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

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 sec

Secretion of macrophage urokinase plasminogen activator

is dependent on proteoglycans

Gunnar Pejler1, Jan-Olof Winberg2, Tram T Vuong3, Frida Henningsson1, Lars Uhlin-Hansen2,

Koji Kimata4and Svein O Kolset5

1

Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden;2Department of Biochemistry, Institute of Medical Biology, University of Tromsø, Norway;3Department of Biochemistry, University of Oslo, Norway;4Institute for Molecular Science of Medicine, Aichi Medical University, Japan;5Institute 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; urokinmetalloprotein-ase; 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)

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 [35S]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), a1

-anti-chymo-trypsin, a1-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 mM

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 mM L-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· 106cells

per well, or in 96-well plates at densities of approximately

1.5· 105 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)1of PMA in the absence or presence of 0.1–2.0 mMHX-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 mM 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 CaCl2 to final concentrations of 0.1M and 10 mM, 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 lL of NaCl/Pi(200 lL final volume) and 20 lL of either substrate S-2288 or S-2444, dissolved

in distilled water at stock concentrations of 20 mM 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 mixed with 150 lL of NaCl/Piin 96-well plates Next, either

of the following protease inhibitors was added at a final concentration of 0.2 lM: PAI-1, a1-anti-chymotrypsin,

a1-protease inhibitor or soybean trypsin inhibitor The effect of phenylmethanesulfonyl fluoride at a final con-centration of 1 mM was also tested After 30 min of incubation, 20 lL of S-2288 (20 mM in H2O) 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 lL of NaCl/Pi and with amiloride at 0.001–10 mM final con-centration (amiloride was diluted from a 100-mM 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 rise to a main b and at 92 kDa and the proMMP-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

was mixed with 3 lL of loading buffer (333 mMTris/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 4C

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 mM Tris/HCl, pH 7.5, 5 mM CaCl2, 0.2 M

NaCl and 0.02% Brij-35) for approximately 20 h at

37C In some cases 10 mMof EDTA was added to both

the washing and assay buffers to block potential

metallo-proteinase activity, but not serine metallo-proteinase activity In

other cases samples were incubated with 10 mM 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 Mr

determin-ation of unknown bands was analyzed with theGELBASE/

GELBLOTTM PRO computer program from Ultra Violet

Products (Cambridge, UK)

In some cases, the serum-free conditioned medium from

J774 cells was incubated with either 0.1MHepes buffer or

24 lgÆmL)1 of trypsin for 15 min at 37C prior to

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· 106) 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/Pifor 1 h at 20C

Next, the membranes were incubated with antiserum

(1 : 200) in 5% milk powder/Tris/NaCl/Pi/0.1% Tween 20,

at 4C 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/Pi/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 incubadilu-tion at

20C, the membranes were again washed extensively with

Tris/NaCl/Pi/0.1% Tween 20, followed by washing with

Tris/NaCl/Pi 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% OsO4/NaCl/Pi, 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 dT25 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 hyb ridized in 0.5Msodium phosphate buffer with 7% SDS and 1 mMEDTA and32P-labelled probes at 65C for 16 h The blots were washed three times at 65C with 40 mM 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 [35S]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 0.15MNaCl and 1% Triton X-100 Both medium and cell fractions were subjected to Sephadex G50 Fine gel chro-matography to remove free [35S]sulfate The chromatograhy was performed in 0.05M Tris/HCl, pH 8.0 with 0.15M 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 TriCarbscintillation counter [35S]Sodium sulfate samples were subjected to chondroitinase ABC treatment to depolymerize chondro-itin sulfate and deaminative cleavage using HNO2 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-xyl or PMA alone or with PMA and HX-HX-xyl in comb ina-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

absence of PMA, resulted in a marked ( threefold)

increase in the synthesis of 35S-labelled macromolecules

(Table 1) After HX-xyl treatment, the major part of the

35S-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 the35S-labelled

macro-molecules in the cell fraction (Table 1).35S-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 35S-labelled

macromolecules released from control and PMA-treated

cells are almost exclusively in proteoglycan form Note also

that the35S-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 35S-labelled

macromolecules to either chondroitinase ABC or

deamin-ative cleavage (HNO2), 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 35S-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 PMA-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 [35S]-labelled macromolecules recovered from medium and cell

fractions of J774 cells Cells were labelled with [35S]sodium sulfate for

20 h with the indicated treatments [ 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 [35S]-radioactivity is from one experiment Four separate

experi-ments showed the same trend.

Treatment

Percentage of [ 35 S]-labelled macromolecules

Total incorporated [35S]-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.

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 a1-protease inhibitor,

a1-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.

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 Mr 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 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,

trypsin was also used a standard in addition to standard 1 and 2 to

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.

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 Mr 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

b and with an approximate Mr 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.

treated with HX-xyl In panel B more cells are shown at a

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

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 a6b1[26] Data presented here suggest an additional

level of regulation of uPA, and also MMP-9, activity in

macrophages, through the dependence of cellular

proteo-glycan expression and secretion

Acknowledgements

The expert technical assistance of Eli Berg and Annicke Stranda is

acknowledged.

This work was supported by grants from The Norwegian Cancer

Society, The Throne-Holst Fund, The Swedish Medical Research

Council (grant no 9913) and from King Gustaf V’s 80th anniversary

Fund.

References

1 Nathan, C.F (1987) Secretory products of macrophages J Clin.

Invest 79, 319–326.

2 Uhlin-Hansen, L., Wik, T., Kjellen, L., Berg, E., Forsdahl, F &

Kolset, S.O (1993) Proteoglycan metabolism in normal and

inflammatory human macrophages Blood 82, 2880–2889.

3 Oynebraten, I., Hansen, B., Smedsrod, B & Uhlin-Hansen, L.

(2000) Serglycin secreted by leukocytes is efficiently eliminated

from the circulation by sinusoidal scavenger endothelial cells in the

liver J Leukoc Biol 67, 183–188.

4 Yeaman, C & Rapraeger, A.C (1993) Membrane-anchored

proteoglycans of mouse macrophages: P388D1 cells express a

syndecan-4-like heparan sulfate proteoglycan and a distinct

chondroitin sulfate form J Cell Physiol 157, 413–425.

5 Clasper, S., Vekemans, S., Fiore, M., Pleb anski, M., Wordsworth,

P., David, G & Jackson, D.G (1999) Inducible expression of the

cell surface heparan sulfate proteoglycan syndecan-2 (fibroglycan)

on human activated macrophages can regulate fibroblast growth

factor action J Biol Chem 274, 24113–24123.

6 Uhlin-Hansen, L., Eskeland, T & Kolset, S.O (1989) Modulation

of the expression of chondroitin sulfate proteoglycan in stimulated

human monocytes J Biol Chem 264, 14916–14922.

7 Forsberg, E., Pejler, G., Ringvall, M., Lunderius, C.,

Tomasini-Johansson, B., Kusche-Gullberg, M., Eriksson, I., Ledin, J.,

Hellman, L & Kjellen, L (1999) Abnormal mast cells in mice

deficient in a heparin-synthesizing enzyme Nature 400, 773–776.

8 Humphries, D.E., Wong, G.W., Friend, D.S., Gurish, M.F., Qiu,

W.T., Huang, C., Sharpe, A.H & Stevens, R.L (1999) Heparin is

essential for the storage of specific granule proteases in mast cells.

Nature 400, 769–772.

9 Pejler, G & Sadler, J.E (1999) Mechanism by which heparin

proteoglycan modulates mast cell chymase activity Biochemistry

38, 12187–12195.

10 Hallgren, J., Spillmann, D & Pejler, G (2001) Structural

requirements and mechanism for heparin-induced activation of a

recombinant mouse mast cell tryptase, mouse mast cell protease-6:

formation of active tryptase monomers in the presence of low

molecular weight heparin J Biol Chem 276, 42774–42781.

11 Tchougounova, E & Pejler, G (2001) Regulation of extravascular

coagulation and fibrinolysis by heparin-dependent mast cell

chy-mase FASEB J 15, 2763–2765.

12 Kolset, S.O., Mann, D.M., Uhlin-Hansen, L., Winberg, J.O &

Ruoslahti, E (1996) Serglycin-binding proteins in activated

macrophages and platelets J Leukoc Biol 59, 545–554.

13 Toyama-Sorimachi, N., Kitamura, F., Habuchi, H., Tobita, Y., Kimata, K & Miyasaka, M (1997) Widespread expression of chondroitin sulfate-type serglycins with CD44 binding ability in hematopoietic cells J Biol Chem 272, 26714–26719.

14 Kolset, S.O., Sakurai, K., Ivhed, I., Overvatn, A & Suzuki, S (1990) The effect of beta- D -xylosides on the proliferation and proteoglycan biosynthesis of monoblastic U-937 cells Biochem.

J 265, 637–645.

15 Halvorsen, B., Aas, U.K., Kulseth, M.A., Drevon, C.A., Christiansen, E.N & Kolset, S.O (1998) Proteoglycans in mac-rophages: characterization and possible role in the cellular uptake

of lipoproteins Biochem J 331, 743–752.

16 Winberg, J.O., Kolset, S.O., Berg, E & Uhlin-Hansen, L (2000) Macrophages secrete matrix metalloproteinase 9 covalently linked

to the core protein of chondroitin sulfate proteoglycans J Mol Biol 304, 669–680.

17 Heussen, C & Dowdle, E.B (1980) Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates Anal Biochem 102, 196–202.

18 Svendsrud, D.H., Loennechen, T & Winberg, J.O (1997) Effect

of adenosine analogues on the expression of matrix metallopro-teinases and their inhibitors from human dermal fibroblasts Biochem Pharmacol 53, 1511–1520.

19 Shively, J.E & Conrad, H.E (1976) Formation of anhydrosugars

in the chemical depolymerization of heparin Biochemistry 15, 3932–3942.

20 Vassalli, J.D & Belin, D (1987) Amiloride selectively inhibits the urokinase-type plasminogen activator FEBS Lett 214, 187–191.

21 Novokhatny, V., Medved, L., Mazar, A., Marcotte, P., Henkin, J.

& Ingham, K (1992) Domain structure and interactions of recombinant urokinase-type plasminogen activator J Biol Chem.

267, 3878–3885.

22 Shapiro, S.D., Campbell, E.J., Senior, R.M & Welgus, H.G (1991) Proteinases secreted by human mononuclear phagocytes.

J Rheumatol 27, 95–98.

23 Jones, C.M., Goldfarb, R.H & Holden, H.T (1983) Macrophage cell lines behave as activated macrophages in the production and regulation of plasminogen activator Cancer Invest 1, 207–213.

24 Carmeliet, P & Collen, D (1996) Gene manipulation and transfer

of the plasminogen and coagulation system in mice Semin Thromb Hemost 22, 525–542.

25 Vassalli, J.D & Pepper, M.S (1994) Tumour biology Membrane proteases in focus Nature 370, 14–15.

26 Khan, K.M & Falcone, D.J (1997) Role of laminin in matrix induction of macrophage urokinase-type plasminogen activator and 92-kDa metalloproteinase expression J Biol Chem 272, 8270–8275.

27 Falcone, D.J., McCaffrey, T.A., Haimovitz-Friedman, A., Vergilio, J.A & Nicholson, A.C (1993) Macrophage and foam cell release of matrix-bound growth factors Role of plasminogen activation J Biol Chem 268, 11951–11958.

28 Opdenakker, G., Van den Steen, P.E & Van Damme, J (2001) Gelatinase B: a tuner and amplifier of immune functions Trends Immunol 22, 571–579.

29 Brunner, G., Reimbold, K., Meissauer, A., Schirrmacher, V & Erkell, L.J (1998) Sulfated glycosaminoglycans enhance tumor cell invasion in vitro by stimulating plasminogen activation Exp Cell Res 239, 301–310.

30 Metkar, S.S., Wang, B., Aguilar-Santelises, M., Raja, S.M., Uhlin-Hansen, L., Podack, E., Trapani, J.A & Froelich, C.J (2002) Cytotoxic cell granule-mediated apoptosis: perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation Immunity 16, 417–428.

31 Spaeny-Dekking, E.H., Kamp, A.M., Froelich, C.J & Hack, C.E (2000) Extracellular granzyme A, complexed to proteoglycans, is

protected against inactivation by protease inhibitors Blood 95,

1465–1472.

32 Schmidt, K., Dartsch, H., Linder, D., Kern, H.F & Kleene, R.

(2000) A submembranous matrix of proteoglycans on zymogen

granule membranes is involved in granule formation in rat

pan-creatic acinar cells J Cell Sci 113, 2233–2242.

33 Lemansky, P & Hasilik, A (2001) Chondroitin sulfate is involved

in lysosomal transport of lysozyme in U937 cells J Cell Sci 114,

345–352.

34 Duval-Jobe, C & Parmely, M.J (1994) Regulation of

plasmino-gen activation by human U937 promonocytic cells J Biol Chem.

269, 21353–21357.

35 Henningsson, F., Ledin, J., Lunderius, C., Wilen, M., Hellman, L.

& Pejler, G (2002) Altered storage of proteases in mast cells from mice lacking heparin: a possible role for heparin in carboxy-peptidase A processing Biol Chem 383, 793–801.

36 Schick, B.P., Gradowski, J.F & San Antonio, J.D (2001) Synthesis, secretion, and subcellular localization of serglycin pro-teoglycan in human endothelial cells Blood 97, 449–458.

37 Hildenb rand, 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.