JunB silencing attenuates TGFβ1-induced changes in cell contractility and cytoskeletal tension, but not induction of markers of smooth muscle differentiation.

<p>(<b>A</b>) BSMC were nucleofected with non-targeting control siRNA or with siRNA against JunB (0.1 µM and 1 µM) and assessed for JunB protein by immunoblotting (left panel, top). Effective knockdown of JunB was observed, with no change in c-Jun levels, demonstrating specificity of the siRNA used. Proliferating cell nuclear antigen (PCNA) expression was used as a loading control. 1 µM JunB siRNA reduced the levels of JunB mRNA by >80%, relative to non-targeting control siRNA, as assessed by semi-quantitative real-time PCR (right panel) (<b>B</b>) Reduction in JunB protein levels by siRNA in BSMC under basal and TGFβ1-stimulated conditions, demonstrated by immunoblotting. JunB levels were normalized to their respective GAPDH levels and expressed as percentage change relative to cells transfected with control siRNA and not subjected to TGFβ1 treatment. A representative immunoblot and its corresponding quantitation are shown. (<b>C</b>) TGFβ1-mediated induction of α-smooth muscle actin (α-SMA) calponin and SM22α, markers of smooth muscle differentiation, was unaffected by silencing of JunB, as shown by immunoblotting (left). Quantification of immunoblots is shown in the graph (right). Gel contraction assays (<b>D</b>) revealed that JunB knockdown significantly reduced both basal and TGFβ1-induced changes in cellular contractility. *p<0.05, t-test (<b>E</b>) Inhibition of JunB inhibits basal and TGFβ1-induced contraction. This inhibition of contraction, measured quantitatively as a reduction of traction (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053430#s2" target="_blank">Methods</a>) was statistically significant (*p<0.05, comparing siCtrl+ TGFβ1 or siJunB-TGFβ1 with siCtrl-TGFβ1; ∧p<0.05 comparing siCtrl+ TGFβ1 with siJunB+ TGFβ1 Kruskal-Wallis test). The median value of traction and the interquartile range across all tested groups is shown.</p

JunB regulates actin polymerization.

<p>(<b>A</b>) JunB silencing in BSMC reduces phospho-cofilin levels under basal and TGFβ1-stimulated conditions, without affecting total cofilin levels. *p<0.05; **p<0.005. Representative immunoblots are indicated in (<b>B</b>). (<b>C</b>) Filamentous (F) and globular (G) actin fractions were purified as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053430#s2" target="_blank">Methods</a>, from pBSMC under vehicle or TGFβ1-treated conditions, following treatment with non-targeting or JunB siRNA. The relative levels of F- and G-actin were subsequently assessed by immunoblotting. Quantification of immunoblot signals from three independent experiments is shown. *p<0.05. Representative immunoblots are indicated in (<b>D</b>).</p

A model depicting the role of JunB in regulation of smooth muscle contractility in response to TGFβ1 signaling.

<p>TGFβ1 induces the expression of JunB as well as other markers of smooth muscle differentiation e.g. α-SMA, calponin and SM22α Additionally, TGFβ1 also promotes smooth muscle contraction via ROCK1-mediated regulation of actin polymerization and acto-myosin crossbridge cycling. JunB mediates this process by promoting the phosphorylation of cofilin, leading to stabilization of filamentous actin and also by regulating the phosphorylation and absolute levels of MLC20, the regulatory light chain of myosin, and its inhibitory phosphatase, MYPT1. Thus, activation of JunB is critical for the changes in contractility and generation of cytoskeletal tension observed upon the TGFβ1-stimulation of smooth muscle cells.</p

JunB levels are increased in BSMC in response to TGFβ1, and in an ex vivo model of rodent bladder distension.

<p>(<b>A</b>) BSMC were treated with TGFβ1 for the indicated times and assessed for JunB levels by immunoblotting. GAPDH is included as a loading control. (<b>B</b>) Immunofluorescence analysis of BSMC showing increased JunB nuclear localization upon TGFβ1 treatment for 24 h. (<b>C</b>) Sections from rat bladders distended ex vivo for 8 h (injured) were stained sequentially with anti-JunB and Cy3-conjugated species-specific secondary antibody. Increased nuclear fluorescent signal for both proteins was evident in the detrusor smooth muscle of stretch-injured specimens, but not of non-distended (control) bladders.</p

TGFβ1induces contractility in bladder smooth muscle cells (BSMC).

<p>(<b>A</b>) Human bladder smooth muscle cells were seeded in collagen gels and treated for 24 h with vehicle (Veh) or 2.5 ng/ml TGFβ1, after which the gels were released from the sides of the well and the resulting decrease in surface area monitored microscopically (top) and quantified (bottom). *p<0.05, t-test. The area of the gel under control conditions is set to 100%. (<b>B</b>) Whisker plot of results from traction force microscopy of BSMC showing an increase in cell traction forces exerted with TGFβ1 treatment. The contractile response, measured quantitatively as enhanced traction (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053430#s2" target="_blank">Methods</a>) was statistically significant (*p<0.05, Kruskal-Wallis test). The median value of traction and the interquartile range for both groups is shown. (<b>C</b>) BSMC were treated for 30 min with inhibitors targeting the PI3-kinase/Akt (PI3K-i, Akt-i) mitogen-activated protein kinases (MEK-i, p38-i, JNK-i) or Rho-kinase (ROCK-i), followed by treatment with vehicle (Control, upper panel of wells) or 2.5 ng/ml TGFβ1 (lower panel) for 24 h and were monitored for changes in gel contractility. Inhibition of signaling via the JNK and ROCK axes abrogated TGFβ1-induced gel contraction. Quantification of changes in gel surface area for the various inhibitors under conditions of TGFβ1 treatment is indicated. (<b>D</b>) A transcription factor ELISA was carried out to assess differences in DNA-binding activities of members of the AP-1 family of transcription factors, using nuclear extracts prepared from BSMC treated with 2.5 ng/ml TGFβ1 for 24 h, or control cells. Fold changes are expressed relative to control which is set to 100%.</p

Separation of BSMCs and VSMCs from the lung using the bi-fluorescent <i>αSMA-hrGFP;NG2-DsRed</i> mouse.

<p>(<b>A</b>) Expression pattern of hrGFP and DsRed in BSMCs and VSMCs in the lung of an <i>αSMA-hrGFP;NG2-DsRed</i> mouse. Both BSMCs and VSMCs are hrGFP⁺. BSMCs are DsRed⁻ whereas VSMCs are DsRed⁺. Asterisk (*) indicates the blood vessel in the lung the airway. Arrows (>) indicate the airway. Scale bar: 20 µm. (<b>B</b>) Algorithm for bronchial and vascular smooth muscle cell identification by flow cytometry. CD31⁺ endothelial cells and CD45⁺ immune cells were separated from dissociated lung preparation (left panel) followed by evaluation of hrGFP and DsRed distribution within CD31⁻CD45⁻ population (middle panel). CD31⁻CD45⁻hrGFP⁺DsRed⁻ population corresponds to BSMCs (pointed by an arrow). CD45⁻CD31⁻hrGFP⁺DsRed⁺ population is enriched in vascular smooth muscle cells (VSMCs). (<b>C</b>) Relative <i>Notch3</i> mRNA expression in isolated singly hrGFP⁺ cells and doubly hrGFP⁺DsRed⁺ cells from lung cell preparation as determined by qPCR followed by normalization to 18S. Data show mean ± SD and are representative of three independent experiments. ***p<0.001.</p

A New Approach for the Study of Lung Smooth Muscle Phenotypes and Its Application in a Murine Model of Allergic Airway Inflammation

<div><p>Phenotypes of lung smooth muscle cells in health and disease are poorly characterized. This is due, in part, to a lack of methodologies that allow for the independent and direct isolation of bronchial smooth muscle cells (BSMCs) and vascular smooth muscle cells (VSMCs) from the lung. In this paper, we describe the development of a bi-fluorescent mouse that permits purification of these two cell populations by cell sorting. By subjecting this mouse to an acute allergen based-model of airway inflammation that exhibits many features of asthma, we utilized this tool to characterize the phenotype of so-called asthmatic BSMCs. First, we examined the biophysical properties of single BSMCs from allergen sensitized mice and found increases in basal tone and cell size that were sustained <i>ex vivo</i>. We then generated for the first time, a comprehensive characterization of the global gene expression changes in BSMCs isolated from the bi-fluorescent mice with allergic airway inflammation. Using statistical methods and pathway analysis, we identified a number of differentially expressed mRNAs in BSMCs from allergen sensitized mice that code for key candidate proteins underlying changes in matrix formation, contractility, and immune responses. Ultimately, this tool will provide direction and guidance for the logical development of new markers and approaches for studying human lung smooth muscle.</p></div

Measurement of changes in physical properties of individual BSMCs from OVA sensitized mice.

<p>BSMCs isolated from PBS-sensitized control and OVA sensitized <i>αSMA-hrGFP;NG2-DsRed</i> mice were allowed to attach to collagen I gel in culture for 72 hrs before assays. (<b>A</b>) Images of hrGFP⁺ BSMCs from PBS control and OVA sensitized mice (insert) and their traction maps on collagen I gels. (<b>B</b>) Contractile moment measurement of individual BSMCs from control and sensitized mice based on their traction maps. The line represents the mean of individual measurements of control (n = 20) and acute asthmatic (n = 22) cells. (<b>C</b>) Measurement of cell size of BSMCs from PBS sensitized control and OVA sensitized mice. The line represents the mean of individual measurements. **p<0.008.</p

Gene sets misregulated in BSMCs from OVA sensitized mice as compared to PBS sensitized control.

<p>The five most significantly down- and up-regulated gene sets among the MSigDB canonical pathways, Gene Ontology categories, and motif gene sets are shown, ordered in each subsection by CAMERA <i>p</i>-value. No motif gene set was up-regulated with p<0.05, so these are excluded. The size column gives the number of genes in the gene set after restricting to members with mouse orthologs represented by probe sets on the arrays.</p