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Abstract
A number of chemical mediators can induce human keratinocytes and epidermal-derived carcinomas to undergo apoptosis, or programmed cell death. Recent evidence suggests pro-inflammatory cytokines, such as interleukin-1 beta or transforming growth factor alpha, protects carcinomas from numerous pro-apoptotic stimuli. Platelet-activating factor (1-alkyl-2-acetyl-3-glycerophosphocholine; PAF) is a lipid mediator with pro-inflammatory effects on numerous cell types. Although PAF can be metabolized to other bioactive lipids, the majority of PAF effects occur through activation of a G protein-coupled receptor. Using a model system created by retroviral transduction of the PAF receptor (PAF-R) into the PAF-R-negative human epidermal cell line KB and the PAF-R-expressing keratinocyte cell line HaCaT, we now demonstrate that activation of the epidermal PAF-R results in protection from apoptosis induced by tumor necrosis factor (TNF) alpha or TNF-related apoptosis-inducing ligand. The PAF-mediated protection was inhibited by PAF-R antagonists, and protection did not occur in PAF-R-negative KB cells. Additionally, we show protection from TNFalpha- or TRAIL-induced apoptosis by PAF-R activation is dependent on the transcription factor nuclear factor (NF)-kappa B, because PAF-R activation-induced NF-kappa B and epidermal cells transduced with a super-repressor form of inhibitor kappa B were not protected by the PAF-R. These studies provide a mechanism whereby the epidermal PAF-R, and possibly other G protein-coupled receptors, can exert anti-apoptotic effects through an NF-kappa B-dependent process.
Highlights
Apoptosis, or programmed cell death, is a fundamental physiological process enabling the removal of damaged or infected cells and in the control of cell populations [1]
Using a model system created by retroviral transduction of the platelet-activating factor (PAF) receptor (PAF-R) into the PAF-R-negative human epidermal cell line KB and the PAF-R-expressing keratinocyte cell line HaCaT, we demonstrate that activation of the epidermal PAF-R results in protection from apoptosis induced by tumor necrosis factor (TNF) ␣ or TNF-related apoptosisinducing ligand
The protective effect of CPAF, but not IL-1, occurred through activation of the PAF-R, because the protection from apoptosis was only seen in PAF-R-expressing KB cells transduced with the PAF-R (KBP) and HaCaT cells, and the protection was inhibited by using PAF-R antagonists
Summary
Reagents—All chemicals were obtained from Sigma unless indicated otherwise. Recombinant human TRAIL/APO2L was purchased from Chemicon (Temecula, CA). Generation of KBP Cells Expressing the Super-repressor IB␣ (IBM)—The IBM containing S32A and S36A mutation of IB␣ has previously been described [31], and the retroviral DNA vector MIEG3, which uses enhanced green fluorescent protein as the selectable marker, was a kind gift of Dr David Williams (Indiana University) [32]. Cell viability was determined using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay (Roche Molecular Biochemicals) and analyzed using a microplate reader (Molecular Devices, Sunnyvale, CA). Caspase-3 Assay—The activation of the caspase proteolytic cascade was measured by the direct assay of caspase-3 enzyme activity in cell lysates using a synthetic fluorogenic substrate (caspase-3 substrate, Ac-Asp-Glu-Val-Asp-AMC (Ac-DEVD-AMC); Alexis Biochemicals, San Diego, CA) as described previously [34]. Luciferase activities were normalized for each transfection using the control -galactosidase activities
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