Secretion of Plasminogen Activator Inhibitor 2 by Human Peripheral Blood Monocytes Occurs via an Endoplasmic Reticulum–Golgi-Independent Pathway

Plasminogen activator inhibitor 2 (PAI-2) is a serine protease inhibitor (serpin) that is secreted and accumulated intracellularly by monocytes. We investigated PAI-2 synthesis by isolated human peripheral blood monocytes and found that a 47-kDa nonglycosylated form of PAI-2 was abundant in conditioned medium from monocytes. Secretion of PAI-2 by monocytes was not inhibited by agents that inhibit either ER–Golgi pathway-dependent secretion, brefeldin A, or N-linked glycosylation, tunicamycin. IL-1β served as a control for a protein that is secreted by an ER–Golgi-independent pathway, and secretion of IL-1β was not inhibited by brefeldin A. This was in contrast to secretion of TNFα, which was dependent on the ER–Golgi pathway. None of the treatments was cytotoxic toward monocytes, as measured by release of the intracellular enzyme lactate dehydrogenase (LDH) into the conditioned medium. Subcellular fractionation revealed that PAI-2 and IL-1β were colocalized. The mechanism for secretion of PAI-2 was not dependent on calcium or intracellular trafficking via the classical vesicular mechanism(s), distinguishing it from IL-1β secretion. These studies show that PAI-2 is secreted by primary human monocytes via an ER–Golgi-independent pathway.     

Mammalian protein secretion without signal peptide removal. Biosynthesis of plasminogen activator inhibitor-2 in U-937 cells

Plasminogen activator inhibitor-2 (PAI-2) is a serine protease inhibitor that regulates plasmin generation by inhibiting urokinase and tissue plasminogen activator. The primary structure of PAI-2 suggests that it may be secreted without cleavage of a single peptide. To confirm this hypothesis we have studied the glycosylation and secretion of PAI-2 in human monocytic U-937 cells by metabolic labeling, immunoprecipitation, glycosidase digestion, and protein sequencing. PAI-2 is variably glycosylated on asparagine residues to yield intracellular intermediates with zero, one, two, or three high mannose-type oligosaccharide units. Secretion of the N-glycosylated species began by 1 h of chase and the secreted molecules contained both complex-type N-linked and O-linked oligosaccharides. Enzymatically deglycosylated PAI-2 had an electrophoretic mobility identical to that of the nonglycosylated precursor and also to that of PAI-2 synthesized in vitro in a rabbit reticulocyte lysate from synthetic mRNA derived from full length PAI-2 cDNA. The amino-terminal protein sequence of secreted PAI-2 began with the initiator methionine residue. These results indicate that PAI-2 is glycosylated and secreted efficiently without the cleavage of a signal peptide. PAI-2 shares this property with its nearest homologue in the serine protease inhibitor family, chicken ovalbumin, and appears to be the first well characterized example of this phenomenon among natural mammalian proteins.     

Inhibition of endothelial secretion of tissue-type plasminogen activator and its rapid inhibitor by agents which increase intracellular cyclic AMP

We investigated the effect of agents which raise intracellular levels of cyclic AMP (cAMP) on the secretion of tissue-type plasminogen activator (t-PA) and type 1 plasminogen activator inhibitor (PAI-1) by cultured human umbilical-vein endothelial cells. Significant inhibition of baseline (unstimulated) t-PA and PAI-1 secretion was observed in response to several agents which, when added exogenously, cause increased intracellular cAMP: cholera toxin, 1-methyl-3-isobutylxanthine (MIX), dibutyryl-cAMP, and prostaglandin E1. These agents also significantly reduced or abolished the previously reported stimulatory effects of thrombin and histamine on t-PA secretion, and, with the exception of MIX, significantly reduced the previously reported stimulatory effect of thrombin on PAI-1 secretion. MIX at a concentration (10 microM) below that required to inhibit t-PA and PAI-1 secretion when tested alone, significantly increased the inhibitory effects of cholera toxin, dibutyryl-cAMP, and prostaglandin E1 on both t-PA and PAI-1 secretion. The data suggest that elevated intracellular levels of cAMP inhibit both spontaneous endothelial secretion of t-PA and PAI-1, and secretion induced by agents (thrombin and histamine) which stimulate endothelial phosphoinositide metabolism, consistent with bidirectional regulation of endothelial fibrinolytic protein secretion by the adenylate cyclase and phosphoinositide signal transduction pathways. The inhibitory effects of cAMP do not appear to be specific for t-PA and PAI-1, since cholera toxin and MIX also inhibited endothelial secretion of the adhesive protein, fibronectin. Significant inhibition of baseline endothelial t-PA and PAI-1 secretion was also caused by the stable prostacyclin analogue iloprost (ZK 36 374) and by arachidonic acid, which is converted by endothelial cells to prostacyclin, suggesting that prostacyclin produced endogenously by endothelial cells may inhibit secretion of fibrinolytic proteins by increasing intracellular cAMP.     

Functional activity of eukaryotic signal sequences in Escherichia coli: the ovalbumin family of serine protease inhibitors

It is widely assumed that the functional activity of signal sequences has been conserved throughout evolution, at least between Gram-negative bacteria and eukaryotes. The ovalbumin family of serine protease inhibitors (serpins) provides a unique tool to test this assumption, since individual members can be secreted (ovalbumin), cytosolic (leukocyte elastase inhibitor, LEI), or targeted to both compartments (plasminogen activator inhibitor 2, PAI-2). The facultative secretion of PAI-2 is mediated by a signal sequence proposed to be inefficient by design. We show here that the same internal domain that promotes an inefficient translocation of murine PAI-2 in mammalian cells is a weak signal sequence in Escherichia coli. In contrast, the ovalbumin signal sequence is much more efficient, whereas the corresponding sequence elements from LEI, maspin and PI-10 are entirely devoid of signal sequence activity in E.coli. Mutations that improve the activity of the PAI-2 signal sequence and that convert the N-terminal regions of maspin and PI-10 into efficient signal sequences have been characterized. Taken together, these results indicate that several structural features contribute to the weak activity of the PAI-2 signal sequence and provide new insights into the plasticity of the "hydrophobic core" of signal sequences. High-level expression of two chimeric proteins containing the PAI-2 signal sequence is toxic, and the reduced viability is accompanied by a rapid decrease in the membrane proton motive force, in ATP levels and in translation. In unc- cells, which lack the F0F1 ATP-synthase, the chimeric proteins retain their toxicity and their expression only affected the proton motive force. Thus, the properties of these toxic signal sequences offer a new tool to dissect the interactions of signal sequences with the protein export machinery.     

Val17Ile single nucleotide polymorphisms similarly as Ala15Thr could be related to the lower secretory dynamics of PAI-1 secretion: theoretical evidence

Plasminogen activator inhibitor (PAI-1) is a fast acting inhibitor of tissue and urokinase plasminogen activators (tPA and uPA). In that way PAI-1 regulates proteolytic activity of many physiological and pathological processes [1-3]. PAI-1 plays an important role in blood coagulation controlling clot lysis which is triggered by tPA activated plasminogen [4]. Only two types of mutations are reported to be associated with PAI-1; one is the frame-shift mutation in exon 4 of PAI-1 gene resulting in a truncated nonfunctional protein and in complete PAI-1 deficiency. The other SNP causes Ala15Thr mutation in the signal peptide. A literature search revealed five variants of polymorphisms during a study of over one thousand individuals. Two are associated with thrombophilia (765 4G/5G and -844 A>G, in the promoter), risk of myocardial infarction and postoperative deep venous thrombosis related to higher than normal levels of PAI-1. The other SNPs associated with PAI-1 deficiency are Ala15Thr, Val17Ile and they are located in the central hydrophobic core of the PAI-1 signal peptide of PAI-1 and Asn195Ile in the 'A' β sheet of the PAI-1. We have analyzed two SNPs not reported to be associated with PAI-1 deficiency. Our analysis suggests that Val17Ile PAI-1 variant might cause slower PAI-1 secretion leading to the deficiency at time and place where it is needed in a similar way as for Ala15Thr SNP. The Asn195Ile mutant may be more stable only as latent form thus no PAI-1 deficiency is expected in this mutant.     

Evidence for intracellular cleavage of plasminogen activator inhibitor type 2 (PAI-2) in normal epidermal keratinocytes

Plasminogen activator inhibitor type 2 (PAI-2) is a serine proteinase inhibitor (serpin), present in high quantities in stratified squamous epithelia. Detergent extracts of human epidermis or cultured keratinocytes contain primarily active, nonglycosylated PAI-2. In keratinocytes, the vast majority of PAI-2 is retained within the cell, supporting the hypothesis that PAI-2 may serve specific intracellular function(s) through interaction with an unknown cytoplasmic proteinase. During interaction with the target proteinase, cleavage of PAI-2 within its reactive site loop leads to the formation of a more stable, "relaxed" conformation (PAI-2r). Using a monoclonal antibody specific for PAI-2r, we demonstrate here that PAI-2r is present in keratinocytes of the granular and basal layers of normal human epidermis. In addition, PAI-2r is detectable in cultured human epidermal keratinocytes, where it is concentrated in a detergent-insoluble fraction within differentiating cells. These data provide evidence for the presence of an endogenous, keratinocyte-derived proteinase that constitutively cleaves intracellular PAI-2 in normal human epidermal keratinocytes. Cleavage of PAI-2 by this proteinase may reflect specific intracellular action of PAI-2 in normal cells. Finally, we demonstrate that a commercially available anti-PAI-2 monoclonal antibody (#3750, American Diagnostica, Greenwich, CT), under native experimental conditions, preferentially recognizes the uncleaved, active form of PAI-2 and does not efficiently detect PAI-2r.     

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 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     

Mutations of the alpha-galactosidase signal peptide which greatly enhance secretion of heterologous proteins by yeast.

The Saccharomyces carlsbergensis MEL1 gene encodes alpha-galactosidase (melibiase; MEL1) which is readily secreted by yeast cells into the culture medium. To evaluate the utility of the MEL1 signal peptide (sp) for the secretion of heterologous proteins by Saccharomyces cerevisiae, an expression vector was constructed which contains the MEL1 promoter and MEL1 sp coding sequence (MEL1sp). The coding sequences for echistatin (Echis) and human plasminogen activator inhibitor type 1 (PAI-1) were inserted in-frame with the MEL1sp. S. cerevisiae transformants containing the resulting expression vectors secreted negligible amounts of either Echis or PAI-1. Using site-directed mutagenesis, several mutations were introduced into the MEL1sp. Two mutations were identified which dramatically increased the secretion of both Echis and PAI-1 to levels similar to those achieved when using the yeast MF alpha 1 pre-pro secretory leader. In particular, increasing the hydrophobicity of the core region plus the addition of a positive charge to the N-terminal domain of the MEL1 sp resulted in the greatest increase in the secretion levels of those two proteins.     

The undecided serpin. The ins and outs of plasminogen activator inhibitor type 2

Plasminogen activator inhibitor type-2 (PAI-2) is a nonconventional serine protease inhibitor (serpin) with unique and tantalizing properties that is generally considered to be an authentic and physiological inhibitor of urokinase. However, the fact that only a small percentage of PAI-2 is secreted has been a long-standing argument for alternative roles for this serpin. Indeed, PAI-2 has been shown to have a number of intracellular roles: it can alter gene expression, influence the rate of cell proliferation and differentiation, and inhibit apoptosis in a manner independent of urokinase inhibition. Despite these recent advances in defining the intracellular function of PAI-2, it still remains one of the most mysterious and enigmatic members of the serpin superfamily.     

Cytoplasmic and nuclear interaction between Rb family proteins and PAI-2: a physiological crosstalk in human corneal and conjunctival epithelial cells

Extracellular plasminogen activator inhibitor type-2 (PAI-2) is a potent inhibitor of urokinase-type plasminogen activator (u-PA) and also acts as a multifunctional protein. However, the biological activity of intracellular PAI-2, as well as its intracellular targets, until now remain an enigma. Here, we show that pRb2/p130 and Rb1/p105, but not p107, interact with PAI-2 in both the cytoplasm and nucleus of normal primary human corneal and conjunctival epithelial cells. We provided the first in vivo evidence that a specific fragment of the PAI-2 promoter is bound simultaneously by pRb2/ p130, PAI-2, E2F5, histone deacetylase 1 (HDAC1), DNA methyltransferase 1 (DNMT1), and histone methyltransferase (SUV39H1), in normal primary human corneal epithelial cells, and by pRb2/p130, PAI-2, E2F5, HDAC1, and DNMT1, in normal primary human conjunctiva epithelial cells. Our results strongly indicate a physiological interaction between pRb family members and PAI-2, suggesting the hypothesis that pRb2/p130 and PAI-2 may cooperate in modulating PAI-2 gene expression by chromatin remodeling, in normal corneal and conjunctival cells.     

Plasminogen activator inhibitor type-1: reactive center and amino-terminal heterogeneity determined by protein and cDNA sequencing

Both the urokinase-type and tissue-type plasminogen activator can convert their approximately 54 kDa type-1 inhibitor (PAI-1) to an inactive form with a lower apparent molecular mass. We have determined the amino-terminal amino acid sequences of human native and converted PAI-1, and isolated PAI-1 cDNA and determined the nucleotide sequence in regions corresponding to the amino-terminus and the cleavage site. The data show that the conversion of the inhibitor consists of cleavage of an Arg-Met bond 33 residues from the carboxy-terminus, thus localizing the reactive center of the inhibitor to that position. In addition, a heterogeneity was found at the amino-terminus, with a Ser-Ala-Val-His-His form and a two-residue shorter form (Val-His-His-) occurring in approximately equal quantities.     

Synergistic stimulating effect between cyclic AMP and phorbol ester on plasminogen activator inhibitor type 2 production in human promyelocytic leukemia cell line PL-21.

We investigated the effect of agents which raise intracellular cyclic AMP (cAMP) and protein kinase C activators on the production of plasminogen activator inhibitor type-2 (PAI-2) by cultured human promyelocytic leukemia cell line, PL-21. As previously reported, PMA, a protein kinase C activator, showed a strong stimulating effect on the PAI-2 production. 1-oleoyl-2-acetyl-sn-glycerol (OAG), another synthetic protein kinase C activator, also showed a stimulating effect, which was, however, much less than that of PMA. The agents which raise intracellular cAMP, dibutyryl cAMP, 8-bromo cAMP, prostaglandin E1, and 3-isobutyl-1-methyl-xanthine, little increased the PAI-2 production when tested alone, but showed significant synergistic effects with PMA or OAG. The synergistic effect between PMA and dibutyryl cAMP was further verified by SDS-PAGE followed by immunoblotting using a monoclonal antibody against the PAI-2. It is interesting that the up-regulation of PAI-2 by cAMP and the synergistic effect with PKC activators forms a contrast to the previous reported bi-directional regulation of endothelial PAI-1 secretion by PKC activator and cAMP.     

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…     

Structural bases of the redox-dependent conformational switch in the serpin PAI-2

Depending on the redox-status, the serpin plasminogen activator inhibitor type 2 (PAI-2) can exist in either a stable monomeric or polymerogenic form. The latter form, which spontaneously forms loop-sheet polymers, has an open beta-sheet A and is stabilized by a disulfide bond between C79 (in the CD-loop) and C161 (at the bottom of PAI-2). Reduction of this bond results in a closing of the beta-sheet A and converts PAI-2 to a stable monomeric form. Here we show that the stable monomeric and polymerogenic forms of PAI-2 are fully interconvertible, depending on redox-status of the environment. Our intramolecular distance measurements indicate that the CD-loop folds mainly on one side of the stable monomeric form of the inhibitor. However, the loop can translocate about 54A to the bottom of PAI-2 so that the C79-C161 disulfide bond can form under oxidizing conditions. We show also that the redox-active C79 can form a disulfide-link to the matrix protein vitronectin, suggesting that vitronectin can stabilize active PAI-2 in extracellular compartments. PAI-2 is therefore a rare example of a redox-sensitive protein for which the activity and polymerization ability are regulated by reversible disulfide bond formation leading to major translocation of a loop and significant conformational changes in the molecule.     

The majority of type 1 plasminogen activator inhibitor associated with cultured human endothelial cells is located under the cells and is accessible to solution-phase tissue-type plasminogen activator

The interactions between exogenously added tissue-type plasminogen activator (t-PA) and the active form of type 1 plasminogen activator inhibitor (PAI-1) produced by and present in cultured human umbilical vein endothelial cells (HUVECs) were investigated. Immunoblotting analysis of the conditioned media obtained from monolayers of HUVECs treated with increasing concentrations of t-PA (less than or equal to 10 micrograms/ml) revealed a dose-dependent formation of both t-PA/PAI-1 complexes, and of a 42,000-Mr cleaved or modified form of the inhibitor. Immunoradiometric assays indicated that t-PA treatment resulted in a fourfold increase in PAI-1 antigen present in the conditioned media. This increase did not result from the release of PAI-1 from intracellular stores, but rather reflected a t-PA-dependent decrease in the PAI-1 content of the Triton X-100 insoluble extracellular matrix (ECM). Although the rate of t-PA-mediated release of PAI-1 was increased by the removal of the monolayer, similar quantities of PAI-1 were removed in the presence or absence of the cells. These results suggest that the cells only represent a semipermeable barrier between ECM-associated PAI-1 and exogenous t-PA. Treatment of HUVECs with t-PA (1 microgram/ml, 2 h) to deplete the ECM of PAI-1 did not affect the subsequent rate of PAI-1 production and deposition into the ECM. Immunogold electron microscopy of HUVECs not only confirmed the location of PAI-1 primarily in the region between the culture substratum and ventral cell surface but failed to demonstrate significant (less than 1%) PAI-1 on the cell surface. Thus, the majority of PAI-1 associated with cultured HUVEC monolayers is present under the cells in the ECM and is accessible to solution-phase t-PA.     

Detection of both type 1 and type 2 plasminogen activator inhibitors in human cells

This report describes the development and use of functional immunoradiometric assays that distinguish the activity of beta-migrating endothelial-type plasminogen activator inhibitor (PAI-1) from that of placental-type plasminogen activator inhibitor (PAI-2). These assays are based upon the binding of PAI-1 and PAI-2 to immobilized single-chain tissue-type plasminogen activator (tPA) and to immobilized urokinase (UK), respectively. The extent of binding of each PAI is quantified by incubating the PAI-PA complex first with rabbit antiserum specific for the individual PAI and then with 125I-labeled goat antirabbit IgG. In control experiments, the assays were shown to be sensitive, dose-dependent over a wide range, and specific for each PAI. These assays were employed to establish the PAI profile of a variety of human cells. Neither PAI-1 nor PAI-2 could be detected in Bowes melanoma cells or in a renal adenocarcinoma cell line (ACHN), while the histiocytic lymphoma cell (U-937) produced only PAI-2. Five cell lines, including two that were previously shown to contain one or the other PAI (e.g., umbilical vein endothelial cells and a fibrosarcoma cell line, HT-1080) in fact contained both PAIs. The cells containing both PAIs were studied in more detail. In each case, SDS treatment of CM was shown to enhance PAI-1 activity (by converting the latent form of this inhibitor into its active form) and to destroy PAI-2 activity. Various compounds including interleukin 1, dexamethasone, and phorbol myristate acetate were found to selectively influence the cellular production of one PAI without concomitantly affecting the production of the other, suggesting that the synthesis of these inhibitors is not coordinately regulated.