Báo cáo khoa học: Secretion of macrophage urokinase plasminogen activator is dependent on proteoglycans potx

Secretion of macrophage urokinase plasminogen activator is dependent on proteoglycans Gunnar Pejler 1 , Jan-Olof Winberg 2 , Tram T. Vuong 3 , Frida Henningsson 1 , Lars Uhlin-Hansen 2 , Koji Kimata 4 and Svein O. Kolset 5 1 Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden; 2 Department of Biochemistry, Institute of Medical Biology, University of Tromsø, Norway; 3 Department of Biochemistry, University of Oslo, Norway; 4 Institute for Molecular Science of Medicine, Aichi Medical University, Japan; 5 Institute for Nutrition Research, University of Oslo, Norway The importance of proteoglycans for secretion of proteolytic enzymes was studied in the murine macrophage cell line J774. Untreated or 4b-phorbol 12-myristate 13-acetate (PMA)-stimulated macrophages were treated with hexyl-b- D -thioxyloside to interfere with the attachment of glycosaminoglycan chains to their respective protein cores. Activation of the J774 macrophages with PMA resulted in increased secretion of trypsin-like serine proteinase activity. This activity was completely inhibited by plasminogen acti- vator inhibitor 1 and by amiloride, identifying the activity as urokinase plasminogen activator (uPA). Treatment of both the unstimulated or PMA-stimulated macrophages with xyloside resulted in decreased uPA activity and Western blotting analysis revealed an almost complete absence of secreted uPA protein after xyloside treatment of either control- or PMA-treated cells. Zymography analyses with gels containing both gelatin and plasminogen confirmed these findings. The xyloside treatment did not reduce the mRNA levels for uPA, indicating that the effect was at the post-translational level. Treatment of the macrophages with xylosides did also reduce the levels of secreted matrix met- alloproteinase 9. Taken together, these findings indicate a role for proteoglycans in the secretion of uPA and MMP-9. Keywords: proteoglycan; xyloside; matrix metalloprotein- ase; urokinase; secretion. The capacity to secrete various compounds is an important property of cells in the monocytoid–macrophage lineage, in addition to the phagocytic and antigen presenting functions [1]. The secretory repertoire includes such molecules as tumor necrosis factor-a, lipoprotein lipase, proteoglycans, leukotrienes, and various proteases [2]. The proteoglycans expressed by monocytes and macrophages have been characterized to some extent. The major product seems to be serglycin, as shown by N-terminal sequencing of proteoglycans released from the cultured monocytic cell lines U937 and THP-1 [2,3]. Moreover, it has been shown that activated murine and human macrophages express syndecan-4 [4] and syndecan-2 [5], respectively, on the cell surface. The release of serglycin from monocytes and macro- phages is the subject of regulation by inflammatory signaling molecules such as interferon-c, transforming growth factor-b, and platelet derived growth factor [2,6]. It is therefore likely that the secretion of proteoglycans in these cells is linked to inflammatory reactions and that its function(s) may be linked to the binding, transport and regulation of other secretory products. Indeed, recent data indicate that mice lacking functional heparin chains attached to their serglycin proteoglycans show severe defects in their capacities to store mast cell proteases in the secretory granules [7,8], clearly demonstrating the importance of intact proteoglycans for normal storage of proteases in these cells. Serglycin proteoglycans have also been implicated in the regulation of mast cell protease activities [9–11]. The biological functions of proteoglycans from activated monocytes and macrophages have not been outlined in any detail. It has however, been shown that serglycin may be associated with chemokines and enzymes after release from the cells [12]. It has furthermore been demonstrated that serglycin may interact with CD44 [13], and possibly engage in cell interactions between immune cells. Considering that serglycin proteoglycans are of critical importance for the secretory granule proteases in mast cells it is reasonable to assume that serglycin proteoglycans may also affect proteases in other cell types. In the present study we have investigated the possible role of proteoglycans in the secretion of proteolytic enzymes by macrophages. For this purpose we made use of b- D -xylosides. These com- pounds have been widely used to study proteoglycan biosynthesis and the role of proteoglycans in different biological processes. b- D -Xylosides will compete with endogenous core protein for access to the glycosaminogly- can biosynthesis machinery [14], resulting in the biosynthesis Correspondence to S.O. Kolset, Institute for Nutrition Research, University of Oslo, Box 1046 Blindern, 0316 Oslo, Norway. Fax: + 47 2285 1398, Tel.: + 47 2285 1383, E-mail: s.o.kolset@basalmed.uio.no Abbreviations: C-ABC, chondroitinase ABC; MMP, matrix metallo- proteinase; HX-xyl, hexyl-b- D -thioxyloside; PMA, 4b-phorbol 12-myristate 13-acetate; uPA, urokinase plasminogen activator; SBTI, soy bean trypsin inhibitor; DMEM, Dulbecco’s modified Eagles medium; PAI-1, plasminogen activator inhibitor 1; tPA/uPA, tissue type/urokinase type plasminogen activators. Enzymes: chondroitinase ABC (EC 4.2.2.4) (Received 17 June 2003, accepted 7 August 2003) Eur. J. Biochem. 270, 3971–3980 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03785.x of free glycosaminoglycan chains attached to the b- D - xyloside rather than intact proteoglycans. Depending on the concentration of xylosides used, endogenous proteoglycan expression may be completely abrogated. b- D -Xylosides seem to be more efficient in abrogating the expression of chondroitin sulfate proteoglycans than heparan sulfate proteoglycans. Results presented here show that the treat- ment of macrophages with b- D -xylosides results in impaired secretion of urokinase plasminogen activatior (uPA), indi- cating that uPA is dependent on proteoglycans. The secretion of matrix metalloproteinase 9 (MMP-9) was also decreased by the xyloside treatment. Materials and methods Materials Sephadex G50 Fine and Superose 6 were from Amer- sham Pharmacia, Uppsala, Sweden. [ 35 S]Sodium sulfate was obtained from Amersham. The chromogenic peptide substrates S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide), S-2444 (pyroGlu-Gly-Arg-p-nitroanilide), S-2390 (H- D -Val-Phe- Lys-p-nitroanilide) and S-2586 (MeO-Suc-Arg-Pro-Tyr-p- nitroanilide) were from Chromogenix, Mo ¨ lndal, Sweden. S-2288 is a general substrate for trypsin-like serine proteinases, whereas S-2444 and S-2390 are relatively specific substrates for plasminogen activators and plas- min, respectively. S-2586 is a substrate for chymotrypsin- like serine proteinases. Hexyl-b- D -thioxyloside (HX-xyl) was used as described previously. This particular xyloside was shown to be one of the most efficient abrogators of proteoglycan biosynthesis in comparison with other xylosides [14,15]. Chondroitinase ABC (C-ABC, EC 4.2.2.4) was bought from Seikagaku Kogyo Co., Tokyo, Japan. Amiloride, soy bean trypsin inhibitor (SBTI), phenylmethanesulfonyl fluoride and gelatin were obtained from Sigma Chemical Co. Plasminogen, human plasminogen activator inhibitor 1 (PAI-1), a 1 -anti-chymo- trypsin, a 1 -protease inhibitor were from Calbiochem- Novabiochem. Cells The murine macrophage cell line, J774 A1 (hereafter called J774), was from the American Type Culture Collection, Rockville, MD, USA. The cells were routinely kept in Dulbecco’s modified Eagles medium (DMEM) with 2 m M L -glutamine and gentamycin (0.1 mgÆmL )1 ), all from Bio Whittaker, Verviers, Belgium. The medium was fortified with 10% fetal bovine serum from Sigma Chemical Co. The human histiocytic lymphoma cell line U937 clone 1 (U937-1) was cultured in RPMI medium with 10% fetal bovine serum, 2 m ML -glutamine and gentamycin (0.1 mgÆmL )1 ), all from Bio Whittaker. Enzyme assays J774 cells were established in medium with serum in 16 mm wells at cell densities between 0.5 and 1.0 · 10 6 cells per well, or in 96-well plates at densities of approximately 1.5 · 10 5 cells per well. After reaching confluency, J774 cells were washed three times in medium without supple- ments to remove serum proteins. The cells were thereafter cultured in the serum-free medium QBSF 51 (Sigma). Cells were incubated with or without 50 ngÆmL )1 of PMA in the absence or presence of 0.1–2.0 m M HX-xyl. No difference in cell numbers could be measured after the different treatments by cell counting after 24 h incubation in serum free media. Maximum effect on the abrogation of proteo- glycan biosynthesis was observed at the 2 m M concentra- tion. This concentration was used in studies on enzyme secretion. After 20 h the conditioned media were harvested, centrifuged to remove nonadherent cells and frozen before further analyses. Media to be used for zymography analyses were frozen after adding Hepes buffer pH 7.4 and CaCl 2 to final concentrations of 0.1 M and 10 m M , respectively. Trypsin-like activities were measured in the recovered conditioned media. 50–100 lL conditioned medium was added to wells of 96-well microtiter plates followed by the addition of 100–150 lLofNaCl/P i (200 lL final volume) and 20 lL of either substrate S-2288 or S-2444, dissolved in distilled water at stock concentrations of 20 m M .The enzyme activities were recorded by reading the absorbance at 405 nm at different time points using a Titertek Multiscan spectrophotometer (Flow Laboratories, Irvine, Scotland). The increase in absorbance showed linear kinetics over a time period of 5 h, indicating that the enzyme was stable for at least this period of time in solution. For inhibition studies, 50 lL of conditioned medium was mixedwith150 lLofNaCl/P i in 96-well plates. Next, either of the following protease inhibitors was added at a final concentration of 0.2 l M :PAI-1,a 1 -anti-chymotrypsin, a 1 -protease inhibitor or soybean trypsin inhibitor. The effect of phenylmethanesulfonyl fluoride at a final con- centration of 1 m M was also tested. After 30 min of incubation, 20 lL of S-2288 (20 m M in H 2 O) was added followed by monitoring of residual trypsin-like activity. The effect of amiloride was tested in a similar fashion. 50 lL of conditioned medium was mixed with 150 lLof NaCl/P i and with amiloride at 0.001–10 m M final con- centration (amiloride was diluted from a 100-m M stock solution in dimethylsulfoxide). Residual activity towards S-2288 was determined after 30 min. Enzymatic determinations were performed in triplicates. Results shown represent the mean ± SD. Zymography SDS/PAGE was performed as described previously [16]. Gels (7.5 cm · 8.5 cm · 0.75 mm) contained 0.1% (w/v) gelatin in both the stacking and the separating gel, which contained 4 and 7.5% (w/v) of polyacrylamide, respectively. In some cases, the separating gel also contained plasmino- gen [16] (10 lgÆmL )1 ) in addition to gelatin that allowed the detection of plasminogen activators [17]. Serum-free med- ium from the monocytic cell line THP-1 was used as a standard because it contains proMMP-9 monomer, giving risetoamainbandat92kDaandtheproMMP-9 homodimer (a minor band at 225 kDa) [16]. In addition, serum-free conditioned medium from normal human skin fibroblasts [18] was used as a source for pro-MMP-2 standard (72 kDa). Ten microlitres of conditioned medium 3972 G. Pejler et al.(Eur. J. Biochem. 270) Ó FEBS 2003 was mixed with 3 lL of loading buffer (333 m M Tris/HCl, pH 6.8, 11% SDS, 0.03% bromophenol blue and 50% glycerol). Six microlitres of this nonheated mixture was applied to the gel, which was run at 20 mA/gel at 4 °C. Thereafter, the gel was washed twice in 50 mL 2.5% (v/v) Triton X-100, and then incubated in 50 mL of assay buffer (50 m M Tris/HCl, pH 7.5, 5 m M CaCl 2 ,0.2 M NaCl and 0.02% Brij-35) for approximately 20 h at 37 °C. In some cases 10 m M of EDTA was added to both the washing and assay buffers to block potential metallo- proteinase activity, but not serine proteinase activity. In other cases samples were incubated with 10 m M of pefabloc (a serine proteinase inhibitor) for 60 min at room temperature. Thereafter the samples were treated as described above. Gels were stained with 0.2% Coomassie Brilliant Blue R-250 (30% methanol) and destained in a solution containing 30% methanol and 10% acetic acid. Gelatinase activity was evident as cleared (unstained) regions. The area of the cleared zones and M r determin- ation of unknown bands was analyzed with the GELBASE / GELBLOT TM PRO computer program from Ultra Violet Products (Cambridge, UK). In some cases, the serum-free conditioned medium from J774 cells was incubated with either 0.1 M Hepes buffer or 24 lgÆmL )1 of trypsin for 15 min at 37 °Cpriorto electrophoresis. Trypsin was thereafter inactivated by the addition of 7 mgÆmL )1 of SBTI. In these experiments, 0.2% of SBTI was also incorporated in both the stacking and separating gels to prevent degradation of the incor- porated gelatin substrate by trace amounts of trypsin that may escape from the inhibitor complex during electro- phoresis. Western blotting Media (5 mL) from nontreated cells (control) and cells treated with PMA and xyloside, respectively, were concen- trated 10 times on Millipore ultrafree-15, NMWL 10 000 (Biomax-10) centrifugal filter device. The concentrated samples were mixed with SDS/PAGE sample buffer, without 2-mercaptoethanol. Cells (1 · 10 6 ) were solubilized by adding 100 lL of SDS/PAGE sample buffer followed by boiling for 3 min. Samples (40 lL) from medium- or cell fractions were subjected to SDS/PAGE on 12% polyacryl- amide gels under reducing conditions. Proteins were subse- quently blotted onto nitrocellulose membranes, followed by blocking with 5% milk powder in NaCl/P i for 1 h at 20 °C. Next, the membranes were incubated with antiserum (1 : 200) in 5% milk powder/Tris/NaCl/P i /0.1% Tween 20, at 4 °C for 20 h. The rabbit anti-(mouse urokinase) Ig was a kind gift from K. Danø, Rigshospitalet, Copenhagen, University Hospital, Denmark. After extensive washing with Tris/NaCl/P i /0.1% Tween 20, the membranes were incubated with secondary Ig conjugated to horseradish peroxidase (Amersham Pharmacia Biotech; 1 : 3000 dilu- tion in TBS/0.1% Tween 20). After 45 min of incubation at 20 °C, the membranes were again washed extensively with Tris/NaCl/P i /0.1% Tween 20, followed by washing with Tris/NaCl/P i without detergent. The membranes were developed with the ECL system (Amersham Pharmacia Biotech) according to the protocol provided by the manu- facturer. Transmission electron microscopy Cells were fixed in 2% glutaraldehyde, incubated in 1% OsO 4 /NaCl/P i , dehydrated and embedded in TAAB-B12 resin. Sections were analyzed at 60 kV in a Philips CM10 microscope and photographed. Isolation of RNA and Northern blotting J774 cells were lysed with Trizol and RNA was extracted with chloroform and precipitated in isopropanol. mRNA was isolated from the precipitate using Dynabeads with oligo dT 25 magnetic beads (Dynal, Oslo Norway), and separated on 1% agarose gels containing formaldehyde and blotted to Hybond N nylon membranes (Amersham Pharmacia Biotech). After prehybridization the blots were hybridizedin0.5 M sodium phosphate buffer with 7% SDS and 1 m M EDTA and 32 P-labelled probes at 65 °C for 16 h. The blots were washed three times at 65 °Cwith40m M sodium phosphate containing 1% SDS, sealed and exposed to phosphorimage screen over night. The obtained screens were analyzed in a phosphorimager (Molecular Dynamics, Amersham Pharmacia Biotech). Probe for murine urokin- ase was a kind gift from L. Hellman, Uppsala University. A probe for the housekeeping gene, 36B4, obtained from H. Nebb, University of Oslo, was used to compare mRNA levels in different samples. Proteoglycan expression To analyze the effects of PMA and HX-xyl treatment on the expression of proteoglycans, J774 cells were labelled with [ 35 S]sodium sulfate for 24 h. PMA and HX-xyl were present only during the labeling period. The media were harvested and loose cells pelleted by centrifugation. The cell fractions were recovered by adding 0.05 M Tris/HCl, pH 8.0 with 0.15 M NaCland1%TritonX-100.Bothmediumandcell fractions were subjected to Sephadex G50 Fine gel chro- matography to remove free [ 35 S]sulfate. The chromatograhy was performed in 0.05 M Tris/HCl, pH 8.0 with 0.15 M NaCl and 0.1% Triton X-100. Material eluting in the void volume was frozen before further analyses. Both medium and cell fractions were analysed by gel chromatography using a Superose 6 column (Pharmacia). Fractions of 1 mL were collected and analysed for content of radioactivity by scintillation counting using a Wallac TriCarb scintillation counter. [ 35 S]Sodium sulfate samples were subjected to chondroitinase ABC treatment to depolymerize chondro- itin sulfate and deaminative cleavage using HNO 2 to degrade heparan sulfate, as previously described [19]. Results Xyloside and proteoglycan expression To analyze the possible importance of proteoglycan expression for the secretion of proteolytic enzyme activities in activated macrophages, J774 cells were treated with HX- xylorPMAaloneorwithPMAandHX-xylincombina- tion. As can be seen in Table 1, PMA treatment resulted in a 50–80% increase in total proteoglycan synthesis. Further, treatment of the cells with HX-xyl, both in the presence or Ó FEBS 2003 Proteoglycans and urokinase (Eur. J. Biochem. 270) 3973 absence of PMA, resulted in a marked ( threefold) increase in the synthesis of 35 S-labelled macromolecules (Table 1). After HX-xyl treatment, the major part of the 35 S-labelled macromolecules expressed was recovered in the culture medium, regardless if PMA was present or not (Table 1). In contrast, control cells and cells treated with PMA retained a major portion of the 35 S-labelled macro- molecules in the cell fraction (Table 1). 35 S-labelled macro- molecules recovered from the medium fractions were analyzed by gel chromatography to discriminate between intact proteoglycans and free glycosaminoglycan chains. Further, samples were analyzed both before and after treatment with alkali (NaOH), a treatment that is known to release glycosaminoglycans from their respective protein cores. In agreement with a previous study [14], treatment with HX-xyl resulted in a shift from synthesis of predomi- nantly intact proteoglycans to an almost exclusive synthesis of free glycosaminoglycan chains (Fig. 1). Note the com- plete shift in elution pattern after alkali treatment in the upper and third panel, showing that the 35 S-labelled macromolecules released from control and PMA-treated cells are almost exclusively in proteoglycan form. Note also that the 35 S-labelled macromolecules in the panels corres- ponding to HX-xyl-treated cells are resistant to alkali treatment, demonstrating the predominance of free glycos- aminoglycan chains. Control- and PMA-treated cells secreted proteoglycans of both chondroitin sulfate and heparan sulfate type, as shown by the partial susceptibility of the secreted 35 S-labelled macromolecules to either chondroitinase ABC or deamin- ative cleavage (HNO 2 ), respectively (first and third panel). In contrast, cells subjected to HX-xyl treatment, in the presence or absence of PMA, secreted predominantly free chondroitin sulfate chains. This was demonstrated by the depolymerization of most of the medium 35 S-labelled macromolecules after treatment with chondroitinase ABC (Fig. 1; panels two and four). However, small amounts of HSPGs can also found in the medium of these cultures. Both heparan and chondroitin sulfate proteoglycans could be detected in the cell fractions of control- and PMA- treated cells, as well as in cells treated with HX-xyl or PMA/ HX-xyl. When these fractions were analyzed by gel chromatography, they displayed almost identical elution profiles (results not shown), irrespective of treatment. The ratio between heparan sulfate and chondroitin sulfate in the cell fractions was therefore not affected by the xyloside treatment. The shift from chondroitin sulfate/heparan sulfate proteoglycans to mostly free chondroitin sulfate chains is, accordingly, only seen in the medium fractions after HX-xyl or PMA/HX-xyl treatment. Xyloside and serine proteinases Conditioned medium collected after 20 h incubation under serum-free conditions did not contain any chymo- trypsin-like activity, as no cleavage of the chromogenic Table 1. [ 35 S]-labelled macromolecules recovered from medium and cell fractions of J774 cells. Cells were labelled with [ 35 S]sodium sulfate for 20hwiththeindicatedtreatments.[ 35 S]-Labelled macromolecules were recovered from cell and medium fractions and the amount determined by scintillation counting. The results presented are the mean values ± SD of three separate measurements. Total incorpor- ated [ 35 S]-radioactivity is from one experiment. Four separate experi- ments showed the same trend. Treatment Percentage of [ 35 S]- labelled macromolecules Total incorporated [ 35 S]-radioactivity (c.p.m.) Cell fraction Medium fraction Control 65 ± 5 35 ± 3 265 000 PMA 60 ± 19 40 ± 6 331 000 HX-xyl 24 ± 5 76 ± 3 723 000 PMA + HX-xyl 20 ± 1 80 ± 18 748 000 Fig. 1. Superose 6 gel chromatography of medium fractions. 35 S-La- belled macromolecules recovered from medium fractions of control cells (Control), HX-xyl-treated cells (HX-xyl), PMA-treated cells (PMA) and cells treated with PMA and HX-xyl (PMA + HX-xyl) were subjected to Superose 6 gel chromatography. Aliquots were also subjected to deaminative cleavage (HNO 2 ) to degrade heparan sulfate, chondroitinase ABC treatment to depolymerize chondroitin/dermatan sulfate or alkali treatment to release free GAG chains and also ana- lyzed by gel chromatography. Equal amounts of radioactivity were taken from the different fractions for analyses by gel chromatography. 3974 G. Pejler et al.(Eur. J. Biochem. 270) Ó FEBS 2003 chymotrypsin substrate S-2586 was observed (result not shown). Considerable activity, however, could be detected when the chromogenic substrate S-2288 was used. This substrate is cleaved by enzymes with trypsin-like substrate specificities. From Fig. 2 it is evident that the secretion of trypsin-like activity was increased approximately twofold when the cells were treated with PMA. When proteoglycan expression was compromised by treatment with HX-xyl, the levels of trypsin-like activities recovered in the conditioned media were reduced both in untreated and in PMA-stimulated cells by  50%. The effects of xyloside varied somewhat between different experiments using different cell batches. In some experi- ments the HX-xyl treatment reduced the secretion of trypsin-like activities to an even larger extent, both in control and PMA-stimulated cells (not shown). The reduc- tion in trypsin-like activity in the medium upon HX-xyl treatment was most pronounced after extended periods of incubation. However, time course studies revealed a clearly noticeable effect already 1 h after the addition of HX-Xyl, with a gradually increased effect up to 20 h of incubation (not shown). Furthermore, in experiments with the human monocytic cell line U937 the presence of trypsin-like activity in supernatants from serum-free cultures could also be demonstrated. The activity was stimulated more than twofold with PMA and was inhibited to a large extent with HX-xyl (not shown). Hence, secretion of trypsin-like pro- teases seems to depend on proteoglycans in both murine J774 macrophage-like cells and in human monocytic U937 cells. Macrophages secrete a wide range of enzymes active at neutral pH, many of which are serine proteinases [1]. However, one prominent serine proteinase in the monocyte/ macrophage system is plasminogen activator (PA). The chromogenic substrate, S-2444, (pyrGlu-Gly-Arg-pNA) is considered to be a relatively specific PA substrate. From Fig. 2 it is apparent that the conditioned media from the J774 cells contained S-2444-cleaving activity, and that the activity towards S-2444 was higher than the activity against S-2288. Further, the S-2444-cleaving activity was stimulated to the same extent by PMA as was the activity towards S-2288, and HX-xyl caused similar inhibitory effects on secretion of S-2444-hydrolyzing activity as was observed for the secretion of activity towards S-2288. These results are thus compatible with the possibility that the cleavage of S-2288 and S-2444 are carried out by the same enzyme activity, and that this activity may be related to plasminogen activator. To characterize the activity further, conditioned media were incubated with various protease inhibitors followed by the measurement of residual trypsin-like activity. The S-2288-hydrolyzing activity, both from control and PMA-stimulated cells, was completely inhibited by phenylmethanesulfonyl fluoride, demonstrating that it was a serine proteinase. Further, the activity was completely inhibited by plasminogen activator inhibitor 1 (PAI-1), but not to any significant extent by neither a 1 -protease inhibitor, a 1 -anti-chymotrypsin nor soybean trypsin inhibitor (Fig. 3). This pattern of inhibition was seen in conditioned media both from control- and PMA-stimulated cells. To verify that the murine macrophage cell line J774 produced plasminogen activators, cell conditioned serum- free medium was subjected to substrate zymography [17]. As shown in Fig. 4 (left panel), a band at approximately 24 kDa was detected in the gel that contained both plasminogen and gelatin, but not in the control gel that contained only gelatin. This indicates that this band is a plasminogen activator. The figure also shows that the presence of PMA resulted in a slight increase in the intensity of this plasminogen activator band, which was verified in other experiments with diluted conditioned medium (data not shown). Figure 4 (left panel) also shows that HX-xyl treatment of the cells resulted in a reduction in the intensity of the plasminogen activator band. This band was also drastically reduced in conditioned medium (control as well as PMA- and Fig. 2. Trypsin-like activities in conditioned media from J774 macro- phages. Equal number of J774 macrophages were incubated with PMA, HX-xyl or both. Conditioned media were harvested and the levels of trypsin-like activities were assayed using the chromogenic substrates, S-2288 or S-2444 (see Materials and methods). Fig. 3. The effect of protease inhibitors on plasminogen activator activity in supernatants from J774 cells. Conditioned media from equal number of untreated and PMA-treated J774 macrophages were incu- bated for 30 min with 0.2 l M of the various macromolecular protease inhibitors, or 1 m M of phenylmethanesulfonyl fluoride, followed by determination of residual trypsin-like activities. Ó FEBS 2003 Proteoglycans and urokinase (Eur. J. Biochem. 270) 3975 HX-xyl-treated) that had been treated with the serine proteinase inhibitor Pefabloc prior to electrophoresis (data not shown). Furthermore, presence of EDTA in the washing and assay buffers had no effect on the intensity of the band (data not shown). Taken together, these data demonstrate that the 24 kDa plasminogen activator band is a serine proteinase. Plasminogen activators may either be of the tissue type (tPA) or urokinase type (uPA). To distinguish between these two types it is possible to use amiloride, which is known to inhibit only the urokinase type [20]. As can be seen in Fig. 5, the enzyme activity in both supernatants was completely inhibited by amiloride, suggesting that most, if not all, of the trypsin-like activity secreted both by control and PMA-activated J774 macrophages is due to uPA. Conditioned media from control and xyloside-treated cells were therefore subjected to Western blotting using an antimurine uPA antibody. As can be seen in Fig. 6, uPA antigen was readily detected in conditioned medium both from control- and PMA-treated cells. Strikingly, in medium from cells incubated with either HX-xyl alone, or with PMA together with HX-xyl, the uPA band was nearly undetectable. The M r of the uPA detected by Western blotting is approximately twice as large as that detected by substrate zymography. The 24 kDa form seen in zymography is most likely the low M r form of uPA consisting of only the active site serine proteinase (SP)-module as described previously [21], while the antibody used in the Western blots only recognized the N-terminal part of uPA. The lack of a band at around 48 kDa in the substrate zymography gel (Fig. 4) indicates that the 48 kDa band seen in the Western blot is the inactive proform of uPA. It is possible that the effect of HX-xyl could be mediated through increased secretion of PAI-1. A decreased activity of uPA due to complex formation with PAI-1 should be evident through formation of a covalent complex with high Fig. 4. Zymographic detection of plasminogen activators and matrix metalloproteinases in supernatants from J774 cells. Supernatants from J774 cells were subjected to SDS/PAGE using gels containing both gelatin and plasminogen (left panel) or only gelatin (right panel). The cells had been treated as described in the legend to Fig. 2 prior to harvesting of the medium. After electrophoresis, the gels were treated as described in Materials and methods. Standard 1 is conditioned medium from human skin fibroblasts, secreting MMP-2 (72 kDa). Standard 2 is conditioned medium from the human monocytic cell line THP-1 containing MMP-9 (92 kDa) and uPA (34 kDa). In some gels, trypsinwasalsousedastandardinadditiontostandard1and2to estimate the M r of uPA in the conditioned media from J774 cells. Fig. 5. The effect of amiloride on plasminogen activator activity in supernatants from J774 cells. Conditioned media from untreated and PMA-treated J774 cells were incubated with increasing concentrations of amiloride for 30 min. Residual trypsin-like activity was measured using the chromogenic substrate S-2288. Fig. 6. Western blotting for urokinase in J774 cells. Conditioned from J774 cells incubated for 20 h with PMA, HX-xyl or both was subjected to SDS/PAGE followed by Western blotting using an antibody against murine urokinase. 3976 G. Pejler et al.(Eur. J. Biochem. 270) Ó FEBS 2003 molecular weight. However, no such complexes could be seen after Western blotting (Fig. 6). Cell fractions were also analyzed by Western blotting. In contrast to the medium fractions, no uPA antigen was detected in any of the four cell fractions analyzed (Result not shown). Furthermore, mRNA was isolated from cells treated with HX-xyl or PMA. As shown in Fig. 7, the levels of mRNA for uPA were not reduced by treatment with xyloside. Xyloside and matrix metalloproteinases The substrate zymography in Fig. 4 revealed that in addition to the uPA band at 24 kDa, the conditioned medium from the J774 cells contained two additional bands. These bands had M r of approximately 250–300 kDa and 112 kDa and were not plasminogen activators, as they were found in both the control gel containing only gelatin as well as in the gel with plasminogen and gelatin. These bands did not appear in gels that were washed and incubated in the presence of EDTA, while the intensity of the bands in harvested media treated with the serine proteinase inhibitor pefabloc prior to electrophoresis was similar to the bands in the untreated control media (data not shown). This indicates that these bands are metalloproteinases, and most likely the dimeric and monomeric forms of metalloproteinase 9 (MMP-9), as macrophages have previously been shown to produce this enzyme [16,22]. Treatment of the conditioned medium with trypsin prior to electrophoresis gave a new bandwithanapproximateM r of 106 kDa (data not shown). This suggests that the metalloproteinase in the J774 medium is most likely the proform of the gelatinase. In the medium from PMA-treated cells, the two MMP bands appeared somewhat stronger compared to the MMP bands in the medium from the control cells (Fig. 4). However, in the media from the HX-xyl-treated cells these two bands were drastically reduced compared to the controls (Fig. 4). Thus, the secretion of metalloproteinases is also affected by HX-xyl treatment. Transmission electron microscopy To investigate if HX-xyl treatment of J774 cells would affect the formation and organization of intracellular granules, cells were subjected to transmission electron microscopy (TEM). From Fig. 8 panel A it is obvious that no striking effects, on neither the number nor the morphology of intracellular vesicles or granules, could be observed in cells Fig. 7. Northern blotting for urokinase in J774 cells. mRNA was iso- lated from cells incubated 20 h with PMA, HX-xyl or both, separated by agarose gel electrophoresis, blotted and hybridized with probes for murine urokinase (upper panel) and the housekeeping gene 36B4. The intensity of the signal for the urokinase measured in a Phosphoimager was related to that of the housekeeping gene. The ratio between the two is given in the lower panel. Fig. 8. Transmission electron microscopy. J774 cells were cultured in the absence and presence of HX-xyl. Both adherent and nonadherent cells were fixed and processed for transmission electron microscopy (A). Magnification is · 2950. B shows more cells (nonadherent) with magnifica- tion · 1200. Ó FEBS 2003 Proteoglycans and urokinase (Eur. J. Biochem. 270) 3977 treatedwithHX-xyl.InpanelBmorecellsareshownata smaller magnification. Discussion In the present paper we show that proteoglycans are important for secreted uPA activity in J774 macrophages. uPA activity has previously been demonstrated in several macrophage cell lines [23] and in human macrophages [22]. Mice lacking uPA expression are not able to recruit sufficient number of macrophages during inflammation [24], suggesting that the enzyme is important in the cellular immune system. Indeed, uPA activity was increased in the medium after PMA treatment, in agreement with the notion that uPA secretion is a characteristic feature of activated macrophages [25]. Additionally, secretion of proteoglycans in monocytes and macrophages increases when the cells are activated [6], as was also apparent in this study. Accord- ingly, secretion of both uPA and proteoglycans increase in activated monocytes and macrophages. Plasmin, generated from the precursor plasminogen through the action of uPA, can cleave matrix proteins such as fibronectin, laminin and aggrecan, and also activate matrix- and membrane associ- ated MMPs, fibroblast growth factor and transforming growth factor b [26]. In atherosclerosis, lipid-rich macro- phages increase uPA and plasmin expression and the release of growth factors from the extracellular matrix [27]. Clearly, the regulation of plasmin formation is important for macrophages and metastasizing tumor cells, and cells involved in tissue repair. Likewise, secretion of MMP-9 from macrophages is important in immune reactions and atherosclerosis [28]. The results presented here thus indicate that proteoglycans secreted from macrophages, e.g. sergly- cin, may regulate the activity or availability of uPA and MMP-9. However, HX-xyl treatment does not lead to a complete inhibition of uPA release from the cells, despite an essentially total abrogation of the synthesis of intact proteoglycans. The reason for this is not known. However, it is possible that preformed uPA and intact proteoglycans are present in the cells and are being released during the course of the experiments. Alternatively, uPA secretion may be only partly dependent on the intact proteoglycans. Control and PMA-stimulated J774 macrophages release proteoglycans of both chondroitin sulfate and heparan sulfate type. In the present study we show that xyloside treatment of both control and PMA-stimulated J774 cells completely blocks the assembly of intact heparan sulfate and chondroitin sulfate proteoglycans that are destined for secretion. Which of the two proteoglycans, heparan sulfate or chondroitin sulfate that is important for the uPA activity/ secretion is at present not known. Importantly, we did not see any reduction in mRNA levels for uPA upon xyloside treatment, indicating that the inhibitory effect of xylosides on extracellular uPA was caused by post-translational mechanisms. However, we do not know at which level uPA is dependent on proteoglycans. One possibility is that uPA is dependent on proteoglycans after release from the cells where the lack of intact proteoglycans may affect the activity or half-life of uPA. It is conceivable that uPA or MMP-9 released to the medium in the J774 system might be inactivated either by other proteases or by protease inhibitors, if no proteoglycans are simultaneously secreted to the medium. In this context it is interesting to note that heparan sulfate has been shown to both protect plasmin from inactivation by protease inhibitors and to stimulate its enzyme activity [29]. In addition, recent findings show that the interaction between serglycin and granzymes in cyto- toxic granules is important to mediate apoptosis in target cells [30]. Granzymes have also been shown to circulate in plasma bound to proteoglycans, whereby they are protected from inactivation by protease inhibitors [31]. Accordingly, based on the findings presented here, one possible function of secreted proteoglycans in macrophages may be to protect and regulate the activity of uPA and MMP-9 expressed and secreted by the same cells. A second possibility could be that the proteoglycans may be important intracellularly in the formation of the secretory vesicles. Each of these two possibilities implies that the protein core of the proteogly- can, or the intact proteoglycan molecule, is an important component of the secretory process, as the xyloside treatment did not reduce the amount of secreted glycos- aminoglycan chains available. The mechanism by which the protein core could influence the secretion of proteolytic enzymes is uncertain. It is possible, for example, that the protein core in some way is involved in intracellular sorting of uPA and MMP-9. Another possibility could be that the protein core is attached to the vesicle membrane, and that such a linkage may be important for formation or structural integrity of the secretory vesicles. In this context it is noted that proteoglycans, possibly GPI-linked to the granule membrane, are important for the formation of zymogen granules in pancreatic acinar cells [32]. Further, proteogly- cans have been suggested to be important for the intracel- lular transport of enzymes to the lysosomes in monocytes [33]. A third possibility would be that the cell-surface proteoglycans participate in the regulation of uPA. HX-xyl- treated cells have reduced levels of cell surface-associated proteoglycans compared to control macrophages. Possibly, this may affect the cell association of uPA after release and/ or the level of activity. In fact, it has been shown previously that cell association of uPA-generating activity enhances the rate of formation of active uPA [34]. An alternative explanation for the effect of the xyloside on uPA and MMP-9 secretion could be that the xyloside treatment reduces the amount of heparan sulfate chains synthesized in favor of chondroitin sulfate, and that uPA and MMP-9 may be specifically dependent on glycosami- noglycans of the heparan sulfate type. In line with such an explanation, it was recently shown that mast cell carboxy- peptidase A expressed by bone marrow-derived mast cells is strictly dependent on heparin glycosaminoglycan for stor- age and processing, whereas mast cell tryptase can be stored and processed also in cells lacking heparin but containing chondroitin sulfate of equal charge density [35]. A dependence of uPA on proteoglycans has to our knowledge not been described previously. However, it has been shown recently that serglycin and tPA colocalize in intracellular granules of endothelial cells, thus giving further support for a role of proteoglycans in the regulation of plasminogen activators [36]. The activity of uPA can be regulated through several mechanisms, including the expression levels, uPA receptor binding and regulation by PAI-1. The expression levels are the subject of regulation through the actions of growth 3978 G. Pejler et al.(Eur. J. Biochem. 270) Ó FEBS 2003 factors and inflammatory mediators [26]. Tumor-associated macrophages have, e.g. been demonstrated to increase the expression of uPA when exposed to transforming growth factor-b [37]. It has also been shown that the expression level of uPA in J774 cells can be regulated through interactions of the cells with extracellular laminin through the integrin receptor a 6 b 1 [26]. 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Hildenbrand,R.,Jansen,C.,Wolf,G.,Bohme,B.,Berger,S.,von Minckwitz, G., Horlin, A., Kaufmann, M. & Stutte, H.J. (1998) Transforming growth factor-beta stimulates urokinase expression in tumor-associated macrophages of the breast. Laboratory Invest. 78, 59–71. 3980 G. Pejler et al.(Eur. J. Biochem. 270) Ó FEBS 2003 . the notion that uPA secretion is a characteristic feature of activated macrophages [25]. Additionally, secretion of proteoglycans in monocytes and macrophages. Secretion of macrophage urokinase plasminogen activator is dependent on proteoglycans Gunnar Pejler 1 , Jan-Olof Winberg 2 , Tram T. Vuong 3 ,