Role of different mechanisms in pro-inflammatory responses triggered by traffic-derived particulate matter in human bronchiolar epithelial cells.

BACKGROUND: Traffic-derived particles are important contributors to the adverse health effects of ambient particulate matter (PM). In Nordic countries, mineral particles from road pavement and diesel exhaust particles (DEP) are important constituents of traffic-derived PM. In the present study we compared the pro-inflammatory responses of mineral particles and DEP to PM from two road tunnels, and examined the mechanisms involved. METHODS: The pro-inflammatory potential of 100 µg/mL coarse (PM10-2.5), fine (PM2.5-0.18) and ultrafine PM (PM0.18) sampled in two road tunnels paved with different stone materials was assessed in human bronchial epithelial cells (HBEC3-KT), and compared to DEP and particles derived from the respective stone materials. Release of pro-inflammatory cytokines (CXCL8, IL-1α, IL-1ß) was measured by ELISA, while the expression of genes related to inflammation (COX2, CXCL8, IL-1α, IL-1ß, TNF-α), redox responses (HO-1) and metabolism (CYP1A1, CYP1B1, PAI-2) was determined by qPCR. The roles of the aryl hydrocarbon receptor (AhR) and reactive oxygen species (ROS) were examined by treatment with the AhR-inhibitor CH223191 and the anti-oxidant N-acetyl cysteine (NAC). RESULTS: Road tunnel PM caused time-dependent increases in expression of CXCL8, COX2, IL-1α, IL-1ß, TNF-α, COX2, PAI-2, CYP1A1, CYP1B1 and HO-1, with fine PM as more potent than coarse PM at early time-points. The stone particle samples and DEP induced lower cytokine release than all size-fractionated PM samples for one tunnel, and versus fine PM for the other tunnel. CH223191 partially reduced release and expression of IL-1α and CXCL8, and expression of COX2, for fine and coarse PM, depending on tunnel, response and time-point. Whereas expression of CYP1A1 was markedly reduced by CH223191, HO-1 expression was not affected. NAC reduced the release and expression of IL-1α and CXCL8, and COX2 expression, but augmented expression of CYP1A1 and HO-1. CONCLUSIONS: The results indicate that the pro-inflammatory responses of road tunnel PM in HBEC3-KT cells are not attributed to the mineral particles or DEP alone. The pro-inflammatory responses seem to involve AhR-dependent mechanisms, suggesting a role for organic constituents. ROS-mediated mechanisms were also involved, probably through AhR-independent pathways. DEP may be a contributor to the AhR-dependent responses, although other sources may be of importance.

Fibrinolytic Changes in Women with Preeclampsia.

OBJECTIVES: Preeclampsia (PE) is a serious complication of pregnancy. The fibrinolytic system play crucial roles regarding placentation and evolution of PE. AIM: To study comprehensively components of the fibrinolytic system and fibrin lysability in women with PE. DESIGN AND METHODS: 117 women with PE and matched controls were included. Tissue type plasminogen activator (t-PA), plasminogen, PAI-1, plasmin inhibitor (PI), D-dimer, the fibrinolytic potential of dextran sulphate euglobulin fraction (DEF), PAI-2, polymere PAI-2, fibrin clot lysability, thrombin activatable fibrinolysis inhibitor (TAFI) and fibrinogen were assessed. RESULTS: Women with PE had significantly increased concentrations of t-PA and PAI-1, whereas the plasma concentration of PAI-2 was significantly lower compared to controls, p < 0.0001. Polymere PAI-2 was detected in both groups. DEF, TAFI and fibrinogen were not different between the groups. D-dimer was significantly increased and plasminogen/PI together with fibrin clot lysability time decreased in the PE-group, p = 0.0004 p = 0.04, p = 0.03, p < 0.0001 respectively. CONCLUSION: This study demonstrates that PE is associated with an affected t-PA/PAI-1 system, decreased PAI-2 and increased fibrin lysability. Furthermore, PAI-2 has the potential to polymerize during pregnancy.

PAI-2/SerpinB2 inhibits proteolytic activity in a P. gingivalis-dominated multispecies bacterial consortium.

OBJECTIVE: The aim of this study was to investigate the ability of the serine protease inhibitor plasminogen activator inhibitor type 2 (PAI-2/Serpin B2) to inhibit proteases produced by a multispecies bacterial consortium in vitro. BACKGROUND: Gingival and periodontal inflammation is associated with an increased flow of protein-rich gingival fluid. This nutritional change in the microenvironment favors bacteria with a proteolytic phenotype, triggering inflammation and associated tissue breakdown. PAI-2 is produced by macrophages and keratinocytes and is present in very high concentrations in gingival crevicular fluid; the highest level in the body. DESIGN: A multispecies bacterial consortium comprising nine bacterial strains, resembling the conditions in a periodontal pocket, was grown planktonically and as a biofilm. After seven days PAI-2 was added to the consortium and the proteolytic activity was assayed with fluorogenic protease substrates; FITC-labeled casein to detect global protease activity, fluorescent H-Gly-Pro-AMC for serine protease activity and fluorescent BIKKAM-10 for Porphyromonas gingivalis-associated protease activity. Protease activity associated with biofilm cells was examined by confocal scanning laser microscopy. RESULTS: PAI-2 inhibited proteolytic activity of the bacterial consortium, as seen by decreased fluorescence of all substrates. PAI-2 specifically inhibited P. gingivalis proteolytic activity. CONCLUSION: To our knowledge, this is the first time that PAI-2 has been shown to inhibit bacterial proteases. Given the high concentration of PAI-2 in the gingival region, our results indicate that PAI-2 might play a role for the integrity of the epithelial barrier.

SerpinB2 (PAI-2) Modulates Proteostasis via Binding Misfolded Proteins and Promotion of Cytoprotective Inclusion Formation.

SerpinB2 (PAI-2), a member of the clade B family of serine protease inhibitors, is one of the most upregulated proteins following cellular stress. Originally described as an inhibitor of urokinase plasminogen activator, its predominant cytoplasmic localisation suggests an intracellular function. SerpinB2 has been reported to display cytoprotective properties in neurons and to interact with intracellular proteins including components of the ubiquitin-proteasome system (UPS). In the current study we explored the potential role of SerpinB2 as a modulator of proteotoxic stress. Initially, we transiently transfected wild-type SerpinB2 and SerpinB2-/- murine embryonic fibroblasts (MEFs) with Huntingtin exon1-polyglutamine (fused C-terminally to mCherry). Inclusion body formation as result of Huntingtin aggregation was evident in the SerpinB2 expressing cells but significantly impaired in the SerpinB2-/- cells, the latter concomitant with loss in cell viability. Importantly, recovery of the wild-type phenotype and cell viability was rescued by retroviral transduction of SerpinB2 expression. SerpinB2 modestly attenuated Huntingtin and amyloid beta fibril formation in vitro and was able to bind preferentially to misfolded proteins. Given the modest chaperone-like activity of SerpinB2 we tested the ability of SerpinB2 to modulate UPS and autophagy activity using a GFP reporter system and autophagy reporter, respectively. Activity of the UPS was reduced and autophagy was dysregulated in SerpinB2-/- compared to wild-type MEFs. Moreover, we observed a non-covalent interaction between ubiquitin and SerpinB2 in cells using GFP-pulldown assays and bimolecular fluorescence complementation. We conclude that SerpinB2 plays an important role in proteostasis as its loss leads to a proteotoxic phenotype associated with an inability to compartmentalize aggregating proteins and a reduced capacity of the UPS.

Anaerobiosis-induced loss of cytotoxicity is due to inactivation of quorum sensing in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic pathogen of clinical importance, causes chronic airway infections in patients with cystic fibrosis (CF). Current literature suggests that pockets with reduced oxygen tension exist in the CF airway mucus. However, virulence features of this opportunistic pathogen under such conditions are largely unknown. Cell-free supernatant of the standard laboratory P. aeruginosa strain PAO1 obtained from anaerobic culture, but not aerobic culture, failed to kill A549 human airway epithelial cells. Further investigation revealed that this reduced cytotoxicity upon anaerobiosis was due to the suppressed secretion of elastase, a virulence factor controlled by P. aeruginosa quorum sensing (QS). Both a lacZ-reporter fusion assay and quantitative real-time PCR (RT-PCR) analysis demonstrated that transcription of the elastase-encoding lasB gene was substantially decreased during anaerobic growth compared with aerobic growth. Moreover, transcription of other genes controlled by the LasI/R QS system, such as rhlR, vqsR, mvfR, and rsaL, was also repressed under the same anaerobic growth conditions. Importantly, synthesis of 3-oxo-C(12)-HSL (PAI-1), an autoinducer molecule that mediates induction of the LasI/R QS system, was >22-fold decreased during anaerobic growth while C(4)-HSL (PAI-2), which mediates RhlI/R QS, was nondetectable under the same growth conditions. Transcription of the lasB gene was restored by exogenous supplementation with autoinducers, with PAI-2 more effective than PAI-1 or Pseudomonas quinolone signal (PQS) at restoring transcription of the lasB gene. Together, these results suggest that anaerobiosis deprives P. aeruginosa of the ability to regulate its virulence via QS and this misregulation attenuates the pathogenic potential of this important pathogen.

Intraperitoneal infusion of recombinant plasminogen activator inhibitor type 2 induced apoptosis in implanted human colon cancer and inhibited its growth and liver metastasis.

Antitumor effects of plasminogen activator (PA) inhibitors (PAls) were analyzed in a mouse model of human colon cancer xenografts. Either recombinant PA inhibitor-1 (rPAI-1) or inhibitor-2 (rPAI-2) was injected intraperitoneally to nude mice bearing human colon cancer xenografts for 6 weeks. Primary tumors in rPAI-2-treated group were smaller (0.45 +/- 0.13 g, n = 16) than in the other two groups (control: 0.73 +/- 0.24 g, n = 15; rPAI-1: 0.62 +/- 0.29 g, n = 19). Primary tumors in the rPAI-2-treated group exhibited less mature ductal structures and were significantly smaller. The apoptotic index was higher in the rPAI-2-treated group (4.64 +/- 2.12%) than in the other groups (control: 1.94 +/- 0.82%; rPAI-1: 2.08 +/- 1.07%). Liver metastasis was less frequent in the rPAI-1 (5/19) and rPAI-2-treated groups (1/16) than in the control group (14/15). PAI-2 more effectively suppressed tumor metastasis and progression, probably by inducing apoptosis; some different unknown mechanism may cause the difference in both antitumor effect and the histological findings. This may indicate the therapeutic potential of these PAls in malignant patients.

Singlet oxygen enhances intrinsic thrombolysis: the intrinsic oxidative clot lysis assay (INOXCLA).

Granulocytes are important cells of inflammation and cellular thrombolysis. They produce urokinase (u-PA) and chloramines. In this study, u-PA/chloramine-mediated fibrinolysis is imitated in a microtiter-plate. Seventy-five microliters plasma are incubated with 50 microL 50% Pathromtin SL, 6% BSA, and 38 mM CaCl2 for 30 minutes (37 degrees C). Then, 50 microL 10 mM chloramine-T in PBS are added. After 30 minutes (37 degrees C), 50 microL 0, 100, or 10 IU/mL u-PA in 6% BSA-PBS are added and the turbidity is determined at 405 nm after 0, 3, or 16 hours. Clot lysis was increased more than tenfold by 0.5 to 1 micromoles chloramine (ED50 after 3h = about 0.25 micromoles = 2 mM final concentration). The normal range for the present intrinsic oxidative clot lysis assay (INOXCLA) is 100% +/- 25% (MV +/- SD; 100 relative % of norm; the normal lysis being 60 absolute %; CVs < 10%). Fifty percent lysis of adherent microclots occurred after 0.75 hours, 2 hours, 14 hours, 13 days, or 17 days when using 1000, 100, 10, 1, or 0 IU/mL u-PA reagent. If the u-PA activity is quenched by PAI-2, no clot lysis appears. Chloramines are important physiologic generators of nonradical excited singlet oxygen and enhance u-PA-mediated lysis of plasma clots. Based on the u-PA/chloramines coaction, a new global fibrinolysis assay has been derived.

Preclinical evaluation of 213Bi-labeled plasminogen activator inhibitor type 2 in an orthotopic murine xenogenic model of human breast carcinoma.

Tumor-associated urokinase plasminogen activator (uPA) is a critical marker of invasion and metastasis, has strong prognostic relevance, and is thus a potential therapeutic target. Experimental data published to date has established the proof-of-principle of uPA targeting by (213)Bi-labeled plasminogen activator inhibitor type 2 (alpha-PAI-2) in multiple carcinoma models. Here, we present preclinical toxicologic and efficacy assessment of alpha-PAI-2 in mice, using both single and multiple-dose schedules, administered by an i.p. route. We also present novel data showing that human PAI-2 inhibited murine uPA and was specifically endocytosed by murine fibroblast cells. This diminishes potential problems associated with species specificity of the targeting reagent in toxicologic assessments as human alpha-PAI-2 should interact with any uPA-expressing host cells. In this model, single bolus doses up to 36 mCi/kg alpha-PAI-2 did not reach the maximum tolerated dose (MTD). The MTD for a multiple fractionated (once daily for 5 days) administration schedule was determined to lie between 4.8 and 6.0 mCi/kg/d x 5. Comparison of the tumor growth rates and survival using sub-MTD single and multiple-dose schedules in an orthotopic human breast carcinoma xenograft murine model indicated that 4.8 mCi/kg/d x 5 was the most efficacious schedule. In conclusion, we have determined a safe dose and schedule of alpha-PAI-2 administration in mice, thus confirming that it is an efficacious therapeutic modality against tumor growth. This will allow detailed safety evaluation in a second species and for the initiation of human studies.

Activation of corneal fibroblast-derived matrix metalloproteinase-2 by tryptase.

PURPOSE: The presence of the active form of matrix metalloproteinase (MMP)-2 and an increased concentration of tryptase are characteristics of tear fluid of individuals with vernal keratoconjunctivitis. Although tryptase does not mediate the activation of purified MMP-2, we have now examined whether it might activate MMP-2 in the presence of cultured human corneal fibroblasts. METHODS: Corneal fibroblasts were cultured in the absence or presence of tryptase, and the activation status of MMP-2 was determined by gelatin zymography. RESULTS: MMP-2 released from corneal fibroblasts was activated by exogenous tryptase. This effect was not mediated by protease-activated receptor 2 or the plasmin-plasminogen system, and it was not apparent on incubation of tryptase with medium conditioned by corneal fibroblasts. It was inhibited by tissue inhibitor of metalloproteinase (TIMP)-2 but not by TIMP-1. CONCLUSIONS: Tryptase activates MMP-2 released from corneal fibroblasts. This action requires the presence of the cells themselves and might be responsible for the presence of activated MMP-2 in tear fluid of individuals with vernal keratoconjunctivitis.

Preclinical studies of targeted alpha therapy for breast cancer using 213Bi-labelled-plasminogen activator inhibitor type 2.

The control of micrometastatic breast cancer remains problematic. To this end, we are developing a new adjuvant therapy based on (213)Bi-PAI2, in which an alpha-emitting nuclide ((213)Bi) is chelated to the plasminogen activator inhibitor-2 (PAI2). PAI2 targets the cell-surface receptor bound urokinase plasminogen activator (uPA), which is involved with the metastatic spread of cancer cells. We have successfully labelled and tested recombinant human PAI2 with the alpha radioisotope (213)Bi to produce (213)Bi-PAI2, which is highly cytotoxic towards breast cancer cell lines. In this study, the 2-day postinoculation model, using MDA-MB-231 breast cancer cells, was shown to be representative of micrometastatic disease. Our in vivo efficacy experiments show that a single local injection of (213)Bi-PAI2 can completely inhibit the growth of tumour at 2 days postcell inoculation, and a single systemic (i.p.) administration at 2 days causes tumour growth inhibition in a dose-dependent manner. The specific role of uPA as the target for (213)Bi-PAI2 therapy was determined by PAI2 pretreatment blocking studies. In vivo toxicity studies in nude mice indicate that up to 100 microCi of (213)Bi-PAI2 is well tolerated. Thus, (213)Bi-PAI2 is successful in targeting isolated breast cancer cells and preangiogenic cell clusters. These results indicate the promising potential of (213)Bi-PAI2 as a novel therapeutic agent for micrometastatic breast cancer.

Plasminogen activator inhibitor type 2: a regulator of monocyte proliferation and differentiation.

We have explored the role of plasminogen activator inhibitor type 2 (PAI-2) in THP-1 monocyte-like cells. These cells possess a mutation in the PAI-2 gene and do not produce an active PAI-2 protein. Transfection of THP-1 cells with plasmids expressing active PAI-2 reduced the cells' inherent adhesive properties and decreased the rate of cell proliferation. THP-1 cells expressing active PAI-2 also displayed an altered phenotype in response to phorbol ester-induced differentiation that was concomitant with a reduction in CD14 expression. THP-1 cells transfected with a variant PAI-2 containing a mutation in the reactive center (PAI-2(Ala380)) displayed no noticeable change in any of these parameters, suggesting the involvement of a PAI-2-sensitive serine protease(s). The antiproliferative effect of PAI-2 was attenuated by treating the PAI-2-expressing THP-1 cells with recombinant urokinase (u-PA), suggesting that PAI-2 was disruptive of a u-PA/u-PA receptor signaling pathway initiated on the cell surface. Consistent with this, treatment of wild-type THP-1 cells with recombinant PAI-2 also caused a reduction in cellular proliferation. These results implicate endogenous PAI-2 as a modulator of monocyte adhesion, proliferation, and differentiation.

213Bi-PAI2 conjugate selectively induces apoptosis in PC3 metastatic prostate cancer cell line and shows anti-cancer activity in a xenograft animal model.

A novel alpha-particle emitting ((213)Bi) plasminogen activator inhibitor type 2 construct, which targets the membrane-bound urokinase plasminogen activator on prostate cancer cells, was prepared and evaluated in vitro and in a xenograft animal model. The PC3 prostate cancer cell line expresses urokinase plasminogen activator which binds to its receptor on the cell membrane; plasminogen activator inhibitor type 2 is bound to urokinase plasminogen activator/urokinase plasminogen activator receptor to form stable complexes. In vitro, the cytotoxicity of (213)Bi-plasminogen activator inhibitor type 2 against prostate cancer cells was tested using the MTS assay and apoptosis was documented using terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labelling (TUNEL) assay. In vivo, antiproliferative effects for tumours and prostate cancer lymph node metastasis were carried out in an athymic nude mouse model with a subcutaneous xenograft of PC3 cells. (213)Bi-plasminogen activator inhibitor type 2 was specifically cytotoxic to PC3 cells in a concentration-dependent fashion, causing the cells to undergo apoptosis. A single local or i.p. injection of (213)Bi-plasminogen activator inhibitor type 2 was able to completely regress the growth of tumours and lymph node metastases 2 days post subcutaneous inoculation, and obvious tumour regression was achieved in the therapy groups compared with control groups with (213)Bi-plasminogen activator inhibitor type 2 when the tumours measured 30-40 mm(3) and 85-100 mm(3). All control animals and one of five (20%) mice treated with 3 mCi kg(-1) (213)Bi-plasminogen activator inhibitor type 2 developed metastases in the lymph nodes while no lymphatic spread of cancer was found in the 6 mCi kg(-1) treated groups at 2 days and 2 weeks post-cell inoculation. These results demonstrate that this novel (213)Bi-plasminogen activator inhibitor type 2 conjugate selectively targets prostate cancer in vitro and in vivo, and could be considered for further development for the therapy of prostate cancer, especially for the control of micro-metastases or in minimal residual disease.

Monocyte plasminogen activator inhibitor 2 (PAI-2) inhibits u-PA-mediated fibrin clot lysis and is cross-linked to fibrin.

Plasminogen activator inhibitor 2 (PAI-2) is a major product of activated human monocytes. Here we show that monocytes inhibited u-PA- but not t-PA-mediated fibrinolysis, by secreting PAI-2 into an overlying fibrin clot. Extracts of arterial and venous human thrombi were found to contain active PAI-2. PAI-2 was cross-linked to fibrin in a reaction catalyzed by two major transglutaminases (TG), tissue TG and factor XIII. The activity of PAI-2 was not affected by such cross-linking. Cross-linking of PAI-2 to fibrin was inhibited by Tridegin, a specific inhibitor of TG, and also by EDTA and iodoacetamide. The use of competitive peptides mimicking the loop between helices C and D of PAI-2 identified Gln 83 and 86 as residues important in cross-linking. This study defines a mechanism by which PAI-2 is localized to fibrin, where it acts as an effective inhibitor of u-PA-mediated fibrinolysis.

Suppression of keratinocyte proliferation by plasminogen activator inhibitor-2.

We have previously shown that urokinase plasminogen activator (uPA) stimulates the growth of human keratinocytes in culture. For this effect, uPA activity is essential to generate the active amino terminal fragment, by an autolytic process. Our findings indicated further that inhibition of uPA may result in the suppression of growth of keratinoytes. Here, we provide evidence that plasminogen activator inhibitor (PAI)-2 has an anti-proliferative effect on keratinocytes. The uPA activity in cultured keratinocytes increased in parallel with cell proliferation, reaching a maximum level at confluency and decreasing gradually thereafter. The analysis of synchronized cells showed that the peak uPA activity in the medium occured just prior to S-phase, suggesting that the production and secretion of uPA is related to cell proliferation. In contrast, PAI-2 levels showed a steady increase, even after confluency. When PAI-2, purified from human cornified cells, was added to synchronized keratinocytes, S-phase was no longer evident and the peak uPA activity was eliminated. In experiments with a bacterially expressed PAI-2 fusion protein, [3H]thymidine incorporation by keratinocytes was significantly suppressed, confirming an anti-proliferative effect of PAI-2. These results strongly suggest that PAI-2 is involved in the regulation of keratinocyte proliferation and differentiation.

Uteroplacental hemostasis in intrauterine fetal growth retardation.

During pregnancy, extensive hemostatic changes occur in the uteroplacental circulation. Invading endovascular trophoblast cells induce physiological adaptations of uterine spiral arteries, required to accommodate the increased maternal blood flow to the intervillous space of the placenta as pregnancy advances. Much of the vascular endothelium and the underlying medial smooth muscle is replaced by trophoblasts, and fibrin or fibrinoid forms a major morphological feature of the arterial walls. Compared with endothelial cells, the trophoblast lining decidual spiral arteries have a reduced capacity to lyse fibrin, and recent studies have shown this to be caused by high levels of plasminogen activator inhibitors (PAI-1 and PAI-2). In pregnancies complicated by intrauterine fetal growth retardation (IUGR), with or without superimposed preeclampsia, a restricted physiological adaptation of uteroplacental spiral arteries is coupled with vascular lesions containing increased fibrin deposition. Significantly higher levels of PAI-1 are found in blood from the uterine vein at delivery and in tissue extracts of the placenta in these pregnancies than are found in normal pregnancy. Recent tissue culture studies have provided new information on the role of trophoblast cells in maintaining hemostatic control in the uteroplacental circulation in pregnancy. Cytotrophoblast cells isolated from the placenta and placental bed from IUGR pregnancies express significantly higher levels of PAI-1, coupled with a significant decrease in plasminogen activator activity, compared with trophoblast cells from normal pregnancy maintained in culture. This localized increased production of PAI-1 may play an important part in restricting endovascular trophoblast invasion in early pregnancy and increasing fibrin deposition and reducing uteroplacental blood flow in pregnancies complicated by IUGR.

Loss of cell viability dramatically elevates cell surface plasminogen binding and activation.

The plasminogen activation cascade is focused at the cell surface by virtue of the presence of plasminogen and plasminogen activator receptors. We have utilized flow cytometric plasminogen (plg) binding and activation assays to examine both plasminogen binding and activation on the surface of specific subpopulations of U937 cells (viable, apoptotic, and dead cells). A direct relationship was found to exist between cell viability (propidium iodide uptake) and the magnitude of lysine-dependent plasminogen binding, with apoptotic and dead subpopulations of cells binding up to 100-fold more plasminogen than viable cells. Despite the high level of lysine-dependent plasminogen binding on dead cells, plasminogen activation was minimal due to low levels of cell-surface urokinase plasminogen activator. Plasminogen activation readily occurred on the surface of apoptotic cells because of a dramatic increase in both lysine-dependent plasminogen binding and endogenous urokinase plasminogen activator. These results indicate that colocalization of plasminogen and urokinase plasminogen activator are paramount for plasminogen activation to proceed on the cell surface. Our data also strongly implicate the involvement of the plasminogen activation cascade in apoptosis, especially on urokinase plasminogen activator-expressing cell types. The current study clearly supports the important role of flow cytometry in cellular plasminogen binding and activation studies.

The serine proteinase inhibitor (serpin) plasminogen activation inhibitor type 2 protects against viral cytopathic effects by constitutive interferon alpha/beta priming.

The serine proteinase inhibitor (serpin) plasminogen activator inhibitor type 2 (PAI-2) is well characterized as an inhibitor of extracellular urokinase-type plasminogen activator. Here we show that intracellular, but not extracellular, PAI-2 protected cells from the rapid cytopathic effects of alphavirus infection. This protection did not appear to be related to an effect on apoptosis but was associated with a PAI-2-mediated induction of constitutive low-level interferon (IFN)-alpha/beta production and IFN-stimulated gene factor 3 (ISGF3) activation, which primed the cells for rapid induction of antiviral genes. This primed phenotype was associated with a rapid development of resistance to infection by the PAI-2 transfected cells and the establishment of a persistent productive infection. PAI-2 was also induced in macrophages in response to viral RNA suggesting that PAI-2 is a virus response gene. These observations, together with the recently demonstrated PAI-2-mediated inhibition of tumor necrosis factor-alpha induced apoptosis, (a) illustrate that PAI-2 has an additional and distinct function as an intracellular regulator of signal transduction pathway(s) and (b) demonstrate a novel activity for a eukaryotic serpin.

Introduction of an RRHR motif into chicken urokinase-type plasminogen activator (ch-uPA) confers sensitivity to plasminogen activator inhibitor (PAI)-1 and PAI-2 and allows ch-uPA-mediated extracellular matrix degradation to be controlled by PAI-1.

Comparison of the amino acid sequence of the chicken and human urokinase-type plasminogen activators (uPAs) revealed that the putative PAI-binding site found in the variable region 1 (VR1) loop of mammalian PAs is absent in the homologous region of ch-uPA. ch-uPA, unlike mammalian PAs, also appears to be refractory to inhibition by human PAIs and as a naturally occurring PAI-resistant variant, constitutes a unique model system for assessing the functional relevance of the PAI-binding site. Therefore, we molecularly constructed a ch-uPA, ch-uPA(RRHR), which contains the putative PAI-binding motif RRHR (residues 192-195) in its VR1 loop. As a result of this substitution, the second-order rate constant of inhibition of PAI-1 increased approximately 700-fold from 4.50 x 10(4) M(-1) x s(-1) for wild-type ch-uPA to 3.02 x 10(7) M(-1) x s(-1) for ch-uPA(RRHR), and the ability to form SDS-stable, uPA-PAI-1 complexes increased approximately 1000-fold. Furthermore, the interaction of ch-uPA(RRHR) with PAI-2 was also substantially enhanced, while the interaction with other members of the serine proteinase inhibitor superfamily, protein nexin 1, alpha1-PI, and C1-inhibitor, was unaffected indicating that the RRHR motif is not a general serine proteinase inhibitor binding site. Finally, we show that extracellular matrix degradation by cells expressing ch-uPA(RRHR) is inhibited by PAI-1 in a dose-dependent manner, while matrix breakdown by cells expressing wild-type ch-uPA is unaffected by PAI-1. Thus acquisition of sensitivity to PAI-1 through a structural motif that enhances the specificity of the protease-inhibitor interaction confers to ch-uPA an added level of regulation in the context of the degradative cellular phenotype.

Proteases and protease inhibitors in tumor progression.

Our understanding of the role of matrix degrading proteases in cancer has dramatically expanded over the last two decades. From correlative observations linking proteases to cancer progression, we have accumulated evidence supporting a causal role for proteases in various steps of tumor progression and have become increasingly aware of the complex interactions that exist among proteases. Specific natural inhibitors of these proteases have also been identified and their role as potent cytostatic agents in cancer has been suggested. In this article some of the concepts on the role of proteases in cancer are discussed and examples of cooperation between matrix metalloproteinases and the plasmin/plasminogen activators system are presented. The role of protease inhibitors such as tissue inhibitor of metalloproteinases-2 (TIMP-2) and plasminogen activator inhibitor-2 (PAI-2) as inhibitors of tumor growth, invasion and metastasis is discussed.