Aim: We aim to investigate in cytotrophoblasts whether these effects on cell viability loss are due to endoplasmic reticulum (ER) stress mediated apoptosis.
Introduction: Placental development relies on a balance between proliferation, differentiation and apoptosis of trophoblasts, a process tightly regulated by growth factors, cytokines and hormones. Endocannabinoids (eCB), such as 2-arachidonoylglycerol (2-AG) and anandamide (AEA) may play a role in these processes. We previously demonstrated that both eCB induced trophoblast cell death.1,2 Here we investigated in cytotrophoblasts whether these effects on cell viability loss are due to endoplasmic reticulum (ER) stress mediated apoptosis.
ER stress is caused by the accumulation of unfolded proteins leading to an unfold protein response (UPR) triggered by transmembrane ER signaling proteins including: pancreatic ER kinase (PKR)-like ER kinase (PERK), inositol-requiring enzyme 1 (Ire1) and Activating transcription factor 6 (ATF6). The dissociation of Grp78 (BiP) from these sensors triggers a series of mechanisms that can restore homeostasis or lead to apoptosis. Placental stress has been implicated in the pathophysiology of pregnancy complications, including growth restriction and pre-eclampsia.
Methods: BeWo cells (ATCC, USA), an accepted model of cytotrophoblast stem cells were treated with AEA or 2-AG (10 micromolar) for 24h. Through quantitative real time polymerase chain reaction (qPCR), we evaluated mRNA levels of ER stress markers: CHOP, Grp78, ATF4 and spliced mXBP1. Protein expression of CHOP was evaluated by western-blot.
Results: After 24h of treatment with both eCB, we found an increase in mRNA levels of ER stress markers: CHOP, Grp78, ATF4 and spliced mXBP1. Protein expression of CHOP also increased in both cases.
Conclusion: These results suggest that cell viability loss promoted by 2-AG and AEA was associated with ER-stress since both PERK and IRE1 arms of UPR are activated. Prolonged ER-stress, contributes to the expression of pro-apoptotic proteins, such as CHOP.
These findings shed light to the impact of endocannabinoids induced-ER stress which may negatively affect trophoblast cell turnover and pregnancy outcomes.
Acknowledgements: This work received support from European Union (FEDER funds through COMPETE) and FCT through project PTDC/DTP-FTO/5651/2014-POCI-01-0145-FEDER-016562; FCT/MEC through national funds and co-financed by FEDER, under PT2020 (UID/01/0145/FERDER/007728) and CCDR-N/NORTE2020/Portugal 2020 (norte-01-0145-FEDER-000024).