β2-Adrenoceptor agonist-induced RGS2 expression is a genomic mechanism of bronchoprotection that is enhanced by glucocorticoids

Abstract

In asthma and chronic obstructive pulmonary disease, activation of G(q)-protein-coupled receptors causes bronchoconstriction. In each case, the management of moderate-to-severe disease uses inhaled corticosteroid (glucocorticoid)/long-acting β(2)-adrenoceptor agonist (LABA) combination therapies, which are more efficacious than either monotherapy alone. In primary human airway smooth muscle cells, glucocorticoid/LABA combinations synergistically induce the expression of regulator of G-protein signaling 2 (RGS2), a GTPase-activating protein that attenuates G(q) signaling. Functionally, RGS2 reduced intracellular free calcium flux elicited by histamine, methacholine, leukotrienes, and other spasmogens. Furthermore, protection against spasmogen-increased intracellular free calcium, following treatment for 6 h with LABA plus corticosteroid, was dependent on RGS2. Finally, Rgs2-deficient mice revealed enhanced bronchoconstriction to spasmogens and an absence of LABA-induced bronchoprotection. These data identify RGS2 gene expression as a genomic mechanism of bronchoprotection that is induced by glucocorticoids plus LABAs in human airway smooth muscle and provide a rational explanation for the clinical efficacy of inhaled corticosteroid (glucocorticoid)/LABA combinations in obstructive airways diseases.

Fig. 1.

Expression of RGS2 in human ASM cells. (A) Primary human ASM cells were treated with combinations of 1 μM dexamethasone (Dex) or 100 nM salmeterol (Salm). Cells were harvested for real-time PCR analysis of RGS2 and GAPDH mRNAs (Upper) or unspliced RGS2 nuclear RNA (nRGS2) and U6 RNA (Lower). (Upper) Data (n = 9), expressed as RGS2/GAPDH, are plotted as fold relative to untreated (at t = 1 h) as means ± SE. (Lower) Data (n = 10) are plotted as nRGS2/U6 as means ± SE. Multiple comparisons at each time were made using ANOVA with a Bonferroni post test. Significance data relative to untreated samples are *P < 0.05, **P < 0.01, and ***P < 0.001. Significance data relative to Dex-treated samples are ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001. Significance data relative to Salm-treated samples are ^dP < 0.05, and ^eP < 0.01. (B) Primary human ASM cells were not stimulated (NS) or treated with Dex (1 μM), Salm (100 nM), or Dex (1 μM) + Salm (100 nM). After 2 h, actinomycin D (10 μg/mL) was added and RNA was harvested at 15, 30, 60, 120, and 240 min for RT-PCR analysis of RGS2 and GAPDH. Data (n = 7 for 15, 30, and 60 min; n = 3 for 120 and 240 min) expressed as RGS2/GAPDH are plotted as a percentage of their respective treatment at t = 0 (i.e., when actinomycin D was added) as means ± SE. (C) Cells as in A were treated with 300 nM fluticasone propionate (FP), 100 nM salmeterol (Salm), and 10 μM forskolin (Forsk) before Western blot analysis of RGS2 and GAPDH. Blots representative of four such experiments are shown. (D) Primary human ASM cells were treated with 100 nM salmeterol plus 300 nM fluticasone propionate (Salm + FP) before Western blotting for RGS2 and GAPDH. Blots representative of four experiments are shown. (E) Primary human ASM cells were left naive or infected with Ad5-PKI or Ad5-LacZ. After 24 h, cells were treated with 300 nM fluticasone propionate (FP) and/or 100 nM salmeterol (Salm) for 6 h before Western blot analysis for RGS2 and GAPDH. Blots representative of three such experiments are shown. Densitometric data for C and D are provided in Fig. S2.

Fig. 2.

Effect of dexamethasone and salmeterol on agonist-enhanced [Ca²⁺]_i in human ASM cells. (A) Primary ASM cells loaded with Fluo4 were stimulated with various concentrations of histamine. Representative traces showing fluorescence/fluorescence at time 0 (F/F₀) are plotted. Peak F/F₀ from four such experiments are plotted as means ± SE. (B) Cells were treated as in A except that 50 nM mepyramine (mep) or 10 μM 2-APB were added 30 min before a 10-μM histamine challenge. Data (mep, n = 6; 2-APB, n = 8) are plotted as means of F/F₀ ± SE. Significance was tested using a Student's t test where *P < 0.05 and ***P < 0.001. (C) Cells were treated as in A, except that 100 nM salmeterol (Salm) and/or 1 μM dexamethasone (Dex) were added 2 or 6 h before 10 μM histamine, 30 μM methacholine (MCh), or a 1-μM U46619 challenge. Data (2 h pretreatment, n = 4; 6 h pretreatment, n = 8–10) expressed as peak F/F₀ are plotted as means ± SE. Multiple comparisons were made using ANOVA with a Bonferroni post test. Significance data relative to untreated samples are ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001. Significance data relative to Dex-treated samples are ^dP < 0.05, ^eP < 0.01, and ^fP < 0.001. Significance between other groups is indicated where ***P < 0.001. (D) Cells were pretreated with Dex (1 μM), Salm (100 nM), or Dex (1 μM) + Salm (100 nM) for the indicated times before challenge with histamine (10 μM) and measurement of fluorescence. Peak F/F_o is plotted as means ± SE. Significance relative to F/F_o induced by histamine was tested by ANOVA with a Dunnett's post test. **P < 0.01; ***P < 0.001.

Fig. 3.

RGS2 attenuates agonist-induced [Ca²⁺]_i primary human ASM cells. Primary human ASM cells were infected with the indicated MOIs of Ad5-RGS2.HA or Ad5-LacZ. (A) After 24 h, cells were harvested for Western blot analysis of HA-tagged RGS2. Blots are representative of four such experiments. (B) Following overnight incubation in serum-free media, cells were loaded with Fluo4 before stimulation with 10 μM histamine. Representative traces of F/F_o are shown and peak F/F_o data from four experiments are plotted as means ± SE. (C) ASM cells were infected with 100 MOI of Ad5-RGS2.HA or Ad5-LacZ or left naive. As in B, cells were challenged with 60 mM potassium chloride (KCl). Peak F/F_o (n = 4) is plotted as means ± SE. Comparisons relative to control (Ad5-LacZ) were made by Student's t test. (D) Primary human ASM cells were transfected with the indicated siRNAs before treatment with 100 nM salmeterol plus 1 μM dexamethasone (Salm + Dex) for 6 h. Western blotting for RSG2 and GAPDH was performed. Blots representative of five experiments are shown. (E) Alternatively, cells were loaded with Fluo4 before a 10-μM histamine challenge. Representative traces of F/F_o from nine such experiments are shown (Fig. S4G).

Fig. 4.

Rgs2 is bronchoprotective. (A) Equilibrated strips of mouse trachealis smooth muscle from wild type (WT) or Rgs2^−/− knockout (KO) animals were challenged with KCl (100 mM). At peak contraction, muscle strips were washed free of KCl and re-equilibrated. Cumulative concentration-response curves (n = 11–15) were constructed to methacholine (MCh) and plotted as a percentage of the KCl-induced response as means ± SE. Trachealis muscles were subsequently Western blotted for Rgs2 and Gapdh. Data from three animal pairings are shown. Comparisons were made using a Mann–Whitney U test. Significance relative to the relevant wild-type control sample is *P < 0.05. (B) Wild type (WT), Rgs2^−/+ heterozygous (Het), and Rgs2^−/− knockout (KO) mice were subjected to lung function analysis. Total lung resistance (Upper panels) and compliance (Lower panels) was measured at baseline (Left panels), following inhalation of nebulized PBS and PBS containing increasing concentrations of methacholine (Center panels) or 5-HT (Right panels). Data (n = 6–16) are plotted as cm H₂O/s/mL (resistance) or ml/cm H₂O (compliance) as means ± SE. (C) Wild type (WT) (Left panels) and Rgs2 knockout (KO) (Right panels) animals were treated by i.t. instillation with 50 μL of 0.9% saline or 0.5 mg/kg formoterol (Form). After 2 h, mice were anesthetized before lung function analysis. Total lung resistance (Upper panels) and compliance (Lower panels) were measured in response to increasing concentrations of nebulized methacholine. Data (n = 4–6) are plotted as cm H₂O/s/mL or ml/cm H₂O as means ± SE. Lungs (n = 4) were subsequently Western blotted for Rgs2 and Gapdh.