World J Mens Health.  2018 May;36(2):139-146. 10.5534/wjmh.170005.

Silencing Histone Deacetylase 7 Alleviates Transforming Growth Factor-β1-Induced Profibrotic Responses in Fibroblasts Derived from Peyronie's Plaque

• Kang DH
• Yin GN
• Choi MJ
• Song KM
• Ghatak K
• Minh NN
• Kwon MH
• Seong DH
• Ryu JK
• Suh JK

• KMID: 2419312
• DOI: http://doi.org/10.5534/wjmh.170005

Abstract

PURPOSE
Epigenetic modifications, such as histone acetylation/deacetylation and DNA methylation, play a crucial role in the pathogenesis of inflammatory disorders and fibrotic diseases. The aim of this study was to study the differential gene expression of histone deacetylases (HDACs) in fibroblasts isolated from plaque tissue of Peyronie's disease (PD) or normal tunica albuginea (TA) and to examine the anti-fibrotic effect of small interfering RNA (siRNA)-mediated silencing of HDAC7 in fibroblasts derived from human PD plaque.
MATERIALS AND METHODS
For differential gene expression study, we performed reverse-transcriptase polymerase chain reaction for HDAC isoforms (1-11) in fibroblasts isolated from PD plaque or normal TA. Fibroblasts isolated from PD plaque were pretreated with HDAC7 siRNA (100 pmol) and then stimulated with transforming growth factor-β1 (TGF-β1, 10 ng/mL). Protein was extracted from treated fibroblasts for Western blotting. We also performed immunocytochemistry to detect the expression of extracellular matrix proteins and to examine the effect of HDAC2 siRNA on the TGF-β1-induced nuclear translocation of Smad2/3 and myofibroblastic differentiation.
RESULTS
The mRNA expression of HDAC2, 3, 4, 5, 7, 8, 10, and 11 was higher in fibroblasts isolated from PD plaque than in fibroblasts isolated from normal TA tissue. Knockdown of HDAC7 in PD fibroblasts inhibited TGF-β1-induced nuclear shuttle of Smad2 and Smad3, transdifferentiation of fibroblasts into myofibroblasts, and abrogated TGF-β1-induced production of extracellular matrix protein.
CONCLUSIONS
These findings suggest that specific inhibition of HDAC7 with RNA interference may represent a promising epigenetic therapy for PD.

Keyword

Extracellular matrix; Fibrosis; Histone deacetylases; Penile induration; Transforming growth factors

MeSH Terms

Blotting, Western
DNA Methylation
Epigenomics
Extracellular Matrix
Extracellular Matrix Proteins
Fibroblasts*
Fibrosis
Gene Expression
Histone Deacetylases*
Histones*
Humans
Immunohistochemistry
Male
Myofibroblasts
Penile Induration
Polymerase Chain Reaction
Protein Isoforms
RNA Interference
RNA, Messenger
RNA, Small Interfering
Transforming Growth Factors
Extracellular Matrix Proteins
Histone Deacetylases
Histones
Protein Isoforms
RNA, Messenger
RNA, Small Interfering
Transforming Growth Factors

Figure

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  Fig. 1 Differential gene expression of histone deacetylases (HDACs) in fibroblasts isolated from human Peyronie's disease (PD) plaque. A representative gel picture shows the gene expression of HDACs in fibroblasts isolated from PD plaque (n=2) or from normal tunica albuginea (TA) tissue from control patients (n=2). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control for reverse-transcriptase polymerase chain reaction. Results were similar from three independent experiments.

• 

  Fig. 2 Histone deacetylase 7 (HDAC7) knockdown inhibits transforming growth factor-β1 (TGF-β1)-induced extracellular matrix protein production in fibroblasts derived from human Peyronie's disease (PD) plaque. (A) Effect of TGF-β1 on HDAC7 expression. Representative Western blot for HDAC7 protein in PD fibroblasts after specific knockdown using small interfering RNA (siRNA) or control siRNA (scramble siRNA). Data are presented as the ratio of the product of protein to that of β-actin. Fibroblasts were transfected with scramble siRNA or siRNA specific to HDAC7 by using Lipofectamine (GIBCO) reagent for 48 hours and were then treated with TGF-β1 (10 ng/mL) for 24 hours. (B) Each bar depicts the mean values (±standard error) from four experiments per group. The relative ratio measured in the no treatment group was arbitrary presented as 1. *p<0.05 compared with no treatment group, †p<0.05 compared with TGF-β1+scramble siRNA group. (C) Representative Western blot for fibronectin, plasminogen activator inhibitor-1 (PAI-1), collagen I, and collagen IV in fibroblasts. Results were similar from four independent experiments.

• 

  Fig. 3 Fluorescent immunocytochemistry showing the inhibition of transforming growth factor-β1 (TGF-β1)-induced extracellular matrix protein expression by histone deacetylase 7 (HDAC7) small interfering RNA (siRNA) in fibroblasts derived from human Peyronie's disease plaque. The cells were washed three times with phosphate-buffered saline and then fixed in 4% paraformaldehyde for 10 minutes at 4℃ and in 100% methanol for 10 minutes at 4℃. Representative fluorescent immunocytochemistry of fibroblasts with antibody against fibronectin, plasminogen activator inhibitor-1 (PAI-1), collagen I, and collagen IV. Nuclei were labeled with the DNA dye 4,6-diamidino-2-phenylindole. Bar indicates 100 µm. Fibroblasts were transfected with scramble siRNA or siRNA specific to HDAC7 by using Lipofectamine (GIBCO) reagent for 48 hours and were then treated with TGF-β1 (10 ng/mL) for 24 hours. Results were similar from four independent experiments.

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  Fig. 4 Histone deacetylase 7 (HDAC7) knockdown inhibits transforming rowth factor-β1 (TGF-β1)-induced myofibroblastic differentiation in fibroblasts derived from human Peyronie's disease plaque. (A) Representative Western blot for alpha-smooth muscle actin (α-SMA). Fibroblasts were transfected with scramble small interfering RNA (siRNA) or siRNA specific to HDAC7 by using Lipofectamine (GIBCO) reagent for 48 hours and were then treated with TGF-β1 (10 ng/mL) for 24 hours. (B) The fibroblasts were serum-starved for 24 hours and transfected with 100 pmol siRNA oligonucleotides targeted specifically to HDAC7 by using Lipofectamine 2000. After transfection, cells were plated and cultured for 48 hours in Dulbecco's modified Eagle's medium. The fibroblasts were then treated with 10 ng/mL TGF-β1 for 24 hours. The cells were washed three times with phosphate-buffered saline and then fixed in 4% paraformaldehyde for 10 minutes at 4℃ and in 100% methanol for 10 minutes at 4℃. Representative fluorescent immunocytochemistry of fibroblasts with antibody against α-SMA (a myofibroblast marker) and F-actin (a cytoskeleton marker). Nuclei were labeled with the DNA dye 4,6-diamidino-2-phenylindole. Bar indicates 100 µm. Results were similar from four independent experiments.

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  Fig. 5 Histone deacetylase 7 (HDAC7) knockdown suppresses transforming growth factor-β1 (TGF-β1)-induced Smad2/3 nuclear translocation in fibroblasts derived from human Peyronie's disease plaque. Representative fluorescent immunocytochemistry of primary human fibroblasts with antibody against total Smad2/3. Fibroblasts were transfected with scramble small interfering RNA (siRNA) or siRNA specific to HDAC7 by using Lipofectamine (GIBCO) reagent for 48 hours and were then treated with TGF-β1 (10 ng/mL) for 1 hour. The cells were washed three times with phosphate-buffered saline and then fixed in 4% paraformaldehyde for 10 minutes at 4℃ and in 100% methanol for 10 minutes at 4℃. Nuclei were labeled with the DNA dye 4,6-diamidino-2-phenylindole (DAPI). Bar indicates 100 µm. Results were similar from four independent experiments.

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