Leptin regulates survival and invasion of trophoblastic cells

En los mamíferos el crecimiento y la supervivencia del feto durante sudesarrollo dependen exclusivamente de la placenta, un órgano altamenteespecializado que produce un gran número de factores de crecimiento. Leptina, unahormona producida principalmente por el tejido adiposo que se asocia con la saciedady el balance energético, presenta un papel importante en reproducción. Se haevidenciado la expresión de leptina y de sus receptores en placenta y se hademostrado que durante la gestación tiene efectos sobre el crecimiento, laangiogénesis y la inmunomodulación, afectando tanto funciones maternas comofetales. Sin embargo, muchos de los mecanismos de acción de leptina sobre laimplantación y el crecimiento embrionario son aún desconocidos. El presente trabajo ha sido desarrollado con el objetivo de investigar la acciónde leptina sobre la supervivencia e invasión de células placentarias. Se utilizaron comomodelos la línea celular Swan-71 y explantos de placenta humana a término bajodistintas condiciones de estrés celular y daño como el hambreado de factores decrecimiento, la radiación UV, la hipertermia y la acidificación del medio de cultivo. Determinamos que leptina promueve la proliferación de células Swan-71, aumentandola expresión de ciclina D1, Ki-67 y p21. Asimismo demostramos que leptina disminuyela muerte celular por apoptosis evidenciada por la disminución de distintos parámetroscomo la activación de caspasa-3, el clivado de PARP-1 y la fragmentación del ADN. También hallamos que leptina modifica los niveles de intermediarios mitocondrialesaumentando la relación Bcl-2/Bax y disminuyendo la expresión de Bid. Al profundizaren el mecanismo involucrado en el efecto de leptina sobre la sobrevida de célulastrofoblásticas, evidenciamos que leptina disminuye los niveles de expresión, actividady fosforilación en serina 46 del factor de transcripción p53, pieza clave en lamodulación del ciclo celular y apoptosis. Leptina además aumenta los niveles de laproteína Mdm-2, reguladora principal de la vida media de p53. Más aún, por ensayoscon cicloheximida demostramos que la vida media de p53 disminuye en presencia deleptina. Por otro lado mediante el uso de inhibidores farmacológicos y de ensayos detransfección transitoria, observamos que las vías de MAPK (ERK 1/2) y PI3K/Aktmedian los efectos de leptina sobre p53. En último lugar, demostramos que leptinapromueve la migración e invasión trofoblástica. Analizamos la expresión de proteínasinvolucradas en la adhesión celular y observamos que en presencia de leptinadisminuye la expresión de E-cadherina y aumentan los niveles de Integrina β1. Probamos también que leptina modula la expresión y actividad de metaloproteasas,favoreciendo la invasión celular. Los resultados obtenidos refuerzan la noción de leptina como una citoquinaplacentaria involucrada en la regulación de procesos de suma relevancia durante lagestación como la proliferación, apoptosis e invasión, sustentando su importancia enla biología de la reproducción.In mammals the growth and survival of the fetus during its developmentdepend exclusively on the placenta, a highly specialized organ that produces severalgrowth factors. Leptin, a hormone produced mainly by adipose tissue that is associatedwith satiety and energy balance, has an important role in reproduction. It has beenfound that leptin and its receptor are expressed in placenta and it has been shown thatduring pregnancy leptin modulates growth, angiogenesis and immunomodulation,affecting both maternal and fetal functions. However, many of the mechanisms ofleptin action on embryo implantation and growth are still unknown. This work has been developed in order to investigate leptin action on survivaland invasion of placental cells. Swan-71 cell line and human placental explants wereused as models and were exposed under different conditions of cellular stress anddamage as starved of growth factors, UV irradiation, hyperthermia and acidification ofthe culture medium. We found that leptin promotes proliferation of Swan-71 cells byincreasing cyclin D1, Ki-67 and p21 expressions. We demonstrated that leptindecreases cell death by apoptosis as evidenced by reduction of various parameterssuch as the activation of caspase-3, cleaved PARP-1 and DNA fragmentation. We alsofound that leptin influences mitochondrial intermediates by augmenting Bcl-2/Baxratio and decreasing Bid expression. By delving into the mechanisms involved in leptineffect on the survival of trophoblast cells, we showed that leptin decreased expression,activity and phosphorylation of serine 46 of p53 transcription factor, a key element inthe modulation of cell cycle and apoptosis. Leptin also increases the levels of Mdm-2,the main regulator of p53 protein half-life. Moreover, using cycloheximide we showedthat p53 half-life decreases in the presence of leptin. Furthermore by usingpharmacological inhibitors and transient transfection assays, we observed that MAPK (ERK 1/2) and PI3K/Akt pathways mediate leptin effects on p53. Moreover, we showedthat leptin promotes trophoblastic migration and invasion. We analyzed the expressionof proteins involved in cell adhesion and found that leptin decreases E-cadherinexpression and increased β1 integrin levels. We also demonstrated that leptinmodulates the expression and activity of metalloproteinases, promoting cell invasion. In summary, our results reinforce the notion of leptin as a placental cytokineinvolved in the regulation of processes extremely important during pregnancy asproliferation, apoptosis and invasion, supporting the importance of leptin in thebiology of reproduction.Fil: Toro, Ayelén Rayen. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Leptin regulates survival and invasion of trophoblastic cells

En los mamíferos el crecimiento y la supervivencia del feto durante sudesarrollo dependen exclusivamente de la placenta, un órgano altamenteespecializado que produce un gran número de factores de crecimiento. Leptina, unahormona producida principalmente por el tejido adiposo que se asocia con la saciedady el balance energético, presenta un papel importante en reproducción. Se haevidenciado la expresión de leptina y de sus receptores en placenta y se hademostrado que durante la gestación tiene efectos sobre el crecimiento, laangiogénesis y la inmunomodulación, afectando tanto funciones maternas comofetales. Sin embargo, muchos de los mecanismos de acción de leptina sobre laimplantación y el crecimiento embrionario son aún desconocidos. El presente trabajo ha sido desarrollado con el objetivo de investigar la acciónde leptina sobre la supervivencia e invasión de células placentarias. Se utilizaron comomodelos la línea celular Swan-71 y explantos de placenta humana a término bajodistintas condiciones de estrés celular y daño como el hambreado de factores decrecimiento, la radiación UV, la hipertermia y la acidificación del medio de cultivo. Determinamos que leptina promueve la proliferación de células Swan-71, aumentandola expresión de ciclina D1, Ki-67 y p21. Asimismo demostramos que leptina disminuyela muerte celular por apoptosis evidenciada por la disminución de distintos parámetroscomo la activación de caspasa-3, el clivado de PARP-1 y la fragmentación del ADN. También hallamos que leptina modifica los niveles de intermediarios mitocondrialesaumentando la relación Bcl-2/Bax y disminuyendo la expresión de Bid. Al profundizaren el mecanismo involucrado en el efecto de leptina sobre la sobrevida de célulastrofoblásticas, evidenciamos que leptina disminuye los niveles de expresión, actividady fosforilación en serina 46 del factor de transcripción p53, pieza clave en lamodulación del ciclo celular y apoptosis. Leptina además aumenta los niveles de laproteína Mdm-2, reguladora principal de la vida media de p53. Más aún, por ensayoscon cicloheximida demostramos que la vida media de p53 disminuye en presencia deleptina. Por otro lado mediante el uso de inhibidores farmacológicos y de ensayos detransfección transitoria, observamos que las vías de MAPK (ERK 1/2) y PI3K/Aktmedian los efectos de leptina sobre p53. En último lugar, demostramos que leptinapromueve la migración e invasión trofoblástica. Analizamos la expresión de proteínasinvolucradas en la adhesión celular y observamos que en presencia de leptinadisminuye la expresión de E-cadherina y aumentan los niveles de Integrina β1. Probamos también que leptina modula la expresión y actividad de metaloproteasas,favoreciendo la invasión celular. Los resultados obtenidos refuerzan la noción de leptina como una citoquinaplacentaria involucrada en la regulación de procesos de suma relevancia durante lagestación como la proliferación, apoptosis e invasión, sustentando su importancia enla biología de la reproducción.In mammals the growth and survival of the fetus during its developmentdepend exclusively on the placenta, a highly specialized organ that produces severalgrowth factors. Leptin, a hormone produced mainly by adipose tissue that is associatedwith satiety and energy balance, has an important role in reproduction. It has beenfound that leptin and its receptor are expressed in placenta and it has been shown thatduring pregnancy leptin modulates growth, angiogenesis and immunomodulation,affecting both maternal and fetal functions. However, many of the mechanisms ofleptin action on embryo implantation and growth are still unknown. This work has been developed in order to investigate leptin action on survivaland invasion of placental cells. Swan-71 cell line and human placental explants wereused as models and were exposed under different conditions of cellular stress anddamage as starved of growth factors, UV irradiation, hyperthermia and acidification ofthe culture medium. We found that leptin promotes proliferation of Swan-71 cells byincreasing cyclin D1, Ki-67 and p21 expressions. We demonstrated that leptindecreases cell death by apoptosis as evidenced by reduction of various parameterssuch as the activation of caspase-3, cleaved PARP-1 and DNA fragmentation. We alsofound that leptin influences mitochondrial intermediates by augmenting Bcl-2/Baxratio and decreasing Bid expression. By delving into the mechanisms involved in leptineffect on the survival of trophoblast cells, we showed that leptin decreased expression,activity and phosphorylation of serine 46 of p53 transcription factor, a key element inthe modulation of cell cycle and apoptosis. Leptin also increases the levels of Mdm-2,the main regulator of p53 protein half-life. Moreover, using cycloheximide we showedthat p53 half-life decreases in the presence of leptin. Furthermore by usingpharmacological inhibitors and transient transfection assays, we observed that MAPK (ERK 1/2) and PI3K/Akt pathways mediate leptin effects on p53. Moreover, we showedthat leptin promotes trophoblastic migration and invasion. We analyzed the expressionof proteins involved in cell adhesion and found that leptin decreases E-cadherinexpression and increased β1 integrin levels. We also demonstrated that leptinmodulates the expression and activity of metalloproteinases, promoting cell invasion. In summary, our results reinforce the notion of leptin as a placental cytokineinvolved in the regulation of processes extremely important during pregnancy asproliferation, apoptosis and invasion, supporting the importance of leptin in thebiology of reproduction.Fil: Toro, Ayelén Rayen. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Leptin is an anti-apoptotic effector in placental cells involving p53 downregulation.

Journal Article; Research Support, Non-U.S. Gov't;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.Grant Support: ART and JLM are supported by a CONICET fellowship. APP is a research fellow supported by the Instituto de Salud Carlos III (CM07/00025). This project was supported by the Universidad de Buenos Aires (UBACYT), the ANPCyT (PICT 2008-0425), the CONICET (PIP 2010-247), the Fundacio´n Florencio Fiorini, Buenos Aires, Argentina and the Instituto de Salud Carlos III (PS09/00119), Spain.Ye

Leptin diminishes BID level in placenta.

<p>A) Swan-71 cells (1×10⁶ cells) were plated in DMEM-F12 media 10% FBS. After 24 h, cells were incubated during 72 h with increasing doses of leptin in DMEM-F12 0% FBS. Cell extracts were prepared as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099187#s2" target="_blank">Materials and Methods</a> and proteins were separated on SDS-PAGE gels. B) Placental explants were processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099187#s2" target="_blank">Material and Methods</a> and incubated in DMEM-F12 0% FBS media supplemented with increasing leptin doses during 24 h. Placental extracts were prepared and proteins were separated on SDS-PAGE gels. In both cases (A and B) BID cleaved fragment was determined by Western blot analysis. DMEM-F12 10% FBS was used as control. Molecular weights were estimated using standard protein markers. Molecular mass (kDa) is indicated at the right of the blot. Loading controls were performed by immunoblotting the same membranes with anti-α-tubulin or anti-β-actin. Bands densitometry is shown in lower panels. Results are expressed as mean ± SD for three independent experiments. Statistical analyzes were performed by ANOVA. Asterisks indicate significant differences from the control according to Bonferroni's multiple comparison <i>post hoc</i> test, relative to FBS 10% (#) or FBS 0% (*). ## p<0.01, * p<0.05, ** p<0.01</p

Leptin enhances BCL-2/BAX relationship in placental cells.

<p>A) Swan-71 cells (1×10⁶ cells) were plated in DMEM-F12 media in the absence of serum and incubated during 72 h with different doses of leptin. DMEM-F12 10% FBS was used as a control. Cell extracts were prepared as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099187#s2" target="_blank">Materials and Methods</a>. Proteins were separated on SDS-PAGE gels and BCL-2 and BAX expression was determined by Western blot analysis. Molecular weights were estimated using standard protein markers. Molecular mass (kDa) is indicated at the right of the blot. Loading controls were performed by immunoblotting the same membranes with anti-α-tubulin. Bands densitometry is shown in lower panels, results are expressed as mean ± SD for three independent experiments. B) Leptin increased BCL-2/BAX relationship. C) BeWo cells were transiently transfected with a plasmid containing a section of BAX promoter (pBax-Luc). After transfection, cells were incubated for 72 h in DMEM-F12 and treated with increasing leptin doses. Cell extracts were prepared as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099187#s2" target="_blank">Materials and Methods</a> and Luciferase activity was normalized to β-galactosidase activity. Activity obtained in the absence of leptin and pBax-Luc was set as control. Statistical analyzes were performed by ANOVA. Asterisks indicate significant differences from the control according to Bonferroni's multiple comparison <i>post hoc</i> test, relative to FBS 10% (#) or FBS 0% (*). ## p<0.01, * p<0.05, *** p<0.001.</p

Leptin enhances BCL-2/BAX relationship in human placental explants.

<p>A) Placental explants were processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099187#s2" target="_blank">Materials and Methods</a> and incubated during 24 h in DMEM-F12 media supplemented with increasing leptin doses. DMEM-F12 10% FBS was used as control. Placental extracts were prepared and proteins were separated on SDS-PAGE gels. BCL-2 and BAX expression were determined by Western blot analysis as indicated in the Figure. Molecular weights were estimated using standard protein markers. Molecular mass (kDa) is indicated at the right of the blot. Loading controls were performed by immunoblotting the same membranes with anti-α-tubulin. Bands densitometry is shown in lower panels, results are expressed as mean ± SD for three independent experiments. B) BCL-2/BAX relationship is shown. Statistical analyzes were performed by ANOVA. Asterisks indicate significant differences from the control according to Bonferroni's multiple comparison <i>post hoc</i> test, relative to FBS 0% (*). * p<0.05, ** p<0.01</p

Leptin reduces p53 levels in placenta.

<p>A) Swan-71 cells (1×10⁶ cells) were plated in DMEM-F12 media in the absence of serum and incubated during 72 h with different leptin concentrations. Cell extracts were prepared as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099187#s2" target="_blank">Materials and Methods</a> and proteins were separated on SDS-PAGE gels. B) Placental explants were processed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099187#s2" target="_blank">Material and Methods</a> and incubated in DMEM-F12 media supplemented with increasing leptin doses during 24 h. Placental extracts were prepared and proteins were separated on SDS-PAGE gels. In both cases (A and B) p53 was determined by Western blot analysis. DMEM-F12 10% FBS was used as a control. Molecular weights were estimated using standard protein markers. Molecular mass (kDa) is indicated at the right of the blot. Loading controls were performed by immunoblotting the same membranes with anti-α-GAPDH. Bands densitometry is shown in lower panels. Results are expressed as mean ± SD for three independent experiments. C) p53 expression in human placental explants was determined by <i>q</i>RT-PCR. RNA was extracted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099187#s2" target="_blank">Materials and Methods</a>. Statistical analyzes were performed by ANOVA. Asterisks indicate significant differences from the control according to Bonferroni's multiple comparison <i>post hoc</i> test, relative to FBS 10% (#) or FBS 0% (*). # p<0.05, ## p<0.01, * p<0.05, ** p<0.01.</p