Anti-apoptotic Actions of the Platelet-activating Factor Acetylhydrolase I α2 Catalytic Subunit

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 ϳ15 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 ␣ 1 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 ␣ 2 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 ␣ 2 homodimer activity by reducing PAF-AH I ␣ 1 expression. These findings identify PAF-AH I ␣ 2 as a potent antiapoptotic protein and describe a new means of pharmacologically targeting PAF-AH I to inhibit PAF-mediated cell death.Platelet-activating factor (PAF, 1 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a key mediator of neuronal death in ischemia, encephalitis, epileptic seizure, meningitis, and human immunodeficiency virus-1 dementia in vivo and participates in etoposide-, prion-, and ␤-amyloid-induced cell death in vitro (1-7). In the periphery, pathological increases in PAF concentrations underlie cytotoxicity in chronic inflammatory dermatoses and lethality in systemic anaphylaxis (8, 9). Although the majority of PAF effects are understood to be transduced by its G-protein-coupled receptor (PAFR) (10), PAFR signaling has been shown to be both pro-and anti-apoptotic. Ectopic PAFR expression exacerbates cell death induced by etoposide and mitomycin C but protects cells from tumor necrosis factor ␣, TRAIL, and extracellular PAF (6,7,11,12). These opposing effects likely depend upon the relative ratio of NF-B-dependent pro-and anti-apoptotic gene products elicited in different cell types in response to the combination of an external apoptotic inducer and PAF (6).Accumulating evidence points to additional PAF signaling pathways transduced independently of PAFR (11, 13-17). PAFR-negative cells undergo apoptosis when extracellular PAF concentrations reach 100 nM and necrosis when PAF levels e...

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“…Cytosolic PAF-AH Ib is an acetylhydrolasespecific complex composed of two 29-kDa ␣ 1 and/or 30-kDa ␣ 2 catalytic subunits and two 45-kDa regulatory LIS1 subunits (15). We have previously shown that overexpression of the ␣ 2 catalytic subunit enhances PAF hydrolysis in cells that endogenously express all three PAF-AH Ib subunits (16). As observed over the course of CNS development (17), the ␣ 1 catalytic subunit was detected at higher levels before differentiation, while the ␣ 2 catalytic and LIS1 regulatory subunits were maximally expressed following terminal differentiation of hNT neurons (Fig.…”

“…To test this directly, we used a biochemical loading approach to elevate intraneuronal C16:0 PAF concentrations in the absence of any other toxic stimuli. PAFs can be internalized following binding to PAFR and clatherin-mediated endocytosis, transglutaminase-dependent transmembrane bilayer movement, engagement with low affinity binding sites at the plasma membrane, or direct physicochemical effects at the plasma membrane (16,18,19). As hNT neurons do not express PAFR (Fig.…”

“…Glycerophospholipids were extracted according to a modified Bligh and Dyer procedure (16). LC-ESI-MS and TLC analyses were performed as described in SI Materials and Methods.…”

Novel isoforms of intracellular platelet activating factor acetylhydrolase (PAFAH1b2) in human testis; encoded by alternatively spliced mRNAs

“…It has been suggested that Paf-ah-Ib's activity in spermatogenesis, apoptosis, and neuronal migration may not rest entirely in its catalytic ability to reduce PAF levels but that it may act as a signaling complex in conjunction with other proteins (4,5,14). The Alpha-2 isoforms described herein may introduce levels of regulation and interaction beyond Pafah1b2's catalytic activity imparting additional, as yet uncharacterized, links to intracellular signaling pathways in testis and other tissues.…”

“…Binding of PAF to surface receptors activates intracellular second messenger signaling cascades, including protein kinase C, tyrosine kinases, and mitogen-activated protein kinases (MAPKs), depending on target cell type (2,3). PAF's actions are not limited to receptor interactions at the cell surface, as it is internalized either bound to receptors or via an as yet uncharacterized non receptor mediated pathway (4,5). Intracellular binding sites for PAF have been found in brain microsomal fractions (6) including a high affinity binding site implicated in transcriptional regulation (7).…”