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 (P < 0.01) was induced by oxidative stress. Overexpression of TBP-2 further strengthens this response and worsens the cell viability (P < 0.01). 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.
Highlights
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]
When lens epithelial cells (LECs) were treated with 50 μM H2O2 for various time periods, cell viability loss was increased in a stepwise manner significantly (P < 0.01) (Figure 1(b))
We evaluated the cell viability and found that the cell viability loss significantly increased in Thioredoxin binding protein-2 (TBP-2) when compared to that of vector group (P < 0.01) (Figure 4(d)). mCherryLC3 puncta assay showed that TBP-2 (28.73 ± 1.606, N = 30) had significantly more mCherry-positive spots than that of vector group (18.40 ± 1.870, N = 30), which indicated higher level of autophagic flux when treated with H2O2 (P < 0.05) (Figure 4(e))
Summary
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]. 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]. TBP2 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]. TBP-2 suppresses cellular proliferation along with cell cycle arrest and has been reported as a tumor suppressor gene [9, 10]
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