblast motility. DISCUSSION: Notch2 is present in differentiated cells of the extravillous trophoblast lineage, such as dCCTs, interstitial and intramural CTBs, suggesting diverse roles of the particular receptor. Notch2 signalling, activated by cell-cell contact of neighbouring dCCTs, could attenuate trophoblast migration.

Notch-dependent RBPJκ inhibits proliferation of human cytotrophoblasts and their differentiation into extravillous trophoblasts.

Abnormal development of invasive trophoblasts has been implicated in the pathogenesis of human pregnancy diseases such as pre-eclampsia. However, critical signalling pathways controlling formation and differentiation of these cells have been poorly elucidated. Here, we provide evidence that the canonical Notch pathway, operating through Notch-dependent activation of its key regulatory transcription factor RBPJκ, controls proliferation and differentiation in villous explant cultures and primary trophoblasts of early pregnancy. Immunofluorescence of first trimester placental tissue revealed expression of RBPJκ and its co-activators, the MAML proteins, in nuclei of proliferative cell column trophoblasts (CCT) and differentiated, extravillous trophoblasts (EVTs). However, RBPJκ expression, transcript levels of the Notch target gene HES1 and activity of a Notch/RBPJκ-dependent luciferase reporter decreased during in vitro differentiation of primary cytotrophoblasts on fibronectin. Silencing of RBPJκ using silencing RNAs (siRNAs) increased proliferation of CCTs in floating villous explant cultures analysed by outgrowth and BrdU labelling. Similarly, down-regulation of the transcription factor enhanced BrdU incorporation in isolated primary cultures. However, motility of these cells was not affected. In addition, gene silencing of RBPJκ increased cyclin D1 expression in the two trophoblast model systems as well as markers of the differentiated, EVT, i.e. integrin α1, ADAM12 and T-cell factor 4. In summary, the data suggest that Notch-dependent RBPJκ activity could be required for balanced rates of trophoblast proliferation and differentiation in human placental anchoring villi preventing exaggerated trophoblast overgrowth as well as premature formation of EVTs.

Review: the ADAM metalloproteinases - novel regulators of trophoblast invasion?

During pregnancy, the extravillous trophoblast (EVT) invades the maternal decidua and remodels spiral arteries reaching as far as the inner third of the myometrium. This process is mandatory to a successful pregnancy since EVTs regulate spiral artery remodeling to achieve maximal vasodilation and thus an adequate nutrient supply to the embryo or communicate with maternal leukocyte populations to guarantee acceptance of the allogeneic conceptus. To achieve this, EVTs undergo a remarkable and unique differentiation process, which yields different phenotypes such as proliferative cell column trophoblasts or growth-arrested, invasive interstitial or endovascular cytotrophoblasts. Matrix metalloproteinases have long been seen as imperative to trophoblast invasion because of their ability to degrade extracellular matrix and therefore allow cellular movement in foreign tissues. However, global gene expression analysis reveals that EVTs also express various members of distintegrin and metalloproteinases (ADAMs). These proteases are associated with the process of proteolytic shedding and activation of surface proteins including growth factors, cytokines, receptors and their ligands rather than extracellular matrix breakdown. While ADAM12 has been associated with chromosomal abnormalities as well as preeclampsia or intrauterine fetal growth restriction, the function of ADAMs in trophoblasts remains elusive. In this article, we review the diverse invasive trophoblast phenotypes, EVT-associated protease systems and related open questions. In addition, we examine recent information about relevant ADAM members and their putative implications for EVT biology.

The role of interleukin-1ß in human trophoblast motility.

The pleiotropic cytokine interleukin-1ß (IL-1ß) can promote physiological cell migration, as well as cancer cell invasion and metastasis. Its role in human trophoblast invasion, however, has not been satisfactorily answered since direct, indirect as well as no effects on trophoblast motility have been published. Therefore, the role of IL-1ß has been re-evaluated by exclusively using human primary trophoblast model systems. Immunofluorescence of first trimester placentae indicated IL-1 receptor 1 (IL-1R1) protein expression in first trimester villous cytotrophoblasts (vCTB) and extravillous trophoblasts (EVT). The latter expressed higher mRNA levels of the receptor as shown by comparative gene chip data of vCTB and EVT. Similarly, Western blot analyses and immunofluorescence revealed a time- and differentiation-dependent increase of IL-1R1 in primary EVT seeded on fibronectin. IL-1ß dose-dependently elevated migration of isolated first trimester EVT through fibronectin-coated transwells, which was inhibited in the presence of IL-1R antagonist (IL-1Ra), whereas proliferation of these cells was not affected. Similarly, the interleukin did not alter proliferation of vCTB and cell column trophoblasts in floating villi of early pregnancy, but promoted migration in villous explant cultures seeded on collagen I. Western blot analyses of supernatants of primary EVT and first trimester villous explant cultures revealed IL-1ß induced secretion of urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI)-1 and PAI-2, which was diminished upon combined IL-1ß/IL-1Ra treatment. In conclusion, these data suggest that IL-1ß directly promotes trophoblast motility of first trimester EVT involving the uPA/PAI system.

IFPA Award in Placentology lecture: molecular regulation of human trophoblast invasion.

Invasion of extravillous trophoblast cell types into maternal uterine tissues is essential for successful human placental development and progression of pregnancy. Whereas endovascular trophoblasts migrate into the maternal spiral arteries, interstitial trophoblasts invade the decidual stroma, colonize the vessels from outside and communicate with diverse uterine cell types such as decidual stromal cells, macrophages and uterine NK cells. For example, interstitial trophoblasts expressing polymorphic human leukocyte antigen-C interact with uterine NK cells through binding to their killer immunoglobulin-like receptors which likely plays a role in trophoblast invasion and reproductive success of pregnancy. Both extravillous trophoblast subtypes are critically involved in the vascular transformation of the spiral arteries into dilated conduits ensuring appropriate blood flow into the intervillous space. Failures in this remodeling process are thought to be associated with severe forms of fetal growth restriction, preeclampsia and other pregnancy complications warranting studies on the molecular regulation of extravillous trophoblast differentiation. Moreover, interstitial trophoblast-derived hormones may regulate diverse biological functions in the decidua. In particular, human chorionic gonadotrophin has been shown to promote angiogenesis and to suppress apoptosis of endometrial stromal cells. In return, decidual cells produce a plethora of soluble factors controlling trophoblast invasion in a time- and distance-dependent manner. However, the underlying mechanisms have not been fully elucidated. Here, we will summarize autocrine as well as paracrine factors regulating invasion of extravillous trophoblasts and discuss critical signaling cascades involved. In addition, we will focus on key regulatory transcription factors controlling cell column proliferation and differentiation of the human extravillous trophoblast.

Trophoblast invasion: assessment of cellular models using gene expression signatures.

Invasive, extravillous trophoblasts (EVT) of the human placenta are critically involved in successful pregnancy outcome since they remodel the uterine spiral arteries to increase blood flow and oxygen delivery to the placenta and the developing fetus. To gain more insights into their biological role different primary cell culture models are commonly utilised. However, access to early placental tissue may be limited and primary trophoblasts rapidly cease proliferation in vitro impairing genetic manipulation. Hence, trophoblastic cell lines have been widely used as surrogates to study EVT function. Although the cell lines share some molecular markers with their primary counterpart, it is unknown to what extent they recapitulate the invasive phenotype of EVT. Therefore, we here report the first thorough GeneChip analyses of SGHPL-5, HTR-8/SVneo, BeWo, JEG-3 and the novel ACH-3P trophoblast cells in comparison to previously analysed primary villous cytotrophoblasts (CTBs) and extravillous trophoblasts (EVTs). Analyses of approximately 14,000 commonly expressed genes revealed that EVTs most closely resemble CTBs with considerable differences to the group of choriocarcinoma cells (JEG-3, BeWo, ACH-3P) and the group of SV40 Large T Antigen-selected cell types (SGHPL-5, HTR-8/SVneo). Similarly, analyses of 912 genes discriminating EVT from CTB, or 370 EVT-specific genes did not unravel a particular cell line with close similarity to any of the primary cell types, although molecular signatures common to EVT and each group of cell lines could be identified. Considering the diversity of mRNA expression patterns it is suggested that molecular studies in trophoblast cell lines require verification of the critical steps in an appropriate primary model system.

Wnt signalling in implantation, decidualisation and placental differentiation--review.

The family of secreted Wingless ligands plays major roles in embryonic development, stem cell maintenance, differentiation and tissue homeostasis. Accumulating evidence suggests that the canonical Wnt pathway involving nuclear recruitment of ß-catenin and activation of Wnt-dependent transcription factors is also critically involved in development and differentiation of the diverse reproductive tissues. Here, we summarise our present knowledge about expression, regulation and function of Wnt ligands and their frizzled receptors in murine and human endometrial and placental cell types. In mice, Wnt signalling promotes early trophoblast lineage development, blastocyst activation, implantation and chorion-allantois fusion. Moreover, different Wnt ligands play essential roles in the development of the murine uterine tract, in cycling endometrial cells and during decidualisation. In humans, estrogen-dependent endometrial cell proliferation, decidualisation, trophoblast attachment and invasion were shown to be controlled by the particular signalling pathway. Failures in Wnt signalling are associated with infertility, endometriosis, endometrial cancer and gestational diseases such as complete mole placentae and choriocarcinomas. However, our present knowledge is still scarce due to the complexity of the Wnt network involving numerous ligands, receptors and non-canonical pathways. Hence, much remains to be learned about the role of different Wnt signalling cascades in reproductive cell types and their changes under pathological conditions.

Signalling pathways regulating the invasive differentiation of human trophoblasts: a review.

The invasive differentiation pathway of trophoblasts is an indispensable physiological process of early human placental development. Formation of anchoring villi, proliferation of cell columns and invasion of extravillous cytotrophoblasts into maternal decidual stroma and vessels induce vascular changes ensuring an adequate blood supply to the growing fetus. Extravillous trophoblast differentiation is regulated by numerous growth factors as well as by extracellular matrix proteins and adhesion molecules expressed at the fetal-maternal interface. These regulatory molecules control cell invasion by modulating activities of matrix-degrading protease systems and ECM adhesion. The differentiation process involves numerous signalling cascades/proteins such as the GTPases RhoA, the protein kinases ROCK, ERK1, ERK2, FAK, PI3K, Akt/protein kinase B and mTOR as well as TGF-beta-dependent SMAD factors. While an increasing number of signalling pathways regulating trophoblast differentiation are being unravelled, downstream effectors such as executing transcription factors remain largely elusive. Here, we summarise our current knowledge on signal transduction cascades regulating invasive trophoblast differentiation. We will focus on cell model systems which are used to study the particular differentiation process and discuss signalling pathways which regulate trophoblast proliferation and motility.

Invasive trophoblasts generate regulatory collagen XVIII cleavage products.

Endostatin, the C-terminal proteolytic fragment of the noncollagenous domain 1 (NC1) of the basement membrane protein collagen XVIII, inhibits cell proliferation and migration. Placental and decidual expression of the peptide suggested a role in angiogenesis and/or extravillous trophoblast differentiation. Here, we demonstrate that supernatants of trophoblastic SGHPL-5 cells, purified first trimester villous trophoblasts and villous explant cultures contain proteases which in vitro cleave 20kDa endostatin from purified, recombinant NC1 domains. However, supernatants of decidual and villous fibroblasts failed to generate the 20kDa endostatin fragment. Moreover, we show that recombinant endostatin inhibits invasion of SGHPL-5 cells through Matrigel invasion chambers. Since mesenchymal cells but not trophoblasts produce collagen XVIII we suspect that invasive trophoblasts may produce endostatin upon contacting the extracellular matrix deposited by decidual stromal cells. Generation of endostatin through trophoblast-derived proteases could play a role in the regulation of trophoblast invasiveness.

Expression pattern of collagen XVIII and its cleavage product, the angiogenesis inhibitor endostatin, at the fetal-maternal interface.

Endostatin, the C-terminal fragment of the basement membrane protein collagen XVIII regulates epithelial cell migration and impairs tumour growth by inhibiting angiogenesis. Here, we investigated the expression pattern of collagen XVIII/endostatin in human placental and decidual tissues of various ages of gestation as well as in primary villous cytotrophoblasts, trophoblast cell lines, and villous explant cultures differentiating along the invasive pathway. RT-PCR analysis revealed production of collagen XVIII mRNA in total placenta and decidua of early and late pregnancy and in SGHPL-5 and HTR-8/Svneo cells. Collagen XVIII transcripts were absent from purified extravillous trophoblasts and syncytialising trophoblast cultures. Accordingly, an antibody against a protein domain common to different collagen XVIII isoforms detected the 180 kDa protein in villous and decidual tissue and cultivated placental fibroblasts but not in the different isolated trophoblast cell types. Immunohistochemical analyses localised collagen XVIII to villous basement membranes and to the endothelium as well as to placental and decidual stromal cells. Interestingly, expression of various forms of endostatin (20 and 26 kDa) was detected in placenta and decidua using Western blot analyses. Moreover, supplementation of recombinant endostatin increased MMP-2 expression in villous explant cultures and SGHPL-5 cells suggesting that the inhibitor may modulate extravillous trophoblast differentiation.

Cytokine-regulated expression of collagenase-2 (MMP-8) is involved in the progression of ovarian cancer.

Matrix metalloproteinases (MMPs) have been implicated in ovarian cancer progression. Among them, MMP-8 that degrades type I collagen may play a crucial role. The aim of our study was to determine MMP-8 expression and regulation in ovarian cancer and its association with other MMPs and tissue inhibitors of metalloproteinases (TIMPs). Tissue microarrays (TMAs) containing tissue cylinders from 302 patients were used for immunohistochemical studies. In addition, MMP-8 expression in vitro was analysed by a specific immunoassay and PCR-analysis. MMP-7 (81%), MMP-8 (95%), MT3-MMP (100%), TIMP-2 (100%), and TIMP-3 (96%) were expressed in all the OVCAs, but the staining intensities varied. MMP-3 (6%), MMP-9 (57%) and TIMP-1 (43%) expressions were more rarely detected. Only MMP-8 expression levels correlated with tumour grade (P<0.01), tumour stage (P<0.01), and a poor prognosis (P<0.05). MMP-8 protein and gene expression in vitro was found to be significantly upregulated by interleukin-1beta (IL-1beta, P<0.01). The data indicate that MMP-8 overexpression in OVCAs is regulated by IL-1beta and that pro-inflammatory cytokines may promote the invasive potential of ovarian cancer.

Heparanase-1 gene expression in normal, hyperplastic and neoplastic prostatic tissue.

Heparanase-1 (Hpa-1) has been implicated in tumour invasion and metastasis. In the present study, we evaluated the clinicopathological significance of Hpa-1 mRNA expression in prostate cancer and non-cancerous prostatic tissue by one-step polymerase chain reaction (PCR) of laser microdissected prostatic gland cells. In addition, cell type-specific expression of Hpa-1 mRNA in prostatic tissue was analysed by in situ hybridisation. Hpa-1 mRNA expression was found in 50% of normal and 40% of hyperplastic prostatic tissue. In situ hybridisation showed that Hpa-1 mRNA was strongly expressed in prostate gland cells. Of the 26 prostate carcinomas tested, 42% were positive for Hpa-1 mRNA. However, in non-cancerous prostatic tissue, Hpa-1 mRNA was significantly more often expressed than in less differentiated or more invasive prostate cancers (P<0.05). In situ hybridisation revealed only focal Hpa-1 mRNA expression in the neoplastic gland cells. Hpa-1 mRNA expression in the tumours significantly correlated with tumour differentiation and tumour stage (P<0.05). Our data indicate that Hpa-1 gene expression may be lost during dedifferentiation of prostatic gland cells.

Regulatory transcription factors controlling function and differentiation of human trophoblast--a review.

In transgenic mice, homozygous mutations of trophoblast-specific transcription factors such as Hand1, Mash-2, I-mfa or GCM1 revealed their key regulatory roles in induction, maintenance or differentiation of distinct placental trophoblast subpopulations in vivo. Descriptive studies have shown that several of these factors are also expressed in the human placenta, suggesting that the molecular mechanisms governing trophoblast differentiation could be similar in mice and men. While an increasing number of putative developmental regulators are being identified in the human placenta, little information is available regarding whether the particular factors play an essential role in trophoblast differentiation processes such as formation of anchoring villi, placental bed invasion or syncytialization. However, expression of abundant trophoblast-specific products such as hormones can be regarded as a hallmark of differentiation, suggesting that the factors controlling their transcription could also be involved in the developmental processes of the placenta. Indeed, studies in different model systems revealed that the human homologues of murine trophoblast-specific transcriptional regulators interact with the promoter regions of typical placental genes such as aromatase P450 (CYP19), chorionic gonadotrophin (CG) or placental lactogen (PL). Additionally, the unique combination of more broadly distributed transcription factors of the Sp or Ap-2 protein family in a particular trophoblast cell type is required to govern mRNA expression in a differentiation-dependent manner. Here, we will summarize our present knowledge on these individual transcription factors that are involved in human trophoblast function and differentiation.