Document Type
Article
Publication Date
2016
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 (5ng/ml) and SAHA (2.5μM) for 24hrs. 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 expression and MMP2 and MMP9 protein activity, 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 expression 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 expression.
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.
Recommended Citation
Gronkiewicz KM, Giuliano EA, Sharma A, Mohan RR. Molecular Mechanisms of Suberoylanilide Hydroxamic Acid (SAHA) in the Inhibition of TGF-β1 Mediated Canine Corneal Fibrosis. Vet Ophthalmol. 2016;19(6):480-487. doi:10.1111/vop.12331.
Copyright
Wiley
Included in
Animal Experimentation and Research Commons, Animals Commons, Musculoskeletal, Neural, and Ocular Physiology Commons, Ophthalmology Commons, Other Pharmacy and Pharmaceutical Sciences Commons, Other Veterinary Medicine Commons, Small or Companion Animal Medicine Commons
Comments
This is the accepted version of the following article:
Gronkiewicz KM, Giuliano EA, Sharma A, Mohan RR. Molecular Mechanisms of Suberoylanilide Hydroxamic Acid (SAHA) in the Inhibition of TGF-β1 Mediated Canine Corneal Fibrosis. Vet Ophthalmol. 2016;19(6):480-487. doi:10.1111/vop.12331.
which has been published in final form at DOI: 10.1111/vop.12331. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.