Mitochondrial fusion protein 2 regulates endoplasmic reticulum stress in preeclampsia

Mitochondrial fusion protein 2 regulates endoplasmic reticulum stress in preeclampsia 线粒体融合蛋白2（Mfn2）调节内质网应激在子痫前期的作用及其机制研究 1 2 3 first-author 1 1 1 1 1 corresp Jihua JIN, s18715296792@163.com Jihua JIN, s18715296792@163.com s18715296792@163.com https://orcid.org/0000-0002-3450-1298 Department of Obstetrics, Jiaxing Municipal Maternal and Child Health Care Hospital, Jiaxing314051, China Department of Obstetrics, Jiaxing Municipal Maternal and Child Health Care Hospital, Jiaxing314051, China Department of Obstetrics, Women and Children Hospital Affiliated to Jiaxing University, Jiaxing314051, China Department of Obstetrics, Women and Children Hospital Affiliated to Jiaxing University, Jiaxing314051, China Department of Obstetrics, Jiaxing Women and Children Hospital Affiliated to Wenzhou Medical University, Jiaxing314051, China Department of Obstetrics, Jiaxing Women and Children Hospital Affiliated to Wenzhou Medical University, Jiaxing314051, China 目的 2 探究线粒体融合蛋白2（Mfn2）调控的内质网应激在子痫前期发生中的作用机制，为临床子痫前期的诊断和治疗提供参考。 创新点 2 本论文着眼于滋养层细胞的内质网应激在子痫前期中具有重要作用这一研究热点，通过对临床样本的检测和细胞水平的研究，阐明了Mfn2调控的内质网应激在子痫前期发生中的重要作用，初步确定该基因可能作为子痫前期的诊断和治疗靶点。该研究具有针对性强、创新高以及满足现实发展需求的意义。 方法 2 采用蛋白质免疫印迹法检测子痫前期和正常胎盘组织中Mfn2的表达差异；活细胞计数（CCK8）法、细胞划痕、Transwell、流式细胞术等实验方法分别用于检测Mfn2敲低对滋养层细胞JEG-3增殖、迁移、侵袭和凋亡的影响。蛋白质免疫印迹法检测内质网应激条件下滋养层细胞中Mfn2的表达，并通过CCK8、流式细胞术和超氧化物歧化酶（SOD）的测定检测Mfn2过表达后对内质网应激条件下滋养层细胞的细胞活力和氧化应激的改善情况。 结论 2 Mfn2在子痫前期胎盘组织中低表达，下调Mfn2可显著抑制滋养层细胞的增殖、迁移和侵袭。子痫前期下内质网应激将抑制Mfn2的表达，Mfn2的稳定表达可以通过调节内质网应激抑制子痫前期发生和发展。 线粒体融合蛋白2（Mfn2） 内质网应激 子痫前期 Preeclampsia (PE) refers to a group of dysfunction syndromes associated with elevated blood pressure and proteinuria in women with previously normal blood pressure after 20 weeks of pregnancy, and it may be accompanied by symptoms including headache, vertigo, nausea and vomiting, and epigastric discomfort ( Steegers et al., 2010 Steegers et al., 2010 ). It is affected by a variety of risk factors and follows a distinctive disease progression, although its pathogenesis is still unknown. Upon termination of a PE pregnancy, the clinical symptoms of PE improve rapidly, suggesting that PE is a disease originating in the placenta. Recent studies have shown that excessive apoptosis of trophoblast cells and reduced trophoblast infiltration capacity can lead to superficial implantation of the placenta, a lack of spiral arteriole remodeling, and insufficient placental perfusion leading to placental ischemia and hypoxia, all of which are important factors that cause the onset of PE ( Eddy et al., 2019 Eddy et al., 2019 ). Therefore, there appears to be a close relationship between excessive trophoblast cell apoptosis and development of PE. Recently, increasing evidence has shown that in patients with PE, endoplasmic reticulum (ER) stress can cause trophoblast cell apoptosis, and this finding has become an important feature of the placental pathology of PE ( Lorenzon-Ojea et al., 2020 Lorenzon-Ojea et al., 2020 ). Mitochondrial fusion protein 2 (Mfn2) is a mitochondrial membrane protein, which is involved in the promotion of mitochondrial fusion, the regulation of mitochondrial morphology and function, cell energy metabolism, signal transduction, proliferation, apoptosis, and many other cellular processes ( Chan, 2006 Chan, 2006 ). The latest research shows that Mfn2 also plays a key role in a variety of ER functions, such as calcium balance and protein synthesis ( Chandhok et al., 2018 Chandhok et al., 2018 ). More than this, studies have found that Mfn2 expression is abnormal in the tissues of patients with PE and that Mfn2 plays an important role in embryo development during the first trimester ( Yu et al., 2016 Yu et al., 2016 ). However, while Mfn2 protein expression levels are known to be altered in the placentas of pregnant women with PE, the role of Mfn2 in trophoblasts and the question of whether it can regulate ER stress to inhibit PE remain unclear. To explore the role of Mfn2 in PE, we analyzed the expression levels of Mfn2 in samples from 20 women with normal pregnancies and 20 women with severe PE using western blot. The clinical characteristics of all patients are summarized in Table 1 Table 1 . The results indicate that the expression of Mfn2 was lower in PE tissues than in normal tissues ( Fig. 1 Fig. 1 ), meaning that decreased Mfn2 levels may be associated with the development of PE. Furthermore, we transfected JEG-3 cells with either a small interfering RNA (siRNA) control or siRNA-Mfn2. The expression levels of Mfn2 were significantly down-regulated in cells transfected with siRNA-Mfn2 compared with the siRNA control group cells after 48 h ( Fig. 2 Fig. 2 a). The cholecystokinin-8 (CCK8) and flow cytometry assays showed that decreasing Mfn2 levels inhibited the proliferation ( Fig. 2 Fig. 2 b) and promoted the apoptosis ( Fig. 2 Fig. 2 e) of JEG-3 cells, while wound healing assays revealed that decreased expression of Mfn2 greatly inhibited the migration of JEG-3 cells ( Fig. 2 Fig. 2 c). In addition, we further tested the effect of Mfn2 on the invasion capacity of trophoblast cells. As shown in Fig. 2 Fig. 2 d, decreased expression of Mfn2 markedly repressed the invasive capacity of JEG-3 cells. Meanwhile, we obtained similar results in JAR cells (Fig. S1). These findings indicate that decreased Mfn2 expression may effectively suppress the proliferation, migration, and invasion of trophoblast cells in vitro. Data (except number, fetal gender, and P ) were expressed as mean±standard deviation. 1 mmHg=0.133 kPa. 2 Group 2 Number 2 Age (year) 2 Gestational age at delivery (week) 4 Blood pressure Systolic (mmHg) Diastolic (mmHg) Birth weight (g) Fetal gender, male/female Preeclampsia 20 30.65±5.16 33.35±3.28 157.45±19.84 99.70±7.36 2445.00±530.62 8/12 Normal 20 29.20±4.27 39.10±0.85 118.95±12.40 71.85±10.64 3626.00±387.49 11/9 P 0.339 <0.001 <0.001 <0.001 <0.001 0.355 ER stress is a characteristic pathological feature of PE ( Chaiworapongsa et al., 2014 Chaiworapongsa et al., 2014 ). To determine whether ER stress affects Mfn2 expression, JEG-3 cells were treated with different concentrations of tunicamycin (Tm) in culture for 24 h, after which the protein expression levels of Mfn2 and ER stress markers such as protein kinase R-like ER kinase (PERK), CCAAT/enhancer-binding protein homologous protein (CHOP), and 78-kDa glucose-regulated protein (GRP78) were detected by western blot, while quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to determine the expression level of Mfn2 messenger RNA (mRNA). The results of these analyses showed that when compared with untreated controls, the expression of Mfn2 in JEG-3 cells was significantly reduced under conditions of ER stress ( Fig. 3 Fig. 3 ). Also, to further determine whether Mfn2 supports trophoblast cell viability and inhibits accumulation of cellular oxidative stress during ER stress, we established a stable Mfn2-overexpression JEG-3 cell line. Puromycin was used to select stably transfected cells. The overexpression of Mfn2 was confirmed by western blot analysis ( Fig. 4 Fig. 4 a). The stable cell lines were then incubated with 2.5 μmol/L Tm for 24 h. Following treatment, cell viability and oxidative stress levels in trophoblast cells were measured using a CCK8 assay ( Fig. 4 Fig. 4 b), flow cytometry ( Fig. 4 Fig. 4 d), and a superoxide dismutase (SOD) detection assay ( Fig. 4 Fig. 4 c). The results of these analyses indicated that Mfn2 overexpression can increase cell viability and inhibit accumulation of cellular oxidative stress in trophoblast cells in the context of ER stress. At present, there are many hypotheses regarding the pathogenesis of PE revolving around immunological factors, genetic influences, oxidative stress, and vascular damage, but these hypotheses cannot fully explain the pathological progression of PE. The symptoms of PE significantly worsen as pregnancy progresses, but upon delivery of the placenta, they improve rapidly, suggesting that PE is related to the presence of the placenta. Placental factors are also considered to be an important cause of PE onset ( Chaiworapongsa et al., 2014 Chaiworapongsa et al., 2014 ). Women suffering from placental trophoblastic disease can have symptoms similar to those of PE, which further suggests that placental trophoblast function is related to the onset of PE. In early pregnancy, the remodeling of spiral arterioles depends on the apoptosis of vascular endothelial cells and smooth muscle cells mediated by extravillous trophoblasts (EVTs). The role EVTs play in promoting the invasion of the spiral arteries, and the destruction of the vascular wall is similar to that of malignant tumor cells, but this invasive behavior is restricted to the first trimester and the inner 1/3 of the myometrium ( Genbacev and Miller, 2000 Genbacev and Miller, 2000 ). When EVTs undergo excessive apoptosis, their invasive ability is weakened, leading to insufficient remodeling of spiral arterioles and shallow implantation of the placenta and also causing local trophoblast cells to be ischemic and hypoxic in the placenta; this drives the development of the first stage of PE. Placental ischemia and hypoxia caused by insufficient spiral arteriole remodeling can induce syncytiotrophoblast cell apoptosis through various apoptotic pathways. This releases syncytiotrophoblast microparticles (STBMs) into the maternal bloodstream, causing inflammation throughout the body. Eventually, the inflammation damages the vascular endothelial cells, causing various clinical symptoms of more progressed PE ( Brosens et al., 2019 Brosens et al., 2019 ). The reduced invasive ability of placental trophoblast cells, along with placental ischemia and hypoxia, is regarded as key events in PE. Excessive apoptosis severely diminishes the invasive ability of trophoblast cells. Trophoblast apoptosis is widespread in placental tissues at different stages of normal pregnancy, making trophoblast apoptosis essential for a normal healthy pregnancy, and this apoptosis is strictly regulated by a series of mechanisms to ensure the normal progression of the pregnancy. However, pathological apoptosis can lead to the occurrence of various diseases. Previous studies examining the mechanisms of apoptosis and related signaling pathways have focused on mitochondrial damage (intrinsic pathway) and extracellular death receptors (extrinsic pathway). In recent years, some studies have reported that the ER may be another important organelle that induces apoptosis and this type of apoptosis may not depend on mitochondria or death receptor pathways. There are many chaperone proteins and folding enzymes present in the ER. When the ER is stimulated by various pathological factors, unfolded or misfolded proteins in the ER lumen will aggregate and damage the normal physiological functions of the cell, leading to ER stress. As a new mechanism of apoptosis, the ER stress apoptotic pathway has been found to be closely related to degenerative neurological diseases, cancers, and osteoporosis. However, there are few studies on the relationship between ER stress-related apoptosis and PE. Burton et al. (2010) Burton et al. (2010) have reported that there is oxidative stress in the placental tissue of patients with PE, and that induction of sustained ER stress will promote excessive apoptosis of trophoblast cells, thereby resulting in maternal-fetal injury. Mfn2 is located on the outer mitochondrial membrane and plays a key role in fusion for this membrane. Chan (2006) Chan (2006) has found that Mfn2 not only participates in the regulation of mitochondrial morphology, but also plays key roles in cell metabolism, proliferation, and apoptosis. Recent studies have also shown that Mfn2 can regulate the morphology of the ER and participate in the regulation of ER stress. Mfn2 expression is increased in the late stages of ER stress, while other mitochondrial fusion-related proteins such as Mfn1, optic atrophy 1 (OPA1), dynamin-related protein 1 (Drp1), and mitochondrial fission 1 (Fis1) are not significantly changed ( Ngoh et al., 2012 Ngoh et al., 2012 ). In Mfn2-knockout mouse embryonic fibroblasts and adult mouse cardiomyocytes, the expression of intracellular chaperone proteins was increased ( Song et al., 2015 Song et al., 2015 ), indicating that Mfn2 can regulate ER stress. Related studies found evidence that mitochondrial dysfunction can also induce ER stress. In order to investigate whether Mfn2 is involved in directly regulating ER stress or indirectly regulating it by modulating mitochondrial morphology and function, Debattisti et al. (2014) Debattisti et al. (2014) used Drosophila with either an OPA1 knockout (OPA1 is an important protein for mitochondrial inner membrane fusion) or an Mfn2 knockout, and found that only the Mfn2 knockout induced ER stress in the flies, indicating that Mfn2 may directly participate in the regulation of ER stress. Muñoz et al. (2013) Muñoz et al. (2013) further demonstrated that Mfn2 can bind to PERK and inhibit its function. When Mfn2 levels are decreased, PERK is continuously activated, resulting in ER stress. Similarly, Mfn2 knockout has also been shown to induce ER stress in hypothalamic anorexigenic pro-opiomelanocortin (POMC) neurons ( Schneeberger et al., 2013 Schneeberger et al., 2013 ). Although previous studies have shown that the expression of Mfn2 in the tissues of patients with PE is abnormal, these studies focused on the mRNA expression level ( Yu et al., 2016 Yu et al., 2016 ). The protein levels of Mfn2 in the placenta of patients with PE and the potential effects of its expression on the biological phenotype of trophoblast cells have not been studied. In the present study, we analyzed the expression of Mfn2 in PE tissues and found that Mfn2 protein expression was significantly decreased in PE tissues compared to healthy placental tissues. We also examined the effects of Mfn2 knockdown on proliferation, invasion, and metastasis of trophoblast cells. The expression of Mfn2 in trophoblasts was significantly decreased following the induction of ER stress via Tm treatment, and the overexpression of Mfn2 rescued trophoblast cell viability and could inhibit accumulation of cellular oxidative stress during ER stress; together, these findings suggest that aberrant placental Mfn2 expression may be involved in the pathogenesis of PE. However, the present study has some limitations. First, we focused only on the influence of Mfn2 on trophoblast cells in vitro and thus were unable to draw conclusions on the effects of Mfn2 in an animal or in vivo model of PE. Second, the study did not thoroughly analyze the molecular mechanism of Mfn2 which regulates ER stress in PE. In addition, the present study was carried out with a relatively small sample size, which only included women with severe PE. It is unknown whether Mfn2 is also decreased in other kinds of PE, such as mild and critical cases. 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Dandan SUN searched the literature, collected the data, and drafted the manuscript. Hanbing WU, Ling AI, Yanting WU, and Hui ZHU performed the statistical analysis. All authors have read and approved the final paper, and they have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the Welfare Technology-Applied Research Project of Zhejiang Province (No. LGF20H 040011), China. 10.1631/jzus.B2000557 1673-1581（2021）02-0165-06 SUN Dandan,ZHU Hui,AI Ling et al., 2021. Mitochondrial fusion protein 2 regulates endoplasmic reticulum stress in preeclampsia. Journal of Zhejiang University-SCIENCE B(Biomedicine & Biotechnology), 22(2):165-170. SUN Dandan,ZHU Hui,AI Ling et al., 2021. Mitochondrial fusion protein 2 regulates endoplasmic reticulum stress in preeclampsia. Journal of Zhejiang University-SCIENCE B(Biomedicine & Biotechnology), 22(2):165-170. 2020-09-17 2020-11-11 2021-02-15 2022-05-09 © Zhejiang University Press 2021 Dandan SUN, Hui ZHU, Ling AI, Hanbing WU, Yanting WU, and Jihua JIN declare that they have no conflict of interest. The present study was approved by the Ethics Committee of Jiaxing Municipal Maternal and Child Health Care Hospital (Jiaxing, China). All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study. Additional informed consent was obtained from all patients for whom identifying information is included in this article. Compliance with ethics guidelines Supplementary information 1 Materials and methods; Fig. S1 浙江大学学报（英文版）（B辑：生物医学和生物技术） 2 22