RESUMEN
Under normal physiologic conditions, tissue factor pathway inhibitor (TFPI) is synthesized primarily by the microvascular endothelium. Using Northern blotting, we studied its transcriptional expression in different organs and compared it with the expression of two other endothelial specific proteins, namely thrombomodulin (TM) and von Willebrand factor (vWF). The order of mRNA expression for each protein was: TFPI-placenta>lung>liver>kidney>heart>skeletal muscle> or =pancreas>brain; TM-heart>pancreas>lung>skeletal muscle>kidney> or =liver>placenta>brain; and vWF-heart>skeletal muscle>pancreas>lung> or =kidney>placenta>brain>liver. Notably, heart expressed TM and vWF mRNA in large amounts and only small amounts of TFPI whereas lung expressed all three mRNAs in significant amounts. Placenta, on the contrary, expressed large amounts of TFPI but only small amounts of TM and vWF mRNAs. Brain by this technique was found to express undetectable amounts of TFPI and TM mRNAs but small amounts of vWF mRNA. The expression of TFPI mRNA in the brain was however detected by RT/PCR and the antigen was localized to the endothelium of microvessels as well as to the astrocytes and oligodendrocytes. Since ultimate expression of proteins is linked to the expression of their mRNAs, our data support a concept that vascular endothelium is made up of phenotypically diverse groups of cells and that endothelial cells of different vascular beds express specific sets of genes that enable them to carry out tissue-specific functions. Importantly, since astrocytes are known to express tissue factor, the TFPI expression by these cells may control coagulation in their microenvironment and their response to injury and inflammation.
Asunto(s)
Lipoproteínas/genética , Trombomodulina/genética , Factor de von Willebrand/genética , Encéfalo/metabolismo , Endotelio Vascular/metabolismo , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Lipoproteínas/metabolismo , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución TisularRESUMEN
Tissue factor pathway inhibitor (TFPI) plays an important role in regulating tissue factor (TF)-initiated blood coagulation. Since serum stimulation of fibroblasts, vascular smooth muscle cells and cardiac myocytes in culture increases the expression of TF mRNA and antigen (Ag) in these cells, we hypothesized that serum may also induce increased synthesis of TFPI in these cells to regulate the TF-induced extravascular clotting at an injury site. To test this concept, we used primary isolates of the following human cell types - fetal and adult lung fibroblasts, pulmonary and aortic smooth muscle cells, and cardiac myocytes. Serum-stimulation of these cells resulted in an increased expression of TF mRNA and Ag (8 to 10-fold). Upon serum stimulation, expression of TFPI mRNA and Ag was also increased in these cells. However, the increase in TFPI-Ag (6 to 8-fold) was significantly greater than the TFPI mRNA (2 to 3-fold). Notably, increased expression of TFPI persisted after the TF expression had declined. Further, increased synthesis of TFPI initially led to the saturation of heparin-releasable binding sites. TFPI-Ag was detected by Western blotting, 35S-metabolic labeling and activity assays on the conditioned media, heparin-released material from cells, and in cell lysates. TFPI-Ag was also detected by immunofluorescence staining of cells. Actinomycin D partially whereas cycloheximide completely prevented the serum-induced increased expression of TFPI synthesis by these cells, suggesting control primarily at the translational but some at the transcriptional level as well. The Mr of undegraded TFPI in all cases was approximately 45 kDa and was of full length. TFPI synthesized locally by fibroblasts, vascular smooth muscle cells and cardiac myocytes could play a significant role in regulating TF-initiated extravascular clotting especially since plasma TFPI that may be available at the injury site lacks a portion of the carboxyl segment and is a less efficient inhibitor.
Asunto(s)
Fibroblastos/metabolismo , Lipoproteínas/biosíntesis , Músculo Liso Vascular/metabolismo , Miocardio/metabolismo , Adulto , Proteínas Sanguíneas , Células Cultivadas , Técnica del Anticuerpo Fluorescente Indirecta , HumanosRESUMEN
Tissue factor pathway inhibitor (TFPI) is the primary physiologic inhibitor of tissue factor-induced clotting. The TFPI gene contains three GATA motifs in the region flanking its transcription initiation sites. GATA motifs present in promoters of other genes bind GATA-2 transcription factor and thereby regulate their transcriptional expression. Both TFPI and GATA-2 transcription factor are synthesized by a variety of normal as well as malignant cells including hepatocellular carcinoma HepG2 and bladder carcinoma ECV304. Here, we studied whether the three GATA motifs flanking the transcription initiation sites regulate TFPI gene expression in HepG2 and ECV304 cells by binding to the GATA-2 transcription factor. Synthetic oligonucleotides containing GATA sequences from the TFPI regulatory region formed DNA-protein complexes with HepG2 and ECV304 nuclear extracts in an electrophoretic mobility shift assay. Using a 740-bp fragment (-496/+244) from TFPI regulatory region, the effect of base substitutions at each of the three GATA motifs was studied in a luciferase reporter gene system. TFPI promoter activity in HepG2 cells was increased 3-fold with mutation in one of the three GATA motifs and in ECV304 cells was essentially unchanged with mutations in all three GATA motifs. Thus, GATA motifs appear to serve a tissue-specific regulatory role in TFPI gene expression in malignant cells.
Asunto(s)
Lipoproteínas/genética , Neoplasias/genética , Proteínas de Unión al ADN/genética , Factor de Transcripción GATA2 , Regulación Neoplásica de la Expresión Génica , Humanos , Lipoproteínas/biosíntesis , Factores de Transcripción/genética , Células Tumorales Cultivadas , Dedos de ZincAsunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Lipoproteínas/genética , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , Proteínas de Unión al ADN/metabolismo , Exones , Regulación de la Expresión Génica , Genes , Humanos , Datos de Secuencia Molecular , Factores de Transcripción NFI , Proteínas Nucleares , Reacción en Cadena de la Polimerasa , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/metabolismo , Proteína 1 de Unión a la Caja YRESUMEN
We have previously described oncolytic adenovirus (Ad) vectors KD3 and KD3-interferon (IFN) that were rendered cancer-specific by mutations in the E1A region of Ad; these mutations abolish binding of E1A proteins to p300/CBP and pRB. The antitumor activity of the vectors was enhanced by overexpression of the Adenovirus Death Protein (ADP, E3-11.6K) and by replication-linked expression of IFN-alpha. We hypothesized that the anticancer efficacy of the KD3-IFN vector could be further improved by expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). E1-deleted Ad vectors were constructed carrying reporter genes for enhanced green fluorescent protein or secreted placental alkaline phosphatase (SEAP) and a therapeutic gene for TRAIL under control of the TetON system. Expression of the genes was increased in the presence of a helper virus and the inducer doxycycline such that up to 231-fold activation of expression for the TetON-SEAP vector was obtained. Coinfection with TetON-TRAIL augmented oncolytic activity of KD3 and KD3-IFN in vitro. Induction of TRAIL expression did not reduce the yield of progeny virus. Combination of TetON-TRAIL and KD3-IFN produced superior antitumor activity in vivo as compared with either vector alone demonstrating the efficacy of a four-pronged cancer gene therapy approach, which includes Ad oncolysis, ADP overexpression, IFN-alpha-mediated immunotherapy, and pharmacologically controlled TRAIL activity.
Asunto(s)
Adenoviridae/genética , Proteínas E3 de Adenovirus/genética , Terapia Genética , Vectores Genéticos , Interferón-alfa/genética , Neoplasias/terapia , Ligando Inductor de Apoptosis Relacionado con TNF/biosíntesis , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Adenoviridae/fisiología , Proteínas E3 de Adenovirus/biosíntesis , Proteínas E3 de Adenovirus/fisiología , Animales , Apoptosis/genética , Línea Celular Tumoral , Doxiciclina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Interferón-alfa/biosíntesis , Interferón-alfa/fisiología , Ratones , Mutación , Neoplasias/genética , Neoplasias/patología , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Tetraciclina/farmacologíaRESUMEN
The E1a region of adenoviruses encodes two early proteins of 289 amino acids (289R) and 243R from two differentially spliced mRNAs of 13 and 12 S, respectively. These E1a proteins of adenoviruses are multifunctional and have been shown to play an essential role in cellular immortalization and transformation. The E1a gene is also known to regulate the expression of certain viral and cellular genes in a positive or negative manner. To identify the domains of the E1a proteins required for their transformation and transcriptional regulatory functions, we have constructed and analyzed several E1a mutations. A region located between amino acid residues 125 and 127 appears to be essential for cell transformation in cooperation with both E1b and the activated cellular oncogene, T24 Har-ras. Mutation at this region does not affect the ability of E1a to trans-activate the Ad2 early E2 promoter significantly. Our experiments have not revealed the domain of E1a most essential for trans-repression. However, the region between amino acid residues 125 and 127 appears to have a small effect on E1a-mediated repression of the immunoglobulin heavy-chain and polyoma enhancers but has no significant effect on the SV40 enhancer. From our results, it appears that the transformation function of E1a can be dissociated from its transcriptional regulatory functions.
Asunto(s)
Transformación Celular Viral , Proteínas Oncogénicas Virales/genética , Oncogenes , Factores de Transcripción/genética , Proteínas Precoces de Adenovirus , Animales , Células Cultivadas , Regulación de la Expresión Génica , Células HeLa , Mutación , Regiones Promotoras Genéticas , Ratas , Relación Estructura-Actividad , Transcripción GenéticaRESUMEN
Human plasma contains a factor Xa-dependent inhibitor of tissue factor/factor VIIa complex termed lipoprotein-associated coagulation inhibitor (LACI). The present study examines the site(s) of LACI synthesis. In this study, cultured hepatocytes isolated from normal human liver were found to be essentially negative in LACI mRNA as revealed by Northern blot analysis using a full-length LACI cDNA as probe. The conditioned media from these cultures were also essentially negative for LACI activity. Similarly, poly(A)+ RNA obtained from normal human liver did not contain detectable LACI mRNA. In contrast, cultured human umbilical vein endothelial cells and human lung tissue (rich in endothelium) both contained abundant amounts of LACI mRNA. Moreover, erythrocyte lysates and culture media from normal monocytes, lymphocytes, or neutrophils did not contain measurable LACI activity; these cells were also negative for LACI mRNA. Platelets, however, contained LACI activity. The likely source of platelet LACI is the megakaryocyte cell since a megakaryocyte cell line (MEG-01) was found to contain LACI mRNA and to secrete small amounts of LACI activity. Additionally, human vascular smooth muscle cells and lung fibroblasts were also found to synthesize only small amounts of LACI. From these observations, we conclude that normal liver does not synthesize LACI and that endothelium is the principal source of plasma LACI. The undegraded LACI synthesized by endothelial cells had a molecular weight of approximately 41,000.
Asunto(s)
Endotelio Vascular/metabolismo , Factor VII/antagonistas & inhibidores , Lipoproteínas/biosíntesis , Hígado/metabolismo , Tromboplastina/antagonistas & inhibidores , Secuencia de Bases , Northern Blotting , Línea Celular , Clonación Molecular , ADN/genética , Factor VII/biosíntesis , Factor VII/metabolismo , Humanos , Lipoproteínas/metabolismo , Hígado/citología , Datos de Secuencia Molecular , Oligonucleótidos , Reacción en Cadena de la Polimerasa , Tromboplastina/biosíntesis , Tromboplastina/metabolismoRESUMEN
We recently proposed that endothelium may represent the primary physiologic site of synthesis of the tissue factor pathway inhibitor (TFPI). In support of this conclusion, we have now found that the poly(A)+ RNAs obtained from rabbit and bovine lung tissues contain abundant amounts of TFPI messenger RNAs (mRNAs), whereas the poly(A)+ RNAs obtained from the liver of these animals contain less than 5% of that found in the lung tissues. Because inflammatory mediators are known to upregulate tissue factor (TF) expression by the endothelium, we have examined the effect of these agents on the TFPI expression by the cultured endothelial cells. When cultured human umbilical vein endothelial cells were stimulated (in 10% fetal bovine serum) with phorbol myristate acetate (PMA), endotoxin, interleukin-1, or tumor necrosis factor-alpha, the TF mRNA increased approximately 7- to 10-fold within 2 to 4 hours. Unstimulated cells constitutively expressed TFPI mRNA and its levels either did not change or increased slightly (up to 1.5-fold) upon stimulation with these inflammatory agents. TF mRNA abruptly declined to a negligible level and the TFPI mRNA returned essentially to the basal level at approximately 24 hours. The membrane-bound TF clotting activity of induced cells peaked between 4 and 8 hours, and finally declined. The cumulative TFPI activity secreted into the media was either unchanged or slightly higher in the induced cell cultures as compared with that present in the noninduced cultures. Endothelial cells were also cultured in 10% heat-inactivated human serum derived from plasma or whole blood. TFPI secreted into the media containing whole blood serum was consistently higher (approximately 1.5-fold at 8 hours) than that secreted into the media supplemented with serum obtained from plasma lacking the formed elements; these cells also expressed similarly increased levels of TFPI mRNA. Moreover, PMA-stimulated cells cultured in whole blood serum expressed modestly increased levels of TFPI mRNA (approximately 1.5-fold); supernatants from these cells also contained similarly increased TFPI activity. Cumulatively, our data indicate that, unlike thrombomodulin and fibrinolytic enzymes synthesized by the endothelial cells, TFPI synthesis is not downregulated and may be slightly upregulated during an inflammatory response. Inspection of the 5' flanking region of the TFPI gene showed a conserved GATA-binding motif located approximately 400 bp upstream of the proposed transcription initiation site(s). This motif by binding to the GATA-2 transcriptional factor may keep the endothelium in an 'on' state for constitutive expression of TFPI.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Endotelio Vascular/metabolismo , Endotoxinas/farmacología , Factor VII/antagonistas & inhibidores , Expresión Génica , Interleucina-1/farmacología , Lipoproteínas/genética , Acetato de Tetradecanoilforbol/farmacología , Tromboplastina/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacología , Animales , Secuencia de Bases , Sangre , Northern Blotting , Bovinos , Línea Celular , Células Cultivadas , Factor VII/genética , Humanos , Inflamación/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Conejos , Tromboplastina/genética , Venas UmbilicalesRESUMEN
In this report, we describe an approach to detect the presence of abnormal alleles in those genetic diseases in which frequency of occurrence of the same mutation is high (e.g., cystic fibrosis and sickle cell disease), and in others in which multiple mutations cause the disease and the sequence variation in an affected member of a given family is known (e.g., hemophilia B). Initially, from each subject, the DNA fragment containing the putative mutation site is amplified by the polymerase chain reaction. For each fragment two reaction mixtures are then prepared. Each contains the amplified fragment, a primer (18-mer or longer) whose sequence is identical to the coding sequence of the normal gene immediately flanking the 5' end of the mutation site, and either an alpha-32P-labeled nucleotide corresponding to the normal coding sequence at the mutation site or an alpha-32P-labeled nucleotide corresponding to the mutant sequence. Single nucleotide primer extensions are then carried out and analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. As predicted by the Watson-Crick base-pair rule, in the wild type only the normal base, in an affected member only the mutant base, and in carriers both the normal and the mutant base are incorporated into the primer. Thus, an essential feature of the present methodology is that the base immediately 3' to the template-bound primer is one of those altered in the mutant, since in this way an extension of the primer by a single base will give an extended molecule characteristic of either the mutant or the wild type. The method is rapid and should be useful in carrier detection and prenatal diagnosis of every genetic disease with a known sequence variation.
Asunto(s)
Fibrosis Quística/genética , Factor IX/genética , Hemofilia A/genética , Mutación , Alelos , Secuencia de Bases , Exones , Femenino , Tamización de Portadores Genéticos , Genotipo , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Factor IX is a multidomain protein essential for hemostasis. We describe a mutation in a patient affecting the first epidermal growth factor (EGF)-like domain of the protein. All exons and the promoter region of the gene were amplified by the polymerase chain reaction method, and sequenced. Only a single mutation (C----G), that predicts the substitution of Pro55 by Ala in the first EGF domain was found in the patient's gene. This mutation leads to new restriction sites for four enzymes. One new site (Nsi) was tested in the amplified exon IV fragment and was shown to provide a rapid and reliable marker for carrier detection and prenatal diagnosis in the affected family. The factor IX protein, termed factor IXHollywood (IXHW), was isolated to homogeneity from the patient's plasma. As compared with normal factor IX (IXN), IXHW contained the same amount of gamma-carboxy glutamic acid but twice the amount of beta-OH aspartic acid. Both IXHW and IXN contained no detectable free -SH groups. Further, IXHW could be readily cleaved to yield a factor IXa-like molecule by factor Xla/Ca2+. However, IXaHW (compared with IXaN) activated factor X approximately twofold slower in the presence of Ca2+ and phospholipid (PL), and 8- to 12-fold slower in the presence of Ca2+, PL, and factor VIIIa. Additionally, IXaHW had only approximately 10% of the activity of IXaN in an aPTT assay. In agreement with the nuclear magnetic resonance-derived structure of EGF, the Chou-Fasman algorithm strongly predicted a beta turn involving residues Asn-Pro55-Cys-Leu in IXN. Replacement of Pro55 by Ala gave a fourfold decrease in the beta turn probability for this peptide, suggesting a change(s) in the secondary structure in the EGF domain of IXHW. Since this domain of IXN is thought to have one high-affinity Ca2+ binding site and may be involved in PL and/or factor VIIIa binding, the localized secondary structural changes in IXHW could lead to distortion of the binding site(s) for the cofactor(s) and, thus, a dysfunctional molecule.