Thioredoxin Binding Protein-2 Regulates Autophagy of Human Lens Epithelial Cells under Oxidative Stress via Inhibition of Akt Phosphorylation
Thioredoxin Binding Protein-2 Regulates Autophagy of Human Lens Epithelial Cells under Oxidative Stress via Inhibition of Akt Phosphorylation
Jiaojie Zhou,1,2 Ke Yao,1 Yidong Zhang,1 Guangdi Chen,3 Kairan Lai,1 Houfa Yin,1 and Yibo Yu1
1Eye Center, 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
2Department of Otolaryngology, 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
3Department of Public Health, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
Received 23 May 2016; Accepted 3 August 2016
Academic Editor: Durga N. Tripathi
Copyright © 2016 Jiaojie Zhou et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Oxidative stress plays an essential role in the development of age-related cataract. Thioredoxin binding protein-2 (TBP-2) is a negative regulator of thioredoxin (Trx), which deteriorates cellular antioxidant system. Our study focused on the autophagy-regulating effect of TBP-2 under oxidative stress in human lens epithelial cells (LECs). Human lens epithelial cells were used for cell culture and treatment. Lentiviral-based transfection system was used for overexpression of TBP-2. Cytotoxicity assay, western blot analysis, GFP/mCherry-fused LC3 plasmid, immunofluorescence, and transmission electronic microscopy were performed. The results showed that autophagic response of LECs with increased LC3-II, p62, and GFP/mCherry-LC3 puncta () was induced by oxidative stress. Overexpression of TBP-2 further strengthens this response and worsens the cell viability (). Knockdown of TBP-2 attenuates the autophagic response and cell viability loss induced by oxidative stress. TBP-2 mainly regulates autophagy in the initiation stage, which is mTOR-independent and probably caused by the dephosphorylation of Akt under oxidative stress. These findings suggest a novel role of TBP-2 in human LECs under oxidative stress. Oxidative stress can cause cell injury and autophagy in LECs, and TBP-2 regulates this response. Hence, this study provides evidence regarding the role of TBP-2 in lens and the possible mechanism of cataract development.
1. Introduction
Age-related cataract is a multifactorial disease that plays a leading role in causing visual impairment (18.4%) and blindness (33.4%) in the world [1]. Although the molecular mechanisms of cataractogenesis still remain unclear, possible risk factors include ultraviolet rays, ionizing radiation, and chemical. All of these environmental factors generate reactive oxygen species (ROS) and disrupt the redox status in human lens epithelial cells (LECs) [2].
Thioredoxin (Trx) is a 12-KD protein which is ubiquitously expressed in all the living cells. Trx reduces a variety of substances to maintain the intracellular redox balance [3, 4]. Thioredoxin binding protein-2 (TBP-2), which is initially known as Vitamin D3 Upregulated Protein 1 (VDUP1) [5], can be regulated by mechanical stress, UV light, heat shock, hypoxia, H2O2, NO, glucose, and insulin [3]. TBP-2 is a negative regulator of the reducing capacities of Trx. TBP-2 binds with the reduced form of Trx and forms an intermolecular disulfide bond at the redox-active catalytic domain [6]. TBP-2 acts as a central regulator of cellular signaling pathways involved in the oxidative stress mechanism. It competes with peroxiredoxin (Prx) and apoptosis signal regulating kinase 1 (ASK1) to bind with Trx [7]. The binding of TBP-2 inactivates Trx, which breaks the Trx-ASK1 complex, allowing for the reactivation of ASK1 activity inducing apoptosis through activating JNK and p38 cascades [8]. Moreover, TBP-2 suppresses cellular proliferation along with cell cycle arrest and has been reported as a tumor suppressor gene [9, 10].
In human lens, previous studies showed that overexpression of TBP-2 increases cells’ apoptosis under oxidative stress, which raises the question of whether TBP-2 might play an important role in cataract development [11]. Further investigation is needed to know the influence of TBP-2 on other aspects of lens cells and its potential therapeutic value in cataract.
Autophagy is a kind of cellular degradation pathway that maintains cellular homeostasis through degradation of intracellular proteins, lipids, and organelles in response to various environmental conditions [12]. Basal autophagy exists in all living cells at a relatively low level and is important for the prevention of aging [13]. Autophagy can be stimulated in numerous conditions, such as nutritional starvation, endoplasmic reticulum (ER) stress [14], increased reactive oxygen species (ROS) [15], reactive nitrogen species (RNS) [16], and senescence [17]. Depending on the circumstances, autophagy sometimes plays a protective role and sometimes accelerates cell damage and de (...truncated)