RGS4 is required for PI3K and Akt-dependent HASM proliferation.

<p>(<b>A</b>) Gel photographs of RT-PCR analysis of RGS4 and GAPDH expression in untreated and PDGF-treated HASM cells infected with lentiviruses encoding either control (<i>ShCnt</i>) or RGS4-specific (<i>ShRGS4</i>) shRNAs. (<b>B</b>) RGS4 depletion attenuates PDGF-mediated HASM proliferation. Untransfected <i>ShRGS4</i> and <i>ShCnt</i> ASM cells were serum-starved for 24 h followed by treatment with serum-free medium or medium containing PDGF for an additional 72 h. Total cell numbers were determined using a Beckman Cell Coulter counter (mean ± SEM of 6 separate experiments performed in 2 cell lines). (<b>C</b>) PDGF-induced cell cycle traversal after 24 h analyzed by FACS analysis of propidium iodide-stained nuclei isolated from <i>ShRGS4</i> and <i>ShCnt</i> HASM cells. (<b>D</b>) Analysis of p85α phosphorylation assessed by immunoblotting of lysates of untreated or PDGF-treated S<i>hCnt</i> or <i>ShRGS4</i> expressing HASM cells. Blots are representative of 3 separate experiments performed in 2 cell lines. (<b>E</b>) Left: Immunoblot analysis of p-p85 PI3K, RGS4 and total p85 PI3K expression in untreated or PDGF-treated cells. Right: Immunoblot analysis of lysates from untreated or PDGF-treated cells immunoprecipitated with indicated antibodies. Blots are representative of 3 separate experiments performed in 3 cell lines. (<b>F</b>) Kinetics of PDGF-mediated Akt phosphorylation were analyzed in lysates of <i>ShRGS4</i> or <i>ShCnt</i> HASM cells by ELISA. Data (mean ± SEM) are expressed as fold change over basal, set as ‘1’ in 5 separate experiments. (<b>G</b>) Akt kinase activity in <i>ShRGS4</i> or <i>ShCnt</i> HASM cells in untreated or PDGF-treated cells. p-Akt (Ser-473) was immunoprecipitated from total cell lysates using a specific antibody followed by incubation with recombinant GSK3. Phospho-GSK-3α/β (Ser-21/9) was quantified by colorimetric assay. Data (mean ± SEM) are fold-change over vehicle-treated <i>ShCnt</i> cells determined in 4 independent experiments measured in triplicate. (<b>H</b>) Total RNA was extracted from <i>ShRGS4</i> and <i>ShCnt</i> HASM cells treated with PDGF or diluent for 8 h followed by analysis of relative <i>cyclin D1</i> expression by real-time PCR. Data are mean ± SEM of 6 independent experiments using 2 cell lines.</p

A schematic illustration of the role of RGS4 in modulating human ASM excitation-contraction coupling and mitogen-induced proliferation.

<p>A model by which regulatory G protein signaling (RGS) molecules modulate agonist-induced contraction and growth factor-induced mitogenesis. Growth factors stimulate receptor tyrosine kinases (RTK) coupled to small G proteins such as Ras and Src. Src then activates phosphoinositide 3-kinase type IA (PI3K) and extracellular receptor kinase 1/2 (ERK1/2). Subsequently, PI3K activates protein kinase B (PKB) and mammalian target of rapamycin (mTOR) that then stimulates S6 kinase (S6K1) phosphorylation. S6K1 phosphorylation induces expression of RGS and other proteins necessary for cell cycle progression and proliferation. The expression of RGS colocalizes with PI3K and promotes prolonged PI3K activation to facilitate cell cycle traversal. Agonists stimulate G protein-coupled receptors (GPCRs) through the activation of specific Gα subunits. PLCβ stimulation then generates IP₃ that mediates calcium release through cytoplasmic calcium stores by binding to the IP₃ and ryanodine receptors. Increased calcium promotes actin-myosin cross bridge cycling in a myosin light chain (MLC) kinase-dependent manner. In parallel, G protein activation stimulates Rho kinase activation that inhibits MLC phosphatase and also promotes regulatory MLC phosphorylation (calcium sensitivity). Agonists and growth factors may activate ASM in a paracrine or autocrine manner. The activation of RTK pathways and the inhibition of agonist-mediated force generation promote ASM hyperplasia that increases ASM mass and may contribute to irreversible airflow obstruction.</p

PDGF markedly inhibits carbachol-induced bronchoconstriction in human small airways.

<p>(<b>A</b>) PCLS were obtained from healthy donors and treated with medium or PDGF (50 ng/ml) for 8 h followed by analysis of carbachol-induced small airway constriction by microscopy. Log EC₅₀ and E_max values (mean ± SEM) were determined in experiments on 16 airways obtained from 4 separate donors as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028504#s2" target="_blank">Methods</a>. (<b>B</b>) PDGF attenuates agonist-induced increases in [Ca²⁺]_i. HASM cells were stimulated for 8 h with 10 ng/ml PDGF or diluent followed by measurement of [Ca²⁺]_i using a Ca²⁺-sensing fluorophore after stimulation with acetylcholine (ACh), histamine or thrombin. Single-cell calcium transients were measured over a period of 300 sec. Table shows mean ± SEM of peak [Ca²⁺]_i levels determined in 30 cells (<i>P</i> values determined by 2-tailed Student's <i>t</i> test). Bottom tracings represent [Ca²⁺]_i in individual cells (blue line = control, diluent-treated; red line = PDGF-treated). Middle tracings represent group mean data ± SEM shown in the shaded segments. (<b>C</b>) RGS4 is required for PDGF-mediated attenuation of agonist-induced [Ca²⁺]_i in HASM cells. The mean responses are shown using thick curves, and the individual cell responses are shown using dashed curves.</p

Clinical and sputum characteristics of patients categorized according to GINA.

<p>GINA: Global Initiative for Asthma; BD: bronchodilator; LABA: long-acting bronchodilator; FEV₁: forced expiratory volume in one second; TCC: total cell counts; BDP: beclomethasone dipropionate; PC₂₀FEV₁: provocative concentration of methacholine to induce a 20% decrease in FEV1;</p><p>*mean (SE);</p>†<p>geometric mean (95% CI);</p>‡<p>median (IQR);</p><p>**<i>P</i><0.05 compared to control.</p

RGS4 is expressed in proportion to lung ASM mass and disease severity in asthma.

<p>(<b>A</b>) RGS4 cells within and adjacent to the asthma ASM bundle. Representative photomicrographs of a bronchial biopsy from a subject with asthma (200×) stained with isotype control antibody (far left) or anti-RGS4 (3 right panels) which include the epithelium, lamina propria and ASM bundle. Far right panel shows staining of a biopsy from a separate patient (400×) stained with antibodies against α-smooth muscle actin and RGS4. Arrows show RGS4⁺ cells within and adjacent to the ASM bundle. (<b>B–C</b>) Dot-plot of the number of RGS4⁺ cells/mm² ASM within the ASM bundle (<b>B</b>) or RGS4⁺ cells/mm² ASM within and adjacent to the ASM bundle (<b>C</b>) in subjects with asthma and healthy controls. Horizontal bars depict the median value (<i>P</i><0.05, Kruskal-Wallis test for all across-group comparisons; <i>P</i> value for Dunn's post test given on figure). (<b>D</b>) Correlation of RGS4⁺ cells/mm² ASM with % predicted FEV₁ in all subjects and those with asthma alone, respectively, with correlation coefficient and <i>P</i> value provided.</p

BAL fluid from ozone-exposed mice induces AHR in slices from naïve mice.

<p>Lung slices from naïve mice were incubated with BAL fluid from mice exposed to ozone (6 ppm; 2 h; T = 0 h), or filtered air. (A) Airways were contracted to carbachol (CCh) to create a concentration-response curve. BAL fluid from ozone-exposed mice (B) decreased log EC₅₀ values; and, (C) increased maximum contraction compared to slices incubated with BAL fluid from air-exposed mice. Data expressed as mean ± SEM shown; * P≤0.05 vs. filtered air control. At least 14 airways were used in each group, and never more than 19.</p

Level of eicosanoids in murine BAL fluid following ozone exposure.

<p>Mice were administered either dexamethasone (2.5 mg/kg) or ABT (50 mg/kg) 18 h prior or indomethacin (10 mg/kg) 2 hr prior to ozone (6 ppm, 2 h; T = 0 h) exposure, with control mice exposed to air. BAL fluid was retrieved and analyzed for eicosanoids using mass spectrometry. Data (n = 4) is expressed as mean ± SEM (nM). ND  =  Not detected.</p

Ozone did not increase CYP enzymes measured.

<p>mRNA was extracted from whole lungs of mice exposed to ozone (6 ppm; 2 h; T = 0 h) and CYP isotypes were quantified using Taq Man qPCR, as described in the methods. Data was run in triplicate and expressed as mean ± SEM (n = 5 in each group).</p

20-HETE increases murine airway sensitivity to carbachol.

<p>Lung slices from naïve mice were incubated over-night with 20-HETE (10 and 1 ng/mL). (A) Airways were contracted to carbachol (CCh) to create concentration-response curves. Incubation with 20-HETE (B) decreased log EC₅₀ values; but, (C) had no effect on maximum contraction compared to control slices. Data expressed as mean ± SEM shown; * P≤0.05 and ** P≤0.01 vs control (exact P values shown). At least 8 airways were used in each group, and never more than 12.</p

Ozone increases murine airway sensitivity to carbachol.

<p>(A) A representive contraction of a murine airway to carbachol (CCh) where A is the airway and V is the blood vessel; and a mean concentration-response curve calculated by changes in the airway lumen area with respect to the baseline image. Slices (n = 13) were prepared as described in the methods. A CCh EC₅₀ value of 0.66 µM and E_max values of 67.1±5.0% were obtained. Airways from mice exposed to ozone at concentrations of (B) 3 ppm or (C) 6 ppm for 2 h, were also contracted to CCh. Mice were sacrificed either immediately (T = 0 h), or the next day (T = 16 h). (D) Area under the curve (AUC) units were used to statistically analyze data. Mean ± SEM shown. NS  =  Non-significant; * P≤0.05; *** P≤0.001 vs filtered air control. At least 6 animals were used in each group, and never more than 8.</p