José Luis Dueñas

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 E2 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 E2 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.

Leptin prevents apoptosis of trophoblastic cells by activation of MAPK pathway

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.

Up-Regulation of Placental Leptin by Human Chorionic Gonadotropin

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.

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.

Intrauterine contraception in nulligravid vs parous women

The performance of MLCu-250 IUDs inserted in 525 nulligravid women was studied for four years and is compared to the results obtained in the same period from 2770 parous women using TCu-200, 7Cu-200 and Nova-T IUDs. In every case the choice of the particular IUD model took into account hysterometry and diameter of the inner cervical orifice in order to aim at the highest theoretical efficacy. The MLCu-250 in nulligravid women produced failure and expulsion rates below those of the other models in parous users; no difference was observed in the case of removal because of bleeding or pain. Finally, there was no case of subsequent infertility or pelvic inflammatory disease (PID) recorded among nulliparae. These results suggest that an adequate choice of the IUD model for nulliparous women with low risk of PID gives as reliable results as in multiparous women.

Role of leptin in female reproduction

Abstract 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.

Regulation of placental leptin expression by cyclic adenosine 5'-monophosphate involves cross talk between protein kinase A and mitogen-activated protein kinase signaling pathways.

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)(2)cAMP], 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 microM. 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)(2)cAMP 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 microm 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)(2)cAMP treatment, and this effect was dose dependent. Finally, we observed that 50 microm 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.

Trends in the use of contraceptive methods and voluntary interruption of pregnancy in the Spanish population during 1997-2007.

BACKGROUND This study was designed to acquire information about the use of contraceptive methods in order to reduce the number of elective abortions. STUDY DESIGN Since 1997, representative samples of Spanish women of childbearing potential (15-49 years) have been surveyed by the Daphne Team every 2 years to gather data of contraceptive methods used. RESULTS During the study period, 1997 to 2007, the overall use of contraceptive methods increased from 49.1% to 79.9%. The most commonly used method was the condom (an increase from 21% to 38.8%), followed by the pill (an increase from 14.2% to 20.3%). Female sterilization and IUDs decreased slightly and were used by less than 5% of women in 2007. The elective abortion rate increased from 5.52 to 11.49 per 1000 women. CONCLUSIONS The factors responsible for the increased rate of elective abortion need further investigation.

Use of contraceptive methods in Spain: results of a national survey.

We conducted a personal interview of 2136 Spanish women aged 15 to 49 years with the aim of determining the contraceptive attitudes of this population. A stratified random sampling was performed to select the women who were to be interviewed, and this sampling design ensured adequate representation of the sample in Spain. At the time of the survey, only 1296 of the 2136 women used some type of contraceptive method (60.6%); the most commonly used method was the condom. The major reason given by the 840 women not using contraceptive methods was the absence of sexual intercourse. Nearly 25% of the Spanish women aged 15-49 years reported that they have no sexual relations.

Activated Translation Signaling in Placenta from Pregnant Women with Gestational Diabetes Mellitus: Possible Role of Leptin

Placentas from gestational diabetes (GDM) suffer from structural and functional changes including overgrowth. That is why we aimed to study [³H]-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. [³H]-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 [³H]-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 effects. These results may provide a possible mechanism for the previously observed increase in placenta growth in GDM.