PAR2於內皮細胞中活化p38 MAPK而誘發IL-8生成之訊息傳遞

Abstract

Protease-activated receptor 2（PAR2）是屬於一個新發現的G protein-coupled receptors家族：protease-activated receptors（PARs）裡的第二個成員。就目前的研究看來，PAR2在發炎以及疼痛的進程中扮演著相當重要的角色，而且在內皮細胞中亦有相當高的表現量。在本篇的研究中，我們發現以具有專一選擇性的PAR2-activating peptide（PAR2-AP）刺激人類臍帶靜脈內皮細胞（human umbilical vein endothelial cells），則會明顯的誘發大量interleukin-8（IL-8）的生成，因此擬探討其中詳細的訊息傳遞機轉。 不論是利用合成的PAR2-AP，或是內生性的PAR2活化劑－胰蛋白酶(trypsin)，均能以時間以及濃度相關性地增加IL-8的生成。利用選擇性的p38 MAPK（p38 mitogen-activated protein kinase ）抑制劑，SB203580，亦能濃度相關性地抑制IL-8的生成。從西方墨點法的實驗結果可以發現，PAR2-AP可以顯著有意義地增加p38 MAPK的磷酸化，而對於p38 MAPK的上游以及下游的訊息蛋白激酶，MAPK kinase 3/6（MKK3/6），eIF– 4E，Mnk-1以及MAPK-activated protein kinase-2（MAPKAPK-2），也都能以時間相關性的模式增加其磷酸化。實驗中再以SB203580處理，亦觀察到除了MAPKAPK-2之外，所有蛋白激酶的磷酸化均能被顯著地抑制。PAR2-AP也能時間相關性地增加 IL-8 mRNA 的表現以及其轉錄調控因子（transcription factor）－activating transcription factor-2的活化；而這些作用均會被SB203580抑制。更進一步地，利用dominant-negative（DN）transfection的技術，發現到不論是DN p38 MAPK，DN MKK3或是 DN MKK6都能有效降低PAR2-AP所造成的IL-8增加現象。綜合上述的結果，在人類臍帶靜脈內皮細胞中，p38 MAPK訊息傳遞途徑對於PAR2所誘發的IL-8生成是相當重要的。

Protease-activated receptor 2（PAR2）is the second member of a new subfamily of G protein-coupled receptors：the protease-activated receptors（PARs）. PAR2 is highly expressed on endothelial cells and plays an important role in inflammation and pain. In this study, we observed that the selective PAR2-activating peptide（PAR2-AP）could significantly induce the interleukin-8（IL-8）production in human umbilical vein endothelial cells（HUVECs）. Therefore, the signaling pathway involved in PAR2-induced endothelial IL-8 production was studied in this paper. Both the PAR2-AP and endogenous PAR2 activator, trypsin, caused concentration- and time-dependent increase of endothelial IL-8 production and this effect was concentration-dependently attenuated by the selective p38 mitogen-activated protein kinase（p38 MAPK）inhibitor, SB203580. Western blotting analysis showed that PAR2-AP induced the phosphorylation of p38 MAPK and its upstream and downstream protein kinases, including MAPK kinase 3/6（MKK3/6）, eIF–4E, Mnk-1 and MAPK-activated protein kinase-2（MAPKAPK-2）, in a time-dependent manner. SB203580 exhibited a significantly decreased phosphorylation of these protein kinases except for MAPKAPK-2. In addition, PAR2-AP caused an elevation of IL-8 mRNA expression and its transcription factor, activating transcription factor-2 activation. As expectedly, these signals were also suppressed by SB203580 in a concentration-dependent manner. Furthermore, introduction of dominant-negative vectors targeting on p38 MAPK, MKK3 and MKK6 resulted in abolishing the IL-8 production by PAR2-AP. In conclusion, our data suggest that the p38 MAPK pathway is important for PAR2-induced IL-8 production in HUVECs.