β3A-Integrin Downregulates the Urokinase-Type Plasminogen Activator Receptor (u-PAR) through aPEA3/ets Transcriptional Silencing Element in the u-PAR Promoter

Abstract

Migration of cells requires interactions with the extracellular matrix mediated, in part, by integrins, proteases, and their receptors. Previous studies have shown that β₃-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 β₃-integrin regulates u-PAR expression. Overexpression of β₃-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 β₃-integrin expression. A region between −398 and −197 bp of the u-PAR promoter was critical for β₃-integrin-induced downregulation of u-PAR promoter activity. Deletion of the PEA3/ets motif at −248 bp substantially impaired the ability of β₃-integrin to downregulate the u-PAR promoter, suggesting that thePEA3/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 β₃-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 β₃A-integrin isoform but not by other β₃-integrin isoforms and required the cytoplasmic membrane NITY⁷⁵⁹ motif. Moreover, overexpression of the short but not the long isoform of the β₃-integrin adapter protein β₃-endonexin blocked u-PAR promoter activity through the PEA3/etsbinding site. Thus, besides the physical interaction of β₃-integrin and u-PAR at the cell surface, β₃ signaling is implicated in the regulation of u-PAR gene transcription, suggesting a mutual regulation of adhesion and proteolysis receptors.