mast cell tryptase controls paracellular permeability of the intestine

Mast Cell Tryptase Controls Paracellular Permeabilityof the Intestine

mast cells signal to colonocytes by release of tryptase and activation of PAR2, and that PAR2 couples to ERK1/2 by a ARR-dependent mechanism to regulate TJs.

ERK1/2extracellular signal-regulated kinases 1/2ARR:-arrestinTJ: tight junction

PAR2TJs

mast cell tryptasePAR2colonocytes

.ERK1/2ARRPAR2

ERK1/2extracellular signal-regulated kinases 1/2ARR:-arrestin

Mast cell:

Model for -arrestin-dependent activation of ERK1/2 by PAR2

microtubule-associated proteins (MAPs) phospholipase A2 (PLA2) Ca2+

FIG. 1. Expression of mRNA encoding PAR2 and trypsinogen IV by colonocytes.

525bp400bp525bp400bpcpcTcpcTNCM460T84PAR2trypsinogenIVNCM460T84

FIG. 2. PAR2-mediated mobilization of [Ca2+]i in colonocytes.

AP:AA-SLIGKV RP:APpeptide-VKGILScontrol

FIG. 3. PAR2-mediated mobilization of [Ca2+]i in colonocytes.

trypsinPAR2 trypsinPAR2c

FIG. 4. Characterization of CHMC.

mast cellIgE bindingdegranuledCHMC

CHMCmast cellSBTI: Soybean trypsin inhibitormast celltryptasedegranule

Flow Cytometry

FIG. 5. Mast cell signaling to colonocytes.

48/80mast cell degranule mast celldegranuletryptasesupernatant formcolonocytes

TER ( transepithelial resistance ) & paracellular permeability

FIG. 6. Effects of PAR2 agonists on paracellular permeability.

PAR2 Activation Increases Paracellular Permeability

HRP

ZO-1E-cadhesinApicalBasalZo-1E-cadhesin

FIG. 7. PAR2-induced redistribution of TJ proteins and F-actin detected by microscopy

ZO-1Claudin-1F-actinoccludinIsotype

PAR2TJ protein F-actin redistribution

FIG. 8. PAR2-induced redistribution of TJ proteins detected by subcellular fractionation and Western blotting.

PAR2TJ proteinsredistributionE-cadherin is a transmembrane glycoprotein that mediates epithelial cell-to-cell adhesion.

FIG. 9. Effects of mast cell products on paracellular permeability.

PAR2paracellular permeability

FIG. 10. ERK1/2-mediated changes in paracellular permeability.

ERK1/2 paracellular permeabilityPAR2ERK1/2PAR2paracellular permeabilityUO126ERK1/2

FIG. 11. Down-regulation of ARR using siRNAARR1ARR2498bp381bpARR cytoplasm-nucleus messengerPAR2ERK1/2 ARR ERK1/2

FIG. 12. ARR-mediated changes in paracellular permeability.

Oligofectamine ARR ERK1/2 bindingPAR2ARR ERK1/2colonocytes

Conclusion

Colonocytes expressed PAR2 mRNA and responded to PAR2 agonists with increased [Ca2+]i

Mast cells signal to colonocytes in a paracrine manner by release of tryptase and activation of PAR2.

ARRERK1/2colonocytes permeability