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Shu-Wen Chang, Tsan-Chi Chen, Chuan-Long Hsu, Chien-Hsueh Lai; Mitomycin C Retards Corneal Fibroblast Cell Migration via Recruitment of FAK/Paxillin Complexes to Lamellipodial Matrix Metallopeptidase-9. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2048.
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To investigate whether mitomycin C (MMC) modulates matrix metallopeptidase-9 (MMP9) expression during corneal fibroblast cell migration.
MMP9 was highly expressed in primary human corneal fibroblasts with a fluorescent green fluorescent protein (GFP)-fused MMP9 construct by using a lentivirus-based pseudovirion infection system. Primary human corneal fibroblasts, GFP-expressing corneal fibroblasts, and MMP9-expressing corneal fibroblasts were treated with MMC at a series of doses ranging from 0 to 0.2 mg/ml for 5 min. Migration rate of MMC-treated cells was studied using the migration assay. MMP9-expressed cell group was compared with the GFP-expressed cell group. Transcript and protein expression of endogenous MMP9 and GFP-tagged MMP9 were analyzed by quantitative real-time PCR and immunoblotting assay, respectively. The intracellular distributions of fluorescent MMP9 were observed using confocal microscopy on the stationary cells. The binding capacity of MMP9 on the focal adhesive kinase (FAK)/paxillin complexes was analyzed by co-immunoprecipitation.
Migration assay indicated that MMC treatment significantly retarded corneal fibroblast migration dose-dependently. MMC increased not only transcript level and protein expression of endogenous MMP9 but also GFP-tagged MMP9 protein expression. MMC retarded the migration of the living fluorescent MMP9-expressing corneal fibroblasts more than that in the living GFP-expressing cells. However, the stationary confocal images revealed that MMC could not change the intracellular distribution of either endogenous MMP9 or fluorescent MMP9 on the lamellipodia/filopodia of corneal fibroblasts. Finally, MMP9 increasingly bound with FAK and paxillin after MMC treatment.
Enhancement of MMP9 expression suppressed the migration rate in MMC-treated human corneal fibroblasts via recruitment of the incompletely active focal adhesion complexes on the focal adhesion sites.
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