TNF-R1 and FADD mediate UVB-Induced activation of K+ channels in corneal epithelial cells

Abstract

The goal of this study was to elucidate the role of Fas, TNF-R1, FADD and cytochrome c in UVB-induced K⁺ channel activation, an early step in UVB-induced apoptosis, in human corneal limbal epithelial (HCLE) cells. HCLE cells were treated with Fas, TNF-R1 or FADD siRNA and exposed to 80 or 150 mJ/cm² UVB. K⁺ channel activation and loss of intracellular K⁺ were measured using whole-cell patch-clamp recording and ion chromatography, respectively. Cytochrome c was measured with an ELISA kit. Cells in which Fas was knocked down exhibited identical UVB-induced K⁺ channel activation and loss of intracellular K⁺ to control cells. Cells in which TNF-R1 or FADD were knocked down demonstrated reduced K⁺ channel activation and decreased loss of intracellular K⁺ following UVB, relative to control cells. Application of TNF-α, the natural ligand of TNF-R1, to HCLE cells induced K⁺ channel activation and loss of intracellular K⁺. Cytochrome c was translocated to the cytosol by 2 h after exposure to 150 mJ/cm² UVB. However, there was no release by 10 min post-UVB. The data suggest that UVB activates TNF-R1, which in turn may activate K⁺ channels via FADD. This conclusion is supported by the observation 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 the UVB-induced K⁺ efflux. The lack of rapid cytochrome c translocation indicates cytochrome c does not play a role in UVB-induced K⁺ channel activation.

Keywords: Apoptosis; Corneal epithelium; Cytochrome c; FADD; FAS; Potassium channel; TNF-R1; Ultraviolet-B.