Phosphatidylserine Efflux and Intercellular Fusion in a BeWo Model of Human Villous Cytotrophoblast
Introduction
The human placenta grows dramatically over a relatively short period during gestation and develops into a highly specialized barrier providing selected transport of nutrients from the maternal to fetal blood. The effective surface area of the placenta is maximized by villus structures that extend from the placental surface and pack the maternal blood space. The external region of a villus consists of a layer of fetal trophoblastic cells, which includes an inner layer of mononuclear cytotrophoblasts and an outer layer of a multinucleate syncytium (syncytiotrophoblast) [1]. The placental surface grows by differentiation and intercellular fusion of the villous cytotrophoblast into the syncytiotrophoblast. The complete mechanism by which villous cytotrophoblast undergo intercellular fusion is unknown.
Phosphatidylserine (PS) externalization appears to be an essential component of the intertrophoblast fusion process. Differentiation of the villous cytotrophoblast results in redistribution of plasma membrane phospholipids with enrichment of PS on the syncytiotrophoblast surface [2], [3], [4], [5], [6], [7]. The importance of exofacial PS enrichment in intertrophoblast fusion is confirmed by observations that monoclonal antibody against a PS-dependent antigen completely blocked the intercellular fusion process in trophoblast models [6].
Phospholipids are normally maintained in an asymmetrical distribution in the plasma membrane of virtually all cells; the cholinephospholipids (about 80 per cent of the sphingomyelin and 75 per cent of the phosphatidylcholine) are located in the outer leaflet, whereas the aminophospholipids (80 per cent of the phosphatidylethanolamine [PE] and almost 100 per cent of the PS) are in the inner leaflet [8]. The asymmetric distribution of aminophospholipids is actively maintained through the action of a Mg/ATP-dependent aminophospholipid translocase that moves errant PS and PE from the cell surface to the endofacial leaflet [9], [10]. The translocase is head group specific, inhibited by vanadate or high concentrations of Ca2+, and has been described in a variety of cells, including choriocarcinoma models of human trophoblast [11]. Phospholipid asymmetry is disrupted when PS is preferentially externalized during apoptosis [12], [13], [14]or intercellular fusion, such as when myoblasts form myotubes in muscle development [15], [16], when sperm and oocyte membranes fuse during fertilization [17], and when villous cytotrophoblasts form the syncytiotrophoblast [2], [5], [6].
Villous cytotrophoblast syncytium formation is also dependent upon insertion into the plasma membrane of a retroviral-like envelope protein (syncytin) encoded by the endogenous retroviral element HERV-W [18], [19], [20]. HERV-W env expression was up-regulated during intercellular fusion in spontaneously differentiating villous cytotrophoblast and in forskolin-treated BeWo. Antibody against syncytin reduced intercellular fusion by approximately 45 per cent. Envelope proteins from infectious retroviruses, such as HIV-1, also mediate formation of large syncytia, with evidence of resultant cell death by apoptosis [21], [22]. Apoptosis in HIV-induced syncytia is mitochondrial-mediated with release of apoptosis-inducing factor (AIF) and cytochrome c, caspase activation, and nuclear apoptosis [23].
Thus, a reasonable argument can be made that physiologic intercellular fusion of villous cytotrophoblast into a large syncytium may be apoptosis related. Both fusion and apoptosis are associated with efflux of PS and can be facilitated by expression of retroviral-like envelope proteins. Indicators of apoptosis have been observed in syncytiotrophoblast layer of isolated placental villous tissue [24], [25], [26]. Nuclei in some areas of the syncytiotrophoblast, particularly near areas of fibrin deposition, were TUNEL positive, suggesting DNA fragmentation. Isolated areas of the syncytium contained pro-apoptotic BAK protein, although BAX was undetectable, and the anti-apoptotic BCL-2 was expressed throughout the syncytium. In addition, activated caspases 3 and 6 were preferentially localized to the syncytiotrophoblast. These data appear to support a relationship between intertrophoblast fusion and apoptosis and have led to the proposal that villous cytotrophoblast syncytialization results from PS externalization induced by initiation of the apoptotic cascade [24], [25], [26].
Although the observations of positive TUNEL reactions, the presence of BAK protein, and activation of caspase 3 and 6 are generally considered indicators of apoptosis in mononuclear cells, their significance in an actively expanding syncytium is unclear. We, therefore, tested the hypothesis that PS efflux and intertrophoblast fusion result from initiation of apoptosis. Using an in vitro model of forskolin-induced differentiation of the choriocarcinoma BeWo, a model of villous cytotrophoblast differentiation, we induced either differentiation using forskolin or apoptosis using staurosporine and investigated the relationship between PS externalization and intercellular fusion. Our data support a model in which intertrophoblast fusion is dependent on PS efflux, but is independent of apoptosis.
Section snippets
Materials
All tissue culture materials and vanadate were purchased from Sigma (St. Louis, MO, USA). The multiple-caspase inhibitor Z-VAD-fmk (carbobenzoxy-valyl-analyl-aspartyl-[O-methyl] fluoromethyl ketone; Enzyme Systems Products, Inc, Livermore, CA, USA) was a generous gift from Dr Thomas Brown, Department of Physiology and Biophysics, Wright State University, Dayton, OH, USA. FITC-annexin V was obtained from BioWhittaker (Walkersville, MD, USA). Polyclonal antibody against hCG was purchased from
PS efflux
Both forskolin and staurosporine treatment of BeWo resulted in similar levels of surface PS expression (P=NS), measured by the binding of FITC–annexin V (Figure 1). The stock solutions of forskolin and staurosporine were prepared in DMSO and diluted in media. Treatment of control BeWo cells with the same final concentrations of DMSO alone did not result in PS externalization (data not shown). We tested whether pretreatment with vanadate or Z-VAD-fmk affected PS efflux. Vanadate is an inhibitor
Discussion
In this study, we used BeWo as a model of villous cytotrophoblast. Differentiation and intercellular fusion were induced by treatment with forskolin, a modulator of cAMP levels [3]. Apoptosis was induced in BeWo using staurosporine. We were then able to compare levels of intercellular fusion and PS efflux under conditions of differentiation versus apoptosis. Although data obtained from any in vitro model system, or even from the use of freshly isolated cytotrophoblast, have caveats that temper
Acknowledgements
The authors would like to thank Ms Patricia Glazebrook and Dr Diana Kunze for assistance in confocal microscopy. This work was supported by a US Public Health Services Award HD23697 from the National Institute of Child Health and Human Development and a grant from the MetroHealth Foundation.
References (58)
- et al.
Monoclonal antiphospholipid antibody reactivity against human placental trophoblast
Journal of Reproductive Immunology
(1992) - et al.
Modulation of phosphatidylserine epitope expression on BeWo cells during forskolin treatment
Placenta
(1993) - et al.
Monoclonal antiphosphatidylserine antibody reactivity against human first-trimester placental trophoblasts
American Journal of Obstetrics and Gynecology
(1995) - et al.
Antiphosphatidylserine antibody removes annexin V and facilitates the binding of prothrombin at the surface of a choriocarcinoma model of trophoblast differentiation
American Journal of Obstetrics and Gynecology
(1997) - et al.
Identification and purification of aminophospholipid flippases
Biochimica et Biophysica Acta
(2000) - et al.
Membrane phospholipid translocase activity in JEG-3, a choriocarcinoma model of cytotrophoblast differentiation
Placenta
(1997) - et al.
Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis
Blood
(1994) - et al.
Myoblast aminophospholipid asymmetry differs from that of fibroblasts
FEBS Letters
(1981) - et al.
Differentiation related differences in the plasma membrane phospholipid asymmetry of myogenic and fibrogenic cells
Biochimica et Biophysica Acta
(1983) - et al.
Apoptosis and syncytial fusion in human placental trophoblast and skeletal muscle
International Review of Cytology
(2001)
Expression of the endogenous retrovirus-3 (ERV-3) induces differentiation of BeWo, a choriocarcinoma model of human placental trophoblast
Placenta
Caspase-8 activation and Bid cleavage contribute to MCF7 cellular execution in a caspase-3-dependent manner during staurosporine-mediated apoptosis
Journal of Biological Chemistry
Bidirectional transbilayer movement of phospholipid analogs in human red blood cells. Evidence for an ATP-dependent and protein-mediated process
Journal of Biological Chemistry
Appearance of phosphatidylserine on apoptotic cells requires calcium-mediated nonspecific flip-flop and is enhanced by loss of the aminophospholipid translocase
Journal of Biological Chemistry
Lipid translocation across the plasma membrane of mammalian cells
Biochimica et Biophysica Acta
Regulation of phospholipid scramblase activity during apoptosis and cell activation by protein kinase C delta
Journal of Biological Chemistry
Regulation of transbilayer plasma membrane phospholipid asymmetry
Journal of Lipid Research
Effects of maturation on RNA transcription and protein expression of four MRP genes in human placenta and BeWo cells
Biochemical and Biophysical Research Communications
ABCA1 and the engulfment of apoptotic cells
Biochimica et Biophysica Acta
Stably transfected ABCA1 antisense cell line has decreased ABCA1 mRNA and cAMP-induced cholesterol efflux to apolipoprotein A1 and HDL
Biochimica et Biophysica Acta
Evaluation of the role of phosphatidylserine translocase activity in ABCA1-mediated lipid efflux
Journal of Biological Chemistry
Characterization of drug transport, ATP hydrolysis, and nucleotide trapping by the human ABCG2 multidrug transporter
Journal of Biological Chemistry
The product of the ABC half-transporter gene ABCG2 (BCRP/MXR/ABCP) is expressed in the plasma membrane
Biochemical and Biophysical Research Communications
The cellular mechanism by which the human endogenous retrovirus ERV-3 env gene affects proliferation and differentiation in a human placental trophoblast model, BeWo
Placenta
Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis
Journal of Biological Chemistry
Electron microscopic observations on the cytotrophoblast contribution to the syncytium in the human placenta
Journal of Anatomy
Expression of phosphatidylserine-dependent antigens on the surface of differentiating BeWo human choriocarcinoma cells
American Journal of Reproductive Immunology
Monoclonal antiphosphatidylserine antibody inhibits intercellular fusion of the choriocarcinoma line, JAR
Biology of Reproduction
Aminophospholipid translocation in erythrocytes: evidence for the involvement of a specific transporter and an endofacial protein
Biochemistry
Cited by (51)
Two types of type IV P-type ATPases independently re-establish the asymmetrical distribution of phosphatidylserine in plasma membranes
2022, Journal of Biological ChemistryFlagging fusion: Phosphatidylserine signaling in cell–cell fusion
2021, Journal of Biological ChemistryCell-surface phosphatidylserine regulates osteoclast precursor fusion
2018, Journal of Biological ChemistryCitation Excerpt :Furthermore, myotube formation in skeletal muscle depends on Anxs A1 and A5 (7) and on Syn-1 (65, 66). Syn-1 (67), PS externalization (63, 68), and Anx A5 (69) also play major roles in formation of placental syncytium. To judge from these similarities, a concerted action of cell-surface PS-associated extracellular Anxs and endogenous retroviral envelope proteins can be a shared mechanistic motif for different cell–cell fusion processes.
The contribution of apoptosis-inducing factor (AIF) to villous trophoblast differentiation
2012, PlacentaCitation Excerpt :The hypothesis on the involvement of early-apoptosis in trophoblast fusion also relies on data that shows that the externalization of PS is required for trophoblast fusion [11]. The mechanisms by which PS externalization occur in trophoblast differentiation have previously been shown to be ATP and protein kinase A (PKA)- dependent but independent of caspase activation [27]. To strengthen our hypothesis that AIF was involved in trophoblast differentiation the translocation of AIF to the nucleus has been shown to temporally coincide with PS-efflux in other cell systems [28].
Mechanisms of Cell Fusion in Cancer
2024, Results and Problems in Cell Differentiation