EP1 receptor-mediated migration of the first trimester human extravillous trophoblast: The role of intracellular calcium and calpain

Context: The root cause of preeclampsia in the human lies in the placenta, where a subpopulation of cytotrophoblast cells called extravillous trophoblasts (EVT), known to be involved in the invasion of the uterine endometrium and utero-placental arteries, become less invasive, resulting in poor perfusion of maternal blood into placenta. Objectives: Because EVT migrate into the prostaglandin (PG) E-2-rich decidua, we tested the roles of PGE(2) and PGE(2)-mediated signaling in EVT migration, using our well-characterized EVT line HTR-8/Svneo as well as first trimester villus explants in culture. Design: mRNA expression of different PGE(2) receptors (EPs) in HTR-8/Svneo cells was studied using RT-PCR. To characterize the functional significance of EP receptors in EVT, different EP receptor agonists and antagonists were used in our migration assay systems and in the measurements of intracellular concentration of Ca2+ ([Ca2+](i)) and calpain activity. Results: Exogenous PGE(2) stimulated EVT migration both in vitro and in the villus explant cultures. Although EVT expressed mRNA for all EP receptors (EP 1-4), a functional predominance of EP1 and EP4 was demonstrated in migration assays using specific EP agonists and antagonists. EP1-receptor-mediated signaling events such as activation of phospholipase C and elevation of cytosolic free [Ca2+](i) were confirmed by the following findings: 1) exogenous PGE2 or an EP1 agonist, but not an EP4 agonist, increased [Ca2+](i), which could be blocked with an EP1 antagonist as well as BAPTA and thapsigargin; 2) phospholipase C inhibitor U73122, BAPTA, and thapsigargin inhibited PGE(2)-mediated migratory response of EVT; and 3) PGE(2)-mediated EVT migration was shown to be dependent on a class of Ca2+-dependent proteases called calpains, known to be involved in cell detachment from substratum during migratory responses. The presence of PGE2 stimulated calpain activity, whereas two calpain inhibitors, calpastatin and N-Ac-Leu-Leu-methioninal (ALLM), blocked EVT migration. Conclusion: PGE(2) stimulates EVT migration by signaling through EP1 receptors, increasing [Ca2+](i), and activating calpain.