Transcription Factor 4 (TCF4) promotes increased corneal endothelial cellular migration by altering microtubules in Fuchs Endothelial Corneal Dystrophy

Abstract

Purpose : Fuchs endothelial corneal dystrophy (FECD) is a complex corneal disease characterized by the progressive decline and morphological changes of corneal endothelial cells (CECs) that leads to corneal edema and vision loss. The most common mutation in FECD is an intronic CTG repeat expansion in transcription factor 4 (TCF4) that leads to its altered expression. Corneal wound healing occurs primarily through cell enlargement and migration, and FECD CECs have been shown to display increased migration speeds. In this study, we aim to determine whether TCF4 can promote cellular migration in FECD CECs.

Methods : We generated novel CEC lines derived from either healthy donor controls or FECD patients that overexpressed different TCF4 isoforms and investigated epithelial-to-mesenchymal (EMT) expression, morphological analysis and cellular migration speeds. To investigate changes in gene expression with TCF4 overexpression, we performed bulk RNA sequencing on FECD CECs after stimulating cellular migration with a scratch assay and identified cytoskeleton regulators as potential mediators of the promigratory phenotype seen in FECD. Cytoskeleton protein expression was validated in normal and FECD ex-vivo specimens.

Results : We found that full length TCF4-B isoform overexpression compared to empty vector control promotes cellular migration (35.8 ± 8.0 % area open vs 50.8 ± 18.0 % area open, p<0.05) in FECD CECs in an EMT-independent manner. RNA-sequencing on our TCF4-B overexpression FECD CECs identified 227 differentially expressed genes (p<0.05, fold change >1.25 and <-1.25). We identified several pathways including the negative regulation of microtubules, with TUBB4A (tubulin beta 4A class IVa) as the top upregulated gene. TUBB4A expression was increased in FECD ex-vivo specimens and there was altered expression of cytoskeleton proteins tubulin and actin compared to normal healthy donor ex-vivo specimens. Additionally, there was increased acetylation and detyrosination of microtubules in FECD supporting that microtubule stability is altered in FECD and could promote cellular migration.

Conclusions : Overexpression of TCF4-B promotes increased cellular migration in an EMT-independent manner by altering microtubules in FECD CECs, and that microtubule stability is dysregulated in FECD.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.