mast cell tryptase controls paracellular permeability of the intestine
DESCRIPTION
Mast Cell Tryptase Controls Paracellular Permeability of 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 β ARR-dependent mechanism to regulate TJs. TJ: tight junction. - PowerPoint PPT PresentationTRANSCRIPT
Mast Cell Tryptase Controls Paracellular Permeability
of 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.
ERK1/2ERK1/2 :: extracellular signal-regulated extracellular signal-regulated kinases 1/2kinases 1/2
βARR:β-arrestinarrestin
TJ: tight junction
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
ERK1/2ERK1/2 :: extracellular signal-regulated kinases 1/2extracellular signal-regulated kinases 1/2
βARR:β-arrestinarrestin
Mast cell:
Model for β-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.
525bp
400bp
525bp400bp
c p c T c 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
NCM460 和 T84 都是種小腸表皮細胞
FIG. 2. PAR2-mediated mobilization of [Ca2+]i in 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+]i in colonocytes.
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。
c
FIG. 4. Characterization of CHMC.
確認 mast cell 與 IgE binding 後,可促進degranuled 。
測不同型態的 CHMC 的螢光強度。
CHMC 是種 mast cell
SBTI: Soybean trypsin inhibitor
證實 mast cell 分泌 tryptase
的方式是 degranule 。
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.
證實 PAR2 Activation Increases Paracellular Permeability
HRP 是種位於細胞質中的酵素。
ZO-1 與 E-cadhesin
Apical
Basal
Zo-1
E-cadhesin
FIG. 7. PAR2-induced redistribution of TJ proteins and F-actin detected by microscopy
ZO-1
Claudin-1
F-actin
occludin
Isotype
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.
結論: PAR2 的確會造成 TJ proteins的 redistribution
E-cadherin is a transmembrane glycoprotein that mediates epithelial cell-to-celladhesion.
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.
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。
UO126 :是種抑制 ERK1/2 磷酸化的藥劑。
FIG. 11. Down-regulation of βARR using siRNA
βARR1βARR2
498bp381bp
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