Phospholipid scramblase 1 (PLSCR1) in villous trophoblast of the human placenta

Abstract

A crucial factor for effective villous trophoblast fusion in the human placenta is the transient deregulation of plasma membrane phospholipid asymmetry leading to externalization of phosphatidylserine to the outer membrane leaflet. Screening of scramblase family members implicated in the collapse of phospholipid asymmetry revealed that phospholipid scramblase 1 (PLSCR1) is strongly expressed in villous trophoblast. Therefore, we assessed the putative role of PLSCR1 in villous trophoblast fusion. Spatio-temporal analysis in first trimester and term placenta showed abundant expression of PLSCR1 in syncytiotrophoblast, macrophages and endothelial cells, while it was virtually absent in villous cytotrophoblasts. For functional studies, BeWo cells, isolated primary term trophoblasts and first trimester villous explants were used. During forskolin-mediated BeWo cell differentiation, neither PLSCR1 mRNA nor protein levels showed significant changes. In contrast, when primary trophoblasts were stimulated with Br-cAMP, a decrease in PLSCR1 mRNA and protein expression was observed. To elucidate a role for PLSCR1 in syncytialization, we used RNA interference and a chemical scramblase inhibitor, R5421 (ethanedioic acid). Silencing of PLSCR1 using siRNA had no effects while inhibition of scramblase activity by R5421 increased GCM-1 mRNA expression, beta-hCG protein secretion and fusion rates of BeWo cells. In primary trophoblasts and villous explants, no effects of siRNA or R5421 treatment on fusion were detected. This study provides data on PLSCR1 localization and general expression in the human placenta. The data make it tempting to speculate on a role of PLSCR1 in negatively regulating trophoblast fusion.

Ethaninidothioic acid (R5421) is not a selective inhibitor of platelet phospholipid scramblase activity

“…Ethaninidothioic acid (R5421) was first described as an inhibitor of scramblase activity in 1998 (Dekkers et al, 1998) and has been used to investigate the roles of phospholipid scrambling in several models (Berghold et al, 2015; Wesseling et al, 2016; Younan et al, 2018). R5421 might therefore provide a useful scaffold for further development of potent and selective TMEM16F inhibitors.…”

“…R5421 (ethaninidothioic acid) was originally reported as an inhibitor of platelet scramblase activity (Dekkers et al, 1998). R5421 has been widely used by other groups to determine the role of scramblase activity in other cells and systems including erythrocytes, viral infection and placental function (Berghold et al, 2015; Wesseling et al, 2016; Younan et al, 2018). No other selective inhibitor of scramblase activity is currently available.…”

Binding of Alphaherpesvirus Glycoprotein H to Surface α4β1-Integrins Activates Calcium-Signaling Pathways and Induces Phosphatidylserine Exposure on the Plasma Membrane

“…Phospholipid scramblases are calcium-dependent proteins that redistribute phospholipids (in particular PS) to the outer leaflet of the plasma membrane ( 44 ). To investigate if scramblase is responsible for the PS exposure, EHV-1-infected cells were treated with R5421, a scramblase inhibitor, which blocks Ca 2+ -induced phospholipid scrambling ( 45 – 47 ). EHV-1-induced PS exposure was significantly inhibited by R5421 in a dose-dependent manner ( Fig.…”

“…Latrunculin B was obtained from Cayman Chemical and diluted in ethanol. The scramblase inhibitor R5421, ethanimidothioic acid N -[( N -butylthio- N -methylamino)-carbonyloxy]-methyl ester (100 µM), was obtained from Endotherm and dissolved in DMSO ( 45 ). The final concentration of DMSO added to the cells was equal to or less than 0.01% of the total volume of the medium in all cases.…”

Vitamin D Receptor Gene Ablation in the Conceptus Has Limited Effects on Placental Morphology, Function and Pregnancy Outcome

“…In profiling placental transcriptomes by microarray, we detected 25 differentially expressed genes between Vdr -/- and Vdr +/+ placentae, a number of which have been shown to be expressed in the human placenta ( Tinf2 [ 49 ], Rgsl7 [ 50 ], Plscr1 [ 51 ] Cd302 [ 52 ]). The greatest gene expression difference observed between Vdr -/- and Vdr +/+ placentae was for Cyp24a1 ( Table 2 ), a gene that directly interacts with VDR in the canonical vitamin D signaling pathway and plays a key role in the vitamin D endocrine system negative feedback loop [ 53 ].…”