Study On Signal Transduction In Regulation Of Human Chorionic Gonadotropin Secretion By Epidermal Growth Factor In Trophoblast Cell

EGF and EGFR are of wide interest in oncogenesis ernbryogenesis and peptide hormone-receptor interactions. Recently, several lines of evidence have suggested possible physiological roles for EGF in feto-placenta development through its mitogenic action as well as its ability to influence differentiated cellular functions. Human chorionic gonadotropin is produced primarily by syncytiotrophoblasts and to some extent by cytotrophoblasts in human placenta. HCG plays a critical role at the early stage of pregnancy. Human placentas are extremely rich in EGFR, suggesting that EGF is important in placental functions. EGF stimulates HCG release in choriocarcinoma cell lines and normal trophoblast in culture. So regulation of HCG secretion by EGF is an important element for normal pregnancy. Objectives 1. To investigate the effects of EGF on secretion of HCG by cultured early placental tissues and the expression of EGFR in trophoblast cells throughout the gestational period. 2. To study the effects of EGF and PKC on JAR trophoblast cell proliferation, HCG secretion and actived ERK1/2 and to discuss the roles of PKC and ERK1/2 on the reguation of cell proliferation and HCG secretion of trophoblast cell. Methods 1. Effects of EGF on the HCG secretion in trophoblasts were detected using a primary culture systerm. Explants of trophoblastic tissue were cultured with or without EGF. Concentration of HCG was determined with ELASA. 2. EGFR expressions in trophoblast cells of different pregnancy phase were localized by immunohistochemical method(ABC) 3. The stimulating activity of EGF, PMA(acute or chronic) and CHEL (a protein kinase C inhibitor) on the proliferation of trophoblast cell was observed by an MTT colorimetric assay. The secretion of HCG stimulated by EGF, PMA(acute or chronic) and CHIEL on trophoblast cell was determined by ELASA. 4. Western blot was used to determine the change of active ERK1/2 of trophoblst cell, when HCG secretion by EGF, PMA(acute or chronic) and CHili was highest. 5. When the combination of EGF and PMA, CHEL , EGTA at their maximum effective dose stimulated JAR cell, cell proliferation was observed by an MTT colorimetric assay and secretion of HCG was determined by ELASA. Results 1. We observed that EGF increased l-ICG secretion of cultured early placental tissue and HCG concentration was highest when culture medium contained I OOng/ml EGF. 2. Expressions of EGFR in early placenta was significantly stronger than that in middle and term placenta, with peak expression observed during the period of 10-12 week placenta throughout the gestational period. 3. EGF stimulated cell proliferation and HCG secretion of trophoblast cell and the effect was in a dose-dependent manner. The concentration of 1 OOng/ml was most effective while phosphorylated ERK1/2 content increased. 4. Treatment with PMA (acute) significantly inhibited cell proliferation and HICG secretion of trophoblast cell. The concentration of 200nM was most effective while the phosphorylation of ERKI/2 was significantly inhibited. PMA (chronic) significantly increased HCG content of JAR cell but fail to influence cell proliferation, and PMA, at maximun effective dose, induced srongly the activation of ERK1/2. Treatment with CI-IEL did not affect J...