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 ...

University of Montreal

Intestinal epithelial cells play an important role in early stages of HIV-1 infection and long-term persistence of the virus. Here we determined the mechanism that regulates HIV-1 activation via prostaglandin J(2) (PGJ(2)) in Caco-2 cells. We showed that treatment of Caco-2 cells with PGJ(2) decreased the infectivity of a luciferase reporter virus, pHXB-luc, as well as HIV production following infection of cells with a X4-tropic virus by antagonizing sodium butyrate, a cellular activator known to induce HIV-1 transcription. Transfection of intestinal epithelial cells such as Caco-2, HT-29 and SW620 cells with full-length HIV-1 LTR (pLTR-luc) revealed that PGJ(2) reduced HIV-1 LTR-mediated reporter gene activity. The involvement of NF-kappaB in the PGJ(2)-dependent down-regulation of HIV-1 transcription was further assessed using the kappaB-regulated luciferase-encoding vectors. In Caco-2 cells, PGJ(2) decreased IKK activity, resulting in reduced NF-kappaB translocation to the nucleus. Since sodium butyrate has been associated with a chronic stress response in AIDS patients, our results suggest that addition of PGJ(2) in the environment of infected intestinal epithelial cells could reduce HIV-1 transcription.

PGJ2 antagonizes NF-kappaB-induced HIV-1 LTR activation in colonic epithelial cells.

Cell-free Human T-cell Leukemia Virus type I (HTLV-I) virions are poorly infectious and cell-to-cell contact is often required to achieve infection. Other factors might thus importantly contribute in increasing infection by HTLV-I. Galectin-1 is a galactoside-binding lectin which is secreted by activated T lymphocytes. Several functions have been attributed to this protein including its capacity to increase cell-to-cell adhesion. Based on previous studies, we postulated that this protein could also accentuate HTLV-I infection.

Induction of galectin-1 expression by HTLV-I Tax and its impact on HTLV-I infectivity.

Several studies have recently demonstrated the existence of human T-cell leukemia virus type 1 (HTLV-1) antisense transcripts, which allow the synthesis of the newly described HBZ protein. Although previous reports have been aimed at understanding the potential role of the HBZ protein in HTLV-1 pathogenesis, little is known as to how this viral gene is regulated. Here, using our K30-3'asLuc reporter construct, we show that the viral Tax protein upregulates antisense transcription through its action on the TRE sequences located in the 3' long terminal repeat. Generation of stable clones in 293T cells demonstrated that Tax-induced HBZ expression is importantly influenced by the integration site in the host genome. The cellular DNA context could thus affect the level of HBZ mRNA expression in infected cells.

Upregulation of human T-cell leukemia virus type 1 antisense transcription by the viral tax protein.

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.

Retroviral gene expression generally depends on a full-length transcript that initiates in the 5' LTR, which is either left unspliced or alternatively spliced. We and others have demonstrated the existence of antisense transcription initiating in the 3' LTR in human lymphotropic retroviruses, including HTLV-1, HTLV-2, and HIV-1. Such transcripts have been postulated to encode antisense proteins important for the establishment of viral infections. The antisense strand of the HIV-1 proviral DNA contains an ORF termed asp, coding for a highly hydrophobic protein. However, although anti-ASP antibodies have been described to be present in HIV-1-infected patients, its in vivo expression requires further support. The objective of this present study was to clearly demonstrate that ASP is effectively expressed in infected T cells and to provide a better characterization of its subcellular localization.

Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells.

HERV (Human Endogenous Retrovirus)-encoded envelope proteins are implicated in the development of the placenta. Indeed, Syncytin-1 and -2 play a crucial role in the fusion of human trophoblasts, a key step in placentation. Other studies have identified two other HERV env proteins, namely EnvP(b) and EnvV, both expressed in the placenta. In this study, we have fully characterized both env transcripts and their expression pattern and have assessed their implication in trophoblast fusion. Through RACE analyses, standard spliced transcripts were detected, while EnvV transcripts demonstrated alternative splicing at its 3' end. Promoter activity and expression of both genes were induced in forskolin-stimulated BeWo cells and in primary trophoblasts. Although we have confirmed the fusogenic activity of EnvP(b), overexpression or silencing experiments revealed no impact of this protein on trophoblast fusion. Our results demonstrate that both env genes are expressed in human trophoblasts but are not required for syncytialization.

Transcriptional and functional studies of Human Endogenous Retrovirus envelope EnvP(b) and EnvV genes in human trophoblasts.

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.

Exosomes are extracellular vesicles that mediate intercellular communication and are involved in several biological processes. The objective of our study was to determine whether endogenous retrovirus group WE, member l (ERVWE1)/syncytin-1 and endogenous retrovirus group FRD, member 1 (ERVFRDE1)/syncytin-2, encoded by human endogenous retrovirus (HERV) envelope (env) genes, are present at the surface of exosomes produced by placenta-derived villous cytotrophoblasts and whether they play a role in cellular uptake of exosomes. In addition, we sought to determine whether these proteins are present in various abundances in serum-derived exosomes from normal pregnant women vs. women with preeclampsia (PE). Isolated exosomes were analyzed for their content by Western blot, a bead-associated flow cytometry approach, and a syncytin-2 ELISA. Binding and uptake were tested through confocal and electron microscopy using the BeWo choriocarcinoma cell line. Quality control of exosome preparations consisted of detection of exosomal and nonexosomal markers. Exosome-cell interactions were compared between cells incubated in the presence of control exosomes, syncytin-1 or syncytin-2-deprived exosomes, or exosomes solely bearing the uncleaved forms of these HERV env proteins. From our data, we conclude that villous cytotrophoblast exosomes are positive for both env proteins and are rapidly taken up by BeWo cells in a syncytin-1- and syncytin-2-dependent manner and that syncytin-2 is reduced in serum-derived exosomes from women with PE when compared to exosomes from normal pregnant women.

Syncytin proteins incorporated in placenta exosomes are important for cell uptake and show variation in abundance in serum exosomes from patients with preeclampsia.

Overexpression of the (+)insert smooth muscle myosin heavy chain (SMMHC) isoform could contribute to airway bronchospasm by increasing the velocity of contraction. Whether the (+)insert isoform is present in the small airways and its expression is reversible in asthma are unknown.

Inhaled corticosteroids modulate the (+)insert smooth muscle myosin heavy chain in the equine asthmatic airways.

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.

A CRE/AP-1-like motif is essential for induced syncytin-2 expression and fusion in human trophoblast-like model.

Myocyte hyperplasia and hypertrophy contribute to the increased mass of airway smooth muscle (ASM) in asthma. Serum-response factor (SRF) is a transcription factor that regulates myocyte differentiation in vitro in vascular and intestinal smooth muscles. When SRF is associated with phosphorylated (p)Elk-1, it promotes ASM proliferation while binding to myocardin (MYOCD) leading to the expression of contractile elements in these tissues. The objective of this study was therefore to characterize the expression of SRF, pElk-1, and MYOCD in ASM cells from central and peripheral airways in heaves, a spontaneously occurring asthma-like disease of horses, and in controls. Six horses with heaves and five aged-matched controls kept in the same environment were studied. Nuclear protein expression of SRF, pElk-1, and MYOCD was evaluated in peripheral airways and endobronchial biopsies obtained during disease remission and after 1 and 30 days of naturally occurring antigenic exposure using immunohistochemistry and immunofluorescence techniques. Nuclear expression of SRF (P = 0.03, remission vs. 30 days) and MYOCD (P = 0.05, controls vs. heaves at 30 days) increased in the peripheral airways of horses with heaves during disease exacerbation, while MYOCD (P = 0.04, remission vs. 30 days) decreased in the central airways of control horses. No changes were observed in the expression of pElk-1 protein in either tissue. In conclusion, SRF and its cofactor MYOCD likely contribute to the hypertrophy of peripheral ASM observed in equine asthmatic airways, while the remodeling of the central airways is more static or involves different transcription factors.

Contribution of SRF, Elk-1, and myocardin to airway smooth muscle remodeling in heaves, an asthma-like disease of horses.

Neutrophils infiltrate the airways of patients with asthma of all severities, yet their role in the pathogenesis of asthma and their contribution to airway remodeling is largely unknown. We hypothesized that neutrophils modulate airway smooth muscle (ASM) proliferation in asthma by releasing bioactive exosomes. These newly discovered nano-sized vesicles have the capacity to modulate immune responses, cell migration, cell differentiation, and other aspects of cell-to-cell communication. The aim of the study is to determine whether bioactive exosomes are released by neutrophils, and, if so, characterize their proteomic profile and evaluate their capacity to modulate ASM cell proliferation. Exosomes were isolated from equine neutrophil supernatants by differential centrifugation and filtration methods, followed by size-exclusion chromatography. Nanovesicles were characterized using electron microscopy, particle size determination, and proteomic analyses. Exosomes were cocultured with ASM cells and analyzed for exosome internalization by confocal microscopy. ASM proliferation was measured using an impedance-based system. Neutrophils release exosomes that have characteristic size, morphology, and exosomal markers. We identified 271 proteins in exosomes from both LPS and unstimulated neutrophils, and 16 proteins that were differentially expressed, which carried proteins associated with immune response and positive regulation of cell communication. Furthermore, neutrophil-derived exosomes were rapidly internalized by ASM cells and altered their proliferative properties. Upon stimulation of LPS, neutrophil-derived exosomes can enhance the proliferation of ASM cells and could therefore play an important role in the progression of asthma and promoting airway remodeling in severe and corticosteroid-insensitive patients with asthma.

Neutrophil-Derived Exosomes: A New Mechanism Contributing to Airway Smooth Muscle Remodeling.

Glucocorticoids (GCs) are the most effective drugs for the treatment of human asthma. However, a subgroup of asthmatic patients with neutrophilic airway inflammation is insensitive to GCs. Interleukin-17 (IL-17), a cytokine upregulated in the airways of a subset of human asthmatic patients, contributes to the recruitment of neutrophils and induces a glucocorticoid resistance in human airway epithelial cells. We hypothesized that IL-17 similarly activates neutrophils and contributes to their persistence in the asthmatic airways in spite of glucocorticoid therapy.

The Interleukin-17 Induced Activation and Increased Survival of Equine Neutrophils Is Insensitive to Glucocorticoids.

Neutrophils are the predominant cells during acute phases of inflammation, and it is now recognized that these leukocytes play an important role in modulation of the immune response. Directed migration of these cells to the sites of injury, known as chemotaxis, is driven by chemoattractants present at the endothelial cell surface or in the extracellular matrix (ECM). Since uncontrolled or excessive neutrophil chemotaxis is involved in pathological conditions such as atherosclerosis and severe asthma, studying the chemical cues triggering neutrophil migration is essential for understanding the biology of these cells and developing new anti-inflammatory therapies. Although several methods have been developed to evaluate neutrophil chemotaxis, these techniques are generally labor-intensive or alter the native form of these cells and their physiological response. Here we report a rapid, non-invasive, impedance-based, and label-free assay for real-time assessment of neutrophil chemotaxis.

Article	Year
PGJ2 antagonizes NF-kappaB-induced HIV-1 LTR activation in colonic epithelial cells. Virology\| PubMed ID: 18755491	2008
Induction of galectin-1 expression by HTLV-I Tax and its impact on HTLV-I infectivity. Retrovirology\| PubMed ID: 19032754	2008
Upregulation of human T-cell leukemia virus type 1 antisense transcription by the viral tax protein. Journal of virology\| PubMed ID: 19073733	2009
Syncytin-2 plays an important role in the fusion of human trophoblast cells. Journal of molecular biology\| PubMed ID: 19616006	2009
Reduced expression of both syncytin 1 and syncytin 2 correlates with severity of preeclampsia. Reproductive sciences (Thousand Oaks, Calif.)\| PubMed ID: 21493955	2011
Polarized expression of the membrane ASP protein derived from HIV-1 antisense transcription in T cells. Retrovirology\| PubMed ID: 21929758	2011
Transcriptional and functional studies of Human Endogenous Retrovirus envelope EnvP(b) and EnvV genes in human trophoblasts. Virology\| PubMed ID: 22277806	2012
A new luciferase-based quantitative assay for the evaluation of human trophoblast fusion. Reproductive sciences (Thousand Oaks, Calif.)\| PubMed ID: 22344723	2012
Syncytin proteins incorporated in placenta exosomes are important for cell uptake and show variation in abundance in serum exosomes from patients with preeclampsia. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 24812088	2014
Inhaled corticosteroids modulate the (+)insert smooth muscle myosin heavy chain in the equine asthmatic airways. Thorax\| PubMed ID: 25205586	2014
A CRE/AP-1-like motif is essential for induced syncytin-2 expression and fusion in human trophoblast-like model. PloS one\| PubMed ID: 25781974	2015
Contribution of SRF, Elk-1, and myocardin to airway smooth muscle remodeling in heaves, an asthma-like disease of horses. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 25979077	2015
Neutrophil-Derived Exosomes: A New Mechanism Contributing to Airway Smooth Muscle Remodeling. American journal of respiratory cell and molecular biology\| PubMed ID: 27105177	2016
The Interleukin-17 Induced Activation and Increased Survival of Equine Neutrophils Is Insensitive to Glucocorticoids. PloS one\| PubMed ID: 27138006	2016
A real-time assay for neutrophil chemotaxis. BioTechniques\| PubMed ID: 27177817	2016

Amandine Vargas

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