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 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 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.

ACKNOWLEDGMENTS

We express our appreciation to Douglas Boyd (M. D. Anderson Cancer Center, Department of Cancer Biology, Houston, Tex.) for his critical appraisal of the manuscript. We are very grateful to M. Kramer (Laboratory of Immunopathology, University of Heidelberg, Heidelberg, Germany) for the u-PAR antibody HD 13.1. We thank L. Miles, The Scripps Research Institute, La Jolla, Calif., for the u-PAR hamster cDNA; J. Loftus, Mayo Clinic, Scottsdale, Ariz., for α_v and β₃ expression constructs; and Mark Ginsberg, The Scripps Research Institute, for the α_IIbβ₃-integrin-expressing A5 CHO cell clone. We thank A. Kopp for critical reading of the manuscript.

This work was supported by the Deutsche Forschungsgemeinschaft (DFG Le 889-4/1 to E.L., M.G., and M.S.), Sanders Stiftung (M.G.), and the Medical Faculty of the Technische Universität München (KKF-8756155 to U.R., E.L., and M.S.).