Abstract
Abstract: :
Purpose: To investigate the expression and role of Notch receptors and their ligands in the human corneal epithelium. Methods: Human corneal epithelial cells were cultured from donor human corneas (n=60). Following RNA extraction from epithelial cells, RT–PCR was performed to determine gene expression of the Notch receptors (N1–4) and their ligands (Delta 1, 3, 4 and Jagged 1, 2). Western blotting and indirect immunofluorescence was then used to confirm protein expression of the genes identified in corneal epithelial cells in vivo and ex vivo respectively. Following serum starvation (0.2% serum for 30 minutes), γ–secretase inhibitor at a concentration of 25µM or 50 µM was added to cultured corneal epithelial cells in triplicate for 4 hours. The consequent changes in expression of Notch1 and Notch2, the proliferation marker: Ki67 and the differentiation marker: Cytokeratin3 were evaluated by Western blotting. Results: Notch 1, 2, Delta 1 and Jagged 1 but not Notch 3, 4, Delta 3, 4 or Jagged 2 genes and proteins were identified in human corneal epithelial cells. Notch 1 and 2 were immunolocalised throughout the corneal epithelium in all suprabasal cell layers, but absent from the basal layer. In contrast, Delta 1 and Jagged 1 appeared to be expressed in cell borders in all cell layers of the corneal epithelium. γ–Secretase inhibition of cultured epithelial cells resulted in a significant decrease in Notch1 and 2 expression (p<0.01), accompanied by a reduced level of Ki67 (p<0.01) and an increase in Cytokeratin3 expression (p<0.01). Conclusions: Notch 1, 2 and their ligands Delta1, Jagged 1, are likely to play a pivotal role in corneal epithelial cell homeostasis. Downregulation of Notch 1 and 2 in corneal epithelial cells is associated with decreased proliferative activity and promotion of epithelial differentiation.
Keywords: cornea: epithelium • cell-cell communication • cornea: basic science