Expression And Role Of LAT1 On Trophblast Cells

Background During mammalian embryo implantation, appropriate invasion of extraembryonic trophoblast into maternal uterus is essential for successful implantation which determines the pregnancy outcome. After blastocyst attachment and invasion into maternal endometrium, invasive extracellular villous trophoblast (extravillous trophoblast, EVT) which is derived from anchoring villi breaks through the basal layer of the endometrial epithelium and migrates into uterus stroma, causing decidualization of stromal cells. Although human decidualization is independent of the trophoblast cells, when in pregnancy, this process involves in more extensive and pronounced changes of all uterine compartments, including uterine spiral arteries, local immune cells as well as other compartments in the epithelialium and stroma. The trophoblast cells which migrate into the uterine spiral arteries eventually replace the endothelial cells and degrade the smooth muscle and elastic membrane to remodel the blood vessel, increasing the utero-placental blood supply, to ensure normal growth and development of the fetus. Trophoblast cell migration, invasion are regulated by hormones, cytokines and immune factors and other factors, the dysfunction of this process can lead to a variety of diseases, such as spontaneous abortion, fetal growth restriction, preeclampsia and choriocarcinoma, etc. Therefore, it is of great clinical implications to investigate the biological behavior of trophoblast cell, especially the trophoblast migration.As a member of L-type amino acid transporter, L-type amino acid transporter 1 (LAT1), is also involved in regulating a variety of tumor cell migration and invasion, besides transporting amino acids. It has been reported that the intervention of gene silencing or 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, (BCH, specific inhibitor of LAT1), can reduce invasion and migration of mouse trophoblast Rs26 TS, suggesting the involvement of LAT1 in trophoblast cell migration and invasion. However, the mechanism of LAT1 to regulate trophoblast migration is still unknown.Our previous founding showed specific expression of LAT1 in trophoblast cells under pathological conditions. Based on similarity biological behavior between trophoblast cells and tumor cells, we speculate that LAT1 may be involved in regulating the biological behavior of trophoblast cells.Objective Investigate the effect of LAT1 on trophoblast cell biological behavior during early placentaion, with in vivo model of mice and in vitro model of trophoblast cell respectively.Methods1 Immunohistochemistry was applied to determine the immnolcaliza-tion of LAT1 protein in mouse uterus on D8. Ectoplacental cones (EPCs) were dissected out from D8 uterus and then cultured in vitro with different concentrations of BCH (0.1μM, 1μM,4μM; specific antagonist of LAT1) and L-Leucine (20μM,200μM,800μM; substrate of LAT1) to detect the role of LAT1 during the EPCs attachment and outgrowth.2 RT-PCR and Western Blotting were applied to detect the expression of LAT1 in HTR8Svneo, JAR and JEG-3. Immunocytochemistry was applied to detect the immnolocalization of LAT1 in HTR8-Svneo, JAR and JEG-3.3 Cells were cultured with additional 20μM,40μM,80μM,160μM concentrations of L-Leucine (substrate of LATI) in complete medium for 24h or 48h, RT-PCR and Western Blotting were applied to detect the expression changes of LAT1 in HTR8-Svneo, JAR and JEG-3.4 Cells were cultured with different concentrations of BCH (0.1μM, 1μM,4μM) for 24h and 48h, MTS assay was used to examine cell proliferation. Flow cytometry was used to analyse cell cycle distribution and apoptosis. Transwell assay and wound healing test were applied to examine the cell migration.Results1 LAT1 protein was highly expressed in secondary decidual zone and also positively in EPCs on D8 in mice. BCH significantly surppressed the EPCs outgrowth (P<0.05). Whereas, L-Leucine showed no significant effect on EPCs outgrowth (P> 0.05).2 The mRNA and protein of LAT1 was highly expressed in HTR8-Svneo, JAR and JEG-3. The protein was localized to the cell membrane and cytoplasm.3 Compared with the control group, additional leucine of different concentratioins had no effect LAT1 expression on HTR8-Svneo, JAR and JEG-3 three trophoblast cells (P>0.05).4 The effect of BCH on proliferation inhibition and apoptosis increase showed concentration and time-dependent pattern. After 48h treatment, the proliferation of various high concentration group was significantly inhibited (P<0.05), HTR8-Svneo and JEG-3 cell cycle were stucked in G2/M phase, and JAR stucked in G0/G1 phase. Meanwhile, the apoptosis rate was significantly increased (P<0.05); Trophoblast migration was significantly inhibited by different concentrations of BCH treatment in HTR8-Svneo cells (P<0.05), but only inhibited by high concentrations of BCH treatment in JAR and JEG-3 cells (P<0.05).Conclusion LAT1 is specifically expressed in trophoblast cells and possibly involved in early placentaion through regulating cell proliferation, apoptosis and migration.