PP2 regulates human trophoblast cells differentiation by activating p38 and ERK1/2 and inhibiting FAK activation.
ABSTRACT Throughout gestation, fetal growth and development depend, in part, on placental transfer of nutrients from the maternal circulation. This latter function depends on multinucleated, terminally differentiated syncytiotrophoblasts. In vitro, freshly isolated cytotrophoblast cells differentiate spontaneously into syncytiotrophoblast in the presence of fetal bovine serum (FBS). We have previously showed that trophoblast differentiation is regulated by ERK1/2 and p38. Moreover, we showed that PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3, 4-d]pyrimidine], a Src family kinase (SFK) specific inhibitor, stimulates biochemical trophoblast cells differentiation while it inhibits cell adhesion and spreading without affecting cell fusion. Therefore, we examined the mechanisms by which PP2 modulates trophoblast cells differentiation. This study shows that PP2 stimulates ERK1/2 and p38 activation after 24h of treatments and up to 3 days while it inhibits focal adhesion kinase (FAK) phosphorylation at many sites including Tyr-397, 407, 576 and 577. Furthermore, we showed that transient activation of ERK1/2 by FBS is independent of SFK and that PP2 induces rapid activation of p38. Moreover, the kinase activity of SFK is negatively regulated by the phosphorylation of their carboxy (C)-terminal regulatory tyrosines by specific proteins called carboxyl-terminal Src kinase (Csk) and Csk homologous kinase (CHK). We showed the expression of Csk and CHK in human trophoblast cells. In summary, this study showed that PP2 stimulates the biochemical differentiation of trophoblast cells by stimulating p38 and ERK1/2 while it inhibits the morphological differentiation by inhibiting FAK activation.
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ABSTRACT: Mitogen-activated protein kinases (MAPKs) control many cellular events from complex programmes, such as embryogenesis, cell differentiation and proliferation, and cell death, to short-term changes required for homeostasis and acute hormonal responses. However, little is known about expression and activation of classical MAPKs, extracellular signal-regulated kinase1/2 (ERK1/2) and p38 in human placenta. Therefore, we examined the expression of ERK1/2 and p38 in trophoblasts from human term placenta, and their implication in differentiation. In vitro, freshly isolated cytotrophoblast cells, cultivated in 10% fetal bovine serum (FBS), spontaneously aggregate and fuse to form multinucleated cells that phenotypically resemble mature syncytiotrophoblasts, that concomitantly produce human chorionic gonadotropin (hCG) and human placental lactogen (hPL). This study shows that the level of ERK1/2 and p38 decreases with increasing days of culture, to reach an undetectable level after 5 days of culture. Moreover, pretreatment of cells with an ERK1/2-specific inhibitor (PD98059) and/or a p38-specific inhibitor (SB203580) suppressed trophoblast differentiation. Our results also demonstrate that the p38 pathway is highly solicited as compared to the ERK1/2 pathway in the differentiation process. Furthermore, ERK1/2 and p38 are rapidly activated upon addition of FBS, but the activation of p38 is delayed compared to that of ERK1/2. In summary, this study showed that ERK1/2 and p38 pathways are essential to mediate initiation of trophoblast differentiation.The Journal of Physiology 08/2005; 566(Pt 2):409-23. · 4.38 Impact Factor
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ABSTRACT: Pyk2 is a protein tyrosine kinase that links G-protein-coupled receptors, inflammatory cytokines, and extracellular stimuli that elevate intracellular calcium concentration with activation of the mitogen-activated protein kinase pathways and regulation of ion channel functions. Here we describe the identification, cloning, and characterization of a new isoform of Pyk2 (Pyk2-H) that is generated by alternative RNA splicing. Pyk2-H is mainly expressed in hematopoietic cells including T-cells, B-cells, and natural killer cells. Engagement of T-cell or B-cell antigen receptors leads to rapid tyrosine phosphorylation of Pyk2-H. Pyk2-H is also activated in response to the chemokines RANTES and macrophage inflammatory protein-1beta in T cells. In addition, we show that glutathione S-transferase fusion proteins containing the carboxyl termini of Pyk2 and Pyk2-H bind to a different set of tyrosine-phosphorylated proteins in thymus lysates. Specific expression of Pyk2-H and its activation by antigens or chemokines in hematopoietic cells may contribute toward the generation of cell type-specific signals involved in host immune responses.Journal of Biological Chemistry 07/1998; 273(23):14301-8. · 4.65 Impact Factor
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ABSTRACT: Highly purified functional cytotrophoblasts have been prepared from human term placentae by adding a Percoll gradient centrifugation step to a standard trypsin-DNase dispersion method. The isolated mononuclear trophoblasts averaged 10 microns in diameter, with occasional cells measuring up to 20-30 microns. Viability was greater than 90%. Transmission electron microscopy revealed that the cells had fine structural features typical of trophoblasts. In contrast to syncytial trophoblasts of intact term placentae, these cells did not stain for hCG, human placental lactogen, pregnancy-specific beta 1-glycoprotein or low mol wt cytokeratins by immunoperoxidase methods. Endothelial cells, fibroblasts, or macrophages did not contaminate the purified cytotrophoblasts, as evidenced by the lack of immunoperoxidase staining with antibodies against vimentin or alpha 1-antichymotrypsin. The cells produced progesterone (1 ng/10(6) cells . 4 h), and progesterone synthesis was stimulated up to 8-fold in the presence of 25-hydroxycholesterol (20 micrograms/ml). They also produced estrogens (1360 pg/10(6) cells . 4 h) when supplied with androstenedione (1 ng/ml) as a precursor. When placed in culture, the cytotrophoblasts consistently formed aggregates, which subsequently transformed into syncytia within 24-48 h after plating. Time lapse cinematography revealed that this process occurred by cell fusion. The presumptive syncytial groups were proven to be true syncytia by microinjection of fluorescently labeled alpha-actinin, which diffused completely throughout the syncytial cytoplasm within 30 min. Immunoperoxidase staining of cultured trophoblasts between 3.5 and 72 h after plating revealed a progressive increase in cytoplasmic pregnancy-specific beta 1-glycoprotein, hCG, and human placental lactogen concomitant with increasing numbers of aggregates and syncytia. At all time points examined, occasional single cells positive for these markers were identified. RIA of the spent culture media for hCG revealed a significant increase in secreted hCG, paralleling the increase in hCG-positive cells and syncytia identified by immunoperoxidase methods. We conclude that human cytotrophoblasts differentiate in culture and fuse to form functional syncytiotrophoblasts.Endocrinology 05/1986; 118(4):1567-82. · 4.72 Impact Factor