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

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!

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of East Anglia

Year dc:date.issued

2014

Author dc:creator

• Elghadban, Salma

Language dc:language

en