Involvement of Notch Signaling in Salivary Cell Differentiation

Notch signaling is involved in directing cell differentiation during embyrogenesis and mediating self-renewal in many organs. Drosophila Notch mutants or flies lacking the Notch ligand Ser do not develop salivary glands. However, the role of Notch signaling in mammalian salivary cell growth and differentiation has not been previously elucidated. Objectives: To examine the role of Notch signaling in a salivary cell differentiation model. Methods: The human salivary cell line HSG expresses differentiation biomarkers when grown on Matrigel^®, an extracellular matrix extract. Homogenates from HSG cells grown on plastic (control) or Matrigel^® were subjected to immunoblot analysis for Notch signaling proteins and the differentiation marker vimentin. Results: All four mammalian Notch receptor homologues (Notch 1-4) and Notch ligands Jagged 1/2 and Delta 1 were expressed in the control cultures, and their expression was not affected by growth on Matrigel^®. However, the activated form of Notch, Notch 1IC, was only detected in cells grown on Matrigel^®. HSG cells expressed vimentin as early as 2 h after seeding on Matrigel^®. The g-secretase inhibitors DAPT and DAPM, which are known to block Notch 1IC formation, inhibited Matrigel^® induced vimentin expression. The expression of a Notch downstream effector Hes-1 (hairy enhancer of split family) was also abolished by DAPT and DAPM. Notch receptor knockdown studies using short interference RNA (siRNA) showed that knockdown of any single Notch receptor was not sufficient to inhibit vimentin induction on Matrigel^®. However, when HSG cells were transfected with all four Notch siRNAs, vimentin expression was inhibited. Conclusion: The current studies suggest that Notch signaling plays an important role in mammalian salivary cell differentiation. Further research on the involvement of the Notch signaling in salivary gland repair and regeneration is warranted (VA Merit Review and NIH/NIDCR R21DE15381).