Catabolism of tissue-type plasminogen activator by the human hepatoma cell line Hep G2. Modulation by plasminogen activator inhibitor type 1

Catalytic activity of tissue-type plasminogen activator (t-PA) in plasma is regulated in part by formation of complexes with specific inhibitors as well as by hepatic clearance. Potential interaction of these two regulatory mechanisms was examined in the human hepatoma cell line Hep G2. These cells secrete plasminogen activator inhibitor type-1 (PAI-1) and initiate catabolism of exogenous t-PA by receptor-mediated endocytosis. Specific binding of 125I-t-PA to cells at 4 degrees C results in dose-dependent formation of a 95-kDa species recognized by monospecific anti-PAI-1 and anti-t-PA antibodies and stable in the presence of low (0.2%) concentrations of sodium dodecyl sulfate (SDS). Specific binding of 125I-t-PA and formation of the 95-kDa SDS-stable species are inhibited in a concentration-dependent manner following preincubation of cells with anti-PAI-1 antibodies. High and low molecular weight forms of urokinase plasminogen activator (u-PA) capable of forming specific complexes with PAI-1 complete for 125I-t-PA binding sites. However, the proenzyme form of u-PA (scu-PA), incapable of forming complexes with PAI-1, does not compete for 125I-t-PA binding sites. The role of the serine protease active site of t-PA in mediating both interaction with PAI-1 and specific binding was examined using 125I-t-PA that had been functionally inactivated with D-phenylalanyl-L-propyl-L-arginyl-chloromethyl ketone (PPACK). 125I-t-PA-PPACK, despite a 6-fold lower affinity than active 125I-t-PA, exhibited specific binding to cells without detectable formation of SDS-stable complexes with PAI-1. Both surface-bound 125I-t-PA and 125I-t-PA-PPACK are internalized and degraded by cells at 37 degrees C. 125I-t-PA is internalized as a stable complex with PAI-1, whereas 125I-t-PA-PPACK is internalized with similar kinetics but without the presence of an SDS-stable complex. Thus, PAI-1 appears capable of modulating t-PA catabolism in the human hepatocyte.     

Regulation of pericellular proteolysis by hepatocyte growth factor activator inhibitor type 1 (HAI-1) in trophoblast cells

Hepatocyte growth factor activator inhibitor type 1/serine protease inhibitor Kunitz type 1 (HAI-1/SPINT1) is a membrane-bound Kunitz-type serine protease inhibitor that is abundantly expressed on the surface of cytotrophoblasts, and is critically required for the formation of the placenta labyrinth in mice. HAI-1/SPINT1 regulates several membrane-associated cell surface serine proteases, with matriptase being the most cognate target. Matriptase degrades extracellular matrix protein such as laminin and activates other cell surface proteases including prostasin. This study aimed to analyze the role of HAI-1/SPINT1 in pericellular proteolysis of trophoblasts. In HAI-1/SPINT1-deficient mouse placenta, laminin immunoreactivity around trophoblasts was irregular and occasionally showed an intense punctate pattern, which differed significantly from the linear distribution along the basement membrane observed in wild-type placenta. To explore the molecular mechanism underlying this observation, we analyzed the effect of HAI-1/SPINT1 knock down (KD) on pericellular proteolysis in the human trophoblast cell line, BeWo. HAI-1/SPINT1-KD BeWo cells had increased amounts of cellular laminin protein and decreased laminin degradation activity in the culture supernatant. Subsequent analysis indicated that cell-associated matriptase was significantly decreased in KD cells whereas its mRNA level was not altered, suggesting an enhanced release and/or dislocation of matriptase in the absence of HAI-1/SPINT1. Moreover, prostasin activation and pericellular total serine protease activities were significantly suppressed by HAI-1/SPINT1 KD. These observations suggest that HAI-1/SPINT1 is critically required for the cell surface localization of matriptase in trophoblasts, and, in the absence of HAI-1/SPINT1, physiological activation of prostasin and other protease(s) initiated by cell surface matriptase may be impaired.     

Expression of hepatocyte growth factor activator inhibitor type 1 in endothelial cells

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is an integral membrane Kunitz-type serine proteinase inhibitor initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA). HGFA is a serum proteinase that is critically involved in the activation of hepatocyte growth factor/scatter factor (HGF/SF) in injured tissue. Previous studies have shown that HAI-1 is expressed on the basolateral surface of various epithelial cells. In this study, we analyzed the expression of HAI-1 in human endothelial cells. Immunohistochemically, HAI-1 protein was observed in the endothelial cells of capillaries, venules and lymph vessels. On the other hand, arterial endothelial cells were poorly stained for HAI-1. Mesothelial cells on the serous surface were also positively immunostained. The endothelial expression of HAI-1 was also examined in cultured human endothelial cells of various origins, such as umbilical vein, microvessels and aorta. Notably, in accordance with the results of immunohistochemistry, HAI-1 mRNA and protein levels were high in the endothelial cells derived from umbilical vein and were hardly detectable in those derived from aorta. A low but distinct level of HAI-1 expression was also observed in endothelial cells from microvessels. As these HAI-1-positive endothelial cells also expressed MET tyrosine kinase, the specific receptor of HGF/SF, it is conceivable that HAI-1 might have an important regulatory role in the HGF/SF-MET signaling axis of endothelial cells, which could be involved in the process of angiogenesis.     

Purification of epidermal plasminogen activator inhibitor

A plasminogen activator inhibitor was purified from human cornified cell extract by DEAE-Sepharose, Sephacryl S-200, and high-performance liquid chromatographies on hydroxyapatite HPHT and anion-exchanger Mono Q at pH 7.2 and 8.0. The purified inhibitor showed Mr 43,000 and pI 5.2 50% inhibition of fibrinolytic activity (1.5 IU) of urokinase and tissue-type plasminogen activator was attained by 0.60 ng and 11.0 ng purified inhibitor, respectively. Synthetic substrate assay demonstrated slow tight-binding inhibition to both urokinase and tissue-type plasminogen activator. The inhibitor did not inactivate plasmin, thrombin, glandular kallikrein or trypsin.     

Regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 in cultured rat Sertoli and Leydig cells

New data are provided to show that (i) rat Sertoli cells produce two types of plasminogen activators, tissue type (tPA) and urokinase type (uPA), and a plasminogen activator inhibitor type-1 (PAI-1); (ii) both tPA (but not uPA) and PAI-1 secretion in the culture are modified by FSH, forskolin, dbcAMP, GnRH, PMA and growth factors (EGF and FGF), but not by hCG and androstenedione (△4); (iii) in vitro secretion of tPA and PA-PAI-1 complexes of Sertoli cells are greatly enhanced by presence of Leydig cells which produce negligible tPA but measurable PAI-1 activity;(iv) combination culture of Sertoli and Leydig cells remarkably increases FSH-induced PAI-1 activity and decreases hCG- and forskolin-induced inhibitor activity as compared with that of two cell types cultured alone. These data suggest that rat Sertoli cells, similar to ovarian granulosa cells, are capable of secreting both tPA and uPA, as well as PAI-1. The interaction of Sertoli cells and Leydig cells is essential for the cells to response to     

Binding of plasminogen activator inhibitor type-1 to extracellular matrix of Hep G2 cells. Evidence that the binding protein is vitronectin.

Catabolism of plasminogen activators by Hep G2 cells is mediated by a specific receptor which recognizes complexes of these serine proteases with their physiological inhibitor, plasminogen activator inhibitor type-1 (PAI-1). This catabolic process is initiated by interaction of exogenous plasminogen activators with bioactive PAI-1, which is secreted and localizes in an active form to the extracellular matrix (ECM) of Hep G2 cells. We now report that vitronectin (VN) mediates the specific binding of PAI-1 to the ECM of these cells. Purified bovine or human VN competes for specific binding of PAI-1 to Hep G2 ECM, and ligand blotting reveals specific binding of PAI-1 to ECM-associated VN. Hep G2 cells secrete both VN and PAI-1, and pulse-chase studies strongly suggest that these proteins associate only following secretion. Although Hep G2 cell-derived VN does not significantly bind to ECM in vitro, 30-40% of endogenous PAI-1 binds to the ECM, even in the presence of human serum, suggesting that ECM-associated VN is entirely derived from bovine serum. PAI-1 was localized by indirect immunofluorescence to ECM beneath cells and at cell margins, whereas VN exhibited a uniform distribution throughout the growth substratum. VN associated with the ECM may confer retention and bioactivity to PAI-1, potentially facilitating both pericellular regulation of plasmin generation and the rapid hepatic clearance of plasminogen activators.     

Regulation of endothelial tissue plasminogen activator and plasminogen activator inhibitor type 1 synthesis by diacylglycerol, phorbol ester, and thrombin

The influence of diacylglycerols, which are physiological activators of protein kinase C, on the production of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1) by human umbilical vein endothelial cells (HUVEC) was studied in order to gain insight into the regulation of fibrinolysis by these cells. 1,2-dioctanoyl-sn-glycerol (diC8) stimulated tPA production in a dose- and time-dependent manner. The tPA antigen in cell supernatants increased from 0.9 ng/10(6) cells in unstimulated cells to 12.4 ng (10(6) cells after incubation with 400 microM diC8 for 24 hours. In contrast, PAI-1 production was not influenced by diC8, whereas phorbol 12-myristate 13-acetate (PMA) or thrombin stimulated both, tPA and PAI-1 production by HUVEC. Staurosporine and H7, which are inhibitors of protein kinase C, inhibited tPA synthesis by HUVEC. The degree of inhibition was dependent on the agonist used. While diC8-induced tPA production was inhibited to more than 80% by H7 (10 microM) and staurosporine (10 nM), higher doses of inhibitors were required to inhibit thrombin- and PMA-induced tPA production. Thrombin-induced PAI-1 production was inhibited to more than 80% by H7 (10 microM) and to about 50% by staurosporine, whereas PMA-induced PAI-1 production was not inhibited by staurosporine, and only to about 50% by higher doses of H7 (30 microM). These data suggest that activation of protein kinase C is a common intracellular trigger mechanism for the induction of tPA synthesis by HUVEC. Protein kinase C is most likely also involved in the regulation of PAI-1 synthesis by HUVEC.     

Receptor-mediated endocytosis of tissue-type plasminogen activator by the human hepatoma cell line Hep G2

Receptor-mediated endocytosis of tissue-type plasminogen activator (t-PA) was characterized with the human hepatoma cell line Hep G2. At 4 degrees C binding of 125I-t-PA to Hep G2 cells is rapid, specific, saturable, and reflective of a homogeneous population of 76,000 high-affinity surface sites per cell (Kd = 3.7 nM). The kinetics of 125I-t-PA binding to its receptor are characterized by rate constants for association (k1 = 1.2 x 10(6) min-1 M-1) and dissociation (k-1 = 0.001 min-1). A specific glycosylation pattern does not appear to be required for binding. Binding does not appear to be mediated by other recognized hepatic receptor systems. At 37 degrees C a single cohort of bound 125I-t-PA molecules disappears rapidly from the cell surface. Ligand then accumulates intracellularly. Thereafter, the intracellular concentration of ligand declines simultaneously with the release of ligand degradation products into the media. In the continued presence of 125I-t-PA at 37 degrees C the concentration of cell-associated ligand plateaus after 30 min with the concomitant appearance of low molecular weight 125I-labeled fragments in the media. Cumulative degradation then increases linearly with time. Under steady state conditions half-maximal ligand uptake and degradation is 26.6 nM and maximal rate of catabolism is 1.2 pmol/10(6) cells/h. At saturating ligand concentrations uptake and degradation by Hep G2 cells continue linearly for at least 6 h even in the absence of protein synthesis. During this period the cumulative ligand uptake exceeds the total cellular capacity of binding sites, consistent with receptor recycling. We conclude that t-PA clearance in human Hep G2 cells involves ligand binding, uptake, and degradation mediated by a novel high-capacity, high-affinity specific receptor system.     

SPARC induces the expression of type 1 plasminogen activator inhibitor in cultured bovine aortic endothelial cells.

SPARC, a Ca(2+)-binding glycoprotein that is expressed during tissue morphogenesis and functions as an inhibitor of cell spreading in vitro, was found to induce the secretion of an Mr = 45,000 protein in bovine aortic endothelial (BAE) cells. This protein was identified as type 1 plasminogen activator inhibitor (PAI-1) on Western blots with anti-PAI-1 antiserum. SPARC stimulated the secretion of PAI-1 protein into the medium of subconfluent BAE cells, but not confluent BAE cells, in a dose- and time-dependent manner. Secretion of PAI-1 into the culture medium was progressive and exhibited an increase of 3- to 7-fold over control values within 24 h after the addition of SPARC. Levels of PAI-1 mRNA were elevated 2-fold within 4 to 24 h after the addition of SPARC and did not increase with higher concentrations of SPARC. Since the induction of PAI-1 mRNA by SPARC was not blocked by cycloheximide, de novo protein synthesis was apparently not required for this stimulation. Control experiments showed that the induction of PAI-1 was not due to contamination of the SPARC preparations with endotoxin. These data demonstrate that SPARC induces the biosynthesis of PAI-1 in BAE cells and suggest a role for SPARC in the regulation of fibrinolysis and in the control of proteolytic events in remodeling tissues.     

The influence of nitric oxide on the regulation of plasminogen activator inhibitor type 1 and tissue-type plasminogen activator expression

Nitric oxide produced in various human tissues by nitric oxide synthase is involved in the regulation of many physiological processes. Mechanism of its action is diverse. The most important physiological activity of nitric oxide is guanylate cyclase activation and an increase of cGMP synthesis. At low concentrations NO plays a pivotal role in vessel relaxation and possesses antithrombotic, antiproliferative and anti-inflammatory features as well. An excessive production of nitric oxide can disturb vascular hemostasis and contribute to development of cardiovascular diseases. Studies provide that NO also participate in fibrynolysis regulation by the influence on the PAI-1 and t-PA expression, what may have important clinical implications. The aim of this review is to present current knowledge about the role of nitric oxide in the regulation of these plasminogen activation system factors.