ROLE OF MYOCARDIN RELATED TRANSCRIPTION FACTOR-A IN TRANSCRIPTION GROWTH FACTOR BETA-INDUCED EPITHELIAL TO MESENCHYMAL TRANSITION OF LENS EPITHELIAL CELLS

Abstract

Transcription growth factor beta (TGFβ) mediated epithelial to mesenchymal transition (EMT) of lens epithelial cells (LEC) is known to cause posterior capsular opacification (PCO). In this work, I have focused on the TGFβ-induced EMT pathway governed by the cellular actin cytoskeleton dynamics. This study is the first to report the involvement of transcription co-factor myocardin related transcription factor-A (MRTF-A) in TGFβ-induced EMT in the lens. Using rat lens epithelial explants, I have conclusively established that in LECs, TGFβ induces nuclear migration of MRTF-A leading to induction of αSMA expression. Furthermore, I have manipulated the intracellular translocation of MRTF-A indirectly using actin binding drugs and established that inhibiting nuclear migration of MRTF-A reduces αSMA production by the cells. In addition, direct manipulation of MRTF-A using adenoviral vectors carrying modified gene constructs show that presence of functional MRTF-A construct in the nucleus is necessary to trigger αSMA expression by causing EMT. In order to understand the involvement of matrix metalloproteinase -9 (MMP-9) in this specific pathway, explants were treated with an MMP2/9 inhibitor and rhMMP9. I have established that rhMMP-9 does not significantly affect the intracellular migration of MRTF-A. Nevertheless, gene expression studies showed that MMP-9 induces the expression of MRTF-A. Taken together, I believe MMP-9 functions through a feedback mechanism controlling MRTF-A expression in the cell. However, the presence of MMP-9 is necessary but not sufficient for induction in MRTF-A nuclear translocation. Overall, the work presented in this thesis demonstrates for the first time the presence of MRTF-A in LECs and successfully shows that the intracellular translocation of MRTF-A as an integral part of TGFβ induced EMT. Therefore, in the future, MRTF-A may be used as a successful target molecule to inhibit in order to prevent EMT leading to PCO.