p16 蛋白質抑制間質金屬蛋白酶-2的轉錄調節機制探討

Transcriptional inhibition of matrix metalloproteinase-2(MMP-2) by p16

王啟鴻(Chi-Hung Wang)

指導教授 ： 洪文俊 ； 黃友利

高雄醫學大學/健康科學院/公共衛生學研究所/碩士(2005年)

摘要

p16蛋白質是一個重要的細胞週期負調節者，在G1期p16會抑制Cyclin D-CDK4/6 complex的活性，使得細胞週期停止在G1期。P16除了在細胞週期中扮演一個負調節者的角色外，在過去的報導裡也被指出可抑制腫瘤細胞的侵犯能力、轉移能力。在本實驗中，我們使用的是A549 (human lung adenocarcinoma cell) 其為一株p16 deletion的細胞株，藉由transfect p16 expression vector，在promoter assay中我們發現p16在A549細胞中會抑制MMP-2的表現。接著我們利用不同片段的MMP-2 promoter，以及site-directed mutation來對於promoter上的特定序列進行分析。

實驗結果我們發現p16對於MMP-2的抑制作用主要是藉由Sp1這一個transcription factor，之後我們利用DNA affinity precipitation assay (DAPA)以及Chromatin immunoprecipitation assay (ChIP)來探討Sp1對於DNA的結合能力是否會被p16所調控，結果顯示Sp1對於DNA的結合能力會被p16所調降。此外我們也發現了Sp1蛋白質其磷酸化的程度會被調降，因此我們推論Sp1對於DNA的結合能力下降，是因為其磷酸化的程度下降所導致的。為了探討影響Sp1蛋白質其磷酸化程度是被何者所調控，我們探討了Erk、Akt、Cyclin A/CDK2等kinase。根據我們的實驗結果，我們可推論p16之所以抑制MMP-2的表現是藉由降低Sp1與Cyclin A/CDK2 complex的結合能力，而使得Sp1磷酸化的程度下降，進而導致了Sp1與DNA的結合能力下降，故抑制了MMP-2的表現。

關鍵字

間質金屬蛋白? ； 轉錄調節

並列摘要

p16 is an important negative regulator of the cell cycle. At G1 phase, it reduces the Cyclin D-CDK4/6 complex activity. As a result, cells are arrested at G1 phase. Besides the negative regulatory role in cell cycle, p16 has been reported to inhibit the invasion and metastasis ability of tumor cell. In our study, we use A549 (human lung adenocarcinoma cell), which is a p16 gene deletion cell line to address the role of p16 in the inhibition of tumor invasion. In the promoter activity assay, we found that p16 can inhibit the expression of MMP-2. Then we use the different MMP-2 promoter deletion and site-directed mutation mutants to analysis the target sequence in MMP-2 promoter. We find that the inhibition of MMP-2 caused by p16 is through the transcription factor, Sp1. In order to check if the DNA binding affinity of Sp1 is regulated by p16, we use DNA affinity precipitation assay (DAPA) and Chromatin immunoprecipitation assay (ChIP) to solve this question. Our data show that the DNA binding affinity of Sp1 is inhibited by p16. Besides we find the phosphorylation level of Sp1 is reduced. We next study the ERK, Akt, and cyclin A/CDK2 kinases which have been reported to phosphorylate Sp1. Our findings document that p16 inhibits Sp1-cyclin A binding affinity and reduces Sp1 phosphorylation which causes reduction of Sp1-DNA binding affinity and down-regulation of MMP-2 transcription.

並列關鍵字

MMP-2 ； p16

參考文獻

Reference List

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