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The GPI-anchor biosynthesis pathway is critical for syncytiotrophoblast differentiation and placental development

ABSTRACT: The glycosylphosphatidylinositol (GPI) biosynthetic pathway in the endoplasmic reticulum (ER) is crucial for generating GPI-anchored proteins (GPI-APs), which are translocated to the cell surface and play a vital role in cell signaling and adhesion. This study focuses on two integral components of the GPI pathway, the PIGL and PIGF proteins, and their significance in trophoblast biology. We show that GPI pathway mutations impact on placental development impairing the development of the syncytiotrophoblast (SynT), and especially the SynT-II layer, which is essential for the establishment of the definitive nutrient exchange area within the placental labyrinth. CRISPR/Cas9 knockout of Pigl and Pigf in mouse trophoblast stem cells (mTSC) confirms the role of these GPI enzymes in syncytiotrophoblast differentiation. Mechanistically, impaired GPI-AP generation induces an excessive unfolded protein response (UPR) in the ER in mTSCs growing in stem cell conditions, akin to what is observed in human preeclampsia. Remarkably, the transcriptomic profile of Pigl- and Pigf-deficient cells separates human patient placental samples into preeclampsia and control groups suggesting an involvement of Pigl and Pigf in establishing a preeclamptic gene signature. Upon differentiation, the impairment of the GPI pathway hinders the induction of WNT signaling for early SynT-II development. Our study unveils the pivotal role of GPI biosynthesis in early placentation and uncovers a new preeclampsia gene expression profile associated with mutations in the GPI biosynthesis pathway, providing novel molecular insights into placental development with implications for enhanced patient stratification and timely interventions.

ORGANISM(S): Mus musculus

PROVIDER: GSE266153 | GEO | 2024/05/19

REPOSITORIES: GEO

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Project description:The syncytiotrophoblast develops as a result of the fusion of placental villous cytotrophoblast cells. These multinuclear cells are essential for fetal-maternal exchange and production of pregnancy-related hormones. Placental deficiency is considered a major risk factor associated with intrauterine growth restriction and preeclampsia. Better understanding of placental patho-physiology requires knowledge on gene expression regulation involved in trophoblast differentiation. Using the well-established in vitro trophoblast differentiation model, we have performed a microarray analysis on mRNA expression in trophoblast and syncytiotrophoblast cell cultures. Dramatic changes in gene expression patterns were detected during trophoblast differentiation. As many as 3524 novel and known genes are found to be up- or down regulated for more than 2-fold. Real-time PCR analysis of a group of genes confirmed the reliability of the microarray data. Overall, this study provided a global view on the molecular events underlying trophoblast differentiation. Subsequent characterization on regulation and function of the identified genes may lead to discovery of new pathways important for placental differentiation and function. Experiment Overall Design: Affymetrix GeneChipTM Human Genome U133 Plus 2.0 microarrays were used for mRNA profiling. RNA samples were subject to Agilent analysis for quality controls. cDNA was prepared from 10 Âµg of RNA, quantified by spectrometry, and used as a template for the synthesis of biotinylated cRNA using RNA transcript labeling reagent. The quality of the cRNA probe was verified by gel electrophoresis as well as a pilot hybridization using the Test-3 arrays. Hybridization solution containing fragmented cRNA probes and control cRNA was supplemented with herring sperm DNA and bovine serum albumin. The probe solution was heated at 99Â° C for 5 min followed by incubation at 45Â° C for 5 min before use. Hybridization was carried out at 45Â° C for 16 hr with constant rotation at 60 rpm. The arrays were washed and stained with streptavidin-phycoerythrin. The GeneChip 5.0 (Affymetrix) program was used to scan and quantitatively document the hybridization signals. Compilation of candidate gene and calculation of changes were performed on SpotFire and Microsoft Excel programs. To minimize the false-positive conclusion, only genes satisfying the following two criteria were considered candidate regulation targets for further analysis: 1) The geneâs hybridization signal reached an absolute level that was significantly higher than that of the background (P<0.05); 2) The changes is more than 2-fold when compared to control group.

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The GPI-anchor biosynthesis pathway is critical for syncytiotrophoblast differentiation and placental development

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