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1.
Bioorg Med Chem Lett ; 23(9): 2595-7, 2013 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-23528297

RESUMEN

By screening directed libraries of serine hydrolase inhibitors using the cell surface form of endothelial lipase (EL), we identified a series of carbamate-derived (EL) inhibitors. Compound 3 raised plasma HDL-C levels in the mouse, and a correlation was found between HDL-C and plasma compound levels. Spectroscopic and kinetic studies support a covalent mechanism of inhibition. Our findings represent the first report of EL inhibition as an effective means for increasing HDL-C in an in vivo model.


Asunto(s)
HDL-Colesterol/sangre , Endotelio Vascular/enzimología , Inhibidores Enzimáticos/química , Lipasa/antagonistas & inhibidores , Tiocarbamatos/química , Animales , Endotelio Vascular/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , Lipasa/metabolismo , Lipoproteínas HDL/deficiencia , Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tiocarbamatos/síntesis química , Tiocarbamatos/farmacología
2.
J Pharmacol Exp Ther ; 298(1): 34-42, 2001 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-11408522

RESUMEN

Human platelets possess two distinct thrombin-activated receptors, PAR-1 (protease-activated receptor-1) and PAR-4, whereas human vascular smooth muscle cells possess only PAR-1. Although such thrombin receptors have been studied extensively in vitro, their physiological roles are still rather ill-defined. We have now employed a potent, selective PAR-1 antagonist, RWJ-58259, to probe the in vivo significance of PAR-1 in thrombosis and vascular injury. RWJ-58259 was examined in two thrombosis models in guinea pigs: the arteriovenous (A-V) shunt assay (monitoring thrombus weight) and the Rose Bengal intravascular photoactivation assay (monitoring time to occlusion). Administration of RWJ-58259 (10 mg/kg, total i.v. dose) did not inhibit thrombus formation in either thrombosis model, although local, intrashunt delivery in the A-V shunt model did elicit a modest antithrombotic effect (thrombus weight reduction from 35 +/- 2 to 24 +/- 4 mg). These results are consistent with the presence of more than one thrombin-sensitive receptor on guinea pig platelets, in analogy with human platelets. Indeed, we were able to establish that guinea pig platelets express three thrombin receptors, PAR-1, PAR-3, and PAR-4. We also examined RWJ-58259 in a vascular restenosis model involving balloon angioplasty in rats. Perivascular administration of RWJ-58259 (10 mg) significantly reduced neointimal thickness (77 +/- 5 microm to 45 +/- 5 microm, P < 0.05), clearly demonstrating an important role for PAR-1 in vascular injury. From these results, it is evident that a PAR-1 antagonist is not especially effective for treating platelet-dependent thrombosis; however, it could well be beneficial for treating restenosis attendant to arterial injury.


Asunto(s)
Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/metabolismo , Oclusión de Injerto Vascular/tratamiento farmacológico , Indazoles/farmacología , Agregación Plaquetaria/efectos de los fármacos , Receptores de Trombina/antagonistas & inhibidores , Urea/farmacología , Angioplastia de Balón , Animales , Arterias Carótidas/fisiología , Arterias Carótidas/cirugía , Técnicas de Cultivo de Célula , Cobayas , Humanos , Indazoles/química , Indazoles/uso terapéutico , Masculino , Agregación Plaquetaria/fisiología , Ratas , Ratas Sprague-Dawley , Receptor PAR-1 , Receptores de Trombina/fisiología , Trombosis/tratamiento farmacológico , Urea/análogos & derivados , Urea/química , Urea/uso terapéutico
3.
Arch Biochem Biophys ; 386(2): 195-204, 2001 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-11368342

RESUMEN

Protease-activated receptor-2 (PAR-2) is a tethered-ligand, G-protein-coupled receptor that is activated by proteolytic cleavage or by small peptides derived from its cleaved N-terminal sequence, such as SLIGRL-NH2. To assess specific PAR activity, we developed an immortalized murine PAR-1 (-/-) cell line transfected with either human PAR-2 or PAR-1. A "directed" library of more than 100 PAR agonist peptide analogues was synthesized and evaluated for PAR-2 and PAR-1 activity to establish an in-depth structure-function profile for specific action on PAR-2. The most potent agonist peptides (EC50 = 2-4 microM) had Lys at position 6, Ala at position 4, and pFPhe at position 2; however, these also exhibited potent PAR-1 activity (EC50 = 0.05-0.35 microM). We identified SLIARK-NH2 and SL-Cha-ARL-NH2 as relatively potent, highly selective PAR-2 agonists with EC50 values of 4 microM. Position 1 did not tolerate basic, acidic, or large hydrophobic amino acids. N-Terminal capping by acetyl eliminated PAR-2 activity, although removal of the amino group reduced potency by just 4-fold. At position 2, substitution of Leu by Cha or Phe gave equivalent PAR-2 potency, but this modification also activated PAR-1, whereas Ala, Asp, Lys, or Gln abolished PAR-2 activity; at position 3, Ile and Cha were optimal, although various amino acids were tolerated; at position 4, Ala or Cha increased PAR-2 potency 2-fold, although Cha introduced PAR-1 activity; at position 5, Arg or Lys could be replaced successfully by large hydrophobic amino acids. These results with hexapeptide C-terminal amides that mimic the native PAR-2 ligand indicate structural modes for obtaining optimal PAR-2 activity, which could be useful for the design of PAR-2 antagonists.


Asunto(s)
Péptidos/química , Péptidos/farmacología , Receptores de Trombina/agonistas , Receptores de Trombina/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Plaquetas/fisiología , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular , Evolución Molecular Dirigida , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Humanos , Ligandos , Ratones , Biblioteca de Péptidos , Péptidos/genética , Péptidos/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Ratas , Receptor PAR-1 , Receptor PAR-2 , Receptores de Trombina/química , Receptores de Trombina/genética , Relación Estructura-Actividad
4.
Proc Natl Acad Sci U S A ; 96(22): 12257-62, 1999 Oct 26.
Artículo en Inglés | MEDLINE | ID: mdl-10535908

RESUMEN

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease alpha-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH(2), RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.


Asunto(s)
Imitación Molecular , Péptidos/síntesis química , Receptores de Trombina/antagonistas & inhibidores , Secuencia de Bases , Línea Celular , Cartilla de ADN , Humanos , Péptidos/metabolismo , Péptidos/farmacología , Inhibidores de Agregación Plaquetaria/síntesis química , Inhibidores de Agregación Plaquetaria/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Ensayo de Unión Radioligante , Receptor PAR-1 , Receptores de Trombina/metabolismo
5.
J Pharmacol Exp Ther ; 288(2): 671-8, 1999 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-9918574

RESUMEN

We developed mice deficient in protease-activated receptor-2 (PAR-2) or PAR-1 to explore the pathophysiological functions of these receptors. In this report, we evaluated mean arterial pressure and heart rate (HR) changes in response to PAR-1 or PAR-2 activation in anesthetized wild-type (WT), PAR-1-deficient (PAR-1(-/-)), and PAR-2-deficient (PAR-2(-/-)) mice. In WT mice, TFLLRNPNDK, a PAR-1 selective activating peptide, caused hypotension and HR decreases at 1 micromol/kg. TFLLRNPNDK also caused secondary hypertension following L-NAME pretreatment. These responses were absent in PAR-1(-/-) mice. In WT mice, SLIGRL, a PAR-2 selective activating peptide, caused hypotension without changing HR at 0.3 micromol/kg. SLIGRL did not induce hypertension following Nomega-nitrol-arginine-methyl ester-HCl (L-NAME). The response to SLIGRL was absent in PAR-2(-/-) mice. SFLLRN, a nonselective receptor activating peptide caused hypotension and HR decreases in WT mice at 0.3 micromol/kg, as well as secondary hypertension following L-NAME. SFLLRN still induced hypotension in PAR-1(-/-) mice, but HR decrease and secondary hypertension following L-NAME were absent. The hypotensive and bradycardic responses to SFLLRN and TFLLRNPNDK in PAR-2(-/-) mice were accentuated compared with WT mice. By using mouse strains deficient in either PAR-1 or PAR-2, we confirmed the in vivo specificity of TFLLRNPNDK and SLIGRL as respective activating peptides for PAR-1 and PAR-2, and the distinct hemodynamic responses mediated by activation of PAR-1 or PAR-2. Moreover, the accentuated response to PAR-1 activation in PAR-2-deficient mice suggests a compensatory response and potential receptor cross-talk.


Asunto(s)
Presión Sanguínea/fisiología , Frecuencia Cardíaca/fisiología , Receptores de Trombina/fisiología , Animales , Presión Sanguínea/efectos de los fármacos , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Ratones , Oligopéptidos/farmacología , Receptor PAR-2 , Receptores de Trombina/deficiencia , Receptores de Trombina/efectos de los fármacos , Especificidad por Sustrato
6.
J Histochem Cytochem ; 46(2): 157-64, 1998 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-9446822

RESUMEN

PAR-2 is a second member of a novel family of G-protein-coupled receptors characterized by a proteolytic cleavage of the amino terminus, thus exposing a tethered peptide ligand that autoactivates the receptor. The physiological and/or pathological role(s) of PAR-2 are still unknown. This study provides tissue-specific cellular localization of PAR-2 in normal human tissues by immunohistochemical techniques. A polyclonal antibody, PAR-2C, was raised against a peptide corresponding to the amino terminal sequence SLIGKVDGTSHVTGKGV of human PAR-2. Significant PAR-2 immunoreactivity was detected in smooth muscle of vascular and nonvascular origin and stromal cells from a variety of tissues. PAR-2 was also present in endothelial and epithelial cells independent of tissue type. Strong immunolabeling was observed throughout the gastrointestinal tract, indicating a possible function for PAR-2 in this system. In the CNS, PAR-2 was localized to many astrocytes and neurons, suggesting involvement of PAR-2 in neuronal function. A role for PAR-2 in the skin was further supported by its immunolocalization in the epidermis. PAR-2C antibody exemplifies an important tool to address the physiological role(s) of PAR-2.


Asunto(s)
Química Encefálica , Sistema Digestivo/química , Epidermis/química , Células Epiteliales/química , Músculo Liso/química , Receptores de Superficie Celular/análisis , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Plaquetas/química , Células Cultivadas , Endotelio/química , Endotelio/citología , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Neuronas/química , Especificidad de Órganos , Receptor PAR-2 , Receptores de Superficie Celular/inmunología , Células del Estroma/química
7.
J Neurochem ; 69(5): 1890-6, 1997 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-9349532

RESUMEN

Protease-activated receptor-2 (PAR-2) is a seven-transmembrane G protein-coupled receptor that possesses a structure and activation mechanism similar to those of the thrombin receptor. It is activated by low concentrations of trypsin (300 pM) and a synthetic hexapeptide [sequence of serine, leucine, isoleucine, glycine, arginine, leucine (SLIGRL), the rodent PAR-2 "tethered ligand"] representing the first six amino acids following the putative PAR-2 cleavage site. Previous studies have indicated that alpha-thrombin and SFLLRN (synthetic hexapeptide sequence of serine, phenylalanine, leucine, leucine, arginine, asparagine; the human thrombin receptor "tethered ligand") induce neurite retraction and neurotoxicity. Because of the strong similarities between thrombin receptor and PAR-2, we have proposed that PAR-2 may also participate in neurodegeneration. In the present study, we used reverse transcriptase polymerase chain reaction and immunocytochemistry to provide the first evidence that PAR-2 is present in the rat hippocampus. Moreover, we found SLIGRL to be toxic to hippocampal neurons in a concentration-dependent manner (> or = 100 microM). Calcium signaling studies were performed to aid in determining the mechanism by which PAR-2 activation is neurotoxic.


Asunto(s)
Hipocampo/metabolismo , Degeneración Nerviosa , Neuronas/fisiología , Receptores de Superficie Celular/biosíntesis , Secuencia de Aminoácidos , Animales , Anticuerpos , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cartilla de ADN , Hipocampo/patología , Humanos , Inmunohistoquímica , Cinética , Datos de Secuencia Molecular , Neuronas/citología , Neuronas/efectos de los fármacos , Oligopéptidos/farmacología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , Reacción en Cadena de la Polimerasa , Ratas , Receptor PAR-2 , Receptores de Superficie Celular/fisiología , Receptores de Trombina/fisiología , Trombina/farmacología , Tripsina/farmacología
8.
Thromb Haemost ; 76(6): 860-6, 1996 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-8972001

RESUMEN

The thrombin receptor (ThrR) is a membrane-bound, G-protein-coupled receptor for the serine protease thrombin. This receptor is expressed in a wide variety of cells and tissues, and elicits a range of physiological responses associated with tissue injury, inflammation, and wound repair. To achieve a better understanding of the physiological role of the ThrR, we have employed homologous recombination to create mice with a disrupted ThrR gene. Following heterozygous (+/-) intercrosses, a total of 351 surviving offspring were genotyped. Only 7% of these offspring were identified as homozygous (-/-) for the disrupted allele, indicating a profound effect on embryonic development. Paradoxically, adult ThrR-/- mice appeared to be normal by anatomical and histological analysis, including their platelet number and function. Similarly, ThrR deficiency had no detectable effect in adult ThrR-/- mice on basal heart rate, arterial blood pressure, vasomotor responses to angiotensin II and acetycholine, and coagulation parameters, even though the ThrR is expressed in many cardiovascular tissue types. In addition, the loss of ThrR function in the peripheral vasculature of adult ThrR-/- mice was confirmed by the absence of various standard hemodynamic effects of the ThrR-activating peptides SFLLRN-NH2 and TFLLRNPNDK-NH2. Our results indicate that ThrR deficiency has a strong impact on fetal development; however. ThrR-/- mice that proceed to full development display surprisingly little change in phenotype compared to the wild-type.


Asunto(s)
Hemodinámica/fisiología , Receptores de Trombina/deficiencia , Animales , Regulación de la Expresión Génica , Ratones , Ratones Mutantes , Fenotipo , Receptores de Trombina/genética
9.
Proc Natl Acad Sci U S A ; 92(20): 9151-5, 1995 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-7568091

RESUMEN

Thrombin receptor activation was explored in human epidermal keratinocytes and human dermal fibroblasts, cells that are actively involved in skin tissue repair. The effects of thrombin, trypsin, and the receptor agonist peptides SFLLRN and TFRIFD were assessed in inositolphospholipid hydrolysis and calcium mobilization studies. Thrombin and SFLLRN stimulated fibroblasts in both assays to a similar extent, whereas TFRIFD was less potent. Trypsin demonstrated weak efficacy in these assays in comparison with thrombin. Results in fibroblasts were consistent with human platelet thrombin receptor activation. Keratinocytes, however, exhibited a distinct profile, with trypsin being a far better activator of inositolphospholipid hydrolysis and calcium mobilization than thrombin. Furthermore, SFLLRN was more efficacious than thrombin, whereas no response was observed with TFRIFD. Since our data indicated that keratinocytes possess a trypsin-sensitive receptor, we addressed the possibility that these cells express the human homologue of the newly described murine protease-activated receptor, PAR-2 [Nystedt, S., Emilsson, K., Wahlestedt, C. & Sundelin, J. (1994) Proc. Natl. Acad. Sci. USA 91, 9208-9212]. PAR-2 is activated by nanomolar concentrations of trypsin and possesses the tethered ligand sequence SLIGRL. SLIGRL was found to be equipotent with SFLLRN in activating keratinocyte inositolphospholipid hydrolysis and calcium mobilization. Desensitization studies indicated that SFLLRN, SLIGRL, and trypsin activate a common receptor, PAR-2. Northern blot analyses detected a transcript of PAR-2 in total RNA from keratinocytes but not fibroblasts. Levels of thrombin receptor message were equivalent in the two cell types. Our results indicate that human keratinocytes possess PAR-2, suggesting a potential role for this receptor in tissue repair and/or skin-related disorders.


Asunto(s)
Endopeptidasas/farmacología , Queratinocitos/metabolismo , Oligopéptidos/farmacología , Receptores de Trombina/metabolismo , Piel/metabolismo , Trombina/farmacología , Tripsina/farmacología , Secuencia de Aminoácidos , Calcio/metabolismo , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Recién Nacido , Cinética , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Oligopéptidos/química , Fosfatidilinositoles/metabolismo , Receptores de Trombina/agonistas , Relación Estructura-Actividad , Trombina/metabolismo
10.
Development ; 119(1): 191-8, 1993 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-8275855

RESUMEN

The serine protease tissue-plasminogen activator (t-PA) has previously been shown to be intracellular in mouse secondary oocytes and extracellular in fertilized eggs. Here we demonstrate that extracellular t-PA activity is bound to the surface of the fertilized egg. The level of t-PA activity associated with preimplantation mouse embryos decreases in the 2-cell stage embryo, then increases in 4-cell and morula stage embryos. However, morulae grown in culture from fertilized eggs lack t-PA activity but are able to bind exogenously added mouse t-PA. Additionally, northern analysis indicates that preimplantation embryos do not contain detectable levels of t-PA mRNA. Therefore, the enzyme activity associated with 4-cell and morula stage embryos in vivo is derived from t-PA present in the oviduct lumen that binds the embryo, and not from protein produced from translation of embryonic mRNA. The binding activity is species and protein specific in that neither mouse urokinase-type plasminogen activator (u-PA) nor human t-PA bind to cultured morulae. Furthermore, binding activity is dose-dependent and saturable, and does not require the active site of t-PA. These data indicate that a cell surface-specific t-PA-binding activity exists in the preimplantation mouse embryo and may localize function and concentrate the proteolytic activity of t-PA in early mouse development.


Asunto(s)
Blastocisto/metabolismo , Receptores de Superficie Celular/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Animales , Blastocisto/fisiología , Ratones , Ratones Endogámicos , Oocitos/metabolismo , Activador de Tejido Plasminógeno/fisiología
11.
Genes Dev ; 6(7): 1202-12, 1992 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-1628827

RESUMEN

The cytoplasmic polyadenylation element (CPE) is an AU-rich sequence in the 3'-untranslated region of many stored maternal mRNAs. The CPE directs the meiotic maturation-specific cytoplasmic polyadenylation and translational activation of these dormant mRNAs in Xenopus. The work presented here demonstrates that the CPE controls a similar regulation in mouse oocytes and utilizes the information to isolate novel maternal mRNAs by polymerase chain reaction (PCR). A degenerate CPE primer was used in an anchored PCR reaction with cDNAs from primary mouse oocytes. Clones were identified that contained the canonical polyadenylation signal AATAAA. A novel PCR test was then used to determine the polyadenylation state of the respective mRNAs before and after meiotic maturation. Two mRNAs, OM-1 and OM-2, are cytoplasmically polyadenylated upon maturation. Another mRNA is not polyadenylated during maturation, although it contains multiple CPE-like elements, indicating that this sequence element is not sufficient for adenylation during this time. Microinjection into primary oocytes of antisense oligodeoxynucleotides directed against OM-1 destroys the mRNA but does not appear to interfere with maturation in vitro. These experiments identify two novel maternal mRNAs and establish a simple strategy for isolating other maternal messages that control meiotic maturation, fertilization, and early mouse development.


Asunto(s)
Citoplasma/metabolismo , Poli A/metabolismo , ARN Mensajero/aislamiento & purificación , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN , Regulación de la Expresión Génica , Ratones , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/farmacología , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Transcripción Genética
12.
Cell ; 69(6): 1021-30, 1992 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-1606611

RESUMEN

Tissue-type plasminogen activator (tPA) mRNA is stored, stable and untranslated, in the cytoplasm of fully grown primary mouse oocytes. Dormancy is associated with an unusually short poly(A) tail, and poly(A) tail elongation controls tPA mRNA translational activation during meiotic maturation. Here we show that the nuclear transcript of this mRNA is extensively polyadenylated and that primary oocytes contain a deadenylating activity capable of silencing the cytoplasmic message. The sequence determinants that control deadenylation and polyadenylation overlap; this AU-rich region thus serves as an adenylation control element (ACE). The translation of a reporter mRNA in primary oocytes is prevented upon inclusion of an ACE in its 3' untranslated region. Therefore, the stage-specific regulation of poly(A) tail length accounts for the regulated synthesis of tPA in oocytes, and reversible deadenylation provides a mechanism for the translational control of dormant mRNAs.


Asunto(s)
Regulación de la Expresión Génica , Oocitos/fisiología , Poli A/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , Animales , Secuencia de Bases , Análisis Mutacional de ADN , Técnicas In Vitro , Meiosis , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , ARN Mensajero/metabolismo , Factores de Tiempo , Activador de Tejido Plasminógeno/genética , Transfección
13.
Mol Cell Biol ; 11(6): 3139-47, 1991 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-1710025

RESUMEN

Tissue plasminogen activator (t-PA) mRNA levels are high in murine brain, lower in kidney, and undetectable in liver. Differences in t-PA mRNA levels are regulated in part at the transcriptional level. Brain, kidney, and liver nuclear extracts direct regulated transcription from the murine t-PA promoter in a manner that reflects the relative levels of t-PA gene expression in these tissues in vivo. Analysis of mutants has defined two GC box motifs as important elements for regulated transcription in vitro. Upon investigation of protein-DNA binding, we detected an activity in brain extracts which was not detected in kidney or liver extracts. An Sp1-like factor also binds to this region in all three tissue types. DNA interference experiments show that the brain-enriched binding activity and the Sp1-like factor contact the same GC-rich sequences. These studies provide additional evidence that brain-enriched DNA-binding activities can interact with sequences also recognized by ubiquitous transcription factors.


Asunto(s)
Encéfalo/enzimología , Regulación Enzimológica de la Expresión Génica , Riñón/enzimología , Hígado/enzimología , Regiones Promotoras Genéticas , ARN Mensajero/genética , Activador de Tejido Plasminógeno/genética , Transcripción Genética , Animales , Secuencia de Bases , Sitios de Unión , Northern Blotting , Núcleo Celular/fisiología , ADN/genética , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Mapeo Nucleótido , Especificidad de Órganos , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero/análisis , Homología de Secuencia de Ácido Nucleico
14.
Mol Cell Biol ; 10(11): 5883-93, 1990 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-2172788

RESUMEN

The induced differentiation of F9 cells by retinoic acid (RA) and cyclic AMP (cAMP) activated transcription of the tissue plasminogen activator (t-PA) gene. This differentiation-responsive regulation of the t-PA promoter was also observed in transient assays. Multiple sequence elements within 243 bp of t-PA DNA contributed to the high level of transcription in retinoic acid- and cyclic AMP-differentiated cells. To investigate the factors involved in controlling t-PA transcription upon differentiation, we used F9 cell extracts to examine proteins that bind two proximal promoter elements. These elements (boxes 4 and 5) are homologous to GC boxes that are known binding sites for transcription factor Sp1. Mobility shift assays in the presence and absence of anti-Sp1 antibodies demonstrated that the proteins which bound to this region were immunologically related to human Sp1. The proteins also had a DNA-binding specificity similar to that of a truncated form of Sp1. Mutations of the GC motif within boxes 4 and 5 that interfered with Sp1 binding reduced in parallel the binding of the F9 cellular factors and lowered transcription in vitro as well as in vivo. Although this proximal region of the t-PA promoter was active in vivo only in differentiated cells, the Sp1-like binding proteins were present in equal concentrations and had similar properties in extracts of both stem and differentiated cells. These data suggest that other cellular elements participate with this Sp1-like factor in controlling differentiation-specific expression.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Factor de Transcripción Sp1/metabolismo , Activador de Tejido Plasminógeno/genética , Tretinoina/farmacología , Animales , Secuencia de Bases , Línea Celular , AMP Cíclico/farmacología , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Teratoma
16.
Mol Cell Biol ; 9(4): 1691-704, 1989 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-2542775

RESUMEN

F9 cells induced to differentiate with retinoic acid (RA) increase transcription of the tissue plasminogen activator (t-PA) gene. Further treatment of these cells with cyclic AMP (cAMP) results in an additional stimulation of t-PA gene transcription. To investigate the mechanism of this two-stage regulation, 4 kilobase pairs (kbp) of 5'-flanking sequence from the murine t-PA gene was isolated. Two major start sites for transcription were found, neither of which depended on a classical TATA motif for correct initiation. By using transient transfection assays, it was determined that 4-kbp of flanking sequence could confer on reporter genes the same two-stage differentiation-specific expression as was observed for the endogenous t-PA gene. Deletion analyses of this 4-kbp fragment showed that 190 bp of flanking sequence was sufficient to bestow the same degree of two-stage regulation on reporter gene constructs. Within this region of DNA, sequence analysis revealed a possible cAMP regulatory element, a CTF/NF-1 recognition sequence, two potential Sp1 sites, and five potential binding sites for transcription factor AP-2. The deletion experiments, coupled with the positions of these potential cis-acting elements, suggest that multiple transcription factors, including those that bind to cAMP regulatory element, CTF/NF-1, Sp1, and AP-2 sites, may be involved in regulation of the t-PA gene during F9 cell differentiation.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , AMP Cíclico/farmacología , Activador de Tejido Plasminógeno/genética , Tretinoina/farmacología , Animales , Secuencia de Bases , Deleción Cromosómica , Mapeo Cromosómico , ADN de Neoplasias/genética , Regulación de la Expresión Génica , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/efectos de los fármacos , Teratoma/genética , Teratoma/patología , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/patología
17.
J Biol Chem ; 263(3): 1563-9, 1988 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-2826484

RESUMEN

Two tissue plasminogen activator (tPA) cDNA clones were isolated from mouse cDNA libraries and characterized. The sequence coding for mature mouse tPA protein including its 29-residue signal peptide, as well as that for the 3' and part of the 5'-nontranslated regions of the mRNA was determined. The mature protein consists of 530 amino acids and is encoded by a transcript of approximately 2800 nucleotides. The mouse tPA protein has 81% homology with its human counterpart, and amino acid sequence conservations suggest that the multidomain-like structure found for human tPA is maintained in the mouse enzyme. The tPA cDNA has been used as a probe to analyze tPA gene expression during F9 cell differentiation. tPA mRNA is not detected in F9 stem cells. When F9 cells are induced to differentiate with retinoic acid and dibutyryl cyclic AMP, tPA mRNA accumulates. We demonstrate that this induction occurs at least in part because of an increase in tPA gene transcription.


Asunto(s)
Clonación Molecular , ADN/metabolismo , Regulación de la Expresión Génica , ARN Mensajero/metabolismo , Teratoma/genética , Activador de Tejido Plasminógeno/genética , Animales , Bucladesina/farmacología , Diferenciación Celular , Ratones , Datos de Secuencia Molecular , Teratoma/metabolismo , Tretinoina/farmacología , Células Tumorales Cultivadas/metabolismo
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