Abstract
PURPOSE: The purpose of the study is to understand how extracellular stresses, such as ultraviolet (UV) irradiation, affect corneal epithelial cells. Cell volume changes, damage to corneal epithelial integrity, and cellular responses were assessed after exposure to UVC stresses. METHODS: Primary human and rabbit corneal epithelial cells were exposed to UVC light in culture conditions. Ultraviolet C irradiation-induced changes in cell size and volume were measured by real-time microscopy and self-quenching of the fluorescent dye calcein, respectively. The effects of UVC irradiation on Src and focal adhesion kinase (FAK) phosphorylation and FAK-dependent integrin signaling were detected by ELISA, immunoblotting, and immunostaining. RESULTS: Ultraviolet C irradiation induced both size and volume shifts in human and rabbit corneal epithelial cells. Ultraviolet C irradiation-induced decrease of cell volume elicited activation of Src and FAK, characterized by increased phosphorylations of SrcY416, FAKY397, and FAKY925. In addition, immunostaining studies showed UVC irradiation-induced increases in phosphorylation of FAK and formation of integrin β5 clustering. Application of Kv channel blockers, including 4-aminopyridine (4-AP), α-DTX, and depressing substance-1 (BDS-1), effectively suppressed UVC irradiation-induced cell volume changes, and subsequently inhibited UVC irradiation-induced phosphorylation of Src/FAK, and formation of integrin β5 clustering, suggesting UVC irradiation-induced volume changes and Src/FAK activation. Hyperosmotic pressure-induced volume decreases were measured in comparison with effects of UVC irradiation on volume and Src/FAK activation. However, Kv channel blocker, 4-AP, had no effect on hyperosmotic pressure-induced responses. CONCLUSIONS: The present study demonstrates that UVC irradiation-induced decreases in cell volume lead to Src/FAK activation due to a rapid loss of K ions through membrane Kv channels.