Figure 5.

FMOD delicately orchestrated Tgfβ1 signal transduction in adult RDFs. Western blotting and qRT-PCR revealed that FMOD reduced Tgfβ1 expression and Tgfβ1 auto-induction (a), and inhibited TGFβ1-dependent Pai1 transcription in adult RDFs (b). Using an AP-1-luciferase reporter system, we further revealed that FMOD significantly downregulated AP-1-mediated non-canonical TGFβ1 signal transduction (which predominately regulates TGFβ1 auto-induction and Pai1 expression) up to 24 h post-treatment (c). Meanwhile, FMOD stimulated adult RDF Smad2 phosphorylation/activation when measured over a short amount of time (2 h) in the presence or absence of TGFβ1, but did not have an obvious effect on Smad2 expression (d). Although FMOD alone downregulated initial Smad3 expression up to 24 h post-treatment, when combined with TGFβ1, FMOD did not affect Smad3 expression in the 48-h experiment period, but it significantly prolonged TGFβ1-responsive Smad3 phosphorylation/activation (e), which was confirmed using a CAGA-box (specific for SMAD3 signal transduction) Luciferase reporter system (f). Dosages: TGFβ1 (100 pM) and FMOD (200 nM). A two-sample t-test was used for statistical analysis. Data were normalized to untreated RDFs at time 0 (dashed lines) and shown as mean±the standard deviation. N=3 (a, b, d, e) or 5 (c, f). *P<0.05; a blue star indicates the significance in comparison with FMOD administration, and a magenta star indicates the significance in comparison with TGFβ1 administration.