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Nuclear factor‐kappa beta (NF‐kB) regulates platelet activating factor (PAF) signaling in fetal ovine pulmonary vascular smooth muscle cells (PVSMC)
Author(s) -
Ibe Basil O.,
Renteria Lissette S
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.626.9
Subject(s) - small interfering rna , platelet activating factor , microbiology and biotechnology , cell growth , transfection , vascular smooth muscle , signal transduction , transcription factor , biology , cell culture , chemistry , biochemistry , endocrinology , gene , smooth muscle , genetics
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 inhibited by both PAFr antagonists. NF‐kB p65 siRNA significantly attenuated cell proliferation compared to 10% FBS and PAF effect. Inclusion of PAF in siRNA 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‐linked NF‐kB p65 pathway. LA Biomed 513292