AP-1 expression in ethanol-treated corneal epithelium in vivo.
OBJECTIVE
To examine the expression pattern of stress-related genes, c-fos and c-jun, both the major components of activator protein-1 (AP-1), in rat corneal epithelium treated with a short-term ethanol exposure. The purpose of the current study was to examine if the ethanol exposure during laser epithelial keratomileusis (LASEK) may stimulate or damage the corneal epithelial cells.


METHODS
Sixty male Wistar rats were used. Fifty microliters of 20% ethanol was placed onto a surface 2.4 mm in diameter of the central corneal epithelium for 30 s. The affected eyes, washed with saline, were then enucleated after various intervals of healing. To know the expression pattern of c-fos and c-jun mRNAs and c-Fos, c-Jun and Jun D proteins, in situ hybridization and immunohistochemistry were carried out. The expression level of c-fos and c-jun mRNAs was determined by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Apoptotic nuclei in the tissue sections were identified by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay.


RESULTS
Thirty to 60 min after the treatment, c-fos and c-jun mRNAs were detected in the corneal epithelium. These signals were no longer evident at 90 min. c-Fos protein was detected in the corneal epithelium around the area of ethanol exposure from 60 to 120 min after the treatment, while c-Jun protein was not detected. Jun D protein was detected in control whole corneal epithelium and not affected by ethanol exposure in the periphery. The levels of c-fos and c-jun mRNAs were increased approximately 8 times at 30 min compared with the control level. TUNEL-positive apoptotic nuclei in the tissue sections were identified.


CONCLUSIONS
Corneal epithelial cells, especially those surrounding the ethanol-exposed area, are transiently transcriptionally activated at a very early phase after the ethanol exposure. mRNA expression for c-fos is followed by protein synthesis, but that of c-jun is not followed by protein synthesis. Resistance of Jun D protein expression to ethanol suggests that it might be a candidate for an AP-1 complex with c-Fos.
Okada
Yuka
Y
Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. yokada@wakayama-med.ac.jp
Saika
Shizuya
S
Miyamoto
Takeshi
T
Shirai
Kumi
K
Ueyama
Takashi
T
Senba
Emiko
E
Ohnishi
Yoshitaka
Y
D000818:Animals; D017209:Apoptosis; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism; D000431:Ethanol / Q000633:toxicity*; D005786:Gene Expression Regulation / Q000187:drug effects; D016762:Genes, fos / Q000235:genetics; D016758:Genes, jun / Q000235:genetics; D007124:Immunoenzyme Techniques; D017403:In Situ Hybridization; D020287:In Situ Nick-End Labeling; D008297:Male; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018808:Transcription Factor AP-1 / Q000235:genetics*
D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D000431:Ethanol