Transcriptome analysis of PPARγ target genes reveals the involvement of lysyl oxidase in human placental cytotrophoblast invasion.

Nadine Segond,Josette Badet,Christine Rouault,Elodie Clouqueur,Philippe Dessen,Danièle Evain-Brion,Thierry Fournier,Bruno Saubamea,Séverine A Degrelle,Sarah Berndt,Keith S K Fong,Katalin Csiszar

doi:10.1371/journal.pone.0079413

Abstract

Human placental development is characterized by invasion of extravillous cytotrophoblasts (EVCTs) into the uterine wall during the first trimester of pregnancy. Peroxisome proliferator-activated receptor γ (PPARγ) plays a major role in placental development, and activation of PPARγ by its agonists results in inhibition of EVCT invasion in vitro. To identify PPARγ target genes, microarray analysis was performed using GeneChip technology on EVCT primary cultures obtained from first-trimester human placentas. Gene expression was compared in EVCTs treated with the PPARγ agonist rosiglitazone versus control. A total of 139 differentially regulated genes were identified, and changes in the expression of the following 8 genes were confirmed by reverse transcription-quantitative polymerase chain reaction: a disintegrin and metalloproteinase domain12 (ADAM12), connexin 43 (CX43), deleted in liver cancer 1 (DLC1), dipeptidyl peptidase 4 (DPP4), heme oxygenase 1 (HMOX-1), lysyl oxidase (LOX), plasminogen activator inhibitor 1 (PAI-1) and PPARγ. Among the upregulated genes, lysyl oxidase (LOX) was further analyzed. In the LOX family, only LOX, LOXL1 and LOXL2 mRNA expression was significantly upregulated in rosiglitazone-treated EVCTs. RNA and protein expression of the subfamily members LOX, LOXL1 and LOXL2 were analyzed by absolute RT-qPCR and western blotting, and localized by immunohistochemistry and immunofluorescence-confocal microscopy. LOX protein was immunodetected in the EVCT cytoplasm, while LOXL1 was found in the nucleus and nucleolus. No signal was detected for LOXL2 protein. Specific inhibition of LOX activity by β-aminopropionitrile in cell invasion assays led to an increase in EVCT invasiveness. These results suggest that LOX, LOXL1 and LOXL2 are downstream PPARγ targets and that LOX activity is a negative regulator of trophoblastic cell invasion.

Highlights

Human placental development relies on trophoblast differentiation along two pathways
To study early human placental development and the regulation of the trophoblastic cell invasion process, we have developed an in vitro invasion model using non proliferative and highly invasive extravillous cytotrophoblasts (EVCTs) primary cells isolated from first-trimester human chorionic villi cultured on MatrigelTM [10,11,12]
lysyl oxidase (LOX) and LOXL1 are largely homologous, do not contain a scavenger receptor cysteine-rich (SRCR) domain, and are secreted as proproteins; this is the subfamily we studied in invasive EVCTs – LOXL2 was considered

Summary

Introduction

Human placental development relies on trophoblast differentiation along two pathways. Trophoblast plugging of the maternal spiral arteries between 6 to 8 weeks of gestation is gradually eliminated between 8 to 12 weeks of gestation, leading to increases in intervillous oxygen tension and placental expression of anti-oxidant enzymes [5,6]. This physiological trophoblastic cell invasion process is tightly controlled during the first trimester and is required for placental development and normal pregnancy outcome. Impaired trophoblast invasion has been implicated in gestational pathologies such as fetal growth restriction and preeclampsia [7,8,9]

Methods

Results

Conclusion