Abstract
Purpose :
Increased oxidative stress is a major cause for the development of age-related cataracts. Lens is formed by anterior epithelial cells and bulky lens fiber cells. However, the role of lens epithelial cells in preserving homeostasis under oxidative stress remains unclear. We hypothesize that connexin hemichannels mediate transport of H2O2 in lens epithelial cells and activate glutathione redox cycle, resulting in reduced reactive oxygen species (ROS) and protection against oxidative stress-induced apoptosis.
Methods :
H2O2 and UVB irradiation were used to induce oxidative stress in both lens epithelial cell line (HLE-B3) and chick primary lens epithelial cells (CLECs). Immunofluorescence and western blot were used to determine connexin expression in these cell models. Cx43 hemichannel activity was determined by ethidium bromide uptake combined with the specific Cx43 hemichannel-blocker, Cx43(E2) antibody. Annexin V and propidium iodide staining were performed to measure the extent of apoptosis and necrosis, respectively. ROS, H2O2 and GSH levels were measured using commercial fluorescence-based assays.
Results :
Among three connexins expressed in the lens, we found rich presence of Cx43 in both HLE-B3 cells and CLECs. H2O2 and UVB induced dye uptake in both cell types, which can be specifically inhibited by Cx43(E2) antibody, suggesting the activation of hemichannels. Inhibition of Cx43 hemichannels by Cx43(E2) antibody led to increased H2O2 or UVB-induced apoptotic cell death compared to non-treated controls. Interestingly, after H2O2 treatment, the initial intracellular H2O2 level was significantly lower in cells with the inhibition of hemichannels, which indicates H2O2 transport activity of hemichannels. However, after initial increase, the H2O2 level rapidly declined in cells with active hemichannel, but not in cells with inhibited hemichannels. Consistently, we observed the sustained, elevated intracellular ROS levels and decreased GSH levels in lens epithelial cells when Cx43 hemichannels were blocked.
Conclusions :
Our results showed that Cx43-hemichannel protect cells from oxidative-induced cell death by mediating H2O2 transport. Modulating intracellular oxidant levels may help reduce extracellular oxidative stress and adaptively adjust intracellular redox level, leading to protecting lens epithelial cells against oxidative damages.
This is a 2020 ARVO Annual Meeting abstract.