Suppression of StarD7 promotes endoplasmic reticulum stress and induces ROS production
Graphical abstract
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.
Section snippets
Antibodies
Mouse monoclonal anti-p-JNK (sc-6254) and anti-JNK (sc-1648); rabbit polyclonal anti-HO-1 (H-105) and goat anti-p53 (FL-393-G) were obtained from Santa Cruz Biotechnology. Rabbit monoclonal anti-phospho-eIF2α (Ser51) (3398) and rabbit polyclonal anti-total-eIF2α (9722) were from Cell Signaling Technology and anti-PARP from Roche. Mouse monoclonal anti-p21 (556,430) was from BD Pharmingen, and mouse monoclonal anti-α-tubulin (Clone B-5-1-2), mouse monoclonal anti-β-actin and rabbit polyclonal
Effects of StarD7 siRNA on HepG2 cell ultrastructure
siRNA gene silencing was used to deplete the expression of StarD7 and to determine its effect on cell ultrastructure. Compared with a non-targeting control siRNA (siC), treatment of HepG2 cells transfected for 72 h with two different specific siRNAs targeting human StarD7 mRNA (siD7 and siD7.2) resulted in a marked depletion of StarD7 protein (Fig. 1A and C). Accordingly, the expression of StarD7 mRNA was reduced by 80% in StarD7 knockdown cells (Fig. 1B). No changes in StarD7 levels were
Discussion
This study demonstrates that the depletion of StarD7 in HepG2 cells: (i) leads to alterations in mitochondria and ER morphology; (ii) causes ER stress response without alterations in apoptosis or authophagy markers; (iii) promotes the destabilization of p53; (iv) stimulates ROS generation in basal conditions; (v) reduces cell viability after H2O2 exposure; and (vi) increases HO-1 and catalase expression levels as well as catalase enzyme activity.
Alterations in the phospholipid metabolism,
Acknowledgements
We gratefully acknowledge Drs. José Bocco, Lucas Trucco, Cecilia Sanchez and Miriam Virgolini for reagents and helpful advice. Also we are very grateful to Dr. Héctor Alex Saka for the correction of the manuscript.
This work was funded by the Consejo Nacional de Investigaciones Científicas y Tecnológicas de Argentina (CONICET), the Agencia Nacional de Promoción Ciencia y Técnica (FONCYT) PICT 2011-0452, and the Secretaría de Ciencia y Técnica de la Universidad Nacional de Córdoba (SECyT-UNC).
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2022, Advances in Botanical ResearchRole of the lipid transport protein StarD7 in mitochondrial dynamics
2021, Biochimica et Biophysica Acta - Molecular and Cell Biology of LipidsCitation Excerpt :In addition, it has been recently reported that HTR-8/SVneo altered their mitochondrial activity during cell invasion induced by placenta-derived mesenchymal stem cells [46]. Here, using the trophoblast-derived HTR-8/SVneo cells, we have demonstrated that the interference with StarD7 expression critically affects not only the mitochondrial morphology as previously reported [26,28,29] but also the mitochondrial dynamics. A reduction in cellular StarD7 expression led to a decrease in Mfn1 and Mfn2 fusion proteins without modification of Drp1 protein level; in this condition, total ROS levels were increased as reported in HepG2 [29] and BEAS-2B cells [28], even though no significant change was detected in mitochondrial ROS.
The START-domain proteins in intracellular lipid transport and beyond
2020, Molecular and Cellular EndocrinologyCitation Excerpt :The mitochondrial integrity function was demonstrated by studies that showed BEAS-2B human bronchial epithelial cells and murine lung epithelial cells had enlarged mitochondria with less cristae, decreased respiration rates, and increased ROS levels when STARD7 was deleted (Yang et al., 2015, 2017). Similar results in STARD7-silenced mouse HEPA-1 or human HepG2 liver cell lines provided strong support for STARD7 maintaining mitochondrial PC levels (Horibata et al., 2016; Flores-Martín et al., 2016). Further, the decrease in mitochondrial PC was due to loss from the inner membrane (Rodriguez-Agudo et al., 2019; Malhi and Kaufman, 2011).