The role of Smad3 in the fibrotic phenotype in human vocal fold fibroblasts

Objectives/Hypothesis To investigate the role of Smad3 as a regulator of transforming growth factor (TGF)-β1-mediated cell activities associated with fibrosis in normal human vocal fold fibroblasts. We also sought to confirm the temporal stability of Smad3 knockdown via small inhibitor ribonucleic acid (siRNA). Vocal fold fibroblasts were employed to determine the effects of Smad3 knockdown on TGF-β1-mediated migration and contraction, as well as regulation of connective tissue growth factor (CTGF). We hypothesized that Smad3 is an ideal candidate for therapeutic manipulation in vivo based on its role in fibrosis. Study Design In vitro. Methods Knockdown of Smad3 via siRNA was performed in our normal human vocal fold cell line. Three-dimensional collagen gel contraction and scratch assays were employed to determine the role of Smad3 on TGF-β1-mediated contraction and migration, respectively. The role Smad3 in the induction of CTGF was characterized via sodium dodecyl sulfate polyacrylamide gel electrophoresis. The effects of Smad3 signaling on Smad7 messenger (m)RNA and protein were also quantified. Results Smad3 knockdown was temporally-stable up to 72 hours (P