Platelet-activating factor induces cyclooxygenase-2 gene expression in corneal epithelium. Requirement of calcium in the signal transduction pathway.
OBJECTIVE
To investigate the effect of the inflammatory mediator platelet-activating factor (PAF) in the induction of the inducible prostaglandin H synthase-cyclooxygenase-2 (COX-2) gene expression in corneal epithelium.


METHODS
Rabbit corneas were incubated in organ culture with or without carbamyl PAF (cPAF, 100 nM). The effects of PAF antagonist BN50730 (10 microM), protein synthesis inhibitor cycloheximide (CHX; 30 micrograms/ml), RNA synthesis inhibitor actinomycin D (50 micrograms/ml), and tumor promoter phorbol ester (TPA); (100 nM) were tested. Total RNA for corneal epithelium was analyzed by Northern blot analysis using mouse COX-2 cDNA fragments labeled with 32P as probes. Western blots were performed using mouse monoclonal antibodies. Primary cultures of rabbit corneal epithelium were loaded with the fluorescent dye fluo-3 AM and changes in intracellular calcium concentration [Ca2+]i were analyzed by laser scanning confocal microscopy.


RESULTS
Platelet-activating factor induction of COX-2 expression was detectable by Northern blot analysis at 2 hours, peaked at 4 hours, and remained increased for as long as 8 hours. At 16 hours, there was a marked increase in COX-2 expression. The effect was abolished by the PAF antagonist. TPA also induced COX-2 gene expression. Neither PAF-nor TPA-induced expression was inhibited by CHX. In a Ca(2+)-free medium, there was a 50% inhibition of COX-2 gene induction by PAF. The calcium ionophore A23187 also caused an increase in expression of COX-2 messenger RNA; this did not occur in Ca(2+)-free medium. Confocal microscopy imaging showed that after the addition of PAF, there was a transient increase in [Ca2+]i in corneal epithelial cells that peaked between 30 and 60 seconds. The increase was inhibited in the presence of BN50730 or in a Ca(2+)-free medium. A23187 also caused a transient increase in [Ca2+]i that was not altered in cells previously treated with PAF or BN50730.


CONCLUSIONS
PAF may enhance prostaglandin synthesis in the corneal epithelium by increasing COX-2 gene expression. This increase is by means of transcriptional activation of the gene and results in increased COX-2 protein formation. Influx of Ca2+ due to PAF stimulation is required to induce the COX-2 gene. A PAF antagonist abolishes all PAF effects and could be of therapeutic value by modulating ocular inflammation at the level of COX-2 gene expression.
Bazan
H E
HE
Louisiana State University Eye Center, Louisiana State University Medical Center, New Orleans 70112, USA.
Tao
Y
Y
DeCoster
M A
MA
Bazan
N G
NG
D000814:Aniline Compounds; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D015152:Blotting, Northern; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D051546:Cyclooxygenase 2; D004790:Enzyme Induction / Q000187:drug effects; D019573:Epithelium, Corneal / Q000187:drug effects / Q000201:enzymology*; D005456:Fluorescent Dyes; D015870:Gene Expression; D007476:Ionophores / Q000494:pharmacology; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D018613:Microscopy, Confocal; D009924:Organ Culture Techniques; D010544:Peroxidases / Q000096:biosynthesis / Q000235:genetics; D010972:Platelet Activating Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D015398:Signal Transduction / Q000502:physiology*; D058924:Thienopyridines; D014230:Triazoles / Q000494:pharmacology; D014966:Xanthenes
D000814:Aniline Compounds; D001381:Azepines; D005456:Fluorescent Dyes; D007476:Ionophores; D007527:Isoenzymes; D010972:Platelet Activating Factor; D012333:RNA, Messenger; D058924:Thienopyridines; D014230:Triazoles; D014966:Xanthenes; C066686:BN 50730; C059715:Fluo-3; D000001:Calcimycin; D010544:Peroxidases; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D002118:Calcium