title: Molecular mechanisms of suberoylanilide hydroxamic acid in the inhibition of TGF-β1-mediated canine corneal fibrosis.
abstract: OBJECTIVE
  To investigate molecular mechanisms mediating anti-fibrotic effect of SAHA in the canine cornea using an in vitro model. We hypothesized that SAHA attenuates corneal fibrosis by modulating Smad-dependent and, to a lesser extent, Smad-independent signaling pathways activated by TGF-β1, as well as matrix metalloproteinase (MMP) activity.
  
  
  METHODS
  Cultured canine corneal fibroblasts (CCF) were incubated in the presence/absence of TGF-β1 (5 ng/mL) and SAHA (2.5 μm) for 24 h. Western blot analysis was used to quantify non-phosphorylated and phosphorylated isoforms of Smad2/3, p38 MAP kinase (MAPK), ERK1/2, and JNK1. Real-time PCR and zymography were utilized to quantify MMP1, MMP2, MMP8, and MMP9 mRNA expressions and MMP2 and MMP9 protein activities, respectively.
  
  
  RESULTS
  TGF-β1 treatment caused a significant increase in phospho-Smad2/3 and phospho-p38 MAPK. SAHA treatment reduced TGF-β1-induced phosphorylation of Smad2/3 but not of p38 MAPK. TGF-β1 did not modulate the phosphorylation of ERK1/2 or JNK1. SAHA caused a significant reduction in phospho-ERK1/2 expression regardless of concurrent TGF-β1 treatment. Neither SAHA alone nor in combination with TGF-β1 altered phospho-JNK1 expression. TGF-β1 significantly increased MMP1 and MMP9 mRNA expressions but did not alter MMP2 mRNA. SAHA treatment attenuated TGF-β1-induced MMP9 mRNA expression while significantly enhancing TGF-β1-induced MMP1 mRNA expression. Zymography detected reduced expression of MMP2 and MMP9 proteins in untreated control CCF. TGF-β1 treatment did not alter their expression, but SAHA treatment +/-TGF-β1 significantly increased MMP2 and MMP9 protein expressions.
  
  
  CONCLUSIONS
  The corneal anti-fibrotic effects of SAHA involve multiple mechanisms including modulation of canonical and non-canonical components of TGF-β1 intracellular signaling and MMP activity.
authors.affiliation: Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO, 65211, USA.
authors.forename: Kristina M
authors.initials: KM
authors.lastname: Gronkiewicz
authors.affiliation: Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO, 65211, USA.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Giuliano
authors.affiliation: Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO, 65211, USA.|Harry S. Truman Memorial Veteran Hospital, Columbia, MO, 65211, USA.
authors.forename: Ajay
authors.initials: A
authors.lastname: Sharma
authors.affiliation: Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO, 65211, USA. mohanr@health.missouri.edu.|Harry S. Truman Memorial Veteran Hospital, Columbia, MO, 65211, USA. mohanr@health.missouri.edu.|Mason Eye Institute, School of Medicine, Columbia, MO, 65211, USA. mohanr@health.missouri.edu.
authors.forename: Rajiv R
authors.initials: RR
authors.lastname: Mohan
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003315:Cornea / Q000473:pathology*; D004285:Dogs; D005355:Fibrosis; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006877:Hydroxamic Acids / Q000494:pharmacology*; D020780:Matrix Metalloproteinase 9 / Q000032:analysis; D053773:Transforming Growth Factor beta1 / Q000037:antagonists & inhibitors*; D000077337:Vorinostat; D048051:p38 Mitogen-Activated Protein Kinases / Q000032:analysis
chemical_list: D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D053773:Transforming Growth Factor beta1; D000077337:Vorinostat; D048051:p38 Mitogen-Activated Protein Kinases; D020780:Matrix Metalloproteinase 9
keywords: Smad; canine; corneal fibrosis; matrix metalloproteinase; mitogen-activated protein kinase; suberoylanilide hydroxamic acid