Smad2/3 signaling involved in urotensin II-induced phenotypic differentiation, collagen synthesis and migration of rat aortic adventitial fibroblasts

Abstract

Objective. To investigate whether Smad2/3 signaling is involved in urotensin II (UII) induced activation of aortic adventitial fibroblasts. Materials and Methods. Growth-arrested adventitial fibroblasts were stimulated with UII in the presence or absence of urotensin II receptor (UT) antagonist SB710411 or transfected with Smad2/3 small inhibitory RNA (siRNA). UII stimulated Smad2/3 phosphorylation, α-smooth muscle actin (α-SMA), and collagen I expression and migration of adventitial fibroblasts were evaluated by western blot analysis, real-time reverse transcription polymerase chain reaction, immunofluorescence, ELISA, and transwell migration assay, respectively. Results. In cultured adventitial fibroblasts, UII time- and dose-dependently stimulated Smad2/3 protein phosphorylation, with maximal effect at 10-8 mol/l (increased by 147.2%, P<0.001). UII stimulated Smad2/3 upregulation and nuclear translocation. SB710411 significantly inhibited these effects. In addition, UII potently induced α-SMA and procollagen 1 protein or mRNA expression (P<0.01), which were completely blocked by Smad2 (decreased by 75.1%, 54.2% in protein, and by 73.3% and 38.2% in mRNA, respectively, P<0.01) or Smad3 siRNA (decreased by 80.3% and 47.0% in protein, and by 72.3% and 47.7% in mRNA, respectively, P<0.01). Meanwhile, Smad2 or smad3 siRNA significantly inhibited the UII-induced collagen 1 secretion and cell migration. Conclusions. UII may stimulate adventitial-fibroblast phenotype conversion, migration, and collagen I synthesis via phosphorylated-Smad2/3 signal transduction pathways.