Elsevier

Placenta

Volume 25, Issue 5, May 2004, Pages 396-407
Placenta

Phosphatidylserine Efflux and Intercellular Fusion in a BeWo Model of Human Villous Cytotrophoblast

https://doi.org/10.1016/j.placenta.2003.11.004Get rights and content

Abstract

Phosphatidylserine (PS) efflux characterizes cytotrophoblast apoptosis and differentiation. To evaluate whether PS externalization and intercellular fusion were secondary to apoptosis, BeWo cells were induced to differentiate by forskolin or undergo apoptosis by staurosporine. PS externalization was measured by FITC–annexin V binding, and intercellular fusion was quantified by counting nuclei in syncytial cells. During forskolin treatment, vanadate decreased PS efflux by 78.0 per cent from 68.0 [5.3] (mean [SD]) to 15.0 [8.8] Lum (×103) (P<0.001), whereas Z-VAD-fmk had no effect (66.5 [7.3]). Vanadate decreased intercellular fusion from 78.1 per cent [4.1] fusion in uninhibited cultures to 23.4 per cent [2.5], compared with 10.0 per cent [1.7] in media alone. Z-VAD-fmk did not affect fusion (80.4 per cent [6.8]). Staurosporine induced PS efflux was not affected by vanadate (69.6 [5.5] Lum ×103), but was inhibited 87.8 per cent by Z-VAD-fmk; from 71.5 [6.2] to 8.7 [3.6] Lum (×103) (P<0.001). Apoptosis was measured by the TUNEL and COMET assays, lamin B fragmentation, activation of procaspase 3, mitochondrial membrane potential, and release of mitochondrial cytochrome c and apoptosis inducing factor. There was no indication of apoptosis associated with differentiation. Thus, PS efflux and intercellular fusion occurred through a vanadate-sensitive mechanism that was independent of apoptosis.

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)

  • L. Lin et al.

    Expression of the endogenous retrovirus-3 (ERV-3) induces differentiation of BeWo, a choriocarcinoma model of human placental trophoblast

    Placenta

    (1999)
  • D. Tang et al.

    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

    (2000)
  • J. Connor et al.

    Bidirectional transbilayer movement of phospholipid analogs in human red blood cells. Evidence for an ATP-dependent and protein-mediated process

    Journal of Biological Chemistry

    (1992)
  • D.L. Bratton et al.

    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

    (1997)
  • E.M. Bevers et al.

    Lipid translocation across the plasma membrane of mammalian cells

    Biochimica et Biophysica Acta

    (1999)
  • S.C. Frasch et al.

    Regulation of phospholipid scramblase activity during apoptosis and cell activation by protein kinase C delta

    Journal of Biological Chemistry

    (2000)
  • D.L. Daleke

    Regulation of transbilayer plasma membrane phospholipid asymmetry

    Journal of Lipid Research

    (2003)
  • L. Pascolo et al.

    Effects of maturation on RNA transcription and protein expression of four MRP genes in human placenta and BeWo cells

    Biochemical and Biophysical Research Communications

    (2003)
  • Y. Hamon et al.

    ABCA1 and the engulfment of apoptotic cells

    Biochimica et Biophysica Acta

    (2002)
  • P. Zheng et al.

    Stably transfected ABCA1 antisense cell line has decreased ABCA1 mRNA and cAMP-induced cholesterol efflux to apolipoprotein A1 and HDL

    Biochimica et Biophysica Acta

    (2001)
  • J.D. Smith et al.

    Evaluation of the role of phosphatidylserine translocase activity in ABCA1-mediated lipid efflux

    Journal of Biological Chemistry

    (2002)
  • C. Ozvegy et al.

    Characterization of drug transport, ATP hydrolysis, and nucleotide trapping by the human ABCG2 multidrug transporter

    Journal of Biological Chemistry

    (2002)
  • E. Rocchi et al.

    The product of the ABC half-transporter gene ABCG2 (BCRP/MXR/ABCP) is expressed in the plasma membrane

    Biochemical and Biophysical Research Communications

    (2000)
  • L. Lin et al.

    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

    (2000)
  • X.-M. Sun et al.

    Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis

    Journal of Biological Chemistry

    (1999)
  • J.D. Boyd et al.

    Electron microscopic observations on the cytotrophoblast contribution to the syncytium in the human placenta

    Journal of Anatomy

    (1966)
  • N.S. Rote et al.

    Expression of phosphatidylserine-dependent antigens on the surface of differentiating BeWo human choriocarcinoma cells

    American Journal of Reproductive Immunology

    (1995)
  • R.R. Adler et al.

    Monoclonal antiphosphatidylserine antibody inhibits intercellular fusion of the choriocarcinoma line, JAR

    Biology of Reproduction

    (1995)
  • J. Connor et al.

    Aminophospholipid translocation in erythrocytes: evidence for the involvement of a specific transporter and an endofacial protein

    Biochemistry

    (1990)
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