The long-term objective of the proposed research is to elucidate the role of the glycerophosphocholine-derived mediator Platelet- activating Factor (PAF) in keratinocyte cytotoxicity. Recent studies have demonstrated that many clinically relevant cytotoxic agents including ultraviolet B (UVB) radiation damage human keratinocytes (HK) in part by inducing oxidative stress and cytokine production. Severe UVB damage to HK can also result in programmed cell death (apoptosis). Because PAF is synthesized in response to oxidative stress and many pro-inflammatory cytokines, and HK express PAF-receptors (PAF-R), involvement of the PAF system in UVB-mediated keratinocyte damage has been assessed using a model system created by retroviral-mediated transduction of the PAF-R into the PAF-R-negative epidermal cell line KB. Expression of the epidermal PAF-R resulted in an increased susceptibility to UVB-induced apoptosis. In addition, oxidative stress to epidermal cells resulted in the production of a PAF-R agonistic activity which mass spectrometric studies revealed was structurally not PAF. Based on the ability of antioxidants to inhibit this activity as well as the augmentation of UVB-induced apoptosis in PAF-R-expressing KB cells, we hypothesize oxidative damage to HK results in the production of oxidized lipids which can modulate apoptosis through PAF-R activation.
Three specific aims will test this hypothesis by 1) Assessing the effects of PAF agonists/antagonists and overexpression of the wild-type or antisense PAF-R constructs on UVB-induced keratinocyte apoptosis in vitro; 2) Assessing the effects of PAF-R antagonists and overexpression of wild-type or antisense PAF-R constructs on UVB-induced apoptosis in SCID/HK xenografts in vivo; and 3) Structural characterization of the lipid species responsible for the UVB-induced non-PAF PAF-R agonistic activity in epidermal cells by mass spectrometry. The project will determine the significance of the PAF system in the oxidative stress response in HK, as well as to structurally define novel biologically active markers of lipid peroxidation, information which could result in potential therapeutic strategies involving PAF-R antagonists for diseases characterized by massive epidermal damage.
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