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J Biol Chem. 2003 Nov 21;278(47):46632-42. Epub 2003 Sep 15.

Loss of activator protein-2alpha results in overexpression of protease-activated receptor-1 and correlates with the malignant phenotype of human melanoma.

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1
Department of Cancer Biology 173, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.

Abstract

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.

PMID:
12975361
DOI:
10.1074/jbc.M309159200
[Indexed for MEDLINE]
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