1: J Biol Chem. 2001 Jul 13;276(28):26340-8. Epub 2001 Apr 30. Integrin alpha(v)beta(3)/vitronectin interaction affects expression of the urokinase system in human ovarian cancer cells. Hapke S, Kessler H, Arroyo de Prada N, Benge A, Schmitt M, Lengyel E, Reuning U. Frauenklinik der Technischen Universitat Munchen, Klinikum rechts der Isar, Ismaninger Strasse 22, D-81675 Munchen, Germany. 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. PMID: 11331280 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Oncogene. 1999 Aug 12;18(32):4554-63. Overexpression of urokinase-type plasminogen activator in pancreatic adenocarcinoma is regulated by constitutively activated RelA. Wang W, Abbruzzese JL, Evans DB, Chiao PJ. Department of Surgical Oncology, The University of Texas MD Andersen Cancer Center, Houston, Texas, TX 77030, USA. The Rel/NF-kappaB transcription factors regulate the expression of many genes. The activity of RelA, a member of the Rel/NF-kappaB transcription factor family, is constitutively activated in the majority of pancreatic adenocarcinomas and cell lines. We report that the urokinase-type plasminogen activator (uPA), one of the critical proteases involved in tumor invasion and metastasis, is overexpressed in pancreatic tumor cells and its overexpression is induced by constitutive RelA activity. The uPA promoter contains an NF-kappaB binding site that directly mediates the induction of uPA expression by RelA. Expression of a dominant-negative IkappaBalpha mutant inhibits kappaB site-dependent transcriptional activation of a uPA promoter-CAT reporter gene. Treating the pancreatic tumor cell lines with the known NF-kappaB inhibitors, dexamethasone and n-tosylphenyalanine chloromethyl ketone (TPCK), abolishes constitutive RelA activity and uPA overexpression. These results show that uPA is one of the downstream target genes induced by constitutively activated RelA in human pancreatic tumor cells, and suggests that constitutive RelA activity may play a critical role in tumor invasion and metastasis. Inhibition of constitutive RelA in pancreatic tumor cells may reduce their invasive and metastatic potential. PMID: 10467400 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Eur J Biochem. 1999 Jan;259(1-2):143-8. Rel transcription factors contribute to elevated urokinase expression in human ovarian carcinoma cells. Reuning U, Guerrini L, Nishiguchi T, Page S, Seibold H, Magdolen V, Graeff H, Schmitt M. Frauenklink der Technischen Universitat Munchen, Germany. ute.reuning@lrz.tu-muenchen.de Elevated levels of the urokinase-type plasminogen activator (uPA) in tumor cells are conductive to tumor cell spread and metastasis. In a previous study we observed that suppression of RelA dramatically reduced endogenous uPA synthesis in the human ovarian cancer cell line OV-MZ-6. Because the uPA promoter contains three potential Rel-like protein binding motifs (RRBE, 5'-NF-kappaB, and 3'-NF-kappaB) we conducted the first thorough systematic uPA promoter analysis to examine the direct impact of Rel proteins on uPA gene transcription. Disruption of RRBE resulted in a approximately 40% decrease in uPA promoter activity, mutation of the 5'-NF-kappaB motif led to an additional 20% decrease. The 3'-NF-kappaB motif was not active. Overexpression of RelA significantly enhanced uPA promoter activity, whereas IkappaB-alpha overexpression reduced uPA promoter activity by 40%. These data were supported by the finding that endogenous uPA was also increased sixfold by overexpression of RelA and decreased by 30% upon overexpression of IkappaB-alpha. Transfection of OV-MZ-6 cells with antisense deoxynucleotides directed to RelA expression reduced uPA promoter activity by at least 40%. Our data clearly suggest that by binding to uPA promoter elements, Rel transcripton factors contribute directly to elevated uPA gene expression in human ovarian cancer cells, thereby promoting the multiple functions of uPA during tumor growth and metastasis. PMID: 9914486 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Nucleic Acids Res. 1995 Oct 11;23(19):3887-93. Inhibition of NF-kappa B-Rel A expression by antisense oligodeoxynucleotides suppresses synthesis of urokinase-type plasminogen activator (uPA) but not its inhibitor PAI-1. Reuning U, Wilhelm O, Nishiguchi T, Guerrini L, Blasi F, Graeff H, Schmitt M. Frauenklinik, Technischen Universitat Munchen, Klinikum rechts der Isar, Germany. The essential role of urokinase-type plasminogen activator (uPA) in tumor invasion and metastasis stresses the necessity of a fine-tuned cellular control over its expression. It has been shown that changes in uPA directly correlate with changes in cell invasiveness. We examined the role of Rel-related proteins in uPA synthesis by human ovarian cancer cells by inhibiting their expression using the antisense (AS) oligodeoxynucleotide (ODN) technology. Exposure of OV-MZ-6 cells to 10 microM phosphorothioate (PS)-derivatized AS-ODN directed to Rel A led to a maximal 50% decrease of uPA antigen in cell lysates and a 70% reduction in cell cultures supernatants accompanied by a significant transient decline in uPA mRNA levels. Antisense-PS-ODN directed to NF-kappa B1 (p50) or c-rel had no effect on uPA protein expression. AS-PS-ODN directed to Rel A also affected the proteolytic capacity of OV-MZ-6 cells reflected by an approximately 70% decrease in the fibrinolytic capacity of the cells within 24 h compared to untreated controls. AS-PS-ODN directed to I kappa B alpha expression increased uPA in cell culture supernatants up to 50%. uPA receptor (uPAR) production and synthesis of plasminogen activator inhibitor type-1 (PAI-1) were not altered by either AS-PS-ODN applied. Western blot and gel retardation analyses revealed constitutive expression of Rel-related proteins in nuclear protein extracts of OV-MZ-6 cells. Thus these proteins seem to be implicated in uPA regulation and may thereby contribute to tumor spread and metastasis. PMID: 7479032 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: J Biol Chem. 1994 Sep 2;269(35):22230-7. Differential DNA sequence specificity and regulation of HIV-1 enhancer activity by cRel-RelA transcription factor. Hansen SK, Guerrini L, Blasi F. Department of Genetics and Microbiol Biology, University of Milano, Italy. The cRel-RelA and NF-kappa B (p50-RelA) transcription factors bind to a kappa B-like sequence termed Rel-related proteins binding element localized in the regulatory region of the human urokinase plasminogen activator (uPA) gene. This sequence is highly conserved in murine and porcine uPA genes where it retained the ability to associate with cRel-RelA. On the other hand, NF-kappa B binding was obtained with the human and porcine elements only. Methylation interference analysis showed that NF-kappa B and cRel-RelA had identical interference patterns. Mutational analysis showed that DNA binding was highly sensitive to mutations within the decameric Rel-related proteins binding element core site. However, alterations of nucleotides flanking the decameric IgK-kappa B motif, which preferentially associated with NF-kappa B, resulted in high affinity cRel-RelA binding both in vitro and in vivo. These data demonstrate that NF-kappa B and cRel-RelA have overlapping but distinct DNA sequence specificities. Bandshift analysis with HeLa and Jurkat cell extracts or with in vitro translated proteins revealed that the SV40-, HIV-1-, and interleukin-2 receptor alpha subunit kappa B elements efficiently associated with cRel-RelA, suggesting that this heterodimer may be involved in the regulation of several genes. Cotransfection studies of HIV-1 long terminal repeat-chloramphenicol acetyltransferase reporter DNA with RelA, cRel, and p50 expression vectors were performed in COS7 and U293 cells to analyze the ability of cRel-RelA to regulate HIV-1 enhancer activity. In vivo formation of the cRel-RelA complex resulted in specific stimulation of the viral enhancer at a level comparable with that obtained with NF-kappa B. These data suggest that activation of cellular cRel-RelA may play a critical role in the regulation of HIV-1 enhancer activity. PMID: 8071349 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: EMBO J. 1992 Jan;11(1):205-13. A novel complex between the p65 subunit of NF-kappa B and c-Rel binds to a DNA element involved in the phorbol ester induction of the human urokinase gene. Hansen SK, Nerlov C, Zabel U, Verde P, Johnsen M, Baeuerle PA, Blasi F. University Institute of Microbiology, Copenhagen, Denmark. The NF-kappa B subunits, p50 and p65, have extensive sequence homology with the c-rel proto-oncogene and the Drosophila morphogen dorsal. It has recently been shown that in vitro translated c-Rel can bind to DNA and form a complex with p50. However, the conditions for DNA binding of c-Rel in vivo and its DNA sequence specificity have not been established. Here we report the identification a novel heterodimeric complex that binds to a kappa B-like, phorbol ester (TPA) responsive DNA sequence, 5'-GGGAAAGTAC-3', in the 5' flanking region of the human urokinase (uPA) gene. This sequence was shown to bind two protein complexes, LC and UC. LC was indistinguishable from NF-kappa B as it reacted with antibodies recognizing the p50 subunit of NF-kappa B, and was shown by UV crosslinking to contain the p50 and p65 subunits of NF-kappa B. UC, on the other hand, strongly reacted with anti-v-Rel, but not with the anti-p50 antibodies, and was shown by crosslinking to contain 75 kDa and 85 kDa protein-DNA adducts. The 75 kDa and the 85 kDa adducts could be immunoprecipitated only by anti-p65 and anti-c-Rel antibodies, respectively, showing that c-Rel formed a heterodimer with p65. Both protein complexes were present in inactive forms in HeLa cell cytosol, and their nuclear translocation was induced by TPA. DNA binding of UC and LC could, furthermore, be inhibited by I kappa B-alpha.(ABSTRACT TRUNCATED AT 250 WORDS) PMID: 1740106 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------