Mast Cell Tryptase Controls Paracellular Permeability of the Intestine

Mast Cell Tryptase Controls Paracellular Permeabilityof the Intestine 生科四甲陳子甯

作者假設： • mast cells signal to colonocytes by release of tryptase and activation of PAR2, and that PAR2 couples to ERK1/2 by a βARR-dependent mechanism to regulate TJs. TJ: tight junction ERK1/2：extracellular signal-regulated kinases 1/2 βARR:β-arrestin

作者目標： • 證實PAR2的活化造成TJs和細胞膜滲透性的改變。 • 證實mast cell 藉由分泌tryptase來活化PAR2，造成colonocytes細胞內鈣離子濃度上升及細胞膜滲透性提高。 • .證實ERK1/2及βARR在PAR2活化的這個機制中，扮演何種角色 ERK1/2：extracellular signal-regulated kinases 1/2 βARR:β-arrestin

Mast cell:

Model for β-arrestin-dependent activation of ERK1/2 by PAR2 microtubule-associated proteins (MAPs) Ca2+ phospholipase A2 (PLA2)

FIG. 1. Expression of mRNA encoding PAR2 and trypsinogen IV by colonocytes. NCM460和T84都是種小腸表皮細胞 525bp 525bp 400bp 400bp p c c c T p c T 確認NCM460和T84這兩株小腸表皮細胞都能夠表現PAR2和trypsinogenIV

FIG. 2. PAR2-mediated mobilization of [Ca2+]iin colonocytes. AP:由六個AA組成-SLIGKV RP:是AP序列相反的peptide-VKGILS。用來當做control組。結論：如果細胞外無鈣離子，細胞仍可利用本身所儲存的鈣離子來進行訊號傳遞。

FIG. 3. PAR2-mediated mobilization of [Ca2+]iin colonocytes. c 結論：trypsin活化PAR2後，的確會讓細胞體內的鈣離子增加。而trypsin的確對PAR2有專一性。

FIG. 4. Characterization of CHMC. 測不同型態的CHMC的螢光強度。確認mast cell與IgE binding後，可促進degranuled。證實mast cell分泌tryptase 的方式是degranule。 CHMC是種mast cell SBTI: Soybean trypsin inhibitor

Flow Cytometry

FIG. 5. Mast cell signaling to colonocytes. 48/80：是種促使mast cell degranule 的藥物。證實mast cell的確是用degranule的方式將tryptase送至supernatant form中，傳至colonocytes

TER ( transepithelial resistance ) & paracellular permeability

FIG. 6. Effects of PAR2 agonists on paracellular permeability. HRP是種位於細胞質中的酵素。證實PAR2 Activation Increases Paracellular Permeability

ZO-1與E-cadhesin Zo-1 Apical Basal E-cadhesin

FIG. 7. PAR2-induced redistribution of TJ proteins and F-actin detected by microscopy ZO-1 occludin Isotype Claudin-1 F-actin 證實了PAR2的確會讓TJ protein 和F-actin redistribution。

FIG. 8. PAR2-induced redistribution of TJ proteins detected by subcellular fractionation and Western blotting. E-cadherin is a transmembrane glycoprotein that mediates epithelial cell-to-celladhesion. 結論：PAR2的確會造成TJ proteins的redistribution

FIG. 9. Effects of mast cell products on paracellular permeability. 證實了PAR2的確會改變paracellular permeability

FIG. 10. ERK1/2-mediated changes in paracellular permeability. UO126：是種抑制ERK1/2磷酸化的藥劑。 ERK1/2 可以調控paracellular permeability，而PAR2可以調控ERK1/2，所以PAR2與調控paracellular permeability有關。

FIG. 11. Down-regulation of βARR using siRNA βARR1 498bp βARR2 381bp βARR是種cytoplasm-nucleus messenger，會與PAR2結合，活化ERK1/2 證實： βARR會活化ERK1/2

FIG. 12. βARR-mediated changes in paracellular permeability. Oligofectamine 可促進βARR與ERK1/2 binding的一種藥劑。證實PAR2活化後，使βARR促進ERK1/2活化，進而改變colonocytes的滲透性。

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. • βARR促進ERK1/2的活化，導致colonocytes的permeability上升

Mast Cell Tryptase Controls Paracellular Permeability of the Intestine