This proposal is a continuation of our ongoing studies of lipid metabolism, focusing on the action of a lipid mediator: 1-0-alkyl-2- acetyl-glycerophosphocholine (platelet-activating factor, PAF). We have found, in corneal epithelium, that PAF activates the expression of the early genes c-fos and c-jun and then the expression of collagenase type 1, the metalloproteinase that degrades interstitial collagen, a major protein of the corneal stroma. A PAF antagonist blocks this effect. We will test two hypotheses: a) that PAF is involved in the mechanism of corneal remodeling and ulcer formation by acting as a lipid second messenger in the transcriptional activation of c-fos, c-jun and the collagenase l gene, and b) that lipoxygenase metabolites formed by the activation of phospholipase A2 have a modulatory effect on PAF. We propose to investigate the PAF signaling pathway and the role of lipoxygenase metabolites in the transcription of collagenase type 1. PAF antagonists and lipoxygenase inhibitors will be evaluated to determine their sites of action and to correlate their biochemical effects with the clinical evolution of corneal ulcers. Another goal of this proposal is to investigate the PAF receptors in the cornea in order to define the sites of action of PAF antagonists in the gene cascade. Powerful analytical procedures such as high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry will be used. Quantification of specific mRNAs will be accomplished by using Northern blots with storage phosphor-imaging. The results obtained will define the involvement of PAF and lipoxygenase metabolites in corneal remodeling and ulcer formation. The new mechanism of action of PAF also can be important in a number of tissue disorders in which normal control of the degradative activity of collagenase appears to be lost, e.a. rheumatoid arthritis, leading to pathological tissue destruction. The action of antagonists can be useful to define new therapeutic tools for control of collagenase type 1 activity.
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