Analysis of the Role of ZEB1 in the Pathogenesis of Posterior Polymorphous Corneal Dystrophy

Abstract

Purpose: : To determine how nonsense mutations in the transcription factor ZEB1 lead to the development of posterior polymorphous corneal dystrophy 3 (PPCD3).

Methods: : Whole cell extracts were obtained from cultured human corneal epithelial cells (HCEpC) as a source of ZEB1 protein. DNA binding assays were performed using the whole cell extract and oligonucleotide probes consisting of the two conserved E2 box motifs and surrounding nucleotides upstream of COL4A3. ZEB1 and COL4A3 mRNA expression in primary human corneal endothelial cells (HCEnC) was measured in PPCD3 and control corneas by RT-PCR. Immunohistochemistry was used to localize ZEB1 and COL4A3 expression in normal human corneas.

Results: : EMSAs and competition EMSAs demonstrated binding of protein(s) in the cultured HCEpC to the E2 box motifs in the probes. The supershift EMSA confirmed that ZEB1, demonstrated to be present in the whole cell extracts, binds to both the proximal and distal E2 box motifs in the COL4A3 promoter region. Both COL4A3 and ZEB1 are expressed in normal HCEnC, although in PPCD3, ZEB1 expression is decreased and COL4A3 expression is increased compared to healthy control corneas.

Conclusions: : ZEB1-mediated alterations in COL4A3 expression are likely associated with the pathogenesis of PPCD3, although COL4A3 expression in healthy adult human corneal endothelial cells raises questions about whether PPCD3 might result from increased expression, rather than simple ectopic expression, of COL4A3 .

Keywords: cornea: endothelium • transcription factors • degenerations/dystrophies