New Transcriptional Reporters to Quantify and Monitor PPAR γ

The peroxisome-proliferator-activated-receptor-γ (PPARγ) is a member of the nuclear receptor superfamily that plays a critical role in diverse biological processes, including adipogenesis, lipid metabolism, and placental development. To study the activity of PPARγ, we constructed two new reporter genes: a fluorescent GFP-tagged histone-2B (PPRE-H2B-eGFP) and a secreted nanoluciferase (PPRE-pNL1.3[secNluc]). This study demonstrates their usage to monitor PPARγ activity in different cell types and screen for PPARγ’s potential ligands

Rôle de PPARgamma dans le développement placentaire humain et l'issue de la grossesse : impact des phtalates et de la mutation de PPARgamma (E352Q) sur la différenciation des trophoblastes

CONTEXTE : les récepteurs activés par les proliférateurs des peroxysomes (PPAR) sont des membres de la superfamille des récepteurs nucléaires qui agissent en tant que facteurs de transcription activés par des ligands. Le PPARgamma joue un rôle essentiel dans la différenciation des adipocytes, le métabolisme des lipides et le développement du placenta. Les phtalates sont des contaminants environnementaux couramment utilisés comme plastifiants dans les produits à base de polychlorure de vinyle (PVC). Récemment, l'exposition de la femme enceinte aux phtalates, qui sont des ligands potentiels de PPARgamma, a été associée à de la prématurité, à des petit poids de naissance et à des interruptions de grossesse. Par ailleurs, des données récentes suggèrent que certaines mutations rares du domaine de liaison du ligand de PPARgamma (LBD) responsables de la lipodystrophie (FPLD3) seraient associées à des complications de la grossesse. OBJECTIFS : notre objectif général était d'évaluer l'activité et le rôle du PPARgamma placentaire durant la grossesse, et en particulier l'impact des phtalates et d'une mutation de PPARgamma associée à FPLD3 (E352Q) dans la différenciation et la fonction des trophoblastes humains in vitro. METHODES : dans ce but, nous avons : 1 / construit des plasmides rapporteurs (PPRE-H2B-eGFP et PPRE-pNL1.3 [secNluc]) pour quantifier l'activité de PPARgamma in vitro ; 2 / étudié l'action du MEHP (mono-2-éthylhexyl-phtalate) sur l'activité de PPARgamma et la différenciation des cytotrophoblastes villeux humains (CTVs) en utilisant des techniques d'immunofluorescence, de mesure de l'activité de PPARgamma, de dosage d'hCG, de western blot et d'analyse lipidomique ; 3 / étudié l'impact de la mutation de PPARgamma (E352Q) sur l'activité de PPARgamma en utilisant un lignée cellulaire NIH/3T3sur la différenciation in vitro des CTVs par analyse fonctionnelle après inactivation avec un siRNA et restitution de l'activité de PPARgamma « wild type » et de PPARgamma muté. RÉSULTATS : les constructions plasmidiques rapporteurs ont permis de quantifier l'activité de PPARgamma dans différents types cellulaires et conditions expérimentales. De plus, nous avons montré que des doses non toxiques de 0,1 µM et 1 µM de MEHP inhibaient significativement l'activité de PPARgamma et la différenciation des CTVs par rapport aux témoins, alors qu'étonnamment, 10 µM avait l'effet opposé. Par ailleurs, l'exposition de CTVs au MEHP inhibe la production d'hCG, altère de manière significative la composition lipidique trophoblastique perturbe sur la voie des MAPK (seul 10 µM de MEHP augmente la pERK alors que 0,1 ,1 et 10 µM diminuent les niveaux de protéine pJNK). Enfin, la mutation E352Q - associée à une grossesse pathologique a non seulement diminué l'activité de PPARgamma, mais également altéré la formation in vitro de syncytiotrophoblastes par fusion des CTVs primaires. CONCLUSION : des études de KO réalisées sur des souris ont montré l'importance de PPARgamma pour le développement du placenta et l'issue de la gestation. En utilisant deux nouveaux systèmes rapporteurs combinés avec un modèle in vitro de différenciation de CTVs, nous avons montré que le MEHP et la mutation E352Q perturbent l'activité de PPARgamma et la physiologie du trophoblaste humain ce qui pourraient expliquer les complications de la grossesse observées in vivo.BACKGROUND: The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily that act as ligand-activated transcription factors. PPARgamma plays critical roles in adipocyte differentiation, lipid metabolism and placental development. Phthalates are environmental contaminants commonly used as plasticizers in polyvinyl chloride (PVC) products. Recently, exposure to phthalates, which are potential PPARgamma ligands, has been associated with preterm birth, low birth-weight, and pregnancy loss. On the other hand, there is emerging evidence that some rare mutations in PPARgamma ligand binding domain (LBD) responsible of human lipodystrophy (FPLD3), might be associated with pregnancy complications. OBJECTIVES: Our general goal was to assess placental PPARgamma activity and role during pregnancy and in particular the impact of phthalates and of a FPLD3-associated mutation of PPARgamma (E352Q) in trophoblast differentiation and functions in vitro. METHODS: In this purpose we have: 1/ constructed reporter plasmids (PPRE-H2B-eGFP & PPRE-pNL1.3[secNluc]) to monitor PPARgamma activity in vitro; 2/ investigated the action of MEHP (mono-2-ethylhexyl-phthalate) on PPARgamma activity and differentiation of primary human villous cytotrophoblasts (VCTs) by using immunofluorescence, PPARgamma activity and hCG assays, western blotting and lipidomics analyses and; 3/ studied the impact of the mutated-PPARgamma (E352Q) on PPARgamma activity in NIH/3T3-cell line, and on VCT differentiation through functional analysis (fusion index) using siRNA knockdown and functional restitution of PPARgamma and mutated PPARgamma in in vitro models. RESULTS: Constructed reporter plasmids allowed us to quantify PPARgamma activity in different cell types and experimental conditions. Furthermore, we showed that non-toxic doses of 0.1 µM and 1 µM MEHP significantly decreased PPARgamma activity and VCT differentiation compared to controls, while, surprisingly, 10 µM had the opposite effect. Also, MEHP exposure inhibited hCG production, significantly altered trophoblastic lipid composition and had significant effects on the MAPK pathway: only 10 µM MEHP increased pERK, whereas 0.1, 1 and 10 µM decreased pJNK protein levels. Finally, the E352Q mutation - associated with pregnancy complication - not only decreased PPARgamma activity, but also altered the in vitro formation of syncytiotrophoblasts by fusion of primary VCTs. CONCLUSION: PPARgamma KO studies in mice showed the importance of PPARgamma for placental development and gestation outcome. Using two novel PPRE reporter systems combined with in vitro model of VCT differentiation, we showed that MEHP and the E352Q mutation impact PPARgamma activity and disrupt human trophoblast physiology that, which could explain the pregnancy complications observed in vivo

Peroxisome proliferator‐activated receptor gamma‐ligand‐binding domain mutations associated with familial partial lipodystrophy type 3 disrupt human trophoblast fusion and fibroblast migration

International audienceThe transcription factor peroxisome proliferator-activated receptor gamma (PPARG) is essential for placental development, and alterations in its expression and/or activity are associated with human placental pathologies such as pre-eclampsia or IUGR. However, the molecular regulation of PPARG in cytotrophoblast differentiation and in the underlying mesenchyme remains poorly understood. Our main goal was to study the impact of mutations in the ligand-binding domain (LBD) of the PPARG gene on cytotrophoblast fusion (PPARGE352Q ) and on fibroblast cell migration (PPARGR262G /PPARGL319X ). Our results showed that, compared to cells with reconstituted PPARGWT , transfection with PPARGE352Q led to significantly lower PPARG activity and lower restoration of trophoblast fusion. Likewise, compared to PPARGWT fibroblasts, PPARGR262G /PPARGL319X fibroblasts demonstrated significantly inhibited cell migration. In conclusion, we report that single missense or nonsense mutations in the LBD of PPARG significantly inhibit cell fusion and migration processes

The Role of Peroxisome Proliferator–Activated Receptor Gamma (PPARγ) in Mono(2-ethylhexyl) Phthalate (MEHP)-Mediated Cytotrophoblast Differentiation

International audienceBACKGROUND: Phthalates are environmental contaminants commonly used as plasticizers in polyvinyl chloride (PVC) products. Recently, exposure to phthalates has been associated with preterm birth, low birth weight, and pregnancy loss. There is limited information about the possible mechanisms linking maternal phthalate exposure and placental development, but one such mechanism may be mediated by peroxisome proliferator-activated receptor c (PPARc). PPARc belongs to the nuclear receptor superfamily that regulates, in a ligand-dependent manner, the transcription of target genes. Studies of PPARc-deficient mice have demonstrated its essential role in lipid metabolism and placental development. In the human placenta, PPARc is expressed in the villous cytotrophoblast (VCT) and is activated during its differentiation into syncytiotrophoblast. OBJECTIVES: The goal of this study was to investigate the action of mono(2-ethylhexyl) phthalate (MEHP) on PPARc activity during in vitro differentiation of VCTs. METHODS: We combined immunofluorescence, PPARc activity/hCG assays, western blotting, and lipidomics analyses to characterize the impacts of physiologically relevant concentrations of MEHP (0.1, 1, and 10 lM) on cultured VCTs isolated from human term placentas. RESULTS: Doses of 0:1 lM and 1 lM MEHP showed significantly lower PPARc activity and less VCT differentiation in comparison with controls, whereas, surprisingly, a 10 lM dose had the opposite effect. MEHP exposure inhibited hCG production and significantly altered lipid composition. In addition, MEHP had significant effects on the mitogen-activated protein kinase (MAPK) pathway. CONCLUSIONS: This study suggests that MEHP has a U-shaped dose-response effect on trophoblast differentiation that is mediated by the PPARc pathway and acts as an endocrine disruptor in the human placenta. https://doi