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PubMed2003

Vascular endothelial growth factor is a chemoattractant for trophoblast cells.

A role for angiogenic growth factors in trophoblast invasion has been postulated. Directional motility (chemotaxis) is an important function of trophoblast cells. We have previously shown that vascular endothelial growth factor (VEGF) increases the random movement of trophoblast cells although placental growth factor (PlGF) has no effect. Heparin inhibited this effect of VEGF. Motility of trophoblast cells has been proposed to be mediated by a nitric oxide (NO) pathway. We hypothesized that VEGF but not PlGF would be chemotactic for trophoblast cells. Chemotaxis of a first trimester extravillous trophoblast cell line, SGHPL-4, and primary isolates of first trimester and term trophoblast cells was measured using a Boyden chamber. Initial experiments to optimize the time of the experiment and identify a positive control were performed. Subsequent experiments ran for 20 h, used 0.5 per cent FBS or 10 ng/ml PDGF as negative and positive controls and were performed in triplicate. VEGF (1, 10 and 100 ng/ml+/-1 microg/ml heparin or +/-100 microM L-NAME) and PlGF (1, 10, 100 ng/ml) were tested. The chamber was placed in a 5 per cent CO(2) in air, 37 degrees C incubator. The number of cells in the lower chamber were counted. There was a dose dependent increase in chemotactic motility of the SGHPL-4 cell line and term trophoblast cells in response to VEGF. PlGF had no effect on the movement of the first trimester trophoblast cell line but did increase the motility of the term trophoblast cells in a dose dependent manner. Heparin increased the cellular motility of both cell types alone. It also further enhanced the chemoactivity of VEGF on the term trophoblast cells but not the cell line. L-NAME did not affect the VEGF-stimulated motility of the first trimester cell line. However, in the term trophoblast cells L-NAME increased the directional cellular motility in the absence of, or in the presence of low concentrations of VEGF. In conclusion, the first trimester and term trophoblast cells appeared to respond differently to the various factors tested in the present study that may reflect differential cellular function as gestation progresses.

Adult↗

PubMed1996

Isolation and characterization of human placental trophoblast subpopulations from first-trimester chorionic villi.

A method for the simultaneous preparation of highly enriched human placental trophoblast populations (villous and extravillous) from first-trimester placental villi (5 to 12 weeks) by using sequential trypsinization, percoll gradient centrifugation, and negative selection with anti-CD9 immunomagnetic separation is described. The purification method resulted in the isolation of four distinct trophoblast populations identified on the basis of morphology and phenotyping: (i) mononuclear villous cytotrophoblast cells which, through differentiation, become committed to syncytium formation; (ii) an extravillous trophoblast population which appeared as a "crazy pavement" and, with subsequent subculturing, differentiated morphologically to mononuclear cells; (iii) an extravillous trophoblast fraction which fused to form multinucleated trophoblast giant cells; and (iv) floating intermediate extravillous trophoblast cells which fused together to form cell clumps and which further differentiated to a mononuclear anchoring intermediate extravillous trophoblast. Short-term cultures of the freshly isolated cell fractions consisted of heterogeneous trophoblasts at different differentiation stages as determined by their varied biochemical and morphological properties. All the isolated trophoblast populations expressed the cytokeratin intermediate filament and the epithelium-specific cell-cell adhesion molecule E-cadherin. The isolated villous trophoblasts in culture expressed integrins alpha 6 and beta 4 and reduced levels of beta 1 subunits, whereas the proliferating extravillous trophoblast cultures expressed alpha 1, alpha 3, and alpha 5 and high levels of beta 1 integrin subunits, vitronectin receptor (alpha V beta 3/beta 5), and major histocompatibility complex class 1 molecules. Furthermore, the isolated trophoblast populations secreted metalloproteases (such as type IV collagenases [mainly 72- and 92-kDa enzymes, i.e., gelatinases A and B]) and urokinase plasminogen activator, as evaluated by substrate gel zymography. This method of isolation should facilitate in vitro studies of trophoblast proliferation, differentiation, invasion, virus interactions, cytokenesis, and immunology.

Cadherins↗

PubMed1995

Regulation of deoxyribonucleic acid synthesis in proliferating and differentiating trophoblast cells: involvement of transferrin, transforming growth factor-beta, and tyrosine kinases.

This report investigates the regulation of DNA synthesis in trophoblast stem cells and differentiating trophoblast cells. Experiments in this study were performed on the Rcho-1 trophoblast cell line, which was established from a transplantable rat choriocarcinoma. Rcho-1 trophoblast cells can be manipulated to proliferate or differentiate along the trophoblast giant cell pathway. DNA synthesis in quiescent trophoblast stem cells (maintained in serum-free medium) was stimulated by fetal bovine serum and donor horse serum or transferrin to a level approximately 30- and 10-fold above the basal level, respectively. Transferrin and horse serum were ineffective at maintaining trophoblast cell proliferation. In contrast, serum-starved differentiating trophoblast cells synthesize DNA at maximal levels and could not be further stimulated by the addition of exogenous factors. Fetal bovine serum-stimulated proliferation was effectively inhibited by transforming growth factor-beta 1. Experiments with the tyrosine kinase inhibitor genistein implicate tyrosine kinase involvement in the regulation of DNA synthesis and proliferation in trophoblast stem cells and DNA synthesis in differentiating trophoblast cells. Proliferating and differentiating trophoblast cells differ in their levels of tyrosine kinase activities and express unique tyrosine-phosphorylated proteins. In summary, DNA synthesis and proliferation in trophoblast stem cells are under extrinsic control, whereas DNA synthesis in differentiating trophoblast cells is under intrinsic control. Both mechanisms require tyrosine kinase activity, but the nature of the tyrosine kinase pathways in each process appears to be distinct.

Animals↗

PubMed2004

Kisspeptin-10, a KiSS-1/metastin-derived decapeptide, is a physiological invasion inhibitor of primary human trophoblasts.

Trophoblast invasion of the uterine extracellular matrix, a critical process of human implantation and essential for fetal development, is a striking example of controlled invasiveness. To identify molecules that regulate trophoblast invasion, mRNA signatures of trophoblast cells isolated from first trimester (high invasiveness) and term placentae (no/low invasiveness) were compared using U95A GeneChip microarrays yielding 220 invasion/migration-related genes. In this 'invasion cluster', KiSS-1 and its G-protein-coupled receptor KiSS-1R were expressed at higher levels in first trimester trophoblasts than at term of gestation. Receptor and ligand mRNA and protein were localized to the trophoblast compartment. In contrast to KiSS-1, which is only expressed in the villous trophoblast, KiSS-1R was also found in the extravillous trophoblast, suggesting endocrine/paracrine activation mechanisms. The primary translation product of KiSS-1 is a 145 amino acid polypeptide (Kp-145), but shorter kisspeptins (Kp) with 10, 13, 14 or 54 amino acid residues may be produced. We identified Kp-10, a dekapeptide derived from the primary translation product, in conditioned medium of first trimester human trophoblast. Kp-10, but not other kisspeptins, increased intracellular Ca(2+) levels in isolated first trimester trophoblasts. Kp-10 inhibited trophoblast migration in an explant as well as transwell assay without affecting proliferation. Suppressed motility was paralleled with suppressed gelatinolytic activity of isolated trophoblasts. These results identified Kp-10 as a novel paracrine/endocrine regulator in fine-tuning trophoblast invasion generated by the trophoblast itself.

Calcium↗

PubMed2001

Expression pattern of the adhesion molecule CEACAM1 (C-CAM, CD66a, BGP) in gestational trophoblastic lesions.

CEACAM1 (CD66a, BGP, C-CAM) is an adhesion molecule of the carcinoembryonic antigen (CEA) family which has been shown to be normally expressed at the apical pole of epithelial cells and to show a dysregulated expression pattern in tumors derived from the latter. The purpose of the present study was to investigate the expression pattern of CEACAM1 in gestational trophoblastic lesions and to compare this expression with the one observed in the normal trophoblast. For this purpose, we performed immunohistochemistry using the 4D1/C2 monoclonal antibody which specifically recognizes CEACAM1 and does not interact with other members of the CEA family. Immunohistochemistry was performed on a total of 20 cases of gestational trophoblastic lesions including complete hydatidiform moles, one placental site trophoblastic nodule (PSN), one placental site trophoblastic tumor (PSTT), and three choriocarcinomas. Immunostaining for cytokeratin, hPL, hCG, and Ki-67 was also performed. Normal placental samples served as a control. CEACAM1 was absent from villous cyto- and syncytiotrophoblast in both normal placenta and hydatidiform molar samples. It was present in the benign extravillous trophoblast, with stronger expression in the proximal extravillous trophoblast of anchoring villi, but was also observed in interstitial and endovascular intermediate trophoblast and chorionic intermediate-like trophoblast. Partial expression was observed in the trophoblast proliferating from the surface of molar villi. In choriocarcinomas, areas of weak expression could be observed along with large areas without CEACAM1 expression. In the PSN and especially in the PSTT, CEACAM1 expression was stronger and more diffuse. The specific localization to extravillous trophoblast and its expression pattern in gestational trophoblastic lesions indicate that CEACAM1 can potentially be a helpful additional diagnostic marker in the differential diagnosis of such lesions.

Antigens, CD↗

PubMed1996

Progression of trophoblast into the endometrium during implantation in the western spotted skunk.

BACKGROUND: In mustelid carnivores, several blastocysts become implanted either approximately 12 days following fertilization or after a delay of implantation. In the western spotted skunk, implantation occurs following a long period of delayed implantation and a brief activation stage. Within each implantation chamber, a large number of trophoblastic plaques form, and the syncytial trophoblast of these plaques adheres to and penetrates into the uterine luminal epithelium. The presence of multiple attachment sites was used to analyse the way in which trophoblast adheres to, penetrates, and removes uterine epithelium and its subsequent association with the subepithelial vascular plexus. METHODS: Implantation chambers from 18 western spotted skunks were collected during the first week postimplantation and the tissue prepared for light microscopy and scanning and transmission electron microscopy. RESULTS: A series of trophoblastic plaques, which form a ring peripheral to the embryonic shield, attach to and penetrate the uterine epithelium. As new trophoblastic plaques are forming, the initial plaques enlarge and spread along the basal lamina of the luminal epithelium, and trophoblastic processes project through this basal lamina. Subsequently there is a stage of consolidation in which cytotrophoblast increases greatly in amount, attachment sites coalesce, and the luminal epithelium is eliminated. Syncytial trophoblast intrudes into endometrial gland lumina and surrounds subepithelial capillaries. CONCLUSIONS: It is suggested that the affinity of syncytial trophoblast for apical junctional complexes of uterine epithelial cells facilitates intrusion of syncytial trophoblast between cells, possibly guided by the marginal ridges of the uterine cells. The trophoblast shows no tendency to adhere to or invade maternal capillaries. This lack of adhesion to endothelial cells suggests either a change in adhesive properties of trophoblast following epithelial penetration or differences in adhesive properties between surface epithelial cells and endothelial cells. Although trophoblast differentiation appears to be chronologically regulated, it could be responding to maternally derived factors.

Animals↗

PubMed1991

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.

Antibodies↗

PubMed1994

Resistance of malignant trophoblast cells to both the anti-proliferative and anti-invasive effects of transforming growth factor-beta.

Human placental trophoblast invasion of the uterus is a highly controlled event. We had shown that transforming growth factor-beta (TGF-beta) produced in the pregnant uterus controls invasiveness and reduces proliferation of first trimester placental trophoblasts in vitro. The anti-invasive effect of TGF-beta was due, at least in part, to induction of tissue inhibitor of metalloproteinases (TIMP)-1. In the present study we compared the effects of TGF-beta on proliferation ([3H]-TdR incorporation) and invasiveness (3-day Matrigel invasion assay) of JAR and JEG-3 choriocarcinoma cells vs normal first trimester human trophoblast cells. Transcripts of type IV collagenases (72- and 92-kDa enzymes, i.e., gelatinases A and B) and their inhibitors (TIMP-1 and TIMP-2) in these cells were measured by Northern analysis, and secretion of gelatinases and plasminogen activators (PAs) was evaluated by gel zymography. The results revealed that: (a) TGF-beta inhibited invasiveness and proliferation of normal trophoblast but not JAR and JEG-3 choriocarcinoma cells; (b) gelatinase A mRNA, expressed by the normal trophoblast and JAR cells, was upregulated in the presence of TGF-beta; (c) gelatinase B mRNA was not detected in the total RNA preparations of treated or untreated normal trophoblast or choriocarcinoma cells; (d) TGF-beta significantly upregulated the levels of TIMP-1 mRNA in the normal trophoblasts, but this transcript was very low in treated as well as untreated choriocarcinoma cells; TGF-beta also upregulated the 3.5-kb TIMP-2 message in the normal trophoblast; (e) gelatin zymography revealed a distinct band of approximately 68-kDa (gelatinase A) in the conditioned media of normal trophoblast and JAR cells; however, TGF-beta did not change the level of secretion of this gelatinase; and (f) the normal trophoblast also exhibited significant PA secretion (casein zymography) which was reduced in the presence of TGF-beta. PA secretion by the malignant trophoblast cells was low and unaffected by TGF-beta. These findings suggest that choriocarcinoma cells may become refractory to the mechanisms which control normal trophoblast proliferation and invasiveness. Concurrent resistance to antiproliferative and anti-invasive molecules such as TGF-beta may be highly relevant to tumor progression.

Cell Division↗

PubMed1990

Mechanisms of trophoblast invasiveness and their control: the role of proteases and protease inhibitors.

Implantation and subsequent placental development in many species including the human are dependent on trophoblast invasion of the uterine epithelium, the underlying basement membrane, connective tissue and blood vessels. However, trophoblast invasion in situ is strictly controlled by the microenvironment provided by the pregnant uterus. Key mechanisms underlying various steps in trophoblast invasion of basement membrane and stroma are similar to those identified in the case of invasive tumor cells: (a) attachment to basement membrane by binding to laminin and possibly other basement membrane components; (b) detachment from the basement membrane matrix prior to its penetration, a process that requires the presence of complex-type oligosaccharides on the cell surface; (c) breakdown of basement membrane components by trophoblast-derived metalloproteases (type IV and interstitial collagenase) and serine proteases (plasminogen activator). Type IV collagenase activity is stimulated by binding to laminin, a molecule also secreted by the trophoblast. Activation of trophoblast-derived metalloproteases appears to be plasmin-dependent. Plasmin results from the cleavage of plasminogen by trophoblast-derived plasminogen activator. Control of trophoblast invasion in situ is mediated by decidua-derived transforming growth factor beta (TGF beta) which in turn induces tissue inhibitor of metalloproteases (TIMP) both in the decidua and the trophoblast. We suggest that this control of trophoblast invasiveness is regulated both spatially as well as temporally during gestation. A preprogrammed decline in trophoblast invasiveness with increasing gestational age remains an additional possibility. The nature of the loss of control of trophoblast invasiveness in choriocarcinoma remains to be identified. Refractoriness to TGF beta action remains to strong possibility.

Basement Membrane↗

PubMedDate not supplied

A fibrin matrix modulates the proliferation, hormone secretion and morphologic differentiation of cultured human placental trophoblast.

Term placental trophoblast epithelialize fibrin deposits attached to villi in vitro and trophoblast cultured on a fibrin matrix form an epithelial bilayer typical of the trophoblast layer on term villi. We compared the morphology of cells grown on fibrin with cells grown on substrates of type IV collagen, laminin, type I collagen, or Matrigel. We also used autoradiography, hormone assays, electron microscopy, and immunofluorescence to determine what functional activities were influenced by trophoblast-fibrin interactions. Cultured cellular trophoblast from term placentae differentiated to form syncytial trophoblast and to secrete estrogen, progesterone, and hCG in the presence or absence of matrices. Trophoblast proliferation was lower in cells grown on matrices and was inversely related to cell height after 24 h in culture. Cells grown on fibrin remained the tallest and had the lowest labelling index. Cells grown for 72 h on fibrin had the most dilated rough endoplasmic reticulum but the lowest media hormone levels. Only cells grown on a fibrin matrix formed a basal lamina-like structure at the trophoblast-substrate interface, and only a fibrin matrix facilitated trophoblast to form an epithelial bilayer in culture. However, this histology was not accompanied by a change in the amount of syncytial trophoblast formed by the cells grown on fibrin. The results suggest that a fibrin matrix uniquely modulates the trophoblast phenotype, away from the secretion of placental specific products like hCG in favour of a repair-oriented phenotype that forms basement membrane and a trophoblast bilayer.

Cell Differentiation↗

PubMed1999

The effects of angiogenic growth factors on extravillous trophoblast invasion and motility.

There is accumulating evidence that deficient trophoblast invasion of the placental bed spiral arteries is crucial to the pathogenesis of pre-eclampsia and intrauterine growth restriction. However, the factors which regulate the process of trophoblast invasion remain unclear. We have investigated whether extravillous trophoblast invasion and motility are mediated by the angiogenic growth factors, vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). The SGHPL-4 extravillous trophoblast cell line was utilized. Expression of mRNA for the receptors of VEGF and PlGF (KDR and flt-1) was determined using the reverse transcriptase polymerase chain reaction. An in vitro model of invasion assessed the number and length of trophoblast processes invading into an extracellular matrix. The motility of cells under standard culture conditions was also quantified. The effect of the addition of VEGF and PlGF (+/-heparin) on trophoblast invasion and motility was determined. The effect of VEGF and PlGF (+/-heparin) on SGHPL-4 cell proliferation was assessed by cell counts at 24, 48 and 72 h post-addition of growth factor. The SGHPL-4 cells expressed mRNA for the flt-1 but not the KDR receptor. The addition of VEGF resulted in a significant decrease in the number of trophoblast processes formed (P< 0.02); this effect was not influenced by the addition of heparin. However, there was no effect on the length of processes formed in response to VEGF (+/-heparin). The addition of PlGF had no effect on either the number or the length of processes formed. The addition of VEGF increased the motility of the SGHPL-4 cells (P< 0.002); the addition of heparin prevented this VEGF-induced increase in motility. The addition of PlGF had no effect on SGHPL-4 motility (+/-heparin). Neither growth factor had any effect on the proliferative ability of SGHPL-4 cells. Contrary to our hypothesis, we did not find that the angiogenic growth factors, VEGF and PlGF, mediated the in vitro invasion of trophoblast cells into an extracellular matrix. However, VEGF did increase trophoblast motility. Our findings of an effect of VEGF on trophoblast motility (and possibly invasion) suggests the presence of functional receptors, which can mediate the actions of VEGF. Caution must be exercised before any extrapolation to the in vivo situation, however, it could be speculated that the increased motility in response to VEGF may be an initial response to attract trophoblast cells to the decidua, and that VEGF might then limit the degree to which trophoblast cells invade.

Cell Movement↗

PubMed1978

Antigens of human trophoblasts: a working hypothesis for their role in normal and abnormal pregnancies.

This report describes the preparation and characterization of antisera to human trophoblast membranes. Rabbit antisera were raised to trophoblast microvilli prepared by differential ultracentrifugation. Antibodies to serum proteins were removed by solid-phase immunoabsorption with normal human serum, and indirect immunofluorescence experiments with cryostat sections of human placentas showed that the absorbed anti-trophoblast sera reacted with trophoblasts as well as with stromal cells and endothelium of chorionic villi. The antisera also produced membrane fluorescence when studied on viable lymphocytes and certain human cell lines. These anti-trophoblast sera were also lymphocytotoxic, and this reaction was abolished by prior absorption of the antisera with leukocytes. The leukocyte-absorbed anti-trophoblast sera retained their ability to react with trophoblasts and certain human cell lines, but no longer reacted with lymphocytes or placental stromal cells and endothelium. Two categories of trophoblast membrane antigens are thus defined: one present on trophoblasts and certain human cells lines (tentatively designated TA(1)), and the other on trophoblasts and lymphocytes, villous fibroblasts, and endothelium (tentatively designated TA(2)). A working hypothesis is proposed stating that normal pregnancy involves the generation of anti-TA(2) subsequent to blastocyst implantation and entrance of trophoblasts into the maternal circulation. This involves a mechanism similar to allogeneic cell stimulation and results in antibodies that block either the recognition or cytotoxicity of TA(1). Failure to mount this response allows TA(1) recognition and trophoblast immunopathology. Experimental and clinical studies in support of this working hypothesis, particularly involving abortion and toxemia, are cited from published reports.

Cell Line↗

PubMed2003

Endovascular trophoblast invasion: implications for the pathogenesis of intrauterine growth retardation and preeclampsia.

Maternal uteroplacental blood flow increases during pregnancy. Altered uteroplacental blood flow is a core predictor of abnormal pregnancy. Normally, the uteroplacental arteries are invaded by endovascular trophoblast and remodeled into dilated, inelastic tubes without maternal vasomotor control. Disturbed remodeling is associated with maintenance of high uteroplacental vascular resistance and intrauterine growth restriction (IUGR) and preeclampsia. Herein, we review routes, mechanisms, and control of endovascular trophoblast invasion. The reviewed data suggest that endovascular trophoblast invasion involves a side route of interstitial invasion. Failure of vascular invasion is preceded by impaired interstitial trophoblast invasion. Extravillous trophoblast synthesis of nitric oxide is discussed in relation to arterial dilation that paves the way for endovascular trophoblast. Moreover, molecular mimicry of invading trophoblast-expressing endothelial adhesion molecules is discussed in relation to replacement of endothelium by trophoblast. Also, maternal uterine endothelial cells actively prepare endovascular invasion by expression of selectins that enable trophoblast to adhere to maternal endothelium. Finally, the mother can prevent endovascular invasion by activated macrophage-induced apoptosis of trophoblast. These data are partially controversial because of methodological restrictions associated with limitations of human tissue investigations and animal studies. Animal models require special care when extrapolating data to the human due to extreme species variations regarding trophoblast invasion. Basal plates of delivered placentas or curettage specimens have been used to describe failure of trophoblast invasion associated with IUGR and preeclampsia; however, they are unsuitable for these kinds of studies, since they do not include the area of pathogenic events, i.e., the placental bed.

Adaptation, Physiological↗

PubMed1989

In vitro metabolism of 25-hydroxyvitamin D3 by human trophoblastic homogenates, mitochondria, and microsomes: lack of evidence for the presence of 25-hydroxyvitamin D3-1 alpha- and 24R-hydroxylases.

In vitro studies were performed to assess the ability of term human trophoblastic tissue to metabolize 25-hydroxyvitamin D3 (25OHD3) and to compare this metabolism to that occurring in porcine renal mitochondria and microsomes. Human trophoblastic homogenates, containing a NADPH-generating system, were able to produce 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] at a rate of 225 pg/mg protein.h, but did not produce detectable quantities (less than 20 pg/mg protein.h) of 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3]. Similarly, mitochondria and microsomes isolated from term human trophoblastic tissue produced 1,25-(OH)2D3 [249 +/- 156 and 199 +/- 82 (mean +/- SD) pg/mg protein.h, respectively] in the presence of an NADPH-generating system, but failed to produce detectable quantities (less than 200 pg/mg protein.h) of 24,25-(OH)2D3. The production of 1,25-(OH)2D3 from the trophoblastic mitochondria and microsomes could be increased by adding 140,000 x g trophoblastic cytosol to the subcellular incubation tubes. This treatment had no effect on the production of 24,25-(OH)2D3. The component(s) present in trophoblastic cytosol responsible for the increased 1,25-(OH)2D3 production by trophoblastic mitochondria and microsomes was shown to be heat labile, trypsin resistant, and less than 1000 mol wt in size. Comparing characteristics of the porcine renal 1 alpha- and 24R-hydroxylase systems with those of the human trophoblastic system revealed that 1) 1,2-dianilinoethane and EDTA totally blocked synthesis of 1,25-(OH)2D3 in trophoblastic mitochondria and microsomes, but had no effect on the synthesis of 1,25-(OH)2D3 by renal mitochondria; and 2) ketoconazole greatly inhibited the synthesis of 1,25-(OH)2D3 and 24,25-(OH)2D3 by renal mitochondria, but had no effect on the production of 1,25-(OH)2D3 by trophoblastic mitochondria or microsomes. Finally, production of 24,25-(OH)2D3 could not be demonstrated in trophoblastic homogenates, mitochondria, or microsomes, while the production of this compound was readily evident in renal mitochondria, but not microsomes. The results of this study question the existence of the 25-hydroxyvitamin D3-1 alpha- and 24R-hydroxylase systems in the trophoblastic portion of the human placenta. This study also suggests that 1,25-(OH)2D3 can be produced in vitro by a mechanism other than enzymatic 1 alpha-hydroxylation. The possibility exists that the mechanism involves the insertion of oxygen at the 1 position of 25-(OH)D3 by free radical chemistry.

Animals↗

PubMed2001

Stimulation of human extravillous trophoblast migration by IGF-II is mediated by IGF type 2 receptor involving inhibitory G protein(s) and phosphorylation of MAPK.

We have earlier shown that migration and invasiveness of first trimester human extravillous trophoblast cells are stimulated by IGF-II, independently of IGF type 1 receptor and that migration stimulation is the primary reason for increased extravillous trophoblast cell invasiveness induced by IGF-II. In the present study we examined the functional role of IGF type II receptor in IGF-II stimulation of extravillous trophoblast cell migration and the underlying signal transduction pathways including the participation of inhibitory G protein(s) and MAPK. The migratory ability of a well characterized in vitro propagated human first trimester extravillous trophoblast cell line expressing the phenotype of extravillous trophoblast cells in situ was quantitated with a Transwell migration assay under different experimental conditions. We found that the extravillous trophoblast cells expressed an abundance of IGF type 2 receptor as detected by immunostaining and Western blots, and recombinant human IGF-II promoted their migration in a dose- and time-dependent manner. Both polyclonal and monoclonal IGF type 2 receptor-blocking antibodies blocked migration-stimulating effects of IGF-II. Two synthetic IGF-II analogs ([Leu27]IGF-II, which can bind to IGF type 2 receptor and IGF-binding proteins, but not IGF type 1 receptor, and [QAYL-Leu27]IGF-II, which can bind to IGFR-II, but neither IGFR-I nor IGF-binding proteins) both stimulated extravillous trophoblast cell migration to levels higher than those induced by wild-type IGF-II. These results reveal that IGF-II action was mediated by IGF type 2 receptor, independently of IGF type 1 receptor and IGF-binding proteins. Treatment of extravillous trophoblast cell membrane preparations with IGF-II decreased adenylyl cyclase activity in a concentration-dependant manner, indicating the participation of inhibitory G proteins in IGF-II action. This was substantiated further with the findings that increasing intracellular cAMP using forskolin or (Bu)2cAMP inhibited basal extravillous trophoblast cell migration and blocked IGF-II stimulation of migration. IGF-II treatment rapidly stimulated phosphorylation of MAPK (ERK-1 and -2), which was blocked by pretreatment of extravillous trophoblast cells with the MAPK kinase (MEK) inhibitor PD98059. Treatment with this inhibitor also blocked extravillous trophoblast cell migration in the presence or absence of IGF-II. These results, taken together, reveal that IGF-II stimulates extravillous trophoblast cell migration by signaling through IGF type 2 receptor, involving inhibitory G proteins and activating the MAPK pathway.

Adenylyl Cyclases↗

PubMed1997

Protein kinase C dependent and independent mechanisms controlling rat trophoblast cell DNA synthesis and differentiation.

Trophoblast giant cells are the steroidogenic cells of the rat placenta. In this study, the role of protein kinase C signalling pathways in the control of DNA synthesis and differentiation-dependent progesterone biosynthesis by trophoblast cells were investigated. Rcho-1 trophoblast cells, derived from a rat choriocarcinoma, can be experimentally manipulated to proliferate or differentiate and provide a useful model for studying trophoblast giant cell endocrine differentiation. The role of protein kinase C signal transduction was examined through the treatment of Rcho-1 trophoblast cells with isoquinolinesulfonamide derivatives (H7, a protein kinase C inhibitor; HA1004, a control compound), chelytherine (a protein kinase C inhibitor), and phorbol esters (protein kinase C activators). Treatment with H7 significantly attenuated DNA synthesis in proliferating and differentiating trophoblast cells and accelerated the acquisition of progesterone biosynthetic capabilities by trophoblast cells. Treatment with HA1004, the related but functionally distinct isoquinolone, did not significantly affect trophoblast DNA synthesis or proliferation and only weakly increased progesterone accumulation. Chelytherine significantly inhibited trophoblast cell proliferation but failed to influence trophoblast progesterone production significantly. The phorbol ester, 12-O-tetradecanoylphorbol acetate, did not significantly influence progesterone accumulation. H7 did not significantly influence the concentration of either P450scc or the mRNA encoding it in Rcho-1 trophoblast cells, or the transcriptional activity of the P450scc gene. The results indicate that signalling pathways sensitive to protein kinase C are involved in the control of trophoblast cell proliferation. Differentiation-dependent production of progesterone is sensitive to H7 but appears to be independent of protein kinase C and occurs at a stage other than P450scc expression.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1996

Cytochrome P450 17 alpha-hydroxylase gene expression in differentiating rat trophoblast cells.

Trophoblast giant cells of the rat placenta express cytochrome P450 17 alpha-hydroxylase (P450c17) and synthesize androgens. The purpose of this study was to investigate androgen production and expression of P450c17 in the Rcho-1 trophoblast cell line. These cells are capable of differentiating along the trophoblast giant cell lineage. Androstenedione production increased approximately 70-fold as Rcho-1 trophoblast cells progressed from the proliferation to the differentiation state. P450c17 enzyme activity and mRNA also showed significant increases associated with trophoblast giant cell differentiation. To study the transcriptional regulation of the P450c17 gene, the activities of a series of P450c17 promoter-luciferase reporter constructs were evaluated following transient transfection into Rcho-1 trophoblast cells. A DNA region located-98 bp upstream of the P450c17 gene transcriptional start site was the shortest promoter DNA construct consistently possessing activity in Rcho-1 trophoblast cells. Activities of longer constructs (-156 to -1560 bp) in this population of cells were significantly greater than the -98 bp promoter-reporter construct. The -476 bp P450c17 construct showed maximal promoter activity in transiently transfected Rcho-1 trophoblast cells and was developmentally activated in stably transfected Rcho-1 trophoblast cells. Activation of the cyclic AMP/protein kinase A pathway did not significantly affect P450c17 promoter activity in Rcho-1 trophoblast cells, in contrast to its effects in mouse MA-10 Leydig cells. In summary, Rcho-1 trophoblast cells are capable of endocrine differentiation and are a useful in vitro system for studying the regulation of trophoblast androgen production and P450c17 gene expression.

Androgens↗

PubMed2006

Invasive trophoblasts stimulate vascular smooth muscle cell apoptosis by a fas ligand-dependent mechanism.

During pregnancy, trophoblasts migrate from the placenta into uterine spiral arteries, transforming them into wide channels that lack vasoconstrictive properties. In pathological pregnancies, this process is incomplete. To define the fundamental events involved in spiral artery remodeling, we have studied the effect of trophoblasts on vascular smooth muscle cells (SMCs). Here we demonstrate for the first time that apoptosis of SMCs can be initiated by invading trophoblasts. When trophoblasts isolated from normal placenta (primary trophoblasts) or conditioned medium was perfused into spiral or umbilical artery segments, apoptosis of SMCs resulted. Culture of human aortic SMCs (HASMCs) with primary trophoblasts, primary trophoblast-conditioned medium, or a trophoblast-derived cell line (SGHPL-4) also significantly increased SMC apoptosis. Fas is expressed by spiral artery SMCs, and a Fas-activating antibody triggered HASMC apoptosis. Furthermore, a Fas ligand (FasL)-blocking antibody significantly inhibited HASMC apoptosis induced by primary trophoblasts, SGHPL-4, or trophoblast-conditioned medium. Depleting primary trophoblast-conditioned medium of FasL also abrogated SMC apoptosis in vessels in situ. These results suggest that apoptosis triggered by the release of soluble FasL from invading trophoblasts contributes to the loss of smooth muscle from the walls of spiral arteries during pregnancy.

Apoptosis↗