INTRODUCTION

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Invasion and metastasis of tumor cells require a complex regulation of different cell surface-associated proteins (4, 53, 55) that facilitate proteolysis and adhesion of cells to the basement membrane and the extracellular matrix (ECM). The invasive process starts with the attachment of cells to the ECM, followed by degradation of various ECM components through proteases, subsequent cell detachment, and migration (5). One important protease involved in these processes is the serine protease urokinase-type plasminogen activator (urokinase) which is bound to a specific receptor (u-PAR). Urokinase converts plasminogen to plasmin, a serine protease with broad substrate specificity for several components of the ECM, including vitronectin, laminin, and fibronectin (5, 27, 43). Together, these adhesive and proteolytic functions of tumor cells facilitate their migration through the basement membrane and the ECM.

Urokinase binds with high affinity to its heavily glycosylated receptor, u-PAR (33, 35), which is composed of three similar protein domains and is linked through a glycosylphosphatidylinositol anchor to the plasma membrane. The amino-terminal domain I of u-PAR interacts with urokinase while the other two domains bind vitronectin (43), a major component of the ECM and an integrin-binding ligand. Binding of urokinase to u-PAR increases the rate of plasmin formation at the plasma membrane (13) and focuses the proteolytic activity onto the leading edge of tumor cells (5). Besides its role in proteolysis, u-PAR is a multifunctional receptor that is involved in chemotaxis, angiogenesis, signal transduction, migration, and adhesion of cells (5, 36).

Expression of u-PAR is controlled mainly at the transcriptional level (26, 29, 48), but posttranscriptional regulation (46) and recycling of u-PAR to the cell membrane (10) represent additional levels of regulation. Transcription of the u-PAR gene gives rise to a 1.4-kb mRNA or an alternatively spliced variant that lacks the carboxy-terminal membrane attachment peptide sequence (37). The importance of transcriptional regulation of the u-PAR promoter by activator protein 1 (AP-1), AP-2, and Sp-1 transcription factors and their corresponding binding sites in the 5'-flanking site of the u-PAR gene have been recently defined (2, 26, 48). However, the aforementioned transcription factor binding sites mediate constitutive and inducible activation of the u-PAR gene, and a negative regulatory element has so far not been characterized. In consideration of the importance of u-PAR for several biological processes, the existence of such a site could be hypothesized.

Recently, proteolysis and adhesion were functionally linked after it had been found that u-PAR binds to vitronectin and is associated with different members of the integrin family (7, 36, 52). Integrins are cell surface heterodimeric transmembrane glycoproteins consisting of  and  subunits that mediate cell-cell and cell-ECM interactions. Each subunit encompasses a large extracellular domain, a membrane-spanning domain, and a short cytoplasmic tail. Most integrins interact with components of the ECM (e.g., vitronectin, fibronectin, and collagen) or blood (e.g., fibrinogen). These integrin ligands cross-link or cluster integrins on the cell surface by binding to adjacent integrin molecules, leading to the formation of focal adhesion contacts, thus activating intracellular signaling pathways ("outside-in signaling") (9). Integrins are implicated in various biological processes including angiogenesis, wound healing, tumor cell invasion, and metastasis (30, 53), but the specificity and mechanisms by which all these functions are regulated are not fully understood. However, there is increasing evidence that the cytoplasmic domain of the integrin  subunits plays a major regulatory role in these processes (24, 40, 44). u-PAR physically interacts on leukocytes with ₂-integrin as shown by colocalization and immunoprecipitation studies (6) and on fibrosarcoma cells with ₁- and ₃-integrins (58). The direct interaction between u-PAR and integrins plays a role in the binding affinity of integrins toward their ligands and might be important for urokinase-mediated signal transduction (47). Although these studies demonstrated that u-PAR physically interacts with -integrins, they do not address the consequence of ₃-integrin expression for u-PAR regulation. Taking into consideration that u-PAR expression is regulated mainly at the transcriptional level (26) and that integrins regulate gene expression via outside-in signaling (39), we undertook a study with the following objectives: (i) to determine if ₃-integrin regulates u-PAR gene activity and (ii) to elucidate the molecular mechanisms by which this occurs. We show for the first time that overexpression of ₃A-integrin downregulates u-PAR transcription via a PEA3/ets binding site in the u-PAR promoter, involving the ₃A-integrin cytoplasmic domain and the adapter protein ₃-endonexin short.

(This study was performed in partial fulfillment of the Ph.D. thesis of S. Hapke.)

	MATERIALS AND METHODS

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Cell culture. CHO cells were obtained from the American Type Culture Collection (CRL 9096). The stable CHO cell clone A5 expresses a high level of _IIb3-integrin (15) and was generously supplied by Mark Ginsberg, The Scripps Research Institute, La Jolla, Calif. The cells were cultivated in MEM alpha medium supplemented with L-glutamine and 10% fetal calf serum (FCS; all from GIBCO, Life Technologies, Grand Island, N.Y.).

Vectors. The _v- and ₃-integrin expression vector constructs (28), a friendly gift from J. Loftus, Mayo Clinic, Scottsdale, Ariz., were cloned into the SalI site of the pBabe Puro plasmid (31). To study the role of the three known ₃-integrin cytoplasmic isoforms on the u-PAR promoter, we used single-chain chimeric receptors which bear CH2 and CH3 portions of human immunoglobulin G1 (IgG1) at the cell surface, the transmembrane domain of CD7, and the full-length cytoplasmic domains of ₃-A-, ₃-B-, and ₃-C-integrins (see Fig. 7A). All constructs were based on previously described chimeric receptors and are in the context of the P5C7 vector, cloned into the MluI/NotI site (23). ₁-integrin NITY and ₃-integrin NPKY are a swap of the cytoplasmic domain of ₁-integrin with the cytoplasmic domain of ₃ and vice versa (see Fig. 7A). A full-length polyomavirus enhancer activator 3 (PEA3) cDNA (56) was inserted between the HindIII and BamHI cloning sites of the cytomegalovirus promoter-driven pcDNA3.1 expression vector (Invitrogen, Leek, The Netherlands).

Confocal laser scanning microscopy. For immunofluorescence, 50,000 untransfected CHO cells or stable ₃-expressing A5 cells were seeded into each well of a four-well chamber slide (Nunc Lab-Tek, Naperville, Ill.). CHO cells were transfected with ₃-integrin or the vector (pBabe Puro) control (31) with TransFast transfection reagent (Promega). At 48 h posttransfection, viable cells were collected, washed briefly in phosphate-buffered saline (PBS), and incubated with a mouse polyclonal antibody against u-PAR (0.75 µg/ml; HD 13.1; a kind gift of M. Kramer, University of Heidelberg, Heidelberg, Germany) or monoclonal antibody against ₃-integrin (0.1 µg/ml; CD61-UNLB; Southern Biotechnology Associates, Birmingham, United Kingdom) in PBS at 4°C for 2 h. After three washes in PBS-2% bovine serum albumin (5 min each), the cells were incubated with 50 ng of Alexa 488-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, Oreg.) secondary antibody/ml for 1 h at room temperature in PBS. Cells were washed three times with PBS (10 min each), and coverslips were examined with a Zeiss Axiovert 35 microscope (Zeiss, Heidelberg, Germany) attached to a laser scanning detection unit (Leica, Bensheim, Germany). For ₃-integrin and u-PAR double staining (see Fig. 2), a ₃-integrin antibody that was coupled with Alexa 488 was used and u-PAR was detected with HD 13.1 followed by the secondary antibody Alexa 568 (goat anti-mouse IgG; Molecular Probes). The monoclonal antibody LM609 against _vv-integrin was supplied by Chemicon (Hofheim, Germany).

Northern blotting. The levels of steady-state u-PAR transcripts were determined by Northern blot analysis (42). Total RNA was extracted from cells with TRIzol (GIBCO), and 15 µg of RNA was electrophoresed in a 1.5% agarose formaldehyde gel and transferred to a positively charged nylon membrane by capillary action, using 10× SSC (1× SSC is 0.15 M NaCl plus 15 mM sodium citrate, pH 7.4). The Northern blot was probed at 65°C with a randomly primed, ³²P-labeled cDNA specific for u-PAR mRNA (14) and subsequently washed at 62°C, using 2× SSC in the presence of 1.0% sodium dodecyl sulfate. Loading efficiencies were checked by reprobing the blot with a radioactive cDNA which hybridizes with the 18S rRNA.

Western blot analysis. Cells were lysed in a buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 10 µg of aprotinin/ml, and 1 mM phenylmethylsulfonyl fluoride; cleared by centrifugation; and electrophoresed by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis under reducing conditions (38). The resolved proteins were transferred to a nitrocellulose membrane (BA-S85; Schleicher & Schuell, Dassel, Germany); the filter was subjected for 1 h to a buffer containing 150 mM NaCl, 5 mM EDTA, 50 mM Tris HCl, 0.25% (wt/vol) gelatin, and 0.5% (vol/vol) Triton X-100; and the filter was incubated sequentially with a rabbit polyclonal antibody directed against u-PAR (40 ng/ml; American Diagnostica no. 3931; Greenwich, Conn.), followed by a mouse horseradish peroxidase-conjugated anti-rabbit IgG. Reactive proteins were visualized by ECL as directed by the manufacturer (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom).

Transfections. Reporter plasmids were transfected with SuperFect transfection reagent (Qiagen, Hilden, Germany) with 3 × 10⁵ CHO cells seeded overnight into six-well plates. The transfection efficiency for CHO cells was about 75% as determined by -galactosidase staining. For the CAT assays, all transient transfections were performed in the presence of 1 µg of u-PAR-CAT reporter constructs, 1 µg of a luciferase expression vector, and, where indicated, 3 µg of an expression plasmid coding for ₃-integrin or equimolar amounts of the control vector. After 3 h, the cells were rinsed twice with PBS, changed to 10% FCS-containing medium, and cultured for another 45 h. The cells were harvested and then lysed by repeated freeze-thaw cycles in 0.25 M Tris-HCl, pH 7.8. Transfection efficiencies were determined by the luciferase activity assay. After normalization for transfection efficiency, CAT activity was measured by incubation of cell lysates at 37°C with 4 µM [¹⁴C]chloramphenicol and 1 mg of acetyl coenzyme A/ml. The mixture was separated by extraction with ethyl acetate, and acetylated products were separated on thin-layer chromatography plates using chloroform-methanol as the mobile phase. The radioactive dots were visualized by autoradiography, and radioactivity was quantified using a Molecular Dynamics 445 SI PhosphorImager (26, 38).

Mobility shift assays. Nuclear extracts were prepared from CHO cells at 80% confluency as described elsewhere (49). Nuclear extracts (7.5 µg) were incubated in a buffer containing 25 mM HEPES (pH 7.5), 0.5 mM EDTA, 0.2 mM dithiothreitol, 100 mM NaCl, 4% glycerol, and 2 µg of poly(dI/dC). Five femtomoles of a Klenow end-labeled ([-³²P]ATP) oligonucleotide (u-PAR PEA3, 5'-TTGGGTCCCACGTTAGGAAGAGAGAGAACTGGG-3') was added to each reaction mixture in the absence or presence of a 100-fold excess of the wild-type (wt) or mutated (mt) competitor sequence (underlined), and binding was allowed at room temperature for 25 min. Subsequently, 1 µg of PEA3 antibody (sc-113X; Santa Cruz Inc., Santa Cruz, Calif.) was added, and incubation was continued at 4°C for 2 h. The reaction mixture was electrophoresed in a 5% polyacrylamide gel, using 0.5× TBE (89 mM Tris, 89 mM boric acid, 1 mM EDTA) running buffer. The gel was dried and exposed to X-ray film overnight at 80°C (26).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

₃-integrin downregulates u-PAR. To define the role of ₃- integrin for u-PAR regulation, we utilized a CHO cell line that does not express the endogenous ₃-integrin subunit (40). As shown in Fig. 1A by Western blot analysis and in Fig. 1B and D by immunofluorescent staining with an antibody directed to the ₃-integrin subunit, CHO cells express no detectable ₃- integrin. However, upon transient (Fig. 1C) and stable (Fig. 1E) transfection of a ₃-integrin expression construct into CHO cells, the ₃-integrin subunit can be readily detected by immunofluorescence. As parent CHO cells have been reported to express endogenous _v-integrin (40), we were interested to know if this endogenous _v-integrin subunit is recruited by exogenous ₃-integrin into a correctly assembled _v3-integrin heterodimer complex. Indeed, immunofluorescence studies with the monoclonal antibody LM609, which recognizes the complexed integrin subunits (_v3), showed that expression of ₃-integrin leads to the surface expression of an _v3-integrin heterodimer (Fig. 1F) in CHO cells. In contrast, no _v3-integrin could be detected in the vector-transfected CHO cells (Fig. 1G).

View larger version (158K): [in this window] [in a new window]   FIG. 1.   Detection of 3-integrin in CHO cells. (A) Western blot. Cell lysates were resolved on a 10% polyacrylamide gel, and 3-integrin was detected by Western blot analysis using an antibody to 3- integrin. The band at ~120 kDa represents 3-integrin. The 3-integrin-expressing ovarian cancer cell line OVMZ-6 served as a positive control. The experiment was repeated twice. (B to G) Immunofluorescence. Untransfected CHO cells (B), CHO cells transiently transfected with 3-integrin (C and F) or the 3-integrin vector (D and G), or a stable 3-integrin-expressing CHO cell clone, A5 (E), was plated on chamber slides and stained by indirect immunofluorescence. CHO cells were incubated with either a monoclonal antibody against 3- integrin (B to E) or an antibody against v3-integrin (F and G) followed by an Alexa 488-conjugated goat anti-mouse secondary antibody. The experiment was repeated twice.

View larger version (210K): [in this window] [in a new window]   FIG. 2.   Transfection of 3-integrin reduces u-PAR protein levels. Immunofluorescence assays were performed. Untransfected CHO cells (A), CHO cells transiently transfected with 3-integrin (B) or the 3-integrin vector (C), or a stable 3-integrin-expressing CHO cell clone, A5 (D), was plated on chamber slides. For panels E and F, first chamber slides were coated with antibody LM609 and then untransfected CHO cells (E) or CHO cells transiently transfected with 3-integrin (F) were plated. For panels G and H, untransfected CHO cells (G) or CHO cells transiently transfected with 3-integrin (H) were plated on chamber slides coated with vitronectin. For panels A to D, cells were double stained: integrin was detected with a monoclonal 3-integrin antibody coupled with Alexa 488, and u-PAR was detected with a polyclonal antibody followed by an Alexa 568-conjugated secondary antibody. For panels E to H, cells were stained by incubating them with an antibody against u-PAR, followed by an Alexa 488-conjugated secondary antibody. The experiments were repeated three to five times.

View larger version (46K): [in this window] [in a new window]   FIG. 3.   Downregulation of u-PAR by 3-integrin on the protein and mRNA levels (A) Western blot. Cell lysates from untransfected and transiently and stably 3-integrin-transfected CHO cells were electrophoresed in a 10% acrylamide gel, and u-PAR was detected by Western blot analysis with a polyclonal antibody to u-PAR. (B) Northern blot analysis of u-PAR mRNA. mRNA was extracted from untransfected and transiently and stably 3-integrin-transfected CHO cells, electrophoresed in a 1.5% formaldehyde-agarose gel, and subsequently transferred to a nylon filter. The filter was probed with cDNAs specific for u-PAR and 18S rRNA transcripts.

Analysis of u-PAR gene transcription by ₃-integrin. The ₃-integrin subfamily includes _IIb3- and _v3-integrin. While the expression of _IIb3-integrin is largely confined to cells of the megakaryocytic lineage and is required for platelet aggregation (12, 60), _v3-integrin is expressed more widely, including in fibroblasts, smooth muscle cells, and endothelial cells. To study the effect of _v- and ₃-integrin on u-PAR promoter activity, we transiently cotransfected CHO cells with a CAT reporter driven by 398 bp of 5'-flanking sequence of the u-PAR promoter. Transient expression of both _v- and ₃-integrin subunits together with the u-PAR promoter resulted in a reduction of u-PAR promoter activity (Fig. 4A). This reduction was brought about by the ₃-integrin expression construct because transfection of _v-integrin alone did not show any effect on the u-PAR promoter. Lysates of CHO cells transfected with the u-PAR promoter-CAT reporter and the ₃-integrin expression vector resulted in 18% [¹⁴C]chloramphenicol conversion, compared to 87% in the absence of ₃-integrin. By contrast, expression of the same amount of _v-integrin showed no effect on the high constitutive activity of the u-PAR promoter. This indicates that the u-PAR promoter contains a transcription factor binding site that might account for the ₃-integrin-mediated repression of u-PAR gene transcription in CHO cells. Dose-response studies demonstrated that ₃-integrin expression resulted in a potent inhibition of u-PAR promoter activity (Fig. 4B). A significant reduction of promoter activity was observed upon transfection with 0.5 to 5 µg of ₃-integrin expression vector, whereas an equimolar amount of the empty expression construct (pBabe Puro) had a minimal effect on reporter activity. Cotransfection of 2 µg of ₃-integrin caused an 80% reduction of u-PAR promoter activity, which, however, could not be further decreased, not even with 5 µg of ₃-expression plasmid. To exclude the possibility that the effect of ₃-integrin is due to a general inhibition of transcriptional activity, we performed a control experiment with the -actin promoter. Compared with the effect of ₃-integrin transfection on a luciferase promoter regulated either by u-PAR or by -actin, the activity of -actin was not affected by the expression of ₃-integrin while the u-PAR promoter activity was inhibited (data not shown).

View larger version (22K): [in this window] [in a new window]   FIG. 4.   3-integrin downregulates u-PAR promoter activity. (A) CHO cells, at 70% confluency, were transiently transfected with 1 µg of a CAT reporter driven by the u-PAR promoter (u-PAR-CAT), a firefly luciferase vector, and the indicated amounts of v and 3-integrin expression vectors. After 3 h, the medium was changed and the cells were cultured for a further 45 h. The cells were lysed, harvested, and assayed for luciferase activity. Cell extracts corrected for differences in transfection efficiency were incubated with [14C]chloramphenicol, and after extraction with ethyl acetate, they were subjected to thin-layer chromatography. The conversion of [14C]chloramphenicol to acetylated derivatives was determined, using a PhosphorImager. The data shown represent the average values and standard deviations for five independent experiments. (B) CHO cells were transiently transfected with 1 µg of bp 398 u-PAR promoter luciferase construct and the indicated amounts of 3-integrin. Luciferase activity was analyzed 48 h after transfection and standardized for Renilla luciferase activity. The data shown represent the average values and the standard deviations for five independent experiments.

Inhibition of the u-PAR promoter activity by ₃-integrin requires a binding site for PEA3/ets. For localization of the region of the u-PAR promoter responsible for ₃-integrin-mediated inhibition of u-PAR promoter activity, CHO cells were cotransfected with a luciferase reporter driven by 5'-deletion fragments of the u-PAR promoter and the ₃-integrin expression vector (Fig. 5A). A dramatic inhibition of the u-PAR promoter was evident with the 1,469-bp and 398-bp u-PAR promoter construct when cotransfected with ₃-integrin while the 197-bp luciferase reporter was not affected by the ₃-integrin. The u-PAR 197 promoter had a low constitutive activity that could not be further downregulated by ₃- integrin. These data suggest that a sequence residing between 398 and 197 bp is critical for the reducing effect of ₃- integrin on u-PAR promoter activity.

View larger version (45K): [in this window] [in a new window]   FIG. 5.   The inhibition of the u-PAR promoter by 3-integrin requires a PEA3/ets binding site in the 5'-flanking sequence of the u-PAR gene. (A) CHO cells were transiently transfected with 1 µg of u-PAR promoter luciferase constructs of different lengths and were indicated with 3 µg of 3-integrin or the control vector. Luciferase activity was analyzed 48 h after transfection and standardized for Renilla luciferase activity. The data shown represent the average values and standard deviations for five independent experiments. (B) CHO cells were transiently cotransfected with 1 µg of a luciferase reporter driven by the 1469 u-PAR promoter or the indicated deletions and mutations (in the context of 1469 u-PAR) with or without 3 µg of 3-integrin or the empty expression vector alone. The data shown represent the average values and the standard deviations for four independent experiments.

View larger version (264K): [in this window] [in a new window]   FIG. 6.   Downregulation of the u-PAR promoter by 3-integrin is mediated by the transcription factor PEA3. (A and B) CHO cells were transiently transfected with the 398 u-PAR promoter (u-PAR CAT) (A) or the same promoter with a deletion of the PEA3/ets site at 248 (u-PAR PEA3 mt 248), a luciferase vector for normalization, and expression vectors encoding PEA3 (3 µg) or 3-integrin (3 µg) in equimolar concentrations (B). For panels A and B, the medium was changed 3 h after transfection, and the cells were cultured for a further 45 h. The cells were harvested and assayed for luciferase activity. Cell extracts corrected for differences in transfection efficiency were incubated with [14C]chloramphenicol and, after extraction with ethyl acetate, subjected to thin-layer chromatography. The conversion of [14C]chloramphenicol to acetylated derivatives was determined by using a PhosphorImager. The data shown represent the average values for two to four independent experiments. (C) Induction of PEA3 DNA-binding activity by 3-integrin. Gel shift assays were performed. Nuclear extracts (n.e.) from CHO cells transfected with 3-integrin or the empty expression vector were incubated with 2 × 104 cpm of a Klenow -32P-end-labeled oligonucleotide spanning the PEA3 site of the u-PAR promoter at 248 bp in the absence or presence of a 100-fold excess of the indicated competitors and electrophoresed in a 5% polyacrylamide gel. The PEA3 mt competitor oligonucleotide has point mutations in the PEA3 site. The arrow indicates a retarded complex of CHO 3-integrin-transfected cells; the asterisks indicate unspecific binding. (D) Nuclear extracts (n.e.) were prepared from the CHO 3-integrin-transfected cells and incubated with a Klenow -32P-end-labeled oligonucleotide spanning the PEA3 site of the urokinase receptor promoter. After 15 min, antibody to PEA3 was added, and the reaction mixture was subsequently subjected to gel electrophoresis. The data are representative of three experiments. (E and F) Immunofluorescence. CHO cells transiently transfected with PEA3 (E) or untransfected (F) were plated on chamber slides. For panels E and F, CHO cells were incubated with a polyclonal antibody against u-PAR, followed by an Alexa 488-conjugated goat anti-mouse secondary antibody. The experiment was repeated three times. (G) Transfection. CHO cells were transiently transfected with 1 µg of the 398 u-PAR promoter (u-PAR-CAT), a firefly luciferase vector, 1 µg of 3-integrin, and the indicated amounts of PEA3 expression vector. The conversion of [14C]chloramphenicol to acetylated derivatives was determined by using a PhosphorImager. The data shown represent the values for two independent experiments.

Regulation of the u-PAR promoter by the cytoplasmic domain of ₃-integrin. The analysis of the regulatory role of ₃-integrin cytoplasmic domains in multiple cellular functions has recently acquired a new level of complexity with the identification of variants of ₃-integrin (50). There are three ₃-integrin isoforms (₃A-, ₃B-, and ₃C-integrin) which result from alternative splicing and differ from one another by their cytoplasmic sequence (Fig. 7A) (21, 60). For a further evaluation of the mechanism of integrin-dependent u-PAR regulation, we created single-chain chimeric receptors (15a) which bear the full-length cytoplasmic domain of ₃-integrin fused to an Ig tag (23) (Fig. 7A). CHO cells were transiently cotransfected with the u-PAR-CAT reporter and the different ₃-integrin isoforms (Fig. 7B). The high constitutive activity of the u-PAR promoter in CHO cells could be downregulated almost completely by the ₃A-integrin while the ₃B-integrin and the IgG1 vector had no effect on the u-PAR promoter activity. Conversion of [¹⁴C]chloramphenicol was reduced from 83%, which mirrors the constitutive activity of the u-PAR promoter, to 10% in cells cotransfected with the ₃A-integrin. The ₃C-integrin isoform reduced the u-PAR promoter activity by 39%.

View larger version (24K): [in this window] [in a new window]   FIG. 7.   Regulation of the u-PAR promoter by the cytoplasmic domain of 3-integrin. (A) Schematic outline of the chimeric receptors used in this study. The amino acid sequences of the cytoplasmic domains of integrin isoforms 3A, 3B, 3C, and 1A and the mutated 3A- and 1A- integrins are shown. Wild-type and mutant intracellular domains of -integrins were expressed in the context of single-chain chimeric receptors, fused to heterologous extracellular and transmembrane domains corresponding to the CH2 and CH3 domains of human IgG1 and the CD7 antigen, respectively. Mutated amino sequences are shown in boldface and underlined. (B) CHO cells were transfected with the indicated wt or mutated integrin isoforms or the appropriate control vector (IgG1). After 3 h, the medium was changed, and the cells were cultivated for a further 45 h. The cells were harvested, lysed, and assayed for CAT activity. The conversion of [14C]chloramphenicol to acetylated derivatives was determined by using a PhosphorImager. The data shown represent the average values and the standard deviations for three independent experiments.

₃-endonexin short downregulates u-PAR promoter activity. Previous studies have shown that the short form of a cytoplasmic protein called ₃-endonexin binds to the NITY⁷⁵⁹ motif within the cytoplasmic domain of the ₃A-integrin (12, 45). This interaction is specific because ₁- and ₂-integrins do not interact with ₃-endonexin. Two isoforms of ₃-endonexin have been discovered, but only the short form interacts with the cytoplasmic domain of ₃A-integrin (22). Since we have shown that the NITY⁷⁵⁹ motif in the cytoplasmic domain of ₃A-integrin is necessary for regulating the u-PAR promoter, we tested whether ₃-endonexin short or its longer isoform is involved in this regulation. CHO cells were cotransfected with the u-PAR promoter-driven CAT reporter along with various amounts of an expression vector that encoded ₃-endonexin short or ₃-endonexin long (Fig. 8A). CAT activity was inhibited almost completely by the short form of ₃-endonexin while the empty expression vector had no effect. An input of the short form of ₃-endonexin diminished the u-PAR promoter activity by 87%. By contrast, the ₃-endonexin long form induced u-PAR promoter activity by 32%. When both ₃-endonexin isoforms were cotransfected with the u-PAR promoter mutated in the PEA3/ets site (u-PAR PEA3 mt 248 bp), the expression of the short form of ₃-endonexin did not reduce the u-PAR promoter activity any further (Fig. 8B). Evidently, the short isoform of ₃-endonexin downregulates the u-PAR gene at least in part through the PEA3/ets site in the u-PAR promoter.

View larger version (34K): [in this window] [in a new window]   FIG. 8.   3-Endonexin short downregulates the u-PAR promoter. CHO cells were transiently transfected, using 1 µg of a CAT reporter driven either by the wt u-PAR promoter (u-PAR CAT) (A) or by the same promoter with a deletion of the PEA3/ets site at 248 (u-PAR PEA3 mt 248), expression vectors encoding the short or long forms of 3-endonexin short, or the empty expression vector (pEGFN1) (B) . Cell extracts normalized for luciferase activity were incubated with [14C]chloramphenicol, extracted with ethyl acetate, and subjected to thin-layer chromatography. The conversion of [14C]chloramphenicol to acetylated derivatives was determined by using a PhosphorImager. The data shown represent the average values and the standard deviations for three (A) or two (B) independent experiments.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Cell migration depends on the coordinated regulation of proteolysis and adhesion at the cell surface and is tightly regulated in several physiologic and pathological conditions. Thus, it is critical to understand how these processes affect each other. In the present study, we provide evidence that overexpression of the adhesion receptor ₃-integrin downregulates the protease receptor, u-PAR. Furthermore, we have demonstrated that this downregulation is mediated, at least in part, through a PEA3/ets site in the u-PAR promoter and involves a NITY⁷⁵⁹ motif in the cytoplasmic domain of the ₃-integrin subunit and the adapter protein ₃-endonexin.

Several of our observations suggest a pivotal role for ₃- integrin in regulating u-PAR. The introduction of ₃-integrin into CHO cells which have a high constitutive u-PAR expression blocks u-PAR protein expression, as is shown by Western blot analysis and immunofluorescence. Equally important, overexpression of ₃-integrin also downregulates u-PAR mRNA and, in both transient and stable transfections, also downregulates u-PAR promoter activity. In contrast, _v-integrin and ₁- integrin alone have no influence on u-PAR regulation. However, upon overexpression of ₃-integrin, _v-integrin is recruited into an _v3-integrin complex, as is evidenced by our immunofluorescence results with an antibody recognizing the ECM ligand binding site of the heterodimer _v3. Clustering of _v3-integrin, which was achieved after ligation with immobilized vitronectin or the antibody LM609, even enhanced the repression of u-PAR in CHO cells. This indicates that the _v3-integrin complex and its ligation (outside-in signaling) and not the single ₃-integrin subunit is required for u-PAR regulation. Thus, besides its involvement in adhesion, migration, and signal transduction (61), _v3-integrin can also inhibit u-PAR transcription.

u-PAR is a multifunctional receptor and not only focuses urokinase enzymatic activity onto the cell surface but is also associated with and modulates the function of ₁-, ₂-, and ₃- integrins (16, 52, 58, 59). It is presently known that u-PAR and integrins coassemble within the plasma membrane and modulate the adhesive and migratory functions of cells. Wei et al. (52) showed that a complex of u-PAR, ₁-integrin, and caveolin affects the adhesive properties of embryonic kidney 293 cells. u-PAR inhibited cell adhesion on fibronectin but promoted cell adhesion to vitronectin, which implies that one effect of the association between u-PAR and integrins is to affect the ligand-binding function of that integrin (7). In addition to the already-described physical interaction between u-PAR and ₃-integrin at the cell surface, we now report that an increased level of ₃-integrin expression results in a reduced transcription of the u-PAR promoter and thus reduced u-PAR protein expression. This helps us to understand how integrins counteract u-PAR action at the cell surface and identifies a new regulatory mechanism between ₃-integrin and the u-PAR. Our findings may explain the observations by Danen et al. (11), who showed that stable overexpression of ₃-integrin in the ₃-negative human melanoma cell line MV3 strongly reduces invasion into Matrigel and also lung colonization but not proliferation in nude mice. Since u-PAR is involved in tumor cell invasion and metastasis, we speculate from our data that the ₃-mediated reduced expression of u-PAR modulates the proteolytic potential of tumor cells required for invasion and metastasis.

The cytoplasmic domain of integrins is fundamental for gene expression, cell proliferation, and cell cycle regulation (9, 39). The membrane-distal NXXY motif within the cytoplasmic domain is highly conserved in ₁-, ₂-, ₃-, ₅-, ₆-, and ₇-integrins and plays a crucial role in integrin-mediated cell functions. For ₃-integrin, this motif (NITY⁷⁵⁹) is important for different functions, including cell adhesion, focal contact formation, and FAK/paxillin tyrosine phosphorylation (40). Our present results show that the transfection of the cytoplasmic domain of ₃-integrin is sufficient to reduce u-PAR gene transcription. This effect was evident when the ₃A-integrin isoform was used, but it was not manifest with the ₃B isoform. Because the NITY⁷⁵⁹ motif is found exclusively within the ₃A-isoform sequence and not in other integrins, we evaluated the importance of the NITY⁷⁵⁹ motif for u-PAR gene regulation and found that deletion and substitution of the NITY⁷⁵⁹ motif through the cytoplasmic NPKY⁷⁷⁵ of ₁-integrin abolish the inhibitory effect of ₃A-integrin on u-PAR expression. However, the NITY⁷⁵⁹ motif itself is necessary but not sufficient to mediate the reduction of the u-PAR promoter activity, because its integration into the ₁-integrin cytoplasmic domain did not inhibit the u-PAR transcription. Probably, other parts of the ₃-integrin cytoplasmic domain in addition to this motif are involved in u-PAR downregulation. This is also suggested by the ability of ₃C-integrin to downregulate the u-PAR promoter, albeit to a lower degree than ₃A-integrin. The ₃A-integrin isoform plays a role in signal transduction and inside-out signaling (1), and now our results point to an additional role as a regulator of u-PAR expression. Because expression of ₃-integrin isoforms might be differentially regulated depending on the functional state of the cell, a change in the pattern of ₃-integrin isoforms might have an impact on ₃-integrin-dependent u-PAR regulation.

The importance of the NITY⁷⁵⁹ motif for u-PAR regulation is also emphasized by the fact that ₃-endonexin, the binding of which relies on a structurally intact NITY⁷⁵⁹ motif (12) in the cytoplasmic domain of ₃A-integrin, downregulates the u-PAR promoter. The NITY⁷⁵⁹ motif is the binding site of ₃A-integrin for its specific cytoplasmic protein ₃-endonexin, which exists in a short and a long isoform, the latter of which does not bind to the cytoplasmic domain of ₃-integrin (45). We established that the short isoform of ₃-endonexin downregulates u-PAR promoter activity, indicating a role for ₃-endonexin short in u-PAR regulation by binding to the cytoplasmic tail of ₃-integrin. The specificity of this regulation is emphasized by the inability of the long form of ₃-endonexin to regulate u-PAR promoter activity. Kashiwagi et al. (22) presented evidence of the interaction of the short form of ₃-endonexin with the ₃ cytoplasmic tail modulating the affinity state of _IIb3- integrin and enhancing fibrinogen-dependent cell aggregation. ₃-endonexin short increases the affinity of individual _IIb3- heterodimers for specific ligands, which suggests inside-out signaling with ₃-endonexin affecting the affinity of _IIb3 for certain ligands (22). Besides these functions, ₃-endonexin also participates in the integrin-mediated gene regulation that starts with activation-binding of ₃-integrin, results in ₃-endonexin recruitment, and eventually affects u-PAR gene regulation via a PEA3/ets binding site in the u-PAR promoter.

Cytoplasmic domains of -integrins mediate downstream signaling events and affect gene regulation. In untransformed cells, integrins induce cell cycle progression via the Ras-mitogen-activated protein kinase (MAPK) pathway (8, 24). Our previous studies indicated that the u-PAR promoter is regulated via a MAPK-dependent signal transduction pathway (25), raising the possibility that ₃-integrin uses this signal transduction pathway to regulate u-PAR (19). However, we did not find any involvement of MAPK in u-PAR gene regulation by ₃-integrin, neither by testing synthetic inhibitors nor with dominant-negative expression vectors for Erk-, Jnk-, or p38-MAPK (data not shown). This implies that integrin-dependent u-PAR gene regulation does not take place via well-documented signaling pathways. Therefore, further studies will be necessary to elucidate the signal transduction pathway(s) involved in the regulation of u-PAR by ₃-integrin and ₃- endonexin.

The involvement of the transcription factor PEA3 in mediating the reduction of the u-PAR promoter by ₃-integrin is supported by several experiments. First, the ability of ₃-integrin to downregulate u-PAR promoter activity is abolished by deletion of the PEA3/ets site at 248 bp. Second, an expression vector encoding the Ets family member PEA3 was sufficient to reduce wt u-PAR promoter activity but not the promoter with a mutation in this consensus sequence. Third, overexpression of PEA3 reduced u-PAR protein expression. Fourth, nuclear extracts of ₃-integrin-overexpressing cells showed enhanced binding activity for the PEA3/ets site, and the Ets family member PEA3 was identified within the DNA-protein complex. Fifth, ₃-endonexin short downregulates only the wild type and not the PEA3/ets-mutated u-PAR promoter. Thus, downregulation of the u-PAR promoter by ₃-integrin and the short form of ₃-endonexin is mediated at least in part through a previously undescribed PEA3/ets silencer region at 248 bp in the u-PAR promoter by PEA3.

Several positive regulatory elements, including AP-1, AP-2, and Sp-1, have been identified in the u-PAR promoter (2, 26, 48), most of them contained within the first 200 bp 5' of the transcriptional start site. We found now that additional positive regulatory elements are located between 400 and 300 bp of the u-PAR promoter, because two deletions in this region (u-PAR del 1 402/350 and u-PAR del 2 349/300) reduced the constitutive activity significantly. Within this region are putative transcription factor binding sites for the transcription factors Sp-1, GATA-2, and NF-1, and further analysis will be necessary to investigate their role in the constitutive and inducible expression of the u-PAR promoter. Deletion of this region had no effect on the regulation of u-PAR by ₃-integrin. Even though the basal promoter activity was lower than the wt u-PAR promoter activity, it could be further reduced by ₃- integrin.

Identification of the PEA3/ets motif represents the first report on a silencing element in the u-PAR promoter. The PEA3/ets site is an oncogene-regulated element and an important positive regulator of gene expression for several matrix-degrading proteolytic enzymes. The Ets family member PEA3, in cooperation with an AP-1 family member, induces the transcription of several MMPs and the serine protease urokinase (3, 20, 38, 51). While all these genes have juxtaposed PEA3/ets-AP-1 elements in their promoters, the PEA3/ets binding site in the u-PAR promoter has no adjacent AP-1 in its vicinity. Our data indicate that PEA3 is not only a transcriptional activator but can also act as a transcriptional repressor, a feature that has not been reported for PEA3 before. However, while this paper was in review, Xing et al. (57) showed that PEA3 can repress HER-2/neu transcription through a PEA3/ets site in the HER-2/neu promoter identical to the one in the u-PAR promoter (5'-TTAGGAAG-3'). The PEA3/ets site in the HER-2/neu promoter functions as a positive regulatory element, because its mutation abrogates constitutive HER-2/neu promoter activity. Therefore, the authors speculate that overexpression of PEA3 displaces a transactivating factor(s) that binds to the PEA3/ets site and then represses HER-2/neu transcription. In contrast, the PEA3/ets site in the u-PAR promoter acts as a weak negative regulatory element inducing constitutive promoter activity when mutated but is crucial for the inhibition of promoter activity by ₃-integrin. The same PEA3/ets site is a positive regulatory element in the HER-2/neu promoter and a negative regulatory element in the u-PAR promoter. Probably, the promoter context is important to define the role of this PEA3/ets motif in a given promoter. PEA3 alone is not the only transcription factor mediating the inhibitory effect of ₃-integrin at the u-PAR PEA3/ets site. While ₃-integrin can abrogate u-PAR promoter activity completely, even overexpression of a large amount of PEA3 could not achieve this, which suggests that another corepressor(s) is also required for this regulation. It seems to be a feature of Ets proteins to form complexes with other unrelated transcription factors, thereby determining whether they have an activating or an inhibitory effect on gene regulation.

Until recently, only a few Ets family members had been described as transcriptional repressors, while an activating function had been outlined in detail for many different Ets proteins (18). Schneikert et al. demonstrated that association of the androgen receptor with ERM, an Ets family member within the PEA3 group, downregulates the MMP-1 promoter activity (41). Goldberg et al. (17) reported that c-Ets-1 can inhibit tetradecanoyl phorbol acetate induction of a PEA3/ets-AP-1 element as shown by in vitro transcription and transient transfections. An inhibitory role for an Ets transcription factor has also been described for Drosophila melanogaster (32). In response to the activation of the epidermal growth factor (EGF) receptor pathway, the Ets transcription factor homologue pointed is overexpressed and an inhibitory signal is generated. pointed reduces the expression of a dorsal follicle marker, rhomboid, that controls patterning in the dorsal region. Our data together with the study by Xing et al. (57) now show that PEA3 can also act as a transcriptional repressor.

In conclusion, we have shown that overexpression of ₃A- integrin downregulates u-PAR at the transcriptional level through a PEA3/ets motif in the u-PAR promoter. The ₃-integrin-dependent downregulation is mediated through the NITY⁷⁵⁹ motif in the ₃A-integrin and through the short form of ₃-endonexin. The negative regulatory role of ₃A-integrin in u-PAR transcription is important in view of the poorly understood interaction and regulation between adhesion and proteolysis. Besides the known physical interaction between ₃A-integrin and u-PAR, data from this study support an additional mechanism in which the activation of ₃A-integrin downregulates the receptor for urokinase at the transcriptional level. This additional regulatory mechanism might be important for coordinating extracellular proteolysis, adhesion, and cell migration at the right time and place. The mutual regulation of both adhesion and proteolysis receptors at different regulation levels may help to achieve this.