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Role of Nuclear Factor-B and Protein Kinase C Signaling in the Expression of the Kinin B₁ Receptor in Human Vascular Smooth Muscle Cells

Faculté de Pharmacie, Université de Montréal, Montréal, Québec, Canada (M.E.M., A.A.); and Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, Ville de Québec, Québec, Canada (M.-T.B., G.M., F.M.).

Kinin B₁ receptor expression was characterized in human umbilical artery smooth muscle cells to further elucidate the function and specificity of three previously proposed pathways [nuclear factor-B (NF-B), protein kinase C, and agonist autoregulation] that regulate this inducible G protein-coupled receptor. Radioligand binding assays, real-time reverser transcription/polymerase chain reaction with an optional actinomycin D treatment period, and NF-B immunofluorescence were primarily employed in these primary cell cultures. Various stimulatory compounds that increase receptor mRNA stability only (human and bovine sera, cycloheximide) or that stimulate NF-B nuclear translocation and both mRNA concentration and stability [interleukin (IL)-1, phorbol 12-myristate 13-acetate (PMA)] all increased the density of binding sites for the tritiated B₁ receptor agonist [³H]Lys-des-Arg⁹-bradykinin (without change in receptor affinity) in cell-based assays. Small interfering RNA assays indicated that NF-B p65 is necessary for the effective expression of the cell surface B₁ receptor under basal or IL-1, fetal bovine serum (FBS), or PMA stimulation conditions. Dexamethasone cotreatment reproduced these effects. IL-1-, FBS-, or PMA-induced stabilization of B₁ receptor mRNA was inhibited by the addition of the protein kinase C inhibitor 3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF-109203x), which also diminished the B_max under FBS or PMA treatment. Lys-des-Arg⁹-bradykinin had little effect on NF-B activation, the B_max, or receptor mRNA abundance or stability. Both NF-B and protein kinase C signaling are required for the effective expression of the kinin B₁ receptor in human umbilical artery smooth muscle cells.

Address correspondence to: Dr. François Marceau, Centre Hospitalier Universitaire de Québec, Centre de recherche en rhumatologie et immunology, 2705 Laurier Blvd, Ville de Québec (Québec), Canada G1V 4G2. E-mail: francois.marceau{at}crchul.ulaval.ca