A mitogen-activated protein kinase (MAP-kinase) cascade is stimulated by platelet activating factor (PAF) in corneal epithelium

Abstract

PURPOSE. The mitogen-activated protein kinases (MAPK) are a family of important proteins that respond to a variety of receptor-mediated stimuli and can link events occurring at the cell membrane with changes in the nucleus. In this study we investigate the effect of platelet activating factor (PAF), a lipid mediator formed in the cornea after injury, on the activation of a MAPK cascade in the rabbit corneal epithelium. METHODS. Rabbit corneas were incubated with or without 500 nM PAF. PAF antagonists BN50730 or 50727 (10 µM) were added 10 min before PAF and the epithelium scraped and homogenated. To determine the enzymatic activity of MAPK and MAPK-kinase (MEK1 and MEK2), a 100,000 × g cytosolic fraction was used directly, fractionated by DE-52 cellulose or immunoprecipitated with antibodies. Activities of MAPK and MEK were assayed in the presence of myelin basic protein (MBP) as substrate (for MAPK) activity or inactive extracellular-signal regulated protein kinase (ERK2 or MAPK). Western blot analysis was performed using anti-ERK2, anti-MEK1, and anti-MEK2 antibodies. RESULTS. Corneal tissue expresses ERK2 or MAPK, and both MEK1 and MEK2, the immediate upstream regulators of MAPK. PAF produces a rapid activation of MEK, as measured by in vitro kinase assays using either inactive ERK2 as substrate or a MAPK fraction obtained by DE-52 chromatography. There was a subsequent activation of MAPK, the maximal activity of which occurs 15 min after stimulation by PAF. PAF antagonists blocked the MEK/MAPK cascade, suggesting that the activation was by a receptor-mediated mechanism. CONCLUSIONS. The evidence presented here, that a MAPK cascade is rapidly activated by PAF in the corneal epithelium, suggests that this signal transduction mechanism can be involved in the increased expression of collagenase and other protease genes, as well as in the activation of phospholipase A 2, events that occur in the corneal epithelium after PAF stimuli.