Tellez Carmen, McCarty Marya, Ruiz Maribelis, Bar-Eli Menashe
Department of Cancer Biology 173, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
J Biol Chem. 2003 Nov 21;278(47):46632-42. doi: 10.1074/jbc.M309159200. Epub 2003 Sep 15.
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. Reexpression 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.
越来越多的证据表明蛋白酶激活受体-1（PAR-1）在人类黑色素瘤的肿瘤侵袭和转移中起作用。在此我们证明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-2α（AP-2）的表达与PAR-1的表达呈负相关。在AP-2阴性的WM266-4黑色素瘤细胞中重新表达AP-2，导致PAR-1的mRNA和蛋白表达降低。PAR-1基因的启动子包含多个转录因子AP-2和特异性蛋白1（Sp1）的推定共有元件。对PAR-1启动子区域bp -365至-329（复合物1）和bp -206至-180（复合物2）进行染色质免疫沉淀分析表明，Sp1在转移性细胞中主要与PAR-1启动子结合，而AP-2在非转移性细胞中与PAR-1启动子结合。对复合物1的体外分析表明，AP-2和Sp1以互斥的方式结合到该区域。用PAR-1启动子荧光素酶构建体的全长和逐步缺失进行转染实验表明，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，外源性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荧光素酶构建体内复合物1的突变分析进一步证明，PAR-1的调节是通过与AP-2和Sp1的相互作用介导的。我们的数据表明，转移性细胞中AP-2的缺失改变了AP-2/Sp1比例，导致PAR-1过表达。综上所述，我们的结果提供了强有力的证据，表明AP-2的缺失与PAR-1的过表达相关，这反过来又有助于人类黑色素瘤恶性表型的获得。
