Integrin alpha(v)beta(3)/vitronectin interaction affects expression of the urokinase system in human ovarian cancer cells.

The urokinase type plasminogen activator (uPA), together with its receptor uPAR and the plasminogen activator inhibitor type-1 (PAI-1) plays a pivotal role during tumor invasion and metastasis. Integrins, via interaction with the extracellular matrix (ECM), control cell adhesion and motility. The two systems are functionally linked because uPAR and PAI-1 bind to the ECM component vitronectin (VN). Because integrin signaling alters gene expression patterns, we investigated whether the expression levels of uPA, uPAR, and PAI-1 are affected by ECM/integrin interactions. Expression of uPA, uPAR, and PAI-1 was significantly enhanced when human ovarian cancer cells (OV-MZ-6) were cultivated on fibronectin or collagen type IV. In contrast, VN induced down-regulation of uPA and uPAR while increasing PAI-1 by up to 4-fold. VN-dependent decrease of uPA protein was paralleled by a significant reduction of uPA promoter activity that was even more pronounced upon alpha(v)beta(3) overexpression and depended on the presence of intact Rel protein-binding sites. The activity of Rel transcription factors was also significantly reduced upon alpha(v)beta(3)-mediated cell adhesion to VN. The activity of the Rel-unresponsive PAI-1 promoter was up to 5-fold induced as a function of alpha(v)beta(3)/VN interaction. Thus, the balance between available concentrations of uPA, uPAR, PAI-1, and integrins in human ovarian cancer cells might provide a switch within the regulation of their invasive phenotype.

beta(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 beta(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 beta(3)-integrin regulates u-PAR expression. Overexpression of beta(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 beta(3)-integrin expression. A region between -398 and -197 bp of the u-PAR promoter was critical for beta(3)-integrin-induced downregulation of u-PAR promoter activity. Deletion of the PEA3/ets motif at -248 bp substantially impaired the ability of beta(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 beta(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 beta(3)A-integrin isoform but not by other beta(3)-integrin isoforms and required the cytoplasmic membrane NITY(759) motif. Moreover, overexpression of the short but not the long isoform of the beta(3)-integrin adapter protein beta(3)-endonexin blocked u-PAR promoter activity through the PEA3/ets binding site. Thus, besides the physical interaction of beta(3)-integrin and u-PAR at the cell surface, beta(3) signaling is implicated in the regulation of u-PAR gene transcription, suggesting a mutual regulation of adhesion and proteolysis receptors.

The integrin alpha 7 cytoplasmic domain regulates cell migration, lamellipodia formation, and p130CAS/Crk coupling.

The integrin alpha(7)beta(1) is the major laminin-binding integrin in skeletal, heart, and smooth muscle and is a receptor for laminin-1 and -2. It mediates myoblast migration on laminin-1 and -2 and thus might be involved in muscle development and repair. Previously we have shown that alpha(7)B as well as the alpha(7)A and -C splice variants induce cell motility on laminin when transfected into nonmotile HEK293 cells. In this study we have investigated the role of the cytoplasmic domain of alpha(7) in the laminin-induced signal transduction of alpha(7)beta(1) integrin regulating cell adhesion and migration. Deletion of the cytoplasmic domain did not affect assembly of the mutated alpha(7)Deltacyt/beta(1) heterodimer on the cell surface or adhesion of alpha(7)Deltacyt-transfected cells to laminin. The motility of these cells on the laminin-1/E8 fragment, however, was significantly reduced to the level of mock-transfected cells; lamellipodia formation and polarization of the cells were also impaired. Adhesion to the laminin-1/E8 fragment induced tyrosine phosphorylation of the focal adhesion kinase, paxillin, and p130(CAS) as well as the formation of a p130(CAS)-Crk complex in wild-type alpha(7)B-transfected cells. In alpha(7)BDeltacyt cells, however, the extent of p130(CAS) tyrosine formation was reduced and formation of the p130(CAS)-Crk complex was impaired, with unaltered levels of p130(CAS) and Crk protein levels. These findings indicate adhesion-dependent regulation of p130(CAS)/Crk complex formation by the cytoplasmic domain of alpha(7)B integrin after cell adhesion to laminin-1/E8 and imply alpha(7)B-controlled lamellipodia formation and cell migration through the p130(CAS)/Crk protein complex.

The role of extracellular and cytoplasmic splice domains of alpha7-integrin in cell adhesion and migration on laminins.

The major laminin-binding integrin of skeletal, smooth, and heart muscle is alpha7beta1-integrin, which is structurally related to alpha6beta1. It occurs in three cytoplasmic splice variants (alpha7A, -B, and -C) and two extracellular forms (X1 and X2) which are developmentally regulated and differentially expressed in skeletal muscle. Previously, we have shown that ectopic expression of the alpha7beta-integrin splice variant in nonmotile HEK293 cells specifically induced cell locomotion on laminin-1 but not on fibronectin. To investigate the specificity and the mechanism of the alpha7-mediated cell motility, we expressed the three alpha7-chain cytoplasmic splice variants, as well as alpha6A- and alpha6B-integrin subunits in HEK293 cells. Here we show that all three alpha7 splice variants (containing the X2 domain), as well as alpha6A and alpha6B, promote cell attachment and stimulate cell motility on laminin-1 and its E8 fragment. Deletion of the cytoplasmic domain (excluding the GFFKR consensus sequence) from alpha7B resulted in a loss of the motility-enhancing effect. On laminin-2/4 (merosin), the predominant isoform in mature skeletal muscle, only alpha7-expressing cells showed enhanced motility, whereas cells transfected with alpha6A and alpha6B neither attached nor migrated on laminin-2. Adhesion of alpha7-expressing cells to both laminin-1 and laminin-2 was specifically inhibited by a new monoclonal antibody (6A11) specific for alpha7. Expression of the two extracellular splice variants alpha7X1 and alpha7X2 in HEK293 cells conferred different motilities on laminin isoforms: Whereas alpha7X2B promoted cell migration on both laminin-1 and laminin-2, alpha7X1B supported motility only on laminin-2 and not on laminin-1, although both X1 and X2 splice variants revealed similar adhesion rates to laminin-1 and -2. Fluorescence-activated cell sorter analysis revealed a dramatic reduction of surface expression of alpha6-integrin subunits after alpha7A or -B transfection; also, surface expression of alpha1-, alpha3-, and alpha5-integrins was significantly reduced. These results demonstrate selective responses of alpha6- and alpha7-integrins and of the alpha7 splice variants to laminin-1 and -2 and indicate differential roles in laminin-controlled cell adhesion and migration.