Platelet activating factor receptors : nuclear localization and signaling in microvascular endothelial cells

It has been postulated that intracellular binding sites for platelet activating factor (PAF) contribute to pro-inflammatory responses to PAF. Isolated nuclei from porcine cerebral microvascular endothelial cells (PCEC) produced PAF-molecular species in response to H2O2 . Using fluorescent-activated cell sorter analysis, we demonstrated the expression of PAF receptors (PAFR) on cell and nuclear surfaces of PCEC. Confocal microscopy studies performed on PCEC, Chinese hamster ovary cells stably overexpressing PAFR and on isolated nuclei from PCEC also showed a robust nuclear distribution of PAFR. Presence of PAFR at the cell nucleus was further revealed in brain endothelial cells by radioligand binding experiments, immunoblotting, and in situ in brain by immunoelectron microscopy. Stimulation of nuclei with PAF evoked a decrease in cAMP production and a pertussis toxin (PTX)-sensitive rise in nuclear calcium, unlike observations in plasma membrane, which exhibited a PTX-insensitive elevation in inositol phosphates. Moreover, on isolated nuclei C-PAF evoked the expression of pro-inflammatory genes inducible nitric oxide synthase and cyclooxygenase-2 (COX-2), and was associated with augmented ERK1/2 phosphorylation and NF-kappaB binding to DNA consensus sequence; COX-2 expression was prevented by MEK/ERK1/2 and NF-kappaB inhibitors. This study describes for the first time the nucleus as a putative organelle capable of generating PAF and expresses its receptor, which upon stimulation induces the expression of pro-inflammatory gene COX-2