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Fil: de Dios, Nataly. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Ciudad Universitaria. Instituto de Quimica Biologica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Quimica Biologica de la Facultad de Ciencias Exactas y Naturales; Argentina

Leptin is a homeostatic regulator in the placenta where it promotes proliferation, protein synthesis and the expression of tolerogenic maternal response molecules such as HLA-G. Leptin also exerts an anti-apoptotic action in placenta controlling the expression of p53 master cell cycle regulator under different stress conditions. On the other hand, leptin is an integrative target of different placental stimuli. The expression of leptin in placenta is regulated by hCG, insulin, steroids, hypoxia and many other growth hormones, suggesting that it might have an important endocrine function in the trophoblastic cells. The leptin expression is induced involving the cAMP/PKA or cAMP/Epac pathways which have profound actions upon human trophoblast function. The activation of PI3K and MAPK pathways also participates in the leptin expression. Estrogens play a central role during pregnancy, particularly 17β-estradiol upregulates the leptin expression in placental cells through genomic and non-genomic actions. The leptin promoter analysis reveals specific elements that are active in placental cells. The transcription factors CREB, AP1, Sp1, NFκB and the coactivator CBP are involved in the placental leptin expression. Moreover, placental leptin promoter is a target of epigenetic marks such as DNA methylation and histone acetylation that regulates not only the leptin expression in placenta during pregnancy but also determines the predisposition of acquiring adult metabolism diseases. Taken together, all these results allow a better understanding of leptin function and regulatory mechanisms of leptin expression in human placental trophoblasts, and support the importance of leptin during pregnancy and in programming adult health. Fil: Schanton, Malena. 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; Argentina Fil: Maymo, Julieta Lorena. 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; Argentina Fil: Pérez Pérez, Antonio. Universidad de Sevilla; España Fil: Sánchez Margalet, Víctor. Universidad de Sevilla; España Fil: Varone, Cecilia Laura. 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; Argentina

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; Argentina Fil: 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; Argentina Fil: Pérez Alcázar, Germán Antonio. Hospital Universitario Virgen Macarena; Fil: Magatti, Marta. Istituto Ospedaliero; Fil: Maskin, Bernardo. Hospital Nacional Professor Dr. Alejandro Posadas; Argentina Fil: 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; Argentina

The steroid hormone 17β-estradiol is an estrogen that influences multiple aspects of placental function and fetal development in humans. During early pregnancy it plays a role in the regulation of blastocyst implantation, trophoblast differentiation and invasiveness, remodeling of uterine arteries, immunology and trophoblast production of hormones such as leptin. Estradiol exerts some effects through the action of classical estrogen receptors ERα and ERβ, which act as ligand-activated transcription factors and regulate gene expression. In addition, estradiol can elicit rapid responses from membrane-associated receptors, like activation of protein-kinase pathways. Thus, the cellular effects of estradiol will depend on the specific receptors expressed and the integration of their signaling events. Leptin, the 16,000MW protein product of the obese gene, was originally considered an adipocyte-derived signaling molecule for the central control of metabolism. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy. The leptin gene is expressed in placenta, where leptin promotes proliferation and survival of trophoblastic cells. Expression of leptin in placenta is highly regulated by key pregnancy molecules as hCG and estradiol. The aim of this paper is to review the molecular mechanisms underlying estrogen functions in trophoblastic cells; focusing on mechanisms involved in estradiol regulation of placental leptin expression.

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 (E(2)) 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 E(2)-induced leptin expression. Moreover, E(2) 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 E(2) 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 E(2) on leptin expression. Moreover, E(2) action probably involves membrane receptors too, as treatment with an estradiol-bovine serum albumin complex partially enhanced leptin expression. The effects of E(2) could be blocked by pharmacologic inhibition of MAPK and the phosphoinositide-3-kinase (PI3K) pathways with 50 microM PD98059 and 0.1 microM Wortmannin, respectively. Moreover, cotransfection of dominant negative mutants of MAP2K or MAPK blocked E(2) induction of leptin promoter. On the other hand, E(2) treatment promoted MAPK1/MAPK3 and AKT phosphorylation in placental cells. In conclusion, we provide evidence suggesting that E(2) 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.

Leptin, the LEP gene product, is produced in placenta where it has been found to be an important autocrine signal for trophoblastic growth during pregnancy. Thus, we have recently described the antiapoptotic and trophic effect of leptin on choriocarcinoma cell line JEG-3, stimulating DNA and protein synthesis. We have also demonstrated the presence of leptin receptor and leptin signaling in normal human trophoblastic cells, activating JAK-STAT, PI3K and MAPK pathways. In the present work we have employed dominant negative forms of MAPK and PKB constructs to find out the signaling pathways that specifically mediates the effect of leptin on protein synthesis. As previously shown, leptin stimulates protein synthesis as assessed by 3H-leucine incorporation. However, both dominant negative forms of MAPK and PKB inhibited protein synthesis in JEG-3 choriocarcinoma cells. The inhibition of PKB and MAPK activity by transfection with the dominant negative kinases prevented the leptin stimulation of p70 S6K, which is known to be an important kinase in the regulation of protein synthesis. Moreover, leptin stimulation of phosphorylation of EIF4EBP1 and EIF4E, which allows the initiation of translation was also prevented by MAPK and PI3K dominant negative constructs. Therefore, these results demonstrate that both PI3K and MAPK are necessary to observe the effect of leptin signaling that mediates protein synthesis in choriocarcinoma cells JEG-3. © 2010 Elsevier Inc. All rights reserved. Fil: Pérez-Pérez, Antonio. Universidad de Sevilla; España Fil: Gambino, Yésica Paola. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Maymo, Julieta Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Goberna, Raimundo. Universidad de Sevilla; España Fil: Fabiani, Fernando. Universidad de Sevilla; España Fil: Varone, Cecilia Laura. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Sánchez Margalet, Víctor. Universidad de Sevilla; España

Leptin, a 16-kDa protein mainly produced by adipose tissue, has been involved in the control of energy balance through its hypothalamic receptor. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in placenta, where it was found to be expressed. In the current study, we examined the effect of cAMP in the regulation of leptin expression in trophoblastic cells. We found that dibutyryl cAMP [(Bu) 2cAMP], a cAMP analog, showed an inducing effect on endogenous leptin expression in BeWo and JEG-3 cell lines when analyzed by Western blot analysis and quantitative RT-PCR. Maximal effect was achieved at 100 μM. Leptin promoter activity was also stimulated, evaluated by transient transfection with a reporter plasmid construction. Similar results were obtained with human term placental explants, thus indicating physiological relevance. Because cAMP usually exerts its actions through activation of protein kinase A (PKA) signaling, this pathway was analyzed. We found that cAMP response element-binding protein (CREB) phosphorylation was significantly increased with (Bu)2cAMP treatment. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor CREB caused a significant stimulation on leptin promoter activity. On the other hand, the cotransfection with a dominant negative mutant of the regulatory subunit of PKA inhibited leptin promoter activity. We determined that cAMP effect could be blocked by pharmacologic inhibition of PKA or adenylyl ciclase in BeWo cells and in human placental explants. Thereafter, we decided to investigate the involvement of the MAPK/ERK signaling pathway in the cAMP effect on leptin induction. We found that 50 μM PD98059, a MAPK kinase inhibitor, partially blocked leptin induction by cAMP, measured both by Western blot analysis and reporter transient transfection assay. Moreover, ERK 1/2 phosphorylation was significantly increased with (Bu)2cAMP treatment, and this effect was dose dependent. Finally, we observed that 50 μM PD98059 inhibited cAMP-dependent phosphorylation of CREB in placental explants. In summary, we provide some evidence suggesting that cAMP induces leptin expression in placental cells and that this effect seems to be mediated by a cross talk between PKA and MAPK signaling pathways. Copyright © 2010 by The Endocrine Society. Fil: Maymo, Julieta Lorena. 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; Argentina Fil: Pérez Pérez, Antonio. Universidad de Sevilla; España Fil: Dueñas, José L.. Hospital Universitario Virgen Macarena; España Fil: Calvo, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Sánchez Margalet, Víctor. Universidad de Sevilla; España Fil: Varone, Cecilia Laura. 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; Argentina

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 transla...

Maternal fever is common during pregnancy and has for many years been suspected to harm the developing fetus. Whether increased maternal temperature produces exaggerated apoptosis in trophoblast cells remains unclear. Since p53 is a critical regulator of apoptosis we hypothesized that increased temperature in placenta produces abnormal expression of proteins in the p53 pathway and finally caspase-3 activation. Moreover, leptin, produced by placenta, is known to promote the proliferation and survival of trophoblastic cells. Thus, we aimed to study the possible role of leptin preventing apoptosis triggered by high temperature, as well as the molecular mechanisms underlying this effect. Fresh placental tissue was collected from normal pregnancies. Explants of placental villi were exposed to 37°C, 40°C and 42°C during 3 h in the presence or absence of 10 nM leptin in DMEM-F12 medium. Western blotting and qRT-PCR was performed to analyze the expression of p53 and downstream effector, P53AIP1, Mdm2, p21, BAX and BCL-2 as well as the activated cleaved form of caspase-3 and the fragment of cytokeratin-18 (CK-18) cleaved at Asp396 (neoepitope M30). Phosphorylation of the Ser 46 residue on p53, the expression of P53AIP1, Mdm2, p21, as well as caspase-3 and CK-18 were significantly increased in explants at 40°C and 42°C. Conversely, these effects were significantly attenuated by leptin 10 nM at both 40°C and 42°C. The BCL2/BAX ratio was also significantly decreased in explants at 40°C and 42°C compared with explants incubated at 37°C, which was prevented by leptin stimulation. These data illustrate the potential role of leptin for reducing apoptosis in trophoblast explants, including trophoblastic cells, triggered by high temperature, by preventing the activation of p53 signaling. Fil: Perez Perez, Antonio. Universidad de Sevilla; España Fil: Toro, Ayelen Rayen. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Vilarino Garcia,Teresa. Universidad de Sevilla; España Fil: Guadix, Pilar. Universidad de Sevilla; España Fil: Maymo, Julieta Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Dueñas, Jose. Universidad de Sevilla; España Fil: Varone, Cecilia Laura. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Sanchez Margalet, Victor. Universidad de Sevilla; España

Insulin and leptin receptors are known to share signaling pathways, such as JAK2/STAT-3 (Janus kinase2/signal transduction and activator of transcription3), MAPK (Mitogen activated protein kinase), and PI3K (phosphoinositide 3-kinase). Both positive and negative cross-talk have been previously found in different cellular systems. Gestational diabetes (GDM) is a pathophysiological state with high circulating levels of both insulin and leptin. We have previously found that these 3 signaling pathways are activated in placenta from GDM patients to promote translation, involving the activation of leptin receptor. Now, we have tested the hypothesis that both leptin and insulin receptors might contribute to this activation in a positive way that may become negative when the system is overactivated. We studied the activation of leptin and insulin receptors in placenta from GDM and healthy pregnancies. We have also performed in vitro studies with insulin and leptin stimulation of trophoblast explants from healthy placenta. We have found that both leptin and insulin receptors are activated in placenta from GDM. In vitro stimulation of trophoblast explants with both leptin and insulin at submaximal doses (0.1 nM) potentiated the activation of signaling, whereas preincubation with maximal concentrations of insulin (10 nM) and further stimulation with leptin showed negative effect. Trophoblastic explants from GDM placenta, which presented high signaling levels, had a negative signaling effect when further incubated in vitro with leptin. In conclusion, insulin and leptin receptors have positive effects on signaling, contributing to high signaling levels in GDM placenta, but insulin and leptin have negative effects upon overstimulation. Fil: Perez Perez, Antonio. Hospital Virgen Macarena; España Fil: Guadix, P.. Hospital Virgen Macarena; España. Hospital Virgen Macarena; España Fil: Maymo, Julieta Lorena. 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; Argentina Fil: Dueñas, Jose Luis. Departamento de Bioquímica Médica y Biología Molecular; España Fil: Varone, Cecilia Laura. 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; Argentina Fil: Sanchez Margalet, M. IVI Sevilla; España Fil: Sanchez Margalet, V.. Hospital Virgen Macarena; España

Reproductive function is dependent on energy resources. The role of weight, body composition, fat distribution and the effect of diet have been largely investigated in experimental female animals as well as in women. Any alteration in diet and/or weight may induce abnormalities in timing of sexual maturation and fertility. However, the cellular mechanisms involved in the fine coordination of energy balance and reproduction are largely unknown. The brain and hypothalamic structures receive endocrine and/or metabolic signals providing information on the nutritional status and the degree of fat stores. Adipose tissue acts both as a store of energy and as an active endocrine organ, secreting a large number of biologically important molecules termed adipokines. Adipokines have been shown to be involved in regulation of the reproductive functions. The first adipokine described was leptin. Extensive research over the last 10 years has shown that leptin is not only an adipose tissue-derived messenger of the amount of energy stores to the brain, but also a crucial hormone/cytokine for a number of diverse physiological processes, such as inflammation, angiogenesis, hematopoiesis, immune function, and most importantly, reproduction. Leptin plays an integral role in the normal physiology of the reproductive system with complex interactions at all levels of the hypothalamic-pituitary gonadal (HPG) axis. In addition, leptin is also produced by placenta, where it plays an important autocrine function. Observational studies have demonstrated that states of leptin excess, deficiency, or resistance can be associated with abnormal reproductive function. This review focuses on the leptin action in female reproduction. Fil: Pérez Pérez, Antonio. Universidad de Sevilla; España Fil: Sánchez Jiménez, Flora. Universidad de Sevilla; España Fil: Maymo, Julieta Lorena. 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; Argentina Fil: Dueñas, José L.. Universidad de Sevilla; España Fil: Varone, Cecilia Laura. 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; Argentina Fil: Sánchez Margalet, Víctor. Universidad de Sevilla; España

Leptin, a peripheral signal synthetized by the adipocyte to regulate energy metabolism, can also be produced by placenta, where it may work as an autocrine hormone. We have previously demonstrated that leptin promotes proliferation and survival of trophoblastic cells. In the present work, we aimed to study the molecular mechanisms that mediate the survival effect of leptin in placenta. We used the human placenta choriocarcinoma BeWo and first trimester Swan-71 cell lines, as well as human placental explants. We tested the late phase of apoptosis, triggered by serum deprivation, by studying the activation of Caspase-3 and DNA fragmentation. Recombinant human leptin added to BeWo cell line and human placental explants, showed a decrease on Caspase-3 activation. These effects were dose dependent. Maximal effect was achieved at 250 ng leptin/ml. Moreover, inhibition of endogenous leptin expression with 2 µM of an antisense oligonucleotide, reversed Caspase-3 diminution. We also found that the cleavage of Poly [ADP-ribose] polymerase-1 (PARP-1) was diminished in the presence of leptin. We analyzed the presence of low DNA fragments, products from apoptotic DNA cleavage. Placental explants cultivated in the absence of serum in the culture media increased the apoptotic cleavage of DNA and this effect was prevented by the addition of 100 ng leptin/ml. Taken together these results reinforce the survival effect exerted by leptin on placental cells. To improve the understanding of leptin mechanism in regulating the process of apoptosis we determined the expression of different intermediaries in the apoptosis cascade. We found that under serum deprivation conditions, leptin increased the anti-apoptotic BCL-2 protein expression, while downregulated the pro-apoptotic BAX and BID proteins expression in Swan-71 cells and placental explants. In both models leptin augmented BCL-2/BAX ratio. Moreover we have demonstrated that p53, one of the key cell cycle-signaling proteins, is downregulated in the presence of leptin under serum deprivation. On the other hand, we determined that leptin reduced the phosphorylation of Ser-46 p53 that plays a pivotal role for apoptotic signaling by p53. Our data suggest that the observed anti-apoptotic effect of leptin in placenta is in part mediated by the p53 pathway. In conclusion, we provide evidence that demonstrates that leptin is a trophic factor for trophoblastic cells. Fil: Toro, Ayelen Rayen. 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; Argentina Fil: Maymo, Julieta Lorena. 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; Argentina Fil: Ibarbalz, Federico Matias. 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; Argentina Fil: Pérez Pérez, Antonio. Universidad de Sevilla; España Fil: Maskin, Bernardo. Hospital Nacional Profesor Alejandro Posadas; Argentina Fil: Faletti, Alicia Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires; Argentina Fil: Sánchez Margalet, Víctor. Universidad de Sevilla; España Fil: Varone, Cecilia Laura. 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; Argentina

Leptin, one of the adipokines that controls energy metabolism via the central nervous system, also has pleiotropic peripheral effects, acting as a proinflammatory cytokine. Leptin is also produced by trophoblastic cells in the placenta, where leptin seems to function as a trophic autocrine hormone. Leptin expression is regulated by various tissue-specific factors, such as insulin, in the adipocyte. However, the complete regulation of leptin production in the placenta is still poorly understood. That is why we investigated the regulation of leptin expression by insulin in JEG-3 trophoblastic cells and human placental explants from normal pregnancies. Western blot analysis and quantitative real time RT-PCR was performed to determine the leptin expression level after treatment of cells or trophoblast explants with different concentrations of insulin (0.1–100 nM). Leptin promoter activity was evaluated by transient transfection with a plasmid construct containing different promoter regions and the reporter luciferase gene. We found a stimulatory, dosedependent effect of insulin on endogenous leptin expression in human placental explants. Maximal effect was achieved at 10 nM insulin, and this effect can be totally prevented both by blocking phosphatidylinositol 3 kinase (PI3K) pathways and mitogen-activated protein kinase (MAPK). Moreover, insulin treatment significantly enhanced leptin promoter activity up to 40% in JEG-3 trophoblastic cells. Deletion analysis demonstrated that a minimal promoter region between � 1951 and � 1546 bp is necessary to achieve insulin effects. In conclusion, we provide evidence suggesting that insulin induces leptin expression in trophoblastic cells, enhancing the activity of leptin promoter region between � 1951 and � 1546 bp, via both PI3Kand MAPK-signaling pathways.

Placentas from gestational diabetes (GDM) suff er from structural and functional changes including overgrowth. That is why we aimed to study [ 3 H]-leucine incorporation into protein in addition to translation signaling in placenta from GDM. Thus, we investigated the expression of leptin and leptin receptor (LEPR), as well as the activation state of signaling proteins regulating protein synthesis, such as mTOR, S6 Kinase, EIF4E-BP1, EIF4E, and eEF2 by measuring protein phosphorylation by immunoblot. [ 3 H]-Leucine incorporation into protein also was determined in trophoblastic placenta explants from GDM and control pregnancy. We found that leptin and LEPR expression are increased in placentas from GDM and the translation machinery activity as well as [ 3 H]-leucine incorporation into protein were higher in placentas from GDM compared with placentas from control pregnancy. In conclusion, protein synthesis rate is increased in placenta from GDM patients, and this may be due, at least in part, by the activation of translation signaling. The increased expression of leptin and LEPR may contribute to these eff ects. These results may provide a possible mechanism for the previously observed increase in placenta growth in GDM. Fil: Pérez Pérez, A.. Universidad de Sevilla; España Fil: Maymo, Julieta Lorena. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Gambino, Yésica Paola. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Guadix, P.. Universidad de Sevilla; España Fil: Dueñas, J. L.. Universidad de Sevilla; España Fil: Varone, Cecilia Laura. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Sánchez Margalet, V.. Universidad de Sevilla; España

Leptin is a 16000 MW protein originally described as an adipocyte-derived signaling molecule for the central control of metabolism. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy. The leptin gene is expressed in placenta, where leptin promotes proliferation and survival of trophoblast cells. Study of the major signaling pathways known to be triggered by leptin receptor has revealed that leptin stimulates JAK/STAT, MAPK and PI3K pathways in placental cells. Leptin also exerts an antiapoptotic action in placenta and this effect is mediated by the MAPK pathway. Moreover, leptin stimulates protein synthesis by activating the translational machinery via both PI3K and MAPK pathways. Expression of leptin in placenta is highly regulated, suggesting that certain key pregnancy molecules participate in such regulation. An important hormone in reproduction, hCG, induces leptin expression in trophoblast cells and this effect involves the MAPK signal transduction pathway. Moreover, the cyclic nucleotide cAMP, which has profound actions upon human trophoblast function, also stimulates leptin expression and this effect seems to be mediated by crosstalk between the PKA and MAPK signaling pathways. Estrogens play a central role in reproduction. 17b-estradiol upregulates leptin expression in placental cells through genomic and non-genomic actions, probably via crosstalk between estrogen receptor-a and the MAPK and PI3K signal transduction pathways. Taken together these findings give a better understanding of the function of leptin and the regulatory mechanisms of leptin expression in human placental trophoblast and further support the importance of leptin in the biology of reproduction. Fil: Maymo, Julieta Lorena. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Pérez Pérez, Antonio. Universidad de Sevilla; España Fil: Gambino, Yésica Paola. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Calvo, Juan Carlos. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Sánchez Margalet, Victor . Universidad de Sevilla; España Fil: Varone, Cecilia Laura. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina

Leptin, the 16,000 molecular weight 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 which it was found to be expressed. In the present work, we have found that recombinant human chorionic gonadotropin (hCG) added to BeWo choriocarcinoma cell line showed a stimulatory effect on endogenous leptin expression, when analyzed by Western blot. This effect was time and dose dependent. Maximal effect was achieved at hCG 100 IU/ml. Moreover, hCG treatment enhanced leptin promoter activity up to 12.9 times, evaluated by transient transfection with a plasmid construction containing different promoter regions and the reporter gene luciferase. This effect was dose dependent and evidenced with all the promoter regions analyzed, regardless of length. Similar results were obtained with placental explants, thus indicating physiological relevance. Because hCG signal transduction usually involves cAMP signaling, this pathway was analyzed. Contrarily, we found that dibutyryl cAMP counteracted hCG effect on leptin expression. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor cAMP response element binding protein repressed leptin expression. Thereafter we determined that hCG effect could be partially blocked by pharmacologic inhibition of MAPK pathway with 50 microM PD98059 but not by the inhibition of the phosphatidylinositol 3-kinase pathway with 0.1 microm wortmannin. Moreover, hCG treatment promoted MAPK kinase and ERK1/ERK2 phosphorylation in placental cells. Finally, cotransfection with a dominant-negative mutant of MAPK blocked the hCG-mediated activation of leptin expression. In conclusion, we provide some evidence suggesting that hCG induces leptin expression in trophoblastic cells probably involving the MAPK signal transduction pathway. Fil: Maymo, Julieta Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina Fil: Pérez Pérez, Antonio. Universidad de Sevilla; España Fil: Sánchez Margalet, Víctor. Universidad de Sevilla; España Fil: Dueñas, José L.. Sistema Sanitario Público de Andalucía. Hospital Universitario Virgen Macarena; España Fil: Calvo, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina Fil: Varone, Cecilia Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina

Leptin (Ob), the peripheral signal produced by the adipocyte to regulate energy metabolism, can also be produced by placenta, where it may work as an autocrine hormone. 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 that mediate the trophic effect of leptin in placenta, by using the human placenta choriocarcinoma JEG-3 cell line, as well as trophoblastic cells from human placenta. We have assayed the early phase of apoptosis, triggered by serum deprivation, by using Annexin V-propidium iodide (PI) labeling and flow cytometric analysis, as well as the late phase of apoptosis by studying the activation of caspase-3. We have studied the major signalling pathways known to be triggered by the leptin receptor, and we have investigated the relative importance of these pathways in the effect of leptin by using pharmacological inhibitors. We have found that leptin stimulates Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway by promoting JAK-2 and STAT-3 tyrosine phosphorylation. We have also demonstrated the activation of mitogen-activated protein kinase (MAPK) pathway by studying phosphorylation of extracellular-signal regulated kinase (Erk) kinase (MEK) and Erk1/2. PI3K pathway is also triggered by leptin stimulation as assessed by the study of protein kinase B (PKB) phosphorylation. These signaling pathways were confirmed in trophoblastic cells obtained from placenta of healthy donors. The effect of leptin on JEG-3 survival was completely reversed by blocking Erk1/2 activation employing the MEK inhibitor PD98059, whereas it was not affected by PI3K inhibition using wortmannin. These data suggest that the leptin antiapoptotic effect in placenta is mediated by the MAPK pathway. Fil: Pérez Pérez, Antonio. Universidad de Sevilla; España Fil: Maymo, Julieta Lorena. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Dueñas, José L.. Universidad de Sevilla; España Fil: Goberna, Raimundo. Universidad de Sevilla; España Fil: Calvo. Juan Carlos. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Varone, Cecilia Laura. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Sánchez Margalet, Victor. Universidad de Sevilla; España

Pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in the placenta, where it works as an autocrine hormone. In this work, we demonstrated that human chorionic gonadotropin (hCG) added to JEG-3 cell line or to placental explants induces endogenous leptin expression. We also found that hCG increased cAMP intracellular levels in BeWo cells in a dose-dependent manner, stimulated cAMP response element (CRE) activity and the cotransfection with an expression plasmid of a dominant negative mutant of CREB caused a significant inhibition of hCG stimulation of leptin promoter activity. These results demonstrate that hCG indeed activates cAMP/PKA pathway, and that this pathway is involved in leptin expression. Nevertheless, we found leptin induction by hCG is dependent on cAMP levels. Treatment with (Bu)2cAMP in combination with low and non stimulatory hCG concentrations led to an increase in leptin expression, whereas stimulatory concentrations showed the opposite effect. We found that specific PKA inhibition by H89 caused a significant increase of hCG leptin induction, suggesting that probably high cAMP levels might inhibit hCG effect. It was found that hCG enhancement of leptin mRNA expression involved the MAPK pathway. In this work, we demonstrated that hCG leptin induction through the MAPK signaling pathway is inhibited by PKA. We observed that ERK1/2 phosphorylation increased when hCG treatment was combined with H89. In view of these results, the involvement of the alternative cAMP/Epac signaling pathway was studied. We observed that a cAMP analogue that specifically activates Epac (CPT-OMe) stimulated leptin expression by hCG. In addition, the overexpression of Epac and Rap1 proteins increased leptin promoter activity and enhanced hCG. In conclusion, we provide evidence suggesting that hCG induction of leptin gene expression in placenta is mediated not only by activation of the MAPK signaling pathway but also by the alternative cAMP/Epac signaling pathway. © 2012 Maymó et al. 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.