Pertussis toxin up-regulates angiotensin type 1 receptors through Toll-like receptor 4-mediated Rac activation

Abstract

Pertussis toxin (PTX) is recognized as a specific tool that uncouples receptors from G(i) and G(o) through ADP-ribosylation. During the study analyzing the effects of PTX on Ang II type 1 receptor (AT1R) function in cardiac fibroblasts, we found that PTX increases the number of AT1Rs and enhances AT1R-mediated response. Microarray analysis revealed that PTX increases the induction of interleukin (IL)-1beta among cytokines. Inhibition of IL-1beta suppressed the enhancement of AT1R-mediated response by PTX. PTX increased the expression of IL-1beta and AT1R through NF-kappaB, and a small GTP-binding protein, Rac, mediated PTX-induced NF-kappaB activation through NADPH oxidase-dependent production of reactive oxygen species. PTX induced biphasic increases in Rac activity, and the Rac activation in a late but not an early phase was suppressed by IL-1beta siRNA, suggesting that IL-1beta-induced Rac activation contributes to the amplification of Rac-dependent signaling induced by PTX. Furthermore, inhibition of TLR4 (Toll-like receptor 4) abolished PTX-induced Rac activation and enhancement of AT1R function. However, ADP-ribosylation of G(i)/G(o) by PTX was not affected by inhibition of TLR4. Thus, PTX binds to two receptors; one is TLR4, which activates Rac, and another is the binding site that is required for ADP-ribosylation of G(i)/G(o).

FIGURE 1.

PTX enhances Ca²⁺ responses by Ang II through AT1R up-regulation. A, average time courses of Ca²⁺ response induced by Ang receptor stimulation with Ang II (100 pm) in control and PTX-treated cells. B and C, peak increases in [Ca²⁺]_i (ΔRatio) plotted against various concentrations of Ang II (B) and ATP (C) in control, PTX-treated, and IL-1β-treated cells. Cells were treated with PTX (100 ng/ml) or IL-1β (10 ng/ml) for 24 h before agonist stimulation. D and E, increases in AT1R density induced by PTX (100 ng/ml) for 24 h. The B_max (D) and K_d (E) values for Ang II binding were calculated with GraphPad Prism software. F, effects of PTX on expression of Gα_q/11 and PLCβ₃. *, p < 0.05 versus PTX-untreated cells. Error bars, S.E.

FIGURE 2.

Involvement of IL-1β production in PTX-induced enhancement of Ca²⁺ response by Ang II stimulation. A, effects of PTX on the expression of AT1R, IL-1α, and IL-1β mRNAs. After cells were treated with PTX (100 ng/ml) for 24 h, total RNA was extracted. The expression of mRNAs was determined with microarray analysis. ID numbers of primer probe sets are shown in parenthesis. B, effects of respective reagents on IL-1β mRNA expression in cardiac fibroblasts. Cells were treated with PTX (100 ng/ml) for 24 h, treated with mastoparan-7 (10 μm) for 12 h, or infected with LacZ, WT Gα_i, Gα_i-ct, RGS4, and GRK2-ct at 300 MOI for 48 h. The -fold increases were calculated by the values of untreated cells (none) set as 1. C, effects of B-oligomer of PTX on Ang II-induced Ca²⁺ releases. Cells were treated with B-oligomer (1 or 10 nm) for 24 h before Ca²⁺ measurement. D, time course of PTX-induced expression of IL-1β protein. E, effects of IL-1β siRNAs, DN-Rac, and wortmannin (WTM) on PTX-induced IL-1β production. Two different siRNAs were used. F and G, effects of IL-1β neutral antibody (F) or IL-1β siRNAs (G) on Ang II-induced Ca²⁺ responses in control, PTX-treated, or IL-1β-treated cells. Cells were treated with PTX (100 ng/ml) or IL-1β (1 ng/ml) for 24 h before Ang II (100 pm) stimulation with or without anti-IL-1β antibody (500 μg/ml). Cells were transfected with IL-1β siRNAs (100 nm) 48 h before PTX treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus PTX-untreated, B-oligomer-untreated, control siRNA-treated, PTX-treated, or IL-1β-treated cells. Error bars, S.E.

FIGURE 3.

Requirement of NF-κB for PTX-induced enhancement of Ca²⁺ response by Ang II stimulation. A, effects of Ro106-9920 and IκBαm on PTX-induced IL-1β mRNA expression. Cells were pretreated for 20 min with Ro106-9920 (1 μm) or infected with IκBαm (100 MOI) for 48 h before the treatment of PTX (100 ng/ml) for 24 h. B, effects of NF-κB inhibitors on Ang II-induced Ca²⁺ responses in PTX-treated cells. C and D, effects of IkBαm and DN-Rac on PTX-induced increase in AT1R density. Cells were infected with adenoviruses expressing IkBαm or DN-Rac 24 h before PTX treatment. AT1R density was determined with receptor binding assay. E, time course of PTX-induced changes in NF-κB-dependent luciferase activity. F, effects of IκBαm, DN-Rac, DN-p47^phox, DPI, and p115-RGS on PTX-induced NF-κB activation. Cells were infected with LacZ, IκBαm, DN-Rac, DN-p47^phox, or p115-RGS at 100 MOI for 48 h or pretreated with DPI (5 μm) for 20 min before the addition of PTX (100 ng/ml) for 6 h. *, p < 0.05; **, p < 0.01 versus PTX-untreated or LacZ-expressing cells. Error bars, S.E.

FIGURE 4.

PTX induces Rac activation. A, time course of Rac activation induced by PTX (100 ng/ml). B, effects of wortmannin on PTX-induced Rac activation. Cells were pretreated with wortmannin (WTM; 100 nm) for 10 min before PTX stimulation. C, localization of GFP-fused wild type Rac and constitutively active Rac (CA-Rac) (G12V) with or without PTX stimulation. D, localization of GFP-fused PX domain of p40^phox (p40^phox-PX), and PI-3-P interaction-deficient mutant (p40^phox-PX (R105K)) with or without PTX treatment. *, p < 0.05 versus PTX-untreated cells. Error bars, S.E.

FIGURE 5.

Effects of DN-Rac on PTX-induced ROS production. A and B, average changes (A) and peak increases (B) in PTX-induced F/F₀ of dichlorofluorescein from time course experiments. The increases in PTX-induced fluorescence of dichlorofluorescein were calculated by the value of maximal fluorescence intensity (F) during 20 min of stimulation and initial value of fluorescence, F₀. *, p < 0.05; **, p < 0.01 versus PTX-untreated or LacZ-expressing cells. Error bars, S.E.

FIGURE 6.

Amplification of Rac-mediated signaling by PTX-induced IL-1β production. A, cells were transfected with siRNAs for Rac1 (si-Rac1) or their randomized controls (si-Cont) for 72 h before 5-min stimulation with IL-1β (10 ng/ml). B, effects of si-Rac1 on the maximal increases in AT1R density by IL-1β stimulation. Cells were treated with IL-1β for 24 h before membrane preparation. C, effects of si-Rac1 on PTX-induced production of IL-1β proteins. Cells were treated with PTX (100 ng/ml) for 90 min. D and E, effects of IL-1β siRNA on PTX-induced Rac activation. Cells were transfected with IL-1β (412) siRNA (100 nm) for 48 h before treatment with PTX (100 ng/ml). F and G, effects of IL-1β (412) siRNA on PTX-induced nuclear localization of the NF-κB p65 subunit. More than 100 cells were scanned and quantified the subcellular localization of p65 using Photoshop (13, 27). *, p < 0.05; ***, p < 0.001 versus IL-1β-treated or control siRNA-treated cells. Error bars, S.E.

FIGURE 7.

Roles of TLR4 in PTX-induced Rac activation and ADP-ribosylation of G_i/G_o. A, effects of TLR4 siRNAs on the expression of TLR4 and AT1R mRNAs. B, effects of TLR4 siRNAs on PTX-induced enhancement of Ang II-induced Ca²⁺ responses. Cells were treated with PTX for 24 h after siRNA treatment for 48 h. C, effects of TLR4 siRNA (si-1002) on PTX-induced Rac activation. D, effects of TLR4 siRNA on PTX-induced ADP-ribosylation of Gα_i proteins. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars, S.E.

FIGURE 8.

Schema of TLR4-mediated AT1R up-regulation induced by PTX. PTX induces ROS production through sequential activation of TLR4, Syk, PI 3-kinase (PI3K), Rac, and NADPH oxidase. Although the mechanism of NF-κB activation induced by ROS is still unknown, ROS mediate NF-κB-dependent expression of IL-1β. Induction of IL-1β also induces Rac activation through IL-1 receptor stimulation, leading to amplification of Rac-dependent signaling. Sustained activation of Rac may be required for PTX-induced AT1R up-regulation in rat cardiac fibroblasts. A-protomer of PTX enters the cells through unidentified binding site, and ADP-ribosylates G_i/G_o proteins.