Role of matriptase in the regulation of epithelial barrier permeability studied using MDCK cells

Elghadban, Salma (2014) Role of matriptase in the regulation of epithelial barrier permeability studied using MDCK cells. Doctoral thesis, University of East Anglia.

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Abstract

The!typeNII!transmembrane!serine!protease!matriptase!plays!an!important!role!in!
the!integrity!of!epithelial!barriers.!However,!the!molecular!mechanisms!underlying!
the!role!of!matriptase!are!unknown.!
To! study! these!mechanisms,! two!variants!of!MadinNDarby!canine!kidney!(MDCK)!
cells!were! used,! together!with! the! “calciumNswitch”!model! of! epithelial! function!
with!measurements!of!transepithelial!electrical!resistance!(TEER).!
Inhibitors!of!matriptase!proteolytic!activity!delayed!the!restoration!of!TEER!after!
calciumNswitch! in!MDCKNI,! which! develop! high! TEER! and! lack! the! “leaky”! tight!
junction!protein!claudinN2,!but!not!MDCKNII.!This!effect!was!confirmed!in!MDCKNI,!
established!to!stably!express!matriptase!targeted!shRNA.!
The!influence!of!matriptase!inhibition!on!MDCKNI!was!shown!not!to!involve!altered!
expression! or! assembly! of! relevant! components! of! paracelluar! junctions! or!
cytoskeleton.! This! excluded! a! role! for! claudinN2! in! the! function! of! matriptase,!
which!had!previously!been!shown!in!human!CacoN2!cells,!and!this!was!confirmed!
using!MDCKNI!cells!stably!overexpressing!claudinN2.!
To! investigate! the! claudinN2Nindependent! function! of! matriptase,! a! candidate!
substrate! approach! was! used.! Proteolytic! activation! of! proNHGF/cNMet,! PARN2,!
ENaC,!EGFR!and!prostasin!by!matriptase!has!effects!on!epithelial!cell!function,!but!
none!were! found! to!have!a! role! in!matriptase! restoration!of!TEER!after! calciumN
switch!in!MDCKNI!cells.!As!the!direct!proteolytic!target!of!matriptase!could!not!be!
identified,! potential! mediators! were! studied! using! arrays! for! phosphorylated!
signalling!proteins!and!inflammatory!cytokines.!ILN1β!and!complement!component!
C5!were! identified!as!genes!downregulated!by!matriptase! inhibition,!while! ILN13!
showed!upregulation.!
This!work!has!confirmed!the!key!role!of!matriptase!activity!in!regulating!epithelial!
barrier!integrity.!The!differential!properties!of!MDCKNI!and!MDCKNII!cells!excluded!
a! role! for! claudinN2.! None! of! the! known! proteolytic! targets! of! matriptase! were!
involved,!however,!changes!in!cytokine!gene!expression!may!be!a!potential!route!
for!matriptase!effects!on!epithelial!barrier!maintenance!

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Brian Watkins
Date Deposited: 22 Jun 2015 13:35
Last Modified: 22 Jun 2015 13:35
URI: https://ueaeprints.uea.ac.uk/id/eprint/53377
DOI:

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