Role of 5'TG3'-interacting factors (TGIFs) in Vorinostat (HDAC inhibitor)-mediated Corneal Fibrosis Inhibition.
OBJECTIVE
We have previously reported that vorinostat, an FDA-approved, clinically used histone deacetylase (HDAC) inhibitor, attenuates corneal fibrosis in vivo in rabbits by blocking transforming growth factor β (TGFβ). The 5'TG3'-interacting factors (TGIFs) are transcriptional repressors of TGFβ1 signaling via the Smad pathway. The present study was designed to explore the expression of TGIFs in human corneal fibroblasts and to investigate their role in mediating the antifibrotic effect of vorinostat.


METHODS
Human corneal fibroblast cultures were generated from donor corneas. RNA isolation, cDNA preparation, and PCR were performed to detect the presence of TGIF1 and TGIF2 transcripts. The cultures were exposed to vorinostat (2.5 µM) to test its effect on TGIF mRNA and protein levels using qPCR and immunoblotting. Myofibroblast formation was induced with TGFβ1 (5 ng/ml) treatment under serum-free conditions. The changes in fibrosis parameters were quantified by measuring fibrosis marker α-smooth muscle actin (αSMA) mRNA and protein levels with qPCR, immunostaining, and immunoblotting. Smad2/3/4 and TGIF knockdowns were performed using pre-validated RNAi/siRNAs and a commercially available transfection reagent.


RESULTS
Human corneal fibroblasts showed the expression of TGIF1 and TGIF2. Vorinostat (2.5 µM) caused a 2.8-3.3-fold increase in TGIF1 and TGIF2 mRNA levels and a 1.4-1.8-fold increase in TGIF1 and TGIF2 protein levels. Vorinostat treatment also caused a significant increase in acetylhistone H3 and acetylhistone H4. Vorinostat-induced increases in TGIF1 and TGIF2 were accompanied by a concurrent decrease in corneal fibrosis, as indicated by a decrease in αSMA mRNA by 83±7.7% and protein levels by 97±5%. The RNAi-mediated knockdown of Smad2, Smad3, and Smad4 markedly attenuated TGFβ1-evoked transdifferentiation of fibroblasts to myofibroblasts. The siRNA-mediated knockdown of TGIF1 and TGIF2 neutralized vorinostat-evoked decreases in αSMA mRNA by 31%-45% and protein levels by 12%-23%.


CONCLUSIONS
Human corneal fibroblasts demonstrate the expression of TGIF1 and TGIF2 transcription factors. These transcriptional repressors are critical, at least partially, in mediating the antifibrotic effect of vorinostat in the cornea.
Harry S. Truman Memorial Veteran Hospital, Columbia, MO ; Veterinary Medicine and Surgery, University of Missouri, Columbia, MO.
Ajay
A
Sharma
Veterinary Medicine and Surgery, University of Missouri, Columbia, MO.
Nishant R
NR
Sinha
Veterinary Medicine and Surgery, University of Missouri, Columbia, MO.
Saad
S
Siddiqui
Harry S. Truman Memorial Veteran Hospital, Columbia, MO ; Mason Eye Institute, School of Medicine, Columbia, MO ; Veterinary Medicine and Surgery, University of Missouri, Columbia, MO.
Rajiv R
RR
Mohan
D054338:Cell Transdifferentiation; D002478:Cells, Cultured; D003315:Cornea / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D003316:Corneal Diseases / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D065306:Corneal Injuries / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005355:Fibrosis; D055785:Gene Knockdown Techniques; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D018398:Homeodomain Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D008954:Models, Biological; D058628:Myofibroblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D034622:RNA Interference; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051785:Smad Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000077337:Vorinostat
D056572:Histone Deacetylase Inhibitors; D018398:Homeodomain Proteins; D006877:Hydroxamic Acids; D012097:Repressor Proteins; D051785:Smad Proteins; C097504:TGIF1 protein, human; C414585:TGIF2 protein, human; D000077337:Vorinostat