The role of the sphingosine-1-phosphate axis in regulating human extravillous trophoblast migration

ABSTRACTThe University of Manchester. Khiria Al-saghir. Doctor of Philosophy. The role of the sphingosine-1-phosphate axis in regulating human extravillous trophoblast migration. February 2014. Failure of trophoblast invasion and remodelling of maternal blood vessels leads to the pregnancy complications pre-eclampsia (PE) and fetal growth restriction (FGR). Metabolomic profiling of placentas from such pregnancies has identified deranged sphingolipid metabolism as one of only a handful of pathways altered in PE/FGR. In other systems, the bioactive sphingolipid, sphingosine-1-phosphate (S1P) controls cell migration therefore this study aimed to determine its effect on extravillous trophoblast (EVT) function.S1P (50 nM? 10 �M) attenuated the migration of the EVT cell lines, Swan-71 and SGHPL-4 (n = 6; p < 0.05) and also the outgrowth of trophoblast from explants of human first trimester placenta. Quantitative PCR and immunolocalisation studies demonstrated that both EVT cell lines and primary EVT express S1P receptors 1, 2 and 3 in similar abundance. Receptor inhibitors were used to reveal S1PR2 as the receptor responsible for mediating the inhibitory effect of S1P inhibitory effect; JTE-013 (100 nM) a specific S1PR2 inhibitor, abolished S1P- attenuated migration (n = 6; p < 0.05 versus S1P alone) whereas treatment with the S1PR1/3 inhibitor, FTY720 (100 nM; n = 6) had no effect on S1P activity. Ligand binding to S1PR2 can activate numerous intracellular signalling pathways via receptor association with the G proteins, G? 12/13, G? q or G? i; however, analysis of Swan-71 cell migration and actin cytoskeleton in the presence of S1P � the Rho kinase inhibitor, Y-27632 (10 �M; n = 6) suggested preferential activation of G? 12/13. Nonetheless, S1P does activate G? i in Swan-71 cells, as demonstrated by analysis of cAMP levels and phosphorylation of downstream signalling molecules; however attempts to shift the balance of intracellular pathway activation towards G? i/Rac using siRNA-mediated knockdown of the Rac inhibitor ARGHGAP22 did not attenuate S1P inhibition of cellular motility, Subsequent experiments explored the possibility of preventing S1P? s actions by modulating EVT S1P receptor isoform expression using factors, including hormones and oxygen, previously reported to affect trophoblast migration or the expression of S1PR in other systems. Neither EGF nor low oxygen levels influenced S1PR expression however both IGF-II (10nM; p<0.05) and vitamin D (10nM; p<0.05) prevented the inhibitory effect of S1P on Swan-71 cell migration, the latter as a result of a significant reduction in S1PR2 expression (4-fold decrease; p<0.05). This study demonstrates that, although EVT express three S1P receptor isoforms, S1P predominantly signals through S1PR2 / G? 12/13 to activate Rho and actin stress fibre formation and thereby acts as potent inhibitor of EVT migration. Strategies aimed at shifting the balance of receptor isoform expression, may provide a mechanism for improving impaired trophoblast migration in compromised pregnancies. Importantly, expression of S1PR2, and therefore S1P function, can be down-regulated by vitamin D. Thus these data suggest that vitamin D deficiency, which is known to be associated with PE, may contribute to the impaired trophoblast migration that underlies this condition.