Loss of activator protein-2α results in overexpression of protease-activated receptor-1 and correlates with the malignant phenotype of human melanoma
Loss of activator protein-2α results in overexpression of protease-activated receptor-1 and correlates with the malignant phenotype of human melanoma
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DOI：
10.1074/jbc.m309159200
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发表时间：
2003-11-21
影响因子：
4.8
通讯作者：
Bar-Eli, M
中科院分区：
文献类型：
--
作者：
Tellez, C;McCarty, M;Bar-Eli, M
越来越多的证据表明，蛋白酶激活受体- 1 （PAR- 1）在人类黑色素瘤的侵袭和转移中起着重要作用。在这里，我们证明了人类黑色素瘤细胞的转移潜能与PAR- 1的过表达相关。我们还提供证据表明，在人类黑色素瘤细胞中，激活蛋白- 2 α (AP- 2)的表达与PAR- 1的表达之间存在负相关。AP- 2在AP- 2阴性的WM266- 4黑色素瘤细胞中重新表达，导致PAR- 1 mRNA和蛋白表达降低。PAR- 1基因的启动子包含多个公认的转录因子AP- 2和特异性蛋白1 （Sp1）的元件。对PAR- 1启动子区bp -365 ~ -329（复合体1）和bp - 206 ~ - 180（复合体2）的染色质免疫沉淀分析表明，Sp1在转移细胞中主要与PAR- 1启动子结合，而AP- 2在非转移细胞中与PAR- 1启动子结合。复合物1的体外分析表明，AP- 2和Sp1以互斥的方式结合到该区域。PAR- 1启动子荧光素酶全长和进行性缺失转染实验表明，与低转移性和非转移性黑色素瘤细胞相比，转移性黑色素瘤细胞具有更高的PAR- 1启动子活性。我们的数据表明，外源AP- 2表达降低了启动子活性，而Sp1的瞬时表达进一步增加了报告基因的表达。对PAR- 1荧光素酶结构中的复合物1的突变分析进一步表明，PAR- 1的调控是通过与AP- 2和Sp1的相互作用介导的。我们的数据表明，转移细胞中AP- 2的缺失改变了AP- 2/ Sp1的比例，导致PAR- 1过表达。综上所述，我们的研究结果提供了强有力的证据，证明AP- 2的缺失与PAR- 1的过表达相关，而PAR- 1反过来又有助于人类黑色素瘤恶性表型的获得。
Increasing evidence implicates the protease- activated receptor- 1 ( PAR- 1) as a contributor to tumor invasion and metastasis of human melanoma. Here we demonstrate that the metastatic potential of human melanoma cells correlates with overexpression of PAR- 1. We also provide evidence that an inverse correlation exists between the expression of activator protein- 2alpha ( AP- 2) and the expression of PAR- 1 in human melanoma cells. Re-expression of AP- 2 in WM266- 4 melanoma cells, which are AP- 2- negative, resulted in decreased mRNA and protein expression of PAR- 1. The promoter of the PAR- 1 gene contains multiple putative consensus elements for the transcription factors AP- 2 and specificity protein 1 ( Sp1). Chromatin immunoprecipitation analysis of the PAR- 1 promoter regions bp -365 to -329 ( complex 1) and bp - 206 to - 180 ( complex 2) demonstrated that Sp1 was predominantly bound to the PAR- 1 promoter in metastatic cells, whereas AP- 2 was bound to the PAR- 1 promoter in nonmetastatic cells. In vitro analysis of complex 1 demonstrated that AP- 2 and Sp1 bound to this region in a mutually exclusive manner. Transfection experiments with full- length and progressive deletions of the PAR- 1 promoter luciferase constructs demonstrated that metastatic melanoma cells had increased PAR- 1 promoter activity compared with low and nonmetastatic melanoma cells. Our data show that exogenous AP- 2 expression decreased promoter activity, whereas transient expression of Sp1 further increased expression of the reporter gene. Mutational analysis of complex 1 within PAR- 1 luciferase constructs further demonstrated that the regulation of PAR- 1 was mediated through interactions with AP- 2 and Sp1. Our data suggest that loss of AP- 2 in metastatic cells alters the AP- 2/ Sp1 ratio, resulting in overexpression of PAR- 1. Taken together, our results provide strong evidence that loss of AP- 2 correlates with overexpression of PAR- 1, which in turn contributes to the acquisition of the malignant phenotype of human melanoma.