Caffeine Inhibits EGF-Stimulated Trophoblast Cell Motility through the Inhibition of mTORC2 and Akt.

Impaired trophoblast invasion is associated with pregnancy disorders such as early pregnancy loss and preeclampsia. There is evidence to suggest that the consumption of caffeine during pregnancy may increase the risk of pregnancy loss; however, little is known about the direct effect of caffeine on normal trophoblast biology. Our objectives were to examine the effect of caffeine on trophoblast migration and motility after stimulation with epidermal growth factor (EGF) and to investigate the intracellular signaling pathways involved in this process. Primary first-trimester extravillous trophoblasts (EVT) and the EVT-derived cell line SGHPL-4 were used to study the effect of caffeine on EGF-stimulated cellular motility using time-lapse microscopy. SGHPL-4 cells were further used to study the effect of caffeine and cAMP on EGF-stimulated invasion of fibrin gels. The influence of caffeine and cAMP on EGF-stimulated intracellular signaling pathways leading to the activation of Akt were investigated by Western blot analysis. Caffeine inhibits both EGF-stimulated primary EVT and SGHPL-4 cell motility. EGF stimulation activates phosphatidylinositol 3-kinase, and Akt and caffeine inhibit this activation. Although cAMP inhibits both motility and invasion, it does not inhibit the activation of Akt, indicating that the effects of caffeine seen in this study are independent of cAMP. Further investigation indicated a role for mammalian target of rapamycin complex 2 (mTORC2) as a target for the inhibitory effect of caffeine. In conclusion, we demonstrate that caffeine inhibits EGF-stimulated trophoblast invasion and motility in vitro and so could adversely influence trophoblast biology in vivo

Transcription factor AP-2α promotes EGF-dependent invasion of human trophoblast

The basic helix-span-helix transcription factor activating protein (AP)-2α is critically involved in cell-specific hormone expression of syncytializing human trophoblasts. Its role in invasive trophoblast differentiation, however, remains largely elusive. Using RT-PCR, Western blotting, and immunofluorescence of first-trimester placentae,wehereshowthat AP-2α is expressed in extravillous trophoblasts (EVTs) both in situ and in vitro as well as in invasive trophoblast cell lines. Its protein expression was increased upon supplementation of epidermal growth factor (EGF) both in primary EVTs and trophoblastic SGHPL-5 cells. Gene silencing of AP-2α using small hairpin microRNA (shRNAmir) did not affect basal invasion of SGHPL-5 cells through Matrigel-coated filters but reduced EGF-stimulated invasion. Similarly, treatment of primary EVTs with AP-2α small interfering RNA decreased EGF-dependent invasion. Proliferation of SGHPL-5 cells and primary EVTs, measured by cumulative cell numbers and 5-bromo-2′-deoxyuridine labeling, respectively, were not affected on loss of AP-2α. EGF-dependent induction of matrix metalloproteinase (MMP)-2, proand active form of urokinase plasminogen activator, and chorionic gonadotropin (CG)-β was noticed in shRNAmir-control cells, whereas these genes were suppressed in EGF-treated shRNAmir-AP-2α cells. Similarly, EGF-stimulated MMP-2 and CGβ protein expression was reduced in AP-2α small interfering RNA-treated primary EVTs. Knockdown of AP-2α also decreased luciferase activity of the CGβ5 promoter in SGHPL-5 cells, which was compensated upon transient overexpression of AP-2α cDNA. In conclusion, we show that AP-2α expression positively affects human trophoblast invasion under EGF-stimulated conditions, likely by inducing critical invasion-promoting genes such MMP-2, urokinase plasminogen activator, and CG

Defining the three cell lineages of the human blastocyst by single-cell RNA-seq

Here, we provide fundamental insights into early human development by single-cell RNA-sequencing of human and mouse preimplantation embryos. We elucidate conserved transcriptional programs along with those that are human specific. Importantly, we validate our RNA-sequencing findings at the protein level, which further reveals differences in human and mouse embryo gene expression. For example, we identify several genes exclusively expressed in the human pluripotent epiblast, including the transcription factor KLF17. Key components of the TGF-β signalling pathway, including NODAL, GDF3, TGFBR1/ALK5, LEFTY1, SMAD2, SMAD4 and TDGF1, are also enriched in the human epiblast. Intriguingly, inhibition of TGF-β signalling abrogates NANOG expression in human epiblast cells, consistent with a requirement for this pathway in pluripotency. Although the key trophectoderm factors Id2, Elf5 and Eomes are exclusively localized to this lineage in the mouse, the human orthologues are either absent or expressed in alternative lineages. Importantly, we also identify genes with conserved expression dynamics, including Foxa2/FOXA2, which we show is restricted to the primitive endoderm in both human and mouse embryos. Comparison of the human epiblast to existing embryonic stem cells (hESCs) reveals conservation of pluripotency but also additional pathways more enriched in hESCs. Our analysis highlights significant differences in human preimplantation development compared with mouse and provides a molecular blueprint to understand human embryogenesis and its relationship to stem cells