Abstract:
PURPOSE: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.
journal_name
Invest Ophthalmol Vis Scijournal_title
Investigative ophthalmology & visual scienceauthors
Chen TC,Lai CH,Chang JL,Chang SWdoi
10.1167/iovs.11-9203subject
Has Abstractpub_date
2012-03-21 00:00:00pages
1539-47issue
3eissn
0146-0404issn
1552-5783pii
iovs.11-9203journal_volume
53pub_type
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