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Agonist-induced down-regulation of platelet-activating factor receptor gene expression in U937 cells.

ABSTRACT: Prolonged exposure (8-24 h) of human promonocytic U937 cells to 100 nM 1-O-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (carbarmyl-PAF), a non-metabolizable analogue of platelet-activating factor (PAF), reduced the numbers of PAF receptors by 50-75%, as determined by the radioligand-binding assay. To clarify whether the down-regulation of receptor numbers is due to decreased expression level of the PAF-receptor gene, the effect of carbamyl-PAF on the steady-state level of PAF-receptor mRNA was examined by a highly sensitive reverse-transcriptase PCR method. A 50% decline in the level of PAF-receptor mRNA was observed in U937 cells pretreated with 100 nM carbamyl-PAF for 24 h. The effect of carbamyl-PAF was dose-dependent, with an EC50 value around 10 nM. PAF-receptor antagonist, SRI-63675, was able to attenuate the effect of carbamyl-PAF. Furthermore lysoPAF, at 1 uM, was unable to induce a significant decrease in PAF-receptor mRNA after incubation for 24 h, indicating that the effect of carbamyl-PAF was specific. The half-life of the PAF-receptor mRNA measured in the presence of actinomycin D was unaffected by carbamyl-PAF treatment. In contrast, nuclear run-off experiments demonstrated that the transcription rate of the PAF-receptor gene in carbamyl-PAF-treated cells was about 65% of that in control cells. These results suggest that the PAF receptor in U937 cells is subject to down-regulation by agonist, at least partly, at the transcriptional level.

SUBMITTER: Chau LY

PROVIDER: S-EPMC1137072 | biostudies-other | 1994 Aug

REPOSITORIES: biostudies-other

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Project description:This data series contains spotted oligo microarray data from 10 different experiments using Agilent Rat v2 microarrays. This data is being made public in support of Fillon S et al. Journal of Immunology, (2006). Proinflammatory bacterial components are at least partially responsible for causing the clinical features of sepsis, a syndrome that causes >100,000 deaths each year in the US (1). In the case of Gram positive infection, a key bacterial element recognized by the innate immune system is the cell wall, a complex network of peptidoglycan covalently linked to teichoic acids, proteins and lipoproteins. The current model of innate immune recognition of Gram positive bacteria suggests bacterial cell wall interacts with host recognition proteins, such as toll-like receptors (TLR) and Nod proteins. We describe an additional recognition system mediated by the platelet activating factor receptor (PAFr) and directed to the pathogen associated molecular pattern (PAMP) phosphorylcholine that results in uptake of bacterial components into host cells. Intravascular choline-containing cell walls bound to endothelial cells and caused rapid lethality in wild type, Tlr2-/- and Nod2-/- mice, but not in Pafr-/- mice. Cell wall exited the vasculature into the heart and brain, accumulating within endothelial cells, cardiomyocytes and neurons in a PAFr-dependent way. Physiological consequences of the cell wall/PAFr interaction were cell specific, being noninflammatory in endothelial cells and neurons, but causing rapid loss of cardiomyocyte contractility that contributed to death. Thus, PAFr shepherds phosphorylcholine-containing bacterial components such as cell wall into host cells from where the response ranges from quiescence to severe pathophysiology. Keywords: Competitive hybridizations The ten experiments in this series comprise of four distinct experiments, two of which were performed as biological triplicates and two as biological duplicates. The table below describes the overall design in detail: File Name Experiment 16011868017643v41_GEO_format.txt RBCEC Replicate 1 16011868017644v41_GEO_format.txt RBCEC Replicate 2 251186821865v41_GEO_format.txt Neuron Replicate 1 16011868021377v41_GEO_format.txt Neuron Replicate 2 251186821690v41_GEO_format.txt CW/Lyt44 Replicate 1 251186821691v41_GEO_format.txt CW/Lyt44 Replicate 2 251186821692v41_GEO_format.txt CW/Lyt44 Replicate 1 251186821693v41_GEO_format.txt CW+TNF/Lyt44+TNF Replicate 1 251186821694v41_GEO_format.txt CW+TNF/Lyt44+TNF Replicate 2 251186829677v41_GEO_format.txt CW+TNF/Lyt44+TNF Replicate 3

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Agonist-induced down-regulation of platelet-activating factor receptor gene expression in U937 cells.

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