Suppression of StarD7 promotes endoplasmic reticulum stress and induces ROS production

Highlights

• StarD7 siRNA leads to alterations in mitochondria and ER morphology.

• StarD7 siRNA causes ER stress response with no apoptosis or autophagy activation.

• StarD7 siRNA promotes destabilization of p53.

• StarD7 siRNA reduces cell viability after H₂O₂ exposure.

• StarD7 siRNA increases HO-1 and catalase protein levels as well as catalase activity.

Abstract

StarD7 is an intracellular lipid transport protein identified as up-regulated in the choriocarcinoma JEG-3 cell line. StarD7 facilitates the delivery of phosphatidylcholine (PC) to the mitochondria, and StarD7 knockdown causes a reduction in phospholipid synthesis. Since inhibition of PC synthesis may lead to endoplasmic reticulum (ER) stress we hypothesized that StarD7 may be involved in maintaining cell homeostasis. Here, we examined the effect of StarD7 silencing on ER stress response and on the levels of reactive oxygen species (ROS) in the human hepatoma cell line HepG2. StarD7 knockdown induced alterations in mitochondria and ER morphology. These changes were accompanied with an ER stress response as determined by increased expression of inositol-requiring enzyme 1α (IRE1α), calnexin, glucose regulated protein 78/immunoglobulin heavy chain-binding protein (Grp78/BiP), protein kinase-like ER kinase (PERK) as well as the phosphorylated eukaryotic translation initiation factor 2, subunit 1α (p-eIF2α). Additionally, a downregulation of the tumor suppressor p53 by a degradation mechanism was observed in StarD7 siRNA cells. Furthermore, StarD7 silencing induced ROS generation and reduced cell viability after H₂O₂ exposure. Decreased expression of StarD7 was associated to increased levels of the heme oxygenase-1 (HO-1) and catalase enzymes as well as in catalase enzymatic activity. Finally, no changes in levels of autophagy and apoptosis markers were observed in StarD7 siRNA treated cells respect to control cells. Taken together, these results indicate that StarD7 contributes to modulate cellular redox homeostasis.

Introduction

StarD7 belongs to the family of START proteins ubiquitously expressed, which are implicated in lipid transport, metabolism, and signaling [1], [2]. StarD7 mRNA was first identified using differential display techniques as a JEG-3 over-expressed gene compared with normal and benign trophoblastic samples [3], [4]. Semiquantitative RT-PCR assays performed in a series of cell lines have shown that StarD7 has widespread expression, predominantly in trophoblast-derived JEG-3, JAR, and HTR8/SVneo cells, as well as in hepatocellular carcinoma HepG2 cells, colorectal adenocarcinoma HT29 and Caco-2 cells [3], [4]. Horibata and Sugimoto demonstrated that StarD7 facilitates the delivery of phosphatidylcholine (PC) to the mitochondria in hepatoma HEPA-1 cells, and suggested that StarD7 extracts PC from the cytoplasmic surface of the ER, Golgi apparatus, or plasma membrane [5]. In addition, we have reported that StarD7 siRNA-transfected JEG-3 cells show decreased phospholipid biosynthesis and ABCG2 expression as well as a reduction in cell migration and proliferation. These observations were accompanied by higher expression of biochemical and morphological differentiation markers [6]. Furthermore, StarD7 gene promoter is activated by Wnt/β-catenin signaling [7], a pathway that promotes proliferation and is frequently altered in cancer cells. Consistently, Ikeda et al. found that miR-193b inhibits pancreatic cancer cell proliferation concomitantly with its ability to target and downregulate StarD7 transcript expression [8]. Additionally, genome wide analysis has shown that StarD7 transcript levels are altered in various conditions such as metabolic state, inflammation processes, and cancer suggesting that its expression must be tightly controlled in normal cell physiology [9]. Recently, Yang et al. demonstrated that the viability of StarD7^−/− embryos was dramatically decreased after embryonic day 10 highlighting an important role of this protein in development [10].

The endoplasmic reticulum (ER) is a compartment composed of a complex network of tubular and planar membranes where the synthesis and export of proteins, carbohydrates, and lipids take place coordinating diverse signaling pathways that regulate cell metabolism, proliferation and death. PC is the most abundant phospholipid in eukaryotic cell membranes synthesized mainly in the ER. The inhibition of PC synthesis results in altered ER morphology leading to the induction of a regulatory program triggered by ER stress, namely “the unfolded protein response” (UPR) [11], [12]. The UPR is an adaptive response composed of three main signaling pathways involving the ER transmembrane sensors: inositol-requiring enzyme 1α (IRE1α), activating transcription factor-6 (ATF6), and protein kinase-like ER kinase (PERK). Each signaling pathway activates transcription factors that mediate the induction of a variety of ER stress response genes connected with ER-associated protein degradation and protein translation attenuation. IRE1α, ATF6 and PERK sense ER stress through Grp78/BiP binding/release via their respective lumenal domains [13]. The UPR inhibits global cap-dependent protein synthesis via PERK-phosphorylated eukaryotic translation initiation factor 2, subunit 1α (p-eIF2α), promotes the upregulation of ER chaperons, and favors the elimination of misfolded proteins via ATF6 and IRE1α. If these adaptive mechanisms are not sufficient to alleviate ER stress, then activation of an apoptotic program involving caspases 7, 12, and 3, JNK, and p53 signaling pathways is initiated [13]. p53 tumor suppressor is a nuclear protein that functions as a regulator of transcription and mediates several biological effects, such as growth arrest, senescence, and apoptosis in response to various forms of stress [14], [15]. Depending on the experimental conditions, ER stress was reported to facilitate decreased [16], [17] or increased p53 levels or activity [18], [19], [20], [21]. Moreover, it was recently reported that p53 regulates ER function in response to stress [22].

Considering the observations that i) StarD7 binds PC, ii) depletion of StarD7 leads to decreased phospholipid biosynthesis, iii) PC synthesis inhibition generates ER stress, and iv) StarD7 increased expression is associated with cancer and cell proliferation, we hypothesize that changes in StarD7 expression causes a dysregulation in cellular homeostasis.

In this study, we demonstrate that StarD7 silencing alters mitochondria and ER morphology initiating an UPR pathway concomitant with increased degradation of p53 protein and ROS generation. We speculate that beyond its role in lipid transport, StarD7 contributes to modulate cellular redox homeostasis.