To investigate the possible importance of serglycin linked to protease secretion, enzyme activ-ities using chromogenic substrates and zymography were measured in cell fractions and serum
Trang 1Madin–Darby canine kidney cells
Lillian Zernichow1, Knut T Dalen1, Kristian Prydz2, Jan-Olof Winberg3and Svein O Kolset1
1 Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Norway
2 Department of Molecular Biosciences, University of Oslo, Norway
3 Department of Biochemistry, Institute of Medical Biology, University of Tromsø, Norway
Studies on proteoglycans (PGs) have, to a large
extent, focused on molecules located in the
extracellu-lar matrix and on cell surfaces [1–3], and their roles
in, for example, the regulation of cell adhesion, cell
migration, proliferation and wound healing However,
PGs located in different intracellular locations are
receiving increasing attention [4,5] In particular, PGs
in storage and secretory granules in cells of the
hae-matopoietic lineage have been the subject of several
recent studies, as, for instance, in the mast cell, where
heparin PG is stored in secretory granules together
with histamine and proteases Generation of mice
with a deleted version of the gene for the heparin-synthesizing enzyme N-deacetylase⁄
N-sulfotransferase-2 (NDST-N-sulfotransferase-2) resulted in the appearance of mast cells with large changes in secretory granule morphology and in greatly reduced levels of the proteases nor-mally confined to these granules [6,7] Heparin PG in mast cells, accordingly, seems to be of fundamental importance for the generation of storage granules Recently, serglycin knockout mice were generated [8] They developed normally and were fertile, but their mast cells were affected in a manner similar to that
of the NDST-2 knockout mice
Keywords
matrix metalloproteinase; MDCK;
plasminogen activator; proteoglycan;
serglycin
Correspondence
S O Kolset, Department of Nutrition,
Institute of Basic Medical Sciences,
University of Oslo, Box 1046 Blindern,
0316 Oslo, Norway
Fax: +47 22 851 398
Tel: +47 22 851 383
E-mail: s.o.kolset@medisin.uio.no
(Received 2 November 2005, accepted
2 December 2005)
doi:10.1111/j.1742-4658.2005.05085.x
Madin–Darby canine kidney (MDCK) cells, which do not normally express the proteoglycan (PG) serglycin, were stably transfected with cDNA for human serglycin fused to a polyhistidine tag (His-tag) Clones with differ-ent levels of serglycin mRNA expression were generated One clone with lower and one with higher serglycin mRNA expression were selected for this study 35S-labelled serglycin in cell fractions and conditioned media was isolated using HisTrap affinity chromatography Serglycin could also
be detected in conditioned media using western blotting To investigate the possible importance of serglycin linked to protease secretion, enzyme activ-ities using chromogenic substrates and zymography were measured in cell fractions and serum-free conditioned media of the different clones Cells were cultured in both the absence and presence of phorbol 12-myristate 13-acetate (PMA) In general, enzyme secretion was strongly enhanced by treatment with PMA Our analyses revealed that the clone with the highest serglycin mRNA expression, level of HisTrap isolated 35S-labelled sergly-cin, and amount of serglycin core protein as detected by western blotting, also showed the highest secretion of proteases Transfection of serglycin into MDCK cells clearly leads to changes in secretion levels of secreted endogenous proteases, and could provide further insight into the biosynthe-sis and secretion of serglycin and potential partner molecules
Abbreviations
cABC, chondroitinase ABC; CS, chondroitin sulfate; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; GAG,
glycosaminoglycan; HS, heparan sulfate; His-tag, polyhistidine tag; MDCK, Madin–Darby canine kidney; MMP, matrix metalloproteinase; NDST-2, N-deacetylase ⁄ N-sulfotransferase-2; NGAL, neutrophil gelatinase-associated lipocalin; PA, plasminogen activator; PG, proteoglycan; PMA, phorbol 12-myristate 13-acetate; PVDF, poly(vinylidene difluoride); uPA, urokinase-type plasminogen activator.
Trang 2Haematopoietic cells either harbour storage granules
or granules destined for constitutive secretion In
human monocytes and macrophages the main PG is
serglycin [9] In these cells, the PG is secreted and
acti-vation results in increased synthesis and secretion of
PGs [9,10], suggesting that secretion of PGs is linked
to inflammatory response Secreted serglycin may
potentially associate with other secreted products from
macrophages in the extracellular environment [11,12],
or team up intracellularly with such molecules during
constitutive secretion In a recent study in murine
macrophages, abrogation of PG biosynthesis with
b-d-xylosides resulted in decreased enzyme secretion [13]
In particular, the secretion of urokinase-type
plasmino-gen activator (uPA) and matrix metalloproteinase-9
(MMP-9, also referred to as 92 kDa gelatinase B and
type IV collagenase) was lowered after xyloside
treat-ment
To further study the interrelationship between
ser-glycin and proteases, both with regard to biosynthesis
and secretion, serglycin was stably transfected into
Madin–Darby canine kidney (MDCK) epithelial cells
A polyhistidine tag (His-tag) was introduced at the
C-terminus to facilitate the isolation of serglycin
Transfectants with vector without serglycin insert were
generated as negative controls It has previously been
shown that MDCK cells secrete MMP-9 [14] and uPA
[15] Secretion of these endogenous proteases was
stud-ied in the transfected cells, in both the absence and
presence of the phorbol ester phorbol 12-myristate
13-acetate (PMA), previously reported to enhance the
secretion of MMP-9 in MDCK cells [14] Results
pre-sented show that the secretion levels of proteases
cor-relate with the levels of serglycin mRNA, HisTrap
isolated 35S-labelled serglycin and serglycin core
pro-tein detected by western blotting The MDCK system
with transfected serglycin is potentially a useful model
to study PGs in relation to secretion of enzymes
important in physiological and pathological
condi-tions
Results
Transfection of serglycin
Clones of MDCK cells with serglycin–His-tag were
obtained, and the levels of serglycin mRNA
deter-mined using northern blotting Cell clones transfected
with the vector without the serglycin insert were
used as negative controls (mock transfectants) No
serglycin mRNA was detected in these clones The
serglycin mRNA expression level in the different
clones was related to the housekeeping gene 36B4
mRNA expression level One clone with a lower ser-glycin mRNA expression level (1–7), one with a higher level (1–10) and one of the mock transfectants were selected for further studies Figure 1 shows nor-thern blots of the selected clones, using 32P-labelled cDNA probes of serglycin, MMP-9, uPA and 36B4, cultured in both absence and presence of PMA The northern blot of the unstimulated cells in Fig 1A shows that the clone with the highest serglycin mRNA level also has the highest mRNA levels of uPA and MMP-9, although the levels are very low for MMP-9 PMA stimulation (Fig 1B) was shown
to lead to an upregulation of the mRNA levels of serglycin, MMP-9 and uPA
Proteoglycan analyses
35S-labelled macromolecules According to Svennevig et al [16] and Erickson & Couchman [17], PGs synthesized by MDCK cells are perlecan, agrin, collagen XVIII, biglycan, bamacan
Fig 1 mRNA expression levels of serglycin, MMP-9, uPA and 36B4 in serglycin-transfected MDCK clones Total RNA was isola-ted from MDCK clones and subjecisola-ted to northern blotting onto the same membrane and hybridized using 32 P-labelled human serglycin, canine MMP-9 and uPA and murine 36B4 cDNA probes (A) Un-stimulated clones (B) PMA-Un-stimulated clones The experiment was repeated independently three times, and the result shown is typical
of the three experiments The northern blots in (A) and (B) for MMP-9 and uPA have been exposed for different periods, due to large differences in expression levels.
Trang 3and versican To investigate the extent to which
expression of serglycin influenced total PG
biosynthe-sis, cells were labelled with 35S sulfate for 24 h The
labelled macromolecules were separated from
unin-corporated 35S-labelled sulfate by Sephadex G-50
Fine gel chromatography It has previously been
shown that the major fraction of 35S-labelled
macro-molecules in MDCK cells are of PG nature [16]
The level of 35S-labelled macromolecules therefore
indicates the level of PG synthesis As can be seen
in Fig 2A, the introduction of serglycin into MDCK
cells increased the amount of 35S-labelled
macromole-cules in the medium, but did not significantly
increase the total amount of 35S-labelled
macromole-cules Radiolabelling was also performed in the
presence of PMA Figure 2B shows that in
PMA-stimulated cells the distribution of PGs changed, so
the majority (78–90%) was secreted into the medium,
whereas the corresponding value for unstimulated
cells was 30–45% The total increase of 35S-labelled
macromolecules in clones 1–7 and 1–10, relative to
the mock, was approximately the same in
unstimu-lated and PMA-stimuunstimu-lated cells
HisTrap isolation of His-tagged serglycin
His-tagged serglycin in cell fractions and
condi-tioned media from unstimulated clones was isolated by
HisTrap affinity chromatography using 35S-labelled
macromolecules obtained by Sephadex G-50 Fine gel
chromatography Figure 3A shows the total amount
of 35S-labelled macromolecules loaded onto the
HisTrap column, whereas Fig 3B shows the total
amount of 35S-labelled macromolecules with affinity
for the column The highest level of 35S-labelled
macromolecules with affinity for the HisTrap column,
in both cell fractions and conditioned media, was
measured in clone 1–10 This level was 2–3 times
higher than for clone 1–7, whereas the level for the
mock transfected clone was found to be negligible
In both clone 1–10 and 1–7 the highest level of
incorporated 35S-labelled sulfate was measured in the
conditioned media As can be seen in Fig 3, serglycin
contributes very little to the total incorporation of
35S-labelled sulfate When comparing the amount of
35S-labelled macromolecules before and after HisTrap
isolation, it was found that only 1 ⁄ 60 of the
radio-activity was associated to serglycin in clone 1–7,
whereas the corresponding value for clone 1–10 was
1 ⁄ 20 Superose 6 gel chromatography of HisTrap
isolated 35S-labelled serglycin from conditioned media
showed that serglycin from clone 1–10 eluted at a
slightly more retarded position compared with clone
1–7 (not shown) Furthermore, analyses of 35S-labelled glycosaminoglycan (GAG) chains obtained from the same material showed a similar trend, with Kav values
of 0.53 and 0.57 for clone 1–7 and 1–10, respectively These findings indicate that the GAG chains of
0 1 2 3 4 5 6 7 8 9
0 1 2 3 4 5 6 7 8 9 1-7
Cell Medium Total
6 )
6 )
A
Medium Total
1,42
1,35
1,0
1,0
Fig 2 35 S-labelled macromolecules in serglycin-transfected MDCK clones Confluent MDCK clones were labelled with 35 S sulfate for
24 h in both absence and presence of PMA, whereupon the cells fractions and conditioned media were harvested and subjected to Sephadex G-50 Fine gel chromatography to remove unincorporated
35
S sulfate Each point represents the mean ± SD of measurement
on material from three separate wells The number on top of the black columns, representing the total 35 S sulfate incorporation, is relative to the mock transfectant (A) Unstimulated clones (B) PMA-stimulated clones The experiment was repeated independ-ently three times, and the result shown is typical of the replicate experiments.
Trang 4serglycin from clone 1–7 are slightly longer than those
of clone 1–10 Analysis of the composition of the
35S-labelled GAGs showed that both clone 1–7 and
1–10 contained 70% chondroitin sulfate (CS) and
30% heparan sulfate (HS) (not shown), indicating that
CS is the dominating GAG, in agreement with previ-ous findings [18]
Western blotting of serglycin
To further analyse for the presence of serglycin, condi-tioned media of the different clones were subjected
to western blotting after chondroitinase ABC (cABC) treatment, using a rabbit polyclonal antibody to human serglycin As evident in Fig 4, clones 1–7 and 1–10 contained the serglycin core protein, with the highest amount in the latter The molecular mass of the serglycin core protein was 35 kDa, in accordance with another study [5] The same results were observed with a mouse monoclonal antibody to the His-tag (not shown)
Enzyme analyses The possible relationship between serglycin and prote-ase secretion was analysed in the different clones using serum-free conditioned media, the chromogenic sub-strates S-2288 and S-2444, and gelatin and plasmino-gen–gelatin zymography Cell fractions were also analysed All cell culture experiments were carried out
in both the absence and presence of PMA, to ensure
0
100
200
300
400
500
600
Medium Total
0
5
10
15
20
25
30
Medium Total
A
B
4 )
4 )
Fig 3 HisTrap isolation of 35 S sulfate-labelled His-tagged
sergly-cin from serglysergly-cin-transfected MDCK clones Cell fractions and
conditioned media from cells exposed to 35S sulfate for 24 h
were buffer exchanged to binding buffer by Sephadex G-50
Fine gel chromatography The samples were further applied to a
1 mL HisTrap column, pre-equilibrated with binding buffer (20 m M
phosphate, 1 M NaCl, 8 M urea and 20 m M imidazole pH 8.0).
After a washing step, the samples were eluted with a solution
containing 20 m M phosphate, 1 M NaCl, 8 M urea and 500 m M
imidazole (pH 8.0) (A) Before HisTrap isolaton (B) After HisTrap
isolation The experiment was repeated independently more than
three times, and the result shown is typical of all the replicate
experiments.
Fig 4 Western blot of serglycin core protein in serglycin-trans-fected MDCK clones Conditioned media from unstimulated MDCK clones were desalted against Milli-Q water by Sephadex G-50 Fine gel chromatography After freeze-drying, the samples were dis-solved in a small volume of Milli-Q water and treated with cABC as described in Experimental Procedures Furthermore, the samples were subjected to SDS ⁄ PAGE followed by western blotting using a rabbit polyclonal antibody to human serglycin The data shown are from a single experiment that was repeated three times with the same results.
Trang 5comparison of basal and stimulated secretion of
pro-teases
Chromogenic substrates
Cell fractions and serum-free conditioned media were
analysed with respect to a broad spectrum of serine
proteases using the chromogenic substrate S-2288
(Fig 5) Media from clone 1–10 showed the highest
enzyme activity in unstimulated cells When cells
were treated with PMA, the enzyme activities in the
media were strongly enhanced Compared with the
media, enzyme activities in cell fractions were found
to be low Media from PMA-treated clones
con-tained 100-fold more protease activity than
medium from unstimulated clones Also after PMA
treatment, medium from clone 1–10 showed the
highest activity, but the difference between the clones
was much less distinct than observed for the
unstim-ulated clones
Further experiments were performed using the
uro-kinase substrate S-2444 Cell fractions and serum-free
conditioned media from the various clones all
con-tained activity towards this substrate, as can be seen in
Fig 6 Again, media from the clone 1–10 had the
high-est enzyme activity Here also PMA treatment resulted
in an 100-fold increase in enzyme activity in the
clones tested
Clearly, the enzyme activities in serum-free
condi-tioned media towards the chromogenic substrates
S-2288 and S-2444 were related to the levels of
sergly-cin, MMP-9 and uPA mRNA expression in the
MDCK-transfected cells The clone with the highest
levels of serglycin, MMP-9 and uPA mRNA and level
of HisTrap isolated 35S sulfate-labelled serglycin, and
amount of serglycin core protein detected by western
blotting, in conditioned media, i.e clone 1–10, also
had the highest secretion of the enzymes analysed with
chromomogenic substrates, both basal and after PMA
treatment
To investigate the nature of the enzyme activities
measured, serum-free conditioned media were
incuba-ted in both the absence and presence of the enzyme
inhibitors amiloride and Pefabloc The enzyme activity
recognizing the substrate S-2288 was inhibited 90%
in the presence of 2 mm Pefabloc, demonstrating that
this activity is of a serine protease nature (Table 1)
Furthermore, an inhibition of 70% of the enzyme
activity was observed in the presence of 2 mm
amilo-ride, indicating that a major part of the serine protease
activity measured with S-2288 is due to plasminogen
activators (PAs) To investigate whether the activity
recognizing the substrate S-2444 is indeed uPA, we
made use of the inhibitor amiloride, considered to be a specific inhibitor of uPA [19] When serum-free condi-tioned media from the different clones were incubated
in the presence of 2 mm amiloride, the activities were inhibited 95%, demonstrating that this activity is of uPA nature (Table 2)
A
B
Fig 5 Analysis of enzyme activities in serglycin-transfected MDCK clones using the chromogenic substrate S-2288 Confluent MDCK clones were cultured for 24 h under serum-free conditions in both absence and presence of PMA Cell fractions and conditioned media were harvested and analysed for enzyme activities by using the chromogenic substrate S-2288 Each point represents the mean and standard deviation of measurement on material from three sep-arate wells (A) Unstimulated clones (B) PMA-stimulated clones The S-2288 activity assay was repeated independently more than three times, and the result shown is typical of all the replicate experiments Note the difference in the scales of the vertical axis for the unstimulated and PMA-stimulated clones.
Trang 6Gelatin and plasminogen–gelatin zymography
To further investigate the relationship between
sergly-cin and protease secretion in MDCK clones, the
possible presence of gelatinases in the serum-free
con-ditioned media of the different clones was investigated
by gelatin zymography The rationale for measuring
gelatinase activities is that these enzymes are known to interact with PGs [20,21]
No gelatinolytic bands could be detected in the serum-free conditioned media of any of the clones tes-ted (Fig 7A) In contrast, when the cells were treates-ted with PMA, several gelatinolytic bands were detected (Fig 7B) By comparing the gelatinolytic bands with those of MMP-9 and MMP-2 standards, it is likely that the 225 and 92 kDa gelatinolytic bands are dimeric and monomeric MMP-9, respectively [22] The highest degree of gelatinolytic activity was evident in clone 1–10, although no particular difference in inten-sity of the 92 kDa (proform) band could be demon-strated between the different clones However, the
78 kDa band, probably an active form, had higher intensity in media from clone 1–10 than from 1–7 and the mock transfected clone The 127 kDa band observed for clone 1–10 may be a complex of mono-meric MMP-9 with neutrophil gelatinase-associated lipocalin (NGAL) [23]
A
B
Fig 6 Analysis of enzyme activities in serglycin-transfected MDCK
clones using the chromogenic substrate S-2444 Confluent MDCK
clones were cultured for 24 h under serum-free conditions in both
absence and presence of PMA Cell fractions and conditioned
media were harvested and analysed for enzyme activities by using
the chromogenic substrate S-2444 Each point represents the mean
and standard deviation of measurement on material from three
sep-arate wells (A) Unstimulated clones (B) PMA-stimulated clones.
The S-2444 activity assay was repeated independently more than
three times, and the result shown is typical of all the replicate
experiments Note the difference in the scales of the vertical axis
for the unstimulated and PMA-stimulated clones.
Table 1 Effect of amiloride and Pefabloc on serine protease activit-ies in transfected MDCK clones Serum-free conditioned media from unstimulated cells were harvested and analysed for enzyme activities using the chromogenic substrate S-2288 in both the absence and presence of amiloride and Pefabloc, both at a final concentration of 2 m M The results are calculated as percentages
of controls Each value represents the mean ± SD of measurement made on three independent wells The assay was repeated inde-pendently three times, and the result shown is typical of the repli-cate experiments.
Inhibitor
1–7
% remaining activity
1–10
% remaining activity
Mock
% remaining activity
Table 2 Effect of amiloride on PA activities in transfected MDCK clones Serum-free conditioned media from unstimulated cells were harvested and analysed for enzyme activities using the chromo-genic substrate S-2444 in both the absence and presence of amilo-ride (2 m M final concentration) The results are calculated as percentages of controls Each value represents the mean ± SD of measurement made on three independent wells The assay was repeated independently three times, and the result shown is typical
of the replicate experiments.
Inhibitor
Clone 1–7
% remaining activity
Clone 1–10
% remaining activity
Mock
% remaining activity
Trang 7The ability to degrade gelatin is not a unique
prop-erty of MMPs The presence of MMP activity in
gela-tin zymography of serum-free conditioned media from
the PMA-treated clones was verified by using 100 nm
galardin, an inhibitor of MMPs [24] After incubation
with galardin, the gelatinolytic bands shown in Fig 7B
were abolished (not shown), indicating that these
gela-tinolytic bands were MMPs
The possible presence of PA activity was also
investi-gated using plasminogen–gelatin zymography Gelatin
gels run without plasminogen were used as controls
against gels containing plasminogen and gelatin Indeed,
when plasminogen was incorporated into the gels, all
the clones displayed PA activity in the 55 kDa region,
the highest activity again being evident in the clone with
the highest mRNA levels for serglycin, MMP-9 and
uPA, i.e clone 1–10 (Fig 7C) Here also PMA
treat-ment resulted in elevated enzyme activities, and two
additional bands with molecular masses of 67 and
35 kDa appeared in the zymogram (Fig 7D) Upon
dilution of media from PMA-stimulated clones, 1–10
showed highest activity (not shown) The PA activity
in serum-free conditioned media from unstimulated
clones was shown to be uPA, because the 55 kDa
band was abolished when including 2 mm amiloride
in all incubation steps after gel electrophoresis In
zymograms of serum-free conditioned media from
PMA-treated cells, both the 55 and 35 kDa bands
were abolished in the presence of amiloride, whereas
the intensity of the 67 kDa band was unaltered The
identity of the 67 kDa band is unknown
Western blotting of MMP-9
In an attempt to identify the nature of the gelatinolytic bands, we performed western blotting using a MMP-9 antibody (Fig 8) Owing to a lack of canine MMP-9 antibody we used a rabbit polyclonal antibody to human MMP-9 As in gelatin zymography, no bands were visualized by western blotting of serum-free con-ditioned media from unstimulated clones (not shown) From the western blot of media from PMA-treated clones in Fig 8, the presence of MMP-9 could be dem-onstrated As for the zymography, there was no partic-ular difference in intensity of the MMP-9 monomer (92 kDa) band between the different clones The rela-tive differences in intensity of the 67 kDa band in the different lanes in Fig 8 are similar to those of the
78 kDa band in Fig 7B These bands may or may not represent the same protein, as the western blotting was performed under reducing conditions, whereas zymography was not
Discussion
Human serglycin has been stably transfected into MDCK cells The results presented show related levels
of serglycin and MMP-9 and uPA, both at mRNA and protein levels In Figs 1, 3 and 8 it can be seen that clone 1–10 has the highest level of serglycin, whereas clone 1–7 has the lowest level The levels of
35S sulfate incorporation were not particularly high in the clone with the highest serglycin mRNA expression,
1-7 1-10 Mock 1-7 1-10 Mock 1-7 1-10 Mock 1-7 1-10 Mock
Mr (kDa)
~ 200
~ 127
~ 92
~ 78
~ 67
~ 55
~ 37
Fig 7 Zymograms of serum-free condi-tioned media from serglycin-transfected MDCK clones Confluent MDCK clones were cultured for 24 h under serum-free conditions in both absence and presence of PMA Conditioned media were harvested and analysed by zymography (A) Gelatin zymography Media from unstimulated clones (B) Gelatin zymography Media from PMA-stimulated clones (C) Plasminogen– gelatin zymography Media from unstimu-lated clones (D) Plasminogen–gelatin zymography Media from PMA-stimulated clones The data shown are from single experiments that were repeated more than three times with the same results.
Trang 8level of HisTrap isolated35S sulfate-labelled serglycin,
and the amount of serglycin core protein detected by
western blotting Figure 3 clearly illustrate that high
serglycin levels do not necessarily translate into high
levels of 35S-labelled macromolecules expressed This
indicates that the higher release of proteases in clone
1–10 is related to the biosynthesis and release of
ser-glycin, and not the endogenous PGs This raises
inter-esting questions concerning the functions of serglycin
in intracellular compartments Our results suggest that
the presence and level of serglycin could be important
for the secretion of different types of proteases
Fur-thermore, the introduction of serglycin into a cell type
not normally expressing this PG, changes the levels of
endogenous protease secretion
We have previously shown increased secretion of
PGs, which is mainly due to increased secretion of
serglycin, in monocytes and macrophages after PMA
stimulation [9,10] An increase in PG secretion in
monocytes and macrophages has also been observed
after stimulation with lipopolysaccharide and gamma
interferon, suggesting that increased serglycin secretion
is linked to inflammatory reactions [10] The biological
functions linked to the release of serglycin from
mono-cytes and macrophages have not been outlined in any great detail, but it has been suggested that serglycin might be important for the binding and release of important inflammatory molecules, such as chemokines [11] It has also recently been shown that abrogation
of PG biosynthesis in the murine macrophage cell line J774 resulted in a decrease in MMP-9 and uPA secre-tion [13] It therefore seems as though further progress
in studies on the biological functions of serglycin will depend, to a certain extent, on a more thorough understanding of interactions with partner molecules
It will furthermore be important to study serglycin secretion in different cell types The processes and regulation leading to the formation of serglycin-containing granules, secretory or storage type, will probably differ to a large extent between different ser-glycin-expressing cells
The transfected MDCK cells generated here can be used as a model system to study possible relations between serglycin and different partner molecules The coordinated levels of serglycin and proteases are important in relation to those cells already known to express serglycin These include haematopoietic cells, such as mast cells, monocytes and macrophages, plate-lets and also endothelial cells and pancreatic acinar cells [4] All these cells have granules which contain a large variety of serglycin-binding molecules, including histamine, chymases, gelatinase, granzymes, platelet factor 4, lactoferrin and procarboxypeptidase With the established MDCK clones we are now able to address questions concerning regulation of serglycin release and interactions with different partner mole-cules
It is of interest to note that histamine, which is an important partner molecule for heparin PG in the mast cell granules, is also important for the genesis of gran-ules Inactivation of the gene encoding histidine decarboxylase, the enzyme converting histidine to histamine, resulted in reduced storage of PG and pro-teases in the granules [25] It therefore seems as though there is cross-talk between the different granule com-ponents during granule formation, and that lack of one important component has serious consequences for this process, and will also affect the amount of partner molecules sorted to such granules Our study shows that introduction of serglycin to MDCK cells leads to changes in secretion levels of proteases, via as yet undefined mechanisms, regulated in relation to the serglycin level This relationship between the levels of serglycin and protease levels could, accordingly, be in support of cross-talk regulatory mechanisms
The fate of serglycin released from different types of immune cells has not been studied to any great extent
Fig 8 Western blot of MMP-9 in serglycin-transfected MDCK
clones Serum-free conditioned media from PMA-stimulated MDCK
clones were subjected to SDS ⁄ PAGE followed by western blotting
using a rabbit polyclonal antibody to human MMP-9 The data
shown is from a single experiment that was repeated three times
with the same results.
Trang 9It has been shown that serglycin may bind to CD44,
and thereby participate in cell–cell interactions [26],
and that it can participate in the delivery of perforin
to target cells [27] Furthermore, it has been shown
that serglycin isolated from macrophages is not
degra-ded when it is addegra-ded back to fresh cultures of
macro-phages, suggesting extracellular functions after release
[28] It has also been shown that serglycin may bind
covalently to a fraction of the MMP-9 secreted from
the monocyte cell line THP-1 [29] This association
was shown to alter the biochemical properties of the
enzyme
There are several possibilities for serglycin to
inter-act with other secreted components, such as enzymes,
growth factors or cytokines, and modulate their
activ-ities [4] Hence, both the generation of secretory
com-plexes during biosynthesis and granule formation and
interactions between secreted components, are
proces-ses in which serglycin is most probably an important
component, worthy of more detailed study
Experimental procedures
Cell culture and transfection
Cell culture reagents were purchased from Sigma (St Louis,
MO), unless otherwise stated MDCK epithelial cells
cul-tures were checked for Mycoplasma infection with
Myco-Alert mycoplasma detection assay (Cambrex, Rockland,
ME) in routine Before each experiment the cells were
grown to confluency (4 days) Triplicate cultures were used
for all experiments
Technologies, Carlsbad, CA) was used to generate the
ser-glycin–His-tag expression vector To obtain inframe
transla-tion into the His sequence, serglycin cDNA was amplified
from human serglycin cDNA [9] by PCR with the following
primers: upper primer (XbaI): 5¢-CTCTAGAGTCATG
ATGCAGAAGCTACTCA-3¢ and lower primer (EcoRI):
5¢-CGAATTCCTTCTAATCCATGTTGACCCAA-3¢ The
obtained PCR product was cloned into a pCRII vector
with the use of TA Cloning Kit (Invitrogen Life
Technol-ogies), cut out with XbaI and EcoRI, both purchased from
cloning of the insert was verified by sequencing Vectors
Myc-His] were stably transfected into MDCK cells with the
DNA-calcium phosphate procedure as described previously
[30,31] Two days after transfection, cells were given
weeks with selective medium, stably transfected single col-onies were picked with cloning rings to obtain homogenous subcell lines stably expressing the serglycin construct
con-trols
Preparation and analysis of RNA
For northern blot analyses, total RNA was extracted from confluent cells using Trizol Reagent (Invitrogen Life
fragments used for generation of serglycin and 36B4 probes were digested and purified from vectors containing human
ribosomal phosphoprotein PO (36B4) Partial cDNAs for canine MMP-9 and uPA were amplified by RT-PCR using total RNA from PMA-stimulated MDCK cells, followed
by PCR using PfuUltra (Stratagene, La Jolla, CA) and cloned into a pPCR-Sript Amp SK(+) vector (Stratagene),
as described previously [32] The following primers were used: For the 5¢-human serglycin: (5¢-CTCTAGAGTCAT GATGCAGAAGCTACTCA-3¢) and 3¢-human serglycin: (5¢-CGAATTCCTTCTAATCCATGTTGACCCAA-3¢) For the 5¢-canine MMP-9: (5¢-TTAGGGAGCACGGAGATG GGTAT-3¢) and 3¢-canine MMP-9: (5¢-GTTGGGCAGA AGCCGTAGAGTTT-3¢), and for the 5¢-canine uPA: (5¢-GTCAGCGCCACACACTGCTT-3¢) and 3¢-canine uPA: (5¢-GCCTTGGGTAGAGCAGACCA-3¢) Correct amplifi-cation was verified by sequencing of the inserts Fragments containing the partial MMP-9 and uPA cDNAs were ampli-fied with PCR using the vectors as template to generate
were separated by electrophoresis in 1% agarose gels and transferred to nylon membranes Probes were generated by
Elmer Life and Analytical Sciences, Boston, MA, USA) using Megaprime DNA labelling systems (Amersham Bio-sciences, Little Chalfont, Bucks, UK) After hybridization, the nylon membranes were washed and further exposed to autoradiography films for detection
Proteoglycan analyses Radiolabelling of macromolecules
For radiolabelling of macromolecules, confluent cells were changed to sulfate-free medium (RPMI 1640) (GibcoBRL Life Technologies, Paisley, UK), containing 2 mm
Trang 10Parallel cell cultures were treated with 50 ngÆmL)1 PMA
during the radiolabelling After 24 h incubation, cell
frac-tions and conditioned media were harvested Cell layers
were recovered by solubilization in a solution containing
4 m guanidine hydrochloride and 50 mm sodium acetate
(pH 6.0) Loose cells were separated from the conditioned
media by centrifugation at 400 g for 3 min Unincorporated
35
S sulfate was removed from cell fractions and media by
Sephadex G-50 Fine gel chromatography Radioactivity was
measured using a liquid scintillation counter
Isolation of His-tagged serglycin
Cell fractions and conditioned media from cells exposed to
35
S sulfate for 24 h were buffer exchanged to binding buffer
containing 20 mm phosphate, 1 m NaCl, 8 m urea and
20 mm imidazole (pH 8.0) by Sephadex G-50 Fine gel
removed during this step The samples were further applied
to a 1 mL HisTrap HP column (Amersham Biosciences),
pre-equilibrated with binding buffer After a washing step,
samples were eluted with a solution containing 20 mm
phosphate, 1 m NaCl, 8 m urea and 500 mm imidazole
(pH 8.0) Fractions containing radioactivity were pooled,
and desalted on PD-10 desalting columns (Amersham
Bio-sciences) The samples were further treated with NaOH to
release GAGs from the serglycin core protein, as described
elsewhere [33] After desalting, the samples were subjected
[16,32] cABC (from Proteus vulgaris) was purchased from
Seikagaku Corporation (Tokyo, Japan) The amounts of
respectively, were calculated from the proportions of
degra-dation products by gel chromatography on Superose 6
(Amersham Biosciences), using 1 m NaCl as the mobile
phase The elution profiles were monitored by liquid
scintil-lation counting
Western blotting of serglycin
Conditioned media from the clones were desalted against
Milli-Q water by Sephadex G-50 Fine gel
chromatogra-phy After freeze-drying, the samples were dissolved in a
small volume of Milli-Q water and treated with cABC at
cABC treatment was performed in the presence of the
serine protease inhibitor Pefabloc SC (Fluka, Buchs,
Switzerland) and the cysteine protease inhibitor
N-ethyl-maleimide (Sigma), both at a 2 mm final concentration
accord-ing to standard procedures Briefly, samples were treated
with 2-mercaptoethanol and separated on 15%
polyacryl-amide gels The proteins in the gels were transferred to
nitrocellulose membranes After blocking with 5%
N Borregaard, Rigshospitalet, Department of Haemato-logy, Copenhagen, Denmark) A mouse monoclonal anti-body to the His-tag (Roche Diagnostics, Mannheim, Germany) was also used to detect the serglycin core pro-tein Bound antibodies were detected using peroxidase-linked secondary antibodies, followed by chemilumines-cence detection Molecular masses were estimated using
Enzyme analyses
For enzyme analyses, confluent cells were changed to serum-free medium (DMEM), containing 2 mm
PMA After 24 h incubation, cell fractions and serum-free conditioned media were harvested Cell layers were
from the serum-free conditioned media by centrifugation at
400 g for 3 min
Chromogenic substrates
Aliquots (100 lL) of the cell fractions and serum-free con-ditioned media were analysed for enzyme activity using the chromogenic substrates H-d-Ile-Pro-Arg-pNA (S-2288) and pyro-Glu-Gly-Arg-pNA (S-2444), essentially as suggested
by the manufacturer (Chromogenix, Milan, Italy) Experi-ments were performed at room temperature in both the absence and presence of the enzyme inhibitors amiloride (Sigma), a selective inhibitor of uPA, and Pefabloc SC, an inhibitor of serine proteases, both used at 2 mm final con-centration in analyses of serum-free conditioned media from unstimulated cells, as previously described [13] Absorbance was read at 405 nm In the analyses performed,
assure that the detected activities were within the linearity
of the assay, absorption measurements were performed regularly up to 1 h for material from PMA-stimulated cells and up to 24 h for material from unstimulated cells The activities, measured as changes in absorption with time, were calculated from the initial linear parts of the different curves As the same amount of either cell fraction or condi-tioned medium was used for the different clones, the result
of the activity measurements is presented as the change in
Gelatin and plasminogen–gelatin zymography
MMP and PA activities were determined by gelatin and