and appears to be mediated by kringles 1-3 and kringle 5 (20,. 21). In contrast to the uPA receptor, at least thee candidate plasminogen receptors have been ...
T H E JOURNAL OF BIOWGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, lnc.
Vol. 269,No. 51, Issue of December 23, pp. 32660-32666, 1994 Printed in U.S.A.
Regulation of Macrophage Receptor-bound Plasminby Autoproteolysis" (Received forpublication, October 4, 1994)
Domenick J. FalconelOn, Wolfgang Borthll,Timothy A. McCaffrey**, Jean Mathew$, and Kimberly McAdaml From the Departments of $.Pathology, $Cell Biology and Anatomy, and **Medicine, Cornell Medical College and the JDiuisionof Hematology, Mt. Sinai School of Medicine, New York, New York 10021
The activation of plasminogen by macrophage is regu- (1-7). Plasmin candirectly cleavethe proteincore of proteoglylated by their expression of receptors for urokinase and cans (81, laminin (91, and fibronectin (9, 10). In addition, plasplasmin(ogen).In these studies we have examined plas- min can activatea family of neutral metalloproteinases,which midogen) binding to adherent human THP-1 macrodegrade collagens and other matrix components (11). In addiphage. Plasmin boundto the THP-1 cellsin a time- and tion to regulating cell movement and matrix remodeling, an dose-dependent manner (K,15.8 t 6.2 m;B , 1.4 0.3 x important biological consequence of plasminogen activation is 106/cell). The lysine analog e-aminocaproic acid competithe releaseof matrix-bound growthfactors. For example, expotively inhibited plasmin binding. Thefraction of mem- sure of endothelial cell monolayers to plasmin resulted in the brane-bound plasmin, however, became increasingly re- release of basic fibroblast growth factor complexed to heparan sistant to displacement with eaminocaproic acid. Over sulfate proteoglycan (12).Treatment of fibroblast and fibrosara 24-h period, membrane-bound plasmin activity fell 80% coma cell-derived matrices with thrombinor plasmin led to the despite the presence of catalytically active plasmin in the incubation media. The lossof receptor-bound plas- release of TGF-P1 (13):We have recently demonstrated that min activity was notdue to proteolytic alterations of its urokinase (uPA)-dependent plasminogen activation regulates ability to liberate basic fibroblast growth factor receptor since '2SI-Lys-plasminogen bound to THP-1 macrophage's cells and TGF-P from endothelial cell matrices (14, 15). pretreated with plasmin with similar affinity as to unMacrophage plasminogen activation is localized to the peritreated cells. Following a 24-h incubation of"'I-Lysplasminogen or '2SI-plasmin with THP-1 cells, several cellular environmentby the expression of a high affinity recepdegradative fragments were apparent in their condi- tor for uPA and relatively low affinity receptor for plasminogen tioned media. The smaller degradative fragments (28 (16-18). Plasminogen binding to its cellular receptor is inhiband 36 kDa) lacked cell binding activity and were dem- ited by the lysine analog eaminocaproic acid (E-ACA)(16, 191, onstrated to be active by casein-zymography.A 48-kDa and appears tobe mediated by kringles 1-3 and kringle 5 (20, fragment bound to cells in a lysine-dependent manner 21). In contrast to the uPA receptor, at least thee candidate but was not active. In contrast, phenylmethylsulfonyl plasminogen receptors have been described: annexin I1 (22), fluoride-inactivated'251-plasminretained its binding ac- a-enolase (23), and gangliosides (24). When plasminogen and tivity over 24 h, and degradative fragments were not uPA are bound to their respective receptors, the eficiency of present in the conditioned media. The binding of lZ5I- plasminogen activation is increased(16-19). Following activaLys-plasmin(ogen)to THP-1 cells was also examined in tion, membrane-bound plasmin is not internalized or readily the presence of excess a2 plasmin inhibitor. Despite the inactivated by its physiologic inhibitors (16, 17, 25, 26). Conabsence of fluid-phase plasminactivity,membranesequently, it would appear that cells do not possess a mecha24 h. At 24 nism to regulate membrane-bound plasmin activity following bound '261-Lys-plasmin(ogen) decreased over h a radiolabeled 48-kDa fragment was observed in the its activation on the cell surface. conditioned media and together with '251-Lys-plasminIn experiments reported here, we have examined the regu(ogen) was bound to cells. Unlike l2'I-Lys-p1asmin, the lation of membrane-bound plasmin(ogen) in human THP-1 48-kDa fragment did not form a complex witha2 plasmin inhibitor. Thus,autoproteolysis of receptor-bound plas- macrophage, Results demonstrate that plasmin autoproteolymin results in fragments with truncated physiologic sis and 6-ACA-resistant binding are novel regulatory mechaproperties that possess either cell binding or catalytic nisms in thecontrol of membrane-bound plasmin activity. activities. We propose that autoproteolysis is a mechaMATERIALS AND METHODS nism forregulating membrane-bound plasmin activity.
*
Cell Culture-Human monocyte-like THP-1 cells (27) were obtained
from American Type TissueCulture (Rockville,MD). THP-1 cells were
The activation of plasminogen is an important regulatory step in cellular movement and connective tissue remodeling associated with a variety of normal and pathologic processes
* This work was supported by Research Grants R01-HL40819, PO1 HL46403, and R29 HL42606 fromthe National Heart, Lung andBlood Institute and Grant4162 from the Austrian Nationalbank.The costsof publication of this article were defrayed inpart by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. 1Towhom correspondence should be addressed: Dept. of Pathology, CornellMedicalCollege,1300YorkAve., New York, NY 10021. Tel.: 212-746-6457; Fax: 212-746-8789.
maintained in suspension culture in Roswell Park memorial medium (RPMI; without HEPES) supplemented with 10% Cellect Gold fetal bovine serum (FBS), penicillin (100 unitdml), streptomycin (100 pg/ml), and glutamine (ICNiFlow Laboratories, McLean, VA). During routine culture a small percentage(- 10%)of THP-1 cells spontaneously differentiate into an adherent cell population.The adherent THP-1cells resemble macrophage, continue to divide, and maintain their adherent phenotypefollowingmechanical harvestingandreplating.Inother The abbreviations used are: TGF-B, transforming growthfactor beta; uPA, urokinase-dependent plasminogen activation;E-ACA, €-aminocaproic acid; FBS, fetal bovine serum; DPBS, Dulbecco's phosphatebuffered saline;MSFM, macrophage serum-free media;PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis.
32660
32661
Regulation of Macrophage Receptor-bound Plasmin
0 .05 . I O .I5 .2 Bound (pMoles) I
0
10
20
30
40
1.5 50
1.00.0
0.5
2.0
Hours
Plasmin (nM)
FIG.1. Dose- and time-dependent binding of plasmin to adherent THP-1 cells. Cells wereplated into 96-well plates (1x 105/well)in RPMI 1640 supplemented with 10%FBS. The next day media were removed and cells washed three times with DPBS. Left panel, cells wereincubated with plasmin (0.14.0 pg/ml) at 37 "C for 2 h (m) or plasmin and 25 m E-ACAto block lysine-dependent binding (0). Right panel, cells were incubated with 1.0 pg/mlplasmin for 0-2 h (m) followed bya 10-minexposure to E-ACAto displace membrane-boundplasmin (0). Membrane-bound plasmin was determined as described under "Materials and Methods." Data represent the mean -c S.E. of six separate wells. studies, we have measured THP-1 expression of uPA and the uPA re- kDa cutoff membrane). SDS sample buffer without mercaptoethanol ceptor.2All of the plasminogen activator activity expressed by THP-1 was added to the media samples and heated for30min at 37"C. Samples and molecular weight markers were electrophoresed in a 12% cells is blocked by anti-uPA IgG? Quantification of Plasmin Actiuity-Plasmin activity was quanti- polyacrylamide gel containing 0.1% casein. The gel was then washed tated by measuring the increase in fluorescence followingthe cleavage (two times) in 2.5% Triton X-100 to remove SDS.The gel wasincubated of the plasmin substrate u-Val-Leu-Lys-aminomethyl coumarin (En- at 37 "C for 40 h in 50 mM Tris-HC1, 10 mM CaCl,, pH 7.8, and stained zyme Systems Products, Dublin, CA) as described previously (15). Ali- with Coomassie Blue.The presence of caseinolytic activity was identiquots of conditioned media were added to microtiter wells containing 82 fied as clear bands on a uniform blue background followingdestaining. WesternBlot for Plasmin-Cell monolayers were lysed with SDS pl of DPBS, 0.05% Tween-20 containing 13 pg of the plasmin substrate. sample buffer without mercaptoethanol and immersed in boiling water Samples were mixed and incubated at 37 "C for 2.5 h. Cleavage of the substrate was monitored in a Fluoroscan microplate reader (excitation, for 5 min. Biotinylated molecular weight markers and samples were electrophoresed in P 1 5 % polyacrylamide gradient gels. Proteins were 330-380 nm; emission, 430-530 nm). Plasmin activity inthetest samples was extrapolated from a standard curve utilizing 0-40 ng/ml transferred to a nitrocellulose membrane, following which the membrane was blocked in Tris-buffered saline, 0.05% Tween-20 (TTBS) conhuman plasmin (American Diagnostica, Greenwich, CT). Membrane-bound plasmin activity was quantitated by incubating taining 5% dry defatted milk for 1h. Following twowashes ("BS), the THP-1 cells with either human plasmin or plasminogen in macrophage membrane was incubated with blocking buffer containing 10pg/ml monoclonal antibody directed against kringles 1-3of human plasmin serum-free media (MSFM,LifeTechnologies, Inc.) for 2 h at 37"C. Unbound plasmin was removed, and cells were washed with DPBS. IgG for 1 h (American Diagnostica). The membrane was washed (two MSFM containing u-Val-Leu-Lys-amino methyl coumarin was added times; TTBS) and incubated for 1 h with 10 pg/ml biotinylated rabbit and allowed to incubate 2.5 h. Fluorescence was monitored in a Fluo- anti-mouse IgG (Pierce) in blocking buffer. The membrane was then roscan microplate reader and extrapolated to the fluorescence gener- washed (two times; TTBS) and incubated 1 h with 4.6 pg/ml avidinated by 0-40 ng/ml plasmin prepared in MSFM. The catalytic efficiency horseradish peroxidase (Pierce) in TBS,0.1% gelatin. Diaminobenziof membrane-bound plasmin has been reported to be increased relative dine was used as a substrate. to fluid-phase plasmin (29). We have not examined this possibility diRESULTS rectly since we were interested inrelative changes in membrane-bound plasmin over time. Nonetheless, the amounts of membrane-bound plasBinding of Plasmin and Plasminogento THP-1 Cells-A vamin may be overestimated since it was calculated by extrapolation from riety of cell types, including monocytes, express large numbers a standard curve of fluid-phase plasmin. It is important to note, however, that thebinding parameters generated via extrapolation of mem- of relatively low affinity binding sites for plasminogen (16-24, 26). The bindingof plasminogen appears tobe lysine dependent brane-bound plasmin activity to plasmin activity in the fluid-phase are similar to that observed with '251-Lys-plasminogen(see "Results"). since the lysine analog E-ACAcompetitively blocks plasminoIodination of Lys-plasminogen and Plasmin-Human Lys-plasminogen binding to cells. In these experiments, we have measured gen (Immuno A.G., Vienna, Austria) and plasmin (American Diagnos- the binding of catalytically active plasmin to adherent THP-1 tics) were iodinated according to the method of McFarlane (30). Iodinated proteins were separated from unincorporated by ''I gel macrophage. As seenin Fig. 1, plasminbinds to adherent chromatography on a PD-10column (Pharmacia Biotech Inc.) pre- THP-1 cells in a dose- and time-dependent manner. The doseequilibrated with HEPES-buffered saline (137 mM NaCl, 4 mM KCl, 11 dependent plasmin binding is completely blocked if E-ACAis m glucose, and 11mM HEPES, pH 7.4) containing 0.5% human albu- present during the incubation period. Based on Scatchard analmin. '251-Plasminwas irreversibly inactivated by treatment with phen- ysis of four separate experiments, the estimated Kd of binding ylmethylsulfonyl fluoride (PMSF). PMSF inactivation of '251-plasmin was determined t o be 15.8 2 6.2 r m (mean 2 S.E.) with 1.4 2 was >90% as determined by cleavage of the plasmin substrate. 0.26 x binding siteskell. The observed binding affinity of Analysis of Plasmin Fragments by SDS-PAGE and Autoradiography-THP-1 cells were incubated with '251-Lys-plasminogen or '251-plas-plasmin t o the THP-1 macrophage was significantly greater min in MSFM for 1and 24 h. Aliquots of conditioned media were diluted than reported for plasminogen binding to either endothelial 1:4 with SDS-sample buffer without P-mercaptoethanol and boiled 3 cells (Kd = 310 2 235 m) (19) or suspension cultures of U937 min. Samples and molecular weight markers were applied to 4 1 5 % monocytes (Kd= 800 500 nM) (16). However, it was of similar gradient gels. Gels werefured in 10%acetic acid for1h andstained with magnitude to that reportedfor plasmin binding to endothelial Coomassie Blue overnight. Following destaining the gels were dried and placed on Kodak XOMATAR film with a Cronex (DuPont) enhanc- cells (Kd= 77 2 18 nM) (19). The plasminogen in the FBS used
lo6
*
ing screen. Enzyme Zymography-Conditioned media were concentrated in an ice bath utilizing an Amicon ultrafiltration chamber with a Y"10 (10 D. J. Falcone, T. A. McCaffrey, W. Borth, J. Mathew, and K. McAdam, manuscript submitted for publication.
to grow the THP-1 cells did not affect plasmin(ogen) binding. Results of at least 20 experiments have demonstrated that THP-1 cells incubated with 10% FBS alone had trace or no detectableplasmin(ogen) on their surfaces by Western blot and/or activity measurements (data notshown). Although E-ACAcompetitively inhibits plasmin binding to
32662
Regulation of Macrophage Receptor-bound Plasmin 0 Total Bound
3
1
b zm
25
-
20-
-
15
-
c
10
-
-0
Kt
5 -
0 1
4
8
24
0
15
30 126
45
.I .2 .3 .4 Bound (pMole8)
BO
76
I-Lys-Plg (nM)
Hours
FIG.2. Loss of c-ACA-sensitive plasmin binding to adherent THP-1 cells. Cells were plated into 96-well plates (1 x 105/well)in RPMI 1640 supplemented with 10% FBS. The next day media were removed and cells washedwith DPBS. Mediawas replaced with MSFM containing 1 pg/ml plasmin and incubated 1-24 h followed by a 10-min exposure to E-ACAto displace membrane-bound plasmin. Membranebound plasmin was determined as described under "Materials and Methods." Data represent the mean * S.E. of six separate wells.
FIG. 3.The effect of plasmin pretreatment on binding of '%SILys-plasminogen to adherent THP-1 cells. Cells were plated into 96-wellplates (1x 105/well)in RPMI 1640supplemented with 10%FBS. Following adherence, the mediawereremoved and replaced with MSFM containing 1pgfml plasmin. The next day cells were washedand incubated with 0.1-6 pdml '261-Lys-plasminogen for 1 h at 4 "C. Following incubation with labeled plasminogen, cells were washed and membrane-bound plasminogen was released by exposure to 25 mM E-ACA(10 min). 1251-Lys-plasminogenresistant to a-ACA displacement was recovered by treating the monolayer with 0.5 NaOH. Data represent the mean -c S.E. of three separate samples.
adherent THP-1 cells (Fig. 1, left panel), the fraction of membrane-bound plasmin that is resistant to subsequent displaceTABLE I ment with E-ACAincreases with time (Fig. 1, right panel). In Loss of plasmin's cell binding activity addition, adherent THP-1 cells were incubated with plasmin Plasmin (1 pgfml) was added to adherent cultures of THP-1 cells for 1-24 h and membrane-bound plasmin determined. There (105/well)in MSFM. Cells were incubated with plasmin for 1 or 24 h was a time-dependent loss of THP-1 membrane-bound plasmin followingwhich the media were recovered and assayed for plasmin activity (Fig. 2). Following 24 h of incubation, -80%of the activity. The cells werewashed; membrane-bound plasmin activity was membrane-bound plasmin activity observed at 1 h was lost. quantitated as described under "Materials and Methods." To determine Furthermore, the remaining membrane-bound plasmin (20%) whether the 24-h conditioned media contained an inhibitor of plasmin binding, fresh plasmin (Pls to) was added and incubated with THP-1 was not susceptible t o displacement by E-ACA. cells for 1 h. Data represent the mean * S.E. of six wells. In an effort to identify the mechanism(s) responsible for the Media activity p g f ml observed loss of enzymatically active plasmin from the THP-1 Pls t, 1000 surface, we determined whether the binding properties of the Pls tZ4 946 f 75 THP-1 plasmin(ogen) receptor weresensitive to plasmincleavMembrane-bound activity ngl ml age. For this purpose, the binding of 1251-Lys-plasminogento THP-1 0.03 * 0.1 cells preincubated with plasmin was determined (Fig. 3). LysTHP-1 + Pls t, 16.69 * 0.05 plasminogen is theplasmin-modified form of Glu-plasminogen THP-1 + Pls h4 0.13 * 0.01 (26, 31). Lys-plasminogen is formed at the cell surface and is THP-1 + Pls h4 13.37 * 0.54 THP-1 + Pls to the form of plasminogen that is preferentially bound (26). The binding of '251-Lys-plasminogen toplasmin-treatedTHP-1 cells was similar to the binding of plasmin to untreated cells (Fig. 1).Based on Scatchard analysis of four separate experi- addition of fresh plasmin. Furthermore, the ability of plasmin ments, the estimated Kd of binding is 14.7 5 1.41 m, and the to bind to cells was lost when plasmin was incubated in the numbers of binding sites is 1.96 5 0.30 x 106/cell. Therefore, absence of cells for 24 h. Therefore, we conclude that plasmin can form an enzymatically active species that is not recognized plasmin-mediated proteolysis of the plasminogenreceptor could not be responsible for the observed loss of plasmin from by the plasmin(ogen) receptors. A possible mechanism for the observed loss in plasmin's cell the cell surface. Autoproteolysis Leads to Catalytically Active Plasmin with- binding properties is autoproteolysis. Therefore, we examined out Cell Binding Activity-We determined whether the region by SDS-PAGE-autoradiographyalterations in'261-Lys-plasminof plasmin responsible for cell binding is lost or modified during ogen incubated with adherent THP-1cells for 1and 24 h. Una n extendedincubation withadherentTHP-1 macrophage. der nonreducing conditions, the major fraction of labeled plasCells were incubated with plasmin (1 pgfml) for 24 h after minogen in the 1-h conditioned media co-migrated with intact which their conditioned media were recovered and assayed for '251-Lys-plasminogen(Fig. 4). Radiolabeled degradative fragremaining plasmin activity. The binding of 24 h conditioned ments between 48 and 40 kDa, which were absent in the startplasmin to freshly plated THP-1cells was compared to control ing material, became visible following 1 h of incubation with plasmin (Table I). THP-1 cells incubated with control plasmin THP-1 cells. In contrast, at 24 h the majority of the radiolafor 1h bound >lOO-fold more plasmin thancells incubated with beled plasminogen had shifted to the 48- and 40-kDa peptide conditioned plasmin. These data suggest that either cells are fragments. Smaller degradative fragments, as well as intact releasing an inhibitorof plasmin binding, or plasmin itself is '251-Lys-plasmin(ogen)were faintly visible at 24 h. Identification of Active Plasmin Fragments by Zymomodified during anextended incubation with the THP-1cells. which of the plasmin fragments In order t o determine whether THP-1cells released a n inhibi- graphy-In order to determine tor of plasmin binding to its receptor, fresh plasmin was added in THP-1-conditioned media were enzymatically active, cells to the 24-h conditioned media and membrane-bound plasmin were incubated with unlabeled plasminogen for 24 h and anadetermined. As seen inTable I, the binding of plasmin to THP-1 lyzed by SDS-PAGE zymography. Plasmin fragments expressincluded inthe cells in the presence of conditioned media was restored by the ing proteolytic activity degrade casein
Regulation of Macrophage Receptor-bound Plasm.in
32663
Hours 1
kD
24
" kD -205 -1 17
'251-Lys-Plg
205 117-
-106
80-
-80
50 -
-50
-80 -50
-33
-28
-33 -28 -19
FIG.5. Zymographiridcmtifie;ationof c.nzyrn:ttic.;ally active plasmin frrcgmrnts. ('rlls w n . plated into 'r-25 flasks x 10") as descrihrd in FIG. 1. Following ndhcwnce the cc~llsw r c - washed and media replnccd with M'SFlI containing 1 &ml pl:lsminogrn. The conditioned mrdium was collrcted 24 h later and concrntratcd 5:l by ultrafiltration as described undrr "Materials and Methods." SDS sample FIG.4. Degradative alterations of "511-Lys-planminopnincu- huffrr was added to aliquots of TIIP-1-conditionrtl mrdium, plasminobated with THP-1 cells. Cells wrre platrd into 12-well plates ( 5 x gen and plasminogen prrincuhatrd (1 h, with high molecular Wright UPA. Samplrs were electrophoresed in composite polyacrylnmide-casc,in I0"iwell) as drscrihedinFig. 1. Following adhcrrnce the crlls were washrd and mrdia replaced with MSFM containing 1 pg/ml 12"I-Lys- gels. Activr plasmin fragmrnls wrre identified by zymomaphy. plasminogen. Conditioned medium was recoverrd at 1 and 24 h. AlterLysate Media ations in molecular weight of "~'1-Lys-Plasminogen was determined by SDS-PACE followed by autoradiography. 1 24 1" 4 (hrs)
polyacrylamide gel. Areas of casein digestion appear unstained following staining of the gel with Coomassie Blue. As positive controls, both plasminogen and plasminogen preincubated with uPA for 1 h were examined. Plasmin activity in the plasminogen preparation was clearly evident in thezymograph (Fig. 5). When plasminogen was activated by 1 h of preincubation with uPA, a wide range (80-20 kDa) of enzymatically active plasmin fragments were formed. In contrast, conditioned media recovered from THP-1 cells incubated with plasminogen for 24 h contained fewerdegradative forms ofenzymatically active plasmin. Several high and two lower molecular weight caseinolytic bands were observed. The lower molecular mass caseinolytic bands (28 and 36 kDa) correspond to the low molecular mass 12'I-labeled plasminogen fragments observed in the media of THP-1 cells a t 24 h (Pig. 4). However, caseinolytic activity did not co-migratewith a prominent radiolabeled fragment of plasminogen tie. 48 kDa). When media derived from THP-1 cells cultured in theabsence of plasminogen were analyzed by SDSPAGE zymography, no caseinolytic bands were observed (data not shown).Taken together, (TableI, Figs. 4 and 5),these data demonstrate thatfollowing activation, plasmin autoproteolysis results in the generation of proteolytically active fragments that do not express cell binding properties. PMSF-inactivated '""I-Plasmin Does Not Lose Its Cell Rinding Properties-If autoproteolysis is responsible for the loss in cell-bound plasmin, then the binding of inactivated l2'II-plasmin to cells would not be expected to change over time. Therefore, we compared the bindingof "%plasmin and PMSF-inactivated "'I-plasmin to adherent THP-1 cells a t 1 and 24 h (Fig. 6). By autoradiography,the radiolabeled plasminpreparation contained a 48-kDa degradative fragment. A similar degradative fragment was observed in the conditioned media when THP-1 cells were incubated withl2'1-Lys-plasminogen for 24 h (Fig. 4). Following a 1-h incubation, both intact plasmin and the 48-kDa degradative fragmentwere found associated with the adherent THP-1 cells. Exposure of the cells with c-ACA released a majority of the intact plasmin and the 48-kDa radiolabeled fragment. As observed utilizing '251-Lys-plasminogen a s a tracer
:1
kD
-200
-46
-30
- + - + Lvsate
E-ACA
Media
j-200 Inactive
1-97
'251-Pls
4-69 -46 ~
-30
- +
-
+
E-ACA
FIG.6. Effect of PMSF inactivation on the binding of '061-plasmin to THP-I cells. (klls wrrr platrd into 6-wll platrs (2 x 10'iiwcll) as descrihcd in Fig. 1. Following adhrrence the cells were washed and media rrplaced with MSFM containing 1 pg/ml ""I-plasmin or PMSFinactivated "'T-plasmin. Conditionrd media and cell lysates were recovered a t 1 and 24 h. Alterations in molrcular weight of "'I-plasmin was determined by SDS-PACE followed hy autoradiomaphy.
(Fig. 4), a t 24 h the majority of the 12'II-plasminwas recovered in the 48-, 40-kDa, and several other smaller degradative fragments (Fig. 6). Associated with the formation of degradative fragments of '2'I-plasmin, a t 24 h '2'I-plasmin recovered from THP-1 cells was dramatically reduced as compared to 1 h. In
Regulation of Macrophage Receptor-bound Plasmin
32664
- 200 - 97 - 66 - 45 24
62 4
1
kD
Media
Lysates 1 24
kD
1 24
~~
204 132-
65 -
PIS
43 -
Hours FIG.7. The loss of anti-kringle IgC binding to plasmin. Cells
30 -
as descrihrd in Fig. 1. were platrd into 12-well plates (0.5x IOfl/'/well~ Following adherence the crlls were washed and media replaced with MSFM containing 1 pg/ml plasmin. Conditioned media wrrr rrcoverrd at 1-24 h and the immunoreactivity of recovered plasmin was determined by Wrstrrn blot utilizing monoclonal anti-kringlr I&.
E-ACA
- + - +
FIG.8.Effect of a2 plasmin inhibitor on the binding of '=!-Lys-
marked contrast, no degradative change in the active site in- plasminogen to THP-1 cells. Crlls wrre incuhatrd with TGF-fil I5 hibited '251-plasmin was observed in the media over 24 h, and nglml) ovrrnight, harvrstrd, and platrd 12-well into plates ( 5 x 105/iwellI similar amountsof PMSF-inactivated 125"Iplasmin were bound as descrihrd in Fig. 1. Following adherence the crlls wrre washed and to cells following 1 and 24 h of incubation (Fig. 6).The e-ACA- media replaced with MSFM contnining TGF-131, 1 pg/ml ""I-l,vs-plasminogen,and 20 pg/ml rr2 plasmininhihitor.Conditionrdmedia resistant fraction of PMSF-plasmin was comparable to thatof wererecovered a t 1 and 24 h. Alterationsinmolrcularweight of I I' c I-Lys-Plasminogen wrrr drtrrminrd by SDS-PAGE followed hy untreated plasmin. These data suggest that autoproteolysis autoradiography. can regulate cell binding. Loss of Anti-kringl~ IgG Binding to Autoproteolyzed of plasmin Plasmin-Figs. 4 and 6 demonstrate the generation Fluid Phose "XFdegradationfragmentsover a 24-h incubationwithTHP-1 4 AP cells. However, in both experiments small quantities of intact plasmin(ogen) were present in the conditioned mediaat 24 h. -c c 4 C The lack of substantial membrane-bound activityat 24 h (Fig. K K 6),despite the presence of material that co-migrates with intact plasmin(ogen),suggeststhatotheralterationsinplasminK (ogen) may have taken place. Since the kringle domains of plasminogen's A-chain mediate its binding to both fibrin and endothelial cells (20, 21), we determined whether plasmin autoproteolysis was associated witha loss of reactivity to a monoclonal anti-kringle I gG. When conditioned media from THP-1 cellsincubatedwithplasminfor 1-24 h were examined by Western blot, a clear time-dependent reduction in kringle imC CotaVtic domain . .-Ap A,aha p,ovnin munoreactivity was observed (Fig.7). Little or no intact immu-c Kringledomain Inhmb.*o' noreactive plasmin could be demonstratedat 24 h. These data Plg suggest that the failureof the remaining intact plasmin(ogen) FIG.9. A model depicting autolytic regulation of receptorin the 24-h conditioned media to bind THP-1 cells was due to bound plasmin activity. I'lasminogrn and uPA hind to their rrspecalterations in its cell-binding dornainb). to tho cell surfacr in a Iysinetivr rrcrptors. l'lasminoyrn initially hinds drpendrntmannrr via itskringles.I'lasminogrn is preferentially Plasmin Autoproteolysis Occurs on the Cell Surface in the Presence of Excess a2 Anti-plasmin-Under physiological con- cleaved hy uPA on the cell surface genrrating catalytically active plasPlasmin which has dissociatrd from its rrcrptoris inhihited by (12 ditions fluid-phase plasminwill be rapidly inactivated by plas- min. plasmin inhihitor. Membrane-hound plasmin is autoprotrolyzed grnermin inhibitors (32). Therefore, i t is not clear from experiment.. ating inactive fragments that rrmain hound to the recrptor. Catalytidescribed above whether autoproteolysis could contribute to cally active fragmrnts released as a consrquencr of autoproteolysis are inhibited by a2 plasmin inhihitor. A fraction of memhranr-hound plasthe physiologic regulation of membrane-bound plasmin activmin hecomes resistant to displacement hy the lysine analog cACA and ity. Consequently,wedeterminedwhetherautoproteolysis is protrctrd from plasmin drgradation. would occur onthe cell surface in the presence of 20-fold molar excess of a2 plasmin inhibitor. THP-1 cells were preincubated with TGF-p1 overnightto up-regulate their expressionof both able in the media samples containing a2 plasmin inhibitor 1 h intact"'I-Lys-plasmin(ogen)was uPA and the uPA receptor.2 However, TGF-p1 does not affect (datanotshown).At found associated with the THP-1 cells. Exposure of the cells plasminogenbinding.2WhenTGF-pl-primedmacrophages wereincubatedwithmediacontaining 12511-Lys-plasminogen with c-ACA released a majority of the bound I:!"I-Lys-plasminand a2 plasmin inhibitor for 1 h, the conditioned media con- ogen, however, these differences are not readily apparent betained two major radiolabeled bands (-80 and 140 kDa). The causefilmswereoverexposedtovisualizeotherradioactive fragments. lower molecular weight band comigrated with intact '"'I-LysThe 24-h conditioned media also contained intact '"I-Lysplasminogen. We have concluded that the higher molecular plasminogen and complexesof 1z51-Lys-plasmina n d a 2plasmin weight band is an SDS-resistant complex of "'I-Lys-plasmin inhibitor; however, the proportion of '2511-Lys-plasminassociand a2 plasmin inhibitor (6,570 kDa) for three reasons: a2 plasmin inhibitor rapidly forms a denaturation-resistant com- ated with a2 plasmin inhibitor was increased. In addition, despite the absence of detectable plasmin activity in the fluidplex with plasmin (32). This band was not observed in the of ""I-Lys-plasminogen were absence of a 2 plasmin inhibitor. Plasmin activity was undetect-phase,degradativefragments
I' I
r
2
Regulation of Macrophage Receptor-bound Plasmin
32665
over time (40). The observed decrease in catalyticactivity was due to autodegradation of the B-chain (26.5 ma) which contains the catalytic domain of plasmin(ogen1 (40). In contrast, most of the A-chain (63 m a ) , which contains plasmin's five kringle domains, remained unaltered (40). Micro-plasmin (intact B-chain; 26.5 kDa) wasobtained by incubating plasmin in a protein-free aqueous bufferat pH 11 (39,401. At an alkaline pH, the B-chain was protected from degradation, whereas the A-chain was partially degraded to a protein of 58 kDa (40,411. This partially degraded A-chain bound lysine-Sepharose demonstrating thepreservation of functional kringle(s) thatmediate lysine-dependent binding (40). Mini-plasminogen (38 kDa) DISCUSSION containing kringle 5 and theB-chain was obtained by digestion The broad substrate specificities of plasmin require that cells of plasminogen with elastase (42). collateral tissue damlocalize its activation in order to prevent Lysine-dependent plasminogen binding to both fibrin and age. The directed activation of plasminogen by macrophage is endothelial cells is inhibited by kringles 1-5 (55 kDa) andminiaffected by their expression of receptors for uPA and plasmin- plasminogen, but not micro-plasminogen or kringles 1-3 (20, ogen (6, 16-18, 33). The regulation of these receptors and the 21,43). Kringle 4 does not exhibit significant binding to either fate of ligands bound to them have great implications for a fibrin or endothelial cells (20,43). These data demonstrate that diverse group of macrophage functions includingmatrix remodkringle 5 playsa pivotal rolein thelysine-dependent binding of eling, migration,and mobilization of matrix-bound growth plasmin(ogen1 to the endothelial cell surface and fibrin. In exfactors. periments reported here, autoproteolysis results in an enzyIn contrast to the uPA receptor (34-37), little has been rematically inactive plasmin fragment (48 kDa) thatbinds cells ported concerning regulation of plasminogen receptors (38).In in a lysine-dependent manner, andcatalytically active plasmin these studies we demonstrate that plasmin issubject to autofragments (-28 and 36 kDa) which are devoid of cell binding proteolysis which leads toa lossin receptor recognition but not catalytic activity. When THP-1 macrophagewere incubated properties. These active fragments may represent micro-plasor plasminogen in serum-freemedia, mem- min or mini-plasmin with an altered kringleconformation. with either plasmin Plasminogen receptors provide a mechanism for localization brane-bound plasmin activity declined dramatically over 24 h despite the presence of catalytically active plasmin in theme- of zymogen, its activation, and protection from inactivation (16, dia (Fig. 2, Table I). Plasmin-mediated proteolysis of the plas- 19, 25). Since fluid-phase plasmin is rapidly inhibited by a2 M -'s -'(321, it is not clear if midogen) receptor was not responsible for the loss in mem- plasmin inhibitor (k,,, 2.8 x brane-bound plasmin activitysincepreincubation of THP-1 autoproteolysis could play a role inregulating membranecells with plasmin did not affect binding of 1251-Lys-plasmino- bound plasmin activity under physiologic conditions. Theregen (Fig.3).When conditioned media from cells incubated with fore, we examined whether autoproteolysis could take place in either '251-Lys-plasminogenor 1251-plasminwere examined by the presence of excess a2 plasmin inhibitor. When THP-1 cells SDS-PAGE and autoradiography, autoproteolysis of the radio- were incubated with '251-Lys-plasminogenand the plasmin inlabeled material had clearly taken place (Figs. 4 and 6). Fol- hibitor, intact membrane-bound '251-plasmin(ogen) decreased lowing a 24-h incubation with THP-1cells, two major degrada- dramatically over 24 h despite theabsence of detectable fluidtive fragments of 'z51-Lys plasmin(ogen1 were visible at 48 and phase plasmin activity (Fig. 8). As observed in the absence of 40 kDa. In addition, faintly visible intact '251-plasmin(ogen) the plasmin inhibitor, intact '251-Lys-plasmin(ogen)and a raand small degradative fragmentswere observed. Similar frag- diolabeled 48-kDa fragment observed in theconditioned media ments were observed when cells wereincubated with '251-plas- were bound to the cells. The binding of both '251-Lys-plasminmin. Both intact '251-plasmin(ogen)and the 48-kDa fragment (ogen) and the 48-kDa fragment were lysine dependent since were found associated with the THP-1cells and could be dis- they were displaced by E-ACA. The failure of the fragment to placed by e-ACA (Fig. 6). Although the 48-kDa plasmin frag- form a complex with a2 plasmin inhibitor corroborates our ment bound THP-1 cells in a lysine-dependent manner, it did conclusion that it lacks an active site. Smaller radiolabeled not appear t o possess plasmin activity as judged by casein fragments which were observed in the absence of a 2 plasmin '251-Lys-plasminogen inhibitor and demonstrated to be active by zymography were zymography (Fig. 5 ) . In contrast to either and 1251-plasmin,degradative fragments didnotformwhen not present. These data demonstrate that in the presence of cells were incubated with PMSF-inactivated 1251-plasmin.Fur- plasmin inhibitors, plasminautoproteolysis is restrictedt o the thermore, similar amounts of PMSF-inactivated '251-plasmin cell surface and generates fragments that possess either cell were bound to cells at 1and 24 h (Fig. 6).Thus, it appears that binding or catalytic activities. under serum-free conditions, autoproteolysis dissociates plasIn these studies, we have also demonstrated that plasmin min into fragments with truncated physiologic properties that binding to THP-1cells is initially E-ACAsensitive but becomes possess either cell binding or catalytic activities. resistant to displacement with E-ACAover time. Following a The modification of structure and biological properties of 24-h incubation withplasmin or '251-Lys-plasminogen,all memnative (Glu'l-plasminogen (92 kDa) by plasmin and otherpro- brane-bound plasmin activity or '251-Lys-plasmin(ogen)was reteases has been described previously. A "preactivated form of sistant to E-ACAdisplacement and further autoproteolysis. It (84 kDa) isobtained by has been recently reported thatendothelial cells in suspension plasminogen termed Ly~~~-plasminogen plasmin-mediated cleavage of the NH,-terminal 76 amino acid cross-linked plasmin to theirsurfaces via expression of transpeptide (39). Ly~~~-plasminogen is formed on the cell surface glutaminase activity (28). However, in these studies the inabiland binds to cells with higher affinity than G1u'-plasminogen ity of E-ACAto displace plasmin bound t o adherent THP-1 was (26, 31). Likewise, the kinetics of Lys-plasminogen conversion not duet o transglutaminase-mediated cross-linking, since cellto L y ~ ~ ~ - p l a s m byi ntissue plasminogen activator is markedly associated large molecular weight complexes containing lZ5Ienhanced (26). When plasmin was incubated in a protein-free Lys-plasmin(ogen) were not observed in SDS-PAGE. Thus, aqueous buffer (pH 6.5), its catalytic activity decreases rapidly E-ACA-resistant and transglutaminase-independent binding of
visible. Similardegradationfragments of greaterintensity were observed in the absence of a2 plasmin inhibitor. The amount of '251-Lys-plasminogen bound to cells at 24 h was markedly reduced. In addition to intact '251-Lys-plasmin(ogen), a radiolabeled fragment also observed in themedia was bound to the cells. Both the intact 1251-Lys-plasminogenand the fragment were displaced by exposure to E-ACAindicating lysinedependent binding.Sincefluid-phase plasmin activity was completely inhibited by the excess of a2 plasmin inhibitor, these data demonstrate that plasmin autoproteolysis occurs on the cell surface despite thepresence of excess plasmin inhibitor.
Regulation of Macrophage Receptor-bound Plasmin
32666
plasminrepresentsan undefined mechanism forlocalizing plasmin to the cell surface. The currentmodel of of plasminogen binding and activation provides mechanisms for localization of zymogen, its regulated activation,and protectionfrom inactivation. However, the model does not provide a mechanism for cell clearance of receptor-bound protease. Based on results of experiments reported here, we propose that autoproteolysis is a mechanism for regulating receptor-bound plasmin activity under conditions of high pericellular plasmin concentration (Fig. 9). REFERENCES 1. Dent, M. A. R., Sumi, R., Morris, J., and Seeley, P. J. (1993) Eur. J. Neurosci.
5,633-647 2. Konno, H., Tanaka, T., Maruo, Y., Nishino, N., Nakamura, S., Baba, S., and Takada, A. (1993) Eur. Surg. Res. 25,239-244 3. Huarte, J., Vassalli, J.-D., Belin, D., and Sakkas, D. (1993) Deu. Biol. 157, 539-546 4. Mohanam, S., Sawaya, R., McCutcheon, I., Mi-Osman, Boyd, D., and Rao, J. S. (1993) Cancer Res. 53,4143-4147 5. McNeill, H., and Jensen, P. J. (1990) Cell Regul. 1, 843-852 6. Estreicher, A,, Muhlhauser, J., Carpentier, J. L., Orci, L., and Vassalli, J. D. (1990) J. Cell Biol. 111, 783-792 7. Schlecte, W., and Brattain, M., and Boyd, D. (1990) Cancer Commun. 2, 173179 8. Richardson, M., Hatton, M. W., and Moore, S. (1988) Clin. Znuest. Med. 11, 139-150 9. Moser, T. L., Enghild, J. J., Pizzo, S. V., and Stack,M. S. (1993) J. Biol. Chem. 268, 18917-18923 10. Wang, L.. Lin, X., Cui, D., Wang, Z., and Chi, C. (1994) J. Biol. Chem. 269, 43324336 11. Murphy, G., Atkinson, S., Ward, R., Gavrilovic, R., and Reynolds, J. J. (1992) Ann. N . Y Acad. Sci. 667,l-12 12. Saksela, O.,and Riflin, D. P. (1990)J. Cell Biol. 110, 767-775 13 Tninale. K.. and Keski-Oia. J . (1992) J. Biol. Chem. 267.25378-25384 ". ".= ~ ~ .J.. . ~ ,. Knli. , ~.,", " ~ . 14. Falcone, D. J., McCaffrey, T. A,, HaimovitzIFriedman, A,, VerGlio, J. A,, and Nicholson, A. C. (1993) J . Biol. Chem. 268, 11951-11958 15. Falcone, D. J., McCaffrey, T. A,, Haimovitz-Friedman, A,, Vergilio, J. A., and Garcia, M. (1993) J . Cell. Physiol. 155, 595-605 16. Plow, E. E , Freany, D. E., Plescia, J., and Miles, L. A. (1990) J. Cell Biol. 103, 2411-2420 17. Kirchheimer, J. C . , and Remold, H. G. (1989) Blood 74, 1396-1402 18. Ellis, V., Behendt, N., and Dano, K (1991) J. Biol. Chem. 266, 12752-12758 " " ~~~~~~~
19. Hajjar, K A., Harpel, P. C., Jaffe, E. A,, and Nachman, R. L. (1986) J. Biol. Chem. 261,11656-11662 20. Wu, H. L., Wu, LS.,Fang, R.-Y., Hau, J. S., Wu, D. H., Chang, B. I., Lin, T. M., and Shi, G. Y. (1992) Biochem. Biophys Res. Commun. 188, 703-711 21. Burge, S. M., Marshall, J. M., and Cederholm-Williams, S. A. (1992) BE J. Dermatol. 126.35-41 22. Hajjar, K. A,, Jacovina,A. T., and Chacko,J. (1994)J. Biol. C h m . 269,2119121197 23. Miles, L. A,, Dahlberg, C. M., Plescia, J., Felez, J., Kato, K., and Plow, E. F. (1991) Biochemistry 30,1682-1691 24. Miles, L. A,, Dahlberg, C. M., Levin, E.G., and Plow, E. F. (1989) Biochemistry 28,9337-9343 25. Hall, S. W., Humphies, J. E., and Gonias, S. L. (1991) J . Biol. Chem. 266, 12329-12336 26. Hajjar, K.A,, and Nachman, R. L. (1988) J. Clin. Inuest. 82, 1769-1778 27. Tsuchiya, S.,Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T., and Tada, K. (1980)Znt. J. Cancer 26, 171-176 28. Bendixen, E., Borth, W., and Harpel, P. C. (1993) J. Biol. Chem. 268, 2196221967 29. Gonzalez-Gronow, M., Stack, S., and Pizzo, S. V (1991) Arch. Biochem. Biophys. 286,625428 30. McFarlane, A. S. (1958) Nature 162.53 31. Silverstein, R.L., Friedlander, R. J., Nicholas, R. L., and Nachman, R.L. (1988) J. Clin. Invest. 82, 194LL1955 32. Travis, J., and Salvesen, G. S. (1983) Annu. Reu. Biochem. 52,655-709 33. Kirchheimer, J. C., and Remold, H. G. (1989) J. Immunol. 143,2634-2639 34. Ploug, M., Ronne, E., Behendt, N., Jensen, A. L., Blasi, F., and Dan0 K. (1991) J. Biol. Chem. 266, 1926-1933 35. Lund, L. R.,Ronne, E., Roldan, A. L., Behendt, N., Romer, J., Blasi, F., and Dano, K. (1991)J . Biol. Chem. 266,5177-5181 36. Picone, R., Kajtaniak, E. L., Nielsen, L. S., Behendt, N., Mastronicola, M. R., Cubellis, M. V.,Stoppelli, M. P., Anderson, S., Dano, K., and Blasi, F. (1989) J. Cell Biol. 108, 693-702 37. Mignatti, P., Mazzieri, R., and Riflrin, D. B. (1991)J. Cell Biol. 113,1193-1201 38. Felez, J., Miles, L. A,, Plescia, and Plow, E. E (1990) J . Cell Biol. 111, 16731683 39. Castellino, F. J., Ploplis, V., Powell, J. R., and Strickland, D. (1981) J. Biol. Chem. 256,4778-4782 40. Wu, L.-H., Shi, G. Y., and Bender, M. L. (1987) Proc. Nutl. Acud. Sci. U. S. A. 84,829%8295 41. Wu, L.-H., Shi, G. Y., Wohl, R. C., and Bender, M. L. (1987) Proc. Natl. Acad. Sci. U. S.A . 84,87934795 42. Sottrup-Jensen, L., Claeys, H., Zajdel, M., Petersen, T. E., and Magnusson, S. (1978) Prog. Chem. Fibrin. Thrombo.3, 191-209 43. Wu, H.-L., Chang, B.-I., Wu, D.-H., Chang, L.-C., Gong, C.-C., Lou, K-L., and Shi, G.-Y. (1990) J. Biol. Chem. 265, 19658-19664
