Iron deficiency in pregnancy can cause fetal growth restriction, which may be due to altered placental function (Black, 2001). Intracellular Ca2+ ([Ca2+]i) regulates many aspects of placental function (Clarson et al. 2002). One of the mechanisms by which [Ca2+]i is maintained is via activation of purinergic receptors such as P2X7 and P2X4 (Roberts & Clarson, 2002). In this study, therefore, we investigated the effect of altered iron status on P2X7 expression in the trophoblast-derived BeWo cell line.
BeWo cells were examined under three conditions: iron depletion (20µM desferioxamine (DFO) for 40h), iron supplementation (5µM iron transferrin (FeTf) for 18h), and iron depletion followed by iron supplementation (DFO +FeTf), and compared to control. Expression of P2X7 and P2X4 was assessed by Western blotting using commercially available antibodies (1:100 dilution, Alomone Laboratories) and normalised to β-actin expression (1:2500 dilution; Sigma). Expression was quantified by denstiometry (ImageJ software).
From Western blotting we identified specific bands for P2X4 and P2X7 at the expected sizes (64 and 120 kDa respectively) in all groups studied. There was no difference in expression of P2X4 between control cells and the 3 conditions investigated (control: 1.0 ± 0.0003, DFO: 0.939 ± 0.265, FeTf: 1.123 ± 0.280, DFO + FeTf: 1.541 ± 0.179, mean ± S.E.M. n = 5). Treatment of BeWo cells with FeTf had no effect on P2X7 expression (control: 1.0 ± 0.0001, FeTf: 1.655 ± 0.389, mean ± S.E.M. n = 6). By contrast, treatment with DFO caused a significant increase in P2X7 expression (control: 1.0 ± 0.0001, DFO: 2.020 ± 0.274, mean ± S.E.M. n = 6, P < 0.05 ANOVA with Bonferroni test). This increase was reversed when DFO treated cells were supplemented with FeTf (DFO: 2.020 ± 0.274, DFO + FeTf: 1.144 ± 0.256, mean ± S.E.M. n = 6, P < 0.05 ANOVA with Bonferroni test).
From this study we have shown that iron deficiency can alter expression of P2X7 purinergic receptor protein which may have an effect on [Ca2+]i in the trophoblast cells of human placenta.
Sponsored by the MRC and SEERAD.