DECIPHERING THE FUNCTION OF THE STOX1 PROTEIN IN THE MANAGEMENT OF OXIDATIVE STRESS IN TROPHOBLAST CELLS

Abstract

Preeclampsia is one of the major diseases of pregnancy, and a major concern for Gynecologists and Obstetricians throughout the World (6). It affects ~5% of pregnancies and is characterized by a pregnancy- induced hypertension accompanied by proteinuria, occurring from the second trimester of gestation. A large corpus of scientific literature connects preeclampsia with oxidative stress (4,5). Free oxygen species combine with Nitric Oxide to generate peroxinitrite ions (ONOO.), which will nitrate proteins. Treating rats with (L-NAME) triggers preeclamptic symptoms, showing the importance of NO as an endothelium protector able to alleviate preeclampsia (8). Preeclampsia has a genetic basis, as shown by epidemiological studies (1), and in 2005, the first gene variants causing preeclampsia were found in Dutch familial forms of preeclampsia in the transcription factor STOX1 (7). In 2013 we have shown that placental overexpression of STOX1 triggers preeclamptic symptoms in pregnant mice (3). A transcriptome study of transgenic and non-transgenic placentas revealed a massive deregulation of genes involved in mitochondrial function and oxidative stress (2) with a massive production of nitrated protein products of nitrosative stress in the transgenic placentas. These results were substantiated by a thorough analysis of mitochondrial physiology in human trophoblast cell models (JEG-3) overexpressing STOX1 or controls. In the present study, we measured by fluorescent markers four molecules involved in oxidative stress: NO, O2., H2O2 and GSH using Diaminofluorescein-2 Diacetate, Dihydroethidium, 2’,7’ Dichlorodihydrofluorescein diacetate, and Monochlorobimane, respectively. The analysis was performed on two control cell lines, a cell line overexpressing STOX1A (AA6) and one overexpressing STOX1B (B10). In addition, twelve drugs were used in order to selectively block specific mitochondrial and oxidative stress pathways. More specifically, O2. production was quenched using Allopurinol (inhibitor of xanthin oxidases), DPI (Inhibitor of NADPH oxidases), rotenone and antimycin (to block the mitochondrial respiratory chain). To evaluate the management of the oxidative stress at later stages, CDD (inhibitor of MnSOD), ATZ (inhibitor of catalase) and BSO (inhibitor of GSH) were used. MnTBAP (simulating MnSOD action), CuDIPS (simulating Cu/ZN SOD action), Catalase, Glutathion (GSH) and NAC were also added before monitoring the four outcomes. Finally the effects of ‘pseudo-hypoxia’ were evaluated by cultivating cells in the presence and in the absence of CoCl2.  Basically, when no drug was added to the culture, STOX1A overexpression increased the production of NO, O2. and H2O2, whereas STOX1B decreased the production of O2. and GSH. The same tendencies were observed after CoCl2 treatment, but at a minor extent. The overproduction of NO and O2. is compatible with the increase of nitrosative stress that we observed in transgenic placentas. Amongst various observations, the treatment of the cells with the different drugs indicated that the major source of O2. in STOX1A overexpressing cells are mitochondria. In the detoxification processes the glutathione appears crucial. Overall these experiments contribute to position STOX1 as a regulator of oxidative stress, which could explain its involvement in diseases such as preeclampsia and Alzheimer’s.