Plasmin Annexin A2 and Inhibited by Inactive by Plasmin-Dependent

Urokinase-Type Plasminogen Activator Stimulation of Monocyte Matrix Metalloproteinase-1 Production Is Mediated by Plasmin-Dependent Signaling through This information is current as Annexin A2 and Inhibited by Inactive of October 2, 2021. Plasmin Yahong Zhang, Zhao-Hua Zhou, Thomas H. Bugge and Larry M. Wahl

J Immunol 2007; 179:3297-3304; ; Downloaded from doi: 10.4049/jimmunol.179.5.3297 http://www.jimmunol.org/content/179/5/3297

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Urokinase-Type Plasminogen Activator Stimulation of Monocyte Matrix Metalloproteinase-1 Production Is Mediated by Plasmin-Dependent Signaling through Annexin A2 and Inhibited by Inactive Plasmin1

Yahong Zhang,* Zhao-Hua Zhou,† Thomas H. Bugge,‡ and Larry M. Wahl2*

Chronic inflammatory diseases are associated with connective tissue turnover that involves a series of proteases, which include the plasminogen activation system and the family of matrix metalloproteinases (MMPs). Urokinase-type plasminogen activator (uPA) and plasmin, in addition to their role in fibrinolysis and activation of pro-MMPs, have been shown to transduce intracellular signals through specific receptors. The potential for uPA and plasmin to also contribute to connective tissue turnover by directly

regulating MMP production was examined in human monocytes. Both catalytically active high m.w. uPA, which binds to the Downloaded from uPAR, and low m.w. uPA, which does not, signiﬁcantly enhanced MMP-1 synthesis by activated human monocytes. In contrast, the N-terminal fragment of uPA, which binds to uPAR, but lacks the catalytic site, failed to induce MMP-1 production, indicating that uPA-stimulated MMP-1 synthesis was plasmin dependent. Endogenous plasmin generated by the action of uPA or exogenous plasmin increased MMP-1 synthesis by signaling through annexin A2, as demonstrated by inhibition of MMP-1 production with Abs against annexin A2 and S100A10, a dimeric protein associated with annexin A2. Interaction of plasmin with annexin A2 resulted in the stimulation of ERK1/2 and p38 MAPK, cyclooxygenase-2, and PGE2, leading to increased MMP-1 production. http://www.jimmunol.org/ Furthermore, binding of inactive plasmin to annexin A2 inhibited plasmin induction of MMP-1, suggesting that inactive plasmin may be useful in suppressing inﬂammation. The Journal of Immunology, 2007, 179: 3297–3304.

onocytes and macrophages are prominent at chronic uPA (HMW-uPA) (7, 8). uPA is comprised of the following three inflammatory lesion sites in which there is extensive domains: an N-terminal growth factor domain, a kringle domain, M degradation and remodeling of connective tissue. Con- and a C-terminal catalytic domain. The first two domains comprise nective tissue turnover is believed to be mediated through the ac- the N-terminal fragment (ATF), which can bind to uPAR, but lacks tion of a series of proteases. These proteases include the plasmin- the catalytic domain and therefore cannot cleave plasminogen to 3 by guest on October 2, 2021 ogen activation system, the matrix metalloproteinase (MMP) generate plasmin. A low m.w. form of uPA (LMW-uPA) is pro- family of enzymes, and lysosomal cathepsins (1–4). In this study, duced as a result of the cleavage of HMW-uPA by plasmin and we examined whether the urokinase plasminogen system, in addi- other proteases (9, 10). LMW-uPA lacks the growth factor domain, tion to the activation of MMPs, also regulated the production of and therefore does not bind to uPAR, but does cleave plasminogen. MMPs by human primary monocytes. The urokinase plasminogen The activity of uPA is regulated by soluble inhibitors, such as activation system has been shown to be an important regulator of plasminogen activator inhibitor 1 (PAI-1). The plasmin generated monocyte migration (5, 6). Urokinase-type plasminogen activator by the action of uPA, in addition to cleaving fibrin, also activates (uPA) when bound to its receptor, uPAR, cleaves cell surface plas- many of the MMPs, thus playing a major role in the facilitation of minogen, resulting in the generation of plasmin. uPA is synthe- connective tissue degradation (11). sized as pro-uPA, a single chain protein, with low catalytic activity, and is activated by a proteolytic cleavage of the Lys158 Ile159 The family of MMP enzymes is comprised of over 20 members peptide bond, resulting in high m.w. two-chain form, high m.w. (12, 13). These enzymes, grouped according to their domain struc- tures and substrate preferences, include collagenases, gelatinases, stromelysins, membrane-type MMPs, and others. Collectively, *Immunopathology Section, †Experimental Medicine Section, and ‡Proteases and MMPs can degrade all extracellular matrix components. MMPs Tissue Remodeling Unit, National Institutes of Dental and Craniofacial Research, have been linked to most diseases that involve tissue destruction, National Institutes of Health, Bethesda, MD 20892 including cancer, rheumatoid arthritis, periodontal disease, and Received for publication May 15, 2007. Accepted for publication June 27, 2007. atherosclerosis (2, 13). The presence of monocytes/macrophages The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance and their production of MMPs at these disease sites may contribute with 18 U.S.C. Section 1734 solely to indicate this fact. to the pathology associated with the degradation of extracellular 1 This work was supported by the Intramural Research Program of National Institutes matrix. A major MMP produced by monocytes is MMP-1 (in- of Health, National Institute of Dental and Craniofacial Research. terstitial collagenase), which degrades fibrillar collagens such 2 Address correspondence and reprint requests to Dr. Larry M. Wahl, Immunopathol- as types I, II, and III. Activation of monocytes/macrophages by ogy Section, National Institute of Dental and Craniofacial Research, National Insti- tutes of Health, Building 30, Room 3A-300, Bethesda, MD 20892. E-mail address: various agonists results in the production of MMP-1 that oc- [email protected] curs, in part, through a PGE2-dependent pathway (14–19) and 3 Abbreviations used in this paper: MMP, matrix metalloproteinase; ATF, N-terminal MAPKs (20). fragment; COX, cyclooxygenase; HMW-uPA, high m.w. uPA; LMW-uPA, low m.w. uPA; PAI-1, plasminogen activator inhibitor 1; uPA, urokinase-type plasminogen Although plasmin has been reported to be involved in the acti- activator. vation of pro-MMP-1, -3, -7, -9, -10, and -13 (11), we explored the www.jimmunol.org 3298 PLASMIN-ANNEXIN A2 INDUCTION OF MONOCYTE MMP-1

possibility that uPA and plasmin may also, through their interaction with cell surface receptors on monocytes, regulate MMP production in monocytes. In this study, we report that uPA plays a major role in inducing MMP-1 production in activated human monocytes, which is mediated by a plasmin-dependent mechanism involving signaling through the annexin A2 heterotetramer. More- over, binding of inactive plasmin to annexin A2 blocks the induction of MMP-1 by active plasmin.

Materials and Methods Reagents HMW-uPA, LMW-uPA, ATF-uPA, and ␣2-antiplasmin were obtained from American Diagnostica. Plasmin (Ն3 U/mg protein) was purchased from Sigma-Aldrich, and inactive plasmin and a stable mutant form of human PAI-1 were from Molecular Innovations. Annexin A2 polyclonal Ab was obtained from Santa Cruz Biotechnology, and mAbs against an- Ј nexin A2 and F(ab )2 Abs against annexin A2 (p36) and S100A10 (p11) were from BD Biosciences. LPS (Escherichia coli 05:B55) was purchased from Sigma-Aldrich. ERK1/2/phospho-ERK1/2 and p38/phospho-p38 Abs were from Cell Signaling Technology. MMP-1 Abs used were rabbit polyclonal Abs against MMP-1 (provided by H. Birkedal-Hansen, National Downloaded from Institute of Dental and Craniofacial Research/National Institutes of Health, Bethesda, MD) and a mouse anti-human MMP-1 mAb (Chemicon Inter- national). Rabbit anti-cyclooxygenase (COX)-2 Abs were obtained from Cayman Chemical, and mouse anti-␤-actin Abs were from Chemicon International.

Purification and culture of human monocytes http://www.jimmunol.org/ Human peripheral blood cells were obtained by leukapheresis of normal volunteers at Department of Transfusion Medicine at the National Insti- tutes of Health. The monocyte fraction was purified by counterflow centrifugal elutriation, as previously described (21, 22), and contained Ͼ90% monocytes, as determined by morphology and flow cytometry. Monocytes were cultured in serum-free DMEM (Cambrex) supplemented with 2 mM L-glutamine (Mediatech) and 10 ␮g/ml gentamicin (Cambrex). Western blot analysis of MMP-1, MAPKs, and COX-2

For MMP-1 determination, purified monocytes were cultured at a density by guest on October 2, 2021 of 5 ϫ 106/ml DMEM in 12-well polystyrene plates (Corning Life Sci- ences). After 36–48 h of treatment with reagents, the conditioned medium was centrifuged and collected. BSA (40 ␮g/ml) was added to the culture supernatants before the precipitation of the proteins with cold ethanol (final concentration, 60%) for at least 15 min at Ϫ70°C. The proteins were pelleted by microcentrifuging at 20,800 ϫ g for 12 min, washed with ethanol, and lyophilized by rotary evaporation. The lyophilized proteins were resuspended in SDS-Laemmli buffer (500 mM Tris-HCl (pH 6.8)/10% SDS/ 0.01% bromphenol blue/20% glycerol), reduced with 5% 2-ME, heated for 4 min at 100°C, and electrophoresed on a 10 or 12% Tris-glycine gel in SDS running buffer (25 mM Tris-HCl (pH 8.3)/192 mM glycine/10% SDS). The proteins were transferred onto 0.45 ␮m nitrocellulose in a buffer containing 25 mM Tris-HCl (pH 8.3)/192 mM glycine/20% methanol and blocked with 50 mM Tris-HCl (pH 7.5)/150 mM NaCl (TBS) containing 5% nonfat dry milk for at least 1 h. The blots were then incubated overnight with Abs against MMP-1. For detection of the levels of activated ERK1/2 and p38 MAPKs, mono- FIGURE 1. Effect of HMW-uPA, LMW-uPA, and ATF-uPA on cytes were cultured in DMEM at 5 ϫ 106/ml in suspension. Ten to 60 min monocyte MMP-1 production. A, Monocytes purified by elutriation after the addition of reagents, the cells were pelleted and lysed with SDS were cultured in serum-free DMEM at 5 ϫ 106 cells/ml in the presence loading buffer (prepared as described above), and the supernatants were or absence of LPS (25 ng/ml) and the indicated concentrations of loaded onto 12% Tris-glycine gels. The blots were then incubated overnight with mouse anti-human Abs against the phosphorylated forms of HMW-uPA. The cultures were harvested at 48 h, and the levels of ERK1/2 and p38 and with rabbit anti-human Abs against total ERK1/2 and MMP-1 were determined in the conditioned medium supernatants by p38 as a measure of equal loading of the gels. Western blot (ACL, active collagenase; PCL, procollagenase). B, All Cell protein isolation for the determination of COX-2 protein levels was data are representative of at least three independent experiments; an prepared, as previously described (23). Briefly, 20 ϫ 106 monocytes in 4 example of this is shown in the comparison of MMP-1 production by ml of DMEM were cultured in suspension in 17-ml polypropylene tubes monocytes from three donors that were cultured in the presence or overnight in the presence or absence of reagents. The cells were then absence of LPS and the indicated concentrations of LMW-uPA, HMW- washed in PBS with protease inhibitors. The cell pellets were resuspended uPA, and ATF-uPA. MMP-1 was determined in the 48-h conditioned culture medium by Western blot. C, MMP-1 mRNA levels were determined by RT-PCR in monocytes that had been cultured for8hinthe in monocytes 8 h after the addition of PAI-1 or HMW-uPA to control presence or absence of LPS and the indicated concentrations of LMW- cultures or exposure of LPS-treated cultures to HMW-uPA or HMW-uPA uPA, HMW-uPA, and ATF-uPA. GAPDH served as an internal control (30 nM) that had been preincubated with PAI-1 (10 ␮g/ml). Data are rep- for RNA levels. D, MMP-1 mRNA levels were determined by RT-PCR resentative of at least three independent experiments. The Journal of Immunology 3299 in 250 mM sucrose-containing protease inhibitors and sonicated (Ultra- sonic Cell Disrupter; Kontes). Nuclear debris and unbroken cells were pelleted at 100 ϫ g, and the supernatant was microfuged at 1500 ϫ g to pellet cell membrane proteins. Equal amounts of protein were loaded onto 12% Tris-glycine gels. The blots were incubated overnight with rabbit anti-COX-2 Abs. Equal loading was measured with mouse anti-␤- actin Abs. Western blots for MMP-1, MAPKs, and COX-2 were incubated overnight with primary Abs, washed, and analyzed by the addition of Alexa Fluor 680 goat anti-rabbit or Alexa Fluor 750 goat anti-mouse Abs (Molecular Probes) and the infrared fluorescence detected with the Odyssey infrared imaging system (LI-COR). Densitometry analysis of the bands on the Western blots was determined with the LI-COR software analysis program or the ImageQuant software analysis program (Amersham Biosciences).

Plasmin activity assay Plasmin activity was determined with the SPECTROZYME PL kit (Amer- ican Diagnostica), which uses the chromogenic substrate H-D-norleucyl- hexahydrotyrosol-lysine-para-nitroanilide diacetate. For detection of cell- associated plasmin activity, monocytes were cultured at 1 ϫ 106/well in

HBSS containing calcium and magnesium (HyClone) with the plasmin Downloaded from substrate. Kinetic absorbance readings were measured at 405 nm.

RT-PCR Total cellular RNA was extracted with the RNeasy Mini Kit (Qiagen) 8 h after stimulation of monocytes with LPS. Transcript levels of MMP-1 were

determined using semiquantitative RT-PCR with GADPH as an internal http://www.jimmunol.org/ control. The primer sets for MMP-1 were 5Ј-TGTGGTGTCTCACAGCT TCC-3Ј and 5Ј-CACATCAGGCACTCCACATC-3Ј, and for GAPDH 5Ј- TCGGAGTCAACGGATTTGGTCGTA-3Ј and 5Ј-ATGGACTGTGGT CATGAGTCC-3Ј. OneStep RT-PCR kit (Qiagen) was used with the following reaction components: 5 ␮lof5ϫ OneStep RT-PCR buffer, 10 mM dNTP, 10 ␮M MMP-1 primer mix, 6 ␮M GAPDH primer mix, 0.5 ␮g of RNA template, 1 ␮l of OneStep RT-PCR enzyme mix, and RNase-free water were added for a total of 25 ␮l. PCR times were as follows: 30 min at 50°C for reverse transcription, 15 min at 95°C for initial PCR, 36 cycles of 40 s at 94°C, 45 s at 57°C, and 1 min at 72°C; and 10 min at 72°C for the final extension. The amplified DNA was separated by 1.7% agarose gel FIGURE 2. uPA stimulation of MMP-1 production by activated mono- by guest on October 2, 2021 electrophoresis and stained with SYTO 60 red fluorescent nucleic acid cytes is mediated through plasmin. A, Monocytes were cultured in 96-well stain (Molecular Probes), and the intensity of the stained bands was ana- plates in the presence or absence of LPS (25 ng/ml) and/or HMW-uPA (30 lyzed with the Odyssey infrared imaging system. nM), LMW-uPA (30 nM), or ATF-uPA (30 nM). Plasmin activity was determined with a Spectrozyme PL kit, with kinetic absorbance readings Cell staining and flow cytometry analysis measured at 405 nM. B, Plasmin was added to monocyte cultures at the indicated concentrations in the presence or absence of LPS. MMP-1 levels Plasmin and BSA were FITC conjugated, as previously described (24), were determined in the 48-h culture supernatants by Western blot. C, using a FluoroTag FITC conjugation kit (Sigma-Aldrich). Purified human MMP-1 mRNA levels were determined by RT-PCR in monocytes 8 h after monocytes were fixed and permeabilized (buffers from eBioscience). A the addition of plasmin in the presence or absence of LPS. GAPDH served total of 3–10 ϫ 105 cells was preincubated with 5 ␮g of human whole IgG as an internal control for RNA levels. Data are representative of at least (Jackson ImmunoResearch Laboratories) in 90 ␮l of PBS containing 0.5% BSA and 2 mM EDTA at 4°C for 30 min to minimize the effect of non- three independent experiments. specific FcR binding sites. FITC-conjugated BSA, plasmin (2.5 ␮gin10 ␮l), or anti-human Abs (1–2.5 ␮g/10 ␮l) then were added, respectively, incubated for 30 min, washed, and analyzed by the FACSCalibur system (BD Biosciences). For annexin A2 binding site blocking experiments, Results polyclonal goat anti-human annexin A2 IgG (Santa Cruz Biotechnology), Catalytically active uPA enhances MMP-1 production by Ј mouse anti-human annexin A2 (p36) IgG F(ab )2, mouse anti-human activated monocytes Ј S100A10 (p11) IgG F(ab )2 (BD Biosciences), and inactive plasmin (10 ␮g/ml), respectively, were preincubated with the cells at 4°C for 30 min To determine the effect of uPA on human monocyte MMP-1 pro- before the addition of 2.5 ␮g/ml plasmin-FITC. For plasmin-FITC binding duction, the catalytically active two-chain form of uPA (HMW- to monocytes, an equal amount of BSA-FITC served as negative control. uPA) was added to control monocytes or monocytes activated by Plasmin and inactive plasmin were also preincubated with monocytes to LPS. HMW-uPA caused a dose-dependent increase in the protein block the binding sites of annexin A2 recognized by anti-human annexin levels of MMP-1 by LPS-stimulated monocytes, whereas it did not A2 Abs. For indirect immunostaining, polyclonal goat anti-human annexin A2 IgG (Jackson ImmunoResearch Laboratories) or goat IgG, as negative induce MMP-1 in unstimulated monocytes (Fig. 1A). The majority control, was added to the cells, followed by the addition of FITC-labeled of MMP-1 produced by monocytes is detected in the active 45- and Ј rabbit anti-goat IgG F(ab )2 as secondary Ab. FACS results were analyzed 43-kDa form (active collagenase). In some experiments, depend- by CellQuest software (BD Biosciences). ing on the time of incubation and the donor, bands corresponding to procollagenase were detected. We next examined whether the Statistical analysis catalytic activity and/or binding to uPAR were required to mediate Comparison between group means was analyzed using ANOVA. The sta- an increase in MMP-1 production by comparing HMW-uPA, tistic data represent the mean Ϯ SEM. A value of p Ͻ 0.01 is regarded as LMW-uPA, and ATF-uPA. All experiments are representative of significant. three independent experiments; an example of this is shown in the 3300 PLASMIN-ANNEXIN A2 INDUCTION OF MONOCYTE MMP-1 Downloaded from http://www.jimmunol.org/

FIGURE 3. Abs against annexin A2 (p36) or S100A10 (p11) block HMW-uPA or plasmin-stimulated MMP-1 production. Monocytes were preincubated by guest on October 2, 2021 Ј for 30 min with a goat Ab against annexin A2 (Gt anti-Ann A2), goat IgG (GtIgG), or the F(ab )2 portion of mAbs against p36 (annexin A2) or p11 (S100A10), a protein associated with annexin A2 involved in the formation of the heterotetramer. LPS was then added to cultures in the absence or presence of HMW-uPA (A and D) or plasmin (B and E). MMP-1 levels were determined 48 h later in the conditioned medium by Western blot. Specific binding of plasmin to annexin A2 and S100A10 was determined by FACS analysis with the addition of FITC-labeled plasmin to monocytes in the presence or Ј absence of polyclonal Abs against annexin A2 or goat IgG as isotype control (data not shown) (C) or the F(ab )2 portion of mAbs against p36 (annexin A2) or p11 (S100A10) (F). Data are representative of at least three individual experiments. comparison of MMP-1 production by monocytes from three do- uPA or LMW-uPA to either control or LPS-stimulated monocytes nors that were cultured in the presence or absence of LPS and the induced a significant increase in plasmin activity (Fig. 2A). In con- indicated concentrations of LMW-uPA, HMW-uPA, and ATF- trast, ATF-uPA caused little, if any, increase in plasmin activity in uPA (Fig. 1B). Catalytically active HMW-uPA, which binds to unstimulated or LPS-stimulated monocytes. uPAR, and LMW-uPA, which does not bind to uPAR, increased We next added plasmin to monocyte cultures to determine its LPS-induced production of MMP-1 protein. In contrast, ATF-uPA, direct effect on MMP-1 production. Plasmin caused a dose-depen- which lacks the catalytic domain, but binds to uPAR, did not stim- dent increase in MMP-1 in LPS-stimulated monocytes, as shown at ulate MMP-1 production. The requirement of catalytically active the protein and mRNA level, but did not induce MMP-1 in control uPA in the induction of MMP-1 expression by monocytes was also (unstimulated) monocytes (Fig. 2, B and C). These findings dem- verified at the mRNA level (Fig. 1C) as well as in experiments onstrate plasmin is generated from cell-associated plasminogen by with a PAI-1 and HMW-uPA complex (Fig. 1D), which binds uPA and that plasmin requires a costimulant, such as LPS, to en- uPAR, but is inactive. These findings demonstrate that the in- hance MMP-1 production by monocytes. creased production of monocyte MMP-1, mediated by uPA, is dependent on the catalytic activity of this enzyme and does not Plasmin induces MMP-1 production in monocytes through require signaling through uPAR. annexin A2 Previous studies have shown that the heterotetramer of annexin A2 uPA stimulation of MMP-1 production is mediated by plasmin can serve as a receptor for plasmin on monocytes (25). To examine Stimulation of MMP-1 by catalytically active HMW-uPA and whether plasmin stimulated MMP-1 production through annexin LMW-uPA, but not ATF-uPA, indicated that uPA was mediating A2, a polyclonal Ab against annexin A2 was added to the mono- its effect through the generation of plasmin from cell-bound plas- cyte cultures. This Ab caused a dose-dependent inhibition of minogen. This possibility was examined with a cell-based assay to HMW-uPA and plasmin enhancement of MMP-1, whereas the iso- Ј determine plasmin levels in monocyte cultures. Addition of HMW- type IgG had no effect (Fig. 3, A and B). Next, F(ab )2 against The Journal of Immunology 3301 Downloaded from http://www.jimmunol.org/ FIGURE 5. ERK1/2 and p38 MAPK pathways are involved in HMW- uPA and plasmin stimulation of MMP-1 production by activated monocytes. A, Monocyte cultures were incubated for 30 min with 10 ␮M PD98059 (PD), an inhibitor of ERK1/2, or 10 ␮M SB203580 (SB), an inhibitor of p38 MAPK, before the addition of LPS, HMW-uPA, or plasmin. The conditioned medium was harvested 48 h later, and MMP-1 levels were determined by Western blot (PCL, procollagenase; ACL, active collagenase). B, Phosphorylation levels of p38 MAPK or ERK1/2 were determined 1 h after stimulation with LPS and compared with total p38 and ERK1/2 as a measure of equal loading. Data are representative of at least by guest on October 2, 2021 three independent experiments. FIGURE 4. Stimulation of MMP-1 by HMW-uPA and plasmin is mediated in part by PGE2. HMW-uPA (30 nM) or plasmin (360 nM) was added to monocyte cultures in the absence or presence of LPS (25 ng/ml), sized as a result of the induction of COX-2 (17). Western blot and the COX-2 protein levels were determined by Western blot at 16 h, analysis of the protein levels of COX-2 in the membranes of the with anti-␤-actin serving as an indicator for equal loading (A) and medium monocytes revealed that the LPS-induced COX-2 was increased levels of PGE2 measured by ELISA at 24 h (B). Monocyte cultures were by HMW-uPA or plasmin (Fig. 4A), which corresponded to in- incubated with indomethacin (Indo) for 30 min before the addition of LPS, creased PGE2 levels in the monocyte culture supernatants (Fig. HMW-uPA, plasmin, or PGE (1 ␮M). MMP-1 levels in the conditioned 2 4B). PGE was also detected in control monocytes treated with medium were determined 48 h later by Western blot (C). Data are repre- 2 HMW-uPA, most likely derived from COX-1 because COX-2 was sentative of at least three individual experiments. not induced. The LPS-stimulated increase in MMP-1 and the further enhancement of MMP-1 by HMW-uPA or plasmin were annexin A2 (p36) and S100A10 (p11), components of the annexin inhibited by indomethacin, which was restored, in part, by the

A2 heterotetramer, was used to determine whether both compo- addition of PGE2 (Fig. 4C). These results demonstrate that nents were involved in the induction of MMP-1. Both Abs inhib- HMW-uPA or plasmin induction of PGE2 is involved in the ited MMP-1 production induced by LPS, and the enhancement by increase of MMP-1. HMW-uPA and plasmin (Fig. 3, D and E). These findings dem- We have previously shown that LPS stimulation of monocyte onstrate that uPA generation of endogenous plasmin or exogenous MMP-1 production is also regulated by the p38 MAPK and plasmin induction of MMP-1 production occurs through compo- ERK1/2 pathways (20). To determine whether HMW-uPA gener- nents that form the annexin A2 heterotetramer. ated plasmin or the direct addition of plasmin also induced MMP-1 Further evidence of the interaction of plasmin with annexin A2 through MAPKs, inhibitors of ERK1/2 (PD98059) (PD) or p38 was determined by FACS analysis. Preincubation of monocytes (SB203580) (SB) were added to monocyte cultures. Both MAPK with the polyclonal Ab against annexin A2 (Fig. 3C) or the mAbs inhibitors suppressed the enhancement of MMP-1 by HMW-uPA against p36 (annexin A2) or p11 (S100A10) (Fig. 3F) blocked the or plasmin (Fig. 5A), indicating that p38 and ERK1/2 were in- binding of FITC-labeled plasmin to monocytes. volved in mediating the induction of MMP-1 by plasmin. This conclusion was further supported by HMW-uPA or plasmin-in- HMW-uPA and plasmin signaling leading to MMP-1 production duced increases in the phosphorylation of p38 and ERK1/2 in LPS- occurs through PGE2 and MAPKs stimulated monocytes (Fig. 5B). HMW-uPA also caused a slight We have previously shown that the production of MMP-1 by ac- increase in the phosphorylation of both MAPKs in unstimulated tivated monocytes is regulated, in part, by PGE2, which is synthe- monocytes. 3302 PLASMIN-ANNEXIN A2 INDUCTION OF MONOCYTE MMP-1 Downloaded from http://www.jimmunol.org/ FIGURE 6. Inactive plasmin binds to annexin A2, blocking the binding of active plasmin, and inhibits monocyte MMP-1 production. A, Inactive plasmin was added to monocyte cultures 30 min before LPS (25 ng/ml) or LPS plus plasmin, and MMP-1 levels were determined 48 h later. B, Plasmin activity was measured with a Spectrozyme PL kit in a cell-free assay to determine whether inactive plasmin affected the activity of plasmin. Plasmin activity was also measured for the mixture of plasmin plus ␣2 anti-plasmin, which inhibits soluble plasmin, as a control for inhibition. C, Monocytes were incubated with FITC-labeled plasmin alone, or pretreated with inactive plasmin for 30 min and then FITC-labeled plasmin, and the intensity of fluorescence was detected by FACS analysis. FITC-labeled BSA served as a control for nonspecific binding. D, Monocytes were incubated with goat anti-human annexin A2, or pretreated with inactive plasmin for 30 min and then goat anti-human annexin A2 (Gt anti-hu Ann A2). FITC-labeled anti-goat IgG was used as the secondary Ab, and goat IgG served as a control for nonspecific binding. Fluorescence intensity was detected by FACS analysis. Data are representative of at least three independent experiments. by guest on October 2, 2021

Inactive plasmin blocks plasmin-induced synthesis of MMP-1 by RAW264.7 cells, a murine macrophage cell line, transfected with monocytes human uPAR (27). Support for uPA signaling through uPAR has We next examined whether catalytically active plasmin was re- been demonstrated, in part, by the effect of ATF-uPA, which binds quired for the induction of MMP-1 synthesis. Inactive plasmin, in to uPAR, but lacks the catalytic domain. Functions affected by which the catalytic site had been irreversibly blocked with a pep- ATF-uPA include proliferation, stimulation of early response tide inhibitor, was added to monocytes before the addition of LPS genes, HIV-1 replication, migration, and apoptosis (28–34). How- or LPS plus plasmin. Inactive plasmin inhibited the production of ever, our findings show that ATF-uPA did not induce signaling MMP-1 by LPS and LPS plus plasmin-treated monocytes (Fig. that led to increased MMP-1 production, nor did it inhibit MMP-1 6A). This inhibition was not related to direct blocking of plasmin production by binding to uPAR due to the rapid turnover of uPAR activity, as shown in a cell-free plasmin activity assay in which the when bound to uPA (35, 36). Rather, only the catalytically active combination of inactive plasmin and plasmin exhibited the same HMW-uPA or LMW-uPA, which does not bind to uPAR, increased activity as plasmin alone (Fig. 6B). FACS analysis revealed that MMP-1 production by activated monocytes. HMW-uPA tended to inactive plasmin blocked the binding of FITC-labeled plasmin induce more MMP-1 than LMW-uPA, possibly due to more efficient (Fig. 6C) and the binding of Abs against annexin A2 to monocytes activation of cell surface plasminogen when uPA is bound to uPAR. (Fig. 6D). These findings demonstrate that inactive plasmin can Collectively, these findings indicated that plasmin generated from the function as an effective inhibitor of plasmin-mediated signaling in cleavage of cell surface-bound plasminogen by uPA was responsible monocytes. for mediating the increase in MMP-1 production. Plasmin has been demonstrated to induce changes in cellular Discussion functions in a variety of cells. Responses and signal pathways af- Studies on uPA and plasmin have, in large part, focused on the role fected in monocytes by plasmin include the following: stimulus of of these enzymes in pericellular matrix degradation. However, the 5-lipoxygenase pathway, chemotaxis through stimulation of there is now increasing evidence that these enzymes may also cyclic guanosine monophosphate, production of cytokines and tis- function to induce cellular activation through various signaling sue factor mediated by AP-1, and NF-␬B activation and expression pathways. Binding of uPA to cell surface uPAR has been shown to of MCP-1 and CD40 via activation of p38 MAPK and JAK/STAT activate several signal transduction pathways, including intracel- pathways (25, 37–39). In our study, we show that plasmin causes lular tyrosine kinases and serine/threonine kinases such as ERK/ a dose-dependent increase in MMP-1 production by LPS-activated MAPK (26). Evidence that uPA can signal through uPAR in mac- monocytes, but has no effect on unstimulated or control cells, dem- rophages, resulting in activation of ERK1/2, has been reported in onstrating that plasmin requires a costimulator, such as LPS, to The Journal of Immunology 3303 exert its effect on MMP-1 production. Moreover, we demonstrate MMP-1 production by LPS-stimulated monocytes, demonstrating that plasmin activity is generated from endogenous cell surface that both of these MAPK pathways are involved in uPA and plas- plasminogen most likely synthesized by monocytes, as a result of min-mediated increase in MMP-1. We reported previously that low levels of plasminogen mRNA that have been previously p38 MAPK regulates MMP-1 primarily through transcription fac- shown in human monocytes (40). Alternatively, plasminogen ac- tors that are affected by PGE2 because inhibition of MMP-1 by p38 quired in vivo may remain bound to the monocytes even though MAPK inhibitors can be reversed by PGE2 or dibutyryl cAMP these cells have undergone extensive washing in the purification (20). In contrast, suppression of MMP-1 by ERK1/2 inhibitors by elutriation and subsequently cultured in the absence of serum. cannot be reversed by PGE2 due to activation of additional tran- Low levels of plasmin activity were detected in control monocytes, scription factors whose regulation is PGE2 independent. which would be in agreement with the constitutive production of Our data demonstrate that the urokinase plasminogen activation uPA by monocytes (41, 42). These findings indicate that the uPA/ system through generation of plasmin has a major role in the reg- plasmin system plays a significant role in the regulation of MMP-1 ulation of monocyte MMP-1 production, which is thought to be production by activated monocytes. involved in the pathology associated with chronic inflammatory The ability of plasmin to increase MMP-1 inferred that it was diseases. Plasmin has also been shown to induce MCP-1 and acting through a specific cell surface receptor. Putative receptors TNF-␣, cytokines associated with inflammatory responses (25, for plasmin on monocyte cell lines and primary monocytes include 38). Our results also show that inactive plasmin blocked the bind- ␣-enolase, TIP49a, and annexin A2 (25, 43–46). Recently, the ing of active plasmin to annexin A2 and the subsequent enhance- annexin A2 heterotetramer has been identified as a receptor for ment of MMP-1 production by activated monocytes. Thus, inac- plasmin-induced signaling in human monocytes (25). Moreover, tive plasmin may be useful in suppressing plasmin-mediated annexin A2 has been linked to plasminogen-dependent matrix in- inflammatory responses without affecting tissue-type PA-mediated Downloaded from vasion by monocytes (46, 47). Annexin A2, a member of a family plasmin activity and its beneficial effects on clot lysis. of proteins that bind anionic phospholipids in a Ca2ϩ-dependent In summary, uPA increases MMP-1 production by LPS-acti- manner, exists in three forms, a monomer (p36), a heterodimer, or vated monocytes through a series of events that include the gen- a heterotetramer. The heterotetramer is comprised of two annexin eration of plasmin, which in turn signals through the components A2 monomers and two S100A10 (p11) monomers. Our data show of the annexin A2 heterotetramer, resulting in the stimulation of that addition of Abs against annexin A2 and S100A10 to activated the COX-2 and MAPK pathways. These findings demonstrate http://www.jimmunol.org/ monocytes in the absence or presence of plasmin inhibited MMP-1 that the urokinase plasminogen activation system plays a major production. 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