Progesterone receptors in the human placenta: Expression, signalling characteristics and functional relevance

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.The human placenta is a transient life sustaining organ which is responsible for mediating all the physiological exchanges between the mother and the fetus. The steroid hormone progesterone, often referred to as the hormone of pregnancy, is critical for the establishment and for maintaining the pregnancy. During the gestation period the human placenta produces progesterone which via interacting with the progesterone receptors exerts its many effects. Specific intracellular progesterone receptors (PRs) have been reported to mediate the genomic signalling of progesterone whereas recently two novel receptor families which are phylogenetically distinct to the nuclear receptor superfamily have been characterised, and shown to mediate progesterone’s non genomic actions. These are the multiple membrane progestin receptors (mPRα, mPRβ, and mPRγ) and progesterone membrane receptor component 1 (PGMRC-1). The rapid progesterone actions mediated via these non-classical progesterone receptors have received attention with main focus on their reproductive functions. Our aim is to elucidate the expression of the receptors in the human placenta, further understand the signalling pathways via which progesterone mediates its effects and lastly examine the functional relevance of these receptors in this organ. Choriocarcinoma cell lines are used frequently as placental models for investigations of steroid hormone actions, but until now little is known about the expression of progesterone receptors (PRs) in these cell lines. Quantitative RT-PCR revealed that in fully syncytialized BeWo cells (treated with 50 µM forskolin for 72 h) there was a significant down-regulation of mPRα and up-regulation of mPRβ and of the PGRMC1 when compared with non-syncytialized BeWo cells. Expression of all the mPR and PGRMC1 mRNAs was significantly lower in JEG-3 cells compared to non-syncytialized BeWo cells. Expression of PR-B was unaltered between the two BeWo states but was significantly higher in JEG-3 cells. Immunofluorescence analysis revealed that mPR proteins are differentially expressed in these choriocarcinoma cell lines as well as in the human placenta. The functionality of mPRs was investigated in vitro, using BeWo and JEG-3 cells that were treated with Org OD-02 (a specific mPR agonist), progesterone (P4) and R5020 (a specific nuclear PR agonist) in the presence or absence of the pro-inflammatory cytokine inteleukin-1β (IL-1β) at a concentration of 10ng/μl. The effect was more exacerbated in JEG-3 cells, where IL-1β induced 40% cell death when compared to BeWo cells that reached a modest but significant 15% cell death. When JEG-3 cells were treated with IL-1β and progesterone, there was a significant decrease in cell death at concentrations of 100nM and 1000nM. When cells were treated with 1000nM progesterone, IL-1β’s effect was completely abolished. Progesterone was also able to induce phosphorylation of ERK1/2 in these cells. Pretreatment of JEG-3 cells with a specific MAPK inhibitor (UO126) inhibited substantially the progesterone’s proliferative effect. Moreover, using the specific mPR agonist Org OD 02-0, we have shown that that the progestin antagonism of apoptotic effects of IL-1β on BeWo cells is mediated through mPRs. Quantitative PCR in clinical samples revealed a 2.8 fold decrease of mPRβ in labouring comparing to non-labouring tissues and 4.6 fold higher levels of mPRγ in preterm mPRγ compared to term placentas. The ratio of mPRα to PR-B was increased in term compared to preterm samples, whereas it was decreased in labour compared to non-labour placentas. There was also a high correlation between mPRα and PGRMC1 expression irrespective of pathologies. This study addressed many fundamental questions regarding how progestins exert their effect at placental level. It is evident that there is a higher order of complexity and changes in the ratios of placental progesterone receptors rather than individual fluctuations might affect subsequent signalling events

Elections and the Cyprus Problem: change or continuity?

Alexandros Zachariades, a research student at LSE, gives us a briefing on the upcoming presidential elections in Cyprus

All hands on deck: the crisis in the Eastern Mediterranean and the need for US leadership

Crisis has returned to the Eastern Mediterranean where Greek and Turkish economic interests, legal claims, and armed forces are squaring off. In this Strategic Update, Alexandros Zachariades explores how the two NATO powers arrived at the current confrontation, how the contest intersects with issues ranging from China to Libya, and how the international community can resolve the tension

Placental DEPTOR as a stress sensor during pregnancy

The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited. Copyright @ 2012 Portland Press. The article has been made available through the Brunel Open Access Publishing Fund.DEPTOR [DEP-domain-containing and mTOR (mammalian target of rapamycin)-interacting protein] is a modulator of mTOR signalling that binds to mTORC (mTOR complex) 1 and mTORC2. However, to date, the precise functions of DEPTOR are not fully elucidated, particularly in reproductive tissues where mTOR acts as a placental nutrient sensor. Pregnancy is associated with major physiological and psychosocial changes and adaptation to these changes is crucial for normal fetal development. In the present study, we tested the hypothesis that maternal stress can affect mTOR signalling at term, and, as a result, influence placental growth. We first investigated the expression of DEPTOR, mTOR, rictor (rapamycin-insensitive companion of mTOR) and raptor (regulatory associated protein of mTOR) from human placentas (n=23) using Q-PCR (quantitative PCR), and correlated these data to days of pregnancy and maternal stress, as well as placental and fetal weight. Maternal and fetal cortisol levels were also measured. JEG-3 and BeWo cells, used as placental in vitro models, were treated with cortisol and DEPTOR expression was assessed using Q-PCR. DEPTOR appears to be the predominant transcript in the human placenta compared with mTOR, rictor and raptor in both term (n=13) and preterm (n=10) placentas as assessed by Q-PCR. There was a significantly lower level only of log-DEPTOR gene expression in the high stress group (-1.34) than in the low stress group (0.07; t₂₀=2.41, P=0.026). Interestingly, mothers with high stress had significantly elevated levels of cortisol (8555 pg/ml) compared with those with low stress (4900 pg/ml). We then tested the hypothesis that cortisol can directly affect DEPTOR expression. When BeWo cells were treated with cortisol 10, 100 and 1000 nM, the expression of DEPTOR was significantly down-regulated by 50, 41 and 39% (all P<0.05) respectively when compared with basal levels. Treatment of JEG-3 cells with cortisol, led to a significant decrease of DEPTOR expression at 100 nM (39%, P<0.05) and at 1000 nM (73%, P<0.01). These novel findings are indicative of a higher order of complexity of DEPTOR signalling in the human placenta that is affected by maternal stress, which could affect pregnancy outcome

Elucidating the role of DEPTOR in Alzheimer's disease

This article has been made available through the Brunel Open Access Publishing Fund.The mammalian or mechanistic target of rapamycin (mTOR) is a Ser/Thr protein kinase that, in response to nutrient stimulation, regulates cellular growth, proliferation, survival, protein synthesis and gene transcription. It has also been implicated in Alzheimer's disease (AD) with neuronal cells and hippocampal slices of AD transgenic mice experiencing dysregulated mTOR and synaptic plasticity in response to treatment with the toxic amyloid β (Aβ1-42) peptide, which has been implicated in AD. DEP domain-containing mTOR-interacting protein (DEPTOR) is a protein which can bind to mTOR and cause its inhibition, and functions as a regulatory protein of mTOR to control its activity. The inhibition of mTOR has been shown to have a neuroprotective effect; in an animal model, it was shown to protect against Aβ-induced neurotoxicity. In the present study, to investigate to role of DEPTOR in a model of AD, we neuronally differentiated the SH-SY5Y cell line and examined the effects of treatment with an Aβ42 peptide, thus mimicking plaque formation. This resulted in a significant increase in mTOR and a significant decrease in DEPTOR expression compared to the unstimulated controls. Moreover, to the best of our knowledge, we demonstrate for the first time a reduction in the protein level of DEPTOR in the precentral gyrus, postcentral gyrus and occipital lobe of a brain with AD compared to a normal control, as well as a significant reduction in DEPTOR expression in samples from late-onset AD (LOAD) compared to early-onset familial AD (EOFAD). The reduction in DEPTOR expression in cases of AD compared to healthy controls can lead to an augmentation of mTOR signalling, leading to Aβ accumulation, which in turn leads to a further reduction in DEPTOR expression. This results in the accumulation of amyloid plaque, shifting the balance from neuroprotection to neurodegeneration

Crucial cross-talk of interleukin-1β and progesterone in human choriocarcinoma

Copyright @ 2012 Spandidos Publications Ltd. This article can be accessed from the links below.This article has been made available through the Brunel Open Access Publishing Fund.Choriocarcinoma is a highly malignant epithelial tumour that is most often associated with hydatidiform mole and presents the most common emergency medical problem in the management of trophoblast disease. We hypothesise that the hormones/cytokines present within the tumour microenvironment play key roles in the development of choriocarcinoma. In this study we assessed the effects of interleukin-1β (IL-1β) on cell death in the presence or absence of the sex hormone progesterone using two choriocarcinoma cell lines (BeWo and JEG-3) as in vitro experimental models. Although IL-1β induced cell death in both cell lines, the effect was more pronounced in JEG-3 cells, where cell death reached 40% compared to 15% in BeWo cells. Cell death of JEG-3 cells in response to IL-1β was significantly decreased by co-treatment with 100 nM and 1000 nM progesterone and completely abolished at a progesterone concentration of 1000 nM. Progesterone was also able to induce phosphorylation of ERK1/2 in these cells. Pretreatment of JEG-3 cells with a specific MAPK inhibitor (UO126) inhibited progesterone's inhibitory effect on cell death. Collectively, these data provide evidence of cross-talk between progesterone and IL-1β in this aggressive and poorly understood tumour that involves activation of a MAPK pathway and involvement of numerous progesterone receptors.This research was funded by a National Institutes of Health Grant ESO12961. This article is made available through the Brunel Open Access Publishing Fund

Immune system function, stress, exercise and nutrition profile can affect pregnancy outcome: Lessons from a Mediterranean cohort

This article is made available through the Brunel Open Access Publishing Fund.Pregnancy is associated with major physiological and future psychosocial changes, and maternal adaptation to these changes is crucial for normal foetal development. Psychological stress in pregnancy predicts an earlier birth and lower birth weight. Pregnancy-specific stress contributes directly to preterm delivery. The importance of nutrition and exercise during pregnancy with regard to pregnancy outcome has long been acknowledged. This importance has only been further emphasized by the recent changes in food quality and availability, lifestyle changes and a new understanding of foetal programming's effects on adult outcomes. We hypothesised that for a successful pregnancy certain events at a nutritional, immune, psycho-emotional and genetic level should be tightly linked. Therefore, in this study we followed an ‘integrative’ approach to investigate how maternal stress, nutrition, pregnancy planning and exercise influence pregnancy outcome. A key finding of our study is that there was a significant reduction in the intake of alcohol, caffeine-containing and sugary drinks during pregnancy. However, passive smoking in the household remained unchanged. In terms of immune profile, a significant inverse correlation was noted between difficulty to ‘fight’ an infection and number of colds (r=-0.289, P=0.003) as well as the number of infections (r=-0.446, P<0.0001) during pregnancy. The vast majority of the pregnant women acquired a more sedentary lifestyle in the third trimester. In planned, but not in unplanned, pregnancies stress predicted infant weight, independent of age and body mass index (BMI). Notably, in mothers with negative attitudes towards the pregnancy, those with an unplanned pregnancy gave birth to infants with significantly higher weights than those with planned pregnancies. Collectively these data suggest that there is a higher order of complexity, possibly involving gene-environment interactions that work together to ensure a positive outcome for the mother as well as the foetus

Differential expression of placental glucocorticoid receptors and growth arrest-specific transcript 5 in term and preterm pregnancies: evidence for involvement of maternal stress.

This article has been made available through the Brunel Open Access Publishing Fund.Pregnancy-specific stress predicts birth outcomes. We hypothesized that there is a maternal stress-GR interaction that can influence fetal birth weight. This study examined the relationship between mothers' stress and attitude towards their pregnancies, placental glucocorticoid receptors (GRs) and growth arrest-specific transcript 5 (GAS5) expression, and the status of GR polymorphism, with their infants' birth weights. GAS5 and GR α were the predominant transcripts in both term and preterm placentas, with GAS5 being primarily localized in the syncytiotrophoblasts. In an attempt to mimic moderate and high stress environment in vitro, BeWo and JEG-3 cytotrophoblast cell lines were treated with 10 nM-1000 nM cortisol. Only expression of GAS5 was significantly upregulated by cortisol in all treatments compared with basal levels, but none of the GRs changed expression significantly. In an attempt to assess a stress versus gene interaction, we studied four GR polymorphisms. In the homozygous group for Tth111I polymorphism, mothers with negative attitudes towards the pregnancy gave birth to infants with significantly lower birth weights compared to women with positive/neutral attitudes. None of the GR splice variants were associated with maternal stress. However, placental GAS5 levels were inversely correlated with maternal stress. This study points towards a potential gene-environment interaction that could be of predictive value for fetal weight.Brunel Open Access Publishing Fun

Expression of mTOR and downstream signalling components in the JEG-3 and BeWo human placental choriocarcinoma cell lines

This article has been made available through the Brunel Open Access Publishing Fund.Emerging data suggest that nutritional status and body weight are related to reproductive function, and nutrient imbalances during pregnancy lead to changes in the expression of fetal genes. Recent studies show that the mTOR acts as a placental growth signalling sensor and its expression is down-regulated in intrauterine growth restriction. To date, very little is known about the expression of this signalling pathway in choriocarcinoma, one of the most lethal germ cell cancers. In this study, cultures of fusigenic (BeWo) and non-fusigenic (JEG-3) human choriocarcinoma cell lines were used to investigate the expression of mTOR and its downstream signalling components. The effects of an inducer of syncytialisation (forskolin) on mTOR, eIF4E binding proteins (4EBPs) and ribosomal protein S6 kinases (S6Ks) in BeWo cells were also assessed. RT-PCR studies revealed that mTOR, 4EBP and S6Ks are expressed at mRNA level in both JEG-3 and BeWo cells. Semi-quantitative RT-PCR analysis revealed that in early stages of syncytialisation (50 µM forskolin for 48 h), the expression of mTOR and 4EBP was down-regulated when compared to unstimulated cells. In fully syncytialised cells (50 µM forskolin for 72 h) the expression of both genes was similar to basal levels. Interestingly, the phosphorylation (Ser371, Thr389) status of p70S6K remained unaltered upon forskolin treatment. These data validate BeWo cells as an experimental model to study the effects of forskolin-induced syncytialisation on mTOR signalling.This article is available through the Brunel Open Access Publishing Fun