Role And Mechanism Of Rho Protein In EGF Induced Human Trophoblast Migration

Background and Objective:Formation of a functional placenta is essential for intrauterine development of the human embryo. Extravillous cytotrophoblasts (EVT) invade the underlying decidua, then surround and migrate into the wall of the uterine spiral arteries, which results in the remodeling of uterine vasculature. This process plays a pivotal role in mammalian placentation and is stringently regulated to ensure a successful pregnancy. Poor invasion of EVT was believed to be associated with insufficient remodeling of the spiral arteries, which was typical of the pathological changes of miscarriage, preeclampsia and intrauterine growth restriction; conversely, excessive invasion leads to placenta percreta, persistent trophoblastic disease or invasive cancer, such as choriocarcinoma. Elucidating the key events of trophoblast invasion is critical to understand these diseases, as well as properly managing them. EVT invasion is a multi-step process involving attachment, degradation through matrix metalloproteinases (MMPs) and subsequent migration through the extracellular matrix. Endocrine factors produced locally or distally play an important role in regulating this process. A collection of endogenous growth factors and cytokines have been implicated in trophoblast migration and/or invasion, however, the mechanisms that link the endocrinal signals to migratory processes are still only partially understood.Epidermal growth factor (EGF) is a multifunctional growth factor that regulates a variety of fundamental cell properties. Maternal EGF deficiency causes fetal hypoglycemia and intrauterine growth retardation in mice. In humans, both the decidua and trophoblast express EGF and its receptor, and the expression of EGF in the trophoblast decreases as the pregnancy progresses, which hints at its important role in early pregnancy. There is evidence that EGF affects the trophoblast cell in many ways, including proliferation, survival, apoptosis, differentiation, secretion, motility, and invasion and/or migration. In recent years, more attention has been paid to the effect and mechanism of EGF on trophoblast cell migration and the majority of these results show that EGF is stimulatory. Binding of EGF to its receptor (EGFR) at the plasma membrane induces dimerization of EGFR, which results in the activation of EGFR tyrosine kinase and trans-autophosphorylation, thus launching a variety of intracellular pathways, including Ras/MAPK, PI(3)K/Akt, PLCg/PKC, and STAT. Rho proteins are part of the extensive Ras superfamily that shuttle between an inactive GDP-bound and an active GTP-bound form and exhibit intrinsic GTPase activities. Rho proteins consist of highly conserved RhoA, RhoB and RhoC isoforms that have been regarded as the master regulators of cytoskeletal reorganization required for cellular migration. Although EGFR activation has been claimed to lead to membrane ruffling and the reorganization of cytoskeleton and focal adhesion through activation of Rho proteins, the details of Rho isoforms involved in the regulation of EGF-induced trophoblast cell migration remain unclear.JEG-3 and JAR cell lines are derived spontaneously from choriocarcinoma and share many properties with the villous trophoblast in terms of their morphology, biochemical markers, and hormone secretion. In addition, EGFR has been reported to be expressed in both JEG-3 and JAR cells. To investigate the roles of Rho protein in epidermal growth factor (EGF)-induced trophoblast cell migration and its mechanism, we examined EGF-mediated stimulation of trophoblast migration, using JEG-3 and JAR and first-trimester human chorionic villus explant cultures on matrigel.Results and conclusions:1. EGF regulates trophoblast cell migration in a dose-dependent manner. Both JEG-3 and JAR cell migration peaked with EGF concentrations of 1ng/ml (P < 0.01); but then decreased with higher concentrations and was down-regulated by the highest concentration (100ng/ml; P < 0.01). Survival, proliferation and apoptosis appear to have no effect on the varied number of cells attributed to cell migration as a result of EGF treatment.2. C3 exoenzyme (5mg/ml), an inhibitor of Rho activity significantly reduced the basal migration rate in both JEG-3 and JAR cells (P < 0.01). Pretreatment with C3 exoenzyme plus EGF also significantly reduced the extent of cell migration (P < 0.01). C3 exoenzyme had no significant effect on survival, proliferation, or apoptosis for JEG-3 or JAR cells (P > 0.05). More over, EGF could significantly stimulate EVT cell outgrowth from explants of human villous tissue (6-9 wk gestation) cultured on matrigel (P < 0.01), and that C3 exoenzyme decreased the outgrowth in the absence or presence of EGF (P < 0.01). 3. Following EGF treatment, in JEG-3 and JAR cells, level of RhoA and RhoC had increased significantly by more than two fold by 12 and 24 h (P < 0.01), whereas the level of RhoB had decreased slightly (P < 0.01). In cultured villous explants, EGF treatment showed a similar pattern to JEG-3 cells. Moreover, EGF significantly increased the amount of the active form of RhoA (P < 0.01) as well as RhoC (P < 0.01) in JEG-3, JAR or cultured villous explants, whereas the level of GTP-bound RhoB was unaffected. These results suggest that RhoA and RhoC may play an important role in trophoblast cell response to EGF stimulation.5. Both RhoA and RhoC siRNA significantly reduced the migration of JEG-3 and JAR cells, and treatment with EGF following siRNA did not restore the migratory capacity of these cells, indicating that both RhoA and C are essential to EGF-stimulated trophoblast cell migration. Neither RhoA nor RhoC siRNA had significant effect on the survival, proliferation, and apoptosis of cells after 24 h in culture.6. Treatment with C3 exoenzyme (5mg/ml, 2h) led to cell shrinkage and disorganization of F-actin, which was not reversed by treatment with EGF after C3 exoenzyme treatment. RhoA siRNA (100nM, 24h) disrupted the F-actin and led to marked cell shrinkage, which was consistent with a decreased migratory ability and looked similar to results after C3 exoenzyme treatment and EGF did not restore the original cell morphology. RhoC siRNA (100nM, 24h) did not destroy the organization of F-actin in JEG-3 and JAR cells.In conclusion, our data confirm that EGF (1ng/ml) not only stimulates JEG-3 and JAR cell migration in serum reduced medium (0.5% FBS), but also stimulates EVT migration from cultured human first trimester villous tissue. We also show that trophoblast migration induced by EGF depends on the increased expression and activation of RhoA and RhoC, but not RhoB. Furthermore, RhoA was found to stimulate the migration by F-actin redistribution, while RhoC was not critical for F-actin redistribution.