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 β (TGF-β) 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-β signalling, in keloid fibroblasts using siRNA.
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.
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-β 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-β signal pathway.
To read this article in full you will need to make a payment
Purchase one-time access:Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
One-time access price info
- For academic or personal research use, select 'Academic and Personal'
- For corporate R&D use, select 'Corporate R&D Professionals'
Subscribe:Subscribe to Journal of Plastic, Reconstructive & Aesthetic Surgery
Already a print subscriber? Claim online access
Already an online subscriber? Sign in
Register: Create an account
Institutional Access: Sign in to ScienceDirect
- The molecular basis of keloid and hypertrophic scar formation.Mol Med Today. 1998; 4: 19-24
- Fibroblasts cocultured with keloid keratinocytes: normal fibroblasts secrete collagen in a keloid like manner.Am J Physiol Cell Physiol. 2002; 283: C212-C222
- On the nature of hypertrophic scars and keloids: a review.Plast Reconstr Surg. 1999; 104: 1435-1458
- Pentoxifylline attenuates tubulointerstitial fibrosis by blocking Smad3/4-activated transcription and profibrogenic effects of connective tissue growth factor.J Am Soc Nephrol. 2005; 16 ([Epub 2005 Jun 29]): 2702-2713
- Identification of novel TGF-beta/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach.J Biol Chem. 2001; 276: 17058-17062
- Transcriptional control by the TGF-beta/Smad signaling system.EMBO J. 2000; 19: 1745-1754
- Sp1 and Smad proteins cooperate to mediate TGF-beta1-induced alpha2 (I) collagen expression in human glomerular mesangial cells.J Biol Chem. 2001; 276: 6983-6992
- Smad3 signalling plays an important role in keloid pathogenesis via epithelial–mesenchymal interactions.J Pathol. 2005; 207: 232-242
- Functions of mammalian Smad genes as revealed by targeted gene disruption in mice.Cytokine Growth Factor Rev. 2000; 11: 49-58
- Smad2 role in mesoderm formation, left–right patterning and craniofacial development.Nature. 1998; 393: 786-790
- Targeted disruption in murine cells reveals variable requirement for Smad4 in transforming growth factor beta-related signaling.J Biol Chem. 2000; 275: 2063-2070
- Distinct oligomeric states of SMAD proteins in the transforming growth factor-beta pathway.J Biol Chem. 2000; 275: 40710-40717
- The gene-silencing efficiency of siRNA is strongly dependent on the local structure of mRNA at the targeted region.Biochem Biophys Res Commun. 2004; 318: 303-310
- Verapamil inhibits interleukin-6 and vascular endothelial growth factor production in primary cultures of keloid fibroblasts.Br J Plast Surg. 2003; 56: 804-809
- The effect of TGF-beta on keloid fibroblast proliferation and collagen synthesis.Plast Reconstr Surg. 1996; 98: 827-833
- The discoidin domain receptor tyrosine kinases are activated by collagen.Mol Cell. 1997; 1: 13-23
- TGF-beta2 activates proliferative scar fibroblasts.J Surg Res. 1999; 82: 319-323
- Differential expression of transforming growth factor-β receptors I and II and activation of Smad 3 in keloid fibroblasts.Plast Reconstr Surg. 2001; 108: 423-429
- Enhanced expression of transforming growth factor-beta type I and type II receptors in wound granulation tissue and hypertrophic scar.Am J Pathol. 1998; 152: 485-493
- Recent advances in fibroblast signaling and biology in scleroderma.Curr Opin Rheumatol. 2004; 16: 739-745
- Differential regulation of TGF-beta signaling through Smad2, Smad3 and Smad4.Oncogene. 2003; 22: 6748-6763
- TGF-beta receptor-mediated signalling through Smad2, Smad3 and Smad4.EMBO J. 1997; 16: 5353-5362
- Smad3 allostery links TGF-beta receptor kinase activation to transcriptional control.Genes Dev. 2002; 16: 1950-1963
- Formation of a stable heterodimer between Smad2 and Smad4.J Biol Chem. 2001; 276: 20688-20694
- The L3 loop and C-terminal phosphorylation jointly define Smad protein trimerization.Nat Struct Biol. 2001; 8: 248-253
- Sedimentation studies reveal a direct role of phosphorylation in Smad3:Smad4 homo- and hetero-trimerization.Biochemistry. 2001; 40: 1473-1482
- Stoichiometry of active smad-transcription factor complexes on DNA.J Biol Chem. 2002; 277: 51008-51016
- From mono- to oligo-Smads: the heart of the matter in TGF-beta signal transduction.Genes Dev. 2002; 16: 1867-1871
- Connective tissue growth factor siRNA modulates mRNA levels for a subset of molecules in normal and TGF-beta 1-stimulated porcine skin fibroblasts.Wound Repair Regen. 2004; 12: 205-216
- Smad3 as a mediator of the fibrotic response.Int J Exp Path. 2004; 85: 47-64
- Transforming growth factor-beta and Smad signalling in kidney diseases.Nephrology (Carlton). 2005; 10: 48-56
- RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells.Proc Natl Acad Sci U S A. 2002; 99: 6047-6052
Published online: January 22, 2007
Accepted: May 9, 2006
Received: November 19, 2005
☆Zimin Wang, Zhongyu Gao and Yi Shi contribute equally to this paper.
© 2006 Published by Elsevier Inc.