Loss of activator protein-2α 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. 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.