Abstract
Purpose:
Investigating the possible role of the high [K+] in tears (25 mM) in protecting the cornea from UVB, we have reported that exposure of human corneal limbal epithelial (HCLE) cells to UVB results in loss of K+ from the cells, activation caspases-8 and -3, and apoptosis. This induction of apoptosis is inhibited by incubation of the cells in medium with elevated [K+] (Exp Eye Res. 92:425, 2011). The present study investigated whether the response to UVB is mediated via Fas ligand-independent activation of Fas (extrinsic pathway) or via caspase-9 (intrinsic pathway).
Methods:
HCLE cells were exposed to 150 mJ/cm2 UVB (302 nm) followed by incubation in medium containing 5.5, 25 or 50 mM K+ for up to 6 hours. Activation of caspases-3, -8 and -9 was measured using fluorometric assays. Control cells, not exposed to UVB, were incubated in medium with 5.5 mM K+. Knockdown of Fas and caspase-8 by siRNA was confirmed by western blot. Activation of Kv3.4 channels by UVB (80 mJ/cm2), determined using BDS-1, was measured by patch-clamp recording in the whole cell, perforated patch mode.
Results:
Knockdown of Fas to <15% of control levels reduced UVB-induced activation of caspase-8 by only 15% and had no effect on caspase-3 activity or activation of K+ channels. Knockdown of caspase-8 to <17% of control levels had no effect on caspase-3 activation by UVB, but complete inhibition of caspase-8 by Z-IEDT-FMK prevented activation of caspase-3 by UVB. Exposure of HCLE cells to UVB activated caspase-9 by 12-fold over control levels within 4 hr. This activation was inhibited in medium with high [K+]. UVB-induced caspase-9 activity is completely inhibited by Z-LEHD-FMK. UVB caused a 6.9-fold increase in caspase-8 activity and a 45.8-fold increase in caspase-3. This activation was reduced to 0% and 12% of maximum, respectively, by inhibition of caspase-9.
Conclusions:
Knockdown of Fas has minimal effect on downstream UVB-induced activation of apoptotic mechanisms, while UVB-induced activation of caspases-8 and -3 is highly dependent on caspase-9 activation. It appears that the Fas-mediated extrinsic pathway is less important than the intrinsic pathway in the response of HCLE cells to UVB. The suppression of UVB-induced caspase-9 activity by high extracellular [K+] supports our overall hypothesis that high [K+] in tears protects the corneal epithelium from UVB by reducing K+ efflux when K+ channels are activated.