Abstract
FURIN is a pro-protein convertase previously shown to be important for placental syncytialisation (Zhou et al. [1]), a process of cell fusion whereby placental cytotrophoblast cells fuse to form a multinucleated syncytium. This finding has been broadly accepted however, we have evidence suggesting the contrary. Spontaneously syncytialising term primary human trophoblast cells and BeWo choriocarcinoma cells were treated with either FURIN siRNA or negative control siRNA or the protease inhibitor, DEC-RVKR-CMK, or vehicle. Cells were then left to either spontaneously syncytialise (primary trophoblasts) or were induced to syncytialise with forskolin (BeWo). Effects on syncytialisation were measured by determining human chorionic gonadotrophin secretion and E-cadherin protein levels. We showed that FURIN is not important for syncytialisation in either cell type. However, in primary trophoblasts another protease also inhibited by DEC-RVKR-CMK, may be involved. Our results directly contrast with those published by Zhou et al. Zhou et al. however, used first trimester villous explants to study syncytialisation, and we used term primary trophoblasts. Therefore, we suggest that FURIN may be involved in syncytialisation of first trimester trophoblasts, but not term trophoblasts. What is more concerning is that our results using BeWo cells do not agree with their results, even though for the most part, we used the same experimental design. It is unclear why these experiments yielded different results, however we wanted to draw attention to simple differences in measuring syncytialisation or flaws in method reporting (including omission of cell line source and passage numbers, siRNA concentration and protein molecular weights) and choice of immunoblot loading controls, that could impact on experimental outcomes. Our study shows that careful reporting of methods by authors and thorough scrutiny by referees are vital. Furthermore, a universal benchmark for measuring syncytialisation is required so that various studies of syncytialisation can be validated.
Highlights
Placental trophoblast syncytialisation is the process of cell fusion whereby the cytotrophoblast cells fuse to form a multinucleated syncytiotrophoblast
BeWo choriocarcinoma cells inhibitor, DEC-RVKR-CMK, decreased FURIN enzyme activity [9], Primary trophoblasts spontaneously syncytialised in culture [10] significantly increased CDH1 protein levels (Fig. 2B; P = 0.04), and and FURIN siRNA transfection successfully decreased FURIN mRNA reduced the percent of nuclei in syncytia (Fig. 2C–F; P = 0.03), it expression by 4.2-fold and protein levels by 3.9-fold at 72 h had no effect on human chorionic gonadotropin (hCG) secretion (Fig. 2A)
We have shown that FURIN does not play a role in spontaneous syncytialisation of term human primary trophoblast cells nor in forskolin-induced syncytialisation of BeWo choriocarcinoma cells
Summary
Placental trophoblast syncytialisation is the process of cell fusion whereby the cytotrophoblast cells fuse to form a multinucleated syncytiotrophoblast. The syncytiotrophoblast is vital for regulating transport of nutrients and wastes between mother and baby, whilst secreting essential pregnancy hormones. Understanding how this occurs is pivotal to understanding placentation. Zhou et al showed that FURIN is increased as primary trophoblasts and BeWo choriocarcinoma cells syncytialise and that knockdown of FURIN using an siRNA or inhibition using the broad protease inhibitor DEC-RVKR-CMK (which inhibits the activity of all PCSKs 1-7 [6, 7]), significantly inhibited syncytialisation in first trimester placental explants and in forskolin-treated BeWo choriocarcinoma cells [1, 8]. Our data show that care needs to be taken in interpreting experimental findings relating to the role of FURIN in human placental syncytialisation
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