During tumor metastasis, a fine-tuned balance between the formation and loosening of adhesive cell contacts has to occur, a process based on the regulated expression of integrins. Human ovarian OV-MZ-6 cancer cells express the integrin alpha(v)beta3, which associates with vitronectin (VN) and correlates with ovarian cancer progression. Adhesion and spreading of OV-MZ-6 cells on VN was accompanied by the formation of focal adhesion contacts and the recruitment of activated tyrosine-phosphorylated focal adhesion kinase. Cultivation of OV-MZ-6 cells on VN resulted in a significantly induced cell proliferation. This VN effect could be mimicked by cultivating cells on the immobilized alpha(v)beta3 directed peptide cyclo-Arg-Gly-Asp-D-Phe-Val (cRGDfV). VN-dependent OV-MZ-6 cell adhesion and proliferation was significantly enhanced by overexpression of alpha(v)beta3 and was accompanied by rapid and transient tyrosine-phosphorylation of p44(erk-1)/p42(erk-2) mitogen-activated protein kinase. Moreover, overexpression of alpha(v)beta3 and OV-MZ-6 cell attachment to VN increased cell motility up to 5-fold accompanied by prominent changes in cytoskeletal organization and cell morphology. Upon alpha(v)beta3/VN interaction, by cDNA expression microarray analysis we identified altered mRNA levels of c-myc, epidermal growth factor receptor (EGF-R), transcription factor Fra-1, prothymosin-alpha (PTMA), integrin-linked kinase (ILK), and the cell adhesion molecule SQM-1, candidates which are possibly involved in changes of the adhesive, migratory, and proliferative phenotype of 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 ␣ v ␤ 3 overexpression and depended on the presence of intact Rel protein-binding sites. The activity of Rel transcription factors was also significantly reduced upon ␣ v ␤ 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 ␣ v ␤ 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.

Migration of cells requires interactions with the extracellular matrix mediated, in part, by integrins, proteases, and their receptors. Previous studies have shown that ␤ 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 ␤ 3 -integrin regulates u-PAR expression. Overexpression of ␤ 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 ␤ 3 -integrin expression. A region between ؊398 and ؊197 bp of the u-PAR promoter was critical for ␤ 3 -integrin-induced downregulation of u-PAR promoter activity. Deletion of the PEA3/ets motif at ؊248 bp substantially impaired the ability of ␤ 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 ␤ 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 ␤ 3 A-integrin isoform but not by other ␤ 3 -integrin isoforms and required the cytoplasmic membrane NITY 759 motif. Moreover, overexpression of the short but not the long isoform of the ␤ 3 -integrin adapter protein ␤ 3 -endonexin blocked u-PAR promoter activity through the PEA3/ets binding site. Thus, besides the physical interaction of ␤ 3 -integrin and u-PAR at the cell surface, ␤ 3 signaling is implicated in the regulation of u-PAR gene transcription, suggesting a mutual regulation of adhesion and proteolysis receptors.

The serine protease urokinase-type plasminogen activator (uPA), its inhibitor PAI-1, and its cellular receptor uPA-R (CD87) are of crucial importance during cellular invasion and migration, required for a variety of physio- and pathophysiological processes. It has become increasingly evident in recent years that the uPA/uPA-R-system has far more functional properties than plasminogen activation alone. This is reflected by its involvement in cellular events such as proliferation, adhesion, migration, and chemotaxis. Since uPA-R lacks a transmembrane domain and thus on its own is not capable of transmitting signals into cells, association and functional cooperation with other signaling molecules/receptors is needed. In this respect, one group of adhesion and signaling receptors, the integrins, have been identified which constitute, together with the uPA/uPA-R-system, an interdependent biological network by which the uPA/uPA-R-system broadly affects integrin functions and vice versa. Moreover, there is a growing body of evidence that cellular uPA, uPA-R, and PAI-1 expression is under control of specific ECM/integrin interactions and also that integrins are regulated by components of the uPA/uPA-R-system. By this multifaceted crosstalk, cells may modulate their proteolytic, adhesive, and migratory activities and monitor ECM integrity in their microenvironment.

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