title: Regulators and mediators of radiation-induced fibrosis: Gene expression profiles and a rationale for Smad3 inhibition.
abstract: OBJECTIVE
  Radiotherapy, an essential modality in cancer treatment, frequently induces fibrotic processes in the skin, including accumulation of extracellular matrix. Transforming growth factor-β is essential in regulating extracellular matrix gene expression and is dependent on Smad3, an intracellular mediator/transcription factor. Our study characterized the genetic expression involved in extracellular matrix accumulation during radiation-induced fibrosis. We performed Smad3 gene silencing in an attempt to abrogate the effects of radiation.
  
  
  METHODS
  Laboratory research.
  
  
  METHODS
  University laboratory.
  
  
  METHODS
  C57 murine dermal fibroblasts were irradiated with 20 Gy RNA isolated (0, 6, 12, 24, 48, 72 hours postirradiation) and mRNA analyzed (reverse transcriptase polymerase chain reaction) for known regulators (Smad3, interleukin-13 [IL-13]), tumor necrosis factor-α [TNF-α]) and mediators of fibrosis (collagen 1A1 [Col1A1]), TGF-β, matrix metalloprotease-1 and -2 (MMP-1, MMP-2), and tissue inhibitor of metalloprotease-1 (TIMP-1). Smad3 gene expression was silenced using siRNA in an effort to restore an unirradiated gene profile.
  
  
  RESULTS
  Following irradiation, there was a steady increase in mRNA expression of Smad3, IL-13, TGF-β, Col1A1, MMP-2, TIMP-1, with peak at 12 to 24 hours and subsequent decline by 72 hours. TNF-α expression remained elevated throughout. MMP-1 showed minimal expression initially, which decreased to negligible by 72 hours. Inhibition of Smad3 significantly decreased expression of Col1A1, TGF-β, MMP-2, and TIMP-1. IL-13 and TNF-α expression was not affected by Smad3 silencing.
  
  
  CONCLUSIONS
  We have characterized the early-phase mRNA expression profiles of the major mediators of radiation-induced fibrosis. Smad3 siRNA effectively abrogated the elevation of Col1A1, TGF-β, TIMP-1, and MMP-2. IL-13 and TNF-α were unaffected by Smad3 silencing and appear to be minor regulators in fibrosis. These findings suggest a therapeutic rationale for Smad3 silencing in vivo.
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, New York University School of Medicine, New York, NY, USA. judy.lee@nyumc.org
authors.forename: Judy W
authors.initials: JW
authors.lastname: Lee
authors.forename: Richard A
authors.initials: RA
authors.lastname: Zoumalan
authors.forename: Cristian D
authors.initials: CD
authors.lastname: Valenzuela
authors.forename: Phuong D
authors.initials: PD
authors.lastname: Nguyen
authors.forename: John P
authors.initials: JP
authors.lastname: Tutela
authors.forename: Benjamin R
authors.initials: BR
authors.lastname: Roman
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Warren
authors.forename: Pierre B
authors.initials: PB
authors.lastname: Saadeh
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D000090602:Collagen Type I, alpha 1 Chain; D005109:Extracellular Matrix / Q000235:genetics; D005347:Fibroblasts / Q000528:radiation effects*; D020869:Gene Expression Profiling; D020868:Gene Silencing; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D017564:Radiation Pneumonitis / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D012867:Skin / Q000473:pathology / Q000528:radiation effects*; D051900:Smad3 Protein / Q000235:genetics* / Q000378:metabolism; D019714:Tissue Inhibitor of Metalloproteinases / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D034741:RNA, Small Interfering; D051900:Smad3 Protein; D019714:Tissue Inhibitor of Metalloproteinases; D016212:Transforming Growth Factor beta; D020778:Matrix Metalloproteinase 2