Abstract
Purpose: :
Recent evidence has shown that the expression of Notch signaling molecules is downregulated in patients with severe dry eye. Our goal was to determine the role of Notch abrogation in the biosynthesis of membrane-associated mucins in a human conjunctival epithelial (HCjE) cell culture model.
Methods: :
HCjE cells were grown in serum-free media to achieve confluence and then switched to serum-containing media for 7 days to promote stratification and differentiation. Cell lysates were extracted in RIPA buffer, and the presence of Notch 1, -2 and -3 receptors was analyzed by western blot. Proliferating, confluent, and stratified HCjE cells were treated for 4, 24, and 48 hours with 10 µM dibenzazepine (DBZ), a gamma-secretase inhibitor that blocks activation of Notch. The biosynthesis of membrane-associated mucins was evaluated by western blot, using antibodies to MUC16 (M11) and MUC1 (214D4). GAPDH was used as housekeeping control.
Results: :
By western blot, Notch 1, -2 and -3 receptors were detected in HCjE cells. Treating HCjE cells with DBZ for 4 hours did not affect the biosynthesis of membrane-associated mucins under any culture conditions. However, treating HCjE cells with DBZ for 24 and 48 hours decreased MUC16 biosynthesis in proliferating and confluent cells. In proliferating cells, levels of MUC16 decreased by 81-100% at 48 hours, whereas in confluent cells MUC16 decreased by 45-84% at 24 hours, and by 30-88% at 48 hours. Interestingly, DBZ did not impair MUC16 biosynthesis in stratified cells. No changes in MUC1 biosynthesis were detected at either time point under these conditions.
Conclusions: :
This study suggests that Notch signaling is involved in the early induction of MUC16 biosynthesis in HCjE cells and could, therefore, contribute to promoting the proper differentiation of ocular surface epithelia into a wet-surface phenotype.Support: Massachusetts Lions Eye Research Fund and NIH EY014847.
Keywords: conjunctiva • cornea: surface mucins • differentiation