c-Jun NH2-Terminal Kinase Regulates Migration of Corneal Epithelial Cells During Wound Closure Through Phosphorylation of Paxillin

Abstract

Purpose:: Migration of corneal epithelial cells plays an important role in healing of corneal epithelial wounds. The role of c-Jun NH₂-terminal kinase (JNK), a member of the family of mitogen-activated protein kinases, in the intracellular signaling responsible for regulation of the formation of focal adhesions and lamellipodia during the migration of corneal epithelial cells was examined.

Methods:: Scratch wounds were introduced into cultured monolayers of simian virus 40-transformed human corneal epithelial (HCE) cells, and the wounded monolayers were incubated in the absence or presence of the JNK inhibitor SP600125. Serial images of the cells were obtained for measurement of the wound area. The phosphorylation of JNK and paxillin was examined by immunoblot analysis. The actin cytoskeleton and focal adhesions were detected by staining of cells with rhodamine-phalloidin and antibodies to paxillin, respectively.

Results:: SP600125 (0.3 to 30 µM) inhibited wound healing in a concentration-dependent manner. Immunoblot analysis showed that wounding increased the level of phosphorylation of JNK and paxillin and that SP600125 inhibited the wounding-induced phosphorylation of both these proteins. Immunofluorescence staining revealed that phosphorylated JNK co-localized with paxillin at focal adhesions formed by HCE cells at the wound margin and that SP600125 inhibited both the formation of focal adhesions and the colocalization of these proteins.

Conclusions:: JNK regulates HCE cell migration by modulating the phosphorylation of paxillin and the consequent formation of focal adhesions. This enzyme may thus play an important role in corneal epithelial wound healing in vivo.

Keywords: cornea: epithelium • cell adhesions/cell junctions • cytoskeleton