Excessive ER-phagy mediated by FAM134B contributes to trophoblast cell mitochondrial dysfunction in preeclampsia

Abstract

Autophagy dysregulation and Ca2+-induced mitochondrial dysfunction in trophoblast cells are proposed to contribute to preeclampsia (PE) development. FAM134B is identified as a receptor associated with endoplasmic reticulum autophagy (ER-phagy). In this study, the placentas of normal pregnant women and PE patients are collected and analyzed by immunohistochemistry, quantitative real-time PCR, and western blot analysis. The effects of ER-phagy are investigated in HTR8/SVneo cells. Significantly increased levels of FAM134B, inositol-1,4,5-triphosphate receptor type 1 (IP3R), calnexin, cleaved caspase 3 and cytochrome C are detected in the PE placenta and sodium nitroprusside (SNP)-treated HTR-8/SVneo cells. Overexpression of FAM134B in HTR-8/SVneo cells results in increased apoptosis, impaired invasion capacity, and diminished mitochondrial function, while an autophagy inhibitor improves mitochondrial performance. Excessive ER-phagy is also associated with an increased concentration of gamma linolenic acid. Our findings suggest that FAM134B contributes to trophoblast apoptosis by mediating ER-mitochondria Ca2+ transfer through mitochondria-associated endoplasmic reticulum membranes (MAMs) and subsequent mitochondrial function, further enhancing our understanding of PE etiology.

1. Introduction

Preeclampsia (PE), a pregnancy-specific disease characterized by hypertension, proteinuria, and multiple organ dysfunction, affects up to 10% of pregnancies worldwide [1]. The primary cause of PE is placental malformation, which is resulted from vascular dysfunction and hypertrophic trophoblastic apoptosis [2–10]. Therefore, maintaining normal trophoblast function plays a vital role in preventing PE.

It is recognized that shallow trophoblast invasion is associated with mitochondrial apoptosis [11,12]. One type of mitochondrial apoptosis is induced by the entry of Ca2+, which relies on the sustained increase in intracellular Ca2+ [13]. The endoplasmic reticulum (ER) is where intracellular Ca2+ is stored [14]. The physical communication site between the ER and mitochondria is defined as mitochondria-associated endoplasmic reticulum membranes (MAMs), which are conserved structures in eukaryotes. MAMs regulate the survival and death of cells by controlling Ca2+ transfer via inositol-1,4,5-triphosphate receptor type 1 (IP3R) and the transport of other metabolites [15]. In recent years, studies have shown that MAMs are essential for autophagy by mediating the formation of autophagosomes, of which cytochrome C (Cyt C) is a biomarker [16]. Autophagy is imperative for maintaining homeostasis and plays a significant role in the onset and succession of PE [17]. Previous studies have revealed that FAM134B (family with sequence similarity 134, member B) is an ER receptor that binds to the autophagy modifiers LC3 and GABARAP, promoting ER-phagy. Overexpression of FAM134B triggers ER fragmentation, whereas downregulation of FAM134B expression in cells results in ER expansion [18]. In addition, FAM134B acts as a suppressor or promoter in different cancer types, such as esophageal cancer [19], colon cancer, and breast cancer [20]. The metabolic function of FAM134B in trophoblast cells has not been investigated despite its known role in tumor cell invasion, migration, and proliferation [21]. Furthermore, maternal lipid metabolism undergoes significant and rapid changes across gestation to fulfil the requirements for fetal development and growth [22]. Fatty acid induces apoptosis and oxidative stress and elevates the cytosolic level of oxidized mitochondrial DNA to activate the inflammasome [21]. However, the underlying mechanism mediated by FAM134B remains poorly understood.

In the present study, we aimed to investigate the role of FAM134B in placentas and HTR-8/SVneo cells, a cell line similar to human extravillous trophoblasts (EVTs). FAM134B expression, as well as its association with changes in fatty acids, was characterized, particularly with regard to its relationship with ER-phagy, Ca2+ flux, and mitochondrial apoptosis.