sirna-transfection-emsa-analyses-on-freshly-isolated-human-villous

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

McGill University

Caveolae are specialized plasma membrane subdomains with distinct lipid and protein compositions, which play an essential role in cell physiology through regulation of trafficking and signaling functions. The structure and functions of caveolae have been shown to require the proteins caveolins. Recently, members of the cavin protein family were found to be required, in concert with caveolins, for the formation and function of caveolae. Caveolins have a paradoxical role in the development of cancer formation. They have been involved in both tumor suppression and oncogenesis, depending on tumor type and progress stage. High expression of caveolins and cavins leads to inhibition of cancer-related pathways, such as growth factor signaling pathways. However, certain cancer cells that express caveolins and cavins have been shown to be more aggressive and metastatic because of their increased potential for anchorage-independent growth. Here, we will survey the functional roles of caveolins and of different cavin family members in cancer regulation.

Caveolin and cavin family members: dual roles in cancer.

Human endogenous retrovirus (HERV)-encoded Syncytin-1 has been suggested to play a major role in trophoblast cell fusion and thereby placenta development. However, recent studies have strongly suggested that other HERV envelope proteins could also be implicated in this process. Based on this premise, herein we compared the expression and functional implication of Syncytin-1 with the more recently described Syncytin-2 protein in various trophoblast cells. Real-time reverse transcription PCR and Western blot analyses in differentiating primary trophoblast cells first indicated a direct correlation between mRNA and protein levels of Syncytin-2 and cell fusion, while an inverse correlation for Syncytin-1 was noted. Similar reverse transcription PCR experiments and promoter studies showed that cell fusion-inducing agents in the trophoblastic BeWo cell line increased the expression of Syncytin-1 but, more importantly, augmented Syncytin-2 expression. Confocal microscopy experiments further revealed that in BeWo cells and in freshly isolated primary human trophoblast cells, Syncytin-1 was present as a cytoplasmic punctuated structure in proximity to regions of cell-to-cell contact. On the other hand, Syncytin-2 presented an inducible signal, which mainly localized to the cytoplasmic membrane. Experiments with siRNA (small interfering RNA)-transfected BeWo and primary human trophoblast cells demonstrated an important diminution in the number of cell fusion events upon repression of Syncytin-2 expression, whereas transfection experiments with Syncytin-1-specific siRNA resulted in a more modest effect. Overall, these results highlight the importance of Syncytin-2 in BeWo and primary human trophoblast cell fusion.

Syncytin-2 plays an important role in the fusion of human trophoblast cells.

Human endogenous retroviruses (HERVs) represent up to 8% of the human genome and express several of its genes in the placenta. Studies have demonstrated that HERV envelope proteins syncytins 1 and 2 play a crucial role in trophoblast fusion and placenta development. Here, we compared the levels of placental expression of syncytins with the severity of preeclampsia (PE) symptoms. Confocal microscopy experiments indicated a pronounced deficiency in cellular fusion in trophoblast cells from patients with PE when compared to controls. As determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot analyses, syncytin mRNA and protein levels were decreased in PE placentas versus controls. Interestingly, syncytin 2 levels were more importantly impaired than syncytin 1. Our results further highlighted the existence of a correlation between the extent of the decrease in the expression levels of both fusogenic proteins and the degree of severity of PE symptoms. These HERV proteins could thereby be used as potential markers for the early diagnosis of PE.

Reduced expression of both syncytin 1 and syncytin 2 correlates with severity of preeclampsia.

Human endogenous retroviruses (HERVs) represent approximately 8% of our genome. HERVs influence cellular gene expression and contribute to normal physiological processes such as cellular differentiation and morphogenesis. HERVs have also been associated with certain pathological conditions, including cancer and neurodegenerative diseases. As HTLV-1 causes adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and has been shown to modulate host gene expression mainly through the expression of the powerful Tax transactivator, herein we were interested in looking at the potential modulation capacity of HTLV-1 Tax on HERV expression. In order to evaluate the promoter activity of different HERV LTRs, pHERV-LTR-luc constructs were co-transfected in Jurkat T-cells with a Tax expression vector. Tax expression potently increased the LTR activity of HERV-W8 and HERV-H (MC16). In parallel, Jurkat cells were also stimulated with different T-cell-activating agents and HERV LTRs were observed to respond to different combination of Forskolin, bpV[pic] a protein tyrosine phosphatase inhibitor, and PMA. Transfection of expression vectors for different Tax mutants in Jurkat cells showed that several transcription factors including CREB appeared to be important for HERV-W8 LTR activation. Deletion mutants were derived from the HERV-W8 LTR and the region from -137 to -123 was found to be important for LTR response following Tax expression in Jurkat cells, while a different region was shown to be required in cells treated with activators. Our results thus demonstrated that HTLV-1 Tax activates several HERV LTRs. This raises the possibility that upregulated HERV expression could be involved in diseases associated with HTLV-1 infection.

Activation of LTRs from different human endogenous retrovirus (HERV) families by the HTLV-1 tax protein and T-cell activators.

The syncytiotrophoblast is a multinuclear cell layer maintained through fusion events with cytotrophoblasts and plays a key role in the properties of the placenta. Monitoring fusion in this cell layer is important in studies aimed at understanding its function. We herein propose a new fusion assay based on the transactivating potential of the human immunodeficiency virus type 1 (HIV-1) Tat protein on its promoter present in the long terminal repeat (LTR) region. We used 2 BeWo cell populations, one stably transfected with the HIV-1 LTR positioned upstream of the luciferase gene and the other stably transfected with a Tat expression vector. Both stable cell lines were responsive to Tat-mediated LTR transactivation and demonstrated normal fusion and human chorionic gonadotropin (hCG) secretion upon stimulation. When both BeWo cell lines were cocultured, forskolin-mediated induction of fusion led to an increase in luciferase activity, which was sensitive to anti-syncytin 1 and -2 antibodies and syncytin 2 small interfering RNAs (siRNAs). Similar results were obtained in primary trophoblasts. Our results highlight the effectiveness and accuracy of this new quantification assay for trophoblast fusion.

A new luciferase-based quantitative assay for the evaluation of human trophoblast fusion.

Caveolae are specialized cell membrane invaginations known to regulate several cancer cell functions and oncogenic signaling pathways. Among other caveolar proteins, they are characterized by the presence of proteins of the cavin family. In this study, we assessed the impact of cavin-1, cavin-2, and cavin-3 on cell migration in a human HT-1080 fibrosarcoma model. We found that all cavin-1, -2 and -3 transcripts were expressed and that treatment with phorbol 12-myristate 13-acetate (PMA), which is known to prime cell migration and proliferation, specifically upregulated cavin-3 gene and protein expression. PMA also triggered matrix metalloproteinase (MMP)-9 secretion, but reduced the global cell migration index. Overexpression of recombinant forms of the three cavins demonstrated that only cavin-3 was able to reduce basal cell migration, and this anti-migratory effect was potentiated by PMA. Interestingly, cavin-3 overexpression inhibited PMA-induced MMP-9, while cavin-3 gene silencing led to an increase in MMP-9 gene expression and secretion. Furthermore, recombinant cavin-3 significantly prevented PMA-mediated dephosphorylation of AKT, a crucial regulator in MMP-9 transcription. In conclusion, our results demonstrate that cellular cavin-3 expression may repress MMP-9 transcriptional regulation in part through AKT. We suggest that the balance in cavin-3-to-MMP-9 expression regulates the extent of extracellular matrix degradation, confirming the tumor-suppressive role of cavin-3 in controlling the invasive potential of human fibrosarcoma cells.

A Role for the Cavin-3/Matrix Metalloproteinase-9 Signaling Axis in the Regulation of PMA-Activated Human HT1080 Fibrosarcoma Cell Neoplastic Phenotype.

Syncytin-2 is encoded by the envelope gene of Endogenous Retrovirus-FRD (ERVFRD-1) and plays a critical role in fusion of placental trophoblasts leading to the formation of the multinucleated syncytiotrophoblast. Its expression is consequently regulated in a strict manner. In the present study, we have identified a forskolin-responsive region located between positions -300 to -150 in the Syncytin-2 promoter region. This 150 bp region in the context of a minimal promoter mediated an 80-fold induction of promoter activity following forskolin stimulation. EMSA analyses with competition experiments with nuclear extracts from forskolin-stimulated BeWo cells demonstrated that the -211 to -177 region specifically bound two forskolin-induced complexes, one of them containing a CRE/AP-1-like motif. Site-directed mutagenesis of the CRE/AP-1 binding site in the context of the Syncytin-2 promoter or a heterologous promoter showed that this motif was mostly essential for forskolin-induced promoter activity. Transfection experiments with dominant negative mutants and constitutively activated CREB expression vectors in addition to Chromatin Immunoprecipitation suggested that a CREB family member, CREB2 was binding and acting through the CRE/AP-1 motif. We further demonstrated the binding of JunD to this same motif. Similar to forskolin and soluble cAMP, CREB2 and JunD overexpression induced Syncytin-2 promoter activity in a CRE/AP-1-dependent manner and Syncytin-2 expression. In addition, BeWo cell fusion was induced by both CREB2 and JunD overexpression, while being repressed following silencing of either gene. These results thereby demonstrate that induced expression of Syncytin-2 is highly dependent on the interaction of bZIP-containing transcription factors to a CRE/AP-1 motif and that this element is important for the regulation of Syncytin-2 expression, which results in the formation of the peripheral syncytiotrophoblast layer.

Chirine Toufaily

Department of Pharmacology and Therapeutics

McGill University

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Chirine Toufaily

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pharmacology and Therapeutics

McGill University

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Department of Pharmacology and Therapeutics