Korean J Urol.  2012 Jan;53(1):44-49. 10.4111/kju.2012.53.1.44.

Activin Receptor-Like Kinase 5 Inhibitor Attenuates Fibrosis in Fibroblasts Derived from Peyronie's Plaque

• Jang JH
• Ryu JK
• Suh JK

• KMID: 1988823
• DOI: http://doi.org/10.4111/kju.2012.53.1.44

Abstract

PURPOSE
Transforming growth factor-beta1 (TGF-beta1) is the key fibrogenic cytokine associated with Peyronie's disease (PD). The aim of this study was to determine the antifibrotic effect of 3-((5-(6-Methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl) methyl)benzamide (IN-1130), a small-molecule inhibitor of the TGF-beta type I receptor activin receptor-like kinase 5 (ALK5), in fibroblasts isolated from human PD plaque.
MATERIALS AND METHODS
Plaque tissue from a patient with PD was used for primary fibroblast culture, and we then characterized primary cultured cells. Fibroblasts were pretreated with IN-1130 (10 microM) and then stimulated with TGF-beta1 protein (10 ng/ml). We determined the inhibitory effect of IN-1130 on TGF-beta1-induced phosphorylation of Smad2 and Smad3 or the nuclear translocation of Smad proteins in fibroblasts. Western blot analyses for plasminogen activator inhibitor-1, fibronectin, collagen I, and collagen IV were performed to evaluate effect of IN-1130 on the production of extracellular matrix proteins.
RESULTS
The treatment of fibroblasts with TGF-beta1 significantly increased phosphorylation of Smad2 and Smad3 and induced translocation of Smad proteins from the cytoplasm to the nucleus. Pretreatment with IN-1130 substantially inhibited TGF-beta1-induced phosphorylation of Smad2 and Smad3 and nuclear accumulation of Smad proteins. The TGF-beta1-induced production of extracellular matrix proteins was also significantly inhibited by treatment with IN-1130 and returned to basal levels.
CONCLUSIONS
Overexpression of TGF-beta and activation of Smad transcriptional factors are known to play a crucial role in the pathogenesis of PD. Thus, inhibition of the TGF-beta signaling pathway by ALK5 inhibitor may represent a promising therapeutic strategy for treating PD.

Keyword

Cells, cultured; Penile induration; TGF-beta type I receptor; Transforming growth factor beta

MeSH Terms

Activin Receptors
Activins
Blotting, Western
Cells, Cultured
Collagen
Cytoplasm
Extracellular Matrix
Extracellular Matrix Proteins
Fibroblasts
Fibronectins
Fibrosis
Humans
Imidazoles
Male
Penile Induration
Phosphorylation
Plasminogen Activators
Protein-Serine-Threonine Kinases
Quinoxalines
Receptors, Transforming Growth Factor beta
Smad Proteins
Transforming Growth Factor beta
Transforming Growth Factor beta1
Activin Receptors
Activins
Collagen
Extracellular Matrix Proteins
Fibronectins
Imidazoles
Plasminogen Activators
Protein-Serine-Threonine Kinases
Quinoxalines
Receptors, Transforming Growth Factor beta
Smad Proteins
Transforming Growth Factor beta
Transforming Growth Factor beta1

Figure

• 

  FIG. 1 Characterization of primary cultured fibroblasts isolated from human Peyronie's disease plaque. Fluorescent immunocytochemistry of cultured cells with antibody against vimentin and smooth muscle α-actin (positive markers) and antibody against desmin and platelet/endothelial cell adhesion molecule-1 (PECAM-1) (negative markers). Nuclei were labeled with the DNA dye 4,6-dimidino-2-phenylindole (DAPI). aSmooth muscle α-actin is a maker for myofibroblasts. Magnification, ×400. Bar indicates 100 µm.

• 

  FIG. 2 Effect of IN-1130 on TGF-β1-induced Smad2 and Smad3 phosphorylation in fibroblasts isolated from human Peyronie's disease (PD) plaque. Representative Western blot for phospho-Smad2 (P-Smad2), total Smad2, phospho-Smad3 (P-Smad3), and total Smad3. Fibroblasts were pretreated for 1 hour with IN-1130 (10 µM) and were then induced with TGF-β1 (10 ng/ml) for 1 hour. Cytosolic and nuclear extracts obtained from fibroblasts were fractionated in a sodium dodecylsulfate-polyacrylamide gel. Hsp90 is a cytosolic marker and lamin is a nuclear marker used for determining the quality of cytosolic and nuclear extracts. Arrows denote target bands. Results were similar from three independent experiments. TGF-β1, transforming growth factor-β1.

• 

  FIG. 3 Effect of 3-((5-(6-Methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol- 2-yl)methyl)benzamide (IN-1130) on transforming growth factor-β1 (TGF-β1)-induced nuclear translocation of Smad2/3 in fibroblasts isolated from human Peyronie's diseas plaque. (A) Fluorescent immunocytochemistry of primary human fibroblasts with antibody against total Smad2 (which also recognizes Smad3). Nuclei were labeled with the DNA dye DAPI. Fibroblasts were pretreated for 1 hour with IN-1130 (10 µM) and were then induced with TGF-β1 (10 ng/ml) for 1 hour. Arrowheads denote nuclear translocation of Smad2/3. Magnification, ×400. Bar indicates 50 µm. (B) Nuclear fluorescence intensity was quantified for all cells. Each bar depicts the mean values (±SD) for n=4 per group. ap<0.01 compared with the other groups. DAPI: 4,6-diamidino-2-phenylindole, TGF-β1: transforming growth factor-β1.

• 

  FIG. 4 Effect of 3-((5-(6-Methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide (IN-1130) on transforming growth factor-β1 (TGF-β1)-induced extracellular matrix protein production in fibroblasts isolated from human Peyronie's disease plaque. (A) Representative Western blot for PAI-1, fibronectin, collagen I, and collagen IV in fibroblasts. Fibroblasts were pretreated for 1 hour with IN-1130 (10 µM) and were then induced with TGF-β1 (10 ng/ml) for 24 hours. Whole-cell extracts were fractionated in a sodium dodecylsulfate-polyacrylamide gel. (B) Data are presented as the relative density of each protein compared with that of β-actin. Each bar depicts the mean values (±SD) from four experiments per group. a: p<0.01, b: p<0.05 compared with the other groups. PAI-1: plasminogen activator inhibitor-1, TGF-β1: transforming growth factor-β1.

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