水解磷酸脂對內皮細胞蛋白質水解酵素作用之研究

Lysophospholipids Induce Matrix Metalloproteinases Expression in Human Endothelial Cells

吳雯婷(WenTing Wu)

指導教授 ： 李心予

國立臺灣大學/生命科學院/動物學研究研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.00393

摘要

摘要
Lysophosphatidic acid (LPA)與 sphingosine-1-phosphate (S1P)是形態與構造十分簡單的小分子水解磷酸脂(lysophopholipid, LPLs)，經由釵h不同種類細胞分泌，具有一些與蛋白質生長因子十分相似的作用，對細胞的生存、複製、增生及分化都扮演很重要的角色。LPA和S1P會經由和細胞表面特異接受器之接合來達成對細胞的作用，目前已有八個水解磷酸脂的接受器在人類基因中被發現，均屬於內皮細胞分化基因(Endothelial differentiation gene)的G蛋白接合型的接受器(G protein-coupled receptor)，進而活化各種中下游之細胞基質，達到傳遞訊息的目的。Matrix metalloproteinases (MMPs)是一群zinc-dependent的蛋白質水解酵素，可分解釵h細胞外基質(extracellular matrix)，在釵h的研究中已發現MMPs參與在組織重建、胚胎發育、傷口修復和血管新生等，也會增加癌細胞侵入(invation)的能力，其中MMP-2不但廣泛表達而且是參與在這些過程的重要成員。在本研究中，使用RT-PCR，西方墨點法和gelatin zymographic analysis等實驗方法，探討LPA與S1P對人類內皮細胞MMP-2表現的調控。發現MMP-2在包括mRNA、蛋白質和酵素活性等各個不同層次的表現皆受到誘導而增加，且生成量的多寡，與處理時間以及濃度皆呈現正相關。又以一些特異訊息傳導路徑化學抑制物質，找出包括MEK/ERK-，NF-κB-，和鈣離子流動所造成的訊息傳遞都參與在這個調控過程中。之後為了更進一步了解LPA和S1P對內皮細胞在生理弁鄋瑤梇情A我們進行細胞侵入能力的實驗，證實了LPA和S1P明顯地增強內皮細胞的侵入能力，而且此作用會被MMP的抑制物所抑制。同時也觀察到在mRNA的層次，S1P能增加MMP的天然抑制物，Tissue inhibitor of metalloproteinases (TIMPs)的表現，包括TIMP-2和TIMP-3。由此推論水解磷酸脂可藉由對MMP-2活性的調控，以增加細胞遷移或侵入的能力，而對MMP活性的調節也可能經由其抑制物來達成。

關鍵字

水解磷酸脂 ； 蛋白質水解酵素 ； 組織蛋白抑制脢 ； 內皮細胞

並列摘要

Abstract
Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are both low molecular weight lysophopholipid (LPLs), which can promote cell proliferation, migration, and invasion via interaction with the endothelial differentiation gene (Edg) family of specific G protein-coupled receptors. Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteolytic enzymes, which are involved in degradation of extracellular matrix, and play critical roles in endothelial cell migration and matrix remodeling during the angiogenesis. Among these MMPs, MMP-2 is an important molecule that is known to trigger the invasion of tumor cells. On the other hand, the nature inhibitors of MMPs, tissue inhibitor of metalloproteinases (TIMPs), are key physiological regulators of MMPs. In our present study, we examine the effects of LPA and S1P on MMPs, especially MMP-2 expression of human endothelial cell. By using RT-PCR, Western blotting, and gelatin zymographic analysis, we showed that LPA and S1P enhanced MMP-2 expression in mRNA, protein levels and also enzymatic activity. The enhancement effects are in a concentration- and time-dependent manner. Next, we evaluated the signal transduction pathway relevant to the MMP-2 expression stimulated by LPLs. Several chemical inhibitors were used including pertussis toxin (Gi blocker), PD98059 (MEK/ERK blocker), U73122 (PLC blocker), LY294002 (PI3 kinase blocker), PDTC (NF-κB blocker), PP2 (Src family kinase blocker), and SKF 96365 (receptor mediate calcium influx blocker). By real-time PCR analysis, the results indicated that these effects are involved in MEK/ERK-, NF-κB-, or calcium influx mediate signaling-dependent pathways. Furthermore, we used chemotaxis chamber to investigate LPL’s effects on cell invasion. Our results showed that endothelial cell invasion was potently increased by LPLs, and the induction can be prevented by preincubation with GM6001, a chemically synthesized MMP inhibitor. Interestingly, we find that S1P also regulated TIMPs expression, including TIMP-2 and TIMP-3. These observations suggest that LPA and S1P may play important roles in endothelial cell invasion by regulating the expression of MMP-2 and may be also MMPs inhibitors.

並列關鍵字

MMPs ； Lysophospholipids ； endothelail ； TIMPs

參考文獻

Abecassis, I., Olofsson, B., Schmid, M., Zalcman, G., and Karniguian, A. (2003) RhoA induces MMP-9 expression at CD44 lamellipodial focal complexes and promotes HMEC-1 cell invasion. Exp Cell Res. 291: 363-376.

Agren, M. S., Mirastschijski, U., Karlsmark, T., and Saarialho-Kere, U. K. (2001) Topical synthetic inhibitor of matrix metalloproteinases delays epidermal regeneration of human wounds. Exp. Dermatol. 10: 337-348.

An, S. Z., Bleu, T., Zheng, Y. H., and Goetzl, E. J. (1998) Recombinant human G protein-coupled lysophosphatidic acid receptors mediate intracellular calcium mobilization. Mol Pharmacol 54: 880-888.

Axelrad, T. W., Deo, D. D., Ottino, P., Van Kirk, J., Bazan, N. G., Bazan, H. E., and Hunt, J. D. (2004) Platelet-activating factor (PAF) induces activation of matrix metalloproteinase 2 activity and vascular endothelial cell invasion and migration. FASEB J. 18: 568-570.

Baker, D. L., Desiderio, D. M., Miller, D. D., Tolley, B., and Tigyi, G. J. (2001) Direct quantitative analysis of lysophosphatidic acid molecular species by stable isotope dilution electrospray ionization liquid chromatography-mass spectrometry. Anal. Biochem. 2001 292: 287-295.