基質金屬蛋白酶-7水解澱粉樣前驅蛋白質之衍生片段參與神經母細胞腫瘤細胞株癌化過程之研究

Abstract

基質金屬蛋白酶-7(MMP-7)是基質金屬蛋白酶家族中最小的一個成員。MMP-7可以切割許多的細胞外基質(extracellular matrix, ECM)，進一步調控在發育過程中細胞的遷移或是受傷組織的修復與重建.除此之外，MMP-7也可切割非細胞外基質的蛋白質而參與許多生理以及致病的過程。在本實驗室過去的研究中發現MMP-7參與了神經母細胞腫瘤細胞株SH-SY5Y的分化以及神經保護的過程。而本篇論文則著重在研究MMP-7經由切割澱粉樣前驅蛋白質(amyloid precursor protein , APP)而進一步參與神經母細胞腫瘤形成的過程中所扮演的角色。APP屬於第一型跨膜蛋白質，APP可經由不同的轉譯後修飾蛋白質水解而參與在神經保護或是神經退化過程。在此，我們推斷MMP-7可以切割APP而其所衍生的片斷可能參與在神經母細胞腫瘤的癌化過程中。 經由西方墨點法分析發現在APP和MMP-7共同轉染的細胞中APP可經由MMP-7多重切割成大小不同的C端片段。利用共軛焦顯微鏡觀察發現經由MMP-7切割之後的C端片段分布在核中。依據分子量和MMP-7的切點，構築APP的截短型:CI和CII，將兩片段於SH SY5Y細胞中表達，經由西方墨點法分析顯示CI和CII分子量大小與APP和MMP-7共同轉染的細胞中所發現的APP衍生片段一致。進一步將細胞的核質分離進行西方墨點法分析，發現在APP和MMP-7共同轉染的細胞的核的部分中有和CII大小一致的片段。而藉由共軛焦顯微鏡的觀察也發現CII片段分佈在核中。而在MMP-7啟動子的報導基因實驗中發現APP在經由MMP-7切割之後所產生的衍生片段會參與調控MMP-7的表現使得MMP-7的表現量增加。這也暗喻了其中有自分泌環(autocrine loop)調控著MMP-7的表現。 綜合以上的結果，我們知道MMP-7可以切割APP產生並且顯露出和致癌有關的片段蛋白質，這些片段蛋白質可以改變細胞的生長行為且很有可能參與在神經母細胞瘤的形成過程中。

Matrix metalloproteinases-7 (MMP-7) is one of the smallest MMPs. MMP-7 is able to degrade the extracellular matrix (ECM) and further regulates cell migration and tissue repair. In addition, MMP-7 processes non-ECM protein and plays important roles in regulating physiological and pathological processes. In our lab, it has previous found that MMP-7 participates in the differentiation and neuroprotection of neuroblatoma cell line SH-SY5Y. Here, we focused on studying the roles of MMP-7 processing amyloid precursor protein (APP) involved in the neuroblastoma formation. APP is a type I transmembrane protein. APP can undergo post-translational proteolytic processing and lead to neuroprotection and neurodegeneration. Here, we hypothesize that MMP-7 is a putative proteolytic processing enzyme of APP and the MMP-7-mediated proteolytic fragments of APP can attribute to the tumorigenicity of neuron crest derived neuronal cells, SH-SY5Y based on the observation of the APP and MMP-7 co-expressing SH-SY5Y cells grow in multiple cell layers in anchorage-independent growth manner. APP can be cleaved by MMP-7 in a multiple cleavage manner based on the existing putative cleavage sites in APP. The APP-derived proteolytic fragments corresponding to the C-terminal fragments are observed in the APP and MMP-7 co-transfected SH-SY5Y cells. The immunofluorescence staining followed by confocal microscopic analysis show that the MMP-7-mediated proteolytic processing of APP C-terminal fragments can translocate into the nucleus. The truncated fragments (CI and CII) of APP corresponding to these MMP-7 mediated proteolytic fragments were constructed and expressed in the SH-SH5Y cells. Based on the Western blot analysis, CI and CII were consistent with the proteolytic fragments generated by the APP/MMP-7 co-transfected SH-SY5Y cells. The nuclear fractionation experiments were performed followed by Western blot analysis; the proteolytic APP C-terminal fragments generated in APP/MMP-7 co-expressing SH-SY5Y cells were similar to the CII fragment. Moreover, the immunofluorescence staining and the confocal microscopic analysis also showed that most of the CII fragments were located in the nucleus. Furthermore, according to the results of MMP-7 promoter activity analysis experiment, the MMP-7 expression is up-regulated in the APP/MMP-7 co-expressing SH-SY5Y cells. This implicates that the possible autocrine loop in modulating MMP-7 expression is associated with this MMP-7-mediated proteolytic processing APP. The above results suggested that MMP-7 is involved in the processing of APP and generate the cryptic oncogenic fragments of APP that changed the cell behaviors and is potentially involved in the neuroblastoma formation.