磷酯質水解酵素A2受體的細胞質區域在第一型磷酯質水解酵素A2調控細胞生理作用中所扮演的角色

Abstract

近年來發現第一型磷酯質水解酵素A2 (簡稱PLA2-I)除了具有分解磷酯質的功能外，尚有促進細胞的增生、細胞的侵入作用、化學增活現象、前列腺素的生成、氣道和血管肌細胞的收縮及受精作用等功能。這些作用都是經由磷酯質水解酵素A2受體(簡稱PLA2-IR)所達成的。目前我們對PLA2-IR的訊息傳遞機制仍不清楚。PLA2-IR的二級結構包括一極大的N端細胞質外區域、一段跨膜區域與一小段的細胞質內區域。此受體的二級結構與巨噬細胞的甘露糖受體很類似，在生理活性上，兩者皆具有將配體內吞的功能。巨噬細胞的甘露糖受體利用N端辨識醣類的區域，將帶有甘露糖殘基的配體或微生物，經內吞作用將之帶入細胞內，進而分解及吸收所結合的配體或微生物。目前已知PLA2-IR亦利用N端似辨識醣類的區域與PLA2-I結合後，同樣利用內吞作用將它帶入細胞，但目前對於此內吞作用與PLA2-I影響細胞生理作用間存在著什麼樣的關係仍不清楚。因此在本實驗中，我們構築老鼠的PLA2-IR基因及針對PLA2-IR細胞質區域的特定胺基酸做突變或截斷C端部份區域，利用轉殖作用將它們穩定地於HEK293細胞株中表現。此外，我們亦挑選了穩定攜帶3個NF-IL6起動子區域的蟲螢光蛋白報導基因之HEK293細胞株。我們期望藉由這樣的實驗設計，能瞭解老鼠的PLA2-IR對pPLA2-I的內化作用是否與其pPLA2-I影響細胞生理作用間有相關連。在表現各種老鼠PLA2-IR基因的HEK293細胞株中，我們觀察到表現mPLAR/L1453A的HEK293細胞株，pPLA2-I會內吞進入細胞並集中在核內；而表現mPLAR/W、mPLAR/TR1451及 mPLAR/S1455A的HEK 293細胞株，pPLA2-I亦會內吞至細胞，但聚集的位置是在細胞核周圍；至於表現mPLAR/TR1434與mPLAR/YY的細胞株，則沒有觀察到pPLA2-I的內吞作用。觀察pPLA2-I促進細胞的增生作用方面，在表現野生株的HEK293細胞株，沒有觀察到明顯的促進細胞的增生作用，但在其它含有mPLA2-IR突變株的細胞株中，卻發現有抑制細胞增生的現象。另外，在穩定攜帶3個NF-IL6起動子區域的蟲螢光蛋白報導基因之HEK293細胞株中，pPLA2-I似乎不會促進第二型環氧合酵素的表現。

Pancreatic phospholipase A2 (PLA2-I), an originally thought to be digestive enzyme, was implicated to play roles in cell proliferation, cell invasion, chemokinesis, eicosanoids production, contraction of vascular and airway smooth muscles, and fertilization. Further studies suggested that these physiological functions were mediated by the PLA2-I receptor (PLA2-IR). Although the signal transduction linking these effects to the event of ligand-receptor interaction remain obscure. The primary sequence alignment reveals that the PLA2-IR is homologous to that of the macrophage mannose receptor, which mediates the uptake of mannose-glycosylated ligands and the phagocytosis of parasitic microorganisms. Although the PLA2-I-inducded PLA2-IR internalization was demonstrated, but the relationship between the PLA2-I-inducded biological effects and the PLA2-IR-dependent internalization is unclear. To demonstrate that PLA2-IR internalization is important for its signal transduction, we constructed mouse PLA2-IR expression plasmids containing wild type, C-terminal truncated and mutations in the cytoplasmic domain of the mPLA2-IR gene, respectively. Each of these plasmids were transfected into HEK293 cell line through lipofection. We found that pPLA2-I did induce the internalization of the wild-type mPLA2-IR. The internalization of mPLA2-IR is determined by the cellular localization of exogenous pPLA2-I in HEK293. Interestingly, we found that pPLA2-I was in the vicinity of nucleus in HEK293 cell lines stably expressed mPLAR/W, mPLAR/TR1451 and mPLAR/S1455A, respectively. mPLAR/L1453A, on the other hand, was found to be located in the nucleus upon pPLA2-I treatment. Internalization of pPLA2-I was not observed in the HEK293 cell line stably expressed mPLAR/TR1434 and mPLAR/YY. In the studies of relationship between mPLA2-IR internalization and cell proliferation, we found that the HEK293 cell line expressed wild-type mPLA2-IR did not exhibit significant PLA2-I-mediated cell proliferation. In the HEK293 cell lines with C-terminal of mPLA2-IR truncated or mutated gene, however, pPLA2-I exhibited any effect to cell proliferation. Besides, we also prepared the HEK293 cell line ,which stably expresses a luciferase reporter gene containing three NF-IL6 responding elements for the examination of effects of mPLA2-IR and its mutants to the PLA2-I-induced cyclooxygenase II expression in HEK293 cell line. The result showed that, in HEK293 cell line, mPLA2-IR did not involve in the regulation of cyclooxygenase II expression.