Abstract
Platelet-activating factor (PAF) is an important mediator of cell loss following diverse pathophysiological challenges, but the manner in which PAF transduces death is not clear. Both PAF receptor-dependent and -independent pathways are implicated. In this study, we show that extracellular PAF can be internalized through PAF receptor-independent mechanisms and can initiate caspase-3-dependent apoptosis when cytosolic concentrations are elevated by approximately 15 pM/cell for 60 min. Reducing cytosolic PAF to less than 10 pM/cell terminates apoptotic signaling. By pharmacological inhibition of PAF acetylhydrolase I and II (PAF-AH) activity and down-regulation of PAF-AH I catalytic subunits by RNA interference, we show that the PAF receptor-independent death pathway is regulated by PAF-AH I and, to a lesser extent, by PAF-AH II. Moreover, the anti-apoptotic actions of PAF-AH I are subunit-specific. PAF-AH I alpha1 regulates intracellular PAF concentrations under normal physiological conditions, but expression is not sufficient to reduce an acute rise in intracellular PAF levels. PAF-AH I alpha2 expression is induced when cells are deprived of serum or exposed to apoptogenic PAF concentrations limiting the duration of pathological cytosolic PAF accumulation. To block PAF receptor-independent death pathway, we screened a panel of PAF antagonists (CV-3988, CV-6209, BN 52021, and FR 49175). BN 52021 and FR 49175 accelerated PAF hydrolysis and inhibited PAF-mediated caspase 3 activation. Both antagonists act indirectly to promote PAF-AH I alpha2 homodimer activity by reducing PAF-AH I alpha1 expression. These findings identify PAF-AH I alpha2 as a potent anti-apoptotic protein and describe a new means of pharmacologically targeting PAF-AH I to inhibit PAF-mediated cell death.
Highlights
Summary—In summary, this study provides mechanistic evidence that sustained exposure to elevated intracellular PAF concentrations is sufficient to elicit apoptosis through a PAFRindependent cell death pathway
To intervene in PAF-mediated death, we describe a novel anti-apoptotic function for PAF-AH I ␣2 and II, and we identify two PAF antagonists that accelerate cytosolic PAF-AH I activity to inhibit PAF-mediated apoptosis
Acknowledgments—We thank Tia Moffat for technical assistance and Jim Bennett for critical reading of this manuscript
Summary
Cell Culture—PC12-AC cells, a clonal derivative of the PC12 pheochromocytoma cell line (American Tissue Culture Collection), were cultured in complete media composed of RPMI 1640 containing 10% horse serum and 5% newborn calf serum at 37 °C in a 5% CO2, 95% air atmosphere. PAF-AH Activity—PAF-AH activities in complete media, serum-free RPMI, PC12-AC cells, PC12-AC conditioned treatment media, and C57Bl/6 mouse brain ϳ3 months of age (positive control) were determined using a commercial PAF-AH assay kit (Cayman Chemicals). Cultures were incubated at 37 °C with 1 M B-PAF in RPMI 1640 containing 0.1% BSA for 0, 5, 15, 30, 45, 60, and 75 min. One ml of methanol acidified with 2% acetic acid was added to each plate, and the extracellular fraction was collected This fraction contained B-PAF in the culture media and uninternalized B-PAF bound to cell surface proteins or associated with the plasma membrane. Transfection efficiency was determined in separate cultures by counting EGFP-positive cells upon transfection with pEGFP-C1 as well as morphological changes using -actin siRNA positive control (Ambion). Following detection of a statistically significant difference in a given series of treatments, post hoc Dunnett’s t-tests or Tukey tests were performed where appropriate. p values under 0.05 were considered statistically significant (shown as * or †); p values under 0.01 were considered highly statistically significant (shown as ** or ††)
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