TGFβ1induces contractility in bladder smooth muscle cells (BSMC).

Abstract

<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