β(3)A-Integrin Downregulates the Urokinase-Type Plasminogen Activator Receptor (u-PAR) through a PEA3/ets Transcriptional Silencing Element in the u-PAR Promoter

Migration of cells requires interactions with the extracellular matrix mediated, in part, by integrins, proteases, and their receptors. Previous studies have shown that β(3)-integrin interacts with the urokinase-type plasminogen activator receptor (u-PAR) at the cell surface. Since integrins mediate signaling into the cell, the current study was undertaken to determine if in addition β(3)-integrin regulates u-PAR expression. Overexpression of β(3)-integrin in CHO cells, which are avid expressers of the receptor, downregulated u-PAR protein and mRNA expression. The u-PAR promoter (−1,469 bp) that is normally constitutively active in CHO cells was downregulated by induced β(3)-integrin expression. A region between −398 and −197 bp of the u-PAR promoter was critical for β(3)-integrin-induced downregulation of u-PAR promoter activity. Deletion of the PEA3/ets motif at −248 bp substantially impaired the ability of β(3)-integrin to downregulate the u-PAR promoter, suggesting that the PEA3/ets site acts as a silencing element. An expression vector encoding the transcription factor PEA3 caused inhibition of the wild-type but not the PEA3/ets-deleted u-PAR promoter. The PEA3/ets site bound nuclear factors from CHO cells specifically, but binding was enhanced when β(3)-integrin was overexpressed. A PEA3 antibody inhibited DNA-protein complex formation, indicating the presence of PEA3. Downregulation of the u-PAR promoter was achieved by the β(3)A-integrin isoform but not by other β(3)-integrin isoforms and required the cytoplasmic membrane NITY(759) motif. Moreover, overexpression of the short but not the long isoform of the β(3)-integrin adapter protein β(3)-endonexin blocked u-PAR promoter activity through the PEA3/ets binding site. Thus, besides the physical interaction of β(3)-integrin and u-PAR at the cell surface, β(3) signaling is implicated in the regulation of u-PAR gene transcription, suggesting a mutual regulation of adhesion and proteolysis receptors.