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• W2012078324 abstract "Since c-src overexpression increases colonic cell invasiveness and because both Src activity and urokinase receptor protein are elevated in invasive colon cancers, the present study was undertaken: 1) to determine if a constitutively active Src regulates urokinase receptor expression and 2) to identify requiredcis-elements and trans-acting factors. SW480 colon cancer cells transfected with an expression plasmid (c-srcY527F) encoding a constitutively active Src protein manifested increased urokinase receptor gene expression and Src activity. Treatment of thesrc transfectants with a Src-inhibitor (PD173955) reduced urokinase receptor protein levels and laminin degradation. Inasmuch as we recently implicated an upstream region of the urokinase receptor promoter (−152/−135) in constitutive urokinase receptor expression, we determined its role for the induction by src. Whereas the activity of a CAT reporter driven by this region was stimulated by c-srcY527F, the u-PAR promoter mutated at the Sp1-binding motif in the −152/−135 region was not. Nuclear extracts from the srctransfectants demonstrated increased Sp1 binding to region −152/−135 compared with those from SW480 cells. Finally, endogenous urokinase receptor protein amounts in 10 colon cancers and corresponding normal colon correlated with Src specific activity. These data suggest that urokinase receptor gene expression is regulated by Src partly via increased Sp1 binding. Since c-src overexpression increases colonic cell invasiveness and because both Src activity and urokinase receptor protein are elevated in invasive colon cancers, the present study was undertaken: 1) to determine if a constitutively active Src regulates urokinase receptor expression and 2) to identify requiredcis-elements and trans-acting factors. SW480 colon cancer cells transfected with an expression plasmid (c-srcY527F) encoding a constitutively active Src protein manifested increased urokinase receptor gene expression and Src activity. Treatment of thesrc transfectants with a Src-inhibitor (PD173955) reduced urokinase receptor protein levels and laminin degradation. Inasmuch as we recently implicated an upstream region of the urokinase receptor promoter (−152/−135) in constitutive urokinase receptor expression, we determined its role for the induction by src. Whereas the activity of a CAT reporter driven by this region was stimulated by c-srcY527F, the u-PAR promoter mutated at the Sp1-binding motif in the −152/−135 region was not. Nuclear extracts from the srctransfectants demonstrated increased Sp1 binding to region −152/−135 compared with those from SW480 cells. Finally, endogenous urokinase receptor protein amounts in 10 colon cancers and corresponding normal colon correlated with Src specific activity. These data suggest that urokinase receptor gene expression is regulated by Src partly via increased Sp1 binding. The urokinase receptor (u-PAR), 1The abbreviations used are: u-PAR, urokinase-type plasminogen activator receptor; AP-1, activator protein-1; AP-2, activator protein-2; CAT, chloramphenicol acetyltransferase; DFP, diisopropyl fluorophosphate; EMSA, electrophoretic mobility shift assay; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; RSV, Rous sarcoma virus; tk, thymidine kinase.1The abbreviations used are: u-PAR, urokinase-type plasminogen activator receptor; AP-1, activator protein-1; AP-2, activator protein-2; CAT, chloramphenicol acetyltransferase; DFP, diisopropyl fluorophosphate; EMSA, electrophoretic mobility shift assay; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; RSV, Rous sarcoma virus; tk, thymidine kinase.a heavily glycosylated, 45–60-kDa cell surface protein, binds the serine protease urokinase specifically and with high affinity (KD ∼0.5 nm) (1Vassalli J. Baccino D. Belin D. J. Cell Biol. 1985; 100: 86-92Crossref PubMed Scopus (586) Google Scholar, 2Stoppelli M.P. Tacchetti C. Cubellis M. Corti A. Hearing V. Cassani G. Appella E. Blasi F. Cell. 1986; 45: 675-684Abstract Full Text PDF PubMed Scopus (274) Google Scholar). The u-PAR (1Vassalli J. Baccino D. Belin D. J. Cell Biol. 1985; 100: 86-92Crossref PubMed Scopus (586) Google Scholar) is comprised of three similar repeats approximately 90 residues each (3Behrendt N. Ploug M. Patthy L. Houen G. Blasi F. Dano K. J. Biol. Chem. 1991; 266: 7842-7847Abstract Full Text PDF PubMed Google Scholar, 4Riittinen L. Limongi P. Crippa M.P. Conese M. Hernandez-Marrero L. Fazioli F. Blasi F. FEBS Lett. 1996; 381: 1-6Crossref PubMed Scopus (19) Google Scholar) with the amino-terminal domain binding the plasminogen activator and the carboxyl-terminal domain tethering the binding protein to the cell surface via a glycosyl phosphatidylinositol anchor (4Riittinen L. Limongi P. Crippa M.P. Conese M. Hernandez-Marrero L. Fazioli F. Blasi F. FEBS Lett. 1996; 381: 1-6Crossref PubMed Scopus (19) Google Scholar). The u-PAR plays an important role in many physiological and pathological functions including wound healing, tissue remodeling, and tumor cell invasion and metastasis (5Romer J. Lund L.R. Eriksen J. Pyke C. Kristensen P. Dano K. J. Invest. Dermatol. 1994; 102: 519-522Crossref PubMed Scopus (147) Google Scholar, 6Sillaber C. Baghestanian M. Hofbauer R. Virgolini I. Bankl H.C. Fureder W. Agis H. Willheim M. Leimer M. Scheiner O. Binder B.R. Kiener H.P. Bevec D. Fritsch G. Majdic O. Kress H.G. Gadner H. Lechner K. Valent P. J. Biol. Chem. 1997; 272: 7824-7832Crossref PubMed Scopus (74) Google Scholar, 7Quattrone A. Fibbi G. Anichini E. Pucci M. Zamperini A. Capaccioli S. Del Rosso M. Cancer Res. 1995; 55: 90-95PubMed Google Scholar, 8Ossowski L. Clunie G. Masucci M. Blasi F. J. Cell Biol. 1991; 115: 1107-1112Crossref PubMed Scopus (201) Google Scholar). The ability of the u-PAR to promote these biological effects is a consequence of its diverse function. First, urokinase bound to the u-PAR activates plasminogen much more efficiently than the fluid-phase plasminogen activator (9Ellis V. Behrendt N. Dano K. J. Biol. Chem. 1991; 266: 12752-12758Abstract Full Text PDF PubMed Google Scholar), thereby facilitating basement membrane degradation. Second, u-PAR clears urokinase-inhibitor complexes from the extracellular space via an α2-macroglobulin receptor-dependent endocytotic mechanism (10Cubellis M. Wun T. Blasi F. EMBO J. 1990; 9: 1079-1085Crossref PubMed Scopus (331) Google Scholar, 11Conese M. Olson D. Blasi F. J. Biol. Chem. 1994; 269: 17886-17892Abstract Full Text PDF PubMed Google Scholar). Third, the u-PAR interacts with the extracellular domain of integrins allowing association with the cytoskeleton, thereby mediating cell adhesion and migration (12Wei Y. Waltz D.A. Rao N. Drummond R.J. Rosenberg S. Chapman H.A. J. Biol. Chem. 1994; 269: 32380-32388Abstract Full Text PDF PubMed Google Scholar, 13Wei Y. Lukashev M. Simon D.I. Bodary S.C. Rosenberg S. Doyle M.V. Chapman H.A. Science. 1996; 273: 1551-1555Crossref PubMed Scopus (696) Google Scholar, 14Yebra M. Parry G.C.N. Stromblad S. Mackman N. Rosenberg S. Mueller B.M. Cheresh D.A. J. Biol. Chem. 1996; 271: 29393-29399Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Fourth, the u-PAR is chemotactic for human monocytes and mast cells, and this may contribute to inflammatory and tissue remodeling processes, which are also often observed in tumor infiltrated areas (6Sillaber C. Baghestanian M. Hofbauer R. Virgolini I. Bankl H.C. Fureder W. Agis H. Willheim M. Leimer M. Scheiner O. Binder B.R. Kiener H.P. Bevec D. Fritsch G. Majdic O. Kress H.G. Gadner H. Lechner K. Valent P. J. Biol. Chem. 1997; 272: 7824-7832Crossref PubMed Scopus (74) Google Scholar,15Resnati M. Guttinger M. Valcamonica S. Sidenius N. Blasi F. Fazioli F. EMBO J. 1996; 15: 1572-1582Crossref PubMed Scopus (302) Google Scholar). The spread of a cancer to distant sites is characterized by extensive tissue remodeling in which surrounding normal tissue and extracellular matrix are proteolytically degraded. Several lines of evidence have implicated the u-PAR in this process. Thus, the overexpression of a human u-PAR cDNA increases the invasion of human osteosarcoma cells through an extracellular matrix-coated porous filter (16Kariko K. Kuo A. Boyd D. Okada S. Cines D. Barnathan E. Cancer Res. 1993; 53: 3109-3117PubMed Google Scholar), whereas down-regulating u-PAR levels using either antisense expression constructs, oligonucleotides, or synthetic compounds reduces the ability of divergent invasive cancers to invade in vitro andin vivo (17Kook Y.H. Adamski J. Zelent A. Ossowski L. EMBO J. 1994; 13: 3983-3991Crossref PubMed Scopus (174) Google Scholar, 18Wilhelm O. Weilde U. Rettenberger S. Schmitt M. Graeff H. FEBS Lett. 1994; 337: 131-134Crossref PubMed Scopus (104) Google Scholar, 19Xing R.H. Mazar A. Henkin J. Rabbani S.A. Cancer Res. 1997; 57: 3585-3593PubMed Google Scholar). Finally, clinical studies on colon and gastric cancers have revealed a correlation between high u-PAR expression and short survival times (20Ganesh S. Sier C.F.M. Heerding M.M. Griffioen G. Lamers C.B.H. Verspaget H.W. Lancet. 1994; 344: 401-402Abstract PubMed Scopus (173) Google Scholar, 21Heiss M.M. Allgayer H. Gruetzner K.U. Funke I. Babic R. Jauch K.-W. Schildberg W. Nat. Med. 1995; 1: 1035-1039Crossref PubMed Scopus (236) Google Scholar). The level of surface-u-PAR is controlled mainly via the regulation of transcription of the seven-exon u-PAR gene located on chromosome 19q13 (22Vagnarelli P. Raimondi E. Mazzieri R. De Carli L. Migantti P. Cytogenet. Cell Genet. 1992; 60: 197-199Crossref PubMed Scopus (10) Google Scholar, 23Casey J.R. Petranka J.G. Kottra J. Fleenor D.E. Rosse W.F. Blood. 1994; 84: 1151-1156Crossref PubMed Google Scholar), although altered mRNA stability and receptor recycling may represent other means of control (24Shetty S. Kumar A. Idell S. Mol. Cell. Biol. 1997; 17: 1075-1083Crossref PubMed Scopus (106) Google Scholar, 25Nykjaer A. Conese M. Cremona O. Gliemann J. Blasi F. EMBO J. 1997; 16: 2610-2620Crossref PubMed Scopus (290) Google Scholar, 26Lund L.R. Ellis V. Ronne E. Pyke C. Dano K. Biochem. J. 1995; 310: 345-352Crossref PubMed Scopus (114) Google Scholar). Regarding transcriptional regulation, Soravia et al. (27Soravia E. Grebe A. De Luca P. Helin K. Suh T.T. Degen J.L. Blasi F. Blood. 1995; 86: 624-635Crossref PubMed Google Scholar) reported that the basal expression of the gene was regulated via Sp1-binding motifs located proximal (−110/−24) to the transcriptional start site. Our laboratory showed that both the constitutive and phorbol 12-myristate 13-acetate-inducible expression of the gene required a footprinted region (−190/−171) of the promoter containing an AP-1 motif (28Lengyel E. Wang H. Stepp E. Juarez J. Doe W. Pfarr C.M. Boyd D. J. Biol. Chem. 1996; 271: 23176-23184Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). A second region of the promoter (−152/−135) bound with an AP-2α-related protein was also demonstrated to be required for the constitutive u-PAR gene expression and moreover for u-PAR mediated extracellular matrix degradation (29Allgayer H. Wang H. Wang Y. Heiss M.M. Bauer R. Nyormoi O. Boyd D. J. Biol. Chem. 1999; 274: 4702-4714Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Since the u-PAR is a key protein in promoting tissue remodeling, it is of essential importance to identify molecules regulating its expression. One possible candidate is the c-src gene, which encodes the pp60c-src protein-tyrosine kinase, since (a) its specific activity is higher in distant metastases as compared with the primary colonic tumors (30Talamonti M.S. Roh M.S. Curley S.A. Gallick G.E. J. Clin. Invest. 1993; 91: 53-60Crossref PubMed Scopus (374) Google Scholar) and (b) this protein-tyrosine kinase stimulates in vitro invasion of rat colonic cells (31Pories S.E. Hess D.T. Swenson K. Lotz M. Moussa R. Steele G. Shibata D. Rieger-Christ K.M. Summerhayes C. Gastroenterology. 1998; 114: 1335-1338Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Therefore, we conducted the present study with the following objectives: 1) to determine whether a constitutively active Src regulates u-PAR gene expression, and 2) to elucidate the molecular mechanisms by which this occurs. Our data show, for the first time, that the urokinase-receptor gene is transcriptionally regulated by this protein-tyrosine kinase and that this induction requires an upstream sequence (−152/−135) bound with Sp1. The u-PAR CAT reporter consists of 449 base pairs of sequence stretching from −398 to +51 (relative to the transcription start site) cloned into theXbaI site of the pCAT-Basic vector (Promega, Madison, WI). The u-PAR-firefly luciferase reporter was generated by cloning the aforementioned sequence into the SmaI site of pGL3 (Promega). The R2 CAT reporter construct, consisting of an oligonucleotide spanning nucleotides −154/−128 of the u-PAR promoter, was as described previously (29Allgayer H. Wang H. Wang Y. Heiss M.M. Bauer R. Nyormoi O. Boyd D. J. Biol. Chem. 1999; 274: 4702-4714Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The urokinase CAT reporter included 2345 base pairs of 5′-flanking region of the urokinase promoter fused directly to the reporter. The c-srcY527F construct contained thec-src coding sequence (32Kmiecik T.E. Shalloway D. Cell. 1987; 49: 65-73Abstract Full Text PDF PubMed Scopus (407) Google Scholar) harboring a tyrosine to phenylalanine substitution at codon 527 and cloned into theHindIII/BamHI cloning site of pcDNA3. The β-actin-renilla-luciferase reporter plasmid was kindly provided by Dr. Menashe Bar-Eli (Department of Cancer Biology, M. D. Anderson Cancer Center, Houston, TX). The v-mos expression construct was as described elsewhere (33Lengyel E. Singh B. Gum R. Nerlov C. Sabichi A. Birrer M. Boyd D. Oncogene. 1995; 11: 2639-2648PubMed Google Scholar). Antibodies to Sp1, Sp2, Sp3, and Sp4 were purchased from Santa Cruz Biotechnology. Oligonucleotides were supplied by Genosys Biotechnologies, The Woodlands, TX. SW480 colon adenocarcinoma cells were grown in McCoy's 5A medium supplemented with 10% fetal bovine serum. Stable constitutively active Src-expressing SW480 clones were generated by transfecting SW480 cells with c-srcY527F using LipofectAMINE (34Ellis L.M. Staley C.A. Liu W. Fleming R.Y.D. Parikh N.U. Bucana C.D. Gallick G.E. J. Biol. Chem. 1998; 273: 1052-1057Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). G418-resistant clones were generated and propagated in the presence of 400 μg/ml G418. Nuclear extracts and EMSA were carried out as described elsewhere (28Lengyel E. Wang H. Stepp E. Juarez J. Doe W. Pfarr C.M. Boyd D. J. Biol. Chem. 1996; 271: 23176-23184Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). EMSA was carried out as described (28Lengyel E. Wang H. Stepp E. Juarez J. Doe W. Pfarr C.M. Boyd D. J. Biol. Chem. 1996; 271: 23176-23184Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 29Allgayer H. Wang H. Wang Y. Heiss M.M. Bauer R. Nyormoi O. Boyd D. J. Biol. Chem. 1999; 274: 4702-4714Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) using 10 μg of nuclear extract, 0.6 μg of poly(dI-dC), and 2 × 104 cpm of a [γ-32P]ATP T4 polynucleotide kinase-labeled oligonucleotide. These were as described by us previously (35Wang H. Skibber J. Juarez J. Boyd D. Int. J. Cancer. 1994; 58: 650-657Crossref PubMed Scopus (40) Google Scholar). Nuclei from approximately 6 × 107cells (SW480, 1D8, and 2C8) were isolated and incubated in the presence of [α-32P]UTP in transcription buffer (150 mm KCl, 5 mm MgCl2, 1 mm MnCl2, 20 mm HEPES, pH 7.9, 10% glycerol, 5 mm dithiothreitol). Nuclei were then treated with DNase I and proteinase K, and the RNA extracted with phenol/chloroform and precipitated. Radioactive RNA (6.6 × 107 cpm) was hybridized to nylon-immobilized u-PAR cDNA, glyceraldehyde-3-phosphate dehydrogenase cDNA, and the linearized empty vector for the u-PAR cDNA (pBC12B1) as a control. Cells were transfected at 60% confluence using poly-l-ornithine as described previously (36Nead M.A. McCance D.J. J. Invest. Dermatol. 1995; 105: 668-671Abstract Full Text PDF PubMed Scopus (31) Google Scholar). CAT assays were performed as described by us previously (28Lengyel E. Wang H. Stepp E. Juarez J. Doe W. Pfarr C.M. Boyd D. J. Biol. Chem. 1996; 271: 23176-23184Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Where indicated, transient transfections were performed in the presence of an RSV-driven luciferase expression vector (4 μg) for the determination of transfection efficiencies. The amount of acetylated [14C]chloramphenicol was determined using a Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using ImageQuant software. Luciferase assays for determining the effect of c-srcY527F on the β-actin promoter were performed using the Dual Luciferase Reporter Assay System by Promega according to the manufacturer's protocol. Western blotting was performed as described previously (29Allgayer H. Wang H. Wang Y. Heiss M.M. Bauer R. Nyormoi O. Boyd D. J. Biol. Chem. 1999; 274: 4702-4714Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 37Gum R. Juarez J. Allgayer H. Mazar A. Wang Y. Boyd D. Oncogene. 1998; 17: 213-226Crossref PubMed Scopus (47) Google Scholar). Briefly, cells were extracted into a Triton X-100-containing buffer supplemented with protease inhibitors. Insoluble material was removed by centrifugation and the cell extract immunoprecipitated with a polyclonal anti-u-PAR antibody. The immunoprecipitated material was subjected to standard Western blotting and the blot probed with 5 μg/ml of an anti-u-PAR monoclonal antibody (3931; American Diagnostica, Greenwich, CT) and an horseradish peroxidase-conjugated goat anti-mouse IgG. Bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech). For the determination of u-PAR by ELISA, resected tissue was prepared and assayed as described by the manufacturer (American Diagnostica). These were carried out as described previously (38Schlechte W. Murano G. Boyd D.D. Cancer Res. 1989; 49: 6064-6069PubMed Google Scholar). Cells were harvested with 3 mmEDTA/phosphate-buffered saline (PBS), washed twice, and seeded (500,000 cells) on radioactive laminin-coated (2 μg/dish) dishes. The cells were allowed to attach overnight. Subsequently, cell surface u-PAR were saturated by incubating the cells at 37 °C for 30 min with 5 nm urokinase and unbound plasminogen activator removed by washing. The cells were then replenished with serum-free medium with, or without, 10 μg/ml plasminogen (final concentration). After varying times at 37 °C, aliquots of the culture medium were withdrawn and counted for radioactivity. Solubilized laminin represents the degraded glycoprotein (38Schlechte W. Murano G. Boyd D.D. Cancer Res. 1989; 49: 6064-6069PubMed Google Scholar). A urokinase receptor binding antagonist (Å5) was developed based on the sequence of amino acids 20–30 of its ligand (urokinase). This peptide was synthesized and cyclized using a covalent linker, as described previously (39.Jones, T. R., Haney, D. N., and Varga, J. (1998) PCT Publication WO 98/21230.Google Scholar). Å5 was tested for its ability to inhibit the binding of 125I-DFP-urokinase to RKO cells. Cells (2 × 104/well) were seeded in a 48-well plate and allowed to attach overnight at 37 °C. The cells were chilled to 4 °C, washed three times with cold PBS, and Å5 (diluted in PBS/0.1% bovine serum albumin) added to the wells at various concentrations (0.1–1000 nm).125I-DFP-urokinase (0.5 nm final concentration) was then added to each well. Unlabeled DFP-urokinase was used as a positive control to calibrate the assay. The plate was incubated for 2 h at 4 °C, and the cells washed extensively, after which they were lysed using 1 m NaOH and radioactivity in each lysate counted using a γ counter. These experiments indicated the IC50 of Å5 to be approximately 11 nm. These assays were performed as described elsewhere (34Ellis L.M. Staley C.A. Liu W. Fleming R.Y.D. Parikh N.U. Bucana C.D. Gallick G.E. J. Biol. Chem. 1998; 273: 1052-1057Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Briefly, cells were rinsed twice with 4 °C phosphate-buffered saline (PBS) and lysed in 250–500 μl of standard radioimmune precipitation buffer, and tumor and mucosal tissues were homogenized in radioimmune precipitation buffer using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). Lysates were additionally homogenized using an 18-gauge needle and clarified by centrifugation at 10,000 ×g. 250 μg of protein was reacted with monoclonal antibody 327 (Oncogene Science Inc., Cambridge, MA) for 1 h for immunoprecipitation of Src. Immune complexes were formed using 6 μg of rabbit-anti-mouse IgG (Oreganon Teknika, Durham, NC) for 1 h, followed by 50 μl of formalin-fixed Pansorbin (Staphylococcus aureus, Cowan strain; Calbiochem, La Jolla, CA) for 30 min. Pellets were washed three times in 0.1% Triton X-100, 150 mm NaCl, and 10 mm sodium phosphate. The kinase reaction was initiated by adding 10 μCi of [γ-32P]ATP, 10 mm Mg2+, 10 μg of rabbit muscle enolase (Sigma) as an exogenous substrate, and 100 μm sodium orthovanadate in 20 mm HEPES. After 10 min, reactions were terminated by adding SDS sample buffer. Products were separated in an 8% polyacrylamide gel and exposed to an x-ray film overnight. The amount of Src protein was determined by immunoblotting. Aliquots (50 μg of protein) of cell lysate were resolved in an 8% SDS-polyacrylamide gels and transferred to a nitrocellulose filter. After blocking, the filter was incubated for 18 h at 4 °C with 1 μg/lane of mAb 327 (Oncogene Science Inc., Cambridge, MA) and washed subsequently. Then, the filter was incubated with rabbit anti-mouse IgG and reactive bands visualized with chemiluminescence (see “Determination of u-PAR Protein Amounts”). Statistical comparison of ELISA and CAT results was done using a two-sided Student's t test (SPSS for Windows statistical software, release 6.1.3, SPSS Inc., Chicago, IL). The correlation between u-PAR amounts and specific or relative Src activity in tumor and colonic mucosa tissues was determined by linear regression analysis and Pearson's correlation coefficient. Statistical significance was defined at p< 0.05. Since both Src activity (30Talamonti M.S. Roh M.S. Curley S.A. Gallick G.E. J. Clin. Invest. 1993; 91: 53-60Crossref PubMed Scopus (374) Google Scholar) and u-PAR mRNA/protein (20Ganesh S. Sier C.F.M. Heerding M.M. Griffioen G. Lamers C.B.H. Verspaget H.W. Lancet. 1994; 344: 401-402Abstract PubMed Scopus (173) Google Scholar, 35Wang H. Skibber J. Juarez J. Boyd D. Int. J. Cancer. 1994; 58: 650-657Crossref PubMed Scopus (40) Google Scholar) amounts are elevated in invasive colon cancers, we hypothesized that this protein-tyrosine kinase could regulate the expression of this receptor. To test this hypothesis, we first transiently co-transfected SW480 cells with a CAT reporter driven by 398 base pairs of 5′-flanking sequence of the u-PAR promoter and increasing amounts of an expression construct encoding a constitutively active Src (c-srcY527F) (Fig.1 A). A significant induction of promoter activity was observed with 0.1–10 μg of c-srcY527F, whereas the empty expression construct (pcDNA3) had minimal effect on reporter activity. To exclude the possibility that the effect of the constitutively active Src on u-PAR promoter activity was due to a general induction of transcriptional activity, we performed a control experiment comparing the effect of c-srcY527F transfection on a luciferase reporter regulated by either the u-PAR or β-actin promoter (Fig. 1 B). β-Actin is a principal component of microfilaments and abundantly expressed in non-muscle cells. In contrast to a strong dose-dependent induction of u-PAR promoter activity, the activity of the β-actin promoter was unchanged by the expression of the c-srcY527F construct. Thus, it is unlikely that the increased u-PAR promoter activity brought about by thesrc expression construct is due to a general effect on transcription. The dose dependence of the src expression construct on u-PAR promoter activity was more evident using the luciferase reporter compared with the CAT reporter. This is probably a consequence of substrate depletion of the radioactive chloramphenicol in the latter assays. To rule out the possibility that the induction of u-PAR expression by the constitutively active Src was due to the introduction of a transforming gene, we performed an experiment in which the u-PAR promoter CAT reporter was co-transfected into SW480 cells with a v-mos expression construct (33Lengyel E. Singh B. Gum R. Nerlov C. Sabichi A. Birrer M. Boyd D. Oncogene. 1995; 11: 2639-2648PubMed Google Scholar). A CAT reporter driven by the urokinase promoter, which previously has been shown to be activated by this construct, served as a positive control (Fig.2). In contrast to the urokinase reporter, which was activated up to 4-fold, no effect of this non-membrane protein kinase on u-PAR promoter activity was observed. Thus, we consider it unlikely that the induction of u-PAR promoter activity in SW480 cells is a mere effect of expressing a transforming gene. We then asked whether the stable expression of the constitutively active Src induced endogenous u-PAR gene expression. Toward this end, we compared the amount of u-PAR protein in SW480 cells, characterized by its very low Src-activity, with two derived clones (1D8, 2C8) stably expressing an exogenous constitutively active Src (Fig.3 A). The growth rates of the untransfected and Src-transfected cells were indistinguishable. Analysis of cell extracts by ELISA indicated that the 2C8 clone expressing the highest level of Src activity (Fig. 3 A) had 8-fold more u-PAR protein (32 ± 5 ng of u-PAR protein/mg of cellular protein) compared with the parental SW480 cells (4 ± 1 ng of u-PAR protein/mg of cellular protein) (Fig. 3 B). The 1D8 clone, characterized by its intermediate Src activity, had an intermediate amount of u-PAR protein (10 ± 2 ng of u-PAR protein/mg of cellular protein). The ELISA data measuring u-PAR protein were corroborated by Western blotting (Fig. 3 C) in which the highest and lowest amount of u-PAR protein was evident in the 2C8 clone and the non-transfected SW480 cells, respectively. The diffuse nature of the immunoreactive bands is probably due to the heavily glycosylated state of the u-PAR protein (40Behrendt N. Ronne E. Ploug M. Luber D. Nielsen L. Schleuning W. Blasi F. Appella E. Dano K. J. Biol. Chem. 1990; 265: 6453-6460Abstract Full Text PDF PubMed Google Scholar). To determine if the increased u-PAR protein was due to a higher transcription rate, we performed nuclear run-on experiments comparing parental SW480 cells and SW480 cells stably expressing either neo (SW480 neo) or the constitutively active Src (1D8 and 2C8) (Fig.3 D). Hybridization of radioactive mRNA from isolated nuclei to a nylon filter-immobilized u-PAR cDNA yielded a signal with 2C8 cells, which was dramatically higher than that achieved with SW480 cells transfected with nothing (SW480) or the neo gene (SW480 neo). Thus, u-PAR gene transcription is induced by an activated Src. One of the functions of u-PAR is to accelerate plasminogen-dependent proteolysis via receptor-bound urokinase. Accordingly, we used laminin degradation as a biological end point to measure the effect of the src transfection on the display of the urokinase binding site. Toward this end, we performed laminin degradation assays comparing the activity of SW480, 1D8, and 2C8 cells. SW480 and 2C8 cells in serum-free medium (▪, ▴) demonstrated minimal solubilization of laminin in the absence of the zymogen, plasminogen (Fig. 4). After addition of plasminogen, SW480 cells showed a moderate degradation of laminin (50,000 cpm/106 cells) in the culture supernatant after 240-min incubation (×). However, addition of plasminogen to thesrc transfectants resulted in a much stronger increase in laminin solubilization indicating plasmin-dependent proteolysis. In this respect, the 2C8 clone, characterized by the highest Src activity, showed the highest amount of solubilized laminin in the supernatant (> 200,000 cpm/106 cells after 240 min; ●) with the 1D8 clone being intermediate (130,000 cpm/106cells; ■). These data suggested that the stable transfection of a low u-PAR-expressing colon cancer cell line with a plasmid encoding a constitutively active Src results in transcriptional activation of the u-PAR gene, the latter in proportion to the amount of Src activity detected in the individual clones. The results presented so far suggested that the expression of the u-PAR gene is transcriptionally induced by expressing a constitutively active Src cDNA in SW480 colon cancer cells. As a corollary to these experiments, we asked whether the inhibition of Src activity in the SW480 cells stably expressing the constitutively active Src would result in a decrease in u-PAR protein amounts, promoter activity, and laminin degradation. 2C8 cells (the SW480 transfected clone showing the highest Src activity) were treated with varying amounts of PD173955 (41Barvian M.R. Klutchko S. Hamby J.M. Kraker A.J. Moore C. Hartl B.G. Lu G.H. Panek R.L. Fry D.W. Doherty A.M. Showalter H.D.H. Proc. Am. Assoc. Cancer Res. 1998; 39: 176Google Scholar), which specifically targets Src family members, and assayed for u-PAR protein by ELISA. Increasing amounts of the PD173955 led to a dose-dependent decrease in the amount of the urokinase receptor (Fig. 5 A). Similarly, an identical concentration range of the Src inhibitor repressed u-PAR promoter activity (Fig. 5 B). We did not observe changes in growth rate or morphological signs of cell toxicity using this concentration r" @default.
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• W2012078324 date "1999-06-01" @default.
• W2012078324 modified "2023-09-27" @default.
• W2012078324 title "Transcriptional Induction of the Urokinase Receptor Gene by a Constitutively Active Src" @default.
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