Mitomycin C retardation of corneal fibroblast migration via sustained dephosphorylation of paxillin at tyrosine 118.
OBJECTIVE
To investigate how mitomycin C (MMC) modulates corneal fibroblast migration and its molecular mechanisms in the wound healing process.


METHODS
After treatment with 0 and 0.2 mg · mL(-1) MMC for 5 minutes, effect of MMC on cell migration of human corneal fibroblasts (HCFs) was examined with a cell migration assay. Both focal adhesion kinase (FAK) and paxillin (PXN) expressions in HCFs were analyzed by semiquantitative real-time PCR, immunoblotting, and immunofluorescence confocal microscopy. Using gene silencing or gene overexpression with lentiviral-based pseudovirion infection, the phosphorylation level of FAK, PXN, and mutated PXNs at tyrosine sites 31 (Y31F-EGFP) and 118 (Y118F-EGFP) were verified in HCFs.


RESULTS
MMC retarded HCF migration at 1 and 2 days posttreatment (dpt). MMC reduced levels of FAK transcript and FAK protein, but increased both transcript and protein expression of PXN at 1 and 2 dpt. Furthermore, MMC upregulated FAK-pY397, which subsequently enhanced PXN-pY31 in a dose-dependent manner at 1 dpt. Concurrently, MMC downregulated PXN-pY118 at 1 dpt. However, MMC treatment resulted in dephosphorylation of FAK-pY397, PXN-pY31, and PXN-pY118 at 2 dpt. The FAK/PXN complex in MMC-treated HCFs was detected at focal adhesion sites more than at the leading edge at 1 and 2 dpt, contributing to retardation of HCF migration. Y118F-EGFP-expressing HCFs exhibited lower mobility than that of PXN-EGFP- or Y31F-EGFP-expressing HCFs.


CONCLUSIONS
The sustained PXN-pY118 dephosphorylation resulted in steadfastness of an incompletely active FAK/PXN complex at focal adhesion sites and played a pivotal role in MMC-retarded HCF migration.
Department of Ophthalmology, Far Eastern Memorial Hospital, Banqiao District, New Taipei City, Taiwan.
Tsan-Chi
TC
Chen
Chien-Hsueh
CH
Lai
Jie-Ling
JL
Chang
Shu-Wen
SW
Chang
D002465:Cell Movement / Q000187:drug effects* / Q000502:physiology; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D015151:Immunoblotting; D016685:Mitomycin / Q000494:pharmacology*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*; D051419:Paxillin / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D060888:Real-Time Polymerase Chain Reaction; D014443:Tyrosine / Q000378:metabolism; D014945:Wound Healing / Q000187:drug effects
D019384:Nucleic Acid Synthesis Inhibitors; D051419:Paxillin; D014443:Tyrosine; D016685:Mitomycin; D051416:Focal Adhesion Protein-Tyrosine Kinases