A role for notch signaling in human corneal epithelial cell differentiation and proliferation.
OBJECTIVE
To identify the role of Notch signaling in the human corneal epithelium.


METHODS
Localization of Notch1, Notch2, Delta1, and Jagged1 in the human corneal epithelium was observed with the use of indirect immunofluorescence microscopy. Gene and protein expression of Notch receptors and ligands in human corneal epithelial cells was determined by RT-PCR and Western blot analysis, respectively. The effects of Notch inhibition (by gamma-secretase inhibition) and activation (by recombinant Jagged1) on epithelial cell proliferation (Ki67) and differentiation (CK3) were analyzed after Western blotting and immunocytochemistry.


RESULTS
Immunofluorescent labeling localized Notch1 and Notch2 to suprabasal epithelial cell layers, whereas Delta1 and Jagged1 were observed throughout the corneal epithelium. Notch1, Notch2, Delta1, and Jagged1 genes and proteins were expressed in human corneal epithelial cells. gamma-Secretase inhibition resulted in decreased Notch1 and Notch2 expression, with an accompanying decrease in Ki67 and increased CK3 expression. The activation of Notch by Jagged1 resulted in the upregulation of active forms of Notch1 and 2 proteins (P < 0.05), with a concurrent increase in Ki67 (P < 0.05) and a decrease in CK3 (P < 0.05) expression. Interestingly, gamma-secretase inhibition in a three-dimensional, stratified corneal epithelium equivalent had no effect on Ki67 or CK3 expression. In contrast, Jagged1 activation resulted in decreased CK3 expression (P < 0.05), though neither Notch activation nor inhibition affected cell proliferation in the 3D tissue equivalent.


CONCLUSIONS
Notch family members and ligands are expressed in the human corneal epithelium and appear to play pivotal roles in corneal epithelial cell differentiation.
Ma
Aihua
A
Cell and Molecular Biology Unit, School of Optometry and Vision Sciences, Cardiff University, Cardiff, Wales, United Kingdom.
Boulton
Mike
M
Zhao
Bojun
B
Connon
Che
C
Cai
Jun
J
Albon
Julie
J
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D000072100:Jagged-1 Protein; D008024:Ligands; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008875:Middle Aged; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism*; D051883:Receptor, Notch2 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000072099:Serrate-Jagged Proteins; D015398:Signal Transduction / Q000502:physiology*
D002135:Calcium-Binding Proteins; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; C000606273:JAG1 protein, human; D000072100:Jagged-1 Protein; D008024:Ligands; D008565:Membrane Proteins; C497761:NOTCH1 protein, human; C497764:NOTCH2 protein, human; D051881:Receptor, Notch1; D051883:Receptor, Notch2; D000072099:Serrate-Jagged Proteins; C064045:delta protein