Abstract
Preeclampsia, a serious hypertensive disorder of pregnancy, is characterized by elevated ceramide (CER) content that is responsible for heightened trophoblast cell death rates via apoptosis and autophagy. Whether trophoblast cells undergo necroptosis, a newly characterized form of regulated necrosis, and the potential role of CER in this process remain to be established. Herein, we report that exposure of both JEG3 cells and primary isolated cytotrophoblasts to C16:0 CER in conjunction with a caspase-8 inhibitor (Q-VD-OPh) promoted necroptotic cell death, as evidenced by increased expression and association of receptor-interacting protein kinases RIP1 and RIP3, as well as phosphorylation of mixed lineage kinase domain-like (MLKL) protein. MLKL activation and oligomerization could be abrogated by pretreatment with the necroptosis inhibitor necrostatin-1 (Nec-1). CER+Q-VD-OPH-treated primary trophoblasts displayed striking necrotic morphology along with disrupted fusion processes as evidenced by maintenance of E-cadherin-stained membrane boundaries and reduced glial cell missing-1 expression, but these events were effectively reversed using Nec-1. Of clinical relevance, we established an increased susceptibility to necroptotic cell death in preeclamptic placentae relative to normotensive controls. In preeclampsia, increased necrosome (RIP1/RIP3) protein levels, as well as MLKL activation and oligomerization associated with necrotic cytotrophoblast morphology. In addition, caspase-8 activity was reduced in severe early-onset preeclampsia cases. This study is the first to report that trophoblast cells undergo CER-induced necroptotic cell death, thereby contributing to the increased placental dysfunction and cell death found in preeclampsia.
Highlights
Necroptosis is a mode of cell death that has overturned the traditional view of necrosis as merely a passive process
Necroptosis is typically initiated by stimuli such as tumor necrosis factor (TNF), Fas ligand or TNF-related apoptosis-inducing ligand (TRAIL) binding to their respective death domain-containing receptor at the cell membrane, leading to activation of receptorinteracting protein kinase 1 (RIP1).[2]
We have recently identified that disruption of sphingolipid metabolism leading to ceramide (CER) accumulation is responsible for increased trophoblast cell death in preeclampsia.[16]
Summary
Necroptosis is a mode of cell death that has overturned the traditional view of necrosis as merely a passive process. Placentae from preeclamptic women are characterized by excessive cell death, including apoptosis[13,14] and autophagy.[15,16,17] the involvement of necroptosis in placental cell death remains unknown. We have recently identified that disruption of sphingolipid metabolism leading to ceramide (CER) accumulation is responsible for increased trophoblast cell death in preeclampsia.[16] CER has a critical role in cell death, in the initiation of apoptosis and autophagy.[18,19] In this study, we sought to examine whether trophoblast cells undergo necroptosis under physiological and pathological conditions of pregnancy, and whether CER acts as a mediator of this process. Cell Death and Disease placentae exhibit higher rates of necroptosis compared with normotensive controls, and we propose that this results from the high CER levels and reduced caspase-8 activity present in these placentae
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