Nuclear factor-kappa beta (NF-kB) regulates platelet activating factor (PAF) signaling in fetal ovine pulmonary vascular smooth muscle cells (PVSMC)

NF-kB a family of transcription factors regulates cell growth in mammalian cells. PAF binds to its GPCR (PAFr) to phosphorylate and induce nuclear translocation of NF-kB p65 leading to PVSMC proliferation. We used siRNA-dependent translation initiation arrest to study a mechanism by which NF-kB p65 regulates PAF stimulation of PVSMC proliferation. Our hypothesis is that a) PAF induces NF-kB p65 DNA binding and b) NF-kB p65 siRNA attenuates PAF stimulation of PVSMC proliferation. For DNA binding, serum starved cells were fed 10nM PAF with and without PAFr antagonists CV 3988 or BN 52021 and incubated for 12 hr. DNA binding was measured by specific ELISA. For NF-kB p65 siRNA effect, starved cells transfected with the siRNA were incubated for 24 hr with and without 10nM PAF. Cell proliferation was measured by DNA synthesis while PAFr protein expression was measured by Western blotting. In both studies, effect of 10% FBS alone was used as the positive control. In general, PAF stimulated DNA binding which was ...