Effects of culture conditions on claudin expression and permselectivity in rat epidermal keratinocytes

Abstract

The claudin family of tight junction proteins is believed to be responsible for the barrier characteristics of tight junctions. A limited number of studies have investigated the structure and function of tight junctions in stratified epithelia. In this project we are investigating, as a function of time and culture conditions, the changes in expression, localization and functional role of claudin tight junction proteins during formation of a model epidermis. Rat epidermal keratinocytes (REKs) were cultured in air-interface (AIC) or liquid-covered (LCC) conditions and on two different supports: uncoated and collagen-coated polycarbonate membranes. The barrier characteristics of tight junctions were investigated by measuring transepithelial electrical resistance (TER) and performing selectivity and permeability assays. Expression of claudin proteins were monitored by western blotting. REK cells grown on uncoated supports and in LCC exhibited a two-fold higher TER value than in AIC. Moreover, electrophysiological measurements revealed a selectivity that favoured the passage of sodium ions, and discriminated against the passage of chloride ions, in both LCC and AIC. For cells grown on collagen-coated supports the resistance increased with time in culture and the electrophysiological measurements revealed the formation of an epidermis that favoured the passage of anions over cations. Throughout the differentiation process only claudin-1, -3, -4, -5, and -7 were expressed. All detected claudins except claudin-7 showed major changes in their expression pattern as a function of time in culture. Interestingly, these changes in claudin expression were not always the same in cells grown on uncoated and collagen-coated supports. Furthermore, the pattern of claudin expression varied markedly between cells in LCC and AIC. These results suggest that, as they differentiate, REK cells modulate the properties of their tight junctions to produce a different ionic selectivity. Our observations further support the hypothesis that specific changes in claudin expression levels may correlate with specific changes in the permselectivity of the epidermis. Furthermore, our results highlighting the importance of the extracellular matrix and culture conditions in tight junction formation.

Date of Award	3 Jan 2013
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Catherine O'Neill (Supervisor) & Martin Steward (Supervisor)