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

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

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

The Murine Bladder Supports a Population of Stromal Sca-1⁺/CD34⁺/lin^- Mesenchymal Stem Cells

<div><p>Bladder fibrosis is an undesired end point of injury of obstruction and often renders the smooth muscle layer noncompliant. In many cases, the long-term effect of bladder fibrosis is renal failure. Despite our understanding of the progression of this disease, little is known about the cellular mechanisms that lead to a remodeled bladder wall. Resident stem (progenitor) cells have been identified in various organs such as the brain, heart and lung. These cells function normally during organ homeostasis, but become dysregulated after organ injury. Here, we aimed to characterize a mesenchymal progenitor cell population as a first step in understanding its role in bladder fibrosis. Using fluorescence activated cell sorting (FACS), we identified a Sca-1⁺/ CD34⁺/ lin^- (PECAM^-: CD45^-: Ter119^-) population in the adult murine bladder. These cells were localized to the stromal layer of the adult bladder and appeared by postnatal day 1. Cultured Sca-1⁺/ CD34⁺/ lin^- bladder cells self-renewed, formed colonies and spontaneously differentiated into cells expressing smooth muscle genes. These cells differentiated into other mesenchymal lineages (chondrocytes, adipocytes and osteocytes) upon culture in induction medium. Both acute and partial obstruction of the bladder reduced expression of CD34 and changed localization of Sca-1 to the urothelium. Partial obstruction resulted in upregulation of fibrosis genes within the Sca-1⁺/CD34⁺/lin^- population. Our data indicate a resident, mesenchymal stem cell population in the bladder that is altered by bladder obstruction. These findings provide new information about the cellular changes in the bladder that may be associated with bladder fibrosis.</p></div

Analysis of gene and protein expression in FACS sorted bladder cells.

<p>(A) qPCR analysis of Sca-1 and CD34 mRNA expression levels in lin^- cells that were FACS sorted based on expression of Sca-1 and CD34. Expression level is normalized to Sca-1^-/CD34^-/lin^- Sca-1 expression levels. (B) qPCR analysis of smooth muscle myosin (SMM) and smooth muscle alpha <b>α</b> actin (ACTA2) of lin^- cells that were FACS sorted based on Sca-1 and CD34 expression levels. Expression level is normalized to Sca-1⁺/CD34⁺/lin^- SMM expression levels. (A, B) Asterisks represent significance values of P < 0.05 * and P < 0.01 ** after 1 Way ANOVA. Graphs represent expression averages from 4 separate sorts with 3–4 CD1 mice pooled per sort. (C, D) Confocal micrographs of Sca-1⁺/CD34⁺/lin^- sorted cells cultured on a glass coverslips after incubation with EdU for the first 24h (D) Two cells that are Sca-1⁺ (green), EdU⁺ (white) but SMM^- (red) at 48h in culture in α-MEM media. (E) Two cells that are Sca-1⁺ (green), EdU⁺ (white) and SMM⁺ (red) at 4d in culture in α-MEM media.</p

Flow cytometric analysis of adult mouse bladder cells.

<p>(A) Density dot plot for bladder cell suspensions sorted based on Sytox red uptake and side-scatter. (B) Gating to exclude doublets. (C) Gating for side scatter versus forward scatter. (D) Gating for side scatter versus lineage (lin) staining (FITC CD31, CD45 and TER-119). (E) Gating of lin^- cells for Sca-1 and CD34 expression.</p

Partial bladder outlet obstruction (pBOO) alters Sca-1 expression while still maintaining a Sca-1⁺/CD34⁺ population in the stromal layer of the bladder.

<p>(A) Immunofluorescence images of Sca-1 and CD34 co-localization in a sham CD1 mouse 1wk post surgery. (B) Immunofluorescence images of Sca-1 and CD34 localization in CD1 mouse 1wk post pBOO surgery. Arrowheads (B) point to localization of Sca-1 to the urothelial layer. Arrowheads (B) point to co-localization of Sca-1 and CD34 within the stromal layer 1 week after pBOO surgery. (C, D) Immunofluorescence images of <i>Sca-1</i>^<i>egfp</i> mice 1 week following sham (C) and (D) pBOO surgery. Arrowheads (D) point to localization of EGFP to the urothelial layer. (E, F) FACS analysis of two unique sorts with sham and pBOO bladder digests. Percentages reported represent percent of total living cells. Asterisks (P, Q) represent significance values P < 0.01 after a Student’s T-Test. (G-L) Quantitative PCR analysis of RNA from Sca-1⁺/CD34⁺/lin^- cells after sham (red cross hatched bars) and pBOO surgeries (blue striped bars). Analysis represents two technical replicates of two separate sorts; one sort had 5 pooled pBOO and 5 pooled sham CD1 mice and the other had pools of 2 pBOO and 2 sham CD1 mice. RNA was collected 7 days post pBOO surgery. Asterisks represent significance values P<0.05 * and P<0.001 *** after Student’s T-Tests. Data is normalized to sham gene expression, and GAPDH was used as the reference gene. (M) qPCR analysis of RNA of Sca-1⁺/CD34^-/lin^- cells from sham surgeries (solid red bar) and pBOO surgeries (solid blue bar).</p

Colony forming assays with Sca-1⁺/CD34⁺/lin^- cells demonstrate that individual cells form colonies and differentiate into cells expressing smooth muscle genes.

<p>(A-D) Confocal micrographs of Sca-1⁺/CD34⁺/lin^- sorted cells cultured on glass coverslips and stained with antibodies to Sca-1 (green) and SMM (red) at varying time points up to 7 days. Arrow in (C) shows a single Sca-1⁺ cell next to a group of SMM⁺ cells (arrowheads) at 4d of culture. (E-H) Confocal micrographs of Sca-1⁺/CD34⁺/lin^- sorted cells cultured on glass coverslips in α-MEM and stained with antibodies to Sca-1 (green) and calponin (red) at varying time points up to 7 days. (I-L) Confocal micrographs of Sca-1⁺/CD34⁺/lin^- sorted cells cultured on glass coverslips in α-MEM and stained with antibodies to Sca-1 (green) and SRF (red) at varying time points. Arrow (L) shows a single cell co-expressing SRF and Sca-1 next to a group of SRF⁺, Sca-1^- cells stained in red. (M-Q) qPCR analysis of Sca-1⁺/CD34⁺/lin^- expression of 5 genes at the time of sort versus after 7 days in α-MEM culture. <i>Sca-1</i>, <i>CD34</i>, and <i>SRF</i> expression levels are normalized to expression levels after 7 d in culture. <i>Myh11</i> and <i>ACTA2</i> are normalized to expression levels at the time of sort. Analysis represents two technical replicates of two separate sorts. Each sort consisted of 5 or 6-pooled CD1 mouse bladder cells. Asterisks (N, P, Q) represent significance values of P < 0.05 * and P < 0.01 ** after Student’s T-test. (R, S) Quantification of spontaneous <i>in vitro</i> differentiation Sca-1⁺/CD34⁺/lin^- cells into SMM and calponin expressing cells. (T) Quantification of <i>in vitro</i> expression of SRF and Sca-1 in Sca-1⁺/CD34⁺/lin^- cells. (R, S, T) Cells from three independent sorting experiments were fixed at 24h, 48h, 4d and 7d. Coverslips were stained for Sca-1 and either SMM, calponin or SRF. Green lines represent cells stained only with Sca-1. Gold lines represent cells stained with Sca-1 and SMM, Sca-1 and calponin or Sca-1 and SRF. Red lines represent cells stained with SMM, calponin or SRF but not Sca-1. Black lines represent cells that did not stain at all. Error bars (R-T) represent standard error of the mean.</p