CONICET | Buscador de Institutos y Recursos Humanos

The process of embryo implantation and trophoblast invasion is currently considered as the most limiting factor for the establishment of pregnancy. Cytokines produced by the maternal endometrium and the developing embryo play an important role in the signaling process to implantation. In particular, leptin has been proposed to play a relevant role in the regulation of the embryo implantation as well as its maintenance. Leptin is a 16000 MW protein product of the obese gene, originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta, where it was found to be expressed. Recently, we have demonstrated that leptin promotes proliferation and survival of trophoblastic cells. In the present work we aimed to study the signal transduction pathways activated by leptin in placenta and to elucidate the mechanisms that mediate the antiapoptotic effect of leptin.Moreover, since leptin could be important in trophoblast cell survival, and therefore in maintenance of the placenta, we attempted to study the regulation of leptin expression in placenta. BeWo and JEG-3 choriocarcinoma cell line, as well as trophoblastic cells from human placenta explants were used.Western blot analyses were carried out to detect leptin expression as well as the phosphorylated form of proteins involved in mayor signaling pathways The early phase of apoptosis, triggered by the lack of serum, was assayed by flow cytometric detection of phosphatidyl serine translocation using FITC-labelled Annexin V and by western blot of caspase-3 fragmentation. Transient transfection assays with a plasmid construction containing different leptin promoter regions and the reporter gene luciferase, and expression vectors for some intermediates of signaling pathways were used to determine the transcriptional regulation of leptin. We have found that leptin stimulates JAK-STAT pathway by promoting JAK2 and STAT3 tyrosine phosphorylation. We have also demonstrated the activation of MAPK pathway by studying phosphorylation of MEK and MAPK (Erk1/2). PI3K pathway is also triggered by leptin stimulation as assessed by the study of PKB phosphorylation. These signaling pathways were confirmed in explants obtained from placenta of healthy donors. The effect of leptin on JEG-3 survival was completely reversed by blocking p42/44 MAPK activation employing the MEK inhibitor PD98059, whereas it was not affected by PI3K inhibition using Wortmannin. We have also found that recombinant human chorionic gonadotropin (hCG) added to BeWo cells showed a stimulatory effect on endogenous leptin expression, when analysed by Western blot. This effect was time and dose-dependent. Maximal effect was achieved at 100 IU hCG/ml. Moreover, hCG treatment enhanced leptin promoter activity up to 12.9 times, evaluated by transient transfection Similar results were obtained with placental explants evidencing physiological relevance. Since hCG signal transduction usually involves cAMP signaling, this pathway was analyzed. We found that dbcAMP counteracted hCG effect on leptin expression. Thereafter we determined that hCG effect could be partially blocked by 50 ìM PD98059 but not by the inhibition of the PI3K pathway with 0.1 ìM Wortmannin. Cotransfection with a dominant negative mutant of MAPK blocked the hCG-mediated activation of leptin expression. Moreover, hCG treatment promoted MEK and ERK1/ERK2 phosphorylation in placental cells.We provide some evidence that suggests that leptin is a trophic and mitogenic factor for trophoblastic cells by promoting proliferation and inhibiting apoptosis. Moreover, we demonstrate that hCG upregulates leptin expression probably involving the MAPK signal transduction pathway. In summary, our results further support the importance of leptin in the biology of reproduction.

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