Leptin promotes cell proliferation and survival of trophoblastic cells

Leptin, the 16-kDa protein product of the obese gene, was 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. In the present work, we studied a possible effect of leptin on trophoblastic cell proliferation, survival, and apoptosis. Recombinant human leptin added to JEG-3 and BeWo choriocarcinoma cell lines showed a stimulatory effect on cell proliferation up to 3 and 2.4 times, respectively, measured by 3H-thymidine incorporation and cell counting. These effects were time and dose dependent. Maximal effect was achieved at 250 ng leptin/ml for JEG-3 cells and 50 ng leptin/ml for BeWo cells. Moreover, by inhibiting endogenous leptin expression with 2 μM of an antisense oligonucleotide (AS), cell proliferation was diminished. We analyzed cell population distribution during the different stages of cell cycle by fluorescence-activated cell sorting, and we found that leptin treatment displaced the cells towards a G2/M phase. We also found that leptin upregulated cyclin D1 expression, one of the key cell cycle-signaling proteins. Since proliferation and death processes are intimately related, the effect of leptin on cell apoptosis was investigated. Treatment with 2 μM leptin AS increased the number of apoptotic cells 60 times, as assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining, and the caspase-3 activity was increased more than 2 fold. This effect was prevented by the addition of 100 ng leptin/ml. In conclusion, we provide evidence that suggests that leptin is a trophic and mitogenic factor for trophoblastic cells by virtue of its inhibiting apoptosis and promoting proliferation. © 2007 by the Society for the Study of Reproduction, Inc.Fil:Kotler, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Calvo, J.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Varone, C.L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

17Beta-estradiol enhances leptin expression in human placental cells through genomic and nongenomic actions

The process of embryo implantation and trophoblast invasion is considered the most limiting factor in the establishment of pregnancy. Leptin was originally described as an adipocyte-derived signaling molecule for the central control of metabolism. However, it has been suggested that leptin is involved in other functions during pregnancy, particularly in the placenta, where it was found to be expressed. In the present work, we have found a stimulatory effect of 17beta-estradiol (E2) on endogenous leptin expression, as analyzed by Western blot, in both the BeWo choriocarcinoma cell line and normal placental explants. This effect was time and dose dependent. Maximal effect was achieved at 10 nM in BeWo cells and 1 nM in placental explants. The E 2 effects involved the estrogen receptor, as the antagonist ICI 182 780 inhibited E2-induced leptin expression. Moreover, E2 treatment enhanced leptin promoter activity up to 4-fold, as evaluated by transient transfection with a plasmid construction containing the leptin promoter region and the reporter gene luciferase. This effect was dose dependent. Deletion analysis demonstrated that a minimal promoter region between - 1951 and -1847 bp is both necessary and sufficient to achieve E2 effects. Estradiol action involved estrogen receptor 1, previously known as estrogen receptor alpha, as cotransfection with a vector encoding estrogen receptor 1 potentiated the effects of E2 on leptin expression. Moreover, E2 action probably involves membrane receptors too, as treatment with an estradiol-bovine serum albumin complex partially enhanced leptin expression. The effects of E2 could be blocked by pharmacologic inhibition of MAPK and the phosphoinositide-3-kinase (PI3K) pathways with 50 μM PD98059 and 0.1 μM Wortmannin, respectively. Moreover, cotransfection of dominant negative mutants of MAP2K or MAPK blocked E 2 induction of leptin promoter. On the other hand, E2 treatment promoted MAPK1/MAPK3 and AKT phosphorylation in placental cells. In conclusion, we provide evidence suggesting that E2 induces leptin expression in trophoblastic cells, probably through genomic and nongenomic actions via crosstalk between estrogen receptor 1 and MAPK and PI3K signal transduction pathways. © 2010 by the Society for the Study of Reproduction, Inc.Fil:Gambino, Y.P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Maymó, J.L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Calvo, J.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Varone, C.L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Leptin stimulates protein synthesis-activating translation machinery in human trophoblastic cells

Leptin was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in placenta, where it may work as an autocrine hormone, mediating angiogenesis, growth, and immunomodulation. Leptin receptor (LEPR, also known as Ob-R) shows sequence homology to members of the class I cytokine receptor (gp130) superfamily. In fact, leptin may function as a proinflammatory cytokine. We have previously found that leptin is a trophic and mitogenic factor for trophoblastic cells. In order to further investigate the mechanism by which leptin stimulates cell growth in JEG-3 cells and trophoblastic cells, we studied the phosphorylation state of different proteins of the initiation stage of translation and the total protein synthesis by [3H]leucine incorporation in JEG-3 cells. We have found that leptin dose-dependently stimulates the phosphorylation and activation of the translation initiation factor EIF4E as well as the phosphorylation of the EIF4E binding protein EIF4EBP1 (PHAS-I), which releases EIF4E to form active complexes. Moreover, leptin dose-dependently stimulates protein synthesis, and this effect can be partially prevented by blocking mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PIK3) pathways. In conclusion, leptin stimulates protein synthesis, at least in part activating the translation machinery, via the activation of MAPK and PIK3 pathways. © 2009 by the Society for the Study of Reproduction, Inc.Fil:Julieta Maymo, Y. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Gambino, É. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Varone, C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Calculation of the bootstrap current profile for the TJ-II stellarator

Calculations of the bootstrap current for the TJ-II stellarator are presented. DKES and NEO-MC codes are employed; the latter has allowed, for the first time, the precise computation of the bootstrap transport coefficient in the long mean free path regime of this device. The low error bars allow a precise convolution of the monoenergetic coefficients, which is confirmed by error analysis. The radial profile of the bootstrap current is presented for the first time for the 100_44_64 configuration of TJ-II for three different collisionality regimes. The bootstrap coefficient is then compared to that of other configurations of TJ-II regularly operated. The results show qualitative agreement with toroidal current measurements; precise comparison with real discharges is ongoing

Proliferation and survival of human amniotic epithelial cells during their hepatic differentiation

Stem cells derived from placental tissues are an attractive source of cells for regenerative medicine. Amniotic epithelial cells isolated from human amnion (hAECs) have desirable and competitive characteristics that make them stand out between other stem cells. They have the ability to differentiate toward all three germ layers, they are not tumorigenic and they have immunosuppressive properties. Although liver transplantation is the best way to treat acute and chronic hepatic failure patients, there are several obstacles. Recently, stem cells have been spotlighted as alternative source of hepatocytes because of their potential for hepatogenic differentiation. In this work, we aimed to study the proliferation and survival of the hAECs during their hepatic differentiation. We have also analyzed the changes in pluripotency and hepatic markers. We differentiated amniotic cells applying a specific hepatic differentiation (HD) protocol. We determined by qRT-PCR that hAECs express significant levels of SOX-2, OCT-4 and NANOG during at least 15 days in culture and these pluripotent markers diminish during HD. SSEA-4 expression was reduced during HD, measured by immunofluorescence. Morphological characteristics became more similar to hepatic ones in differentiated cells and representative hepatic markers significantly augmented their expression, measured by qRT-PCR and Western blot. Cells achieved a differentiation efficiency of 75%. We observed that HD induced proliferation and promoted survival of hAECs, during 30 days in culture, evaluated by 3H-thymidine incorporation and MTT assay. HD also promoted changes in hAECs cell cycle. Cyclin D1 expression increased, while p21 and p53 levels were reduced. Immunofluorescence analysis showed that Ki-67 expression was upregulated during HD. Finally, ERK 1/2 phosphorylation, which is intimately linked to proliferation and cell survival, augmented during all HD process and the inhibition of this signaling pathway affected not only proliferation but also differentiation. Our results suggest that HD promotes proliferation and survival of hAECs, providing important evidence about the mechanisms governing their hepatic differentiation. We bring new knowledge concerning some of the optimal transplantation conditions for these hepatic like cells.Fil: Maymo, Julieta Lorena. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Riedel, Rodrigo Nicolas. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Pérez Alcázar, Germán Antonio. Hospital Universitario Virgen Macarena;Fil: Magatti, Marta. Istituto Ospedaliero;Fil: Maskin, Bernardo. Hospital Nacional Professor Dr. Alejandro Posadas; ArgentinaFil: Dueñas, José Luis. Hospital Universitario Virgen Macarena;Fil: Parolini, Ornella. Istituto Ospedaliero;Fil: Sánchez-Margalet, Víctor. Hospital Universitario Virgen Macarena;Fil: Varone, Cecilia Laura. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin