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

Abstract

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 < 0.001), diminished TGF-β1-mediated collagen gel contraction and migration, and blunted induction of CTGF, but it had no effect on TGF-β1-mediated Smad7 mRNA or protein induction.

Conclusion: Transforming growth factor-β1 stimulated profibrotic cell activities in our cell line and these actions were largely reduced with Smad3 knockdown. These data provide continued support for therapeutic targeting of Smad3 for vocal fold fibrosis because it appears to regulate the fibrotic phenotype.

Level of evidence: N/A. Laryngoscope, 126:1151-1156, 2016.

Keywords: Smad3; TGF-β; Voice; fibrosis; scar; vocal fold.

Figure 1

Schematic of TGF-β signaling via Smad phosphorylation and nuclear translocation.

Figure 2

Temporal stability of Smad3 knockdown with siRNA compared to nonsense sequence over 72 hours following transfection (n=3; *p<0.05).

Figure 3

The effects of TGF-β1 on three-dimensional collagen gel contraction (n=4; A; mean+/−SEM). TGF-β1 stimulated a statistically-significant increase in the contractile phenotype (p=0.024). This response was blunted by Smad3 knockdown (*p<0.05 relative to control; #p<0.05 relative to TGF-β1 stimulation). Representative gels are shown in B.

Figure 4

The effects of TGF-β1 on cell migration (A; mean+/−SEM). TGF-β1 had a dose-dependent effect on cell migration at both 10 and 20ng/mL (p=0.031 and p=0.002, respectively). Migratory rate increased in response to TGF-β1 in the context of Smad3 knockdown; this response, however, was not statistically significant when compared to control/untreated cells (p=0.347 at 10ng/mL and p=0.319 at 20ng/mL). Representative images are shown in B (nonsense siRNA not shown).

Figure 5

Representative SDS-PAGE gels probed for CTGF at 12 and 24 hours. TGF-β1 stimulated CTGF expression and this response was attenuated by Smad3 knockdown at both time points.

Figure 6

Smad7 mRNA expression in response to Smad3 knockdown +/− TGF-β1 at both 6 and 24 hours (A; *p<0.05; n=3; mean+/−SEM); Smad7 mRNA expression increased in response to TGF-β1. Smad3 knockdown had little effect on this inherent anti-fibrotic response, as confirmed via protein analysis (B; representative gel).