Abstract
Purpose:
In studies designed to investigate the possible role of the high K+ levels in tears (25 mM), we have previously reported that exposure of human corneal limbal epithelial (HCLE) cells to UVB results in activation of K+ channels, loss of K+ from the cells and activation of the initiator caspase, caspase-8, and the activator caspase, caspase-3, leading to apoptosis. This activation of the apoptotic pathway is inhibited by incubation of the cells in medium with elevated [K+] (Ubels JL et al. Exp Eye Res. 92:425, 2011). Caspase-6 is activated by caspase-3 and has a positive feedback effect on caspase-8. The purpose of this study was to determine whether caspase-6 is activated when cells are exposed to UVB, whether caspase-3 is involved in this activation and if elevated [K+] inhibits this activation.
Methods:
HCLE cells were exposed to 150 or 200 mJ/cm2 UVB (302 nm) followed by incubation in culture medium containing isosmotic 5.5, 25, 50 or 100 mM K+ for up to 6 hours. Activation of apoptosis was determined by measurement of caspase-6 activity using a colorimetric assay. Controls were cells not exposed to UVB and incubated in 5.5 mM K+.
Results:
Maximal UVB-induced activation of caspase-6 occurred 3 - 5 hours post-exposure in cells incubated in 5.5 mM K+. At 5 hours caspase-6 activity was 138% or 181% of control after exposure to 150 or 200 mJ/cm2 UVB, respectively. The caspase-6 inhibitor, Z-VEID-FMK, prevented this UVB-induced activation of the enzyme. After exposure of cells to UVB the caspase-3 inhibitor, Z-DEVD-FMK, also prevented UVB-induced activation of caspase-6. Incubation of cells in medium with 100 mM K+ after exposure to UVB resulted in a significant reduction in UVB-induced caspase-6 activation, as compared to cells incubated in 5.5 mM K+.
Conclusions:
Caspase-6 is involved in the pathway leading to apoptosis of HCLE cells treated with UVB at doses relevant to ambient outdoor exposure. The loss of this response when caspase-3 is inhibited suggests that caspase-6 is involved in a loop that amplifies the response to UVB. Reduction of UVB-induced caspase-6 activation by high extracellular [K+] suggests that the high [K+] in tears reduces loss of intracellular K+ from ocular surface cells in response to UVB, helping to prevent apoptotic damage.
Keywords: 482 cornea: epithelium •
426 apoptosis/cell death •
486 cornea: tears/tear film/dry eye