The membrane-associated MUC1 improves adhesion of salivary MUC5B on buccal cells. Application to development of an in vitro cellular model of oral epithelium

Significance Statement

The mucosal pellicle is a thin layer of salivary proteins lining epithelial oral cells. This pellicle has a protective function of oral mucosae against abrasion, pathogenic microorganisms or chemical xenobiotics. However, the currently available cellular models of oral mucosa do not consider this layer. We therefore developed a model based on TR146 cells, stably transfected in order to express the membrane-associated mucin MUC1. From a morphological point of view, the cells exhibited the appearance of non-keratinized oral mucosal cells, with typical membrane folds. The transfected cells could retain a higher quantity of the salivary mucins MUC5B than the native cells, highlighting that the membrane-associated mucins favour binding of salivary mucins to epithelial oral cells. We also showed that confluent transfected cells incubated with human saliva were lined with a “pellicle”, i.e. a thin filamentous salivary network. This in vitro model is a promising tool to investigate biological events occurring at the surface of oral mucosae, such as permeability to drugs or microorganism invasion. It may also be useful to study molecular interactions between food constituents and mucosae at the origin of some sensory properties of food products (astringency, aroma persistence…).

Journal Reference

Arch Oral Biol. 2016 Jan;61:149-55.

Ployon S¹, Belloir C¹, Bonnotte A², Lherminier J³, Canon F¹, Morzel M⁴.

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CNRS, UMR 6265 Centre des Sciences du GoÛt et de l’Alimentation, F-21000 Dijon, France; INRA, UMR1324 Centre des Sciences du GoÛt et de l’Alimentation, F-21000 Dijon, France; Université de Bourgogne, UMR Centre des Sciences du GoÛt et de l’Alimentation, F-21000 Dijon, France.
Université de Bourgogne, UMR 1347 Agroécologie, Plateforme DImaCell, Centre de Microscopie INRA/Université de Bourgogne, F-21000 Dijon, France.
INRA, UMR1347 Agroécologie, ERL CNRS 6300, Plateforme DImaCell, Centre de Microscopie INRA/Université de Bourgogne, F-21000 Dijon, France.
CNRS, UMR 6265 Centre des Sciences du GoÛt et de l’Alimentation, F-21000 Dijon, France; INRA, UMR1324 Centre des Sciences du GoÛt et de l’Alimentation, F-21000 Dijon, France; Université de Bourgogne, UMR Centre des Sciences du GoÛt et de l’Alimentation, F-21000 Dijon, France. Electronic address: [email protected]

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Abstract

OBJECTIVES:

The mucosal pellicle is a thin layer of salivary proteins, mostly MUC5B mucins, anchored to epithelial oral cells. This pellicle is involved in protection of oral mucosae against abrasion, pathogenic microorganisms or chemical xenobiotics. The present study aimed at studying the involvement of MUC1 in mucosal pellicle formation and more specifically in salivary MUC5B binding using a cell-based model of oral epithelium.

DESIGN:

MUC1 mRNAs were not detected in TR146 cells, and therefore a stable cell line named TR146/MUC1 expressing this protein was developed by transfection. TR146 and TR146/MUC1 were incubated with human saliva in order to evaluate retention of MUC5B by epithelial cells.

RESULTS:

The cell surface of both TR146 and TR146/MUC1 was typical of a squamous non-keratinized epithelium, with the presence of numerous microplicae. After incubation for 2h with saliva diluted in culture medium (1:1) and two washes with PBS, saliva deposits on cells appeared as a loose filamentous thin network. MUC5B fluorescent immunostaining evidenced a heterogeneous lining of confluent cell cultures by this salivarymucin but with higher fluorescence on TR146/MUC1 cells. Semi-quantification of MUC5B bound to cells confirmed a better retention by TR146/MUC1, evaluated by Dot Blot (+34.1%, p<0.05) or by immunocytochemistry (+44%, p<0.001).

CONCLUSION:

The membrane-bound mucin MUC1 is a factor enhancing the formation of the mucosal pellicle by increasing the binding of salivaryMUC5B to oral epithelial cells. An in vitro model suitable to study specifically the function and properties of the mucosal pellicle is proposed.

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