The endocannabinoid 2-arachidonoylglycerol promotes endoplasmic reticulum stress in placental cells

Marta Almada,Bruno Fonseca,Natércia Teixeira,Lia Costa,Patrícia Alves,Daniela Gonçalves,Georgina Correia-Da-Silva,Jorge Braga

doi:10.1530/rep-19-0539

Abstract

Proliferation, differentiation and apoptosis of trophoblast cells are required for normal placental development. Impairment of those processes may lead to pregnancy-related diseases. Disruption of endoplasmic reticulum (ER) homeostasis has been associated with several reproductive pathologies including recurrent pregnancy loss and preeclampsia. In the unfolded protein response (UPR), specific ER-stress signalling pathways are activated to restore ER homeostasis, but if the adaptive response fails, apoptosis is triggered. Protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1) and Activating transcription factor 6 (ATF6) are central players in UPR and in ER-stress-induced apoptosis, as well as downstream transcription factors, as C/EBP homologous protein (CHOP). Our previous studies have shown that the endocannabinoid 2-arachidonoylglycerol (2-AG) modulates trophoblast cell turnover. Nevertheless, the role of ER-stress on 2-AG induced apoptosis and cannabinoid signalling in trophoblast has never been addressed. In this work, we used BeWo cells and human primary cytotrophoblasts isolated from term-placenta. The expression of ER-stress markers was analysed by qRT-PCR and Western blotting. ROS generation was assessed by fluorometric methods, while apoptosis was detected by the evaluation of caspase -3/-7 activities and Poly (ADP-ribose) polymerase (PARP) cleavage. Our findings indicate that 2-AG is able to induce ER-stress and apoptosis. Moreover, the eukaryotic initiation factor 2 (eIF2α)/CHOP pathway involved in ER-stress-induced apoptosis is triggered through a mechanism dependent on cannabinoid receptor CB2 activation. The results bring novel insights on the importance of ER-stress and cannabinoid signalling on 2-AG mechanisms of action in placenta.

Highlights

Placental development comprises tightly controlled processes of proliferation, differentiation and apoptosis of the trophoblast cells by the interplay of hormones, growth factors and other signalling mediators
We have previously reported that 2-AG induces apoptosis in BeWo cells, by a mechanism involving reactive oxygen species (ROS)/RNS generation associated with caspase -3/-7 activation (Costa et al 2014a)
These results suggest that the pro-apoptotic unfolded protein response (UPR) response is activated by 2-AG treatment but is dependent on cannabinoid signalling through cannabinoid receptor type 2 (CB2) activation

Summary

Introduction

Placental development comprises tightly controlled processes of proliferation, differentiation and apoptosis of the trophoblast cells by the interplay of hormones, growth factors and other signalling mediators. Protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1) and Activating transcription factor 6 (ATF6) are ER-stress transducers that sense the protein folding status and transmit the information to the cytosol and nucleus (Xu et al 2005, Szegezdi et al 2006) These proteins enroll UPR-mediated pathway, an adaptive mechanism that includes up-regulation of the ER-protein folding machinery, ER-associated protein degradation and inhibition of protein synthesis. The cellular adaptive mechanisms, including the ER-stress-induced coping responses, are physiologically important for a normal placental development, since trophoblast cells undergo complex processes of cellular turnover.

Materials and methods

Results

Discussion