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Jonathan Tovey, Kristina M Gronkiewicz, Elizabeth A Giuliano, Saad Siddiqui, Ajay Sharma, Justin L Brooke, Rajiv R Mohan; Role of cellular kinases and Smads in the modulation of SAHA-mediated corneal fibrosis inhibition. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5147.
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© ARVO (1962-2015); The Authors (2016-present)
We have shown that SAHA, an FDA approved histone deacetylase inhibitor, prevents TGFb1-driven corneal fibrosis in humans, canines and equines in vitro, and rabbits in vivo. The present study sought to delineate underlying mechanism of SAHA anti-fibrotic response in the cornea using an in vitro model.
Donor human and canine corneas were used to generate primary human and canine corneal fibroblast cultures. The fibroblast cultures were exposed to SAHA (2.5 µM) in +/- of TGFb1 (5ng/ml) for 24 hours under serum-free conditions. Total cellular lysates were prepared to perform immunoblotting for quantification of total and phosphorylated ERK1/2, JNK, p38 MAPK, Smad7, NFkB and IkB. Cultures were fixed in fresh 4% paraformaldehyde to perform immunofluorescence studies of listed signaling proteins.
TGFb1 treatment of human and canine fibroblasts caused a notable increase in phospho-Smad2/3 and phoshpo-p38MAPK protein levels. SAHA significantly attenuated TGFb1-evoked increase in phospho-Smad2/3 but did not inhibit TGFb1-induced phospho-p38MAPK. Significant levels of phospho-ERK, phospho-JNK were detected in human and canine fibroblasts. TGFb1 treatment did not cause any further increase in the phosphorylation of ERK or JNK suggesting that TGFb1 does not significantly activate these pathways in the corneal fibroblast. Most interestingly, irrespective of TGFb1 presence, SAHA treatment strongly inhibited the phospho-ERK and phospho-JNK levels in the human and canine corneal fibroblasts.
SAHA inhibits corneal fibrosis by modulating TGFb1-indcued activation of pro-fibrogenic Smad2/3 pathway. Additionally, inhibition of ERK, JNK phosphorylation evoked by other cytokines released during corneal injury help explain SAHA’s remarkable anti-fibrotic effect.
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