ABSTRACT
Carcinoma progression is associated with the loss of epithelial features, and the acquisition of mesenchymal characteristics and invasive properties by tumour cells. The loss of cell-cell contacts may be the first step of the epithelium mesenchyme transition (EMT) and involves the functional inactivation of the cell-cell adhesion molecule E-cadherin. Repression of E-cadherin expression by the transcription factor Snail is a central event during the loss of epithelial phenotype. Akt kinase activation is frequent in human carcinomas, and Akt regulates various cellular mechanisms including EMT. Here, we show that Snail activation and consequent repression of E-cadherin may depend on AKT-mediated nuclear factor-kappaB (NF-kappaB) activation, and that NF-kappaB induces Snail expression. Expression of the NF-kappaB subunit p65 is sufficient for EMT induction, validating this signalling module during EMT. NF-kappaB pathway activation is associated with tumour progression and metastasis of several human tumour types; E-cadherin acts as a metastasis suppressor protein. Thus, this signalling and transcriptional network linking AKT, NF-kappaB, Snail and E-cadherin during EMT is a potential target for antimetastatic therapeutics.
Subject(s)
Carcinoma, Squamous Cell/pathology , Proto-Oncogene Proteins c-akt/metabolism , Transcription Factor RelA/metabolism , Transcription Factors/biosynthesis , Urinary Bladder Neoplasms/pathology , Animals , Cadherins/genetics , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Cell Line, Tumor , Disease Progression , Epithelium/pathology , Homeodomain Proteins/biosynthesis , Homeodomain Proteins/genetics , Mesoderm/pathology , Promoter Regions, Genetic , Rats , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Signal Transduction , Snail Family Transcription Factors , Transcription Factor RelA/biosynthesis , Transcription Factor RelA/genetics , Transcription Factors/genetics , Transcription, Genetic , Transfection , Up-Regulation , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/metabolism , Zinc Finger E-box Binding Homeobox 2ABSTRACT
The identification and characterization of components of the transforming growth factor beta (TGFbeta) signalling pathway are proceeding at a very fast pace. To illustrate a number of our activities in this field, we first summarize our work aiming at the selection from a large collection of single residue substitution mutants of two activin A polypeptides in which D27 and K102, respectively, have been modified. This work has highlighted the importance of K102 and its positive charge for binding to activin type II receptors. Activin K102E, which did not bind to high-affinity receptor complexes, may be a valuable beta chain, when incorporated in recombinant inhibin to unambiguously detect novel inhibin binding sites at the cell surface. We then illustrate how Smad5 knockout mice and an overexpression approach with a truncated TGFbeta type II receptor in the mouse embryo can contribute to the identification of a novel TGFbeta-->TbetaRII/ALK1-->Smad5 pathway in endothelial cells in the embryo proper and the yolk sac vasculature. We conclude with a summary of our results with a Smad-interacting transcriptional repressor but focus on its biological significance in the vertebrate embryo.
Subject(s)
Signal Transduction/physiology , Transforming Growth Factor beta/physiology , Activin Receptors/metabolism , Activins/genetics , Activins/metabolism , Animals , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Drug Interactions , Homeodomain Proteins/pharmacology , Neovascularization, Physiologic/drug effects , Phosphoproteins/metabolism , Phosphoproteins/physiology , Repressor Proteins/pharmacology , Smad5 Protein , Trans-Activators/metabolism , Trans-Activators/physiology , Vertebrates/embryology , Zinc Finger E-box Binding Homeobox 2ABSTRACT
Osteogenic protein-1 (OP-1) or bone morphogenetic protein-7 (BMP-7) stimulates cartilage formation in mouse bone rudiments in vitro but arrests terminal differentiation of prehypertrophic chondrocytes into hypertrophic chondrocytes. In this study we report that these effects of OP-1 depend on the developmental stage of the bone rudiment, early stages (E14 and E15 metatarsals) being most responsive. E17 metatarsals that already contained a hypertrophic area that had initiated mineralization were no longer affected by OP-1. We then investigated whether the sensitivity of the early long bone rudiments to OP-1 correlated with high expression of the OP-1 binding type I serine/threonine kinase receptors (activin receptor-like kinase: ALK-2/ActR-I, ALK-3/BMPR-IA or ALK-6/BMPR-IB) at this early stage. We did not find any significant difference in overall mRNA levels of these ALKs between stages E14 through E17 as assessed by RNase protection assays. However, by immunohistochemistry we found that ALK-6 staining was strong in E14 early cartilage primordium and its future perichondrium but dropped sharply to low levels in these cell types until onset of chondrocyte (pre)hypertrophy at E16. By contrast, ALK-2 and ALK-3 immunostainings in E14 were barely detectable. We also examined by immunohistochemistry the local synthesis of OP-1. OP-1 was present in E14 early chondrocytes and forming perichondrium but in low amounts; however, production of OP-1 increased in these cell types with age. All three receptor types as well as OP-1 were present in significant amounts in prehypertrophic chondrocytes and late hypertrophic chondrocytes including those undergoing mineralization. The temporary high immunostaining for ALK-6 in the early proliferating chondrocytes and future perichondrium of E14 bone rudiments, and its absence in older bones correlated with the sensitivity of chondrocytes and perichondrium to (exogenous) OP-1. We therefore propose that the effects of OP-1 on these cells in vitro are mediated by ALK-6/BMPR-IB. We furthermore conclude that locally produced OP-1 is a potential autocrine/paracrine growth factor. Increased local production of OP-1 may be partially responsible for the age-related decrease in responsiveness to exogenous OP-1 with respect to hypertrophy and mineralization of cartilage.
Subject(s)
Bone Development/physiology , Bone Morphogenetic Proteins/pharmacology , Protein Serine-Threonine Kinases/metabolism , Receptors, Growth Factor/metabolism , Receptors, Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/metabolism , Activin Receptors , Animals , Bone Morphogenetic Protein 7 , Bone Morphogenetic Protein Receptors, Type I , Cell Differentiation , Cell Division , Chondrocytes/physiology , Embryo, Mammalian , Female , Immunohistochemistry , Mice , Organ Culture Techniques , Organ SpecificityABSTRACT
Activation of transforming growth factor beta receptors causes the phosphorylation and nuclear translocation of Smad proteins, which then participate in the regulation of expression of target genes. We describe a novel Smad-interacting protein, SIP1, which was identified using the yeast two-hybrid system. Although SIP1 interacts with the MH2 domain of receptor-regulated Smads in yeast and in vitro, its interaction with full-length Smads in mammalian cells requires receptor-mediated Smad activation. SIP1 is a new member of the deltaEF1/Zfh-1 family of two-handed zinc finger/homeodomain proteins. Like deltaEF1, SIP1 binds to 5'-CACCT sequences in different promoters, including the Xenopus brachyury promoter. Overexpression of either full-length SIP1 or its C-terminal zinc finger cluster, which bind to the Xbra2 promoter in vitro, prevented expression of the endogenous Xbra gene in early Xenopus embryos. Therefore, SIP1, like deltaEF1, is likely to be a transcriptional repressor, which may be involved in the regulation of at least one immediate response gene for activin-dependent signal transduction pathways. The identification of this Smad-interacting protein opens new routes to investigate the mechanisms by which transforming growth factor beta members exert their effects on expression of target genes in responsive cells and in the vertebrate embryo.
Subject(s)
DNA-Binding Proteins/metabolism , Homeodomain Proteins/metabolism , Repressor Proteins/metabolism , Trans-Activators/metabolism , Xenopus Proteins , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , COS Cells , Cloning, Molecular , DNA, Complementary , Down-Regulation , Homeodomain Proteins/chemistry , Homeodomain Proteins/genetics , Molecular Sequence Data , Promoter Regions, Genetic , Protein Binding , Repressor Proteins/chemistry , Repressor Proteins/genetics , Sequence Homology, Amino Acid , Xenopus , Zinc FingersABSTRACT
We describe a novel expression cloning strategy in the fission yeast for the isolation of mammalian transcription factors using a mammalian promoter as target. This strategy is possible because of the conservation between mammalian cells and Schizosaccharomyces pombe of the mechanism that leads to the selection of the transcription start site. It also opens new perspectives to investigate the transcriptional regulation of genes for which detailed promoter analysis is difficult.
Subject(s)
Cloning, Molecular/methods , Schizosaccharomyces/genetics , Transcription Factors/genetics , Transcription Factors/isolation & purification , Cells, Cultured , DNA, Complementary/genetics , Gene Expression , Humans , Promoter Regions, Genetic , Transcription, Genetic , Transfection , Vascular Cell Adhesion Molecule-1/geneticsABSTRACT
We have analyzed the transcriptional activity of the human plasminogen activator inhibitor-1 promoter in the fission yeast Schizosaccharomyces pombe. This promoter is active in S. pombe, and the initiation site of transcription corresponds to the site identified previously in mammalian cells. Mutations in the AP-1-binding site (PAI-1 A box) or the HLTF-binding site (the B box), which reduced the basal and phorbol ester-induced levels of PAI-1 expression in human cells, also decreased the transcriptional activity in S. pombe. Gel retardation assays showed that an S. pombe protein binds specifically to this B box element and displays the same B box sequence requirement as HLTF. Furthermore, this yeast protein binds specifically to other HLTF-binding sites in the human immunodeficiency virus-1 long terminal repeat (LTR) and the simian virus 40 (SV40) enhancer. The B box (but not a mutated B box) strongly stimulated transcription when combined with adh downstream promoter elements, indicating that the S. pombe B box-binding protein, like HLTF, is a transcriptional activator. We conclude that the transcriptional activity of the nonviral PAI-1 promoter is controlled by the same promoter elements in S. pombe as in mammalian cells. In addition, mammalian trans-acting factors that bind to these promoter elements were shown to have counterparts with conserved DNA-binding activity in S. pombe. These results further illustrate the conservation of the mechanism of transcription between mammalian cells and fission yeast.
Subject(s)
DNA, Fungal/genetics , Plasminogen Activator Inhibitor 1/genetics , Promoter Regions, Genetic/genetics , Schizosaccharomyces/genetics , Trans-Activators/metabolism , Animals , Binding Sites , DNA, Fungal/metabolism , DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic/genetics , Genetic Vectors/genetics , HIV Long Terminal Repeat/genetics , Humans , Mammals , Mutation , Simian virus 40/genetics , Transcription Factors/metabolism , Transcription, Genetic/geneticsABSTRACT
A 2.4-kilobase (kb) DNA fragment, located 7.1 kb upstream from the human tissue-type plasminogen activator (t-PA) gene (t-PA2.4), acts as an enhancer which is activated by glucocorticoids, progesterone, androgens, and mineralocorticoids. Transient expression of t-PA-chloramphenicol acetyltransferase reporter constructs in HT1080 human fibrosarcoma cells identified a glucocorticoid responsive unit with four functional binding sites for the glucocorticoid receptor, located between bp -7,501 and -7,974. The region from bp -7,145 to -9,578 (t-PA2.4) was found to confer a cooperative induction by dexamethasone and all-trans-retinoic acid (RA) to its homologous and a heterologous promoter, irrespective of its orientation. The minimal enhancer, defined by progressive deletion analysis, comprised the region from -7.1 to -8.0 kb (t-PA0.9) and encompassed the glucocorticoid responsive unit and the previously identified RA-responsive element located at -7.3 kb (Bulens, F., Ibañez-Tallon, I., Van Acker, P., De Vriese, A., Nelles, L., Belayew, A., and Collen, D. (1995) J. Biol. Chem. 270, 7167-7175). The amplitude of the synergistic response to dexamethasone and RA increased by reducing the distance between the enhancer and the proximal t-PA promoter. The synergistic interaction was also observed between the aldosterone and the RA receptors. It is postulated that the t-PA0.9 enhancer might play a role in the hormonal regulation of the expression of human t-PA in vivo.
Subject(s)
Enhancer Elements, Genetic , Gene Expression Regulation, Enzymologic , Tissue Plasminogen Activator/genetics , Cell Line , Chromosome Mapping , Dexamethasone/pharmacology , Humans , Receptors, Androgen/physiology , Receptors, Glucocorticoid/physiology , Receptors, Steroid/physiology , Signal Transduction , Transcription, Genetic , Tretinoin/pharmacologyABSTRACT
We report the characterization of two vertebrate homologs of Drosophila mothers against dpp (Mad) isolated from the mouse and the Xenopus embryo, named MusMLP (mad-like protein) and XenMLP, respectively, together with a summary of their expression patterns in the embryo. Overexpression of XenMLP causes ventralization of Xenopus embryos and we demonstrate that the C-terminal domain is necessary and sufficient to confer this biological effect. This domain also has the potential for transcriptional activation, as shown in one-hybrid assays in mammalian cells. We further demonstrate that MLPs are multidomain proteins by showing a cis-negative effect of the N-terminal domain on the transactivation by the C-terminal domain and that the proline-rich, middle domain maximizes the activity of the C-terminal domain. We also mapped the MusMLP gene to a region on mouse chromosome 13 that corresponds to a region on human chromosome 5q that contains cancer-related genes.
Subject(s)
DNA-Binding Proteins/physiology , Nuclear Proteins/genetics , Trans-Activators , Transcription Factors/genetics , Xenopus laevis/embryology , Amino Acid Sequence , Animals , Cell Nucleus/metabolism , Chromosome Mapping , Mice , Molecular Sequence Data , Morphogenesis , Polymerase Chain Reaction/methods , Sequence Alignment , Smad Proteins , Structure-Activity Relationship , Transcription, Genetic , Transcriptional Activation , Xenopus laevis/geneticsABSTRACT
A 5.4-kb cDNA encoding the protein that binds to the B Box of the plasminogen activator inhibitor-1 (PAI-1) gene was isolated and sequenced. The protein, named helicase-like transcription factor (HLTF), contains a DNA-binding domain, a RING finger domain, and seven helicase domains and is homologous to SWI/SNF proteins. Two HLTF mRNAs of 5.5 and 4.5 kb were detected in most human tissues, a single gene was located on chromosome 3q24-25, and the protein was located in the nucleoplasm. Two HLTF proteins differing in translation start site (Met-1 or Met-123) were obtained by in vitro translation in reticulocyte lysate or by immunoprecipitation from HeLa cell nuclear extracts. In vitro transcription from the PAI-1 promoter in HeLa cell extracts was inhibited by HLTF antibodies and by the HLTF DNA binding domain. Over-expression of HLTF or HLTFMet123 produced a three-fold induction of PAI-1-LUC transient expression in HeLa cells. Mutation of the PAI-1 B Box led to an eight-fold reduction of basal PAI-1-LUC expression in these cell lines, but did not affect the four- to six-fold induction by phorbol esters.
Subject(s)
DNA-Binding Proteins/genetics , Gene Expression Regulation/physiology , Plasminogen Activator Inhibitor 1/genetics , Transcription Factors/genetics , Amino Acid Sequence , Base Sequence , Cell Nucleus/chemistry , Chromosomes, Human, Pair 3 , Cloning, Molecular , DNA/metabolism , DNA Helicases/genetics , DNA, Complementary/genetics , DNA-Binding Proteins/analysis , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , Molecular Sequence Data , Molecular Weight , Muscle, Skeletal , Organ Specificity , Protein Binding , RNA, Messenger/analysis , Regulatory Sequences, Nucleic Acid/genetics , Sequence Analysis, DNA , Transcription Factors/analysis , Transcription Factors/chemistry , Transcription Factors/metabolism , Transcription, Genetic/genetics , Zinc Fingers/geneticsABSTRACT
All-trans-retinoic acid (RA) and retinoids induce synthesis of tissue-type plasminogen activator (t-PA) in endothelial and neuroblastoma cells in vitro and in rats in vivo. In HT1080 fibrosarcoma cells, induction of t-PA-related antigen secretion and t-PA mRNA steady state levels by RA were found to depend on de novo protein and mRNA synthesis. Fragments derived from the 5'-flanking region of the t-PA gene (+197 to -9578 base pairs (bp)) were linked to the chloramphenicol acetyltransferase gene. Transfection studies demonstrated that the region spanning bp -7145 to -9578 mediated induction by RA. A functional retinoic acid response element (RARE), consisting of a direct repeat of the GGGTCA motif spaced by 5 nucleotides (t-PA/DR5), was localized at -7.3 kilobases. The t-PA/DR5 element interacted with the heterodimer composed of retinoic acid receptor alpha and retinoid X receptor alpha in vitro, whereas its mutation abolished induction by RA in transient expression. In human EA.hy926 hybrid endothelial and in SK-N-SH neuroblastoma cells, the activity of t-PA/DR5 was found to be independent of the intervening sequence (-632 to -7144 bp) and of its distance from the transcription initiation site. Staurosporine, an inhibitor of protein kinase activity, inhibited induction by RA, suggesting that it required protein phosphorylation.
Subject(s)
Gene Expression Regulation, Enzymologic/drug effects , Gene Expression/drug effects , Repetitive Sequences, Nucleic Acid/drug effects , Tissue Plasminogen Activator/biosynthesis , Tissue Plasminogen Activator/genetics , Tretinoin/pharmacology , Alkaloids/pharmacology , Animals , Base Sequence , Cell Line , Chloramphenicol O-Acetyltransferase/biosynthesis , DNA/chemistry , Dose-Response Relationship, Drug , Endothelium/enzymology , Enzyme Induction , Fibrosarcoma , Humans , Molecular Sequence Data , Mutagenesis , Neuroblastoma , Oligodeoxyribonucleotides , Protein Kinase C/antagonists & inhibitors , Protein Multimerization , Rats , Receptors, Retinoic Acid/metabolism , Recombinant Proteins/biosynthesis , Retinoic Acid Receptor alpha , Retinoid X Receptors , Sequence Deletion , Staurosporine , Transcription Factors/metabolism , Transfection , Tumor Cells, CulturedABSTRACT
In an effort to modify the fibrinolytic and/or pharmacokinetic properties of recombinant low M(r) single-chain urokinase-type plasminogen activator (rscu-PA-32k), mutants were prepared by site-directed mutagenesis of clusters of charged amino acids with the highest solvent accessibility. The following mutants of rscu-PA-32k were prepared: LUK-2 (Lys 212, Glu 213 and Asp 214 to Ala), LUK-3 (Lys 243 and Asp 244 to Ala), LUK-4 (Arg 262, Lys 264, Glu 265 and Arg 267 to Ala), LUK-5 (Lys 300, Glu 301 and Asp 305 to Ala) and LUK-6 (Arg 400, Lys 404, Glu 405 and Glu 406 to Ala). The rscu-PA-32k moieties were expressed in High Five Trichoplasiani cells, and purified to homogeneity from the conditioned cell culture medium, with recoveries of 0.8 to 3.7 mg/l. The specific fibrinolytic activities (220,000 to 300,000 IU/mg), the rates of plasminogen activation by the single-chain moieties and the rates of conversion to two-chain moieties by plasmin were comparable for mutant and wild-type rscu-PA-32k moieties, with the exception of LUK-5 which was virtually inactive. Equi-effective lysis (50% in 2 h) of 60 microliters 125I-fibrin labeled plasma clots submerged in 0.5 ml normal human plasma was obtained with 0.7 to 0.8 microgram/ml of wild-type or mutant rscu-PA-32k, except with LUK-5 (no significant lysis with 16 micrograms/ml). Following bolus injection in hamsters, all rscu-PA-32k moieties had a comparably rapid plasma clearance (1.3 to 2.7 ml/min), as a result of a short initial half-life (1.4 to 2.5 min).(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Mutagenesis, Site-Directed , Recombinant Fusion Proteins/genetics , Urokinase-Type Plasminogen Activator/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cricetinae , Half-Life , Molecular Sequence Data , Mutagenesis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/pharmacokinetics , Urokinase-Type Plasminogen Activator/biosynthesis , Urokinase-Type Plasminogen Activator/pharmacokineticsABSTRACT
The role of glycosylation on the enzymatic properties of single chain urokinase-type plasminogen activator (scu-PA) was investigated by site-specific mutagenesis of the glycosylated Asn-302 residu to Gln. In addition, the role of the NH2-terminal polypeptide chain and of the Cys-148 to Cys-279 interchain disulphide bond on the activity of non-glycosylated scu-PA was investigated. Therefore, variants of recombinant scu-PA (rscu-PA) were produced by transfecting Chinese hamster ovary cells with cDNA encoding rscu-PA N302Q (rscu-PA with Asn-302 to Gln mutation), rscu-PA C279A,N302Q (rscu-PA with Cys-279 to Ala and Asn-302 to Gln mutations) or rscu-PA del(N2-F157)C279A,N302Q (rscu-PA C279A,N302Q with deletion of Asn-2 through Phe-157). These mutants were purified to homogeneity from conditioned cell culture medium and were obtained essentially as single chain molecules with specific activities on fibrin plates of (mean +/- S.E.; n = 6) 45,000 +/- 5000. IU/mg, 19,000 +/- 800 IU/mg and < or = 100 IU/mg for rscu-PA N302Q, rscu-PA C279A,N302Q and rscu-PA del(N2-F157)C279A,N302Q, respectively, as compared to 64,000 +/- 2600 IU/mg for wild-type rscu-PA obtained in the same expression system. Plasmin quantitatively converts rscu-PA N302Q and rscu-PA C279A,N302Q to amidolytically active two-chain derivatives with a specific activity of 56,000 IU/mg and 32,000 IU/mg, respectively, as compared to 75,000 IU/mg for wild-type rscu-PA. Plasminogen activation as a function of time was comparable for rscu-PA N302Q and wild-type rscu-PA, and somewhat slower for rscu-PA C279A,N302Q. In a human plasma milieu in vitro, consisting of a 125I-fibrin labeled plasma clot submerged in plasma, 50 percent clot lysis in 2 h required 2.2 micrograms/ml rscu-PA N302Q and 6.0 micrograms/ml rscu-PA C279A,N302Q, as compared to 3.2 micrograms/ml wild-type rscu-PA. In contrast, rscu-PA del(N2-F157)C279A,N302Q was not converted to an amidolytically active two chain derivative by plasmin, and did not induce significant plasminogen activation in purified systems or clot lysis in a human plasma milieu. Following bolus injections in hamsters, the initial half-lives (1.8-2.6 min) and the plasma clearances (0.6-1.5 ml min-1) were comparable for wild-type rscu-PA and for the three rscu-PA mutants. These results suggest that the fibrinolytic activity in a plasma milieu in vitro and the in vivo turnover of rscu-PA are not markedly affected by the absence of carbohydrate.(ABSTRACT TRUNCATED AT 400 WORDS)
Subject(s)
Urokinase-Type Plasminogen Activator/chemistry , Animals , Base Sequence , Cricetinae , Enzyme Activation , Fibrinolysin/pharmacology , Fibrinolysis , Glycosylation , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides/chemistry , Plasminogen/metabolism , Recombinant Proteins , Structure-Activity Relationship , Urokinase-Type Plasminogen Activator/metabolism , Urokinase-Type Plasminogen Activator/pharmacokineticsABSTRACT
Phorbol 12-myristate 13-acetate induces a 3- and 10-fold induction of chloramphenicol acetyltransferase (CAT) activity in HT1080 and HeLa cells, respectively, following transient transfection of a 336-base pair plasminogen activator inhibitor-1 (PAI-1) promoter fragment linked to a CAT reporter gene. Substitution mutations in the regions encompassing nucleotides -78 to -69 (TGGGTGGGGC) or -61 to -54 (TGAGTTCA), but not in the regions -155 to -149 (TGCCTCA) or -84 to -76 (AGTGAGTGG) reduced this induction. Gel electrophoresis of double-stranded -65 to -50 oligonucleotides of the PAI-1 promoter region and nuclear extracts from Hela cells produced a gel shift pattern similar to that obtained with a AP-1 consensus oligomer, and excess unlabeled AP-1 oligomer reverted binding, suggesting that this region of the PAI-1 promoter is an AP-1-like binding site. Gel electrophoresis of double-stranded -82 to -65 oligonucleotides with HeLa nuclear extracts revealed a gel shift pattern of three bands; Sp1 consensus oligomer competed with the binding to two of these bands and AP-2 consensus sequence oligomer with the binding to the third band. The -82 to -65 oligomer also bound to purified AP-2 and Sp1 proteins. Southwestern blotting of HeLa nuclear extracts revealed that the labeled oligomer spanning region -82 to -65 bound to proteins with molecular masses of 52 and 72 kDa. Consensus AP-2 oligonucleotides competed for binding of the labeled -82 to -65 oligonucleotide to the 52-kDa protein, but consensus Sp-1 oligonucleotides did not compete for binding to the 72-kDa compound. The 72-kDa component binding to the -82 to -65 region may represent a new protein involved in transcriptional regulation.
Subject(s)
DNA-Binding Proteins/metabolism , Plasminogen Inactivators/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Sp1 Transcription Factor/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Transcription Factors/metabolism , Base Sequence , Chloramphenicol O-Acetyltransferase/biosynthesis , Chloramphenicol O-Acetyltransferase/genetics , Electrophoresis , Enzyme Induction , HeLa Cells , Humans , Immunoblotting , Molecular Sequence Data , Mutagenesis, Site-Directed , Plasmids , Promoter Regions, Genetic , Transcription Factor AP-2 , Transfection , Tumor Cells, CulturedABSTRACT
Retinoic acid induces tissue-type plasminogen activator (t-PA) but not plasminogen activator inhibitor-1 (PAI-1) expression in cultured human umbilical vein endothelial cells (HUVEC). To further investigate the relation between the structure of the retinoids and their ability to induce t-PA synthesis in vitro, 11 analogues were studied in HUVEC culture. The retinoid analogues were classified into one of three groups according to their t-PA-inducing potential. Group 1 showed little induction (0.9- to 1.9-fold after 48 h) at concentrations between 10(-8) and 10(-6) M. Group 2, which includes all-trans-retinoic acid, induced t-PA threefold to fivefold at 10(-6) M but had little effect at 10(-8) M (less than threefold). Group 3, which comprises arotinoid acid (RO-13-7410) and RO-13-6307, induced t-PA antigen secretion fivefold at 10(-8) M. The retinoids of groups 2 and 3 had a terminal carboxyl group and alkyl substitution of the lipophylic head of the retinoid skeleton. The group 3 retinoids also contained an aromatic ring. The t-PA-inducing activity of these third-generation retinoids correlates to some extent with other activities, including regression of papilloma, keratinization in vivo, and clonal inhibition of tumor cell lines in vitro. Some of the retinoids caused a small but significant (up to 1.5-fold at 24 h) increase in PAI-1 antigen secretion. The group 3 retinoids appear to be sufficiently potent inducers of t-PA secretion to warrant further investigation in in vivo animal models.
Subject(s)
Endothelium, Vascular/drug effects , Retinoids/pharmacology , Tissue Plasminogen Activator/biosynthesis , Cell Survival/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Humans , Plasminogen Inactivators/metabolism , Tissue Plasminogen Activator/metabolism , Umbilical VeinsABSTRACT
rt-PA P47G, K49N, a substitution variant of recombinant human tissue-type plasminogen activator (rt-PA), in which proline at position 47 and lysine at position 49 were replaced by glycine and asparagine respectively, was previously described by Ahern et al. (J Biol Chem 1990; 265:5540-5) to have an extended in vivo half-life with unaltered in vitro fibrinolytic properties. Because this variant might possess an increased in vivo thrombolytic potency, we have constructed its cDNA, expressed it in Chinese hamster ovary cells and determined its biochemical, thrombolytic and pharmacokinetic properties relative to those of home-made rt-PA and of alteplase (Actilyse). The specific fibrinolytic activities on fibrin plates were 160,000 +/- 17,000, 210,000 +/- 88,000 and 460,000 +/- 72,000 IU/mg (mean +/- SEM) for rt-PA P47G, K49N, rt-PA and alteplase, respectively, while the catalytic efficiencies for plasminogen activation (k2/Km) in the absence of fibrin were comparable (1.1 to 1.7 x 10(-3) microM-1s-1). Fibrin enhanced the rate of plasminogen activation by rt-PA P47G, K49N 100-fold and by both wild-type molecules 390-fold. Binding of the variant rt-PA to fibrin was significantly reduced, but its affinity for lysine-Sepharose was unaltered. In an in vitro clot lysis system, consisting of a radiolabeled human plasma clot submersed in plasma, 50% clot lysis in 2 h required 0.67 +/- 0.14 micrograms/ml rt-PA P47G, K49N, 0.36 +/- 0.01 micrograms/ml rt-PA and 0.17 +/- 0.01 micrograms/ml alteplase, respectively (mean +/- SEM; n = 3 or 4). At these doses residual fibrinogen levels at 2 h were in excess of 80%.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Tissue Plasminogen Activator/genetics , Animals , Base Sequence , Cricetinae , DNA/genetics , Fibrinogen/metabolism , Fibrinolysis/drug effects , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Pulmonary Embolism/drug therapy , Recombinant Proteins/genetics , Recombinant Proteins/pharmacokinetics , Recombinant Proteins/pharmacology , Tissue Plasminogen Activator/pharmacokinetics , Tissue Plasminogen Activator/pharmacologyABSTRACT
The contribution of the NH2-terminal polypeptide chain and of the Cys148-Cys279 interchain disulphide bond to the enzyme activity of urokinase-type plasminogen activator (u-PA) was studied using site-specific mutagenesis. Recombinant single-chain u-PA (rscu-PA) variants were produced by transfecting Chinese hamster ovary cells with cDNA encoding des(Asn2-Phe157)rscu-PA (rscu-PA with deletion of Asn2-Phe157), [Ala279]rscu-PA (rscu-PA with Cys279----Ala mutation) or des(Asn2-Phe157)[Ala279]rscu-PA [des(Asn2-Phe157)rscu-PA with Cys279----Ala mutation]. Des(Asn2-Phe157)rscu-PA, [Ala279]rscu-PA and des(Asn2-Phe157)[Ala279]rscu-PA, purified from conditioned cell culture medium, were obtained as nearly homogeneous single-chain molecules with Mr approximately 30,000, 54,000 and 30,000, and specific fibrinolytic activities on fibrin plates of (mean +/- SD; n = 3) 860 +/- 150 IU/mg, 43.0 +/- 2.5 IU/micrograms and 240 +/- 20 IU/mg, respectively, compared to 69.0 +/- 4.3 IU/micrograms for wild-type rscu-PA obtained in the same expression system. The plasminogen activating potential in a buffer milieu of [Ala279]rscu-PA was somewhat lower than that of rscu-PA, but that of both deletion mutants was virtually abolished. In a human plasma milieu in vitro, consisting of a radiolabelled human plasma clot submerged in plasma, 50% clot lysis in 2 h required 6.5 micrograms/ml [Ala279]rscu-PA or 3.4 micrograms/ml rscu-PA, whereas with both deletion mutants no significant clot lysis was observed with up to 16 micrograms/ml. Treatment of [Ala279]rscu-PA or rscu-PA with plasmin resulted in quantitative conversion to two-chain molecules and was associated with an increase in specific amidolytic activity from about 600 IU/mg to 62.5 IU/micrograms for [Ala279]rscu-PA as compared to an increase from about 0.3 IU/micrograms to 75.0 IU/micrograms for rscu-PA. In contrast, no significant amidolytic activity could be generated by treatment of des(Asn2-Phe157)rscu-PA or des(Asn2-Phe157)[Ala279]rscu-PA with plasmin. The u-PA B-chain, isolated from plasmin-treated [Ala279]rscu-PA, had enzymic properties which were comparable to those of rtcu-PA, with respect to specific fibrinolytic activity, amidolytic activity, kinetics of plasminogen activation and clot-lysis activity in a human plasma milieu in vitro. Following bolus injection into hamsters, the plasma clearances were comparable (0.7-1.1 ml/min) for wild-type rscu-PA and for the three truncated rscu-PA mutants.(ABSTRACT TRUNCATED AT 400 WORDS)
Subject(s)
Recombinant Proteins/metabolism , Urokinase-Type Plasminogen Activator/metabolism , Amino Acid Sequence , Animals , Base Sequence , CHO Cells , Chromosome Deletion , Cricetinae , Fibrinolysin/metabolism , Fibrinolysis , Humans , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides , Plasminogen/metabolism , Thrombin/metabolism , Transfection , Urokinase-Type Plasminogen Activator/geneticsABSTRACT
A recombinant chimeric plasminogen activator, MA-15C5Hu/scu-PA-32k, composed of a humanized fibrin fragment-D-dimer-specific monoclonal antibody (MA-15C5Hu) and a recombinant low-molecular-mass single-chain urokinase-type plasminogen activator, comprising amino acids Leu144-Leu411 (scu-PA-32k), was produced by cotransfecting Chinese hamster ovary (CHO) cells with the cDNA encoding the MA-15C5Hu light-chain sequence and the cDNA encoding the MA-15C5Hu heavy-chain sequence fused with the cDNA encoding scu-PA-32k. Purified MA-15C5Hu/scu-PA-32k migrated as a 215-kDa band on non-reducing SDS/PAGE, which is consistent with a molecule composed of one antibody and two scu-PA-32k moieties. However, the chimera was obtained as a mixture of single-chain u-PA-32k (37%) and amidolytically inactive (50%) and active (13%) two-chain u-PA-32k, the latter of which was removed by immunoadsorption on a monoclonal antibody specific for two-chain urokinase. The fragment-D-dimer affinity and enzymatic properties of MA-15CHu/scu-PA-32k were similar to those of MA-15C5Hu or of scu-PA-32k. In an in vitro system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated human plasma, MA-15C5Hu/scu-PA-32k had a 12-fold higher fibrinolytic potency than scu-PA-32k: 50% lysis in 2 h required 0.43 +/- 0.12 micrograms u-PA-32k equivalent of the chimera/ml versus 5.4 +/- 0.3 micrograms/ml of scu-PA-32k (mean +/- SEM, n = 4). Addition of purified fibrin fragment-D dimer reduced the fibrinolytic potency of MA-15C5Hu/scu-PA-32k in a concentration-dependent way, indicating that the increased potency is the result of antibody targeting. Thus, a recombinant humanized antifibrin antibody/u-PA chimera has been obtained in which only the variable domains of the antibody moiety are of non-human origin. The chimera has intact antigen-binding capacity, u-PA enzymatic activity and a significantly increased fibrinolytic potency in a plasma medium in vitro.
Subject(s)
Antibodies, Monoclonal/genetics , Fibrin/genetics , Plasminogen Activators/genetics , Receptors, Peptide , Recombinant Fusion Proteins/genetics , Urokinase-Type Plasminogen Activator/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , CHO Cells , Cricetinae , DNA/genetics , Dithioerythritol/chemistry , Electrophoresis, Polyacrylamide Gel , Fibrin/metabolism , Fibrinolysin/pharmacology , Genetic Vectors , Humans , Hydrolysis , Molecular Sequence Data , Oxidation-Reduction , Peptide Fragments/genetics , Plasminogen Activators/metabolism , Polymerase Chain Reaction , Receptors, Cell Surface/metabolism , Recombinant Fusion Proteins/metabolismABSTRACT
K1K2Pu, a recombinant t-PA/u-PA chimera with increased thrombolytic potency in animal models of venous and arterial thrombosis, which consists of amino acids 1 to 3 and 87 to 274 of human tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of human single chain urokinase-type plasminogen activator (scu-PA), was produced and conditioned for use in patients. Chinese hamster ovary cells were transfected with an expression plasmid containing the K1K2Pu cDNA, high producer cell lines were selected and scaled up in 800 cm2 roller bottles, and 350 ml conditioned cell culture medium was harvested 3 to 7 times at 2 to 5 day intervals. Batches of 21 +/- 4 liter (mean +/- SD, n = 28) containing 1.8 +/- 0.6 mg/l of K1K2Pu related antigen were purified by chromatography on Copper chelate-Sepharose and immunoadsorption on an insolubilized murine monoclonal antibody (MA-1C8). Yields were 8.6 +/- 3.4 mg K1K2Pu per batch with a specific activity of 83,000 +/- 44,000 IU/mg. The final material, obtained at a concentration of approximately 0.7 mg/ml, was dialyzed against 0.3 M NaCl, 0.02 M Tris-HCl buffer, pH 7.5, containing 0.01% Tween 80 and 10 KIU/ml aprotinin. It was homogeneous on SDS-PAGE, contained 6.5 +/- 6.9 percent two chain material and the contamination with murine monoclonal antibody was less than 0.1 percent. After filtration of pools of 3 to 5 selected batches on 0.22 microns Millipore filters the material was sterile and virus free by routine screening; it was obtained at a concentration of approximately 0.5 mg/ml with a specific activity of 110,000 +/- 16,000 IU/mg (mean +/- SD, n = 3) and an endotoxin content of 0.5 to 7 units/mg. Bolus injection at a dose of 1 mg/kg in mice did not produce weight loss within 8 days. Thus, this material appears to be suitable for the investigation on a pilot scale of the pharmacokinetic and thrombolytic properties of K1K2Pu in patients with thromboembolic disease.
Subject(s)
Fibrinolytic Agents/isolation & purification , Plasminogen Activators , Recombinant Fusion Proteins/isolation & purification , Tissue Plasminogen Activator/isolation & purification , Urokinase-Type Plasminogen Activator/isolation & purification , Amino Acid Sequence , Animals , Base Sequence , CHO Cells , Cricetinae , DNA/genetics , Fibrinolytic Agents/pharmacology , Fibrinolytic Agents/toxicity , Humans , Mice , Molecular Sequence Data , Polymerase Chain Reaction , Protein Engineering , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/toxicity , Thrombolytic Therapy , Tissue Plasminogen Activator/genetics , Tissue Plasminogen Activator/pharmacology , Tissue Plasminogen Activator/toxicity , Urokinase-Type Plasminogen Activator/genetics , Urokinase-Type Plasminogen Activator/pharmacology , Urokinase-Type Plasminogen Activator/toxicityABSTRACT
A mutant of recombinant tissue-type plasminogen activator (rt-PA), obtained by deletion of residues Lys296 to Gly302 [rt-PA del(K296-G302)], was previously shown to be resistant to inhibition by plasminogen activator inhibitor-1 (PAI-1) (Madison et al, Nature 339:721, 1989). This mutant was obtained by expression of its cDNA in Chinese hamster ovary cells and purification to homogeneity from conditioned cell culture medium. It was obtained as a single chain molecule with amidolytic activity, specific fibrinolytic activity, and binding to fibrin and lysine, which were comparable or somewhat lower than those of wild-type rt-PA obtained in the same expression system. The plasminogen-activating potential of rt-PA del(K296-G302) in the presence of CNBr-digested fibrinogen was about twofold lower than that of wild-type rt-PA. The inhibition rate of rt-PA del(K296-G302) by recombinant PAI-1 (rPAI-1) was more than 500-fold lower than that of wild-type rt-PA. In a human plasma milieu in vitro, rt-PA del(K296-G302) induced dose-dependent lysis of a 125I-fibrin-labeled plasma clot; equi-effective concentrations (causing 50% clot lysis in 2 hours) were 0.28 micrograms/mL and 0.36 micrograms/mL for mutant and wild-type rt-PA, respectively. In this system, addition of rPAI-1 to the plasma resulted in a concentration-dependent reduction of the fibrinolytic potency of rt-PA del(K296-G302) and of rt-PA; a 50% reduction required 2.4 micrograms/mL and 0.15 micrograms/mL rPAI-1, respectively. Continuous infusion of mutant or wild-type rt-PA over 60 minutes in hamsters with a 125I-labeled plasma clot in the pulmonary artery resulted in dose-dependent clot lysis, with a thrombolytic potency (percent clot lysis per milligram of compound administered per kilogram of body weight) and a specific thrombolytic activity (percent clot lysis per microgram per milliliter steady state rt-PA-related antigen level in plasma) that were not significantly different. Bolus injection in hamsters of 1 mg/kg rPAI-1 followed by bolus injection of 1 mg/kg rt-PA del(K296-G302) or wild-type rt-PA resulted in neutralization of the thrombolytic potency of wild-type rt-PA, while the mutant retained approximately half of its thrombolytic potency. These results indicate that rt-PA del(K296-G302), with a known resistance to inhibition by rPAI-1 in purified systems, maintains this property both in a plasma milieu in vitro and in an experimental animal model of thrombolysis in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)
Subject(s)
Tissue Plasminogen Activator/metabolism , Amino Acid Sequence , Animals , Chemical Phenomena , Chemistry, Physical , Chromogenic Compounds , Cricetinae , DNA/genetics , Fibrin/metabolism , Fibrinogen/metabolism , Fibrinolysis , Gene Expression , Humans , Hydrolysis , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligopeptides/metabolism , Plasminogen/metabolism , Plasminogen Inactivators/pharmacology , Pulmonary Embolism/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Tissue Plasminogen Activator/antagonists & inhibitors , Tissue Plasminogen Activator/geneticsABSTRACT
The synthesis of plasminogen activators and inhibitors in endothelial cells is highly regulated by hormones, drugs and growth factors. The present study evaluates the effect of retinoic acid on the synthesis of tissue-type plasminogen activator (t-PA) and of plasminogen activator inhibitor-1 (PAI-1) by cultured human umbilical vein endothelial cells (HUVEC). Retinoic acid produced a time- and concentration-dependent increase in the secretion of t-PA-related antigen but not of PAI-1 related antigen into the culture medium. A maximal sevenfold increase of t-PA antigen after 24 h was observed with 10 microM and a half-maximal increase with 0.1 microM retinoic acid. Retinoic acid induced a time-dependent increase of the t-PA mRNA, with a maximum at 8 h and returning to normal at 24 h. The protein kinase inhibitor H7 decreased the t-PA antigen induced by both retinoic acid and phorbol 12-myristate 13-acetate. These results suggest that treatment of HUVEC with retinoic acid increases t-PA production by a pathway which, at some level, involves protein kinases. Thus, retinoic acid induces t-PA synthesis in the absence of altered PAI-1 synthesis, which may enhance the fibrinolytic potential of the endothelium.