Manipulation of CD98 expression affects both trophoblast cell fusion and amino acid transport activity during syncytialization of human placental BeWo cells

Abstract

The physiological importance of CD98 surface antigen in regulating placental trophoblast cell fusion and amino acid transport activity has been studied in parallel in a cell model of syncytialization (the cytotrophoblast cell line BeWo following increased intracellular cAMP by forskolin treatment) using antisense oligonucleotides. CD98 protein abundance (determined by Western blot) was decreased by 50 % following antisense oligonucleotide transfection. Transfection with antisense oligonucleotide altered the responses of BeWo to forskolin. Cell fusion (determined by a quantitative flow cytometry assay) was inhibited by 57 %, and both human chorionic gonadotropin secretion and L-leucine influx through system L were suppressed. These findings show that CD98 is involved in the process of cell fusion necessary for syncytiotrophoblast formation and that during this physiologically important event, amino acid transport activity is also regulated through expression of this membrane protein.

Figure 1. Effect on cell fusion of antisense oligonucleotide to CD98

BeWo cells expressing either H2B-GFP or Mit-DsRed2 were mixed, cultured and transfected without (•, ○) or with antisense (▾) or mismatch (▿) oligonucleotide. Cells were further cultured with 100 μM forskolin (○, ▾, ▿) or vehicle (•) for the time indicated. Cell fusion was analysed on a flow cytometer, as described in Methods. Data represent the means ± S.D. of three separate experiments.

Figure 2. Effect on its protein expression level of antisense oligonucleotide to CD98

BeWo cells expressing either H2B-GFP or Mit-DsRed2 were mixed, cultured and transfected without or with antisense or mismatch oligonucleotide. CD98 protein levels in the cellular extracts before (control) and after a further 48 h treatment with 100 μM forskolin or with vehicle were analysed as described in Methods. A, Western blot (under reducing conditions). The results presented are from a single representative experiment. B, quantification of CD98 protein levels. The intensity of each band was quantified using an image documentation and analysis system. Data represent the means ± S.D. of three separate experiments, expressed as percentage of control (i.e. values without culture). * Significantly different from control. † Significantly different from values for cells cultured with vehicle alone. ‡ Significantly different from values for cells cultured with forskolin alone. As, antisense oligonucleotide; Mis, mismatch oligonucleotide.

Figure 3. Effect on L-leucine influx of antisense oligonucleotide to CD98

BeWo cells expressing either H2B-GFP or Mit-DsRed2 were mixed, cultured and transfected without or with antisense or mismatch oligonucleotide. L-Leucine influx was measured over a 3 min period before treatment (control) and after a further 48 h treatment with 100 μM forskolin or with vehicle in a medium containing 2 μM L-[ H]leucine with or without 2 mM 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) or 2 mM L-leucine in the absence of Na⁺. A, carrier-mediated influx rate defined by subtracting the diffusional component (determined by measuring the influx of 2 μM L-[³H]leucine in the presence of 20 mM unlabelled L-leucine) from the total influx. B, system L-mediated influx rate defined as the difference between the influx in the presence and absence of BCH. Data represent the means ± S.D. of three separate experiments of triplicate assays. * Significantly different from control. † Significantly different from values for cells cultured with vehicle alone. ‡ Significantly different from values for cells cultured with forskolin alone. As, antisense oligonucleotide; Mis, mismatch oligonucleotide.

Figure 4. Confocal microscopy of human term placenta showing immunofluorescence localization of CD98

The section shows two adjacent chorionic villi cut transversely. There is positive immunostaining of (syncytio- and cyto-) trophoblast, as well as of components in the villus core (fetal capillary endothelium; fetal cells present within the capillary lumen). Scale bar represents 10 μm.