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1.
Figure 2

Figure 2. RGS4 is expressed in proportion to lung ASM mass and disease severity in asthma.. From: An RGS4-Mediated Phenotypic Switch of Bronchial Smooth Muscle Cells Promotes Fixed Airway Obstruction in Asthma.

(A) 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–C) Dot-plot of the number of RGS4+ cells/mm2 ASM within the ASM bundle (B) or RGS4+ cells/mm2 ASM within and adjacent to the ASM bundle (C) in subjects with asthma and healthy controls. Horizontal bars depict the median value (P<0.05, Kruskal-Wallis test for all across-group comparisons; P value for Dunn's post test given on figure). (D) Correlation of RGS4+ cells/mm2 ASM with % predicted FEV1 in all subjects and those with asthma alone, respectively, with correlation coefficient and P value provided.

Gautam Damera, et al. PLoS One. 2012;7(1):e28504.
2.
Figure 1

Figure 1. Human airway smooth muscle (HASM) mitogens induce RGS4 expression.. From: An RGS4-Mediated Phenotypic Switch of Bronchial Smooth Muscle Cells Promotes Fixed Airway Obstruction in Asthma.

(A) HASM cells express RGS mRNAs. RGS expression was determined by real-time PCR in HASM cells left untreated or treated with PDGF for 2 h. Data presented are mean ± SEM of 3 separate experiments performed in triplicate using Gadph as an internal control, relative to RGS2 in untreated cells, set as ‘1’. (B) Analysis of RGS4 expression in HASM cells treated with mitogens such as EGF (1 ng/ml), PDGF (10 ng/ml), thrombin and cytokines by real-time PCR. Data (mean ± SEM) of 5 independent experiments. (C) Kinetics of PDGF-mediated RGS4 mRNA expression as a function of time. Values (mean ± SEM of 3 separate experiments performed in triplicate) are relative to those of untreated cells, set as ‘1’ (D) RGS4 expression in untreated (top) or PDGF-treated (bottom) HASM cells immunostained with polyclonal anti-RGS4 followed by Alexa Fluor 488-conjugated anti-IgG and DAPI. Left: High power (650×) visualization of immunostaining with an anti-RGS4 antibody shows plasmalemma staining of RGS4 protein after PDGF. These data are representative of triplicate wells in 2 separate cell lines. Bars represent 50 microns.

Gautam Damera, et al. PLoS One. 2012;7(1):e28504.
3.
Figure 4

Figure 4. PDGF markedly inhibits carbachol-induced bronchoconstriction in human small airways.. From: An RGS4-Mediated Phenotypic Switch of Bronchial Smooth Muscle Cells Promotes Fixed Airway Obstruction in Asthma.

(A) 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 Emax values (mean ± SEM) were determined in experiments on 16 airways obtained from 4 separate donors as described in Methods. (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 (P values determined by 2-tailed Student's t 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. (C) 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.

Gautam Damera, et al. PLoS One. 2012;7(1):e28504.
4.
Figure 5

Figure 5. A schematic illustration of the role of RGS4 in modulating human ASM excitation-contraction coupling and mitogen-induced proliferation.. From: An RGS4-Mediated Phenotypic Switch of Bronchial Smooth Muscle Cells Promotes Fixed Airway Obstruction in Asthma.

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.

Gautam Damera, et al. PLoS One. 2012;7(1):e28504.
5.
Figure 3

Figure 3. RGS4 is required for PI3K and Akt-dependent HASM proliferation.. From: An RGS4-Mediated Phenotypic Switch of Bronchial Smooth Muscle Cells Promotes Fixed Airway Obstruction in Asthma.

(A) Gel photographs of RT-PCR analysis of RGS4 and GAPDH expression in untreated and PDGF-treated HASM cells infected with lentiviruses encoding either control (ShCnt) or RGS4-specific (ShRGS4) shRNAs. (B) RGS4 depletion attenuates PDGF-mediated HASM proliferation. Untransfected ShRGS4 and ShCnt 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). (C) PDGF-induced cell cycle traversal after 24 h analyzed by FACS analysis of propidium iodide-stained nuclei isolated from ShRGS4 and ShCnt HASM cells. (D) Analysis of p85α phosphorylation assessed by immunoblotting of lysates of untreated or PDGF-treated ShCnt or ShRGS4 expressing HASM cells. Blots are representative of 3 separate experiments performed in 2 cell lines. (E) 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. (F) Kinetics of PDGF-mediated Akt phosphorylation were analyzed in lysates of ShRGS4 or ShCnt HASM cells by ELISA. Data (mean ± SEM) are expressed as fold change over basal, set as ‘1’ in 5 separate experiments. (G) Akt kinase activity in ShRGS4 or ShCnt 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 ShCnt cells determined in 4 independent experiments measured in triplicate. (H) Total RNA was extracted from ShRGS4 and ShCnt HASM cells treated with PDGF or diluent for 8 h followed by analysis of relative cyclin D1 expression by real-time PCR. Data are mean ± SEM of 6 independent experiments using 2 cell lines.

Gautam Damera, et al. PLoS One. 2012;7(1):e28504.

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