Role of Nuclear Factor-κB and Protein Kinase C Signaling in the Expression of the Kinin B₁Receptor in Human Vascular Smooth Muscle Cells

Abstract

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-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 B 1 receptor agonist [3 H]Lys-des-Arg 9 -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 B 1 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 B 1 receptor mRNA was inhibited by the addition of the protein kinase, which also diminished the B max under FBS or PMA treatment. Lys-des-Arg 9 -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 B 1 receptor in human umbilical artery smooth muscle cells.

Fluorescent Ligands of the Bradykinin B1 Receptors: Pharmacologic Characterization and Application to the Study of Agonist-Induced Receptor Translocation and Cell Surface Receptor Expression

“…The cells were washed twice with the binding medium and filled with 0.5 ml of prewarmed (37°C) binding medium. The binding medium was serum-free culture Medium 199 (Invitrogen) supplemented with 0.1% bovine serum albumin and sodium azide [0.02% (w/v)] for the peptide radioligand of the B 1 receptor only; 1 M amastatin, 1 M captopril, and 1 M phosphoramidon (Sigma-Aldrich) were added to the binding medium (as in our previous reports; Sabourin et al, 2002;Moreau et al, 2007). The experiments dealt with the effect of cell coincubation of the radioligand at a fixed concentration (1 nM) with a panel of unlabeled drugs.…”

“…The institutional research ethics board approved the anonymous use of human umbilical cord segments obtained after elective cesarean section deliveries. Primary cultures of human umbilical artery smooth muscle cells, which express endogenous kinin B 1 receptors (Morissette et al, 2006;Moreau et al, 2007), were obtained and propagated as described. To quantify the calcium mobilization induced or influenced by several B 1 receptor ligands in naturally expressed receptors of human origin, Fura-2 fluorometry (Invitrogen, Carlsbad, CA) was applied to the smooth muscle cells maintained in serum-containing medium, detached using trypsin-ethylenediamine tetraacetic acid, suspended in complete culture medium containing fetal bovine serum (to neutralize the trypsin), counted, centrifuged for 5 min at 1100 rpm at room temperature, and resuspended in Hanks' balanced salt solution (1ϫ, pH 7.4, prepared from 10ϫ concentrate; Wisent Canadian Laboratories, St. Bruno, QC, Canada) with 10 mM HEPES and 1.6 mM CaCl 2 .…”

“…This G protein-coupled receptor has two properties of interest: the vigorous regulation of its expression and an original adaptation mechanism to agonist stimulation. First, unlike the widely distributed and preformed B 2 receptors, the expression of B 1 receptors is induced under inflammatory or tissue injury conditions (e.g., in injured or ischemic vascular cells in vivo, in circulating peripheral T cells during multiple sclerosis flares, in cultured cells of various types stimulated with inflammatory cytokines; Agata et al, 2000;Emanueli et al, 2002;Prat et al, 2005;Moreau et al, 2007;Bawolak et al, 2008b). Current evidence indicates that this G protein-coupled receptor is not submitted to agonist-induced phosphorylation or internalization, contrary to the related bradykinin B 2 receptor (Leeb-Lundberg et al, 2005).…”

A ligand-based approach to investigate the expression and function of angiotensin converting enzyme in intact human umbilical vein endothelial cells

“…To evaluate the localization of ACE in freshly isolated human umbilical blood vessels, immunostaining was applied to paraffin section of the vein and artery using the anti-ACE The translocation of NFmaintained in their regular culture medium 60-90 min after drug treatments using immunofluorescence as described [22]. …”

Up-regulation of bradykinin receptors in rat bronchia via IκB kinase-mediated inflammatory signaling pathway

“…Data obtained previously have demonstrated that NF-κB signaling plays an important role in the process up-regulation of the bradykinin B 1 receptor (Moreau et al, 2007;Ni et al, 1998;Sabourin et al, 2002;Schanstra et al, 1998). Others have also shown that the NF-κB pathway is important for inflammatory cytokine enhanced expression of bradyinin B 1 and B 2 receptors in osteoblasts and fibroblasts (Brechter et al, 2008) ; TPCA-1 is a highly selective IκB kinase 2 inhibitor, the results from 57 assays gave a mean IC 50 = 17.9 nM on IKK-2 and has 22-fold selectivity over IKK-1 (Podolin et al, 2005).…”