Syncytin Expression Research Articles

Relevance of Wnt10b and activation of β-catenin/GCMa/syncytin-1 pathway in BeWo cell fusion.

To study the involvement of specific Wnt(s) ligand during trophoblastic BeWo cell differentiation. BeWo cells on treatment with forskolin/human chorionic gonadotropin (hCG) were studied for cell fusion by desmoplakin I+II staining and/or hCG secretion by ELISA. Levels of Wnt10b/β-catenin/glial cell missing a (GCMa)/syncytin-1 were studied by qPCR/Western blotting in forskolin-/hCG-treated control siRNA and Wnt10b silenced BeWo cells. BeWo cells on treatment with hCG (5IU/mL) led to a 94-fold increase in Wnt10b transcript. Wnt10b silencing showed significant decrease in forskolin-/hCG-mediated BeWo cell fusion and/or hCG secretion. It led to down-regulation of β-catenin (nuclear and cytoplasmic), GCMa and syncytin-1 expression. Treatment of BeWo cells with H89, protein kinase A (PKA) signaling inhibitor, significantly reduced forskolin-/hCG-induced Wnt10b, β-catenin, and syncytin-1 expression, which also resulted in reduced cell fusion. Wnt10b is involved in forskolin/hCG-mediated BeWo cell fusion via β-catenin/GCMa/syncytin pathway, which may also involve activation of PKA.

DNA hypomethylation and aberrant expression of the human endogenous retrovirus ERVWE1/syncytin-1 in seminomas

BackgroundSyncytin-1 and 2, human fusogenic glycoproteins encoded by the env genes of the endogenous retroviral loci ERVWE1 and ERVFRDE1, respectively, contribute to the differentiation of multinucleated syncytiotrophoblast in chorionic villi. In non-trophoblastic cells, however, the expression of syncytins has to be suppressed to avoid potential pathogenic effects. Previously, we have shown that the transcriptional suppression of ERVWE1 promoter is controlled epigenetically by DNA methylation and chromatin modifications. In this study, we describe the aberrant expression of syncytin-1 in biopsies of testicular germ cell tumors.ResultsWe found efficient expression and splicing of syncytin-1 in seminomas and mixed germ cell tumors with seminoma component. Although another fusogenic gene, syncytin-2 was also derepressed in seminomas, its expression was significantly lower than that of syncytin-1. Neither the transcription factor GCM1 nor the increased copy number of ERVWE1 were sufficient for this aberrant expression of syncytin-1 in seminomas. In accordance with our recent finding of the highly increased expression of TET1 dioxygenase in most seminomas, the ERVWE1 promoter was significantly hypomethylated in comparison with the matched controls. In contrast, 5-hydroxymethylcytosine levels were not detectable at the ERVWE1 promoter. We further describe that another endogenous retroviral element adjacent to ERVWE1 remains transcriptionally suppressed and two additional HERV-W family members are only slightly upregulated in seminomas.ConclusionsWe conclude that DNA demethylation of the ERVWE1 promoter in seminomas is a prerequisite for syncytin-1 derepression. We propose the spliced syncytin-1 expression as a marker of seminoma and suggest that aberrant expression of endogenous retroviruses might be a correlate of the hypomethylated genome of seminomas.

Expression of Leukocytic Syncytin-1 in B-Cell Acute Lymphoblastic Leukemia and Acute Myeloid Leukemia Patients.

Syncytin-1 is improperly expressed in several cancers. However, its expression profile across leukocytes in leukemia patients has not yet been analyzed. A total of 50 AML cases and 14 B-cell ALL patients were consecutively recruited. Bone marrow samples were subjected to flow cytometry. Statistical analysis was applied to compare syncytin-1 expression between B-cell ALL and AML across granulocytes, leukemia cells, and T-lymphocytes (including CD3+, CD4+, and CD8+ subsets thereof) and to correlate syncytin-1 expression to leukemia cells and lymphocytes with the T-cell subset percentages. The syncytin-1-expressing leukemia cell% in AML patients was significantly higher than that in B-cell ALL patients (p < 0.05). The CD8+ T-cell% in AML patients was significantly higher than that in B-cell ALL patients (p < 0.05). The syncytin-1 expression rate on leukemia cells was positively correlated with the CD8+ T-cell percentage (r = 0.289, p < 0.05), while the syncytin-1 expression rate on lymphocytes was negatively correlated with the CD3+, CD4+, and CD8+ T-cell percentages (r = -0.273, -0.450, and -0.307, respectively; p < 0.05). The percentage of syncytin-1-expressing leukemia cells in AML - due to its positive correlation with the CD8+ suppressor T-cell percentage - shows potential as an indicator of poorer long-term immunity in AML patients.

Hypermethylation suppresses expression of human syncytin 2 in placenta of preeclampsia

Our previous retrospective cohort study found a significant increase of preeclampsia in pregnancies conceived through in vitro fertilization - embryo transfer (IVF-ET) compared to naturally conceived pregnancies (OR, 1.9, 95% CI 1.5-2.5). IVF-ET manipulates gametes and embryos during the pivotal stage of methylation reprogramming, so it is likely to induce inappropriate methylation and lead to preeclampsia. Considering syncytin 2 encoded by HERV-FRD plays a key role in trophoblast cell fusion and placenta invasion, deficiency of syncytin 2 might contribute in the development of preeclampsia.

Silver nanoparticles exhibit size-dependent differential toxicity and induce expression of syncytin-1 in FA-AML1 and MOLT-4 leukaemia cell lines

Human endogenous retrovirus (HERV) sequences make up ~8% of the human genome and increased expression of some HERV proteins has been observed in various pathologies including leukaemia and multiple sclerosis. However, little is known about the function of these HERV proteins or environmental factors which regulate their expression. Silver nanoparticles (AgNPs) are used very extensively as antimicrobials and antivirals in numerous consumer products although their effect on the expression of HERV gene products is unknown. Cell proliferation and cell toxicity assays were carried out on human acute T lymphoblastic leukaemia (MOLT-4) and Fanconi anaemia associated acute myeloid leukaemia (FA-AML1) cells treated with two different sizes of AgNPs (7nm and 50nm diameter). Reverse-transcriptase polymerase chain reaction and western blotting were then used to the assess expression of HERV-W syncytin-1 mRNA and protein in these cells. FA-AML1 cells were more sensitive overall than MOLT-4 to treatment with the smaller 7nm sized AgNp's being the most toxic in these cells. MOLT-4 cell were more resistant and showed no evidence of differential toxicity to the different sized particles. Syncytin-1 mRNA and protein were induced by both 7 and 50nm AgNPs in both cell types yet with different kinetics. In summary, the observation that AgNPs induce expression of syncytin-1 in FA-AML1 and MOLT-4 cells at doses as little as 5 µg/ml is grounds for concern since this protein is up-regulated in both malignant and neurodegenerative diseases. Considering the widespread use of AgNPs in the environment it is clear that their ability to induce syncytin-1 should be investigated further in other cell types.

Upregulation of Leukocytic Syncytin-1 in Acute Myeloid Leukemia Patients.

BackgroundSyncytin-1, a cell membrane-localizing fusogen, is abnormally expressed in several cancers, including endometrial cancer, breast cancer, and leukemia. Although abnormal syncytin-1 expression has been detected in two-thirds of leukemia blood samples, its expression profile in acute leukemia patients has not yet been analyzed.Material/MethodsBone marrow samples from 50 acute myelogenous leukemia (AML) cases and 14 B-cell acute lymphocytic leukemia (B-cell ALL) patients were subjected to flow cytometry to assess leukocyte type distributions and leukocytic syncytin-1 surface expression. RT-PCR was applied to assess leukocytic syncytin-1 mRNA expression. Statistical analysis was applied to compare syncytin-1 expression between AML and B-cell ALL patients across blasts, granulocytes, lymphocytes, and monocytes as well as to determine clinical factors statistically associated with changes in syncytin-1 expression.ResultsThe leukocyte type distributions of the AML and B-cell ALL cohorts highly overlapped, with an observable difference in blast distribution between the 2 cohorts. The AML cohort displayed significantly greater syncytin-1 surface and mRNA expression (p<0.05). Syncytin-1 surface and mRNA expression was significantly increased across all 4 leukocyte types (p<0.05). The percentage of syncytin-1-expressing blasts was significantly greater in AML patients (p<0.05), with blasts showing the largest fold-change in syncytin-1 expression (p<0.05). M5, M5a, and M5b AML patients displayed significantly higher syncytin-1 surface expression relative to all other AML French-American-British (FAB) classifications (p<0.05).ConclusionsThese findings suggest leukocytic syncytin-1 expression may play a role in the development and/or maintenance of the AML phenotype and the acute monocytic leukemia phenotype in particular.

Expression of Syncytin 1 (HERV-W), in the preimplantation human blastocyst, embryonic stem cells and trophoblast cells derived in vitro.

As Syncytin 1 (human endogenous retrovirus (HERV-W)) is crucial for human embryo placentation is it expressed during preimplantation embryo development? Syncytin 1 was expressed mainly in trophoblast cells of the blastocyst particularly in cells underlying the inner cell mass (ICM). Syncytin 1 (along with HERV-FRD or Syncytin 2) is expressed in first-trimester placenta and required for cell-cell fusion to enable formation of syncytiotrophoblast and effective placentation. Preimplantation human embryos donated for research were cultured in vitro and protein expression of Syncytin 1 at the blastocyst stage of development investigated. Comparisons were made with protein (Syncytin 1) and mRNA (Syncytin 1 and 2) expression in human embryonic stem cells (hESCs) undergoing differentiation to trophoblast-like cells in vitro. In total, 10 blastocysts (×3 or 4 replicates) were analysed and 4 hESC lines. The study was terminated after consistent observations of embryos were made. Donated embryos were thawed and cultured to blastocyst, fixed with 4% w/v paraformaldehyde. Syncytin 1 protein expression was determined by immunofluorescent localisation and confocal microscopy. Additionally, hESCs were differentiated to trophoblast-like cells in standard and conditioned culture medium with growth factors (bone morphogenetic protein 4 (BMP4) or fibroblast growth factor 4 (FGF4) and assessed for mRNA (Syncytin 1 and 2) by quantitative polymerase chain reaction (qPCR) and protein expression by immunolocalization and western blot. Syncytin 1 was expressed in cytoplasm and on the cell surface of some trophoblast cells, and consistently the trophectoderm underlying the ICM of the blastocyst. There was weak but consistent expression of Syncytin 1 in cells on the periphery of the ICM also displaying pluripotency antibody marker (Tra-1-60). Three-dimensional reconstruction of confocal slice data provided good visualization of expression. The time course of expression of Syncytin 1 was replicated in hESCs differentiated in vitro confirming the embryo observations and providing statistically significant differences in protein and mRNA level (P= 0.002) and (P< 0.05), respectively. Culture of a limited number of embryos to blastocyst in vitro may not replicate the range and quality of development in situ. Probes (antibodies, PCR) were tested for specificity, but might have non-specific reactions. Syncytin expression is a prerequisite for embryo implantation and placentation. Understanding when expression first occurs during embryo development may be informative for understanding conditions of abnormal gestations such as pre-clampsia. The study was supported partly by an ERASMUS training grant and grant G0801059 from the Medical Research Council, U.K. There were no competing interests.

Altered expression of human endogenous retroviruses syncytin-1, syncytin-2 and their receptors in human normal and gestational diabetic placenta.

Syncytins belong to the Human Endogenous Retrovirus family. The syncytin-1 receptor, SLC1A5, and syncytin-2 receptor, MFSD2, interact with their respective syncytin proteins to induce syncytiotrophoblast formation. However, there is no information about syncytins in gestational diabetic placenta. Therefore, we studied the expression and localization of syncytins and their receptors during normal placental development and in gestational diabetic placenta. Immunohistochemistry and Western-blot methods were performed with antibodies against syncytin-1, syncytin-2, SLC1A5 and MFSD2 in human first trimester placental tissues, normal term and gestational diabetic placentas. Syncytin-1, syncytin-2 and MFSD2 mRNA transcripts were determined by qRT-PCR in normal and diabetic term placentas. Cytoplasmic syncytin-1, syncytin-2, SLC1A5 and MFSD2 immunoreactions were observed in the trophoblastic layers in all placental samples. Some of the stromal cells showed strong cytoplasmic punctate staining. There were significantly weak syncytin-2 and MFSD2 immunoreaction intensities in diabetic placentas by ImageJ analysis, in parallel with decreased syncytin-2 and MFSD2 proteins in diabetic placentas by Western-blot. Protein expression of SLC1A5 increased dramatically in early pregnancy compared to term placenta. Syncytin-1, syncytin-2 and MFSD2 mRNA transcripts showed similar relative expression pattern by qRT-PCR. Syncytins were localized not only in cytotrophoblast cells and the basement membrane of the syncytiotrophoblast but also in the apical microvillous membrane and cytoplasm of syncytiotrophoblast, and some of the stromal cells and endothelium. Decreased syncytin-2 and MFSD2 proteins in gestational diabetic placentas might cause abnormal syncytiotrophoblast formation and possibly be involved in the pathology. Therefore, our study highlights an important potential relationship between syncytins and gestational diabetic placenta.

Expression patterns of ERVWE1/Syncytin-1 and other placentally expressed human endogenous retroviruses along the malignant transformation process of hydatidiform moles

IntroductionUp to 20% of hydatidiform moles are followed by malignant transformation in gestational trophoblastic neoplasia and require chemotherapy. Syncytin-1 is involved in human placental morphogenesis and is also expressed in various cancers. We assessed the predictive value of the expression of Syncytin-1 and its interactants in the malignant transformation process of hydatidiform moles. MethodsSyncytin-1 glycoprotein was localized by immunohistochemistry in hydatidiform moles, gestational trophoblastic neoplasia and control placentas. The transcription levels of its locus ERVWE1, its interaction partners (hASCT1, hASCT2, TLR4 and DC-SIGN) and two loci (ERVFRDE1 and ERV3) involved the expression of other placental envelopes were assessed by real-time PCR. ResultsSyncytin-1 glycoprotein was expressed in syncytiotrophoblast of hydatidiform moles with an apical enhancement when compared with normal placentas. Moles with further malignant transformation had a higher staining intensity of Syncytin-1 surface unit C-terminus but the transcription level of its locus ERVWE1 was not different from that of moles with further remission and normal placentas. hASCT1 and TLR4, showed lower transcription levels in complete moles when compared to normal placentas. ERVWE1, ERVFRDE1 and ERV3 transcription was down-regulated in hydatidiform moles and gestational trophoblastic neoplasia. ConclusionsVariations of Syncytin-1 protein localization and down-regulation of hASCT1 and TLR4 transcription are likely to reflect altered functions of Syncytin-1 in the premalignant context of complete moles. The reduced transcription in gestational trophoblastic diseases of ERVWE1, ERVFRDE1 and ERV3, which expression during normal pregnancy is differentially regulated by promoter region methylation, suggest a joint dysregulation mechanism in malignant context.

Promoter Hypermethylation and Decreased Expression of Syncytin-1 in Pancreatic Adenocarcinomas.

Syncytin-1 is a member of human endogenous retroviral W gene family (HERVW1). Known to be expressed in human placental trophoblast, syncytin-1 protein mediates the fusion of cytotrophoblasts for the formation of syncytiotrophoblasts, the terminally differentiated form of trophoblast lineage. In addition, in vitro studies indicate that syncytin-1 possessed nonfusogenic functions such as those for immune suppression, cell cycle regulation and anti-apoptotic activities. Overexpression of syncytin-1 has been observed in various malignant tissues including breast, endometrial and ovarian cancers. It was reported that syncytin-1 gene expression is associated with dynamic changes of DNA hypomethylation in the 5’ LTR. In this study, applying the real-time PCR, Western blot analysis and immunohistochemistry methods, we demonstrate a constitutive expression of syncytin-1 in normal pancreas tissues as well as normal tissues adjacent to cancer lesions. Moreover, a reduced expression is found in the pancreatic adenocarcinoma tissues. The expression levels of syncytin-1 are not correlated with the stage, historical grade and gender, but inversely correlated with patients’ age. Furthermore, COBRA and bisulfite sequencing results indicated that the lower expression of syncytin-1 is correlated with the hypermethylation of two CpG dinucleotides in the 5’ LTR of syncytin-1 gene. The nonfusogenic function of syncytin-1 in normal pancreas as well as its role(s) in the pathogenesis and progression of pancreatic cancers remains to be investigated. Identification of the two CpG dinucleotides around transcription start site as key epigenetic elements has provided valuable information for further studies on the epigenetic regulation of syncytin-1 in pancreatic cancer cells.

ETS1 induces human trophoblast differentiation.

A possible role for the transcription factor v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS1) in human trophoblast cell differentiation was examined using a highly enriched fraction of human mononuclear cytotrophoblast cells (CTBs) that differentiate spontaneously in vitro to a multinucleated syncytiotrophoblast cell (STB) phenotype. ETS1 mRNA and protein levels were abundant in freshly isolated CTBs and decreased as the cells differentiated. Silencing of ETS1 expression in freshly prepared CTBs markedly attenuated syncytialization, as demonstrated by desmoplakin staining, and blocked the induction of syncytin, the transcription factor activator protein-2α, placental lactogen, and other STB-specific genes. Conversely, overexpression of ETS1 in primary trophoblast cells induced STB marker gene mRNAs and transactivated each of the gene proximal promoters. Taken together, these findings strongly suggest a critical role for ETS1 in the induction of human villus CTB differentiation. The effect of ETS1 on syncytialization likely results, at least in part, from inhibition of syncytin expression, whereas the induction of STB marker genes likely results in part from transactivation by activator protein-2α.

Involvement of nephrin in human placental trophoblast syncytialization.

The placenta has numerous functions, such as transporting oxygen and nutrients and building the immune tolerance of the fetus. Cell fusion is an essential process for placental development and maturation. In human placental development, mononucleated cytotrophoblast (CTB) cells can fuse to form a multinucleated syncytiotrophoblast (STB), which is the outermost layer of the placenta. Nephrin is a transmembrane protein that belongs to the Ig superfamily. Previous studies have shown that nephrin contributes to the fusion of myoblasts into myotubes in zebrafish and mice, presenting a functional conservation with its Drosophila ortholog sticks and stones. However, whether nephrin is involved in trophoblast syncytialization remains unclear. In this study, we report that nephrin was localized predominantly in the CTB cells and STB of human placenta villi from first trimester to term pregnancy. Using a spontaneous fusion model of primary CTB cells, the expression of nephrin was found to be increased during trophoblast cell fusion. Moreover, the spontaneous syncytialization and the expression of syncytin 2, connexin 43, and human chorionic gonadotropin beta were significantly inhibited by nephrin-specific siRNAs. The above results demonstrate that nephrin plays an important role in trophoblast syncytialization.

Correction: Reduced Syncytin-1 Expression Levels in Placental Syndromes Correlates with Epigenetic Hypermethylation of the ERVW-1 Promoter Region

Correction: Reduced Syncytin-1 Expression Levels in Placental Syndromes Correlates with Epigenetic Hypermethylation of the ERVW-1 Promoter Region

MicroRNA-378a-5p targets cyclin G2 to inhibit fusion and differentiation in BeWo cells.

MicroRNAs are expressed abundantly in the placenta throughout pregnancy. We have previously reported that microRNA (miR)-378a-5p promoted trophoblast migration and invasion. To further understand the role of miR-378a-5p during placental development, we investigated whether it may regulate the differentiation of syncytiotrophoblast (STB). Using a choriocarcinoma cell line, BeWo, we found that miR-378a-5p was down-regulated during forskolin-induced STB differentiation. Transfection of a miR-378a-5p mimic into BeWo cells decreased the formation of multinucleated STB, increased E-cadherin, and decreased the expression level of STB marker genes. On the other hand, transfection of anti-miR-378a-5p resulted in an increase in formation of multinucleated STB and expression of STB marker genes, as well as the loss of E-cadherin. Bioinformatic analysis revealed that miR-378a-5p has four potential binding sites at the 3' untranslated region (UTR) of cyclin G2 (CCNG2). Using luciferase reporter assays, we showed that miR-378a-5p decreased the luciferase activity of reporter constructs that contain CCNG2 3' UTR. In addition, miR-378a-5p decreased, whereas anti-miR-378a-5p increased, CCNG2 mRNA levels. Overexpression of CCNG2 increased the expression of syncytin-1 and fusion index and reversed the inhibitory effects of miR-378a-5p. In contrast, silencing of CCNG2 using siRNA increased E-cadherin and decreased syncytin-1 levels. These findings provide initial evidence that CCNG2 promotes STB differentiation and suggest that miR-378a-5p exerts an inhibitory role in STB differentiation, in part, by down-regulating CCNG2 expression, in the BeWo cell model.

Syncytin-1 in differentiating human myoblasts: relationship to caveolin-3 and myogenin.

Myoblasts fuse to form myotubes, which mature into skeletal muscle fibres. Recent studies indicate that an endogenous retroviral fusion gene, syncytin-1, is important for myoblast fusions in man. We have now expanded these data by examining the immunolocalization of syncytin in human myoblasts induced to fuse. Additionally, we have compared the localization of syncytin with the localization of caveolin-3 and of myogenin, which are also involved in myoblast fusion and maturation. Syncytin was localized to areas of the cell membrane and to filopodial structures connecting myoblasts to each other and to myotubes. Weaker staining was present over intracellular vesicles and tubules. Caveolin-3 was detected in the sarcolemma and in vesicles and tubules in a subset of myoblasts and myotubes. The strongest staining occurred in multinucleated myotubes. Wide-field fluorescence microscopy indicated a partial colocalization of syncytin and caveolin-3 in a subset of myoblasts. Super-resolution microscopy showed such colocalization to occur in the sarcolemma. Myogenin was restricted to nuclei of myoblasts and myotubes and the strongest staining occurred in multinucleated myotubes. Syncytin staining was observed in both myogenin-positive and myogenin-negative cells. Antisense treatment downmodulated syncytin-1 expression and inhibited myoblast cell fusions. Importantly, syncytin-1 antisense significantly decreased the frequency of multinucleated myotubes demonstrating that the treatment inhibited secondary myoblast fusions. Thus, syncytin is involved in human myoblast fusions and is localized in areas of contact between fusing cells. Moreover, syncytin and caveolin-3 might interact at the level of the sarcolemma.

Decreased Expression and Altered Methylation of Syncytin-1 Gene in Human Placentas Associated with Preeclampsia

Syncytin-1 is a protein coded by a human endogenous retrovirus (HERV) gene of the HERV-W family (HERVWE1). Syncytin- 1 mediates formation of syncytiotrophoblasts through fusion of cytotrophoblasts, a hallmark of terminal differentiation of placental trophoblast linage. Syncytin-1 also possesses nonfusogenic functions and regulates cell cycle progression. While decreased syncytin-1 expression and syncytium deficiency are considered important pathological changes in preeclampsia, the molecular mechanism(s) underlying syncytin-1 downregulation remains unclear. In this study, we confirmed that expression levels of syncytin-1 mRNA and protein were significantly lower in preeclamptic placentas compared to normal controls. Human chorionic somatomammotropin expression, a marker for syncytium function, was also decreased in preeclamptic placentas. The mRNA levels of ASCT2, the syncytin-1 receptor involved in cell fusion process, and GCMa, a transcriptional factor known to regulate syncytin-1 expression, were not significantly altered. Methylation in the 5'LTR of syncytin-1 promoter was quantified by COBRA, methylation-specific PCR, and DNA sequencing. Results from all three assays indicated significantly hypermethylated syncytin-1 promoter in preeclamptic placentas compared to normal controls. Methylation levels were inversely correlated with syncytin-1 mRNA levels, suggesting that hypermethylation may lead to syncytin-1 downregulation. Further experiments indicated that DNMT1 and DNMT3B3 mRNA and protein levels were increased in preeclamptic placentas, suggesting that higher DNA methyltransferase activity may contribute to the hypermethylation of syncytin-1 in preeclamptic placentas. These results indicated that aberrant hypermethylation is involved in downregulation of syncytin-1, and epigenetic alterations may play a significant role in the development of preeclampsia.

Decreased expression and DNA methylation levels of GATAD1 in preeclamptic placentas

Expression of syncytin-1, or the human endogenous retroviral family W member 1 (HERVWE1) in human placental trophoblasts is regulated by DNA methylation. Increased DNA methylation and decreased expression of syncytin-1 have been observed in preeclamptic placentas. The syncytin-1-mediated fusogenic as well as non-fusogenic activities, e.g., cell cycle promotion, anti-apoptosis, and immune suppression, are implicated in the pathogenic changes in preeclamptic placentas. It is noteworthy that in a close vicinity to syncytin-1 there are two genes, peroxisome biogenesis factor 1 (PEX1) and GATA zinc finger domain containing 1 (GATAD1), as well as multiple CpG islands around these genes. In this study we determined if these adjacent genes might, like syncytin-1, subject to epigenetic regulation in preeclamptic placentas. Data from quantitative real-time PCR and Western blotting indicated that while PEX1 expression remained stable, GATAD1 expression was significantly decreased in the third-trimester placentas associated with preeclampsia than those associated with normal pregnancy. Immunohistochemistry detected high GATAD1 expression in trophoblast linage, and confirmed its reduced levels in preeclamptic placentas. However, COBRA and bisulfate sequencing detected decreased DNA methylation in levels in the 3 [prime] region of GATAD1 gene in preeclamptic placentas. The positive correlation between 3 [prime] methylation and GATAD1 expression was confirmed by treatment of choriocarcinoma JAR cells with DNMT inhibitor. These data pointed to a potential role of GATAD1 for the syncytium deficiency often associated with preeclamptic placentas. The sharp contrast of the methylation alterations for the closely positioned GATAD1 and HERVWE1 may provide a useful model for studying the accurate control of DNA methylation as well as their positive and negative impact on gene expression in placental trophoblasts.

Reduced syncytin-1 expression in choriocarcinoma BeWo cells activates the calpain1-AIF-mediated apoptosis, implication for preeclampsia.

Placentas associated with preeclampsia are characterized by extensive apoptosis in trophoblast lineages. Syncytin-1 (HERVWE1) mediates the fusion of cytotrophoblasts to form syncytiotrophoblasts, which assume the placental barrier, fetal-maternal exchange and endocrine functions. While decreased syncytin-1 expression has been observed in preeclamptic placentas, it is not clear if this alteration is involved in trophoblast apoptosis. In the current study, we found that siRNA-mediated knockdown of syncytin-1 led to apoptosis in choriocarcinoma BeWo, a cell line of trophoblastic origin. Characterization of the apoptotic pathways indicated that this effect does not rely on the activation of caspases. Rather, decreased syncytin-1 levels activated the apoptosis inducing factor (AIF) apoptotic pathway by inducing the expression, cleavage, and nuclear translocation of AIF. Moreover, calpain1, the cysteine protease capable of cleaving AIF, was upregulated by syncytin-1 knockdown. Furthermore, treatment with calpain1 inhibitor MDL28170 effectively reversed AIF cleavage, AIF nuclear translocation, and cell apoptosis triggered by syncytin-1 downregulation, verifying the specific action of calpain1-AIF pathway in trophoblast apoptosis. We confirmed that preeclamptic placentas express lower levels of syncytin-1 than normal placentas, and observed an inverse correlation between syncytin-1 and AIF/calpain1 mRNA levels, a result consistent with the in vitro findings. Immunohistochemistry analyses indicated decreased syncytin-1 and increased AIF and calpain1 protein levels in apoptotic cells of preeclamptic placentas. These findings have for the first time revealed that decreased levels of syncytin-1 can trigger the AIF-mediated apoptosis pathway in BeWo cells. This novel mechanism may contribute to the structural and functional deficiencies of syncytium frequently observed in preeclamptic placentas.

Human endogenous retroviral syncytin exerts inhibitory effect on invasive phenotype of B16F10 melanoma cells.

Fusogenic endogenous retroviral syncytin plays an important role in the formation of syncytiotrophoblasts in human placenta. Apart from its expression in placenta, brain and testis, syncytin has also been found in many cancers. Although syncytin has been proposed to serve as a positive prognostic marker in some cancers, the underlying mechanism is unclear. The aim of this study is to evaluate the effects of syncytin expression on the invasive phenotype of melanoma cells. The eukaryotic expression plasmid for syncytin-EGFP was constructed and transfected into B16F10 melanoma cells. The effect of syncytin on the invasion potential of tumor cells was evaluated in B16F10 subline cells that stably expressed syncytin-EGFP fusion protein or EGFP alone. The B16F10 sublines that stably expressed syncytin-EGFP or EGFP alone were established respectively and confirmed by immunofluorescent and immunoblotting assay. Syncytin expression in B16F10 cells was associated with decreased cell proliferation, migration and invasion. Multinucleated giant cells that contained as many as five nuclei were induced in syncytin-expressing cells. In addition, syncytin expression did not alter the sensitivity of B16F10 cells to trichosanthin, a toxin that damages syncytiotrophoblasts more efficiently than other tissues. These results suggest that syncytin expression in some cancers may confine their invasion potential and thus serve as a positive prognostic factor.

Expression of Human Endogenous Retrovirus-W Including Syncytin-1 in Cutaneous T-Cell Lymphoma

The pathomechanism of mycosis fungoides (MF), the most common type of primary cutaneous T-cell lymphomas (CTCLs) and a malignancy of non-recirculating, skin-resident T-cells, is unknown albeit underlying viral infections have been sought for. Human endogenous retroviruses (HERVs) are ancient retroviral sequences in the human genome and their transcription is often deregulated in cancers. We explored the transcriptional activity of HERV sequences in a total of 34 samples comprising MF and psoriasis skin lesions, as well as corresponding non-malignant skin using a retrovirus-specific microarray and quantitative RT-PCR. To identify active HERV-W loci, we cloned the HERV-W specific RT-PCR products, sequenced the cDNA clones and assigned the sequences to HERV-W loci. Finally, we used immunohistochemistry on MF patient and non-malignant inflammatory skin samples to confirm specific HERV-encoded protein expression. Firstly, a distinct, skin-specific transcription profile consisting of five constitutively active HERV groups was established. Although individual variability was common, HERV-W showed significantly increased transcription in MF lesions compared to clinically intact skin from the same patient. Predominantly transcribed HERV-W loci were found to be located in chromosomes 6q21 and 7q21.2, chromosomal regions typically altered in CTCL. Surprisingly, we also found the expression of 7q21.2/ERVWE1-encoded Syncytin-1 (Env) protein in MF biopsies and expression of Syncytin-1 was seen in malignant lymphocytes, especially in the epidermotropic ones, in 15 of 30 cases studied. Most importantly, no Syncytin-1 expression was detected in inflammatory dermatosis (Lichen ruber planus) with skin-homing, non-malignant T lymphocytes. The expression of ERVWE1 mRNA was further confirmed in 3/7 MF lesions analyzed. Our observations strengthen the association between activated HERVs and cancer. The study offers a new perspective into the pathogenesis of CTCL since we demonstrate that differences in HERV-W transcription levels between lesional MF and non-malignant skin are significant, and that ERVWE1-encoded Syncytin-1 is expressed in MF lymphoma cells.