Loss of activator protein-2alpha results in overexpression of protease-activated receptor-1 and correlates with the malignant phenotype of human melanoma.
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.
Tellez
Carmen
C
Department of Cancer Biology 173, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
McCarty
Marya
M
Ruiz
Maribelis
M
Bar-Eli
Menashe
M
D001665:Binding Sites; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000172:deficiency* / Q000378:metabolism / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008545:Melanoma / Q000209:etiology* / Q000473:pathology; D010641:Phenotype; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D044463:Receptor, PAR-1 / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology; D016329:Sp1 Transcription Factor / Q000378:metabolism; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000172:deficiency* / Q000378:metabolism / Q000502:physiology
D004268:DNA-Binding Proteins; D044463:Receptor, PAR-1; D016329:Sp1 Transcription Factor; D050656:Transcription Factor AP-2; D014157:Transcription Factors