Interaction between plasminogen activator inhibitor type-2 and pre-mRNA processing factor 8

Plasminogen activator inhibitor type-2 (PAI-2), a mem-ber of the ovalbunin family of serine protease inhibitor (ov-serpin) super family, is a multifunctional protein which isinvolved in the regulation of fibrinolysis, invasion andmetastasis of cancer cells, and regulation of apoptosis. HeLacells transfected with PAI-2 were protected from TNF-α-induced apoptosis. Like other members of ov-serpin family,PAI-2 contains a non-conserved 33 amino acid residuesloop region between its C and D he…     

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     

Differences between uterine and melanoma forms of tissue plasminogen activator

Tissue plasminogen activator purified from human uterine tissue exhibits differences in N-terminal starting positions in relation to the melanoma cell plasminogen activator usually studied. A new starting position is compatible with an additional N-terminal processing apart from those already known. Like the melanoma activator, the uterine activator was found to yield protein chains starting at either of two positions. One of these was identical between uterine and melanoma activators, whereas the other was unique in each case. The most abundant starting position for the uterine preparation was at a valine residue, apparently from cleavage of a Gln-Val bond, and corresponding to Val-7 of the longest form of the melanoma activator chain.     

Complex formation between plasminogen activator inhibitor 1 and vitronectin in purified systems and in plasma

Functionally active PAI-1 is bound to a discrete binding or carrier protein in plasma, which was recently identified as vitronectin. In the present study, the interaction between PAI-1 and vitronectin has been studied in purified systems and in plasma by agarose gel electrophesis using non-denaturing conditions and by crossed immunoelectrophoresis using an antiserum produced towards purified PAI-1/vitronectin complex. Both methods revealed a clearly distinguishable complex with electrophoretic mobility in between the parent molecules. Virtually all of the purified vitronectin, which did not contain any appreciable amounts of polymerized material, and almost all of the vitronectin in plasma, had the capacity to form a complex with PAI-1. The results suggested a stoichiometry of 1:1 as the most likely ratio between the two molecules in the complex. In contrast to functionally active PAI-1, latent or chloramine T-inactivated PAI-1 did not form such a complex with vitronectin.     

Expression of plasminogen activator and plasminogen activator inhibitor mRNA in human fibroblasts grown on different substrates

mRNA levels for urokinase type plasminogen activator (uPA), tissue type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) were examined in human diploid (neonatal foreskin) fibroblasts grown in 200-ml microcarrier suspension culture. Four different substrates were used. These included gelatin-coated polystyrene plastic, DEAE-dextran, glass-coated polystyrene plastic and uncoated polystyrene plastic. Our previous studies have shown that culture fluids from diploid fibroblasts grown on DEAE-dextran contained higher levels of plasminogen-dependent fibrinolytic activity than culture fluids from the same cells grown on other substrates. The increased plasminogen activator activity was due largely to elevated amounts of tPA (In Vitro Cell. Develop. Biol. 22: 575–582, 1986). The present study shows that there is a corresponding elevation of tPA mRNA in diploid fibroblasts cultured on DEAE-dextran relative to the other substrates. There does not appear to be any difference in uPA mRNA or in mRNA for PAI-1 or PAI-2 produced by the same cells on the four substrates. These data suggest that the influence of the substrate on plasminogen activator production is mediated at the genetic level.     

Plasminogen activator inhibitor 2: expression and role in differentiation of epidermal keratinocyte

Plasminogen activator inhibitor 2 (PAI-2) is an enzyme inhibitor which is involved in cell differentiation, tissue growth and regeneration. In this study, immunocytochemistry, in situ hybridization and confocal laser scanning microscopy were used to investigate the expression and role of PAI-2 in differentiation of keratinocytes in vitro. The result showed that in the mono-layer differentiated keratinocytes induced by high calcium concentration, the expression of PAI-2 and its mRNA increased significantly, accompanied by expression increase of the differentiation marker keratin 10; and in the multi-layer differentiated keratinocytes induced by high calcium, PAI-2 expressed strongly mainly in the keratinocytes of middle as well as upper stratified layers, while K10 expressed in the keratinocytes of all stratified layers. Furthermore, the changes of the parameters related to keratinocyte differentiation were detected after inhibition of PAI-2 functions by its antibody, and the data showed that when treated by PAI-2 antibody, involucrin in the keratinocytes envelope expressed increasingly with an altering distribution from part to the whole envelope area. Our results indicate that during differentiation of epidermal keratinocyte, PAI-2 expresses mainly in the more differentiated keratinocytes and may protect the terminal differentiated keratinocytes from prematuration through inhibiting involucrin expression in cornified envelope.     

Kinetic analysis of plasminogen activator inhibitor type-2: urokinase complex formation and subsequent internalisation by carcinoma cell lines

The overexpression of urokinase (uPA), which plays a key role in tumour invasion and metastasis, is an established prognostic marker and potential therapeutic target. Plasminogen activator inhibitor type 2 (PAI-2), an efficient and specific inhibitor of uPA, has been shown to selectively deliver potent cytotoxins to tumour cells. However, a direct quantitative analysis of both the inhibition kinetics and subsequent fate of PAI-2 upon interaction with cell-surface uPA has not been previously undertaken. In this study, we analysed specific PAI-2 binding to receptor-bound uPA on human breast and prostate cancer cell lines to directly measure inhibition kinetics. Cell-surface uPA:PAI-2 complex formation, which is reflective of complete uPA inhibition, was found to be very efficient (inactivation constant [K(I)] = 60-80 pM, depending on cell line used) and rapid (inactivation rate constant [k(inact)] = 0.32-0.47 min(-1) at 37 degrees C, depending on cell line used). To directly quantify and visualise cellular internalisation and localisation, we developed a novel assay based on the use of PAI-2 labelled with Alexa(488) fluorochrome and a polyclonal antibody to quench Alexa(488) fluorescence. The efficient and rapid formation of uPA:PAI-2 complexes was thus shown to be associated with specific and rapid internalisation of PAI-2, which could be localised within endosomes and lysosomes. PAI-2 was subsequently degraded, presumably within lysosomes. This study is the first to provide definitive evidence for uPA/uPAR-mediated PAI-2 endocytosis.     

Expression of the catalytic activity of plasminogen activator under physiologic conditions

We have investigated the factors governing the plasminogen-dependent fibrinolysis catalyzed by the serine proteinase, plasminogen activator (EC 3.4.21.-), under physiologic conditions. We found that live rabbit fibroblasts digested much less fibrin than predicted by cell-free assay of the secreted plasminogen activator. The reduced catalytic activity of plasminogen activator expressed by cells growing on fibrin was regulated by the salt concentration of culture medium. The plasminogen activators of cells from several mammalian species were inhibited by physiologic salt concentrations (0.15 M NaCl) in cell-free assays. CaCl₂ and KCl, but not D-glucose, were also effective inhibitors. The catalytic activity of purified human urokinase and of plasmin was unaffected by increased ionic strength. Plasminogen activators secreted both spontaneously and in response to stimulation by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate, were inhibited by 0.15 M NaCl. Physiologic salt concentration appeared to function by interacting with plasminogen activator, or plasminogen, and a third component, possibly a reversible inhibitor. One consequence of this regulation of plasminogen activator under physiologic conditions is the limitation of plasminogen-dependent fibrin degradation by living cels.     

The cellular target for the plasminogen activator,urokinase, in human fibroblasts - 66 000-dalton protein

The effects of urokinase, the plasminogen activator of human urine, on the isolated substratum-attached pericellular matrices of cultured human lung fibroblasts were studied in serum-free conditions. Pericellular matrices were prepared from dense cultures of human lung fibroblasts after labelling of the cultures with radioactive glycine. Extraction of the cultures with sodium deoxycholate and hypotonic buffer gave a reproducible pattern of polypeptides when analyzed in polyacrylamide gels. The isolated pericellular matrix was subsequently exposed to affinity chromatography-purified urokinase. Urokinase affected a 66 000-dalton protein but none of the other matrix polypeptides. The appearance of a 62 000-dalton protein that remained attached to the matrix was seen concomitantly suggesting that it was derived from the 66 000-dalton protein. The 66 000-dalton protein is the first known cellular target for urokinase.     

Inhibition of apoptosis and caspase-3 in vascular smooth muscle cells by plasminogen activator inhibitor type-1

Increased expression of plasminogen activator inhibitor type 1 (PAI-1) is associated with decreased apoptosis of neoplastic cells. We sought to determine whether PAI-1 alters apoptosis in vascular smooth muscle cells (VSMC) and, if so, by what mechanisms. A twofold increase in the expression of PAI-1 was induced in VSMC from transgenic mice with the use of the SM-22alpha gene promoter (SM22-PAI+). Cultured VSMC from SM22-PAI+ mice were more resistant to apoptosis induced by tumor necrosis factor plus phorbol myristate acetate or palmitic acid compared with VSMC from negative control littermates. Both wild type (WT) and a stable active mutant form of PAI-1 (Active) inhibited caspase-3 amidolytic activity in cell lysates while a serpin-defective mutant (Mut) PAI-1 did not. Similarly, both WT and Active PAI-1 decreased amidolytic activity of purified caspase-3, whereas Mut PAI-1 did not. WT but not Mut PAI-1 decreased the cleavage of poly-[ADP-ribose]-polymerase (PARP), the physiological substrate of caspase-3. Noncovalent physical interaction between caspase-3 and PAI-1 was demonstrable with the use of both qualitative and quantitative in vitro binding assays. High affinity binding was eliminated by mutations that block PAI-1 serpin activity. Accordingly, attenuated apoptosis resulting from elevated expression of PAI-1 by VSMC may be attributable, at least in part, to reversible inhibition of caspase-3 by active PAI-1.     

Proteomic profiling of proteins associated with urokinase plasminogen activator receptor in a colon cancer cell line using an antisense approach

Expression of urokinase plasminogen activator (uPA) and its receptor (uPAR) strongly correlates with a malignant tumour cell phenotype. In the multistep process of metastasis, uPA binding to uPAR influences different cellular functions. In the present study, a highly metastatic colon cancer cell line, HCT116 was transfected with an expression vector containing a 5' uPAR cDNA fragment in an antisense orientation. This construct was most effective in reducing uPAR cell surface expression as confirmed by flow cytometry analysis. Antisense transfection of HCT116 cells had no effect on proliferation but the following effects were observed: (1) a 1.3-fold decreased adhesion; (2) a two-fold decreased Erk MAP kinase activity; (3) a 2.7-fold decrease in Src kinase activity; (4) a 1.5- and two-fold decrease in uPA cell surface expression and secretion; (5) abrogation of promatrix metalloproteinase-9 secretion; and (6) a complete suppression of plasminogen-dependent matrix degradation. Using proteomic analysis, we demonstrate loss of approximately 200 proteins and quantitative differences in the expression of 141 other proteins in an antisense-clone compared to wild-type and mock-transfected control. Such changes in protein expression with the down-regulation of uPAR may be an important contributor in colon cancer progression and metastasis and may not only provide a basis to develop a proteomic data bank of uPAR-mediated signaling molecules but may also lead to the development of therapeutic approaches for the cure and better management of colon cancer.     

Induction of plasminogen activator inhibitor-2 is associated with suppression of invasive activity in TPA-mediated differentiation of human prostate cancer cells

We previously reported that 12-O-tetra-decanoylphorbol-13-acetate (TPA) induces microglia-like differentiation and decreases malignancy in human prostate cancer TSU-Pr1 cells. To investigate the mechanism underlying differentiation and decrease of malignancy in TSU-Pr1 cells treated with TPA, we attempted to identify genes expressed differentially during the differentiation using differential display. We successfully detected plasminogen activator inhibitor type-2 (PAI-2) as one gene up-regulated by TPA treatment. The change in expression of PAI-2 by TPA was blocked by treatment with protein kinase C or mitogen-activated protein kinase inhibitors. We also found that secretion of PAI-2 protein was increased by TPA treatment. Moreover, we demonstrated that suppression of invasive activity of TSU-Pr1 cells by TPA treatment was blocked by co-treatment with anti-PAI-2 antibody. These results suggest that induction of PAI-2 is associated with suppression of invasive activity in TSU-Pr1 cells treated with TPA.     

Structural basis of the cofactor function of denatured albumin in plasminogen activation by tissue-type plasminogen activator

Certain denatured proteins function as cofactors in the activation of plasminogen by tissue-type plasminogen activator. The present study approached the structural requirements for the cofactor activity of a model protein (human serum albumin). Heat denaturation of 100-230 microM albumin (80 degrees C and 60-90 min) reproducibly yielded aggregates with radius in the range of 10-150 nm. The major determinant of the cofactor potency was the size of the aggregates. The increase of particle size correlated with the cofactor activity, and there was a minimal requirement for the size of the cofactor (about 10 nm radius). Similar to other proteins, the molecular aggregates with cofactor function contained a significant amount of antiparallel intermolecular beta-sheets. Plasmin pre-digestion increased the cofactor efficiency (related to C-terminal lysine exposure) and did not affect profoundly the structure of the aggregates, suggesting a long-lasting and even a self-augmenting cofactor function of the denatured protein.     

Plasminogen activators and plasminogen activator inhibitors in connective tissues and connective tissue cells: influence of the neuropeptide substance P on expression

Tissue segments isolated from ligament, epiligament, and synovial tissues from mature female New Zealand White Rabbits were demonstrated to consitutively secrete a plasminogen activator. Several tissues were also observed to constitutively secrete a plasminogen activator inhibitor which was detected in the form of a PA-PAI complex. Heterogeneity was observed in PA and PAI activity between the different connective tissues. Heterogeneity also existed between and within the medial collateral (MCL), lateral collateral (LCL), and the anterior cruciate (ACL) ligaments. In addition to the differences in constitutive expression of PA and PAI activity, differences in the responsiveness to the neuropeptide substance P (10^?5?10^?9 M) were also detected. This responsiveness to substance P was displayed by an increase in PA and PAI activity in the conditioned medium. The pattern of responsiveness reflected the degree of innervation of these tissues. That is, synovium and epiligament tissue were the most responsive tissues to substance P while the MCL, LCL and ACL were less responsive to the neuropeptide. Parallel results were obtained using cell culture with fibroblasts isolated from the above mentioned tissues. That is, the pattern of responsiveness was similar between cells and tissue segments. More specifically, cells isolated from both synovium and epiligament increased their both their PA (slightly) and PAI activity following exposure to substance P. This was demonstrated at both the protein and RNA level. Thus, cells within a tissue maintain their phenotype when removed from their three-dimensional matrix. These results are unique in demonstrating that normal ligament and synovial cells and tissue respond to substance P by altering the expression of PA and PAI activity. This investigation further supports the concept that innervation may be important in normal connective tissue function.     

Enhanced secretion of tissue plasminogen activator by simultaneous use of retinoic acid and ascorbic acid from tissue cultured gastroepiploic artery

Tissue-type plasminogen activator (tPA) is a key enzyme in the fibrinolysis system and the regulation of its expression has been extensively studied in cultured vascular endothelial cells. Many kinds of supplements including growth factors are needed, however, to keep endothelial cells viable, which leads the culture condition far from the physiological milieu. Using a new device of amorphous calcium phosphate coated culture plate, we succeeded in culturing ring-cut gastroepiploic artery in a basic medium of RPMI 1640 containing 10% fetal calf serum. The overall normal vessel architecture and the antigenicity of von Willebrand factor, tPA and plasminogen activator inhibitor type 1 (PAI-1) were retained for at least 9 days. tPA was constantly secreted into the conditioned medium at least up to day 12. Employing this organ culture technique, we analyzed the effects of two well-known profibrinolytic vitamins of retinoic acid (Vit. A) and ascorbic acid (Vit. C) on the release of tPA and PAI-1. The cultured artery responded well and the tPA secretion was enhanced by factors of 1.5 fold by Vit. A, 1.7 fold by Vit C and 3.2 fold by their combination, whereas none of these stimuli increased PAI-1 secretion. These results suggested that the cultured ring-cut artery retained functional endothelial cells for at least 9 days and was suitable in analyzing the regulatory mechanism of protein synthesis and secretion from the vascular wall. Using this method, vitamins A and C were shown to lead the intravascular condition to a profibrinolytic state.     

Expression of active plasminogen activator inhibitor-1 reduces cell migration and invasion in breast and gynecological cancer cells

Urokinase-type (uPA) plasminogen activator is regulated by serine protease inhibitors (serpins), especially plasminogen activator inhibitor-1 (PAI-1). In many cancers, uPA and PAI-1 contribute to the invasive phenotype. We examined the in vitro migration and invasive capabilities of breast, ovarian, endometrial, and cervical cancer cell lines compared to their plasminogen activator system profiles. We then overexpressed active wild-type PAI-1 and an inactive "substrate" P14 form of PAI-1 (T333R) using stable transfection and adenoviral gene delivery. We also upregulated endogenous uPA and PAI-1 in these cells by treatment with transforming growth factor-beta. Some breast and ovarian cancer cell lines with natural expression of uPA, PAI-1, and urokinase receptor showed substantial migration and invasion compared to other cell lines that lack expression of these proteins. However, overexpression of active wild-type PAI-1, but not P14-PAI-1 (T333R), in these cell lines showed reduced migration and invasion. Since vitronectin binding by both forms of PAI-1 is equivalent, these results imply that PAI-1-vitronectin interactions are less critical in altering migration and invasion. Our results show that the in vitro migratory and invasive phenotype in these breast and ovarian cancer cell lines is reduced by active PAI-1 due to its ability to inhibit plasminogen activation.     

Binding of tissue-type plasminogen activator (t-PA) to Neisseria meningitidis and Haemophilus influenzae

Abstract Forty-nine bacterial strains representing five species known to interact with human plasminogen were tested for the ability to bind the two major human plasminogen activators, t-PA and urokinase. The bacterial species tested included Haemophilus influenzae, Neisseria meningitidis, Streptococcus pyogenes, Streptococcus equisimilis and human group G streptococci. All N. meningitidis and 11 of 14 H. influenzae strains displayed substantial binding of t-PA with values in the range of 20–46%. On the contrary, none of the streptococcal strains bound significant amounts of tPA. With urokinase no binding could be found for any of the bacterial species tested. Scatchard analysis with a selected H. influenzae strain (HI23354) demonstrated 10 000 receptors per bacterium for t-PA with a K _d value of about 20 nmol l⁻¹. The corresponding values with a selected N. meningitidis strain (Mo 52) was 8500 receptors per bacterium and 70 nmol l⁻¹. t-PA binding could be reduced about 40% by the addition of 10 nmol l⁻¹ of the lysine analogue ϵ-aminocaproic acd (EACA) whereas no inhibitory effect could be demonstrated with arginine. Addition of 2 μmol l⁻¹ of plasminogen which is enough to occupy all bacterial sites for plasminogen did not interfere with the t-PA binding, suggesting that the receptors for t-PA and plasminogen are distinct. Using very high plasminogen concentrations however, t-PA binding could be reduced by about 50% possibly due to an interaction between t-PA and plasminogen in the fluid phase. Our results demonstrate the occurrence of a previously unknown type of bacterial receptor that is capable of specifically binding t-PA.     

Structural similarity of the covalent complexes formed between the serpin plasminogen activator inhibitor-1 and the arginine-specific proteinases trypsin, LMW u-PA, HMW u-PA, and t-PA: use of site-specific fluorescent probes of local environment

We have used two fluorescent probes, NBD and dansyl, attached site-specifically to the serpin plasminogen activator inhibitor-1 (PAI-1) to address the question of whether a common mechanism of proteinase translocation and full insertion of the reactive center loop is used by PAI-1 when it forms covalent SDS-stable complexes with four arginine-specific proteinases, which differ markedly in size and domain composition. Single-cysteine residues were incorporated at position 119 or 302 as sites for specific reporter labeling. These are positions approximately 30 A apart that allow discrimination between different types of complex structure. Fluorescent derivatives were prepared for each of these variants using both NBD and dansyl as reporters of local perturbations. Spectra of native and cleaved forms also allowed discrimination between direct proteinase-induced changes and effects solely due to conformational change within the serpin. Covalent complexes of these derivatized PAI-1 species were made with the proteinases trypsin, LMW u-PA, HMW u-PA, and t-PA. Whereas only minor perturbations of either NBD and dansyl were found for almost all complexes when label was at position 119, major perturbations in both wavelength maximum (blue shifts) and quantum yield (both increases and decreases) were found for all complexes for both NBD and dansyl at position 302. This is consistent with all four complexes having similar location of the proteinase catalytic domain and hence with all four using the same mechanism of full-loop insertion with consequent distortion of the proteinase wedged in at the bottom of the serpin.     

Fibrinolytic system of cultured endothelial cells: regulation by plasminogen activator inhibitor

Cultured bovine aortic endothelial cells have a relatively complex fibrinolytic system that is responsive to both the physiological state of the cell itself and to a variety of agents added to the culture medium. The fibrinolytic activity of these cells results from the production of both urokinase-type and tissue-type plasminogen activators and is regulated by an inhibitor capable of neutralizing their activities. The properties of these fibrinolytic components will be reviewed, and their respective roles in initiating and regulating the fibrinolytic activity of the cells will be summarized. A cDNA coding for the inhibitor has been isolated, and its sequence will be compared to that of other serine proteinase inhibitors.     

5-Hydroxytryptamine 2A receptor signaling cascade modulates adiponectin and plasminogen activator inhibitor 1 expression in adipose tissue

Knowledge of the regulatory factors associated with down-regulation of adiponectin gene expression and up-regulation of PAI-1 gene expression is crucial to understand the pathophysiological basis of obesity and metabolic diseases, and could establish new treatment strategies for these conditions. We showed that expression of 5-HT(2A) receptors was up-regulated in hypertrophic 3T3-L1 adipocytes, which exhibited decreased expression of adiponectin and increased expression of PAI-1. 5-HT(2A) receptor antagonists and suppression of 5-HT(2A) receptor gene expression enhanced adiponectin expression. Activation of Gq negatively regulated adiponectin expression, and inhibition of mitogen-activated protein kinase reversed the Gq-induced effect. Moreover, the 5-HT(2A) receptor blockade reduced PAI-1 expression. These findings indicate that antagonism of 5-HT(2A) receptors in adipocytes could improve the obesity-linked decreases in adiponectin expression and increases in PAI-1 expression.