Topological localization of plasminogen activator inhibitor type 2

Topological Localization of Plasminogen ActivatorInhibitor Type 2

Michael A. Liew,1* Virginia McPhun,2 and Mark S. Baker1

1Department of Biological Sciences, University of Wollongong, Wollongong, NSW, Australia2Division of Molecular Medicine, John Curtin School of Medical Research, Canberra, Australia

Received 21 April 1999; Revision Received 12 November 1999; Accepted 28 December 1999

Background: Plasminogen activator inhibitor type 2(PAI-2) is a member of the serine protease inhibitor (SER-PIN) superfamily and forms stable complexes with uroki-nase type plasminogen activator (uPA). uPA can be foundon the cell surface attached to its specific receptor(uPAR), allowing for controlled degradation of the extra-cellular matrix by the activation of plasminogen into plas-min. The aim of this study was to evaluate if PAI-2 couldalso be detected on the cell surface, providing a means ofregulating the activity of cell surface uPA.Methods: Intact or permeabilized cell lines or humanperipheral blood leukocytes were assayed by flow cytom-etry 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-linkedimmunosorbent assay (ELISA) or Western blotting forPAI-2 antigen.Results: By flow cytometry, cell surface PAI-2 was notdetected on monocytes from human peripheral blood,

MM6, or HaCaT cells. Cell surface PAI-2 was only detectedvery weakly on the surface of U937 cells. In contrast,PAI-2 could be detected in all of these cells when fixedand permeabilized. By ELISA, PAI-2 was very abundant inthe cytosol-enriched preparations of U937, MM6, and Ha-CaT cells, but was present in lower amounts in the plasmamembrane-enriched preparations. By Western blotting,monomeric nonglycosylated PAI-2, but not uPA/PAI-2complexes, could be detected in the cytosol and plasmamembrane-enriched preparations.Conclusions: These results indicate that PAI-2 cannot bedetected on the surface of PAI-2-expressing cells, andconfirm that PAI-2 is predominantly a cytosolic protein.Cytometry 40:32–41, 2000. © 2000 Wiley-Liss, Inc.

Key terms: leukocytes; urokinase; plasma membrane; cellsurface

The plasminogen activation cascade has been demon-strated to be important in processes involving extracellu-lar matrix (ECM) remodeling. This occurs directly by plas-min cleaving components of the ECM (e.g., fibrin) orindirectly through the activation of other proteases (e.g.,procollagenase). Such ECM remodeling is important inprocesses including cell migration, angiogenesis, fibrino-lysis, inflammation, and pregnancy (7,36,44). Plasmin for-mation from plasminogen is under the regulation of twoactivators, tissue type plasminogen activator (tPA) andurokinase type plasminogen activator (uPA). These twoactivators are regulated by at least two specific inhibitors,plasminogen activator inhibitor types 1 and 2 (PAI-1 andPAI-2).

PAI-2, a member of the serine protease inhibitor (SER-PIN) superfamily, was first isolated by Kawano et al. (21)from human placental tissue and later from the monocyticcell line U937 (24). PAI-2 exists in at least two isoforms, anonglycosylated 47-kDa and a glycosylated 60-kDa form(46). PAI-2 forms SDS stable complexes with uPA in a 1:1ratio (for reviews, see references 3,8,26).

PAI-2 is expressed during physiological processes suchas pregnancy (25) and by normal tissue, e.g., normalhuman epidermis (29). Variations in PAI-2 expression areassociated with certain pathological conditions includingintrauterine growth retardation (14) and cancer (4). Cellsof a human monocytic lineage including K562 (32,38,41),THP-1 (41), and U937 (15,41) and the HaCaT cells of ahuman keratinocyte line (5,39) have been shown to ex-press PAI-2 in vitro. The K562 and THP-1 cells are onlyable to synthesize PAI-2 after stimulation with phorbolmyristate acetate (PMA; 15,38,41). It has also been ob-served that a particular subclone of THP-1 cells express atruncated form of PAI-2 mRNA (15). U937 cells expresscell-associated and secreted PAI-2 at low levels withoutstimulation as detected by enzyme-linked immunosorbentassay (ELISA) of cell lysates (15,41). The HaCaT cell linehas been demonstrated to secrete PAI-2 without stimula-

*Correspondence to: Michael Liew, Department of Biological Sciences,University of Wollongong, Wollongong, NSW, Australia.

E-mail: [email protected]

© 2000 Wiley-Liss, Inc. Cytometry 40:32–41 (2000)

tion (39). Most studies to date have focused on intracel-lular PAI-2 as being involved in the prevention of apopto-sis (10,19,27) or on secreted PAI-2 being involved in theinhibition of ECM degradation (1,28).

There is much evidence that suggests that the plasmin-ogen activation system is localized specifically at the cellsurface (7,36,44). Plasminogen can bind to the surface ofcells by numerous low-affinity plasminogen receptors(33–35). tPA can also bind to plasminogen receptors onU937 cells (11,12), whereas uPA binds to the cell surfacethrough its specific receptor (uPAR; 31,44,45).

Because plasminogen activators have been detected onthe cell surface, it would be intuitive that PAIs might alsopossess a secreted or cell surface topology. Although PAIshave been observed to be associated with cellular mem-branes, their precise topological expression remains to beclearly addressed. The consequence of having plasmamembrane- associated PAIs could be to enable more effi-cient inhibition of cell surface plasminogen activators.This study investigates whether endogenous nonglycosy-lated PAI-2 can be found on the cell surface. The cellularmodel implemented were monocytic cell lines known tosynthesize PAI-2. The PAI-2 topology was studied usingtwo approaches. The first approach was to examine PAI-2expression by flow cytometry on intact viable cells and infixed permeabilized cells. The second approach was toisolate plasma membranes from the cells and examinetheir PAI-2 content by ELISA and Western blotting.

MATERIALS AND METHODSMaterials

The monoclonal antibodies directed against human uPA(no. 394) and human PAI-2 (no. 3750) were obtained fromAmerican Diagnostica Inc. (ADI; Greenwich, CT). Theimmunoglobulin G2a (IgG2a) isotype control was pro-vided by PharMingen (San Diego, CA) and was directedagainst trinitrophenol (TNP). The IgG1 isotype directedagainst dinitrophenol (DNP) and OKT3 directed againsthuman T-cell receptor were isolated from hybridoma su-pernatants by NH4SO4 precipitation and protein G affinitypurification. Fluorescein isothiocyanate (FITC)-labeled af-finity isolated F(ab)’2 fragments of anti-mouse immuno-globulin and peroxidase (POD) conjugated affinity iso-lated anti-sheep/goat immunoglobulin were obtainedfrom Silenus (Hawthorn, Australia). Goat polyclonal anti-human PAI-2 (GAH-PAI-2) and recombinant human PAI-2were a kind gift from Biotech Australia (Roseville, Austra-lia). Human urokinase was provided by Serono AustraliaPty. Ltd. (Frenchs Forest, Australia). Propidium iodide(PI), saponin, phenylmethylsulfonyl fluoride (PMSF), O-phenyldiamine (OPD), and deoxycholic acid were fromSigma Chemical Co. (St. Louis, MO). Polyscreen polyvinyli-dene difluoride (PVDF) membrane was provided by Du-pont (Sydney, Australia). Biotinylated broad range sodiumdodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) standards and DC protein assay kit were fromBio-Rad Laboratories (North Ryde, Australia). SeeBlueprestained SDS-PAGE standards were from Novex Austra-

lia Pty. Ltd. (Terrey Hills, Australia). The enhanced chemi-luminescence (ECL) kit and high-performance autoradiog-raphy film (Hyperfilm) were from Amersham Ltd.(Buckinghamshire, UK). CNBr-activated Sepharose 4B andFPLC Superdex 75 column were from Amrad PharmaciaBiotech (Boronia, Australia). Microsep centrifugal concen-trators were obtained from Filtron Technology Corp.(Northborough, MA). The Immunopure Plus ActivatedPeroxidase kit was provided by Pierce (Rockford, IL).Avidin-POD was from Calbiochem-Novachem Pty. (Alex-andria, Australia).

Cell Culture

All cell lines were incubated in a humidified incubatorat 37°C in 5% (v/v) CO2. The human cell lines K562,THP-1, U937, MM6, and Jurkat were cultured in RPMI 110% (v/v) fetal calf serum (FCS). The human keratinocytecell line HaCaT was cultured in Dulbecco’s modified Eaglemedium (DMEM) 1 10% (v/v) FCS (39). In addition, theMM6 cells (48) were cultured under lipopolysaccharide(LPS)-free conditions by baking all glassware overnight at150°C, as well as autoclaving the glassware and glassdistilled water before preparation of media. The HaCaTcell line (39) was chosen to act as a positive control forcell surface expression of PAI-2.

The K562 cell line was originally derived from a patientwith chronic myeloid leukemia and is a pluripotent stemcell that is the least differentiated toward a monocyticphenotype (13,16,22). The U937 cell line was derivedfrom a patient with human histiocytic lymphoma and islike a promonocyte (24,47) The THP-1 cell line was de-rived from a patient with acute monocytic leukemia and ismonocyte like (43,47). The MM6 cell line was derivedfrom a pateint with monoblastic leukemia. It is the mostdifferentiated and the closest to a mature monocyte (48).

Flow Cytometry of Cell Surface Antigens

Cell lines were harvested and cell viability estimatedusing the trypan blue exclusion method. HaCaT cells wereharvested by treatment with phosphate-buffered saline(PBS) 1 0.05% (w/v) ethylenediaminetetraaectic acid(EDTA) for 20 min at 37°C followed by 1–2 min in trypsin-EDTA at 37°C. 106 cells were stained with either isotypeor uPA or PAI-2-specific primary antibody (in PBS 1 0.1%[w/v] bovine serum albumin [BSA]) at a saturating con-centration of 20mg/ml for 30 min at 4°C. Bound primaryantibody was detected using a 1/50 dilution of FITC-labeled affinity isolated F(ab)’2 fragments of anti-mouseimmunoglobulin for 30 min at 4°C. Cells were washedwith PBS 1 0.1% (w/v) BSA after each step. Prior toanalysis on a FACSort (Becton Dickinson, San Jose, CA),the cells were stained with PI (5mg/ml) for 5 min to allowdiscrimination of viable cells from apoptotic or dead cells.Viable cells were defined as those cells that exclude stainand exhibit no PI fluorescence. A total of 20,000 eventswere acquired. Experiments were carried out at leastthree times for each cell line.

33PAI-2 AND THE CELL SURFACE

Flow Cytometry of Permeabilized Cells

To examine “total” antigen expression (a combinationof cell surface and intracellular expression), similar studieswere performed on permeabilized cells. Cells were fixedin 4% (w/v) paraformaldehyde in PBS for 30 min at 4°C. Allsubsequent incubations and washes were conducted infreshly prepared PBS 1 0.1% (w/v) BSA 1 0.1% (w/v)saponin. Staining of antigens was as described above.These experiments were repeated three times.

Human Peripheral Blood Leukocytes (PBL)

Blood was obtained by venipuncture from human vol-unteers under University of Wollongong Human Ethicsapproval and placed in anticoagulant (85 mM trisodiumcitrate, 65 mM citric acid, 110 mM dextrose, 1.6 ml/10 mlblood). Cells were lyzed in 168 mM NH4Cl, 10 mMKHCO3, and 110 mM EDTA, pH 7.3 (16 ml/ml of blood),for 5min at room temperature and the PBL pelleted (200gfor 5 min at 4°C). The cells were washed once with coldPBS and cell viability estimated using the trypan blueexclusion method. Cells (5 3 105) were stained as for thecell lines and analyzed by flow cytometry as describedabove. The three groups of PBL, i.e., lymphocytes, mono-cytes, and granulocytes, were identified by their differentsizes and granularities on a forward versus side scatter dotplot. As a control to confirm that instrument settings werecorrect, the PBL preparation was stained with OKT3 at thesame time as the other test antibodies to ensure that theCD31 T-lymphocyte population was visualized. A total of50,000 events were acquired. These experiments wererepeated on PBL obtained from three different volunteers.

Plasma Membrane Isolation

Plasma membranes were isolated as previously reportedusing a two-phase polyethylene glycol (PEG)/dextran sys-tem (6,37). Protein concentrations were determined bythe BioRad DC protein assay kit using BSA as a standard.Plasma membrane preparations were resuspended in 10mM Tris-HCl, pH 7.4, 1% (v/v) Triton-X 100, and 1% (w/v)deoxycholic acid and stored at -20°C. About 1% of theprotein recovered was in the plasma membrane-enrichedfraction for the suspension cells. For the HaCaT cells, anadherent cell line, the percent of protein recovered in theplasma membrane fraction was about 20%.

Immunopurification and Peroxidase Labeling ofGAH-PAI-2

GAH-PAI-2 was purified by affinity chromatography on aPAI-2 antigen column. Recombinant human PAI-2 wasimmobilized on CNBr-activated Sepharose 4B according tothe manufacturer’s instructions. The column was washedwith 10 bed volumes of the following buffers in thefollowing order: (a) 10 mM Tris, pH 7.5; (b) 100 mMglycine, pH 2.5; (c) 10 mM Tris, pH 8.8; and (d) 10 mMTris, pH 7.5. GAH-PAI-2 was loaded onto the PAI-2 antigencolumn by batch loading for 60 min with rocking at roomtemperature. The column was washed with 10 mM Tris,pH 7.5, until a stable baseline was achieved. Bound anti-

body was eluted with 100 mM glycine, pH 2.5, into onebed volume of 1 M Tris, pH 9.0. Collected antibody wasdialyzed against PBS and concentrated using centrifugalmicroconcentrators to approximately 6 mg/ml and storedat 20°C until further use.

Immunopurified GAH-PAI-2 was conjugated to POD(GAH-PAI-2-POD) using the Immunopure Plus ActivatedPeroxidase kit (Pierce). The GAH-PAI-2-POD conjugatewas further purified using an FPLC Superdex 75 column toremove unincorporated POD and stored at 220°C untilrequired. The GAH-PAI-2-POD was successfully labeled asjudged by visualization on a 10% SDS-PAGE gel undernonreducing conditions and Western blotting.

PAI-2 ELISA

Ninety-six-well microtiter trays were coated with unpu-rified GAH-PAI-2 (5 mg/ml) in 35 mM NaHCO3, 14 mMNa2CO3, pH 9.5, overnight at 4°C. The plates were thenwashed three times with PBS 1 0.05% (v/v) Tween 20followed by three washes in distilled water. Plates wereblocked using PBS containing 1% (w/v) skim milk powderand 0.05% (v/v) Tween 20 (PBS-SMT) for 2 h at roomtemperature. PAI-2 standards and samples were applied tothe plate diluted in PBS-SMT and incubated overnight at4°C. For plasma membrane preparations and standards,deoxycholic acid was added to the PBS-SMT to a finalconcentration of 1 mM to aid solubilization. Plates werewashed and GAH-PAI-2-POD added at a final concentrationof 4 mg/ml for 2 h at room temperature. After washing, thesubstrate buffer (0.2 M Na2HP04, pH 5.0 with citric acid)containing 400 mg/ml OPD1, 0.012% (v/v) H202 wasadded, then the absorbance determined at 490 nm on aBioRad plate reader (model 3550). Wells with no captureantibody but with sample added were analyzed to deter-mine nonspecific background. Samples and standardswere analyzed in duplicate throughout and all sampleswere tested twice at different times. The ELISA was linearfrom 0.39 to 3.1 ng/ml PAI-2.

Western Blotting

uPA/PAI-2 complexes were formed by incubating 1 mgof each protein together in PBS for 45 min at room tem-perature. PAI-2 standards, uPA/PAI-2 complexes, cytosol-enriched fractions, and plasma membrane-enriched frac-tions were separated on a 12% SDS-PAGE under eitherreducing or nonreducing conditions. Separated proteinswere transferred onto a PVDF membrane at 30 V over-night at 4°C.

The membrane was blocked for 2 h at room tempera-ture using PBS containing 5% (w/v) powdered skim milkand 0.05% (v/v) Tween 20. The membrane was nextincubated in GAH-PAI-2-POD (2 mg/ml) overnight at 4°Cwith shaking. The membrane was then washed four timeswith PBS 1 0.05% (v/v) Tween 20 and probed with PODconjugated affinity isolated anti-sheep/goat immunoglob-ulin diluted 1/2,000 in blocking buffer for 2 h at roomtemperature with shaking to increase the sensitivity of theWestern blot. The blot was then washed again prior todevelopment by ECL. SeeBlue markers were used to en-

34 LIEW ET AL.

FIG. 1. Relationship between cell surface expression of uPA or PAI-2 and viability of U937 cells. Graphs on the left represent cell surface uPA expressionand graphs on the right represent cell surface PAI-2 expression. (Top) Dot plots showing isotype controls (IgG1 and IgG2a, respectively) staining versusPI staining (cell viability). (Middle) Dot plots showing uPA and PAI-2 monoclonal antibody staining versus PI staining. (Bottom) Histogram plots of cellsprobed with the two antibodies (solid line) and isotype controls (dotted line). A gate is set on the PI negative (viable cells) in the lower two quadrantsof the dot plots.


sure proper transfer of protein to the membrane andbiotinylated markers were used to obtain an accurateassessment of the molecular weight. Biotinylated markerswere detected using avidin-POD diluted 1/1,000 in PBS 10.05% (v/v) Tween 20 1 0.1% (w/v) BSA. Western blotswere repeated twice.

Statistical Analysis

To perform statistics, the mean fluorescence was logtransformed and statistical significance was determinedusing an unpaired Student’s t-test with P , 0.05 taken assignificant. The mean fluorescence of cells stained witheither the uPA or PAI-2 antibody was compared to themean fluorescence of the appropriate isotype control.Statistics were performed on measurements from threeexperiments.

RESULTSFlow Cytometry of Cell Surface Antigens

Viable cells were determined by exclusion of PI (locat-ed in the lower two quadrants, Fig. 1, top, middle). Theviability of the nonadherent cells ranged from 62 to 97%.

The HaCaT cell line showed the lowest viability, rangingfrom 33 to 77%. This indicated substantial cell injury, mostlikely due to the rigorous method required to preparethese cells as a single cell suspension for flow cytometricanalysis. Positive expression of antigen was observed as ashift to the right (Fig. 1).

All of the human cell lines tested were positive for cellsurface uPA when compared to the isotype control stain-ing (Fig. 2a). Jurkat, HaCaT, and K562 cells were weaklypositive for cell surface uPA. The other myeloid lines,MM6, U937, and THP-1, were found to be strongly posi-tive for cell surface uPA. In human PBL preparations, onlythe monocytes and granulocytes were positive for cellsurface uPA.

Only the U937 cell line was weakly positive for cellsurface PAI-2 when compared to the isotype control stain-ing (Fig. 2b). None of the cells in the PBL preparationwere positive for cell surface PAI-2 expression (Figs. 2band Fig. 3). No correlation was found between uPA (orPAI-2) expressed on the cell surface and variations in thedifferentiation status of the myeloid cell lines along themonocyte/macrophage differentiation pathway.

FIG. 2. Expression of cell surface uPA andPAI-2 on viable cells. Cell viability was deter-mined by the exclusion of PI. Open bars arethe isotype control stains for each cell type.Closed bars are staining for (a) uPA and (b)PAI-2. Results are expressed as mean fluores-cence, n53; error bars represent the SD.* P,0.05, ** P,0.01.

36 LIEW ET AL.

Flow Cytometry of Permeabilized Cells

In order to confirm those cells that express PAI-2 withan intracellular topology, and so that the antibody em-ployed in our studies can recognize PAI-2, the total (cellsurface and intracellular) expression of uPA and PAI-2 wasdetermined.

The distribution of uPA in permeabilized cells was fairlyuniform, and all cell lines examined were positive for totaluPA compared to the isotype control staining. Permeabil-ized lymphocytes, monocytes, and granulocytes were alsopositive for total uPA (Fig. 4a). The distribution of expres-sion of intracellular PAI-2 within populations was found tobe bimodal, containing a strongly positive population anda negative population in the PAI-2-expressing cells. Theresults for total PAI-2 were derived from the stronglypositive peak. Permeabilized MM6, U937, and HaCaT celllines were strongly positive for PAI-2 (Fig. 4b). The THP-1cell line was weakly positive for PAI-2 expression. Inhuman peripheral blood, monocytes were strongly posi-tive for PAI-2 whereas lymphocytes and granulocyteswere weakly positive for PAI-2 (Fig. 5). Again, no correla-tion could be drawn between total uPA and PAI-2 expres-sion, or between the differentiation status of the myeloidcell lines with total expression of either uPA or PAI-2antigen.

ELISA of Plasma Membrane-Enriched Preparations

By ELISA, both the Jurkat and THP-1 subclone employedin this study were negative for PAI-2 in both the cytosoland the plasma membrane-enriched fractions (Table 1). Incontrast, HaCaT, U937, and MM6 cells were found to bepositive for PAI-2 antigen in both cytosol and plasmamembrane-enriched fractions. Increasing levels of PAI-2were observed in the following rank order: HaCaT, MM6,and U937 cell lines.

Western Blotting of Plasma MembranePreparations

Western blotting experiments were performed to deter-mine the relative sizes of the PAI-2 found in each of thetwo subcellular fractions. Under nonreducing or reducingconditions, the PAI-2 detected had several bands. Therecombinant PAI-2 standard (Fig. 6a, lane 1) under reduc-ing conditions shows four distinct bands with molecularweights of 38, 47, 110, and 137 kDa. The 47-kDa band ismonomeric recombinant PAI-2, whereas the 110 and 137-kDa bands are believed to represent dimeric and trimericserpin, respectively. There was also evidence of a degra-dation product in the PAI-2 preparation as visualized bythe 38-kDa band. Under nonreducing conditions, thePAI-2 trimer was not visible (Fig. 6b, lane 1).

FIG. 3. (a) Forward scatter versus side scat-ter dot plot of viable human PBL. L, lympho-cytes; M, monocytes; G, granulocytes. (b) Aside scatter versus viability dot plot of viablehuman PBL. Viable monocytes are showngated by the square. (c) Cell surface PAI-2expression on viable monocytes. There isonly weak reactivity of 3750 (solid line) com-pared to the IgG2a isotype (dotted line).


The polyclonal sera used were capable of detectingPAI-2 and uPA/PAI-2 complexes. Complexes between uPAand PAI-2 (Fig. 6, lane 2) can be visualized as three distinctbands of 73, 98, and 177 kDa molecular weight. Theremaining bands seen corresponded to uncomplexedPAI-2. The 73-kDa band was probably a complex between47 kDa PAI-2 and 33 kDa uPA, the 98-kDa band a complexbetween 47 kDa PAI-2 and 55 kDa uPA, and the 177-kDaband a complex between trimeric PAI-2 and 33kDa uPA.There was no difference in the molecular weights of thebands under reducing or nonreducing conditions.

Under reducing conditions (Fig. 6a), the preparationsfrom the Jurkat cell line were used as negative controls. Inagreement with the ELISA results, the preparations fromthe Jurkat cells (lanes 3 and 4) were completely negativefor PAI-2 under reducing conditions. In contrast, a 47-kDaband was detected in both the cytosol (lane 5) and plasma

membrane-enriched preparations (lane 6) of U937 cellsunder reducing conditions. A 47-kDa band could be seenin the cytosol-enriched fraction (lane 7) from the MM6 cellline but no band was observed in the plasma membrane-enriched fraction (lane 8), probably because it was belowthe detection limit of the Western blot. The 47-kDa bandcorresponds to the molecular weight of the nonglycosy-lated recombinant 47-kDa PAI-2 standard (lane 1). Nobands comigrating with uPA/PAI-2 complexes were evi-dent in any of the preparations when compared to theuPA/PAI-2 complex standards (lane 2).

Under nonreducing conditions (Fig. 6b), the prepara-tions from the THP-1 cell line were used as negativecontrols. The preparations from THP-1 cells were negativefor PAI-2 (lanes 3 and 4). The 47-kDa PAI-2 band was stillevident in the cytosol-enriched preparations of both U937and MM6 cells (lanes 5 and 7). The major difference

FIG. 4. Expression of uPA and PAI-2 in per-meabilized cells. Open bars are the isotypecontrol stains for each cell type. Closed barsare staining for (a) uPA and (b) PAI-2. Resultsare expressed as mean fluorescence, n53;error bars represent the SD. * P,0.05,** P,0.01.

38 LIEW ET AL.

observed was that the 47-kDa PAI-2 band present in theU937 plasma membrane-enriched preparation under non-reducing conditions was replaced by a high molecularweight band around 180 kDa (lane 6). There was also thesame band appearing weakly in the U937 cytosol-enrichedpreparation (lane 5). This molecular weight correspondsto multimers of PAI-2, but may also represent an interac-tion of PAI-2 with other proteins. This interaction is sen-sitive to reduction. As was observed under reducing con-ditions, no bands comigrating with the uPA/PAI-2complex standard (lane 2) were evident in any of thepreparations, under nonreducing conditions.

DISCUSSIONBy flow cytometry, only viable U937 cells were weakly

positive for cell surface PAI-2. PAI-2 antigen was detect-able in plasma membrane-enriched preparations from the

HaCaT, MM6, and U937 cell lines. There was no enrich-ment for PAI-2 in the plasma membrane-enriched frac-tions, meaning that PAI-2 is not a classical plasma membraneprotein. The molecular weight of the PAI-2 determined inthe plasma membrane (47 kDa) was identical to that of theintracellular form of PAI-2. Although stringent washeswere implemented to ensure the purity of the prepara-tions, it cannot be ruled out that the PAI-2 present in theplasma membrane-enriched fractions was a contaminationfrom the cytosol. These data together indicate that PAI-2 ispredominantly found in the cytosolic compartment.

The HaCaT cell line was originally used in these exper-iments as a positive control because it has been reportedpreviously to express cell surface PAI-2 detectable by flowcytometry (39). In contrast, this study found that using a“gate” set strictly on viable HaCaT cells, no PAI-2 wasdetectable on the cell surface. If PAI-2 is on the cellsurface, it is possible that the epitope recognized by thePAI-2 monoclonal antibody used in this study was masked,because a different monoclonal antibody was used in thestudy by Reinartz et al. (39). It also appeared that moreprotein was isolated in the plasma membrane prepara-tions from the HaCaT cells compared to the other cells.This is probably attributable to a loss of PAI-2 from thedead cells during the harvesting procedure. This wouldalso explain why there is a strong PAI-2 signal in individualHaCaT cells by flow cytometry, but a lower amount whenthe cells are homogenized and analyzed by ELISA.

The use of the PAI-2 monoclonal antibody was validatedfor flow cytometry studies in fixed permeabilized cells be-cause only cell lines that have been reported to express PAI-2were found to be positive in the flow cytometry assay usedin this study. The low levels of PAI-2 detected in the perme-abilized lymphocytes and granulocytes could aid in the pres-ervation of ECM deposited in a wound site. PermeabilizedTHP-1 cells were positive for PAI-2 by flow cytometry butnegative by ELISA because the flow cytometry is more se-nisitive. It has also been reported previously that the Jurkatcell line expresses PAI-2 (40). In contrast, results from thisstudy found that Jurkat cells were negative for PAI-2.

There have been observations of PAI-2 being bound tothe membranes of tissues. Indeed, active PAI-2 was a partof a large molecular weight species isolated from mem-

FIG. 5. (a) Forward scatter versus side scatter dot plot of permeabilizedhuman PBL. L, lymphocytes; M, monocytes; G, granulocytes. Monocytesare gated. (b) Histogram plot of total PAI-2 expression in monocytes fromhuman peripheral blood. The vertical axis is the cell number and thehorizontal axis represents PAI-2 expression. Staining with 3750 (solidline) is compared to the IgG2a isotype (dotted line).

Table 1ELISA Results for PAI-2 Content of Cytosolic and Plasma

Membrane-Enriched Preparations From the Cell Lines Tested*

Cell line

Cytosol-enrichedfraction

[PAI-2](ng/mg)

Plasma membrane-enriched fraction[PAI-2](ng/mg)

Jurkat ,0.39 ,0.39THP-1 ,0.39 ,0.39HaCaT 41 3MM6 65 9U937 290 17

*Units are expressed as nanograms of PAI-2 per milligram oftotal protein in the cytosolic or plasma membrane-enriched prep-arations, respectively.


brane preparations of syncytiotrophoblasts (17). Jensen etal. (20) observed that some of the PAI-2 produced bydifferentiated keratinocytes was covalently incorporatedinto the cornified envelopes. Also, in skin, PAI-2 has beendetected associated with the periphery of cells located inthe suprabasal granular cell layer of normal human epider-mis (29). There have also been observations of PAI-2 onthe surface of cells. Reinartz et al. (39) observed PAI-2 onthe cell surface of HaCaT cells by flow cytometry that wascomplexed with uPA. Baker et al. (2) originally observedthat PAI-2 was found on the cell surface of U937 cells byflow cytometry. They suggested that the PAI-2 found onthe surface was active using a monoclonal antibody thatdistinguished formation of uPA/PAI-2 complexes from ac-tive PAI-2. Results from this study found PAI-2 to be associ-ated with the cytosolic side of the plasma membrane.

Observations throughout the literature of PAI-2 associatingwith the membranes of tissues may be a result of PAI-2 beingcross-linked by transglutaminase activity (18) to the ECMclose to the cell surface, as opposed to integral plasmamembrane components. If the PAI-2 is cross-linked to the

ECM, this could provide an additional means of protectingthe ECM from proteolysis. It could also provide a stop signalfor migrating cells because it has been found that migratingcells of a monocytic-macrophage lineage produce uPA (42).Under the correct conditions, monocytic cells may be able tocross-link PAI-2 because U937 cells and monocytes areknown to express tissue transglutaminase (9,30) and theU937 cells express factor XIII on their cell surface (23).

In conclusion, PAI-2 is predominantly a cytosolic pro-tein. In terms of uPA inhibition at the cell surface, therewas no evidence of cell surface-bound PAI-2 on the celllines investigated, to regulate plasminogen activation. Thiscould mean that the PAI-2 observed in close proximity tothe surface of cells may potentially be associated with theECM adjacent to the cell. Future work will focus onexamining the localization of PAI-2 in the ECM.

ACKNOWLEDGMENTSThe Jurkat cell line was the kind gift of Dr. Charles

Harvey (St. Vincent’s Hospital). The HaCaT cell line wasthe kind gift of Professor Norbert E. Fusenig (Deutsches

FIG. 6. Western blot analysis of cytosolicand plasma membrane-enriched preparationsof U937 and MM6 cells. (a) Reducing West-ern blot. (b) Nonreducing Western blot. Thepositions of standards are indicated on theright in kDa. Lanes are as follows: (1) 1 mgrecombinant human PAI-2; (2) complex ofhuman recombinant PAI-2 (1 mg) and humanuPA (1 mg); (3) negative control (non-PAI-2-expressing) cell line cytosol; (4) negativecontrol cell line plasma membrane; (5) U937cytosol; (6) U937 plasma membrane; (7)MM6 cytosol; (8) MM6 plasma membrane.The PAI-2 and uPA/PAI-2 standards undernonreducing conditions are from a shorterexposure from the same blot. The arrows onthe left of the blots indicate the molecularweights of the uPA/PAI-2 complexes of 73,98, and 177 kDa. Jurkat cells used as a nega-tive control for the reducing Western blot,whereas THP-1 cells were used for the non-reducing Western blot.

40 LIEW ET AL.

Krebsforschungszentrum). The MM6 cell line was thekind gift of Dr. H.W.L. Ziegler-Heitbrock (University ofMunich). The authors thank Nurse Sheena McGee for herassistance with the blood sampling, as well as all of thevolunteers.

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Topological localization of plasminogen activator inhibitor type 2