Inhibition of Akt activity and calcium channel function coordinately drive cell-cell fusion in the BeWO choriocarcinoma placental cell line

To establish a simple and quantitative live cell fusion assay for placental syncytialization, we generated stable GFP and dsRed expressing fusogenic BeWo cell lines. Fluorescent Activated Cell Sorting was shown to provide a quantitative determination of forskolin (cAMP-mediated) fusion in a time and...

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Bibliographic Details
Published in:PloS one 2012-01, Vol.7 (1), p.e29353-e29353
Main Authors: Vatish, Manu, Tesfa, Lydia, Grammatopoulos, Dimitris, Yamada, Eijiro, Bastie, Claire C, Pessin, Jeffrey E
Format: Article
Language:English
Subjects:
1-Phosphatidylinositol 3-kinase
AKT protein
Analysis
Biology
Blotting, Western
Calcium
Calcium (intracellular)
Calcium - metabolism
Calcium channels
Calcium Channels - metabolism
Cell Fusion
Cell Line, Tumor
Cell lines
Choriocarcinoma - metabolism
Chorionic Gonadotropin, beta Subunit, Human - metabolism
Class I Phosphatidylinositol 3-Kinases - metabolism
Colforsin - pharmacology
Cyclic adenosine monophosphate
Cyclic AMP
Cyclic AMP - metabolism
Endocrinology
Female
Flow Cytometry
Fluorescence
Forskolin
Humans
Hypoxia
Inhibition
Kinases
Lentivirus - genetics
Medicine
Microscopy, Confocal
Pharmacology
Pituitary (anterior)
Placenta
Placenta - cytology
Placenta - metabolism
Placental hormones
Polymerase Chain Reaction
Potentiation
Preeclampsia
Pregnancy
Proteins
Proto-Oncogene Proteins c-akt - metabolism
Secretion
Signaling
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Summary:To establish a simple and quantitative live cell fusion assay for placental syncytialization, we generated stable GFP and dsRed expressing fusogenic BeWo cell lines. Fluorescent Activated Cell Sorting was shown to provide a quantitative determination of forskolin (cAMP-mediated) fusion in a time and concentration dependent manner consistent with the increased secretion of beta human chorionic gonadotrophin (β-HCG) and appearance of multi-nucleated cells. Analyses of the fusion process demonstrated that in addition to increased cAMP levels, simultaneous reduction of intracellular calcium and inhibition of Type 1 phosphatidylinositol 3 kinase (PI3K)/Akt signaling also resulted in cell fusion. Although individual blockade of calcium channel function or PI3K/Akt signaling was without effect, the combination with forskolin resulted in a potentiation of cell fusion. These data demonstrate syncytialization is a complex process that depends upon the regulation of distinct signaling inputs that function in concert with each other.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0029353