Role of nuclear factor-kappaB and protein kinase C signaling in the expression of the kinin B1 receptor in human vascular smooth muscle cells.
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-kappaB (NF-kappaB), 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-kappaB 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-kappaB nuclear translocation and both mRNA concentration and stability [interleukin (IL)-1beta, 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-kappaB p65 is necessary for the effective expression of the cell surface B1 receptor under basal or IL-1beta, fetal bovine serum (FBS), or PMA stimulation conditions. Dexamethasone cotreatment reproduced these effects. IL-1beta-, 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 Bmax under FBS or PMA treatment. Lys-des-Arg9-bradykinin had little effect on NF-kappaB activation, the Bmax, or receptor mRNA abundance or stability. Both NF-kappaB and protein kinase C signaling are required for the effective expression of the kinin B1 receptor in human umbilical artery smooth muscle cells.
Moreau
Marie Eve
ME
Faculté de Pharmacie, Université de Montréal, Canada.
Bawolak
Marie-Thérèse
MT
Morissette
Guillaume
G
Adam
Albert
A
Marceau
François
F
D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000737:chemistry*; D032389:Myocytes, Smooth Muscle / Q000737:chemistry*; D016328:NF-kappa B / Q000502:physiology*; D011493:Protein Kinase C / Q000502:physiology*; D021381:Protein Transport; D012333:RNA, Messenger / Q000032:analysis; D034741:RNA, Small Interfering / Q000494:pharmacology; D043783:Receptor, Bradykinin B1 / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007375:Interleukin-1; D016328:NF-kappa B; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D043783:Receptor, Bradykinin B1; D003907:Dexamethasone; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate