title: Halofuginone down-regulates Smad3 expression and inhibits the TGFbeta-induced expression of fibrotic markers in human corneal fibroblasts.
abstract: OBJECTIVE
  Due to its ability to disrupt transforming growth factor beta (TGF-β) signaling, halofuginone has been successfully used to treat various fibrotic disorders. Here we investigated the antifibrotic potential of halofuginone in human corneal fibroblasts.
  
  
  METHODS
  Human corneal fibroblasts were isolated from human donor corneas for in vitro experiments. TGF-β was used to stimulate pro-fibrotic responses from corneal fibroblasts under halofuginone treatment. The expression of alpha smooth muscle actin (α-SMA) and fibronectin was analyzed by western blots. Phalloidin toxin was used to stain cultures for stress fiber assemblies. Quantitative reverse transcription PCR (qRT-PCR) and immunostaining were used to analyze the expression of type I collagen mRNA and protein, respectively. The expression of Smad2, Smad3, phospho-Smad2, and phospho-Smad3 was determined by western blots.
  
  
  RESULTS
  Halofuginone was well tolerated by human corneal fibroblasts up to 10 ng/ml as demonstrated by a cell viability assay. At this concentration, TGF-β-induced expression of the fibrotic markers α-SMA, fibronectin, and type I collagen was significantly reduced. Interestingly, under our experimental conditions, halofuginone treatment led to reduced protein expression of Smad3, which was both dose- and time-dependent.
  
  
  CONCLUSIONS
  Our results suggest that halofuginone may exert its antifibrotic effects in the cornea via a novel molecular mechanism and may be used as an antifibrotic agent for corneal fibrosis treatment.
authors.affiliation: Department of Ophthalmology, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Elizabeth F
authors.initials: EF
authors.lastname: Nelson
authors.forename: Craig W
authors.initials: CW
authors.lastname: Huang
authors.forename: Jillian M
authors.initials: JM
authors.lastname: Ewel
authors.forename: Angela A
authors.initials: AA
authors.lastname: Chang
authors.forename: Ching
authors.initials: C
authors.lastname: Yuan
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D003315:Cornea / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D005355:Fibrosis / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D010880:Piperidines / Q000494:pharmacology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D052999:Quinazolinones / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000009:adverse effects
chemical_list: D000199:Actins; D015415:Biomarkers; D024042:Collagen Type I; D005353:Fibronectins; D010880:Piperidines; D011500:Protein Synthesis Inhibitors; D052999:Quinazolinones; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1; C010176:halofuginone