Prostaglandin E2-mediated migration of human trophoblast requires RAC1 and CDC42.

The invasion of maternal decidua and uterine spiral arteries by a trophoblast subpopulation called extravillous trophoblast (EVT) is essential for the establishment of a normal placenta and an adequate blood flow toward the fetus. Derangements in these processes underlie pregnancy-related diseases like preeclampsia and intrauterine growth restriction. Many growth factors, growth factor binding proteins, and extracellular matrix components can positively or negatively regulate the proliferation, migration, and/or invasiveness of these EVT cells. RHO GTPases, including RHOA, RAC1, and CDC42, are ubiquitous proteins that control cytoskeletal changes by forming stress fibers and projecting lamellipodia and filopodia during cellular migration. We had previously shown that prostaglandin (PG) E(2) produced in abundance by the decidua promotes the migration of first-trimester human EVTs by increasing the intracellular concentration of calcium and activating calpain. Using our well-characterized immortalized EVT cell line, HTR-8/SVneo, as well as villus explants from first-trimester placentae, this study examined the role of RHO GTPases RAC1 and CDC42 in PGE(2)-mediated migratory responses of these cells. Though a RAC1 inhibitor, NSC23766 as well as RAC1 knockdown by siRNA decreased the migration of HTR-8/SVneo cells in a Transwell migration assay, this inhibition could not be restored by PGE(2) or 17-phenyl trinor PGE(2) (PGE receptor PTGER1 agonist) or PGE(1) Alcohol (PGE receptor PTGER4 agonist). Similar results were noted for EVT cell spreading in villus explants. Furthermore, CDC42 silencing using siRNA inhibited PGE(2)-induced migration of HTR-8/SVneo cells. Finally, the treatment of EVT cells with PGE(2), PTGER1 agonist, or PTGER4 agonist activated RAC1 and CDC42 at 10 min, suggesting that RAC1 and CDC42 play an essential role in PGE(2)-mediated migration of human EVTs.

Roles of Rho guanosine 5'-triphosphatase A, Rho kinases, and extracellular signal regulated kinase (1/2) in prostaglandin E2-mediated migration of first-trimester human extravillous trophoblast.

Prostaglandin (PG) E(2) may regulate invasiveness of human placenta because we previously reported stimulation of migration of placental trophoblasts by PGE(2) acting through PGE receptor (EP)-1 and activating calpain. RhoA GTPase and its important effector Rho kinase (ROCK) have also been previously shown to regulate trophoblast migration. Using immortalized HTR-8/SVneo trophoblast cells and first-trimester human chorionic villus explant cultures on matrigel, we further examined the role of RhoA/ROCK and MAPK (ERK1/2) pathways on PGE(2)-mediated stimulation of trophoblast migration. Migration of cytotrophoblasts was shown to be inhibited by treatment of the trophoblast cell line and chorionic villus explants with either cell-permeable C3 transferase or selective RhoA small interfering RNA. These inhibitions were significantly mitigated by the addition of PGE(2), an EP1/EP3 agonist or an EP3/EP4 agonist, suggesting that RhoA plays an important role in trophoblast migration but may not be obligatory for PGE(2) action. Treatment of HTR-8/SVneo cells with nonselective ROCK inhibitor Y27632 or ROCK small interfering RNAs inhibited migration of these cells, which could not be rescued with PGE(2) or the other two EP agonists, suggesting the obligatory role of ROCK in PGE(2)-induced migratory response. Furthermore, U0126, an inhibitor of MAPK kinases MEK1 and MEK2, abrogated PGE(2)-induced migration of trophoblasts, and PGE(2) or the other two EP agonists stimulated ERK1/2 activation in trophoblasts, which was not abrogated by pretreatment with C3 transferase, indicating that ERK signaling pathway is an efficient alternate pathway for RhoA in PGE(2)-mediated migration of trophoblasts. These results suggest that ROCK and ERK1/2 play more important roles than RhoA in PGE(2)-mediated migration stimulation of first-trimester trophoblasts.

Rho guanosine 5'-triphosphatases differentially regulate insulin-like growth factor I (IGF-I) receptor-dependent and -independent actions of IGF-II on human trophoblast migration.

Both IGF-I and IGF-II stimulate migration of human extravillous trophoblast (EVT) cells. Although IGF-I is known to signal through IGF type 1 receptor (IGF1R), IGF-II signals through IGF1R as well as in an IGF1R-independent manner. The purpose of this study was to investigate the roles of Rho GTPases in IGF1R-independent and -dependent actions of IGF-II on EVT cell migration. To distinguish IGF1R-dependent and -independent actions, we used picropodophyllin, a selective inhibitor of IGF1R tyrosine kinase, and IGF analogs with differential affinities for IGF1R, IGF-II/cation-independent mannose 6-phosphate receptor, and IGF-binding proteins. IGF1R-dependent actions of IGF-II were confirmed by showing the effects of IGF1R-selective agonist Des1-3 IGF-I. We used pharmacological inhibitors or selective small interfering RNAs to investigate the roles of RhoA, RhoC, Rac1, Cdc42, and Rho effector kinases called ROCK-I and -II in IGF-induced EVT cell migration. Although basal migration of EVT cells required each member of the Rho GTPase family studied, IGF1R-dependent and -independent EVT cell migration exhibited differential requirements for these enzymes. IGF1R-mediated EVT cell migration was found to depend on RhoA and RhoC but not on Rac1 or Cdc42. However, IGF1R-independent effect of IGF-II on EVT cell migration required ROCKs but not RhoA, RhoC, Rac1, or Cdc42. Most importantly, IGF1R-independent action of IGF-II was found to be exaggerated when RhoA or RhoC was down-regulated. Thus, different members of the Rho GTPase family regulate IGF-II-mediated EVT cell migration differentially, depending upon whether it signals through IGF1R or in an IGF1R-independent manner.

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) E2-rich decidua, we tested the roles of PGE2 and PGE2-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 PGE2 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 PGE2 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 PGE2-mediated migratory response of EVT; and 3) PGE2-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: PGE2 stimulates EVT migration by signaling through EP1 receptors, increasing [Ca2+]i, and activating calpain.