TNF-R1 and FADD mediate UVB-Induced activation of K+channels in corneal epithelial cells.
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/cm2UVB. 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/cm2UVB. 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.
Department of Biology, Calvin College, 3201 Burton St. SE, Grand Rapids, MI 49546, USA; Department of Physics and Astronomy, Calvin College, 3201 Burton St. SE, Grand Rapids, MI 49546, USA.
Peter M
PM
Boersma
Department of Physics and Astronomy, Calvin College, 3201 Burton St. SE, Grand Rapids, MI 49546, USA.
Loren D
LD
Haarsma
Department of Biology, Calvin College, 3201 Burton St. SE, Grand Rapids, MI 49546, USA.
Mark P
MP
Schotanus
Department of Biology, Calvin College, 3201 Burton St. SE, Grand Rapids, MI 49546, USA. Electronic address: jubels@calvin.edu.
John L
JL
Ubels
D017209:Apoptosis; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D003580:Cytochromes / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism* / Q000528:radiation effects; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism*; D006801:Humans; D020287:In Situ Nick-End Labeling; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000378:metabolism*; D012313:RNA / Q000235:genetics; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism*; D015398:Signal Transduction; D014466:Ultraviolet Rays; D019014:fas Receptor / Q000378:metabolism
D003580:Cytochromes; C505533:FADD protein, human; C491901:FAS protein, human; D053200:Fas-Associated Death Domain Protein; D015221:Potassium Channels; D047888:Receptors, Tumor Necrosis Factor, Type I; D019014:fas Receptor; D012313:RNA; D011188:Potassium
Apoptosis; Corneal epithelium; Cytochrome c; FADD; FAS; Potassium channel; TNF-R1; Ultraviolet-B