title: Analysis of the role of ZEB1 in the pathogenesis of posterior polymorphous corneal dystrophy.
abstract: OBJECTIVE
  To determine how nonsense mutations in the transcription factor ZEB1 lead to the development of posterior polymorphous corneal dystrophy type 3 (PPCD3).
  
  
  METHODS
  Whole-cell extracts were obtained from cultured human corneal epithelial cells (HCEpCs) 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 (HCEnCs) was assayed in both PPCD3 and control corneas by RT-PCR. Immunohistochemistry was used to localize ZEB1 and COL4A3 expression in normal human cornea.
  
  
  RESULTS
  Electromobility shift assays (EMSAs) and competition EMSAs demonstrated binding of protein(s) in the cultured HCEpCs 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 HCEnCs, although in PPCD3, ZEB1 expression is decreased and COL4A3 expression is increased compared with levels of both genes in healthy control corneas.
  
  
  CONCLUSIONS
  Inversely related HCEnC expression levels of ZEB1 and COL4A3 in PPCD3 indicate that ZEB1-mediated alterations in COL4A3 expression are most likely associated with the pathogenesis of this corneal endothelial dystrophy. However, the demonstration of COL4A3 expression in healthy adult primary HCEnCs suggests that PPCD3 is more likely to involve an alteration in the timing and/or degree of COL4A3 expression than to result from the dichotomous change implied by the previously proposed ectopic expression model.
authors.affiliation: Jules Stein Eye Institute, David Geffen School of Medicine at the University of California, Los Angeles, California, USA.
authors.forename: Vivek S
authors.initials: VS
authors.lastname: Yellore
authors.forename: Sylvia A
authors.initials: SA
authors.lastname: Rayner
authors.forename: Catherine K
authors.initials: CK
authors.lastname: Nguyen
authors.forename: Rajendra K
authors.initials: RK
authors.lastname: Gangalum
authors.forename: Zhe
authors.initials: Z
authors.lastname: Jing
authors.forename: Suraj P
authors.initials: SP
authors.lastname: Bhat
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Aldave
mesh_terms: D000328:Adult; D000368:Aged; D001324:Autoantigens / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D018389:Codon, Nonsense; D024141:Collagen Type IV / Q000235:genetics / Q000378:metabolism; D003317:Corneal Dystrophies, Hereditary / Q000235:genetics* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004728:Endothelium, Corneal / Q000378:metabolism; D019573:Epithelium, Corneal / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology*; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D008875:Middle Aged; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics*; D000071799:Zinc Finger E-box-Binding Homeobox 1
chemical_list: D001324:Autoantigens; D018389:Codon, Nonsense; D024141:Collagen Type IV; D018398:Homeodomain Proteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D014157:Transcription Factors; C486307:ZEB1 protein, human; D000071799:Zinc Finger E-box-Binding Homeobox 1; C057112:type IV collagen alpha3 chain