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.     

Differentiating cells of murine stratified squamous epithelia constitutively express plasminogen activator inhibitor type 2 (PAI-2)

 In stratified squamous epithelia a critical balance among cell proliferation, differentiation, and death must be maintained in order for these tissues to fulfill their barrier function. Previous studies have demonstrated that plasminogen activator inhibitor 2 (PAI-2) is a product of differentiating epidermal keratinocytes, suggesting a role for this inhibitor during squamous differentiation. Furthermore, in certain tumor cell lines, overexpression of PAI-2 confers resistance to the induction of programmed cell death, suggesting cytoprotective function(s). In the present study we demonstrate that PAI-2 mRNA and protein are constitutively and uniquely expressed in differentiating cells of murine stratified squamous epithelia, including epidermis, esophagus, vagina, oral mucosa, and tongue. PAI-2 immunohistochemical localization patterns suggest a predominantly cytosolic distribution, consistent with biochemical identification of the major PAI-2 species as a 43-kDa, presumably non-glycosylated protein. Functional analysis shows that the majority of epithelial PAI-2 is active. In contrast to the high levels of PAI-2 expression in stratified squamous epithelia, little or no PAI-2 is detectable in simple epithelia. These findings suggest that epithelial PAI-2 may mediate inhibition of intracellular proteinases associated with events during terminal differentiation and death that are unique to stratified squamous epithelia. Accepted: 29 June 1998     

Interaction of plasminogen activator inhibitor (PAI-1) with vitronectin

Immobilized vitronectin was found to bind both purified plasminogen activator inhibitor type 1 (PAI-1) and the PAI-1 in conditioned culture medium of human sarcoma cells. Similarly, immobilized PAI-1 bound both purified vitronectin and vitronectin from normal human serum. These interactions were demonstrated using both enzyme immunoassay and radioiodinated proteins. Solid-phase vitronectin bound PAI-1 with Kd 1.9 x 10(-7) M, and the reverse interaction gave a Kd 5.5 x 10(-8) M. Evidence was also found for a second type of binding with a Kd below 10(-10) M. The molar ratios of the two proteins in the complex at the saturation levels were approximately one molecule of soluble PAI-1 bound per three molecules of immobilized vitronectin and approximately one molecule of soluble vitronectin being bound per one molecule of immobilized PAI-1. Binding of PAI-1 to vitronectin did not lead to an irreversible loss of the ability of PAI-1 to inhibit urokinase (u-PA) and tissue-type plasminogen activator (t-PA). Active u-PA released vitronectin-bound 125I-labeled PAI-1 radioactivity, suggesting that u-PA interacts with the complex. The Mr 50,000 urokinase cleavage product of PAI-1 also bound to vitronectin, but this bound fragment did not inhibit u-PA. Binding of PAI-1 to vitronectin did not interfere with the ability of vitronectin to promote the adhesion and spreading of cells. These results suggest that the interaction between vitronectin and PAI-1 may serve to confine pericellular u-PA activity to focal contact sites where cells use proteolysis in regional detachment.     

Functional interaction of plasminogen activator inhibitor type 1 (PAI-1) and heparin.

Plasminogen activator inhibitor type 1 (PAI-1), the fast-acting inhibitor of tissue-type plasminogen activator (t-PA) and urokinase (u-PA), is a member of the serpin superfamily of proteins. Both in plasma and in the growth substratum of cultured endothelial cells, PAI-1 is associated with its binding protein vitronectin, resulting in a stabilization of active PAI-1. Recently, it has been demonstrated that the PAI-1-binding site on vitronectin is adjacent to a heparin-binding site (Preissner et al., 1990). Furthermore, it can be deduced that the amino acid residues, proposed to mediate heparin binding in the serpins antithrombin III and heparin cofactor II, are conserved in PAI-1. Consequently, here we have investigated whether PAI-1 also interacts with heparin. At pH 7.4, PAI-1 quantitatively binds to heparin-Sepharose and can be eluted with increasing [NaCl]. Binding of PAI-1 to heparin-Sepharose can be efficiently competed with heparin in solution (IC50, 7 microM). In the presence of heparin, the protease specificity of PAI-1 toward thrombin is substantially increased. This is shown by (i) quenching of thrombin activity of PAI-1 in the presence of heparin and (ii) induction of the formation of SDS-stable complexes between thrombin and PAI-1 by heparin. In a dose response curve, both effects reached a maximum at approximately 1 unit/mL and then diminished again upon further increasing the heparin concentration, strongly suggesting a template mechanism as an explanation for the observed effect. In contrast to vitronectin, heparin does not stabilize the active conformation of PAI-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidermal plasminogen activator inhibitor (PAI) is immunologically identical to placental-type PAI-2

Plasminogen activator inhibitor (PAI) purified from human epidermis [(1986) FEBS Lett. 408, 273-277] was immunologically identified as placental-type PAI-2. In both fibrinolytic and synthetic substrate assays inhibitory activity of epidermal PAI was neutralized by anti-PAI-2, but not by anti-endothelial type PAI-1. Immunoblotting technique confirmed that the purified epidermal PAI is reactive with anti-PAI-2, but not with anti-PAI-1. Consequently PAI in human epidermis was demonstrable by immunohistochemical technique.     

Association of plasminogen activator inhibitor type 2 (PAI-2) with proteasome within endothelial cells activated with inflammatory stimuli

Quiescent endothelial cells contain low concentrations of plasminogen activator inhibitor type 2 (PAI-2). However, its synthesis can be rapidly stimulated by a variety of inflammatory mediators. In this study, we provide evidence that PAI-2 interacts with proteasome and affects its activity in endothelial cells. To ensure that the PAI-2·proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after (a) transfection of HeLa cells with pCMV-PAI-2 and coimmunoprecipitation of both proteins with anti-PAI-2 antibodies and (b) silencing of the PAI-2 gene using specific small interfering RNA (siRNA). Subsequently, cellular distribution of the PAI-2·proteasome complexes was established by immunogold staining and electron microscopy analyses. As judged by confocal microscopy, both proteins appeared in a diffuse cytosolic pattern, but they also could be found in a dense perinuclear and nuclear location. PAI-2 was not polyubiquitinated, suggesting that it bound to proteasome not as the substrate but rather as its inhibitor. Consistently, increased PAI-2 expression (a) abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-2 and pd2EGFP-N1, (b) prevented degradation of p53, as evidenced both by confocal microscopy and Western immunoblotting, and (c) inhibited proteasome cleavage of specific fluorogenic substrate. This suggests that PAI-2, in endothelial cells induced with inflammatory stimuli, can inhibit proteasome and thus tilt the balance favoring proapoptotic signaling.     

Plasminogen activator inhibitor type 2 (PAI-2) is present in normal human conjunctiva

The purpose was to characterize plasminogen activator inhibitor type 2 (PAI-2) expression in normal human conjunctiva in vivo and in vitro. PAI-2 antigen was assayed by immunostaining and immunoblotting of extracts from normal human conjunctival epithelial lysates and conditioned media (CM) of cultured human conjunctival keratinocytes. Immunostaining of normal human conjunctival epithelia revealed that PAI-2 was found consistently in the superficial keratinocytes and, in some biopsies, also in the lower keratinocyte layers. In all cases, PAI-2 was concentrated around the cell periphery. In extracts of conjunctival epithelia and cultured conjunctival keratinocytes, PAI-2 had an apparent molecular weight of 45 kDa, consistent with the non-glycosylated form. The majority of PAI-2, approximately 90%, was cell associated, however, a small percentage of PAI-2 was released into the CM in a linear manner with time. PAI-2 in the conditioned medium had a higher molecular weight, consistent with a glycosylated form. Conjunctival PAI-2 was active, as shown by its ability to complex with a target enzyme, urokinase plasminogen activator (uPA). Although PAI-2 was detectable both in monolayer (i.e., relatively undifferentiated) conjunctival keratinocyte cultures as well as in stratified (i.e., more differentiated) cultures, steady state levels of PAI-2 were greater in the latter. PAI-2 is constitutively expressed by normal human conjunctival epithelial cells. The expression of PAI-2 throughout all epithelial layers in some biopsies of conjunctiva in vivo contrasts with the previously established distribution of PAI-2 in corneal epithelia, where it is present exclusively in the most superficial (i.e. most highly differentiated) cells. The role of PAI-2 in either tissue is unclear. However, we speculate that its distinct distribution in conjunctival versus corneal epithelia underscores inherent differences between these tissues, and may reflect specific functions of this proteinase inhibitor in both conjunctival and corneal epithelial cells.     

High-level synthesis of biologically active human plasminogen activator inhibitor type 1 (PAI-1) in Escherichia coli

Segments of a cDNA encoding human plasminogen activator inhibitor type 1 (PAI-1) were subcloned into a highly regulated and inducible Escherichia coli expression system. A plasmid encoding the mature form of human endothelial PAI-1 produced a functional recombinant molecule, as indicated by its ability to inhibit tissue plasminogen activator's enzymatic activity. In contrast to PAI-1 isolated from human fibrosarcoma cells, the biological activity of the recombinant PAI-1 was not dependent on pretreatment with denaturing agents. A construct encoding a polypeptide lacking the first 80 amino acids of PAI-1 also produced elevated levels of the truncated recombinant protein. However, this truncated product was functionally inactive, indicating that an intact N terminus is required for activity.     

Non-muscle alpha-actinin-4 interacts with plasminogen activator inhibitor type-1 (PAI-1)

PAI-1 modulates many biological processes involving fibrinolysis, cell migration or tissue remodelling. In addition to inhibiting serine proteases (mainly tPA and uPA), PAI-1 interacts with vitronectin (Vn), fibrin or alpha(1)-acid glycoprotein, interactions which are important for PAI-1-mediated effects in inflammation, tumor invasion and metastasis. To further identify proteins interacting with PAI-1, the yeast two-hybrid strategy was employed. Screening of a human placenta cDNA library identified--in addition to the C-terminal region of cytokeratin 18 (CK18(182-430))--a large C-terminal fragment of alpha-actinin-4 (Act-4) as a binding partner for PAI-1. Two different cDNA clones encoding Act-4(287-911) and Act-4(330-911) respectively, were isolated. An Act-4(330-911)/GST-fusion protein, but not GST alone, was immunoprecipitated together with active PAI-1. In solid phase binding assays, active wild-type PAI-1 as well as the PAI-1 variant Q123K (which does not interact with multimeric Vn) was found to bind to Act-4(330-911)/GST. Latent PAI-1, latent Q123K, and the inactive PAI-1 variant Q55P did not display any binding activity. Act-4 is mainly present intracellularly and is involved in cellular motility via interaction with the actin cytoskeleton, thus probably affecting the metastatic potential of tumor cells. However, an extracellular Act-4-derived fragment (mactinin) has previously been identified, which (i) is generated by proteolytic action of uPA, (ii) displays significant chemotactic activity for monocytes, and (iii) promotes monocyte/macrophage maturation. We suggest that PAI-1, via interaction with both Act-4 and uPA, may function as a modulator of this mononuclear phagocyte response, not only in inflammation but also in tumor invasion and metastasis.     

Interaction of plasminogen activator inhibitor type-1 (PAI-1) with vitronectin.

The serpin plasminogen activator inhibitor type 1 (PAI-1) plays an important role in physiological processes such as thrombolysis and fibrinolysis, as well as pathophysiological processes such as thrombosis, tumor invasion and metastasis. In addition to inhibiting serine proteases, mainly tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, PAI-1 interacts with different components of the extracellular matrix, i.e. fibrin, heparin (Hep) and vitronectin (Vn). PAI-1 binding to Vn facilitates migration and invasion of tumor cells. The most important determinants of the Vn-binding site of PAI-1 appear to reside between amino acids 110-147, which includes alpha helix E (hE, amino acids 109-118). Ten different PAI-1 variants (mostly harboring modifications in hE) as well as wild-type PAI-1, the previously described PAI-1 mutant Q123K, and another serpin, PAI-2, were recombinantly produced in Escherichia coli containing a His(6) tag and purified by affinity chromatography. As shown in microtiter plate-based binding assays, surface plasmon resonance and thrombin inhibition experiments, all of the newly generated mutants which retained inhibitory activity against uPA still bound to Vn. Mutant A114-118, in which all amino-acids at positions 114-118 of PAI-1 were exchanged for alanine, displayed a reduced affinity to Vn as compared to wild-type PAI-1. Mutants lacking inhibitory activity towards uPA did not bind to Vn. Q123K, which inhibits uPA but does not bind to Vn, served as a control. In contrast to other active PAI-1 mutants, the inhibitory properties of A114-118 towards thrombin as well as uPA were significantly reduced in the presence of Hep. Our results demonstrate that the wild-type sequence of the region around hE in PAI-1 is not a prerequisite for binding to Vn.     

Topological localization of plasminogen activator inhibitor type 2

BACKGROUND: Plasminogen activator inhibitor type 2 (PAI-2) is a member of the serine protease inhibitor (SERPIN) superfamily and forms stable complexes with urokinase type plasminogen activator (uPA). uPA can be found on the cell surface attached to its specific receptor (uPAR), allowing for controlled degradation of the extracellular matrix by the activation of plasminogen into plasmin. The aim of this study was to evaluate if PAI-2 could also be detected on the cell surface, providing a means of regulating the activity of cell surface uPA. METHODS: Intact or permeabilized cell lines or human peripheral blood leukocytes were assayed by flow cytometry for cell surface uPA or PAI-2. Plasma membrane-enriched preparations prepared from Jurkat, HaCaT, THP-1, U937, or MM6 cells were assayed by enzyme-linked immunosorbent assay (ELISA) or Western blotting for PAI-2 antigen. RESULTS: By flow cytometry, cell surface PAI-2 was not detected on monocytes from human peripheral blood, MM6, or HaCaT cells. Cell surface PAI-2 was only detected very weakly on the surface of U937 cells. In contrast, PAI-2 could be detected in all of these cells when fixed and permeabilized. By ELISA, PAI-2 was very abundant in the cytosol-enriched preparations of U937, MM6, and HaCaT cells, but was present in lower amounts in the plasma membrane-enriched preparations. By Western blotting, monomeric nonglycosylated PAI-2, but not uPA/PAI-2 complexes, could be detected in the cytosol and plasma membrane-enriched preparations. CONCLUSIONS: These results indicate that PAI-2 cannot be detected on the surface of PAI-2-expressing cells, and confirm that PAI-2 is predominantly a cytosolic protein.     

Identity between the placental protein PP10 and the specific plasminogen activator inhibitor of placental type PAI-2

The highly specific plasminogen activator inhibitor of placental type, PAI-2, occurs in the placenta in a low molecular mass form of 46.6 kDa, and in pregnancy plasma in a (possibly glycosylated) high molecular mass form of 60 kDa. Extensive knowledge is available about the functional properties of PAI-2 as a plasminogen activator inhibitor and about its molecular biology and regulation. Of the several placenta proteins (PP) isolated, one of them, PP10, has a molecular mass of 48 kDa and its occurrence in malignancy and in complications during pregnancy has been the topic of a number of studies, though its properties and physiological significance are unknown. The present findings constitute evidence of immunological identity between PP10 and PAI-2. The sections of the amino acid sequence of PP10 analysed here were found to have identical counterparts in the sequence of the low molecular mass form of PA1-2, but in several preparations PP10 was found to occur in an inactive two-chain form due to cleavage of an Arg-Thr bond, the two peptide chains being linked to each other by a disulphide bridge. The cleavage site is identical to that observed in the reaction between PAI-2 and urokinase. The results make it possible to coordinate and correlate the findings of many separate studies and our own observations on PP10 and PAI-2.     

Thrombin inhibits apoptosis of monocytes and plasminogen activator inhibitor 2 (PAI-2) is not responsible for this inhibition

Plasminogen activator inhibitor 2 (PAI-2) has been shown to inhibit apoptosis in transfected cells. We have investigated this phenomenon in activated human monocytes, which are a physiological source of intracellular PAI-2. Apoptosis of monocytes was rapidly induced by removal of serum, addition of hydrogen peroxide, or binding of a monoclonal antibody to Fas. Treatment of monocytes with thrombin or lipopolysaccharide (LPS) inhibited apoptosis of monocytes and also up-regulated intracellular PAI-2. Increased apoptosis was accompanied with increased activity of caspases 3 and 8. Thrombin or LPS treatment of monocytes decreased the activity of both caspases, which correlated with protection from apoptosis. The role for PAI-2 in protection of monocytes from apoptosis was studied. Monocytes were transfected with antisense oligonucleotides that blocked PAI-2 antigen, and antisense for PAI-2 had no effect on apoptosis of monocytes. No interaction was evident between PAI-2 and recombinant caspases 3 and 8 in vitro. PAI-2 was not a substrate for caspases during apoptosis of monocytes, although some cleavage of recombinant PAI-2 by caspase 3 was evident in vitro. This study shows that thrombin or LPS protected monocytes from apoptosis and that PAI-2 did not mediate this inhibitory effect.     

Xanthoangelols isolated from Angelica keiskei inhibit inflammatory-induced plasminogen activator inhibitor 1 (PAI-1) production

The folk medicine Angelica keiskei (Ashitaba) exhibits antitumor, antioxidant and antidiabetic activities and it has recently attracted attention as a health food. Ashitaba is thought to have antithrombotic properties, but this has not yet been scientifically proven. The elevation of plasma plasminogen activator inhibitor 1 (PAI-1), an inhibitor of fibrinolysis results in a predisposition to the risk of thrombosis. The present study showed that Ashitaba exudates injected intraperitoneally and orally administered over long-term suppressed the lipopolysaccharide (LPS) induced PAI-1 increase in mouse plasma. We also found that xanthoangelol, xanthoangelols B and D, the components of Ashitaba exudates, significantly inhibited TNFα-induced PAI-1 production from human umbilical vein endothelial cells (HUVECs). These findings suggest that Ashitaba can decrease elevated PAI-1 production, and that daily consumption of Ashitaba product might maintain anticoagulant status by inhibiting elevations in PAI-1 under inflammatory conditions.     

Activation of human endothelial cell-type plasminogen activator inhibitor (PAI-1) by negatively charged phospholipids

The endothelial cell-type plasminogen activator inhibitor (PAI-1) may exist in an inactive, latent form that can be converted into an active form upon treatment of the protein with denaturants, such as sodium dodecyl sulfate, guanidine HCl, or urea. The present paper demonstrates that latent PAI-1 can be activated by lipid vesicles containing the negatively charged phospholipids phosphatidylserine (PS) or phosphatidylinositol. The presence of a net negative charge on the phospholipid headgroup is essential for activation, since lipid vesicles consisting exclusively of zwitterionic phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, do not activate PAI-1. In the presence of PS vesicles, PAI-1 inhibited tissue-type plasminogen activator 50-fold more effectively than in the absence of phospholipids, whereas sodium dodecyl sulfate enhanced PAI-1 activity by 25-fold. In mixed phospholipid vesicles containing PS and phosphatidylcholine in various molar ratios, the extent of PAI-1 activation was directly related to the PS content of the phospholipid membrane. Ca2+ ions interfered with the inhibitory activity of PS-activated PAI-1, suggesting that Ca2+ ions may regulate PAI-1 activity in the presence of negatively charged phospholipids. An important consequence of these findings is that, as in blood coagulation, negatively charged phospholipids may play an important regulatory role in controlling the fibrinolytic system by activating an inhibitor of tissue-type plasminogen activator.     

Critical role of type 1 plasminogen activator inhibitor (PAI-1) in early host defense against nontypeable Haemophilus influenzae (NTHi) infection

Respiratory systems are constantly being challenged by pathogens. Lung epithelial cells serve as a first line of defense against microbial pathogens by detecting pathogen-associated molecular patterns (PAMPs) and activating downstream signaling pathways, leading to a plethora of biological responses required for shaping both the innate and adaptive arms of the immune response. Acute-phase proteins (APPs), such as type 1 plasminogen activator inhibitor (PAI-1), play important roles in immune/inflammatory responses. PAI-1, a key regulator for fibrinolysis and coagulation, acts as an APP during acute phase response (APR) such as acute lung injury (ALI), inflammation, and sepsis. However, the role of PAI-1 in the pathogenesis of these diseases still remains unclear, especially in bacterial pneumonia. In this study, we showed that PAI-1 expression is upregulated following nontypeable Haemophilus influenzae (NTHi) infection. PAI-1 knockout (KO) mice failed to generate early immune responses against NTHi. Failure of generating early immune responses in PAI-1 KO mice resulted in reduced bacterial clearance and prolonged disease process, which in turn led to enhanced inflammation at late stage of infection. Moreover, we also found that NTHi induces PAI-1 via activation of TLR2–MyD88–MKK3–p38 MAPK signaling pathway. These data suggest that PAI-1 plays critical role in earl host defense response against NTHi infection. Our study thus reveals a novel role of PAI-1 in infection caused by NTHi, one of the most common gram-negative bacterial pathogens in respiratory systems.