Platelet-derived factors dysregulate placental sphingosine-1-phosphate receptor 2 in human trophoblasts

RESEARCH QUESTION: Sphingosine-1-phosphate (S1P) is an essential and bioactive sphingolipid with various functions, which acts through five different G-protein coupled receptors (S1PR1-5). This study investigates the localisation of S1PR1-S1PR3 in the human placenta and analyses the influence of different flow rates, various oxygen concentrations and platelet-derived factors on the expression profile of S1PRs in trophoblasts. DESIGN: Expression dynamics of placental S1PR1-S1PR3 were determined in human first trimester (n=10), pre-term (n=9) and term (n=10) cases. Furthermore, we investigated the expression of these receptors in different primary cell types isolated from human placenta, verified the findings with publicly available single cell RNA-Seq data from first trimester and immunostaining of human first trimester and term placentas. Moreover, we tested whether the placental S1PR subtypes are dysregulated in differentiated BeWo cells under different flow rates, different oxygen concentrations or presence of platelet-derived factors. RESULTS: qPCR revealed that S1PR2 is the predominant placental S1PR in first trimester and vanishes towards term. All other S1P receptors increased from first trimester towards term. S1PR1 could be localised in endothelial cells, whereas S1PR2 and S1PR3 are predominantly found in villous trophoblasts. Furthermore, S1PR2 was found to be significantly downregulated in BeWo cells when co-incubated with platelet-derived factors. CONCLUSION: Our study suggests that the placental S1PR repertoire is differentially expressed across gestation. S1PR2 expression in villous trophoblasts is negatively influenced by platelet-derived factors, which could contribute to downregulation of placental S1PR2 over time of gestation as platelet presence and activation in the intervillous space increases from mid of first trimester onwards

Fluid shear stress induces a shift from glycolytic to amino acid pathway in human trophoblasts

BACKGROUND: The human placenta, a tissue with a lifespan limited to the period of pregnancy, is exposed to varying shear rates by maternal blood perfusion depending on the stage of development. In this study, we aimed to investigate the effects of fluidic shear stress on the human trophoblast transcriptome and metabolism. RESULTS: Based on a trophoblast cell line cultured in a fluidic flow system, changes caused by shear stress were analyzed and compared to static conditions. RNA sequencing and bioinformatics analysis revealed an altered transcriptome and enriched gene ontology terms associated with amino acid and mitochondrial metabolism. A decreased GLUT1 expression and reduced glucose uptake, together with downregulated expression of key glycolytic rate-limiting enzymes, hexokinase 2 and phosphofructokinase 1 was observed. Altered mitochondrial ATP levels and mass spectrometry data, suggested a shift in energy production from glycolysis towards mitochondrial oxidative phosphorylation. This shift in energy production could be supported by increased expression of glutamic-oxaloacetic transaminase variants in response to shear stress as well as under low glucose availability or after silencing of GLUT1. The shift towards amino acid metabolic pathways could be supported by significantly altered amino acid levels, like glutamic acid, cysteine and serine. Downregulation of GLUT1 and glycolytic rate-limiting enzymes, with concomitant upregulation of glutamic-oxaloacetic transaminase 2 was confirmed in first trimester placental explants cultured under fluidic flow. In contrast, high fluid shear stress decreased glutamic-oxaloacetic transaminase 2 expression in term placental explants when compared to low flow rates. Placental tissue from pregnancies with intrauterine growth restriction are exposed to high shear rates and showed also decreased glutamic-oxaloacetic transaminase 2, while GLUT1 was unchanged and glycolytic rate-limiting enzymes showed a trend to be upregulated. The results were generated by using qPCR, immunoblots, quantification of immunofluorescent pictures, padlock probe hybridization, mass spectrometry and FRET-based measurement. CONCLUSION: Our study suggests that onset of uteroplacental blood flow is accompanied by a shift from a predominant glycolytic- to an alternative amino acid converting metabolism in the villous trophoblast. Rheological changes with excessive fluidic shear stress at the placental surface, may disrupt this alternative amino acid pathway in the syncytiotrophoblast and could contribute to intrauterine growth restriction

A genome-wide Drosophila epithelial tumorigenesis screen identifies Tetraspanin 29Fb as an evolutionarily conserved suppressor of Ras-driven cancer.

Oncogenic mutations in the small GTPase Ras contribute to ~30% of human cancers. However, Ras mutations alone are insufficient for tumorigenesis, therefore it is paramount to identify cooperating cancer-relevant signaling pathways. We devised an in vivo near genome-wide, functional screen in Drosophila and discovered multiple novel, evolutionarily-conserved pathways controlling Ras-driven epithelial tumorigenesis. Human gene orthologs of the fly hits were significantly downregulated in thousands of primary tumors, revealing novel prognostic markers for human epithelial tumors. Of the top 100 candidate tumor suppressor genes, 80 were validated in secondary Drosophila assays, identifying many known cancer genes and multiple novel candidate genes that cooperate with Ras-driven tumorigenesis. Low expression of the confirmed hits significantly correlated with the KRASG12 mutation status and poor prognosis in pancreatic cancer. Among the novel top 80 candidate cancer genes, we mechanistically characterized the function of the top hit, the Tetraspanin family member Tsp29Fb, revealing that Tsp29Fb regulates EGFR signaling, epithelial architecture and restrains tumor growth and invasion. Our functional Drosophila screen uncovers multiple novel and evolutionarily conserved epithelial cancer genes, and experimentally confirmed Tsp29Fb as a key regulator of EGFR/Ras induced epithelial tumor growth and invasion