Inhibition of Smad3 expression decreases collagen synthesis in keloid disease fibroblasts.
BACKGROUND
Keloids represent a dysregulated response to cutaneous wounding that result in an excessive deposition of extracellular matrix (ECM), especially types I and III collagen. In keloid scars, the ratio of 'type I to type III collagen' varies compared to normal skin. Transforming growth factor beta (TGF-beta) plays a central role in the pathogenesis of fibrosis by inducing and sustaining activation of keloid fibroblasts. However, the underlying mechanisms are poorly understood. RNA interference (RNAi) is an evolutionally conserved mechanism for repressing targeted gene expression. In mammalian cells, RNAi is mediated by small interfering RNA (siRNA). In this paper, we examined the function of Sma and Drosophila mothers against decapentaplegic homolog 3 (Smad3), recently characterized as intracellular effector of TGF-beta signalling, in keloid fibroblasts using siRNA.


METHODS
Dermal fibroblasts obtained from one female patient aged 21 years were maintained in Dulbecco's modified Eagle's medium. Cells (<6 passages) were treated with or without Smad3 siRNA and the expression levels of related genes were examined by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western Blot. Statistical analysis was performed using one-way ANOVA (Dunnett correction) and the Excel 7.0 software, with significance set at p<0.05.


RESULTS
The knockdown of Smad3 expression in mRNA and protein levels was confirmed using RT-PCR and Western Blot. Compared to blank, the mRNA levels of types I and III procollagen were also significantly and uniquely decreased following the reduction of Smad3 by siRNA (p<0.05). The results indicate that Smad3 plays an important role in the TGF-beta induced fibrosis in keloid. Downregulation of Smad3 expression in keloid fibroblasts can significantly decrease procollagen gene expression. SiRNA targeting Smad3 was an efficient reagent to reduce ECM deposition and attenuate process of fibrosis. It could be a new promising therapeutic approach to improve skin wound healing and inhibit progression of fibrotic conditions by interrupting the TGF-beta signal pathway.
Wang
Zimin
Z
Department of Orthopaedics, Changhai Hospital, Second Military Medical University, Shanghai 200433, China. wzmsmmu@yahoo.com
Gao
Zhongyu
Z
Shi
Yi
Y
Sun
Yi
Y
Lin
Zihao
Z
Jiang
Hua
H
Hou
Tiesheng
T
Wang
Qiugen
Q
Yuan
Xiangbin
X
Zhu
Xiaohai
X
Wu
Hong
H
Jin
Youxin
Y
D000328:Adult; D002478:Cells, Cultured; D024042:Collagen Type I / Q000096:biosynthesis*; D024061:Collagen Type III / Q000096:biosynthesis*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D007627:Keloid / Q000235:genetics* / Q000473:pathology; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051900:Smad3 Protein / Q000235:genetics* / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000502:physiology
D024042:Collagen Type I; D024061:Collagen Type III; D034741:RNA, Small Interfering; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta