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

El Chami, CĂ©cile

[Thesis]. Manchester, UK: The University of Manchester; 2012.

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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.

Bibliographic metadata

Type of resource:
Content type:
Form of thesis:
Type of submission:
Degree programme:
MPhil Physiology
Publication date:
Location:
Manchester, UK
Total pages:
100
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.
Thesis main supervisor(s):
Thesis co-supervisor(s):
Thesis advisor(s):
Language:
en

Institutional metadata

University researcher(s):

Record metadata

Manchester eScholar ID:
uk-ac-man-scw:170104
Created by:
El Chami, Cecile
Created:
17th September, 2012, 14:04:47
Last modified by:
El Chami, Cecile
Last modified:
6th October, 2017, 08:12:14

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