Abstract
Purpose :
Long-lived lens fiber cells require strong cellular protective function against oxidative insults for maintaining their hemostasis and viability; however, the underlying molecular mechanism remainly large elusive. We hypothesize that mechano-activated connexin (Cx) hemichannels (HCs) and glutathione (GSH) transport protect lens fiber cells against oxidative insults.
Methods :
Site-directed mutagenesis was used to prepare recombinant RCAS(A) DNA constructs containing different Cx50 site mutations. High-titer recombinant RCAS(A) retrovirus was prepared and used to infect chick embryonic fibroblast cells and lens primary cells to express exogenous Cxs. Western blot was performed to determine expressions of Cxs. Fluid flow shear stress was used to apply mechanical stimulation. Cx HC activity was determined by Lucifer yellow/rhodamine dextran uptake assay. Hydrogen peroxide and ultraviolet B radiation were used to induce oxidative stress. Intracellular GSH and reactive oxygen species were determined by using commercial fluorescence-based kits. Cell apoptosis and necrosis were measured by Annexin V and PI staining. Single lens fiber cells were isolated by enzymatic digestion and cell dissociation.
Results :
We found that mechano-activated Cx HCs mediated the transport of GSH into lens fiber cells, leading to the decrease of the accumulation of intracellular reactive oxygen species induced by both hydrogen peroxide and ultraviolet B radiation, and protection of lens fiber cells against cell apoptosis and necrosis. In addition, HCs formed by both homomeric Cx50, Cx46 and heteromeric Cx50/Cx46 were mechanosensitive and facilitated the transport GSH into the cell. Among them, mechano-activated Cx50 HCs showed higher Km than Cx46 HCs (Km: Cx50: 0.72 ± 0.12 mM; Cx46: 0.43 ± 0.14 mM) and Vmax (Cx50: 44.50 ± 2.00 AU/20 min; Cx46: 17.18 ± 1.34 AU/20 min) in transporting GSH. Consistently, deficiency of Cx50 in single lens fiber cells exhibited a higher level of oxidative stress. Moreover, outer cortical short lens fiber cells expressing full length Cxs were more resistant to oxidative injury than central core long lens fibers
Conclusions :
Activation of Cx HCs by interstitial fluid flow in lens fiber cells is essential in cellular uptake of reductants and protection of lens fiber cells against oxidative stress-induced cell death.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.