Regulation of trophoblast beta1-integrin expression by contact with endothelial cells

BACKGROUND: In human and non-human primates, migratory trophoblasts penetrate the uterine epithelium, invade uterine matrix, and enter the uterine vasculature. Invasive trophoblasts show increased expression of β1 integrin. Since trophoblast migration within the uterine vasculature involves trophoblast attachment to endothelial cells lining the vessel walls, this raises the possibility that cell-cell contact and/or factors released by endothelial cells could regulate trophoblast integrin expression. To test this, we used an in vitro system consisting of early gestation macaque trophoblasts co-cultured on top of uterine microvascular endothelial cells. RESULTS: When cultured alone, trophoblasts expressed low levels of β1 integrin as determined by quantitative immunofluorescence microscopy. When trophoblasts were cultured on top of endothelial cells for 24 h, the expression of trophoblast β1 integrin was significantly increased as determined by image analysis. β1 Integrin expression was not increased when trophoblasts were cultured with endothelial cell-conditioned medium, suggesting that upregulation requires direct contact between trophoblasts and endothelial cells. To identify endothelial cell surface molecules responsible for induction of trophoblast integrin expression, trophoblasts were cultured in dishes coated with recombinant platelet endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), or αVβ3 integrin. Trophoblast β1 integrin expression (assessed by immunofluorescence microscopy and Western blotting) was increased when PECAM-1 or αVβ3 integrin, but not ICAM-1, was used as substrate. CONCLUSIONS: Direct contact between trophoblasts and endothelial cells increases the expression of trophoblast β1 integrin

Early Developmental and Evolutionary Origins of Gene Body DNA Methylation Patterns in Mammalian Placentas

Over the last 20-80 million years the mammalian placenta has taken on a variety of morphologies through both divergent and convergent evolution. Recently we have shown that the human placenta genome has a unique epigenetic pattern of large partially methylated domains (PMDs) and highly methylated domains (HMDs) with gene body DNA methylation positively correlating with level of gene expression. In order to determine the evolutionary conservation of DNA methylation patterns and transcriptional regulatory programs in the placenta, we performed a genome-wide methylome (MethylC-seq) analysis of human, rhesus macaque, squirrel monkey, mouse, dog, horse, and cow placentas as well as opossum extraembryonic membrane. We found that, similar to human placenta, mammalian placentas and opossum extraembryonic membrane have globally lower levels of methylation compared to somatic tissues. Higher relative gene body methylation was the conserved feature across all mammalian placentas, despite differences in PMD/HMDs and absolute methylation levels. Specifically, higher methylation over the bodies of genes involved in mitosis, vesicle-mediated transport, protein phosphorylation, and chromatin modification was observed compared with the rest of the genome. As in human placenta, higher methylation is associated with higher gene expression and is predictive of genic location across species. Analysis of DNA methylation in oocytes and preimplantation embryos shows a conserved pattern of gene body methylation similar to the placenta. Intriguingly, mouse and cow oocytes and mouse early embryos have PMD/HMDs but their placentas do not, suggesting that PMD/HMDs are a feature of early preimplantation methylation patterns that become lost during placental development in some species and following implantation of the embryo

The MUC1 extracellular domain subunit is found in nuclear speckles and associates with spliceosomes.

MUC1 is a large transmembrane glycoprotein and oncogene expressed by epithelial cells and overexpressed and underglycosylated in cancer cells. The MUC1 cytoplasmic subunit (MUC1-C) can translocate to the nucleus and regulate gene expression. It is frequently assumed that the MUC1 extracellular subunit (MUC1-N) does not enter the nucleus. Based on an unexpected observation that MUC1 extracellular domain antibody produced an apparently nucleus-associated staining pattern in trophoblasts, we have tested the hypothesis that MUC1-N is expressed inside the nucleus. Three different antibodies were used to identify MUC1-N in normal epithelial cells and tissues as well as in several cancer cell lines. The results of immunofluorescence and confocal microscopy analyses as well as subcellular fractionation, Western blotting, and siRNA/shRNA studies, confirm that MUC1-N is found within nuclei of all cell types examined. More detailed examination of its intranuclear distribution using a proximity ligation assay, subcellular fractionation, and immunoprecipitation suggests that MUC1-N is located in nuclear speckles (interchromatin granule clusters) and closely associates with the spliceosome protein U2AF65. Nuclear localization of MUC1-N was abolished when cells were treated with RNase A and nuclear localization was altered when cells were incubated with the transcription inhibitor 5,6-dichloro-1-b-d-ribofuranosylbenzimidazole (DRB). While MUC1-N predominantly associated with speckles, MUC1-C was present in the nuclear matrix, nucleoli, and the nuclear periphery. In some nuclei, confocal microscopic analysis suggest that MUC1-C staining is located close to, but only partially overlaps, MUC1-N in speckles. However, only MUC1-N was found in isolated speckles by Western blotting. Also, MUC1-C and MUC1-N distributed differently during mitosis. These results suggest that MUC1-N translocates to the nucleus where it is expressed in nuclear speckles and that MUC1-N and MUC1-C have dissimilar intranuclear distribution patterns

Differential Effects of Sodium Butyrate and Lithium Chloride on Rhesus Monkey Trophoblast Differentiation

<div><p>Trophoblast differentiation during early placental development is critical for successful pregnancy and aberrant differentiation causes preeclampsia and early pregnancy loss. During the first trimester, cytotrophoblasts are exposed to low oxygen tension (equivalent to~2%-3% O₂) and differentiation proceeds along an extravillous pathway (giving rise to invasive extravillous cytotrophoblasts) and a villous pathway (giving rise to multinucleated syncytiotrophoblast). Interstitial extravillous cytotrophoblasts invade the decidua, while endovascular extravillous cytotrophoblasts are involved in re-modelling uterine spiral arteries. We tested the idea that sodium butyrate (an epigenetic modulator) induces trophoblast differentiation in early gestation rhesus monkey trophoblasts through activation of the Wnt/β-catenin pathway. The results show that syncytiotrophoblast formation was increased by butyrate, accompanied by nuclear accumulation of β-catenin, and increased expression of EnvV2 and galectin-1 (two factors thought to be involved in trophoblast fusion). Surprisingly, the expression of GCM1 and syncytin-2 was not affected by sodium butyrate. When trophoblasts were incubated with lithium chloride, a GSK3 inhibitor that mimics Wnt activation, nuclear accumulation of β-catenin also occurred but differentiation into syncytiotrophoblast was not observed. Instead the cells differentiated to mononucleated spindle-shaped cells and showed molecular and behavioral characteristics of endovascular trophoblasts. Another highly specific inhibitor of GSK3, CHIR99021, failed to induce endovascular trophoblast characteristics. These observations suggest that activation of the Wnt/β-catenin pathway correlates with both trophoblast differentiation pathways, but that additional factors determine specific cell fate decisions. Other experiments suggested that the differential effects of sodium butyrate and lithium chloride might be explained by their effects on TNFα production. The results provide valuable tools to manipulate trophoblast differentiation <i>in vitro</i> and to better understand the differentiation pathways that occur during early gestation.</p></div

Effect of sodium butyrate on the formation of multinucleated syncytiotrophoblast.

<p>Trophoblasts were incubated in the presence or absence of sodium butyrate (0.5 mM) for 7 days. (A) Phase contrast images of live cells. (B) Merged immunofluorescence images of cells stained using an antibody against E-cadherin (green) and DAPI (blue). (C) Graph showing the fusion indices for cells cultured in the absence or presence of sodium butyrate. The fusion index was calculated as described in Methods. The results are means ± SEM from three experiments. The asterisk indicates the value is significantly different (p<0.05) from the control.</p

Expression of extravillous trophoblast marker proteins.

<p>Cells were cultured for 7 days (in the absence of sodium butyrate or lithium chloride) after which the expression of the selected adhesion molecules was detected by Western blotting as described in Methods. The graphs below the Western blot show densitometric quantitation of protein bands and values are shown as means ± SEM (n = 3). The asterisks indicate values that are significantly different (p<0.05) from the respective controls. ND, not detected.</p