TGFß INDUCED KFM TRNASFORMATION IN HUMAN CORNEAL FIBROBLASTS IS MODULATED BY YAP AND TAZ

Abstract

Purpose : Corneal stromal wound healing is a complex process which integrates a cascade of events including elaboration and remodeling of the extracellular matrix (ECM) and transdifferentiation of quiescent cells in the stroma (keratocytes, to activated fibroblasts and subsequently myofibroblasts-KFM transformation). Transforming Growth Factor ß1 (TGFß1) is elaborated in wounds after injury and promotes KFM transformation. TGFß causes cellular changes through smad transcriptional modulators which result in the phosphorylation of smad2/3 and association with smad4 relocates these proteins to the cell nucleus, accelerating the transcriptional cascade. The novel mechanotransduction factors Yes-associated protein (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ) are strongly implicated in the matrix stiffness. Here, we examined the effects of knockdown of YAP and TAZ on smad2/3/4, TGFß1 induced KFM transformation of human corneal fibroblasts (HCFs).

Methods : We used a knockdown approach to silence YAP and TAZ separately in immortalized human corneal stromal fibroblasts. 24h post siRNA transfection, cells were cultured in the presence or absence of 10ng/ml TGFß1 for 72 hr. The expression of Smad2/3/4 and Alpha Smooth Muscle Actin (αSMA) were assessed by immunostaining and Western blotting. Images were quantitatively analyzed using MATLAB.

Results :
TGFß1 stimulation resulted in the translocation of YAP and TAZ to the nucleus suggesting inhibition of phosphorylation. Specific knockdown of YAP and TAZ decreased TGFß1 induced αSMA (effect of YAP knockdown>TAZ) suggesting YAP and TAZ to play a role in KFM transdifferentiation.

Conclusions : Our findings suggest that YAP and TAZ function as modulators of TGFß1 induced KFM transformation. These results suggest YAP and TAZ represent novel therapeutic targets for improving corneal wound healing outcomes for patients.
I think modulation is more accurate for the state of the investigations to date. Vijay---your thoughts? Also: the data are what the data are but we will need to reconcile the differing results prior to publication. Again, Vijay, your thoughts welcome in devising a work plan towards pblication

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.