- 學位論文
Src致癌基因對間質性金屬蛋白酶-2的轉錄調控機制探討
Transcriptional regulation of matrix metalloproteinase-2 (MMP2) by Src oncogene
摘要
中文摘要 v-Src 與細胞內相似的原致癌基因c-Src 均屬於非受器型酪胺酸 激酶(non-receptor tyrosine kinases)。酪胺酸激酶的活化已被指出可調 節多種生物功能的反應，包括細胞增生、分化、存活、侵犯、轉移等。 Src 蛋白在許多上皮癌細胞均有較高的活性表現，且這樣的癌症通常 較惡性。Src 酪胺酸激酶也可藉由調升細胞間質蛋白酶 (matrix proteases) 表現以促進癌症的轉移。在實驗中，我們發現相較於 C3H10T1/2 細胞株，v-Src 轉型的IV5 細胞株有較高的間質性金屬蛋 白酶2 (Matrix Metalloproteinase 2；MMP2)表現。promoter assay 的結 果指出，v-Src 藉由活化轉錄作用以提升MMP2 的表現。接著縮短 promoter 片段，與在promoter 特定序列上進行突變進行分析，發現距 離起始區上游-91 到-84 位置的Sp1 結合區，是v-Src 最主要的調節 區。我們的結果也指出v-Src 可藉由調節ERK 訊息傳遞路徑來活化 MMP2。而在EMSA，RT-PCR 與Zymography 的結果顯示，加入Src 抑制劑PP1 與MEK1 抑制劑PD98059 後，能有效降低Sp1 與DNA 間的結合力，MMP2 mRNA 的表現與MMP2 的酵素活性。此外降低 Src 激酶的活性，也會導致ERK1/2 的磷酸化活性下降。綜合以上結 果我們可推論v-Src 藉由ERK/Sp1 訊息調節路徑來提升MMP2 表現。
並列摘要
Abstract The v-Src and its cellular protooncogene homolog c-Src are non-receptor tyrosine kinases. Activation of the Src tyrosine kinase has been implicated in the regulation of many biological responses including cell proliferation, differentiation, survival, invasion and migration. In addition, Src protein expression and activity are elevated in many epithelial cancers, and often associated with malignant potential. The Src tyrosine kinase also contributes to the cancer metastasis through uprgulation of matrix proteases. In this study, we demonstrated that expression of MMP2 was increased in v-Src transformed IV5 cell line compared with the parental C3H10T1/2 cell line. Promoter activity assays showed that v-Src up-regulated MMP2 via transcriptional activation. Deletion and mutation of MMP2 promoter demonstrated that the SP1 site at -91 to -84 base pairs from transcription start site is the major response element regulated by v-Src. Our results also indicated that v-Src activated MMP2 via the extracellular signal-regulated kinase (ERK) pathway. EMSA assay and zymography also showed that Src kinase inhibitor, PP1 and MEK1 inhibitor, PD98059 reduced the Sp1 DNA 4 binding affinity and MMP2 enzymatic activity. Besides, Inhibition of Src kinase activity reduced the ERK1/2 phosphorylation level. Collectively, these results suggest that v-Src upregulates the MMP2 expression via the ERK/SP1-mediated signal pathway.