Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 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

The role of the urokinase receptor (uPAR) in the internalization of the urokinase-plasminogen activator inhibitor type-1 (uPA.PAI-1) complex has been investigated. First, exploiting the species specificity of uPA binding, we show that mouse LB6 cells (that express a mouse uPAR) were unable to bind or degrade the human uPA.PAI-1 complex. On the other hand, LB6 clone 19 cells, which express a transfected human uPAR, degraded uPA.PAI-1 complexes with kinetics identical to the human monocytic U937 cells. We also show by immunofluorescence experiments with anti-uPA antibodies that in LB6 clone 19 cells, the uPA.PAI-1 complex is indeed internalized. While at 4 degrees C uPA fluorescence was visible at the cell surface, shift of the temperature to 37 degrees C caused a displacement of the immunoreactivity to the cytoplasmic compartment, with a pattern indicating lysosomal localization. If uPA.PAI-1 internalization/degradation is mediated by uPAR, inhibition of uPA.PAI-1 binding to uPAR should block degradation. Three different treatments, competition with the agonist amino-terminal fragment of uPA, treatment with a monoclonal antibody directed toward the binding domain of uPAR or release of uPAR from the cell surface with phosphatidylinositol-specific phospholipase C completely prevented uPA.PAI-1 degradation. The possibility that a serpin-enzyme complex receptor might be primarily or secondarily involved in the internalization process was excluded since a serpin-enzyme complex peptide failed to inhibit uPA.PAI-1 binding and degradation. Similarly, complexes of PAI-1 with low molecular mass uPA (33 kDa uPA), which lacks the uPAR binding domain, were neither bound nor degraded. Finally we also show that treatment of cells with uPA.PAI-1 complex caused a specific but partial down-regulation of uPAR. A similar result was obtained when PAI-1 was allowed to complex to uPA that had been previously bound to the receptor. The possibility therefore exists that the entire complex uPA.PAI-1-uPAR is internalized. All these data allow us to conclude that internalization of the uPA.PAI-1 complex is mediated by uPAR.
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PMID:Internalization of the urokinase-plasminogen activator inhibitor type-1 complex is mediated by the urokinase receptor. 131 48

In this study, we investigated the mechanism of angiotensin II (Ang II) induced secretion of plasminogen activator inhibitor-1 (PAI-1) from astroglial cells prepared from 21-day-old rat brain. Competition-inhibition experiments with the use of selective antagonists for Ang II receptor subtypes indicated that astroglial cells contain chiefly Ang II type 1 (AT1) receptors. The interaction of Ang II with AT1 receptors resulted in a time- and concentration-dependent stimulation of PAI-1 gene expression. A maximal, 20-fold induction of PAI-1 messenger RNA (mRNA) steady-state levels was observed with 10 nM Ang II. This effect of Ang II was blocked by DuP753, an AT1 receptor antagonist, but not by PD123177, an AT2 receptor antagonist. Raise in PAI-1 mRNA levels was followed by an elevation in PAI-1 concentration in culture media reaching its maximum after 24 h. Interaction of Ang II with AT1 receptors also resulted in a time- and concentration-dependent stimulation of inositol phospholipid (IP) hydrolysis. A maximal, 3- to 5-fold stimulation of IP hydrolysis was observed with 10 nM Ang II. The time course experiments indicated that Ang II-induced stimulation of IP hydrolysis precedes the stimulation of PAI-1 mRNA. This suggested that activation of phospholipase C, IP hydrolysis system and possibly protein kinase C (PKC) may mediate Ang II's effect on PAI-1 mRNA. Direct stimulation of PKC by phorbol ester, phorbol 12,13-dibutyrate (PDB), resulted in a time- and concentration-dependent elevation of PAI-1 mRNA levels, similar to that caused by Ang II (maximal stimulation of 20-fold with 100 nM PDB for 4 h). This effect was totally blocked by the protein kinase C inhibitor, H7. In addition, Ang II stimulation of PAI-1 mRNA was also blocked by H7. In contrast, Ang II did not elevate PAI-1 mRNA levels in astroglial cultures from neonatal rat brains. However, treatment of neonatal cultures with PDB increased levels of this mRNA species. These observations indicate that the coupling of AT1 receptors with IP hydrolysis and PKC activation may be important for Ang II stimulation of PAI-1 gene expression. The lack of Ang II's effect on PAI-1 mRNA in neonatal astroglia may be explained either by a low coupling efficiency between AT1 receptors and the second messenger system, or by a low AT1 to AT2 receptor level ratio.
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PMID:Angiotensin II stimulation of plasminogen activator inhibitor-1 gene expression in astroglial cells from the brain. 153 91

Activation of protein kinase C leads to a strong induction of tissue-type plasminogen activator (t-PA) expression in endothelial cells. Using endothelial cells from human umbilical vein (HUVECs) and human aorta (HAECs), we have studied this regulation of t-PA and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), at the mRNA level and have compared their induction with the expression of platelet-derived growth factors A and B (PDGF-A and PDGF-B) and the proto-oncogenes c-jun and c-fos. Treatment of HUVECs with exogenous bacterial phospholipase C or the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol led to a threefold and a twofold increase, respectively, in t-PA concentrations in 24-hour-conditioned medium. Similarly, the more stable protein kinase C activator 4 beta-phorbol-12-myristate-13-acetate (PMA) caused about a 10-fold increase in t-PA antigen levels. This effect of PMA is maximal between 8 and 16 hours at a concentration of 10 nM and is fully accounted for by parallel increases in t-PA mRNA levels. An increase in intracellular cyclic adenosine monophosphate levels by forskolin (10 microM) slightly diminished t-PA expression but further enhanced the PMA-induced increases in t-PA synthesis and mRNA levels by at least twofold. PMA also enhanced the mRNA levels of two other important endothelium-expressed genes, PDGF-A and PDGF-B, with a time profile similar to that of t-PA, with peak values about fivefold higher than control values. Forskolin did not further stimulate this PMA-induced PDGF expression in HUVECs, which suggests a regulatory mechanism different from that of t-PA. Qualitatively very similar induction patterns of t-PA, PDGF-A, and PDGF-B were seen with HAECs. In contrast to t-PA and PDGF, PAI-1 mRNA and antigen levels increased only slightly after PMA treatment of HUVECs or HAECs; forskolin alone or in combination with PMA diminished the expression of PAI-1. The induction of t-PA mRNA by PMA was dependent on protein synthesis and was preceded by a strong transient increase in c-jun and c-fos mRNA levels; the induction of c-fos but not of c-jun was potentiated by forskolin. Because the products of these two proto-oncogenes form dimeric complexes for which specific binding sites are present in the t-PA promoter region, they may mediate the protein kinase C-dependent increase in t-PA gene expression, including the stimulating action of cyclic adenosine monophosphate.
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PMID:Role of protein kinase C and cyclic adenosine monophosphate in the regulation of tissue-type plasminogen activator, plasminogen activator inhibitor-1, and platelet-derived growth factor mRNA levels in human endothelial cells. Possible involvement of proto-oncogenes c-jun and c-fos. 164 85

Vitronectin (VN; = complement S-protein), a plasma glycoprotein that is also associated with extracellular sites, was identified in washed human platelets contaminated with less than 0.05% of plasma VN. A specific enzyme-linked immunosorbent assay (ELISA) for VN has been developed and was used to detect and to quantitate VN in detergent extracts of washed platelets with 8.1 +/- 4.6 micrograms/10(9) platelets (n = 10), representing about 0.8% of the plasma VN pool. Platelet and plasma VN were similar by immunochemical criteria using Western-blot analysis, although platelet VN was mainly found as partially proteolyzed polypeptide. Total release of platelet VN occurred at optimal doses of Ca-ionophore 23187 or thrombin, whereas no VN was released by platelet treatment with digitonin or Staphylococcus alpha-toxin. During stimulation of washed platelets with various concentrations of thrombin, the nearly concomitant release of VN and plasminogen activator inhibitor-1 (PAI-1) together with platelet factor 4 indicated the association of VN with inner-platelet storage granules. Furthermore, platelet VN and PAI-1 in Ca-ionophore releasates comigrated during ultracentrifugation in high mol wt fractions of sucrose density gradients, indicating a possible association of both components. Complex formation of platelet VN and PAI-1 was verified by a sensitive enzyme-linked immunosorbent assay (ELISA) and accounts at least in part for a high molecular form of platelet VN. The identification of platelet VN and its binding to platelet PAI-1 raises the possibility that VN, in contrast to other adhesive proteins, may participate in localized regulatory functions of blood coagulation and fibrinolysis in platelet-matrix interactions and the protection of the matrix against proteolysis.
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PMID:Identification of and partial characterization of platelet vitronectin: evidence for complex formation with platelet-derived plasminogen activator inhibitor-1. 247 18

Transforming growth factor-beta (TGF-beta) is a multifunctional peptide that elicits a wide variety of responses in cells. TGF-beta binds to cell surface receptors that contain cytoplasmic serine/threonine kinase domains. Here we provide evidence that both phospholipase C and protein kinase C (PKC) are involved in the TGF-beta activation of transcription and luciferase expression from the p3TP-Lux plasmid. Down-regulation of PKC prevents TGF-beta 1 induction of luciferase expression. Staurosporin and Calphostin C, inhibitors of PKC, block the ability of TGF-beta 1 to initiate transcription of the luciferase gene. Further, D609, an inhibitor of phosphatidylcholine-phospholipase C (PC-PLC), and secondarily PKC also blocks TGF-beta 1-induced transcription of the transgene in A549 cells while the phosphatidylinositol-PLC pathway inhibitor U73122 is without effect. TGF-beta elevates steady-state mRNA levels for the endogenous PAI-1 and fibronectin genes. Treatment of cells with calphostin C or D609 prevents the TGF-beta-induced increase in these mRNAs. Together, these results suggest that PC-PLC and PKC are in a TGF-beta signaling pathway that results in elevated gene expression.
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PMID:Evidence for involvement of phosphatidylcholine-phospholipase C and protein kinase C in transforming growth factor-beta signaling. 777 10

Urokinase-type plasminogen activator (u-PA), its receptor (u-PAR), and type 1 inhibitor (PAI-1) in cultured human mesangial cells were investigated. Treatment with phospholipase C (PLC) released plasminogen activators [with relative mol wt (M(r)) of 55,000 and 100,000] and u-PAR into the culture medium. By Western blot, both u-PA and PAI-1 were present in the M(r) 100,000 band. Since PAI-1 binds only active, two-chain u-PA (tcu-PA), formation of the M(r) 100,000 band reflects conversion of the single-chain, proenzyme form of u-PA (scu-PA) to tcu-PA. Immunofluorescence staining of whole cells demonstrated the presence of PAI-1, u-PA, and u-PAR. Immunofluorescence staining and Western blot analysis showed enrichment of PAI-1, u-PA, and u-PAR in a preparation of substratum-attached extracellular matrix and membrane proteins termed adhesion plaques. Using a chromogenic assay, we found that PAI-1 expression in adhesion plaques exceeded that of u-PA. We conclude that cultured human mesangial cells produce receptor-bound u-PA/PAI-1 complexes localized to adhesion plaques.
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PMID:Two-chain urokinase, receptor, and type 1 inhibitor in cultured human mesangial cells. 838 15

In a previous study (Latron et al. 1991. Arterioscler. Thromb. 11: 1821-1829) we have shown that oxidized low density lipoproteins (ox-LDL) stimulated the synthesis and secretion of plasminogen activator inhibitor-1 (PAI-1) by human umbilical vein endothelial cells (HUVEC) in culture. The present study is intended to give insight into the intracellular process responsible for this stimulation. The HUVEC lipids were labeled for 16 h with [3H]arachidonate and incubated either with native LDL (n-LDL) or ox-LDL for various times (15, 30, 60 min). Compared with unstimulated cells (no LDL added), ox-LDL induced a significant increase in the intracellular level of unesterified [3H]arachidonate, concomitantly with a significant decrease of the phosphatidylinositol fraction. The most marked effect was observed at 30 min and was significantly much less with n-LDL. Phospholipase inhibitors (4-bromophenacylbromide and mepacrine) added to the culture medium completely prevented the ox-LDL-induced stimulation of phosphatidylinositol degradation, [3H]arachidonate release, and PAI-1 secretion. HUVEC possess both phospholipase C and A activities and a high lysophospholipase activity, the phospholipase A pathway being in vitro more sensitive to inhibition by 4-bromophenacylbromide than the phospholipase C pathway. These results suggest that the stimulation of PAI-1 secretion by ox-LDL is mediated by the hydrolysis of membrane phosphatidylinositol through the activation of phospholipase A.
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PMID:Phosphatidylinositol turnover during stimulation of plasminogen activator inhibitor-1 secretion induced by oxidized low density lipoproteins in human endothelial cells. 844 34

We have previously demonstrated that vitronectin (VN), a morphoregulatory protein in the vessel wall, is internalized and translocated to the subendothelial matrix by an integrin-independent mechanism (J. Histochem. Cytochem. 41, 1823-1832, 1993). The cell surface component which mediates the initial contact of VN with endothelial cells is defined here. The specific binding of VN to endothelial cells demonstrated the following properties: a threefold increase after phorbol ester treatment; 85% inhibition by pretreatment of cells with phosphatidylinositol-phospholipase C to release glycolipid-anchored surface proteins; a 90% inhibition by urokinase (u-PA) receptor blocking antibody. u-PA increased VN binding to cells due to an eightfold increase in the affinity of VN for the u-PA receptor. Structure-function studies showed that the amino-terminal fragment of u-PA, devoid of any proteolytic activity, mediated this effect. Active plasminogen activator inhibitor-1 (PAI-1), but not inactivated PAI-1, inhibited VN binding to cells and displaced VN that was prebound to endothelial cell monolayers. Similarly, VN binding to purified (immobilized) u-PA receptor, but not to integrin, was enhanced by u-PA and inhibited by PAI-1. Hence, the binding of soluble VN to endothelial cell surfaces is mediated by the u-PA receptor, and the relative concentrations of u-PA and PAI-1 are able to regulate the strength of this interaction. Endothelial cell adhesion to immobilized VN was found to be integrin-mediated without any involvement of the VN-uPA-receptor system. Hence, the interaction of VN with the u-PA receptor may be involved in the regulation of cellular processes necessary for endothelial cell invasion and migration at VN-rich extracellular matrix sites.
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PMID:The urokinase receptor is a major vitronectin-binding protein on endothelial cells. 861 11

Fig. 1 depicts our current thinking about the ways in which Mo1 and p150,95 form cis interactions with other leukocyte receptors. With respect to the associations of Mo1 with Fc gamma RIIIB and uPAR, the inhibitory effect of saccharides such as NADG suggests a lectin-carbohydrate interaction that may involve the recognition of Mo1's beta-glucan site for N-linked carbohydrates4 that are expressed by both Fc gamma RIIIB and uPAR. This hypothesis is supported by the results of Stockl et al., who showed that the binding of C-terminal-specific mAb VIM12 to Mo1, which enhances the phospholipase C-mediated release of Fc gamma RIIIB, was inhibited by NADG. However, unlike the sample lectin-carbohydrate interaction that appears to govern the association between Mo1 and Fc gamma RIIIB, effective Mo1-dependent uPAR signaling also depends on the binding of intact uPA to uPAR (the receptor-binding ATF of uPA proving insufficient to prime neutrophils for an enhanced burst response to FMLP). We speculate that ATF (residues 6-135) binds to uPAR while the carboxyl terminal fragment (residues 136-411), which includes a glycosylation site at residue 144, binds to the lectinlike site of Mo1, thus fostering the linkage between the two receptors. In support of this model is the fact that exposure of neutrophils to ATF reduced the degree of molecular proximity between Mo1 and uPAR (the latter probably occupied by endogenous intact uPA) and increased the molecular association between Mo1 and Fc gamma RIIIB (both as detected by quantitative RET). This hypothesis is analogous to the concept proposed by Nykjaer et al in which plasminogen activator inhibitor-1 initially binds to uPA to form a complex that secondarily binds to the alpha 2 macroglobulin receptor, leading to internalization of the complex. Whereas the contribution of intact uPA to the interaction between Mo1 and uPAR remains speculative (based on the indirect data available), no such ambiguity exists for the role of the LPS/LBP ligand in regulating the association between Mo1 and CD14. In this circumstance, no physical linkage exists between the two receptors without the ligand complex. This observation is consistent with the previously described affinity of the beta 2 integrins for LPS, leading to the notion that the LPS portion of the LPS/LPB complex binds to Mo1, serving to link it with LPS/LBP bound to CD14. The observed reversibility of the interactions between the integrin glycoproteins and uPAR or CD14 illustrates the fact that these associations can be highly dynamic and tied to cellular processes that include directed motility (Mo1-uPAR), adherence to substrates (Mo1-CD14), and energy metabolism (p150,95-uPAR). We speculate that the GPI-anchored receptor proteins serve as rapidly diffusible, expendable "scouts" for the beta 2 integrins, which serve to expand their ligand binding repertoire in a cis-acting fashion.
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PMID:Beta 2 (CD11/CD18) integrins can serve as signaling partners for other leukocyte receptors. 914 45


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