Role of JNK-dependent serine phosphorylation of paxillin in migration of corneal epithelial cells during wound closure.
OBJECTIVE
Migration of corneal epithelial cells plays an important role in healing of corneal epithelial wounds. The role of c-Jun NH(2)-terminal kinase (JNK), a member of the family of mitogen-activated protein kinases, in the intracellular signaling responsible for the migration of corneal epithelial cells during wound closure was examined.


METHODS
Scratch wounds were introduced into cultured monolayers of simian virus 40-transformed human corneal epithelial (HCE) cells in the absence or presence of the JNK inhibitor SP600125. The phosphorylation and localization of JNK and paxillin during wound closure were examined by immunoblot and immunofluorescence analyses. The effects of a small interfering RNA (siRNA) specific for JNK and of a mutant form of paxillin on HCE cell migration were determined by transfection.


RESULTS
SP600125 inhibited wound healing in a time- and concentration-dependent manner. Immunoblot analysis showed that wounding increased the phosphorylation of JNK and of paxillin on serine (Ser) 178 in a manner sensitive to SP600125. Immunofluorescence staining revealed that phosphorylated JNK colocalized with paxillin at focal adhesions formed by HCE cells at the wound margin and that SP600125 inhibited the formation of such adhesions. Expression of JNK siRNA or of a paxillin mutant in which Ser178 is replaced by alanine inhibited HCE cell migration during wound closure.


CONCLUSIONS
JNK regulates HCE cell migration by modulating the phosphorylation of paxillin and the consequent formation of focal adhesions. A JNK-paxillin signaling pathway may thus play an important role in corneal epithelial wound healing in vivo.
Kimura
Kazuhiro
K
Department of Ocular Pathophysiology, Yamaguchi University Graduate School of Medicine, 111 Minami-Kogushi, Ube City, Yamaguchi, Japan. k.kimura@yamaguchi-u.ac.jp
Teranishi
Shinichiro
S
Yamauchi
Junji
J
Nishida
Teruo
T
D000873:Anthracenes / Q000494:pharmacology; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000502:physiology*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015151:Immunoblotting; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D008856:Microscopy, Fluorescence; D051419:Paxillin / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D034741:RNA, Small Interfering / Q000494:pharmacology; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014945:Wound Healing / Q000187:drug effects / Q000502:physiology*
D000873:Anthracenes; D004791:Enzyme Inhibitors; C496061:PXN protein, human; D051419:Paxillin; D034741:RNA, Small Interfering; C432165:pyrazolanthrone; D012694:Serine; D048031:JNK Mitogen-Activated Protein Kinases