Apoptosis of alcohol-exposed human placental cytotrophoblast cells is downstream of intracellular calcium signaling

Abstract

Background: Apoptosis is induced by ethanol (EtOH) in human placental trophoblast cells, possibly disrupting placentation and contributing to intrauterine growth restriction in fetal alcohol spectrum disorder (FASD). EtOH induces programmed cell death in several embryonic tissues by raising intracellular Ca(2+) . Therefore, the role of Ca(2+) signaling in EtOH-induced apoptosis was examined using human first trimester cytotrophoblast cell lines, examining the hypothesis that apoptosis is dependent on intracellular Ca(2+) signaling.

Methods: Using HTR-8/SVneo and SW.71 cytotrophoblast cell lines, real-time intracellular Ca(2+) concentration was monitored by fluo-4 epifluorescence microscopy and apoptosis was assessed by flow cytometry of cells fluorescently labeled for DNA fragmentation (TUNEL) and annexin V binding.

Results: Intracellular Ca(2+) concentrations increased synchronously in all cells within 10 seconds of exposure to 50 mM EtOH, but not at lower EtOH concentrations (10 to 25 mM) incapable of inducing apoptosis. Trophoblast cells treated with inhibitors of Ca(2+) signaling (BAPTA-AM, U73122, xestospongin D, BAPTA, SKF-96365) produced no intracellular Ca(2+) transients after exposure to 50 mM EtOH and were protected from cell death induced by EtOH.

Conclusions: EtOH-induced apoptosis in human cytotrophoblast cells, identified by DNA fragmentation and externalized phosphatidylserine, was dependent upon Ca(2+) signaling. Both intracellular Ca(2+) mobilization and extracellular Ca(2+) influx were required, as well as phosphatidylinositol signaling. Inhibition by SKF-96365 suggests that the capacitative Ca(2+) entry mechanism that utilizes TRPC channels was activated by EtOH. Apoptosis occurs downstream of Ca(2+) signaling in trophoblasts and may contribute to placental insufficiency and poor fetal growth associated with FASD.

Keywords: Alcohol; Apoptosis; Calcium; Fetal Alcohol Spectrum Disorder; Intracellular Signaling; Intrauterine Growth Restriction; Trophoblast.

Figure 1. Effect of ethanol on apoptosis in human cytotrophoblast cells

Cytotrophoblasts were exposed to 50 mM EtOH and assessed for apoptosis using TUNEL and Annexin V-binding methods. Apoptosis was assessed in HTR-8/SVneo cells using both the TUNEL (A) and Annexin V (B) procedures. Annexin V binding was also assessed in SW.71 cytotrophoblast cells (C). Experiments were repeated three times and the averages are shown with error bars indicating the SEM. *, p < 0.05 compared to control (0 min).

Figure 2. Effects of ethanol on intracellular Ca²⁺ concentration in HTR-8/SVneo human cytotrophoblast cells

A. Ethanol at the indicated concentrations was added to fluo-4-loaded cells after 90 s while the intracellular Ca²⁺ concentration was monitored in real time at 10-s intervals. Average Ca²⁺ concentrations were calculated from confluent fields for a total of 5 minutes. Experiments were repeated three times and the averages with error bars indicating the SEM are shown. B. Examples of fluo-4 fluorescence 20 s after adding 0 mM (upper panel) or 50 mM (lower panel) ethanol to a field of cytotrophoblast cells. C. Individual cells were monitored for 5 min after exposure (arrow) to 50 mM ethanol. Intracellular Ca²⁺ concentration was monitored to illustrate the variability of Ca²⁺ transients among cytotrophoblast cells. D. Initial Ca²⁺ transients and subsequent smaller transients were observed when intracellular Ca²⁺ concentration was monitored for 1 h after cells were exposed (first arrow) to 50 mM or 0 mM ethanol, as indicated. Averages and SEMs are shown for three experiments. After 1 h, cells were exposed to 5 nM ionomycin (second arrow) to demonstrate that the fluorescent dye was still responsive to the intracellular Ca²⁺ concentration. Insert shows examples of individual cell tracings for 50 mM (upper) or 0 mM (lower) ethanol treatments.

Figure 3. Source of ethanol-induced Ca²⁺ mobilization

HTR-8/SVneo cells were treated with BAPTA-AM, U73122, U73343, BAPTA or SKF-96365, as indicated, and simultaneously loaded with 5 mM fluo4-AM for 30 min at 37°C. Cell fluorescence was monitored at 10 s intervals with addition of 50 mM ethanol after 90 s (arrow). Ca²⁺ levels were monitored for a total of 5 min. Experiments were repeated three times and the averages with error bars indicating the SEM are shown.

Figure 4. Requirements of Ca²⁺ signaling for ethanol-induced apoptosis

HTR-8/SVneo (A-B) or SW.71 (C-D) cytotrophoblast cells were preincubated with vehicle or the indicated Ca²⁺ signaling inhibitors, as in Fig. 3, and then treated with vehicle or 50 mM ethanol. After 60 min, cells were assessed for apoptosis by the TUNEL (A, C) or Annexin V-binding (B, D). Experiments were repeated three times and the averages with error bars indicating the SEM are shown. *, p < 0.05 compared to vehicle treatment.