Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The binding, internalization, and degradation of tissue-type plasminogen activator (t-PA) were studied in a rat hepatoma (Novikoff) cell line. Binding of t-PA to specific saturable high affinity binding sites (Kd = 12 nM, 54,000 sites/cell) was followed by internalization and degradation and did not require a functional active site. The catabolism of t-PA was not inhibited by an excess of urokinase-type plasminogen activator (u-PA), and t-PA bound to Novikoff membranes was not complexed to PAI-1, suggesting a mechanism independent of PAI-1. Additionally, a mannose receptor is not involved since t-PA binding was not influenced by an excess of mannose, galactose, ovalbumin, or EDTA. Furthermore, the degradation of t-PA was not influenced by 10 mM 6-aminohexanoic acid, a lysine analogue. The t-PA receptor binds to and can be eluted from wheat germ agglutinin-Sepharose. Cross-linking of t-PA with partially purified receptor and ligand blot analysis, suggest that t-PA binds to two proteins, a principal one of 55 kDa and a minor one of 43 kDa. Novikoff cells are able also to bind (Kd = 1.4 nM, 25,000 sites/cell) and degrade u-PA. The binding was inhibited by pro-u-PA and the amino-terminal fragment of u-PA, but not by an excess of t-PA. The u-PA receptor, but not the t-PA receptor, was removed by treatment with phosphatidylinositol-specific phospholipase C. Our results show that the clearance receptor for t-PA on Novikoff cells is different from the mannose receptor and the PAI-1-dependent receptor described in other cells. The rat hepatoma cells are thus a good model to study the PAI-1 independent hepatocyte-specific clearance of t-PA.
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PMID:Demonstration of a specific clearance receptor for tissue-type plasminogen activator on rat Novikoff hepatoma cells. 131 32

We have characterized a transcriptional enhancer of the human urokinase-type plasminogen activator (uPA) gene and found a regulatory element required for co-operation between a PEA3--AP-1 element and an AP-1 site in the enhancer. We designated this regulatory element co-operation mediator (COM). Both the PEA3--AP-1 element, the AP-1 site and the COM are required for efficient phorbol ester induction of transcription from the uPA promoter in the HepG2 hepatoma cell line. We show that the COM is also required for co-operation between the PEA3--AP-1 element and a glucocorticoid response element, both in the presence or absence of TPA, indicating that the COM is generally capable of mediating synergism between inducible enhancer elements. The COM contains multiple overlapping binding sites for nuclear proteins, designated uPA enhancer factors 1-4 (UEF-1-4). We have identified putative binding sites for UEF-1, -2 and -3. The UEF-1 and -3 sites in the uPA enhancer are highly conserved between species. We demonstrate the binding of UEF-3 to the NIP element, a previously characterized regulatory element in the human interleukin-3 and stromelysin promoters, suggesting that this factor plays a role in regulation of a variety of genes.
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PMID:A regulatory element that mediates co-operation between a PEA3-AP-1 element and an AP-1 site is required for phorbol ester induction of urokinase enhancer activity in HepG2 hepatoma cells. 133 May 39

The human hepatoma HuH-7 cell line was shown to constitutively express both a plasminogen activator (PA) and a plasminogen activator inhibitor (PAI). Four sublines of the HuH-7 cell line were analyzed and found to express differing amounts of both PA and PAI. The plasminogen activator produced by these cells was identified as urokinase based upon molecular weight, inhibition of activity with anti-UK but not anti-t-PA antibodies, adherence to an anti-UK affinity column and by Northern blotting demonstrating positive hybridization with the cDNA for UK, but not with the t-PA cDNA. The inhibitor produced by HuH-7 cells was identified as PAI-1 by molecular weight, immunoblotting techniques, adherence to an anti-PAI-1 affinity column, and by Northern blotting demonstrating positive hybridization with the cDNA for PAI-1, but not with the PAI-2 cDNA. The expression of both UK and PAI-1 by HuH-7 cells could be modulated by cytokines known to influence the acute phase response. The addition of interleukin-1 (IL-1) induced the expression of both UK and PAI-1. The increase of PAI-1 was due to an increase in amount of the PAI-1 mRNA. The presence of both interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF) also increased UK and PAI-1 levels, although not as dramatically as IL-1. The addition of IL-1 together with IL-6 produced a slight synergistic response with respect to PAI-1 expression. This suggests that PAI-1 is able to respond to mediators which aid in the induction of the acute phase response. These studies demonstrate that cells of liver origin are able to produce components of the fibrinolytic system. The synthesis of these components can be altered by inflammatory mediators and thus may be involved in hepatic regulation of fibrinolysis in both normal and diseased states.
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PMID:Human HuH-7 hepatoma cells express urokinase and plasminogen activator inhibitor-1: identification, characterization and regulation by inflammatory mediators. 137 1

The binding of tissue-type plasminogen activator (t-PA) to membranes prepared from human liver was investigated, and a specific, saturable, high-affinity binding site (Kd = 3.4 nM) was identified. The binding of t-PA to liver membranes was not affected by an excess of D-mannose or D-galactose, or by active urokinase (u-PA), whereas binding of t-PA to membranes prepared from human HepG2 hepatoma cells was inhibited by u-PA. HepG2-membrane-bound t-PA was fully complexed to PA inhibitor 1 (PAI-1), whereas liver-membrane-bound t-PA was not complexed. Gel filtration on Sephacryl S300 of membrane proteins solubilized in deoxycholate revealed that high-affinity t-PA binding activity elutes at an apparent molecular mass of 40 kDa. Monoclonal antibodies specific for the growth factor and the kringle 2 domains inhibited the binding of t-PA to liver membranes and the catabolism of t-PA by rat hepatoma cells. Human liver membranes also bound u-PA; binding was inhibited by pro-u-PA, the N-terminal fragment of u-PA, but not by the 33 kDa form of u-PA or by t-PA. Our results show that human liver membranes contain a specific 40 kDa binding protein for t-PA that is different from the PAI-1-dependent receptor described on HepG2 cells and the mannose receptor isolated from human liver.
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PMID:Characterization of the binding of plasminogen activators to plasma membranes from human liver. 144 49

Co-secretion of plasminogen activator inhibitor type 1 (PAI-1) and urokinase-type plasminogen activator was identified in short-term cultures of primary type II pneumocytes isolated from adult rats. After separation by sodium dodecyl sulfate (SDS)-PAGE and reverse fibrin autography (reverse FA) of serum-free conditioned medium (SFCM), cellular lysate, and extracellular matrix (ECM), the inhibitor was seen as a zone of spared lysis at an apparent molecular mass of 46 to 48 kD. The plasminogen activator (PA) activity could only be visualized when human instead of bovine fibrin was used in the indicator gel. It presented as a single band of lysis at an apparent molecular mass of 45 kD when tested by regular FA and was found adjacent to PAI-1 when examined by reverse FA. Immunoblot analysis of type II pneumocyte SFCM, cellular lysate, and ECM revealed two bands at 46 and 48 kD, consistent with the apparent molecular masses (Mr) reported for rat PAI-1 from HTC hepatoma cells. Type II pneumocyte PAI-1 formed SDS-resistant complexes with tissue-type and urokinase-type plasminogen activator and was found to be stable to acid, to short-term exposure to heat, and to the denaturants guanidine HCl and SDS, while being sensitive to treatment with alkali and urea. When levels of type II pneumocyte PAI-1 activity were monitored over time during short-term culture conditions, the level of PAI-1 in SFCM remained stable, whereas activity in the lysate accumulated and activity in the ECM declined.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Plasminogen activator inhibitor type 1 production by rat type II pneumocytes in culture. 154 Mar 77

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor that inhibits both tissue-type and urokinase-type plasminogen activators. Expression of PAI-1 is regulated by growth factors, cytokines, and hormones. To determine the molecular mechanisms involved in the basal expression of the rat PAI-1 gene, we have analyzed the cis-acting sequences and the trans-acting factors involved in the transcription of this gene in the HTC rat hepatoma cell line. DNase I protection analyses revealed eight regions within the first 764 base pairs of 5'-flanking sequence that interact specifically with HTC cell nuclear proteins. The proteins that bind to five of the eight footprinted sites were identified as PEA3-, Sp1-, and CTF/NF-1-like proteins using competition electrophoretic mobility shift assays. The expression of fusion genes containing progressive 5' deletions of the rat PAI-1 promoter linked to the chloramphenicol acetyltransferase reporter gene were analyzed in transient transfection experiments in HTC cells. These studies demonstrated the Sp1 and CTF/NF-1 sites to be important for transcriptional activation. Two of the footprinted sites contain the sequence 5'-TTTGn(n)TCAAT-3' and were shown in competition electrophoretic mobility shift assays to bind the same or related protein(s). Sequences containing these sites, from -764 to -628 base pairs, and from -266 to -188 base pairs, were identified in functional studies as repressor elements of transcription.
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PMID:Regulatory sequences and protein-binding sites involved in the expression of the rat plasminogen activator inhibitor-1 gene. 160 87

Hyperthermia is a clinical sign of inflammation and constitutes in itself an adaptive defense mechanism. The fibrinolytic system, a highly regulated proteolytic system, is involved in inflammatory processes. Plasminogen activator inhibitor 1 (PAI-1) is the principal inhibitor of the two activators of the fibrinolytic system: tissue- and urokinase-type PAs (t-PA and u-PA). Our present paper provides the first evidence that hyperthermia can directly induce PAI-1. A moderate heat stress, sufficient to induce heat shock protein 70 mRNA approximately 100-fold, resulted in a two- to three-fold increase in functionally active PAI-1 in the conditioned medium of human HT-1080 fibrosarcoma and Hep G2 hepatoma cells. Exposure of these cells to 42 degrees C led to a similar two-fold and two- to five-fold induction of PAI-1 mRNA expression in HT-1080 and Hep G2 cells, respectively, as has been determined by using both oligo d(T) selected and total RNA preparations. These results suggest that the observed increase in PAI-1 accumulation is due to an induction of PAI-1 biosynthesis. Run-on transcription analysis indicates that the induction of PAI-1 biosynthesis by hyperthermia is mediated by a stimulation of PAI-1 gene transcription. No significant effect of hyperthermia was found on t-PA or u-PA at the level of antigen accumulation, mRNA, and gene transcription in human HT-1080 fibrosarcoma cells. These results point to an additional regulatory mechanism of fibrinolysis in the context of inflammation.
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PMID:Induction of plasminogen activator inhibitor 1 biosynthesis by hyperthermia. 165 90

The binding of t-PA-PAI-1 to human hepatocytes at 4 degrees C reached a maximum at 2 h. Scatchard analysis indicated 74,000 +/- 11,000 high-affinity binding sites for complex per human hepatocyte, with a Kd of 0.87 +/- 0.09 nM. Almost identical results were achieved with the human hepatoma cell line Hep G2. Binding of [125I]t-PA-PAI-1 complex was unaffected by high concentrations of unlabelled t-PA, PAI-1, u-PA or u-PA-PAI-1 complex; only t-PA-PAI-1 complex competed for binding. Hepatocyte-bound t-PA-PAI-1 was internalized and degraded at 37 degrees C. Thus, hepatocytes have a specific t-PA-PAI-1 receptor that participates in clearance of this complex.
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PMID:The receptor for tissue plasminogen activator (t-PA) in complex with its inhibitor, PAI-1, on human hepatocytes. 184 16

Interleukin-1 (IL-1) release from monocyte-macrophages (Mo) appears dependent on pericellular proteolysis mediated by plasmin. Thus plasminogen activator inhibitors (PAI) which bind the serine proteases responsible for the conversion of plasminogen to plasmin, may inhibit IL-1 release from Mo. We have examined the effect of purified PAI from a hepatoma cell line Hep G2, on IL-1 release from Mo with secondary effects on lymphocyte proliferation in vitro. Fast acting inhibitors of both urokinase (u-PA) and tissue plasminogen activator (two chain t-PA) were noted in harvest fluids of Hep G2 cells. These inhibitors were stable at pH 3 but lost activity at 45 degrees C. They were SDS-stable and migrated with Mr53 and 104 kDa. These properties conformed to characteristics of type-1 plasminogen activator inhibitor (PAI-1). Partially purified PAI-1 added to human Mo cultured on 125I fibrin layer both in the presence and absence of plasminogen inhibited secretion of IL-1 by Mo in response to LPS. This effect, however, did not correlate with the inhibition of plasminogen dependent fibrinolysis. This suggested a degree of sequestration and inaccessibility of membrane bound u-PA of LPS activated Mo to PAI-1. PAI-1, in addition, inhibited mitogen stimulated peripheral blood mononuclear cell (PBMC) proliferation at similar concentration ranges. This effect was abrogated by the addition of specific antisera to PAI-1. PAI-1 may be released as part of an acute phase response. In addition to influencing fibrinolysis, PAI-1 may constitute a negative feedback pathway on Mo IL-1 release and subsequent immune activation in vivo.
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PMID:Monocyte-macrophage release of IL-1 is inhibited by type-1 plasminogen activator inhibitors. 196 70

Human Hep G2 hepatoma and HT 1080 fibrosarcoma cells were cultured in large scale under conditions which allowed enhanced secretion of plasminogen activator inhibitor-1 (PAI-1). A modified urokinase was obtained by reacting urokinase with phenylmethylsulfonyl fluoride followed by alkali treatment. The resulting product, called anhydrourokinase, was found to reversibly bind the PAI-1 when immobilized on cyanogen bromide-activated Sepharose 4B beads. Using this affinity absorbent, we have purified PAI-1 from the cell-conditioned media. A number of differences have been observed during Hep G2 and HT 1080 PAI purification. 1) The PAI activity in Hep G2 medium concentrate is more stable, and the concentrate depleted of active PAI-1 showed spontaneous regeneration of PAI-1 activity. In contrast, the PAI activity in HT 1080 medium concentrate declines rapidly on standing. 2) Hep G2 PAI-1 invariably copurified with an adhesive protein, vitronectin or its NH2-terminal fragment, while pure HT 1080 PAI-1 alone was obtained by affinity purification on anhydrourokinase-Sepharose 4B. 3) Based on specific activity measurement and complex formation analysis using a sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis technique, the purified Hep G2 PAI-1 appears completely active while the HT 1080 PAI-1 is only one-fourth as active. SDS was found to exert dual effects on purified PAI-1s. SDS treatment partially inactivated a fully active Hep G2 PAI-1 and a moderately active HT 1080 PAI-1 but partially activated an HT 1080 PAI-1 whose activity had previously been allowed to decay to a very low level. Purified vitronectin was found to enhance and stabilize the PAI-1 activity of the partially active HT 1080 PAI-1. It is concluded that fully active PAI-1 in association with vitronectin can be isolated by anhydrourokinase-Sepharose 4B chromatography and that vitronectin is a binding protein for PAI-1 which activates and stabilizes PAI-1.
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PMID:Affinity purification of active plasminogen activator inhibitor-1 (PAI-1) using immobilized anhydrourokinase. Demonstration of the binding, stabilization, and activation of PAI-1 by vitronectin. 247 Jul 35


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