TGFβ1induces contractility in bladder smooth muscle cells (BSMC).

(A) 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) 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 Methods) was statistically significant (*p<0.05, Kruskal-Wallis test). The median value of traction and the interquartile range for both groups is shown. (C) 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. (D) 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%.