Mechanism by which nuclear factor-kappa beta (NF-kB) regulates ovine fetal pulmonary vascular smooth muscle cell proliferation

摘要

Abstract Platelet activating factor (PAF) modulates ovine fetal pulmonary hemodynamic. {PAF} acts through its receptors (PAFR) in pulmonary vascular smooth muscle cells (PVSMC) to phosphorylate and induce nuclear translocation of NF-kB p65 leading to {PVSMC} proliferation. However, the interaction of NF-kB p65 and {PAF} in the nuclear domain to effect {PVSMC} cell growth is not clearly defined. We used siRNA-dependent translation initiation arrest to study a mechanism by which NF-kB p65 regulates {PAF} stimulation of {PVSMC} proliferation. Our hypotheses are: (a) {PAF} induces NF-kB p65 {DNA} binding and (b) NF-kB p65 siRNA attenuates {PAF} stimulation of {PVSMC} proliferation. For {DNA} binding, cells were fed 10 nM {PAF} with and without {PAFR} antagonists {WEB} 2170, {CV} 3988 or {BN} 52021 and incubated for 12 h. {DNA} binding was measured by specific ELISA. For NF-kB p65 siRNA effect, starved cells transfected with the siRNA were incubated for 24 h with and without 10 nM PAF. Cell proliferation was measured by {DNA} synthesis while expression of NF-kB p65 and {PAFR} protein was measured by Western blotting. In both studies, the effect of 10% {FBS} alone was used as the positive control. In general, {PAF} stimulated {DNA} binding which was inhibited by {PAFR} antagonists. siRNAs to NF-kB p65 and {PAFR} significantly attenuated cell proliferation compared to 10% {FBS} and {PAF} effect. Inclusion of {PAF} in siRNA-treated cells did not reverse inhibitory effect of NF-kB p65 siRNA on {DNA} synthesis. {PAFR} expression was inhibited in siRNA-treated cells. These data show that PAF-stimulation of {PVSMC} proliferation occurs via a PAFR-NF-kB p65 linked pathway.