Expression of 1-cys peroxiredoxin in the corneal wound-healing process.
OBJECTIVE
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.


CONCLUSIONS
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.
Pak
Jhang Ho
JH
Department of Ophthalmology and Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center, 388-1 Pungnap-2 dong, Songpa-gu, Seoul 137-756, Korea.
Choi
Hyun-jeung
HJ
Choi
Chul Young
CY
Tchah
Hungwon
H
D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques; D002470:Cell Survival; D003315:Cornea / Q000601:surgery; D003319:Corneal Stroma / Q000166:cytology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D015151:Immunoblotting; D054018:Lasers, Excimer; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D016685:Mitomycin / Q000494:pharmacology; D010544:Peroxidases / Q000235:genetics / Q000378:metabolism*; D054464:Peroxiredoxins; D010766:Phosphorylation; D018903:Photorefractive Keratectomy; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014945:Wound Healing / Q000502:physiology*
D000477:Alkylating Agents; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D016685:Mitomycin; D010544:Peroxidases; D054464:Peroxiredoxins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3