Figure 5.

Latent TGF-β1 activation by mechanical stress requires a functional cytoskeleton. (A) To control whether stretching interferes with TGF-β1 reporting activity, TMLC were directly grown on a silicon membrane and stretched by 10% in the presence and absence of 10 pM TGF-β1. Reporter activity was compared with TMLC grown on coverslips to protect from stretching and placed upside-down on the membrane during 10% stretching. (B) This configuration was repeated in the sandwich assay, in which lung Mfs were cultured for 7 d on silicone membranes that were stretched 3–10%. After stretching, TMLC were cultured separately for further analysis. Active TGF-β1 was measured as a function of stretch applied to TMLC/Mf sandwich cultures (C), TX-100 cytoskeletons, and Mf-derived ECM (DOC; D). Active TGF-β1 is expressed as the percentage of nonstretched control (con) corrected for TMLC baseline reporter activity. Phosphorylation of Smad2 (pSmad2) was assessed by immunofluorescence detecting pSmad2 in nonstretched (E) and 10% stretched (F) conditions and by Western blotting of Mf culture extracts equilibrated for total Smad2 (H). Intensity of pSmad2 labeling in the nuclei (G) and of pSmad2 bands on the Western blot (I) was quantified by densitometric analysis. Error bars represent SD of the mean. Bar, 10 μm.