E-GEOD-28426 - LOXs in invasive trophoblasts
Released on 4 December 2013, last updated on 2 June 2014
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 gamma (PPARG) plays a major role in placental development, and activation of PPARG by its agonists results in inhibition of EVCT invasion in vitro. To identify PPARG 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 PPARG 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 PPARG. 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 beta-aminopropionitrile in cell invasion assays led to an increase in EVCT invasiveness. These results suggest that LOX, LOXL1 and LOXL2 are downstream PPARG targets and that LOX activity is a negative regulator of trophoblastic cell invasion. Microarray analysis was performed using GeneChip technology in EVCTs isolated from first trimester human placentae. Gene expression in rosiglitazone-treated primary EVCT cultures was compared with matched untreated controls. A total of 175 probe sets identified differentially regulated genes. One of these genes, lysyl oxidase (LOX) which was up-regulated, was further analyzed. Expression of the LOX family was determined by real time Q-PCR, Western blotting, immunohistochemistry and immunofluorescence. LOX, LOXL and LOXL2 mRNA expression was significantly upregulated in PPARg-activated EVCTs. LOX and LOXL2 protein were present throughout the trophoblast cytoplasm, while LOXL was localized to the nucleus and nucleolus. Cell invasion assays on Matrigel Transwells showed that specific inhibition of LOX activity by ß-aminopropionitrile led to an increase in EVCT invasiveness, showing the basal inhibitory effect of LOX and LOX-like in the trophoblastic cells invasion process. Together these results show that LOX family as PPARg-target genes participate to the regulation of trophoblast invasion. Total RNA from EVCT were labelled according to the standard Affymetrix protocol. Five independent targets per treatment vs control were generated and hybridized on a GeneChip.
transcription profiling by array
Severine Aude Degrelle <firstname.lastname@example.org>, A Chissey, B Saubamea, C Rouault, D Evain-Brion, E Clouqueur, J Badet, K Csiszar, K Fong, N Segond, P Dessen, S A Degrelle, S Berndt, T Fournier