PLATELET-ACTIVATING-FACTOR INDUCES CYCLOOXYGENASE-2 GENE-EXPRESSION IN CORNEAL EPITHELIUM - REQUIREMENT OF CALCIUM IN THE SIGNAL-TRANSDUCTION PATHWAY

Purpose. To investigate the effect of the inflammatory mediator platel et-activating factor (PAF) in the induction of the inducible prostagla ndin H synthase-cyclooxygenase-2 (COX-2) gene expression in corneal ep ithelium. Methods. Rabbit corneas were incubated in organ culture with or without carbamyl PAF (cPAF, 100 nM). The effects of PAF antagonist BN50730 (10 mu M), protein synthesis inhibitor cycloheximide (CHX; 30 mu g/ml), RNA synthesis inhibitor actinomycin D (50 mu g/ml), and tum or promoter phorbol ester (TPA); (100 nM) were tested. Total RNA for c orneal epithelium was analyzed by Northern blot analysis using mouse C OX-2 cDNA fragments labeled with P-32 as probes. Western blots were pe rformed 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 f actor induction of COX-2 expression was detectable by Northern blot an alysis at 2 hours, peaked at 4 hours, and remained increased for as lo ng as 8 hours. At 16 hours, there was a marked increase in COX-2 expre ssion. The effect was abolished by the PAF antagonist. TPA also induce d COX 2 gene expression. Neither PAF- nor TPA-induced expression was i nhibited by CHX. In a Ca2+-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 i n Ca2+-free medium. Confocal microscopy imaging showed that after the addition of PAF, there was a transient increase in [Ca2+](i) in cornea l epithelial cells that peaked between 30 and 60 seconds. The increase was inhibited in the presence of BN50730 or in a Ca2+-free medium. A2 3187 also caused a transient increase in [Ca2+](i) that was not altere d in cells previously treated with PAF or BN50730. Conclusions. PAF ma y enhance prostaglandin synthesis in the corneal epithelium by increas ing COX-2 gene expression. This increase is by means of transcriptiona l activation of the gene and results in increased COX-2 protein format ion. Influx of Ca2+ due to PAF stimulation is required to induce the C OX-2 gene. A PAF antagonist abolishes all PAF effects and could be of therapeutic value by modulating ocular inflammation at the level of CO X-2 gene expression.