Role of nuclear factor-κB and protein kinase C signaling in the expression of the kinin B1 receptor in human vascular smooth muscle cells

Kinin B 1 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-kappa B (NF-kappa 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-kappa 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-kappa B nuclear translocation and both mRNA concentration and stability [ interleukin (IL)-1 beta, phorbol 12-myristate 13-acetate (PMA)] all increased the density of binding sites for the tritiated B 1 receptor agonist [H-3] Lys-des-Arg(9)-bradykinin (without change in receptor affinity) in cell-based assays. Small interfering RNA assays indicated that NF-kappa B p65 is necessary for the effective expression of the cell surface B 1 receptor under basal or IL-1 beta, fetal bovine serum (FBS), or PMA stimulation conditions. Dexamethasone cotreatment reproduced these effects. IL-1 beta-, FBS-, or PMA-induced stabilization of B-1 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-Arg(9)-bradykinin had little effect on NF-kappa B activation, the B-max, or receptor mRNA abundance or stability. Both NF-kappa B and protein kinase C signaling are required for the effective expression of the kinin B-1 receptor in human umbilical artery smooth muscle cells.