Elevated β1,4-galactosyltransferase I in highly metastatic human lung cancer cells -: Identification of E1AF as important transcription activator
Elevated β1,4-galactosyltransferase I in highly metastatic human lung cancer cells -: Identification of E1AF as important transcription activator
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DOI：
10.1074/jbc.m413631200
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发表时间：
2005-04-01
影响因子：
4.8
通讯作者：
Gu, JX
中科院分区：
文献类型：
--
作者：
Zhu, XY;Jiang, JH;Gu, JX
与低转移性肺癌PGLH7细胞相比，在高转移性肺癌PGBE1细胞中检测到β 1,4-半乳糖转移酶I （GalT I; EC 2.4.1.38）水平升高。通过小干扰RNA降低GalT I表面表达或通过突变干扰GalT I表面功能抑制细胞对层粘连蛋白的粘附、体外侵袭电位和局灶黏附激酶酪氨酸磷酸化。高转移性细胞中GalT I活性上调的机制尚不清楚。为了研究GalT I的表达调控，我们克隆了GalT I基因转录起始点（-1653至-52）两侧的5'区。同时转染GalT I启动子/荧光素酶报告子和Ets家族蛋白E1AF表达质粒，荧光素酶报告子活性呈剂量依赖性增加。通过缺失和突变分析，我们在GalT I启动子的-205和-200核苷酸之间发现了一个ets结合位点，这对E1AF的反应性至关重要。在PGLH7细胞和COS1细胞中，通过电泳迁移位移实验发现E1AF可以结合并激活GalT I启动子。E1AF对DNA的亲和力较强，导致PGBE1细胞中GalT I的表达升高。稳定转染E1AF表达质粒后，PGLH7细胞中GalT - I表达增加，且稳定转染物迁移速度快于对照细胞。同时，稳定转染的PGLH7细胞表面β 1,4- Gal分支的含量增加。表皮生长因子和显性活性Ras、JNK1和ERK1也可诱导GalT I表达。这些数据表明E1AF在高转移性肺癌细胞中激活人GalT I基因中起重要作用。
The elevated levels of beta 1,4-galactosyltransferase I ( GalT I; EC 2.4.1.38) are detected in highly metastatic lung cancer PGBE1 cells compared with its less metastatic partner PGLH7 cells. Decreasing the GalT I surface expression by small interfering RNA or interfering with the surface of GalT I function by mutation inhibited cell adhesion on laminin, the invasive potential in vitro, and tyrosine phosphorylation of focal adhesion kinase. The mechanism by which GalT I activity is up-regulated in highly metastatic cells remains unclear. To investigate the regulation of GalT I expression, we cloned the 5'-region flanking the transcription start point of the GalT I gene (-1653 to -52). Cotransfection of the GalT I promoter/luciferase reporter and the Ets family protein E1AF expression plasmid increased the luciferase reporter activity in a dose-dependent manner. By deletion and mutation analyses, we identified an Ets-binding site between nucleotides -205 and -200 in the GalT I promoter that was critical for responsiveness to E1AF. It was identified that E1AF could bind to and activate the GalT I promoter by electrophoretic mobility shift assay in PGLH7 cells and COS1 cells. A stronger affinity of E1AF for DNA has contributed to the elevated expression of GalT I in PGBE1 cells. Stable transfection of the E1AF expression plasmid resulted in increased GalT I expression in PGLH7 cells, and stable transfectants migrated faster than control cells. Meanwhile, the content of the beta 1,4- Gal branch on the cell surface was increased in stably transfected PGLH7 cells. GalT I expression can also be induced by epidermal growth factor and dominant active Ras, JNK1, and ERK1. These data suggest an essential role for E1AF in the activation of the human GalT I gene in highly metastatic lung cancer cells.