Purpose: To study the regulation by mitomycin C (MMC) of 1-cys peroxiredoxin (Prx) expression during the corneal wound-healing process and its induction pathway in cultured bovine keratocytes (BKs).
Methods: Rat corneas were excised at 4 hours, 12 hours, 1 day, 3 days, and 7 days after photorefractive keratectomy (PRK). Expression of 1-cys Prx in the corneas was examined by Northern blot and immunoblot analyses. Cultured BKs were exposed to 0.02% MMC for 5 minutes and maintained under normal culture conditions for different time periods. Subsequently, levels of 1-cys Prx and extracellular signal-regulated kinase (ERK)1/2 expression were measured by immunoblot analysis using polyclonal 1-cys Prx, ERK1/2, or phospho-ERK1/2 antibodies. To inhibit ERK1/2 activation, the BKs were pretreated with 50 micromol/mL PD98059 for 1 hour before MMC exposure and incubated in complete medium with or without PD98059 for 24 hours. MMC-induced cytotoxicity was determined by colorimetric cell-counting kit-8 assay.
Results: Increased levels of 1-cys Prx expression were seen in wounded rat corneas at 12 hours after injury and reached the highest level between 1 and 3 days, during which time active proliferation occurred. Induction of 1-cys Prx expression was obvious in proliferating BKs stimulated by growth factors. MMC treatment in cultured BKs resulted in increased expression of 1-cys Prx and phospho-ERK1/2 in a time-dependent manner. Treatment with 50 micromol/mL PD98059 significantly inhibited the active ERK1/2 and 1-cys Prx expression induced by MMC, leading to synergistic cytotoxicity in the BKs.
Conclusion: 1-Cys Prx may function as an important enzyme in cell proliferation during the postinjury corneal wound-healing process. Furthermore, the induction of 1-cys Prx expression through the ERK1/2 signaling pathway may contribute to cellular defense against cytotoxic agents, thus playing an important role in cell survival.