N-glycosylation Affects E-cadherin-mediated Cell-Cell Adhesion and Differentiation of Oral Keratinocytes

Oral epithelium is a self-renewing tissue, organized into zones of proliferation and early and late stages of differentiation. E-cadherin, the classical calcium-dependent N-glycoprotein cell-cell adhesion receptor, has been reported to affect the progression of keratinocyte morphogenesis and differentiation. We have shown that N-glycans directly affect the stability of E-cadherin and the nature of E-cadherin-mediated cell-cell contacts. Highly N-glycosylated E-cadherin mediates the formation of weak cell-cell contacts, while hypo-N-glycosylated E-cadherin forms stable junctional complexes and promotes cell polarization and differentiation. The N-glycosylation status of E-cadherin is regulated by the activity of ALG7, the gene that initiates N-glycosylation in the endoplasmic reticulum. Objectives: Our goal was to investigate the role of E-cadherin N-glycosylation in oral epithelial cell growth and differentiation. Methods: Oral keratinocytes, OKB8, were grown in a keratinocyte serum-free low calcium (0.4 mM) medium in which E-cadherin is not active; calcium concentration was then increased to 1.2 mM to activate E-cadherin and induce differentiation. At different time points, cells were collected, lysed, and analyzed for levels of the ALG7 gene product, GPT, and for the abundance and N-glycosylation status of E-cadherin. In addition, expression and phosphorylation of E-cadherin interacting proteins, b-catenin and a-catenin, and linkage to actin cytoskeleton were determined and correlated with the expression of differentiation markers: involucrin, loricrin, and profilaggrin. Results: Our data show that following calcium switch, E-cadherin was abundant and highly N-glycosylated, consistent with cell proliferation and subsequent migration. After 48-72 hours in high calcium medium, E-cadherin decreased in abundance and became hypo-N-glycosylated with increased binding of b-catenin and a-catenin. This correlated with expression of differentiation markers. Conclusions: Our data indicate that oral keratinocyte growth and differentiation are accompanied by highly regulated N-glycosylation, expression, and adhesive activity of E-cadherin. Supported by NIH grants RO1 DE10183 and RO1 DE14437 (MAK) and T35 DE07268.