Involvement of a serine protease in the synthesis of platelet-activating factor by endothelial cells stimulated by tumor necrosis factor-alpha or interleukin-1 alpha.
It has been shown that production of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by endothelial cells (EC) stimulated with tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 alpha requires the synthesis of new proteins and is regulated by anti-proteinases. Here, we demonstrate that TNF-alpha and IL-1 alpha induce the expression by EC of a 34-kDa diisopropyl fluorophosphate-binding protein immunoprecipitated by an anti-human elastase antibody. This protein is released in the medium and cleaves the chromogenic substrate N-methoxysuccinyl- Ala-Ala-Pro-Val p-anilide, which is specific for elastase. The generation of this elastase-like protein seems to be important for the synthesis of PAF induced by TNF-alpha and IL-1 alpha, as suggested by the following observations: (a) it precedes the synthesis of PAF; (b) the inhibitors of serine protease and anti-human elastase antibody prevent the synthesis of PAF and the activation of 1-O-alkyl-2-lyso-glycerophosphocholine acetyl-CoA: acetyltransferase, which is a key enzyme of the PAF remodelling pathway; (c) elastase, at concentrations similar to that detectable in the medium of cytokine-activated EC, elicits a rapid synthesis of PAF by EC. High-performance liquid chromatography-tandem mass spectrometric analysis of bioactive PAF demonstrates that the molecular species produced after stimulation of EC with TNF-alpha, IL-1 alpha or elastase are similar, with a predominant synthesis of the alkyl species. These results indicate that TNF-alpha and IL-1 alpha stimulate the production of a serine protease which is critical in the activation of enzymes involved in PAF synthesis, suggesting the potential involvement of this mechanism in the regulation of EC functions.
Bussolino
F
F
Dipartimento di Genetica Biologia e Chimica Medica, Università di Torino, Italy.
Arese
M
M
Silvestro
L
L
Soldi
R
R
Benfenati
E
E
Sanavio
F
F
Aglietta
M
M
Bosia
A
A
Camussi
G
G
D000123:Acetyltransferases / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000494:pharmacology*; D010196:Pancreatic Elastase / Q000378:metabolism; D010972:Platelet Activating Factor / Q000096:biosynthesis*; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D007375:Interleukin-1; D010972:Platelet Activating Factor; D011480:Protease Inhibitors; D014409:Tumor Necrosis Factor-alpha; D000123:Acetyltransferases; C046035:1-alkylglycerophosphocholine acetyltransferase; D012697:Serine Endopeptidases; D010196:Pancreatic Elastase