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:
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]. 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.,Plebanski,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
Ó FEBS 2003 Proteoglycans and urokinase (Eur. J. Biochem. 270) 3979
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. 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