Cell type–specific regulation of von Willebrand factor expression by the E4BP4 transcriptional repressor

Blood ◽  
2005 ◽  
Vol 105 (4) ◽  
pp. 1531-1539 ◽  
Author(s):  
Christine Hough ◽  
Carla D. Cuthbert ◽  
Colleen Notley ◽  
Christine Brown ◽  
Carol Hegadorn ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Promoter Activity ◽  
Transcriptional Repressor ◽  
Binding Motif ◽  
Cell Type ◽  
Response Element ◽  
Cell Type Specific ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractMechanisms of tissue-restricted patterns of von Willebrand factor (VWF) expression involve activators and repressors that limit expression to endothelial cells and megakaryocytes. The relative transcriptional activity of the proximal VWF promoter was assessed in VWF-producing and -nonproducing cells, and promoter activity was highest in endothelial cells followed by megakaryocytes. Only basal VWF promoter activity was seen in nonendothelial cells. Here we identify a negative response element located at nucleotides (nts) +96/+105 and demonstrate, using chromatin immunoprecipitation (ChIP) analysis, that in vivo this sequence interacts with the E4BP4 transcriptional repressor. Differences in size and relative abundance of nuclear E4BP4 were observed. In HepG2 cells, low levels of larger forms of E4BP4 are present that directly interact with the negative response element. In VWF-expressing cells, high levels of smaller forms predominate with no evidence of direct DNA binding. However, in endothelial cells, mutation of the VWF E4BP4 binding motif not only restores but also further elevates VWF promoter activity, suggesting that E4BP4 may be part of a coordinated binding complex. These observations implicate this binding motif in repressing both activated and basal levels of VWF transcription by different cell type–specific mechanisms, and support the hypothesis that E4BP4 sequesters negative regulators of transcription, thereby enhancing activated gene expression.

scholarly journals Ionizing irradiation increases transcription of the von Willebrand factor gene in endothelial cells

Blood ◽  
1996 ◽  
Vol 88 (10) ◽  
pp. 3801-3814 ◽  
Author(s):  
N Jahroudi ◽  
AM Ardekani ◽  
JS Greenberger
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Promoter Activity ◽  
Core Promoter ◽  
Irradiation Damage ◽  
Mrna Levels ◽  
Ionizing Irradiation ◽  
Von Willebrand ◽  
Willebrand Factor

Ionizing irradiation damage to the vasculature results in an increase in procoagulant activity of endothelial cells, including elevated von Willebrand factor (vWf) secretion. We investigated the mechanism of irradiation induction of vWf release and demonstrated that vWf mRNA levels were increased when either human or bovine endothelial cells were exposed to 20 Gy irradiation. This response to irradiation was independent to de novo protein synthesis, but required new transcription. Nuclear run-on experiments indicated that increased vWf transcriptional activity was partly responsible for the higher levels of the mRNA accumulation. Transfection analyses with plasmids in which a human growth hormone structural gene was under the control of the endothelial-cell-specific vWf promoter demonstrated that irradiation increased promoter activity. Deletion analyses demonstrated that sequences necessary for irradiation induction of the promoter activity were located within the 112-bp sequences (-90 to +22) that constitute the non-endothelial-cell-specific core promoter region of the vWf gene. Results of gel mobility assays and deletion analyses demonstrated that a site in the vWf promoter other than the putative NF-kB binding site is involved in the mechanism of irradiation induction of the vWf.

HUMAN HEPATIC ENDOTHELIAL CELLS AND HEPATOCYTES IN CULTURE: MORPHOLOGICAL FEATURES, AND PRODUCTION OF VON WILLEBRAND FACTOR AND FIBRINOGEN

1987 ◽  
Author(s):  
R Harrison
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Human Liver ◽  
Liver Cells ◽  
Mixed Population ◽  
Dependent Manner ◽  
Cell Type ◽  
Von Willebrand ◽  
Willebrand Factor

Liver cells were derived from cadaveric organ donors. Pieces of human liver 5 to 50 grams were minced, washed, and incubated in collagenase at 37 degrees C. After washing, the cell suspension was plated into culture vessels that had been briefly pre-treated with an extract derived from human liver. A mixed population of liver cells, including endothelial cells, hepatocytes, and Kupffer cells, attached within hours. At the end of 2 to 3 weeks there developed clusters of densely packed cells of two types. The most numerous cells were initially fusiform but grew as a monolayer even when densely packed. As density increased they assumed a polygonal form; cells with this morphological appearance stained immunocytochemically for von Willebrand factor antigen. They were relatively small and resembled cells derived from human umbilical vein except that the cytoplasm was more filmy in appearance. The second prominent cell type was significantly larger and likewise replicated to form clusters. These large cells sometimes contained multiple nuclei, exhibited a relatively low nuclear to cytoplasmic ratio, and immunocytochemically stained for human fibrinogen. A more distinct nuclear membrane and prominent nucleoli were characteristics of hepatocytes that were useful light microscopically in distinguishing these cells from sinusoidal endothelial cells. Ultrastructurally, endothelial cells were characterized by small size, holes in and among the cells that probably were the in vitro analogue of fenestrae, and numerous Weibel-Palade bodies in the cytoplasm, which otherwise was relatively bland. Hepatocytes, by contrast, had an active appearing cytoplasm containing more organelles. Canaliculi and typical tight junctions formed between adjacent hepatocytes. Levels of vWF and fibrinogen increased in a time dependent manner in media overlying this mixed population of cells. Human factor VIII has not yet been detected in the media overlying these mixed cells derived from human liver, and factor VIII antigen has not yet been demonstrable immunocytochemically in either cell type.

Requirement for Both D Domains of the Propolypeptide in von Willebrand Factor Muitimerization and Storage

1993 ◽  
Vol 70 (06) ◽  
pp. 1053-1057 ◽  
Author(s):  
Agnès M Journet ◽  
Simin Saffaripour ◽  
Denisa D Wagner
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Von Willebrand Factor ◽  
Deletion Mutants ◽  
Relative Importance ◽  
D2 Domain ◽  
Constitutive Secretion ◽  
Von Willebrand ◽  
And Storage ◽  
Willebrand Factor

SummaryBiosynthesis of the adhesive glycoprotein von Willebrand factor (vWf) by endothelial cells results in constitutive secretion of small multimers and storage of the largest multimers in rodshaped granules called Weibel-Palade bodies. This pattern is reproduced by expression of pro-vWf in heterologous cells with a regulated pathway of secretion, that store the recombinant protein in similar elongated granules. In these cells, deletion of the vWf prosequence prevents vWf storage. The prosequence, composed of two homologous domains (D1 and D2), actively participates in vWf multimer formation as well. We expressed deletion mutants lacking either the D1 domain (D2vWf) or the D2 domain (D1vWf) in various cell lines to analyze the relative importance of each domain in vWf muitimerization and storage. Both proteins were secreted efficiently without being retained in the endoplasmic reticulum. Despite this, neither multimerized past the dimer stage and they were not stored. We conclude that several segments of the prosequence are jointly involved in vWf muitimerization and storage.

Adverse Influence of Cigarette Smoking on the Endothelium

1993 ◽  
Vol 70 (04) ◽  
pp. 707-711 ◽  
Author(s):  
Andrew D Blann ◽  
Charles N McCollum
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Tobacco Smoke ◽  
Lipid Peroxides ◽  
Short Exposure ◽  
Acute Effects ◽  
Adverse Influence ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe effect of smoking on the blood vessel intima was examined by comparing indices of endothelial activity in serum from smokers with that from non-smokers. Serum from smokers contained higher levels of von Willebrand factor (p <0.01), the smoking markers cotinine (p <0.02) and thiocyanate (p <0.01), and was more cytotoxic to endothelial cells in vitro (p <0.02) than serum from non-smokers. The acute effects of smoking two unfiltered medium tar cigarettes was to briefly increase von Willebrand factor (p <0.001) and cytotoxicity of serum to endothelial cells in vitro (p <0.005), but lipid peroxides or thiocyanate were not increased by this short exposure to tobacco smoke. Although there were correlations between von Willebrand factor and smokers consumption of cigarettes (r = 0.28, p <0.02), number of years smoking (r = 0.41, p <0.001) and cotinine (r = 0.45, p <0.01), the tissue culture of endothelial cells with physiological levels of thiocyanate or nicotine suggested that these two smoking markers were not cytotoxic. They are therefore unlikely to be directly responsible for increased von Willebrand factor in the serum of smokers. We suggest that smoking exerts a deleterious influence on the endothelium and that the mechanism is complex.

Epinephrine Induces von Willebrand Factor Release from Cultured Endothelial Cells: Involvement of Cyclic AMP-dependent Signalling in Exocytosis

1997 ◽  
Vol 77 (06) ◽  
pp. 1182-1188 ◽  
Author(s):  
Ulrich M Vischer ◽  
Claes B Wollheinn
Keyword(s):  
Endothelial Cells ◽  
Adenylate Cyclase ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Free Calcium ◽  
Camp Content ◽  
Von Willebrand ◽  
Willebrand Factor

Summaryvon Willebrand factor (vWf) is released from endothelial cell storage granules after stimulation with thrombin, histamine and several other agents that induce an increase in cytosolic free calcium ([Ca2+]i). In vivo, epinephrine and the vasopressin analog DDAVP increase vWf plasma levels, although they are thought not to induce vWf release from endothelial cells in vitro. Since these agents act via a cAMP-dependent pathway in responsive cells, we examined the role of cAMP in vWf secretion from cultured human umbilical vein endothelial cells. vWf release increased by 50% in response to forskolin, which activates adenylate cyclase. The response to forskolin was much stronger when cAMP degradation was blocked with IBMX, an inhibitor of phosphodiesterases (+200%), whereas IBMX alone had no effect. vWf release could also be induced by the cAMP analogs dibutyryl-cAMP (+40%) and 8-bromo-cAMP (+25%); although their effect was weak, they clearly potentiated the response to thrombin. Epinephrine (together with IBMX) caused a small, dose-dependent increase in vWf release, maximal at 10-6 M (+50%), and also potentiated the response to thrombin. This effect is mediated by adenylate cyclase-coupled β-adrenergic receptors, since it is inhibited by propranolol and mimicked by isoproterenol. In contrast to thrombin, neither forskolin nor epinephrine caused an increase in [Ca2+]j as measured by fura-2 fluorescence. In addition, the effects of forskolin and thrombin were additive, suggesting that they act through distinct signaling pathways. We found a close correlation between cellular cAMP content and vWf release after stimulation with epinephrine and forskolin. These results demonstrate that cAMP-dependent signaling events are involved in the control of exocytosis from endothelial cells (an effect not mediated by an increase in [Ca2+]i) and provide an explanation for epinephrine-induced vWf release.

VON WILLEBRAND FACTOR (vWF) PRO-POLYPEPTIDE IS REQUIRED FOR vWF MULTIMER FORMATION

1987 ◽  
Author(s):  
C L Verweij ◽  
M Hart ◽  
H Pannekoek
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Vascular Endothelial Cells ◽  
Expression System ◽  
Proteolytic Processing ◽  
Information Encoding ◽  
Interchain Interactions ◽  
Von Willebrand ◽  
Willebrand Factor

The von Willebrand factor (vWF) is a multimeric plasma glycoprotein synthesized in vascular endothelial cells as a pre-pro-polypeptide with a highly repetitive domain structure, symbolized by the formula:(H)-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-(0H).A heterologous expression system, consisting of a monkey kidney cell line (C0S-1), transfected with full-length vWF cDNA, is shown to mimic the constitutively, secretory pathway of vWF in endothelial cells. The assembly of pro-vWF into multimers and the proteolytic processing of these structures is found to oro-ceed along the following, consecutive steps. Pro-vWF subunits associate to form dimers, a process that does not involve the pro-polypeptide of pro-vWF. This observation is derived from transfection of C0S-1 cells with vWF cDNA, lacking the genetic information encoding the pro-polypeptide, composed of the domains D1 and D2. Pro-vWF dimers are linked intracellularly to form a regular series of multimeric structures that are secreted and cannot be distinguished from those released constitutively by endothelial cells. The presence of the pro-polypeptide, embedded in pro-vWF, is obligatory for multimerization since the deletion mutant lacking the D1 and D2 domains fails to assemble beyond the dimer stage. It is argued that the D domains are involved in interchain interactions.

scholarly journals von Willebrand factor released from Weibel-Palade bodies binds more avidly to extracellular matrix than that secreted constitutively

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1531-1534 ◽  
Author(s):  
LA Sporn ◽  
VJ Marder ◽  
DD Wagner
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Immunofluorescent Staining ◽  
Cultured Endothelial Cells ◽  
Human Foreskin Fibroblasts ◽  
Platelet Binding ◽  
The Matrix ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Large multimers of von Willebrand factor (vWf) are released from the Weibel-Palade bodies of cultured endothelial cells following treatment with a secretagogue (Sporn et al, Cell 46:185, 1986). These multimers were shown by immunofluorescent staining to bind more extensively to the extracellular matrix of human foreskin fibroblasts than constitutively secreted vWf, which is composed predominantly of dimeric molecules. Increased binding of A23187-released vWf was not due to another component present in the releasate, since releasate from which vWf was adsorbed, when added together with constitutively secreted vWf, did not promote binding. When iodinated plasma vWf was overlaid onto the fibroblasts, the large forms bound preferentially to the matrix. These results indicated that the enhanced binding of the vWf released from the Weibel-Palade bodies was likely due to its large multimeric size. It appears that multivalency is an important component of vWf interaction with the extracellular matrix, just as has been shown for vWf interaction with platelets. The pool of vWf contained within the Weibel-Palade bodies, therefore, is not only especially suited for platelet binding, but also for interaction with the extracellular matrix.

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