Cell biology of tissue factor, the principal initiator of blood coagulation.
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Animals↗
Biomedical subjects
H Prydz
Publications and source records attributed to H Prydz.
Sequencing and analysis of a 35.4 kb region on the right [corrected] arm of chromosome IV from Saccharomyces cerevisiae reveal 23 open reading frames.
The complete DNA sequence of cosmid clone 31A5 containing a 35 452 bp segment from the right [corrected] arm of chromosome IV from Saccharomyces cerevisiae, was determined from an ordered set of subclones in combination with primer walking on the cosmid. The sequence contains 23 open reading frames (ORFs) of more than 100 amino acid residues and the tRNA-Va12a gene. Five ORFs corresponded to the known yeast genes SNQ2, SES1, GCV1, RPL2B and RPS18A. The DNA sequence for RPS18A is interrupted by an intron. One ORF corresponded to a part of the yeast gene HEX2 at the end of the cosmid insert. Four ORFs encoded putative proteins which showed strong homologies to other previously known proteins, three of yeast origin and one of non-yeast origin. Two ORFs were classified as having borderline homologies: one had similarity to two protein families and another to two protein products of unknown function from other species. The remaining 11 ORFs bore no significant similarity to any published protein.
Amino Acid Sequence↗
Notes on the cell biology of tissue factor.
Tissue factor is both a trigger of blood coagulation and a true receptor inducing an intracellular signal upon binding of its ligand factor VII to its extracellular parts. When induced in endothelial cells more than 75% of tissue factor antigen was found on the apical side of the cell.
Animals↗
Tissue specific expression of the human LCAT gene in transgenic mice and its effect on blood lipids.
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Animals↗
Structural organization and mapping of the human TCF11 gene.
We recently cloned human cDNA representing several mRNA isoforms of human TCF11, a transcription factor of the basic-region, leucine-zipper (bZIP) family located on chromosome 17q22 as well as a genomic clone of this gene. We have now determined the complete genomic organization of the TCF11 gene, which consists of 9 exons distributed over 15 kb of genomic DNA. Pulsed-field gel electrophoresis was used to construct a physical map around TCF11, to characterize a 450-kb YAC clone that contains the gene, and to link TCF11 physically to BTR, a marker on chromosome 17.
Amino Acid Sequence↗
Sequencing of the rat light neurofilament promoter reveals differences in methylation between expressing and non-expressing cell lines, but not tissues.
The DNA methylation pattern of the promoter (pNF-L) region of the rat light-neurofilament-encoding gene (NF-L), a neuron-specific gene, was assessed in NF-L expressing and non-expressing cell lines and tissues by genomic sequencing using PCR amplification of bisulfite-modified DNA. We analysed twenty-five potential CpG methylation sites between nucleotide (nt) positions -311 and +103 of pNF-L, containing Sp1- and AP-2-binding sites, a CGCCCCCGC box and a cAMP-responsive element. Six out of 25 possible CpG methylation sites are within these elements. The pNF-L promoter was unmethylated in NF-L-expressing rat brain, as well as in liver not expressing NF-L. In NF-L-expressing PC12 cells, the promoter was unmethylated, whereas in non-expressing glioma C6 cells intensive methylation occurred. A cluster of methylated CpG dinucleotides spanned the region from nt -176 to -67 bp. Thus, methylation of this promoter region could play a role in silencing NF-L in the glioma cell line in vitro, but not in liver tissue in vivo. In a non-CpG sequence, in the CpApG trinucleotide at nt position -114, cytosine was found to be partially methylated. It is thus possible to describe the methylation state of each cytosine present in the area of genomic DNA of interest.
Animals↗
Regulation of lecithin:cholesterol acyltransferase by TGF-beta and interleukin-6.
The human hepatoma derived HepG2 cells were treated with transforming growth factor-beta (TGF-beta) or interleukin-6 (IL-6) +/- dexamethasone. The effects of treatment on lecithin:cholesterol acyltransferase (LCAT) catalytic activity and mRNA level as well as on the apolipoprotein A-I (apo A-I) mRNA level were determined. Both the LCAT activity in medium from treated HepG2 cells and the LCAT mRNA level were decreased by TGF-beta. There was no significant effect of IL-6 +/- dexamethasone, neither on the LCAT activity nor on LCAT mRNA levels. Treatment with dexamethasone alone resulted in a decreased LCAT activity in spite of a slight increase in LCAT mRNA level. The apo A-I mRNA level was reduced after treatment with TGF-beta and increased after treatment with IL-6 +/- dexamethasone and dexamethasone alone. To analyze if the effects on mRNA levels were caused by transcriptional or post-transcriptional mechanisms, run-on experiments on isolated nuclei from treated HepG2 cells and mRNA degradation experiments were performed. The transcription rate of the LCAT gene was not affected by TGF-beta, but was increased (50-100%) after treatment with IL-6 +/- dexamethasone and dexamethasone alone. The transcription rate of the apo A-I gene was reduced (20%) by TGF-beta and increased (30-60%) by IL-6 +/- dexamethasone and dexamethasone alone. Both dexamethasone and TGF-beta increased the rate of LCAT mRNA degradation. These results show that the reduced LCAT mRNA level after treatment with TGF-beta was caused by post-transcriptional mechanisms.
Apolipoprotein A-I↗
Binding of human factor VIIa to tissue factor induces cytosolic Ca2+ signals in J82 cells, transfected COS-1 cells, Madin-Darby canine kidney cells and in human endothelial cells induced to synthesize tissue factor.
Tissue factor (TF) is the most potent trigger of blood clotting known. It activates factor VII (FVII) thereby initiating a cascade of proteolytic reactions resulting in thrombin production. The cloning of TF revealed its structural characteristics to be those of a receptor related to the class 2 cytokine receptor superfamily, but until now no intracellular signal has been discovered related to binding of the ligand (FVIIa) to the putative receptor. We have studied possible intracellular signaling effects of the FVIIa-TF interaction by measuring cytosolic free Ca2+ in single fura-2-loaded cells and found that 200 nM FVIIa caused Ca2+ transients in about 30% of human umbilical vein endothelial cells treated with interleukin-1 beta to express TF, compared to below 5% in uninduced cells. A gradual increase of the basal Ca2+ level was also caused by binding of FVIIa. In the human bladder carcinoma cell line J82, which has a high constitutive TF activity, similar results were found. An antibody neutralizing TF activity decreased the response rate to control levels. COS-1 cells which do not make TF did not respond to FVIIa as opposed to COS-1 cells expressing TF after transfection with a human TF cDNA construct. The canine kidney cell line MDCK, a constitutive TF producer, responded especially well; up to 100% of the cells examined showed Ca2+ oscillations which were dose dependent with regard to frequency, latency, maximal amplitude, and recruitment of responding cells. The frequency was reduced by inhibition of Ca2+ influx with 100 microM LaCl3. In confluent MDCK cells the Ca2+ oscillations were synchronous, constituting the first evidence of a synchronous cytosolic Ca2+ oscillator generated by global application of agonist. Thus, TF mediates a cytosolic Ca2+ signal upon interaction with its ligand FVIIa, thereby suggesting a more complex biological role for TF.
Animals↗
Physical linkage of the gene cluster containing the LCAT gene to the DNA marker D16S124 at human chromosome region 16q22.1.
Pulsed-field gel electrophoresis has been used to construct a long-range restriction map spanning more than 900 kb in the q22.1 region of human chromosome 16. The gene cluster containing the lecithin:cholesterol acyl transferase (LCAT) gene is located less than 480 kb from the anonymous DNA marker D16S124 in this map. The results suggest three putative CpG islands within 125 kb, in addition to the island previously shown to be located within the gene cluster. This implies a clustering of both genes and CpG islands in this chromosomal region.
Chromosome Mapping↗
Evaluation of the exon predictions of the GRAIL software.
Identification of transcribed sequences within genomic regions has been a major rate-limiting step in the pursuit of genes. We have tested the ability of GRAIL 1 and GRAIL 2 to locate coding exons in the 41 longest human genomic sequences available in release 38 of the EMBL database. Of the total annotated exons GRAIL 1 correctly identified 68% of coding exons. GRAIL 2 correctly identified 71%. Of the 686 predictions made by GRAIL 1, 46.4% did not correspond to an annotated exon. The corresponding figures for GRAIL 2 were 556 and 30.5%. The GRAIL 2 software predicted as excellent a significantly higher proportion of the coding exons than did GRAIL 1, and also gave a significantly lower figure for false predictions. GRAIL 2 thus represents a significant improvement over GRAIL 1.
Exons↗
1 kb of the lactase-phlorizin hydrolase promoter directs post-weaning decline and small intestinal-specific expression in transgenic mice.
Adult-type hypolactasia is a genetic condition making approximately one half of the human population intolerant to milk because of abdominal symptoms. The cause is a post-weaning down-regulation of the intestinal-specific enzyme lactase-phlorizin hydrolase (LPH) reducing the intestinal capacity to hydrolyze lactose. We here demonstrate that the stretch -17 to -994 in the pig LPH-promoter carries cis-elements which direct a small intestinal-specific expression and a post-weaning decline of a linked rabbit beta-globin gene. These data demonstrate that the post-weaning decline of LPH is mainly due to a transcriptional down-regulation.
Age Factors↗
Molecular cloning of a putative novel human bZIP transcription factor on chromosome 17q22.
We have cloned and characterized cDNA clones representing several mRNA isoforms generated by alternative splicing of a single gene localized to chromosome 17q22. Sequence analysis showed that the predicted translational product of the longest open reading frame (2316 nucleotides, 772 amino acids) is related to transcription factors of the basic leucine zipper (bZIP) class. The sequence contained several regions characteristic of transcriptional regulatory domains. A cluster of amino acids flanking the bZIP region on both sides was highly conserved between TCF11 and p45 NF-E2, a subunit of the human globin locus control region-binding protein, NF-E2. These same regions showed remarkable homology to two invertebrate proteins, CNC and skin-1, postulated to regulate embryonic development in Drosophila melanogaster and Caenorhabditis elegans, respectively.
Amino Acid Sequence↗
Mice transgenic for the human LCAT gene.
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Animals↗
Polar expression of tissue factor in human umbilical vein endothelial cells.
Endothelial cells grown on filters developed junctional complexes that reduced diffusional transport and increased electrical resistance over the cell layer. Induction of tissue factor by recombinant interleukin-1 beta led to a highly polarized tissue factor expression on the apical cell surface only. After prolonged growth to allow deposition of matrix, removal of the endothelial cells by collagenase or by 0.1 mol/L NH4OH left behind some cellular material as well as tissue factor, which was only detectable in the upper compartment. A human bladder carcinoma cell line, which does not form tight junctions and expresses tissue factor constitutively, showed essentially no polarity. Endothelial cell secretory compounds like von Willebrand factor, tissue plasminogen activator, and plasminogen activator inhibitor-1 were constitutively released to both sides. The added secretion due to recombinant interleukin-1 beta stimulation of the endothelial cells observed for von Willebrand factor and tissue plasminogen activator was, however, localized to the apical surface. The availability of tissue factor on the luminal surface of endothelial cells, ie, allowing contact with factor VII in the flowing blood, has potentially very significant pathophysiological consequences.
Cell Polarity↗
An improved method for detection of 5-methylcytosine by PCR-based genomic sequencing.
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5-Methylcytosine↗
A tight cluster of five unrelated human genes on chromosome 16q22.1.
A locus on human chromosome 16q22.1 contains at least five tightly clustered genes which are unrelated by sequence homology and apparently unrelated by function. The genes for a putative proteasome subunit (MECL 1), a chymotrypsin-like protease (CTRL), a protein serine kinase (PSKH1), the previously cloned lecithin:cholesterol acyl transferase (LCAT) and a protein of unknown function are found within 40 kb of genomic DNA. Exons from the former four genes are located within a 12 kb region including a CpG island associated with the putative proteasome gene. Three of the genes are widely expressed, whereas the genes for the protease and LCAT are highly tissue specific. The distance between the transcriptional units of the gene upstream of LCAT and LCAT is only 199 bp. Alternative polyadenylation of the protease transcripts creates a transcription unit which overlaps with the oppositely oriented kinase gene. The selective advantage of this unusual gene clustering may involve transcriptional interference(s) and coregulatory events not yet understood. Given the current estimate of about 100,000 genes in the human genome, our findings support the notion that genes are not evenly distributed.
Amino Acid Sequence↗
A methylated CpG island 3' in the apolipoprotein-E gene does not repress its transcription.
CpG islands are always associated with the 5' end of housekeeping genes, covering their promoters and transcription start sites. CpG islands associated with genes of limited expression are less uniformly localized; the genes for apolipoprotein-E and -AI contain CpG islands corresponding to their last exons. As expected, the CpG island in the apo-AI gene is unmethylated in DNA from all tissues analyzed, expressing as well as non-expressing apolipoprotein-AI. In contrast, the apo-E CpG island is methylated in DNA from all tissues analyzed except sperm. The apo-E gene is transcribed in many tissues and is not repressed by this methylation. This establishes a functional difference between 5' and 3' CpG islands, because methylation of the former invariably leads to transcriptional repression. A similar methylation pattern was seen in the rat apo-E gene, which implies that this pattern probably was established before the divergence of rodents and primates. The numerous human apo-E alleles resulting from CpG to TpG/CpA mutations in the CpG island (i.e. deamination of methylated cytosine to thymine) suggest that this island is less protected from methylation in germ line than typical CpG islands.
Alleles↗
The genetic defect of the original Norwegian lecithin:cholesterol acyltransferase deficiency families.
Three of the original Norwegian lecithin:cholesterol acyltransferase (LCAT) deficiency families have been investigated for mutations in the gene for lecithin:cholesterol acyltransferase by DNA sequencing of the exons amplified by the polymerase chain reaction. A single T----A transversion in codon 252 in exon 6 converting Met(ATG) to Lys(AAG) was observed in all homozygotes. In spite of the identical mutation, the disease phenotypes differed in severity. This was not reflected in the expression of LCAT in the heterozygotes.
Female↗