Glucose inhibits human placental GH secretion, in vitro

peer reviewedHuman placenta specifically expresses the GH-V gene leading to the production of placental Growth Hormone (PGH). During pregnancy, PGH levels increase progressively in maternal blood, but its regulation remains unknown. In this study the effect of glucose on PGH secretion by human term placenta was tested, in vitro, by means of two different experimental models: organ culture of villous tissue and primary culture of isolated cytotrophoblasts. PGH was assayed in the culture medium by an immunoradiometric assay using a specific PGH monoclonal antibody. The presence of glucose (25 mmol/L) in the culture medium significantly inhibited (p < 0.001) the secretion of PGH by either placental villous explants or by cultured trophoblast cells. This inhibitory effect of glucose on PGH secretion was dose-dependent. More than 50% inhibition being observed with 5.5 mmol/L. In the same conditions, the daily production of hPL and hCG, were unmodified. Furthermore, the glucose-induced inhibition of PGH secretion was more effective when cultured trophoblast cells are differentiated into syncytiotrophoblast. This study demonstrates, for the first time, that among the gestational polypeptide hormones secreted by the human placenta, only PGH secretion is modulated by glucose, suggesting a key metabolic role for this hormone during pregnancy

The Expression and Localization of N-Myc Downstream-Regulated Gene 1 in Human Trophoblasts

The protein N-Myc downstream-regulated gene 1 (NDRG1) is implicated in the regulation of cell proliferation, differentiation, and cellular stress response. NDRG1 is expressed in primary human trophoblasts, where it promotes cell viability and resistance to hypoxic injury. The mechanism of action of NDRG1 remains unknown. To gain further insight into the intracellular action of NDRG1, we analyzed the expression pattern and cellular localization of endogenous NDRG1 and transfected Myc-tagged NDRG1 in human trophoblasts exposed to diverse injuries. In standard conditions, NDRG1 was diffusely expressed in the cytoplasm at a low level. Hypoxia or the hypoxia mimetic cobalt chloride, but not serum deprivation, ultraviolet (UV) light, or ionizing radiation, induced the expression of NDRG1 in human trophoblasts and the redistribution of NDRG1 into the nucleus and cytoplasmic membranes associated with the endoplasmic reticulum (ER) and microtubules. Mutation of the phosphopantetheine attachment site (PPAS) within NDRG1 abrogated this pattern of redistribution. Our results shed new light on the impact of cell injury on NDRG1 expression patterns, and suggest that the PPAS domain plays a key role in NDRG1's subcellular distribution. © 2013 Shi et al

Widespread DNA hypomethylation at gene enhancer regions in placentas associated with early-onset pre-eclampsia.

Pre-eclampsia is a serious complication of pregnancy that can affect both maternal and fetal outcomes. Early-onset pre-eclampsia (EOPET) is a severe form of pre-eclampsia that is associated with altered physiological characteristics and gene expression in the placenta. DNA methylation is a relatively stable epigenetic modification to DNA that can reflect gene expression, and can provide insight into the mechanisms underlying such expression changes. This case-control study focused on DNA methylation and gene expression of whole chorionic villi samples from 20 EOPET placentas and 20 gestational age-matched controls from pre-term births. DNA methylation was also assessed in placentas affected by late-onset pre-eclampsia (LOPET) and normotensive intrauterine growth restriction (nIUGR). The Illumina HumanMethylation450 BeadChip was used to assess DNA methylation at >480 000 cytosine-guanine dinucleotide (CpG) sites. The Illumina HT-12v4 Expression BeadChip was used to assess gene expression of >45 000 transcripts in a subset of cases and controls. DNA methylation analysis by pyrosequencing was used to follow-up the initial findings in four genes with a larger cohort of cases and controls, including nIUGR and LOPET placentas. Bioinformatic analysis was used to identify overrepresentation of gene ontology categories and transcription factor binding motifs. We identified 38 840 CpG sites with significant (false discovery rate 12.5% methylation difference compared with the controls. Significant sites were enriched at the enhancers and low CpG density regions of the associated genes and the majority (74.5%) of these sites were hypomethylated in EOPET. EOPET, but not associated clinical features, such as intrauterine growth restriction (IUGR), presented a distinct DNA methylation profile. CpG sites from four genes relevant to pre-eclampsia (INHBA, BHLHE40, SLC2A1 and ADAM12) showed different extent of changes in LOPET and nIUGR. Genome-wide expression in a subset of samples showed that some of the gene expression changes were negatively correlated with DNA methylation changes, particularly for genes that are responsible for angiogenesis (such as EPAS1 and FLT1). Results could be confounded by altered cell populations in abnormal placentas. Larger sample sizes are needed to fully address the possibility of sub-profiles of methylation within the EOPET cohort. Based on DNA methylation profiling, we conclude that there are widespread DNA methylation alterations in EOPET that may be associated with changes in placental function. This property may provide a useful tool for early screening of such placentas. This study identifies DNA methylation changes at many loci previously reported to have altered gene expression in EOPET placentas, as well as in novel biologically relevant genes we confirmed to be differentially expressed. These results may be useful for DNA- methylation-based non-invasive prenatal diagnosis of at-risk pregnancies

RhoE Is Regulated by Cyclic AMP and Promotes Fusion of Human BeWo Choriocarcinoma Cells

Fusion of placental villous cytotrophoblasts with the overlying syncytiotrophoblast is essential for the maintenance of successful pregnancy, and disturbances in this process have been implicated in pathological conditions such as pre-eclampsia and intra-uterine growth retardation. In this study we examined the role of the Rho GTPase family member RhoE in trophoblast differentiation and fusion using the BeWo choriocarcinoma cell line, a model of villous cytotrophoblast fusion. Treatment of BeWo cells with the cell permeable cyclic AMP analogue dibutyryl cyclic AMP (dbcAMP) resulted in a strong upregulation of RhoE at 24h, coinciding with the onset of fusion. Using the protein kinase A (PKA)-specific cAMP analogue N6-phenyl-cAMP, and a specific inhibitor of PKA (14–22 amide, PKI), we found that upregulation of RhoE by cAMP was mediated through activation of PKA signalling. Silencing of RhoE expression by RNA interference resulted in a significant decrease in dbcAMP-induced fusion. However, expression of differentiation markers human chorionic gonadotrophin and placental alkaline phosphatase was unaffected by RhoE silencing. Finally, we found that RhoE upregulation by dbcAMP was significantly reduced under hypoxic conditions in which cell fusion is impaired. These results show that induction of RhoE by cAMP is mediated through PKA and promotes BeWo cell fusion but has no effect on functional differentiation, supporting evidence that these two processes may be controlled by separate or diverging pathways

A one-nucleotide difference in a cAMP and phorbol ester response element leads to differential regulation of the human chorionic somatomammotropin A and B gene transcription

Human chorionic somatomammotropin (hCS) is encoded by two highly related genes, hCS-A and hCS-B, located in the hGH/hCS gene locus. Both genes are expressed in the syncytiotrophoblast layer of the placenta and hCS release from trophoblast cells is known to be increased by cAMP and phorbol esters. However, it remains unclear whether this regulation acts at the level of hCS gene expression or secretion and whether both genes are affected. We examined the effects of cAMP and phorbol 12-myristate 13-acetate (PMA) on the transcription of the hCS-A and hCS-B genes. Transient expression experiments revealed a 7 bp cAMP- and PMA-responsive element (CRElhCS-A) spanning nucleotides-1102 to -1096 upstream of the hCS-A gene. In contrast, the homologous sequence upstream of hCS-B (CRElhCS-B), differing from CRElhCS-A by a single substitution, shows little or no response to cAMP. In band-shift assays, the CRElhCS-A oligonucleotide was shown to bind two factors related to CREBP and AP-1, whereas CRElhCS-B only competes for one of these complexes. Finally, Southwestern analysis revealed that the CRElhCS-A element binds two ubiquitous proteins of 100 kDa and 47 kDa respectively, whereas CRElhCS-B interacts only with the 47 kDa protein. Taken together, these results suggest that a 47 kDa protein binding to the CRElhCS-A and CRElhCS-B elements is involved in the PMA response of the hCS-A and hCS-B genes, and a 100 kDa protein plays a crucial role in cAMP regulation of the hCS-A gene

Efficient lipofection of human trophoblast cells in primary cultures

Human choriosomatomammotropic hormone, also known as placental lactogen, is expressed in syncytiotrophoblast cells of the placenta. Studying transcriptional regulation of the genes coding for this hormone, we became interested in transfecting primary cultures of these trophoblast cells. In this study, we show that it is possible to transfect, by the lipofection method, these giant cells in an efficient and reproducible manner. We show the presence of an enhancer region downstream from the hCS-B gene, functionally active in these cells; furthermore, we demonstrate the placenta-specific characteristic of this enhancer, previously identified in a human choriocarcinoma cell line

Differential expression of the TEF family of transcription factors in the murine placenta and during differentiation of primary human trophoblasts in vitro.

We describe the molecular cloning of murine (m) Transcriptional Enhancer Factor (TEF)-5 belonging to the TEF family of transcription factors. We show that mTEF-5 is specifically expressed in trophoblast giant cells and other extra-embryonic structures at early stages of development. At later stages, mTEF-5 is specifically expressed in the labyrinthine region of the placenta and in several embryonic tissues. We further show that the other mTEFs are differentially expressed in extraembryonic structures and in the mature placenta. Interestingly, human (h)TEF-5 is specifically expressed in the differentiated syncytiotrophoblast of the human term placenta and its expression is upregulated during the differentiation of cytotrophoblasts to syncytiotrophoblast in vitro, whereas that of hTEF-1 is down-regulated. Together with previous results describing hTEF-binding sites in the human placental lactogen-B gene enhancer, these novel observations support a role for hTEF-5 in the regulation of this gene. We further propose that the hTEF factors may play a more general role in placental gene regulation and development

Effect of human parathyroid hormone on the cAMP production and the endocrine functions of trophoblast cells from first trimester placenta

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