Taurine Attenuates Calpain-2 Induction and a Series of Cell Damage via Suppression of NOX-Derived ROS in ARPE-19 Cells

Abstract

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) are key transmembrane proteins leading to reactive oxygen species (ROS) overproduction. However, the detailed roles of NOXs in retinal pigment epithelial (RPE) cell metabolic stress induced by Earle's balanced salt solution (EBSS) through starvation remain unclear. In this study, we investigated what roles NOXs play in regard to calpain activity, endoplasmic stress (ER), autophagy, and apoptosis during metabolic stress in ARPE-19 cells. We first found that EBSS induced an increase in NOX2, NOX4, p22phox, and NOX5 compared to NOX1. Secondly, suppression of NOXs resulted in reduced ER stress and autophagy, decreased ROS generation, and alleviated cell apoptosis. Thirdly, silencing of NOX4, NOX5, and p22phox resulted in reduced levels of cell damage. However, silencing of NOX1 was unaffected. Finally, taurine critically mediated NOXs in response to EBSS stress. In conclusion, this study demonstrated for the first time that NOX oxidases are the upstream regulators of calpain-2, ER stress, autophagy, and apoptosis. Furthermore, the protective effect of taurine is mediated by the reduction of NOX-derived ROS, leading to sequential suppression of calpain induction, ER stress, autophagy, and apoptosis.

Figure 1

Earle's balanced salt solution (EBSS) increases NADPH oxidative activity and ROS generation in ARPE-19 cells in a time-dependent manner. (a–c) A Western blot analysis was carried out to detect the expression levels of NOXs. (d, e) Intracellular ROS was determined by flow cytometry. (f, g) Earle's balanced salt solution (EBSS) increases cytoplasmic calcium in ARPE-19 cells. The intracellular calcium was detected with Fluo-3 AM. TM stands for tunicamycin. The data are presented as the means ± SEM of three independent experiments. ^#p < 0.05 and ^##p < 0.01 compared to control (0 h).

Figure 2

ROS scavenging and NOX suppression attenuate EBSS-induced loss of calcium homeostasis and ER stress in ARPE-19 cells. (a–c) The expression levels of NOX proteins were examined by Western blot. (d–g) The expression levels of calpains and the ER stress-related proteins were examined by Western blot. The data are presented as the means ± SEM of three independent experiments. ^##p < 0.01 compared to the control group. ^∗p < 0.05 and ^∗∗p < 0.01 compared to the EBSS group.

Figure 3

ROS scavenging and NOX suppression attenuate EBSS-induced autophagy, apoptosis, and ROS generation. (a–d) The expression of autophagy-related proteins and apoptosis-related proteins was detected by Western blot. (e, f) Intracellular ROS was evaluated by flow cytometry. The data are presented as the means ± SEM of three independent experiments. ^##p < 0.01 compared to the control group. ^∗p < 0.05 and ^∗∗p < 0.01 compared to the EBSS group.

Figure 4

NOX4, p22phox, and NOX5 modulate loss of calcium homeostasis and ER stress in ARPE-19 cells. (a–c) Silencing of NOX2/NOX4/p22phox/NOX5 decreases its protein level. (d–g) The activities of ER stress and calpain-2 were suppressed by siRNA knockdown of NOX4, p22phox, and NOX5. The data are presented as the means ± SEM of three independent experiments. ^##p < 0.01 compared to the control group. ^∗p < 0.05 and ^∗∗p < 0.01 compared with the EBSS group.

Figure 5

NOX4, p22phox, and NOX5 modulate autophagy and apoptosis in ARPE-19 cells. (a–d) NOX4, p22phox, or NOX5 silencing inhibits autophagy and apoptosis. (e, f) siRNA knockdown of NOX4, p22phox, or NOX5 decreases cell apoptosis. (g, h) siRNA knockdown of NOX4, p22phox, or NOX5 decreases ROS production. Intracellular ROS was evaluated by flow cytometry. The data are presented as the means ± SEM of three independent experiments. ^##p < 0.01 compared to the control group. ^∗p < 0.05 and ^∗∗p < 0.01 compared to the EBSS group.

Figure 6

Effects of taurine on NOX expression and ROS generation induced by EBSS. (a–c) Western blot analysis was carried out to determine the expression of NOX proteins in ARPE-19 cells. (d, e) Intracellular ROS was evaluated by flow cytometry. (f, g) Confocal images of ROS labelled with DCFH-DA (green) and nuclear stained with DAPI (blue). Scale bar = 20 μm. The data are presented as the means ± SEM of three to five independent experiments. ^##p < 0.01 compared to the control group. ^∗p < 0.05 and ^∗∗p < 0.01 compared to the EBSS group. T: taurine.

Figure 7

Schematic diagram of the effects of taurine in ARPE-19 cells. The protective effect of taurine is regulated by the reduction of intracellular ROS from NOXs, leading, in turn, to sequential suppression of calpain induction, ER stress, autophagy, and apoptosis. NADPH oxidases (NOXs) are transmembrane proteins that are localized either in intracellular granules and vesicles or on the cell surface membranes.