1: J Cell Biochem. 2004 Oct 15;93(3):437-53. Transforming growth factor-beta1-dependent activation of Smad2/3 and up-regulation of PAI-1 expression is negatively regulated by Src in SKOV-3 human ovarian cancer cells. Wakahara K, Kobayashi H, Yagyu T, Matsuzaki H, Kondo T, Kurita N, Sekino H, Inagaki K, Suzuki M, Kanayama N, Terao T. NetForce Co. Ltd., Taiko 3-1-18, Nakamura, Nagoya, Aichi 453-0801, Japan. The net balance between urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) has been implicated in tumor cell invasion and metastasis. To elucidate the mechanism of the transforming growth factor-beta1 (TGF-beta1)-dependent up-regulation of PAI-1 expression, we investigated which signaling pathway transduced by TGF-beta1 is responsible for this effect. Here, we show (1) nontoxic concentrations of TGF-beta1 up-regulates uPA expression in HRA and SKOV-3 human ovarian cancer cells, (2) TGF-beta1 activates Smads (phosphorylation of Smad2 and nuclear translocation of Smad3) and subsequently up-regulates PAI-1 expression in HRA cells, whereas TGF-beta1 neither activates Smads nor up-regulates PAI-1 in SKOV-3 cells, (3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment significantly induces TGF-beta1-dependent activation of Smads, leading to PAI-1 synthesis, compared with controls, in SKOV-3 cells, (4) combination of TGF-beta1 and PP2, which activates PAI-1 expression and reduces uPA expression in SKOV-3, results in decreased invasiveness, (5) pharmacological inhibitors for mitogen-activated protein kinase (MAPK) (PD98059) and phosphoinositide-3-kinase (PI3K) (LY294002 and wortmannin) or AS-PI3K ODN transfection do not affect TGF-beta1-induced Smad signaling and up-regulation of PAI-1 expression in SKOV-3 cells pretreated with PP2, and (6) the induction of PAI-1 protein was partially inhibited by an inhibitor of Sp1-DNA binding, mithramycin, implicating, at least in part, Sp1 in the regulation of this gene by TGF-beta1. In conclusion, TGF-beta1-dependent activation of Smad2/3, leading to PAI-1 synthesis, may be negatively regulated by Src, but not its downstream targets MAPK and PI3K in SKOV-3 cells. These data also reflect the complex biological effect of uPA-PAI-1 system. Copyright 2004 Wiley-Liss, Inc. PMID: 15372629 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Blood. 2004 Jul 1;104(1):256-62. Epub 2004 Mar 18. MAPK and JNK transduction pathways can phosphorylate Sp1 to activate the uPA minimal promoter element and endogenous gene transcription. Benasciutti E, Pages G, Kenzior O, Folk W, Blasi F, Crippa MP. Laboratory of Molecular Genetics, S. Raffaele Scientific Institute and Universita Vita-Salute, Milan, Italy. Two upstream regions of the human urokinase (uPA) gene regulate its transcription: the minimal promoter (MP) and the enhancer element. The activity of the minimal promoter is essential for basal uPA transcription in prostate adenocarcinoma PC3 cells. Binding of a phosphorylated Sp1 transcription factor is, in turn, essential for the activity of the MP. Here we report that the Jun kinase (JNK) pathway is required for the basal activity of the MP and for the expression of the endogenous uPA gene in PC3 cells and for activated transcription in LNCaP cells. On the other hand, the p42/p44 mitogen-activated protein kinase (MAPK) pathway activates uPA gene expression through Sp1 phosphorylation in HeLa, LNCaP, and CCL39-derivative cells that do not typically express uPA in basal conditions. In HeLa cells the dominant-negative form of JNK interferes with the p42/p44 MAPK activation of the uPA-MP. The results suggest that the stress-activated protein kinase (SAPK)/JNK pathway plays an important role in the phosphorylation of Sp1, which, in turn, leads to basal or activated transcription from the uPA-MP element. PMID: 15031204 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Blood. 2002 Nov 1;100(9):3325-32. Binding of Sp1 to the proximal promoter links constitutive expression of the human uPA gene and invasive potential of PC3 cells. Ibanez-Tallon I, Ferrai C, Longobardi E, Facetti I, Blasi F, Crippa MP. Laboratory of Molecular Genetics, Department of Biological and Technological Research (DIBIT) and Universita Vita-Salute, S. Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy. Activated transcription of the urokinase-type plasminogen activator (uPA) gene depends on the enhancer, located approximately 2 kb from the start of transcription. The proximal promoter, driving basal transcription, contains a GC-/GA-rich sequence immediately upstream of the TATA box. We have investigated the role played by this element in the transcription of the uPA gene in HeLa and PC3 cells, which do not express or constitutively express the gene, respectively. This region binds either Sp1 or Sp3, as monomers or multimers, but not a combination of the 2 proteins. The more efficient binding of Sp1 to the proximal promoter in PC3 cells is correlated to its phosphorylation state. Polymerase chain reaction (PCR)-coupled, chromatin immunoprecipitation experiments with anti-Sp1 antibodies indeed show an enrichment of proximal promoter sequences in PC3 cells and support the observed difference in transcription levels from proximal promoter constructs in HeLa versus PC3 cells. Furthermore, overexpression of Sp1 increases transcription from the reporter construct in HeLa cells, whereas in PC3 cells, overexpression of Sp3 does not reduce transcription from the same construct, indicating that the Sp1/Sp3 balance cannot be shifted. We conclude that the GC-/GA-rich element of the uPA regulatory region is an independent functional element, regulated by Sp family proteins. Phosphorylation of Sp1 determines the presence in vivo and the functionality of this element in PC3 cells. Thus, the cellular context determines the relevance of the GC-/GA-rich region in uPA gene transcription, which contributes to constitutive gene expression, related, in turn, to the invasive phenotype. PMID: 12384434 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Mol Endocrinol. 2001 Oct;15(10):1677-92. Transactivation via RAR/RXR-Sp1 interaction: characterization of binding between Sp1 and GC box motif. Shimada J, Suzuki Y, Kim SJ, Wang PC, Matsumura M, Kojima S. Laboratory of Molecular Cell Sciences, Tsukuba Institute, RIKEN, Koyadai, Tsukuba, Ibaraki 305-0074, Japan. Modulation of Sp1 activity by nuclear receptors is a novel mechanism by which fat-soluble hormones regulate gene expression. We previously established that upon autoinduction of RARs by RA, RARs/RXRs physically interact with Sp1, potentiate Sp1 binding to the GC box motifs, and thus enhance transactivation of the urokinase promoter, which lacks a canonical RAR-responsive element/RXR-responsive element. Here, we examined whether a similar mechanism might participate in transcriptional regulation of other key RA-inducible genes in endothelial cells and characterized binding between Sp1 and GC box motifs. Northern blot analyses showed that in addition to urokinase, after induction of RARs, RA up-regulates GC-rich region-dependent mRNA expression of transglutaminase, TGF beta 1, and types I and II TGF beta receptors. RA failed to alter the expression of Sp1 at both mRNA and protein levels. Reporter and gel shift assays and Western blot analyses suggested that either RA-treatment or RAR/RXR-overexpression enhances transactivation of these genes through a GC-rich region and strengthens the affinity of Sp1 to GC box motifs, accompanying a potential conformational change of Sp1 as reflected in its increased immunogenicity. Detailed analyses of the GC box motifs within the urokinase and other promoters indicate that interaction between RAR/RXR and Sp1 does not occur in the presence of nonfunctional GC box motifs containing five tandem purine or pyrimidine bases at the 3'-flanking region of hexanucleotide core sequence. These findings provide insight into the molecular mechanisms underlying RARE/RXRE-independent transactivation of RA-inducible gene promoters. PMID: 11579201 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Int J Oncol. 2001 Apr;18(4):877-83. Decreased tissue plasminogen activator and increased plasminogen activator inhibitors and increased activator protein-1 and specific promoter 1 are associated with inhibition of invasion in human A375 melanoma deprived of tyrosine and phenylalanine. Pelayo BA, Fu YM, Meadows GG. Pharmacology and Toxicology Graduate Program, Department of Pharmaceutical Sciences, College of Pharmacy and The Cancer Prevention and Research Center, Washington State University, Pullman, WA 99164-6510, USA. We previously found that dietary tyrosine (Tyr) and phenylalanine (Phe) restriction significantly decreased the metastatic phenotype of the pigmented murine B16BL6 melanoma in vivo and decreased the in vitro invasion of these cells. Here we report that invasion and chemoinvasion through GFR Matrigel of the human amelanotic A375 melanoma also is significantly inhibited by Tyr and Phe deprivation in vitro. Deprivation of these two amino acids decreased the secretion and protein expression of tissue-type plasminogen activator (tPA) while expression and secretion of plasminogen activator inhibitor (PAI-1 and PAI-2) were increased. Moreover, nuclear extracts of Tyr- and Phe-deprived cells exhibited increased binding of the transcription factors, activator protein-1 (AP-1) and specific promoter-1 (Sp1), to consensus oligonucleotides as determined by electrophoretic mobility shift assay. Nuclear binding activity to the oligonucleotide consensus sequence for AP-1 was inhibited by antibody against c-Fos and more effectively inhibited by an antibody against c-Jun. We conclude that decreased invasion and chemoinvasion of A375 melanoma cells deprived of Tyr and Phe are related to decreased secretion of tPA and increased secretion of PAIs. Increased AP-1 and Sp1 binding implicates these transcription factors in the regulation of PAI expression. PMID: 11251188 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: J Biol Chem. 2000 Aug 25;275(34):26333-42. Cellular localization of membrane-type serine protease 1 and identification of protease-activated receptor-2 and single-chain urokinase-type plasminogen activator as substrates. Takeuchi T, Harris JL, Huang W, Yan KW, Coughlin SR, Craik CS. Department of Pharmaceutical Chemistry and Biochemistry and Biophysics, Cardiovascular Research Institute, University of California, San Francisco, California 94143, USA. Membrane-type serine protease 1 (MT-SP1) was recently cloned, and we now report its biochemical characterization. MT-SP1 is predicted to be a type II transmembrane protein with an extracellular protease domain. This localization was experimentally verified using immunofluorescent microscopy and a cell-surface biotinylation technique. The substrate specificity of MT-SP1 was determined using a positional scanning-synthetic combinatorial library and substrate phage techniques. The preferred cleavage sequences were found to be (P4-(Arg/Lys)P3-(X)P2-(Ser)P1-(Arg)P1'-(Ala)) and (P4-(X)P3-(Arg/Lys)P2-(Ser)P1(Arg) P1'(Ala)), where X is a non-basic amino acid. Protease-activated receptor 2 (PAR2) and single-chain urokinase-type plasminogen activator are proteins that are localized to the extracellular surface and contain the preferred MT-SP1 cleavage sequence. The ability of MT-SP1 to activate PARs was assessed by exposing PAR-expressing Xenopus oocytes to the soluble MT-SP1 protease domain. The latter triggered calcium signaling in PAR2-expressing oocytes at 10 nm but failed to trigger calcium signaling in oocytes expressing PAR1, PAR3, or PAR4 at 100 nm. Single-chain urokinase-type plasminogen activator was activated using catalytic amounts of MT-SP1 (1 nm), but plasminogen was not cleaved under similar conditions. The membrane localization of MT-SP1 and its affinity for these key extracellular substrates suggests a role of the proteolytic activity in regulatory events. PMID: 10831593 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Cancer Res. 2000 Mar 15;60(6):1546-51. Coordinate up-regulation of Sp1 DNA-binding activity and urokinase receptor expression in breast carcinoma. Zannetti A, Del Vecchio S, Carriero MV, Fonti R, Franco P, Botti G, D'Aiuto G, Stoppelli MP, Salvatore M. Centro Medicina Nucleare, Consiglio Nazionale delle Ricerche, Dipartimento di Scienze Biomorfologiche e Funzionali, Universita Federico II, Naples, Italy. The regulatory mechanisms underlying the overexpression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in highly invasive breast carcinomas remain poorly understood. In this study, we have simultaneously determined the level of uPAR and the activity of the transcription factor Sp1 in 14 breast carcinomas and 5 benign lesions. We found that uPAR levels and Sp1-binding activity are coordinately elevated in malignant tumors (r, 0.94; P < 0.001). On the contrary, undetectable or only barely detectable levels of uPAR and Sp1 activity were found in benign breast lesions. Finally, the engagement of uPAR by catalytically inactive uPA in the MDA-MB-231 breast carcinoma cell line results in a rapid up-regulation of Sp1-binding activity followed by an increase of uPAR protein. These results, taken together, suggest the existence of a uPA-dependent positive regulatory loop that may progressively enhance malignant breast cell invasiveness. PMID: 10749121 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: J Biol Chem. 1999 Jun 25;274(26):18428-37. Transcriptional induction of the urokinase receptor gene by a constitutively active Src. Requirement of an upstream motif (-152/-135) bound with Sp1. Allgayer H, Wang H, Gallick GE, Crabtree A, Mazar A, Jones T, Kraker AJ, Boyd DD. Department of Cancer Biology, M.D. Anderson Cancer Center, Houston, Texas 77030, USA. 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 required cis-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 the src 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 src transfectants 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. PMID: 10373450 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Blood. 1999 Jun 15;93(12):4264-76. Physical interaction between retinoic acid receptor and Sp1: mechanism for induction of urokinase by retinoic acid. Suzuki Y, Shimada J, Shudo K, Matsumura M, Crippa MP, Kojima S. Laboratory of Molecular Cell Sciences, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Tsukuba, Ibaraki, Japan. Induction of urokinase plasminogen activator (uPA) by retinoic acid (RA) is the initial event preceding certain subsequent biological changes in vascular endothelial cells. We investigated the molecular mechanism by which RA stimulates the expression of uPA, which lacks a canonical RA receptor (RAR)-responsive element, in bovine and human aortic endothelial cells. Upon stimulation with RA, mRNA levels of RARalpha and beta transiently increased in parallel with the induction of uPA, and this increase was inhibited by cycloheximide. Results of transient transfection of RAR/RXR cDNAs and experiments using specific agonists and antagonists suggested that uPA induction is dependent upon RAR (initially, RARalpha) with the help of RXRalpha. Deletion analysis of the uPA promoter suggested that RAR/RXR acts on GC box region within the uPA promoter. This was further supported by inhibition of Sp1 binding to this region. Coimmunoprecipitation studies, glutathione S-transferase pull-down experiment, and mammalian two-hybrid assays suggested a physical interaction between RAR/RXR and Sp1. Furthermore, gel shift studies showed that the binding of Sp1 to the uPA GC box is significantly potentiated in the presence of RARs/RXRs. Finally, Sp1 and RAR/RXR synergistically enhanced the transactivation activity of the uPA promoter. These results suggest that (1) RA induces RARs mainly via RARalpha and that (2) RAR/RXR physically and functionally interact with Sp1, resulting in a potentiation of uPA transcription. PMID: 10361124 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Mol Cell Endocrinol. 1996 Mar 25;117(2):167-73. The same sequence mediates activation of the human urokinase promoter by cAMP in mouse Sertoli cells and by SV40 large T antigen in COS cells. Grimaldi P, Geremia R, Albanesi C, Rossi P. Dipartimento di Sanita Pubblica e Biologia Cellulare, Universita di Roma Tor Vergata, Italy. Cell-specific activation by follicle-stimulating hormone and its intracellular mediator, cAMP, of the human urokinase promoter in mouse Sertoli cells requires overlapping purine-rich and GC-rich sequences between -54 and -42 from the transcriptional start site. We have previously shown that binding of unidentified nuclear factors to these sequences is induced by cAMP stimulation, and that sequences from the enhancerless SV40 replication origin can interfere with the binding, whereas consensus Sp1 binding sites are ineffective. We now show that sequences within the SV40 origin able to compete for the formation of cAMP-induced DNA-protein complexes in Sertoli cell nuclear extracts are binding sites for the SV40 large T antigen. Large T antigen expressed in COS cells binds the cAMP-responsive sequences of the human urokinase gene and transactivates the proximal promoter, thus mimicking the effect of nuclear factors induced by cAMP in Sertoli cells. We show that Egr-1 is one of the factors present in cAMP-induced DNA-protein complexes formed between the human urokinase promoter and Sertoli cell nuclear extracts. However, Egr-1 levels are similar in unstimulated and cAMP-treated Sertoli cells, suggesting that this factor interacts with a different GC-box binding factor, that we have previously shown to be strongly induced by cAMP treatment of Sertoli cells. We propose that SV40 large T antigen in COS cells can mimick the action of heterodimers formed in cAMP stimulated Sertoli cells between Egr-1 and a cell specific cAMP-induced GC-box binding factor. PMID: 8737376 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Blood. 1995 Jul 15;86(2):624-35. A conserved TATA-less proximal promoter drives basal transcription from the urokinase-type plasminogen activator receptor gene. Soravia E, Grebe A, De Luca P, Helin K, Suh TT, Degen JL, Blasi F. Department of Genetics and Microbiology, University of Milano, Italy. The urokinase-type plasminogen activator receptor (uPAR) focuses at the cell surface the activation of pro-uPA and, hence, the formation of plasmin, thus enhancing directional extracellular proteolysis. To characterize the transcriptional regulatory mechanisms that control receptor expression, we have cloned an uPAR DNA segment containing upstream regulatory sequences from both the human and murine genomes. We report that a proximal promoter, contained within 180 bp from the major transcription start sites of the human uPAR gene, drives basal transcription. This region lacks TATA and CAAT boxes and contains relatively GC-rich proximal sequences. A subregion of this sequence, highly conserved between human and murine genes, contains most of the promoter activity and is specifically bound by HeLa nuclear proteins, one of which belongs to the SP1 class. PMID: 7605992 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: DNA Cell Biol. 1994 Nov;13(11):1063-9. Regulation of the urokinase gene by the retinoblastoma protein. Novak U, Paradiso L, Hamilton JA. University of Melbourne, Department of Medicine, Royal Melbourne Hospital, Parkville, Victoria, Australia. The promoter of the human urokinase plasminogen activator (uPA) gene contains a sequence identical with the retinoblastoma control element (RCE) of the murine c-fos gene, as well as several Sp1 binding sites. In a number of cell lines, the uPA promoter is activated during enforced expression of the retinoblastoma protein, pRB. Electrophoretic mobility-shift assays revealed that the RCE sequence of the uPA gene forms only one specific DNA-protein complex that does not contain pRB. The formation of the RCE-protein complex can be inhibited by 20 molar excess of the unlabeled RCE sequences and by 5 molar excess of the unlabeled E2F binding site. The RCE of the human uPA gene interacts specifically with a protein, which appears to be distinct from members of the E2F family of proteins, Sp1, ATF2, and Elf-1, which are all transcription factors shown to be regulated by pRB. PMID: 7702750 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: J Biol Chem. 1994 Oct 21;269(42):25992-8. The murine urokinase-type plasminogen activator receptor gene. Suh TT, Nerlov C, Dano K, Degen JL. Division of Basic Science Research, Children's Hospital Research Foundation, Cincinnati, Ohio 45229. The murine urokinase-type plasminogen activator receptor (uPAR) gene has been isolated and its complete nucleotide sequence established. The gene is organized into seven exons comprising 9.5% of the 13,207-base pair region that spans the interval between the transcription initiation and polyadenylation sites. The region upstream of the transcription initiation site lacks TATA- or CCAAT-like elements but is flanked by a G+C-rich region, which contains a number of potential regulatory elements including Sp1 and AP1 binding motifs. The close association of both Sp1 and AP1 sites within the proximal promoter region is consistent with the observation that the murine uPAR gene is inducible by phorbol esters. The major functional domains of the encoded protein, including the signal peptide, three cysteine-rich internal repeats, and the glycolipid anchor attachment motif, are encoded by separate exons. Based on the organization of the murine uPAR gene and the distribution of protein domains within the exons in the Ly-6 family of genes, it appears that the uPAR gene evolved secondarily to two internal duplication events within a Ly-6-like ancestral gene. The cloned and sequenced murine uPAR gene will be a valuable tool in understanding the regulation and biological roles of uPAR in that it will permit detailed studies of gene expression and uPAR-dependent processes in vitro, as well as the generation of both gain-of-function and loss-of-function mutants in transgenic mice. PMID: 7929309 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Cell Growth Differ. 1991 Jul;2(7):335-41. Autocrine and paracrine regulation of tissue inhibitor of metalloproteinases, transin, and urokinase gene expression in metastatic and nonmetastatic mammary carcinoma cells. Korczak B, Kerbel RS, Dennis JW. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. Acquisition of metastatic competence by tumor cells is frequently accompanied by increased expression of extracellular proteases capable of degrading basement membrane and extracellular matrix. However, very little is known about how the genes encoding these enzymes and their inhibitor proteins are regulated in metastatic versus nonmetastatic cells. In this report, we have compared autocrine and paracrine regulation of tissue inhibitor of metalloproteinases (TIMP), transin, and urokinase plasminogen activator (uPA) genes in genetically related nonmetastatic SP1 and metastatic A3a cell lines. Compared to SP1 cells, metastatic A3a cells showed 15-20-fold higher transin, 3-5-fold less TIMP mRNA, and comparable levels of uPA mRNA. A qualitatively similar shift in expression of these genes was rapidly (i.e., 4-8 h) induced in nonmetastatic SP1 cells following the addition of conditioned medium from A3a cells. The gene-regulating activity present in A3a conditioned medium was heat-labile, suggesting that it was protein in nature. The responsiveness of SP1 cells to the factor(s) secreted by A3a conditioned medium was inhibited by cycloheximide. Basic fibroblast growth factor mimicked the effect of the A3a conditioned medium as an inducer of transin expression in the tumor cells. Although medium conditioned by the tumor cells did not affect uPA expression, addition of epidermal growth factor to the tumor cells transiently induced expression of uPA with a biphasic response that differed in SP1 and A3a cells. Initial induction of uPA at 2-4 h was similar for both cell lines, but after 24 h of exposure to epidermal growth factor, SP1 cells showed a net reduction in uPA, whereas metastatic cells returned to the unstimulated levels.(ABSTRACT TRUNCATED AT 250 WORDS) PMID: 1782152 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Nucleic Acids Res. 1988 Aug 11;16(15):7527-44. Multiple nuclear factors interact with promoter sequences of the urokinase-type plasminogen activator gene. von der Ahe D, Pearson D, Nakagawa J, Rajput B, Nagamine Y. Friedrich Miescher-Institut, Basel, Switzerland. To characterize proteins that bind to the cyclic AMP inducible promoter of the urokinase-type plasminogen activator gene, we performed a DNAase I footprinting analysis. Within 500 nucleotides upstream of the transcription start site we found eight protected regions due to at least four different binding proteins. Among these is a single binding site for the transcription factor CTF/NF1, which is flanked on each side by two conserved binding sites for the transcription factor Sp1. A region at -380, which shares a similarity with sequences observed in the corresponding regions of other cyclic AMP regulated genes, was protected. This binding site contains a sequence of ten nucleotides which is repeated further upstream at -480 and also protected against DNAase I digestion. Comparisons of extracts from four different cell lines revealed that all DNA binding factors are present in nuclei of uPA expressing and nonexpressing cells. Mechanism underlying hormonal regulation of the gene is discussed. PMID: 3412894 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Biochemistry. 1987 Dec 15;26(25):8270-9. The murine urokinase-type plasminogen activator gene. Degen SJ, Heckel JL, Reich E, Degen JL. Division of Basic Science Research, Children's Hospital Research Foundation, Cincinnati, Ohio 45229. The murine urokinase-type plasminogen activator (uPA) gene has been isolated from a BALB/c liver DNA cosmid library and its nucleotide sequence established. The gene is organized into 11 exons comprising 34.7% of the 6710 base pair (bp) region spanning the interval between the presumed transcription initiation and polyadenylation sites. The transcription initiation site is flanked by common RNA polymerase II promoter elements, including a TATA box and a potential transcription factor Sp1 binding site. A large polypurine tract of the structure (AG)22(AGGG)16(AG)28 is located 79 bp upstream of the 5'-terminus. It was highly sensitive to the single-strand-specific nuclease S1, suggesting a non-B-DNA conformation of unknown significance. Consistent with the well-documented influence of adenosine cyclic 3',5'-phosphate (cAMP) on uPA gene expression, there is a dodecanucleotide homologous to proposed regulatory sequences identified in other cAMP-modulated genes. Comparison of the murine uPA gene to the previously described porcine and human uPA genes revealed an unusually high degree of evolutionary (interspecies) sequence conservation that was not limited to exons but included introns and flanking sequences as well. PMID: 2831940 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------