Abstract
Purpose :
Exposure to ultraviolet B (UVB) radiation causes a K+ efflux from corneal epithelial cells due to activation of K+ channels. This loss of intracellular K+ is an early step in UVB-induced apoptosis, and inhibition of K+ efflux results in decreased rates of apoptosis in corneal epithelial cells following UVB exposure (Ubels et al. Exp Eye Res. 92:145; 145:26). Ligand-independent activation of tumor necrosis factor receptor-1 (TNF-R1) by UVB results in apoptosis (Sheikh et al. Oncogene 17:2555). This suggests that UVB may activate K+ channels via TNF-R1. This study was designed to investigate the roles of TNF-R1 and Fas-associated protein with death domain (FADD), which is activated by TNF-R1, in the initiation of the UVB-induced K+ efflux.
Methods :
Ion chromatography was used to measure K+ loss from human corneal limbal epithelial (HCLE) cells following exposure to 150 mJ/cm2 UVB radiation or incubation with 50 ng/mL TNF-α. Activation of K+ channels by 80 mJ/cm2 UVB was measured by whole-cell voltage-clamp current recordings using standard amphotericin-B perforated patch techniques. siRNA was used to knock down TNF-R1 and FADD in HCLE cells.
Results :
Exposure to UVB caused an increase in K+ channel currents in less than 5 minutes. Knockdown of TNF-R1 resulted in a 50% reduction in UVB-induced K+ current, while FADD knockdown resulted in complete inhibition of UVB-induced K+ channel activation. Cells exposed to UVB lost 45% of intracellular K+ within 20 minutes of exposure. After TNF-R1 knockdown UVB-induced K+ loss was eliminated, while FADD knockdown reduced UVB-induced K+ loss to 15%. Cells incubated with TNF-α for 20 minutes exhibited a 17% reduction of intracellular K+. These cells recovered normal intracellular K+ levels by 45 minutes.
Conclusions :
The data suggest that UVB directly activates TNF-R1, which in turn may activate K+ channels via FADD. This conclusion is supported by the fact that TNF-α also causes loss of intracellular K+. This signaling pathway appears to be integral to UVB-induced K+ efflux, since knockdown of TNF-R1 or FADD inhibits UVB-induced K+ efflux.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.