EB病毒第一型後期膜蛋白對第三型金屬蛋白酶抑制劑抑制作用之分子機制

Molecular mechanism of EBV latent membrane protein-1 induced inhibition of tissue inhibitor of metalloproteinase-3

張淑慧(Shu-Hui Chang)

指導教授 ： 洪文俊

高雄醫學大學/健康科學院/公共衛生學研究所/碩士(2006年)

https://doi.org/10.6832/KMU.2006.00140

摘要

EB 病毒屬於一種γ型疱疹病毒，大多數的成年人都被感染過。EB病毒感染與許多惡性腫瘤有關，包括：Burkitt’s lymphoma 、Hodgkin’s disease 及鼻咽癌(Nasopharyngeal Carcinoma )有關。 EB病毒後期膜蛋白LMP-1 (Latent membrane protein 1) 被認為是主要造成鼻咽癌的致癌蛋白。研究顯示在EB 病毒感染的C33A人類上皮細胞株中發現，當LMP大量表現時，會誘發間質金屬蛋白酶MMP-9的表現。更多相關報導指出，許多鼻咽癌病人有淋巴結轉移的現象。
在生理狀況下，組織中間質金屬蛋白酶( MMP )的活性是由體內天然的組織間質金屬蛋白酶抑制劑TIMP來調控。目前為止，已經有四種TIMP被發現，分別為TIMP-1、TIMP-2、TIMP-3、TIMP-4 等。在生理功能上，TIMP-3較其他TIMP有不同的功能。首先，它可以抑制ADAM-17 (a disintergrin and metalloproteinases) 、ADAMT-4與ADAMT-5 (ADAM with thrombospondin domain)，其中ADAM-17又稱為TACE (TNF-αconverting enzyme )可以抑制TNF-α由細胞釋出，調控細胞的發炎反應。第二，TIMP-3具有誘使細胞走向凋亡(proapoptosis)的能力。第三，TIMP-3具有對抗血管新生之功能進而抑制癌細胞的轉移 。第四，在許多的腫瘤中發現它們的TIMP-3 promoter有Hypermethylation的現象，導致TIMP-3的表現量下降，顯示出TIMP-3抑制腫瘤生長作用的重要性。
在本實驗中，主要探討EB病毒的後期膜蛋白LMP-1對於TW04細胞株中TIMP-3的分子調控機制，我們發現藉由誘導大量的LMP-1表現會抑制 TIMP-3的表現，接著利用Promoter activity assay的方法進一步尋找 LMP-1調控 TIMP-3的作用區域，結果發現介於TIMP-3 promoter 上游的第44個base pair處至第28個base 之間包含了AML-1a、Sp1與c-Myb等轉錄因子結合位，可能是其調控區域。並藉由加入不同的protein kinase inhibitors，來找出其訊息傳導途徑。結果發現，加入p38激酶抑制(SB203580)後，TIMP-3 mRNA的表現有回升的現象。在探討TIMP-3 對於細胞侵犯能力上，我們發現誘導細胞LMP-1表現會明顯增加細胞侵犯能力，而同時表現TIMP-3 的TW04細胞株，其細胞移行與侵犯的能力明顯的受到抑制。
根據以上結果推測LMP-1可能是經由p38 路徑而抑制TIMP-3的表現，進而達到增強細胞轉移與侵犯的能力。

關鍵字

ＥＢ病毒後期膜蛋白 ； 金屬蛋白酶抑制劑

並列摘要

Epstein-Barr virus is a prototype gamma herpes virus which is widely spreaded infection in the adults. EBV has been implicated in the pathogenesis of several human malignancies such as Burkitt’s lymphoma, Hodgkin’s disease and nasopharyngeal carcinoma (NPC). Latent membrane protein 1 (LMP-1) is the oncoprotein of EBV associated NPC. Some evidences showed that LMP-1 enhanced the MMP-9 expression in the EBV-infected human epithelial cell lines C33A. In addition, numerous studies indicated that many NPC patients have lymph-node metastasis.
In the physical condition, MMPs are regulated by natural inhibitor called tissue inhibitor of metalloproteinases (TIMPs). Until now, there are four kinds of TIMPs: TIMP-1, TIMP-2, TIMP-3 and TIMP-4. The biological function of TIMP-3 is different from the others. First, it could inhibit ADAM-17 (a disintergrin and metaloproteinases) , ADAMT-4 and ADAMT-5 (ADAM with thrombospondin domain). TIMP-3 could inhibit TNF-α release from the cells and regulate cellular inflammation. Second, TIMP-3 could promote cancer cells to undergo apoptosis. Third, TIMP-3 could inhibit the metastasis and angiogenesis of cancer cells. Fourth, TIMP-3 played a key role in the inhibition of tumor growth. Some evidences showed that the TIMP-3 promoter was hypermethylated in the tumor, and its expression was significantly down-regulated in tumors.The goal of our study is to elucidate the molecular mechanism by which LMP-1 regulates TIMP-3 in the NPC cell line TW04. Our results showed that TIMP-3 was decreased in LMP-1-expressing TW04 cells. Promoter activity assays indicated that the -44 / +28 region of TIMP-3 promoter was regulated by LMP-1. By using different kinds of protein kinase inhibitors, it was showed that the TIMP-3 mRNA level was restored after treatment with p38 kinase inhibitor SB203580. We found that the LMP-1 induced TIMP-3 inhibition could increase the migration and invasion of TW04 cells. While co-expression with TIMP-3 attenuated LMP-1-evoked the migration and invasion. According to our results, we suggest that LMP-1 might increase the cell migration and invasion through p38 mediated down-regulation of TIMP-3.

並列關鍵字

EBV latent membrane protein ； issue inhibitor of metalloproteinase

參考文獻

Reference

1. Raab-Traub, N., Flynn, K. ,Klein, Pizza G., De Vinci C., Occhiuzzi L.(1987) EBV associated malignancies. J Exp Pathol 3,449-56.

2. Weiss, L. M., Chen, Y. Y., Liu, X. F., and Shibata, D. (1991). Epstein-Barr virus and Hodgkin's disease. A correlative in situ hybridization and polymerase chain reaction study. Am J Pathol 139, 1259-1265.

3. Raab-Traub, N., Flynn, K., Pearson, G., Huang, A., Levine, P., Lanier, A., and Pagano, J. (1987). The differentiated form of nasopharyngeal carcinoma contains Epstein-Barr virus DNA. Int J Cancer 39, 25-29.

4.Nakamura S, Ueki T, Yao T, Ueyama T, Tauneyoshi M.(1994) Epstein-Barr virus in gastric carcinoma with lymphoid stroma. Special reference to its detection by the polymerase chain reaction and in situ hybridization in 99 tumors, including a morphologic analysis. Cancer 333,724-6.