Bradykinin stimulates NF-kappaB activation and interleukin 1beta gene expression in cultured human fibroblasts.

The guanine nucleotide-binding regulatory protein α-subunit, Gα16, is primarily expressed in hemopoietic cells, and interacts with a large number of seven-membrane span receptors including chemoattractant receptors. We investigated the biological functions resulting from Gα16 coupling of chemoattractant receptors in a transfected cell model system. HeLa cells expressing a κB-driven luciferase reporter, Gα16, and the formyl peptide receptor responded to fMLP with a ∼7- to 10-fold increase in luciferase activity. This response was accompanied by phosphorylation of IκBα and elevation of nuclear κB-DNA binding activity, indicating activation of NF-κB. In contrast to Gα16, expression of Gαq, Gα13, and Gαi2 resulted in a marginal increase in κB luciferase activity. A GTPase-deficient, constitutively active Gα16 mutant (Q212L) could replace agonist stimulation for activation of NF-κB. Furthermore, expression of Gα16 (Q212L) markedly enhanced TNF-α-induced κB reporter activity. The Gα16-mediated NF-κB activation was paralleled by an increase in phospholipase C-β activity, and was blocked by pharmacological inhibitors of protein kinase C (PKC) and by buffering of intracellular Ca2+. The involvement of a conventional PKC isoform was confirmed by the finding that expression of PKCα enhanced the effect of Gα16, and a dominant negative PKCα partially blocked Gα16-mediated NF-κB activation. In addition to formyl peptide receptor, Gα16 also enhanced NF-κB activation by the C5a and C3a receptors, and by CXC chemokine receptor 2 and CCR8. These results suggest a potential role of Gα16 in transcriptional regulation downstream of chemoattractant receptors.

Section: Discussionmentioning

confidence: 77%

Gα16 Couples Chemoattractant Receptors to NF-κB Activation

Yang

Sang

Rahman

et al. 2001

“…Nuclear extracts were prepared by a modified method of Dignam et al (24) and EMSA were performed as previously described (25). Oligonucleotides and their complementary strands for EMSAs were from Promega.…”

Section: Emsamentioning

confidence: 99%

Up-Regulation of Functional Kinin B1 Receptors in Allergic Airway Inflammation

Christiansen

Eddleston

Woessner

et al. 2002

B1 receptors are known to be induced during allergic airway inflammation in animal models. However, little is known regarding in vivo B1 receptor expression in humans. We examined B1 receptor mRNA expression in nasal tissue samples from allergic rhinitis and normal subjects. Allergic rhinitis subjects displayed significantly higher expression of B1 receptor mRNA than did the normal subjects, and nasal allergen challenge increased B1 receptor mRNA expression at 8 to 24 h time points in allergic rhinitis subjects. No significant difference was found in B2 receptor expression. To confirm B2 and B1 receptor functional activity, subjects were challenged with kinin agonists. Nasal challenge with the B1 receptor ligand, Lys-des-Arg-bradykinin (BK), activated extracellular signal-regulated kinase in allergic rhinitis, but not normal, subjects. Nasal challenge with the B2 receptor ligand, BK, activated extracellular signal-regulated kinase in both allergic rhinitis and normal subjects. The consequences of B1 receptor activation were investigated using the human airway epithelial cell lines A549 and BEAS-2B. We demonstrated that Lys-des-Arg-BK activates the transcription factor AP-1. Taken together, these results show that functional B1 receptors are induced in the airway during allergic inflammation and suggest that they participate in the regulation of gene expression.

“…Several G protein-couple receptors, including the B2 BK receptor, have been shown to mediate NF-B activation and cytokine gene transcription in human epithelial cells and fibroblasts (16,17). BK-stimulated NF-B activation has been demonstrated to be mediated through B2 BK receptors coupled to the G i /G o class of heterotrimeric G proteins (16).…”

mentioning

confidence: 99%

“…BK-stimulated NF-B activation has been demonstrated to be mediated through B2 BK receptors coupled to the G i /G o class of heterotrimeric G proteins (16). Activation of the small GTP-binding protein, Rho A, and PI3K are involved in BK-stimulated NF-B activation in A549 cells (17,18).…”

mentioning

confidence: 99%

Bradykinin B2 Receptor Mediates NF-κB Activation and Cyclooxygenase-2 Expression via the Ras/Raf-1/ERK Pathway in Human Airway Epithelial Cells

Chen

Yu²,

Lei³

et al. 2004

In this study, we investigated the signaling pathways involved in bradykinin (BK)-induced NF-κB activation and cyclooxygenase-2 (COX-2) expression in human airway epithelial cells (A549). BK caused concentration- and time-dependent increase in COX-2 expression, which was attenuated by a selective B2 BK receptor antagonist (HOE140), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), an NF-κB inhibitor (pyrrolidine dithiocarbate), and an IκB protease inhibitor (l-1-tosylamido-2-phenylethyl chloromethyl ketone). The B1 BK receptor antagonist (Lys-(Leu8)des-Arg9-BK) had no effect on COX-2 induction by BK. BK-induced increase in COX-2-luciferase activity was inhibited by cells transfected with the κB site deletion of COX-2 construct. BK-induced Ras activation was inhibited by manumycin A. Raf-1 phosphorylation at Ser338 by BK was inhibited by manumycin A and GW 5074. BK-induced ERK activation was inhibited by HOE140, manumycin A, GW 5074, and PD 098059. Stimulation of cells with BK activated IκB kinase αβ (IKKαβ), IκBα phosphorylation, IκBα degradation, p65 and p50 translocation from the cytosol to the nucleus, the formation of an NF-κB-specific DNA-protein complex, and κB-luciferase activity. BK-mediated increase in IKKαβ activity and formation of the NF-κB-specific DNA-protein complex were inhibited by HOE140, a Ras dominant-negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Our results demonstrated for the first time that BK, acting through B2 BK receptor, induces activation of the Ras/Raf-1/ERK pathway, which in turn initiates IKKαβ and NF-κB activation, and ultimately induces COX-2 expression in human airway epithelial cell line (A549).