From the Sutton Rheumatism Research Laboratory, Royal North Shore
Hospital, St. Leonards, New South Wales 2065, Australia
Human gelatinase B is involved in tissue
remodeling and angiogenesis. It is thought to be synthesized and
rapidly secreted as an inactive precursor. In this report, we have
shown that human endothelial cells accumulate active forms of
gelatinase B in the cytosol. Microvascular but not macrovascular
endothelial cells dramatically increased the expression of cytosolic
gelatinase B in response to phorbol myristate acetate. Western blotting
showed that tissue inhibitor of metalloproteinase-1 (TIMP1) was also present in the cytosol. Whereas gelatinase B was complexed with TIMP1
in the conditioned medium, it existed as a free enzyme in the cytosol,
suggesting that the formation of gelatinase B and TIMP1 complex occurs
after their secretion. Immunogold electron microscopy revealed that
gelatinase B was localized in secretory vesicles which were especially
prominent in invading pseudopodia. In contrast, TIMP1 was found
throughout the cytoplasm but was not present in the gelatinase
vesicles. The accumulation of intracellular activated gelatinase B,
ready for rapid release, may facilitate the migration of microvascular
endothelial cells during angiogenesis.
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INTRODUCTION |
The formation of capillaries from pre-existing microvessels
(angiogenesis) occurs in a variety of normal and pathological conditions, including wound healing, tumor growth and metastases, and
arthritis (1). During angiogenesis, microvascular endothelial cells
secrete proteases, migrate through the underlying extracellular matrix,
and proliferate to organize into new blood vessels. The initial step of
angiogenesis requires focalized degradation of the basement membrane
(2). The process is carried out by at least two matrix
metalloproteinases (MMPs),1
gelatinase A and gelatinase B, both of which degrade basement membrane
collagens (3) and are produced by many cell types, including
endothelial cells (4, 5). They are unique among the MMPs in that their
latent forms are thought to be secreted in physical association with
their natural inhibitors, the tissue inhibitors of MMPs (TIMPs) (6, 7).
Pro-gelatinase B binds to TIMP1, whereas pro-gelatinase A complexes
with TIMP2, both via the COOH-terminal domain of the enzymes (6,
8).
With the exception of neutrophil gelatinase B, which is stored in
cytoplasmic granules (9), MMPs are thought to be synthesized and
rapidly secreted as latent precursors that must be processed extracellularly to their active forms to express enzymatic activity (10). Gelatinase A binds to and can be activated by the membrane-type MMP (MT1-MMP) at the cell surface of several tumors, transformed and
normal cell types (11, 12). The activation of gelatinase B is thought
to occur in the extracellular milieu, although the mechanism is not
clearly understood. Recent reports have suggested that stromelysin-1
may be responsible (6, 14, 15). Ginestra et al. (13) have
recently shown that membrane vesicles shed from human HT1080
fibrosarcoma cells contained active gelatinase B. In this report, we
have demonstrated that microvascular endothelial cells are capable of
accumulating active gelatinase B in the cytoplasm. Furthermore, this
active enzyme is separately compartmentalized from TIMP1 by being
located in secretory vesicles.
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EXPERIMENTAL PROCEDURES |
Cells--
Human microvascular endothelial cells (FSE) derived
from neonatal foreskin obtained after circumcision were isolated using Ulex europaeus 1-coated Dynabeads as described previously
(16). Macrovascular endothelial cells were obtained from human
umbilical veins (HUVE) isolated as described by Jaffe (17). FSE were
grown and maintained in Biorich medium (ICN Biomedicals, Aurora, OH) containing 30% normal pooled human serum (derived from healthy volunteers) plus 100 µg/ml endothelial cell growth supplement prepared as described by Maciag et al. (18) and 50 µg/ml
heparin (Sigma). HUVE were grown in Biorich containing 20% fetal calf serum plus 50 µg/ml endothelial cell growth supplement and 50 µg/ml
heparin. Cells were used at passage 4.
Experimental Protocol--
HUVE and FSE (3 cell lines each) were
cultured on 60-mm dishes (Becton Dickinson) at a density of 1.5 × 106 cells/dish in growth medium. Endothelial cells were
washed twice with Hanks' balanced salt solution and preincubated in
basal medium (Biorich plus 1% normal pooled serum) for 6 h. The
cells were then replaced with fresh basal medium and incubated in the
absence or presence of 100 ng/ml phorbol myristate acetate (PMA)
(Sigma) for 24 h. Human serum was necessary to maintain an intact
confluent monolayer of microvascular cells throughout the course of the experiment (5). Gelatinases present in human serum were removed by
running the serum through a gelatin-Sepharose affinity column (3)
(Pharmacia Biotech Inc.).
Conditioned Media and Triton X-114 Cell Extracts--
After
incubation, the conditioned medium was collected and the cytosolic
fraction was separated from the membrane fraction by Triton X-114
extraction as described by Lewalle et al. (19). The extract
was partitioned into the detergent (membrane fraction) and aqueous
phases (cytosolic fraction) at 37 °C for 5 min and centrifuged at
5000 × g for 2 min. The aqueous phase was then collected. To eliminate any carry-over effect from the aqueous fraction, the detergent phase was repartitioned three times in 1.5%
Triton X-114. It was then concentrated by mixing with 30 µl of packed
gelatin-Sepharose (Pharmacia) with end-over-end rotation for 30 min at
4 °C. The bead suspension was centrifuged at 8000 × g for 2 min and the pellet was then analyzed by gelatin
zymography.
Flow Cytometric Analysis--
Flow cytometry was performed as
described previously (20) with modifications. Cells were grown to
confluence in 35-cm2 dish and treated with either basal
medium alone or basal medium containing 100 ng/ml PMA for 24 h.
The cells were then detached from the monolayers using 20 mM EDTA in washing buffer (2% fetal calf serum in
phosphate-buffered saline) for 10 min at 37 °C and scraped using a
rubber policeman. After centrifugation at 1100 rpm for 10 min, the
pellet was resuspended and fixed in freshly prepared 2%
paraformaldehyde in phosphate-buffered saline for 30 min at room
temperature. The cells were then washed and permeabilized for 10 min
with 10% Triton X-100, washed, centrifuged, and the primary monoclonal
mouse antibody against gelatinase B (1:50 dilution, Oncogene Science,
Uniondale, NY) was added for 60 min at room temperature. Fluorescein
isothiocyanate-conjugated secondary antibody was then applied for 30 min and the cells were then analyzed by flow cytometry (Coulter Elite,
Hialeah, FL).
Zymography--
Samples were analyzed by zymography under
nonreducing conditions as described previously (21).
Immunoblotting--
MMPs and TIMP1 were detected by immunoblot
analysis after SDS-polyacrylamide gel electrophoresis. Antibodies to
stromelysin-1, gelatinase B, and TIMP1 were used at 1 µg/ml (Oncogene
Science).
Immunogold Electron Microscopy--
FSE (7 × 104) were seeded onto type I collagen gel and prepared as
described previously (22) in an culture well insert for 24 h
before PMA (100 ng/ml) was added for a further 24 h. The gel was
removed, fixed with 2% paraformaldehyde and 0.05% glutaraldehyde in
phosphate-buffered saline, pH 7.4, for 1 h at 4 °C, and then dehydrated in ethanol and embedded in LR White acrylic resin (Emgrid, Australia). Single and double immunogold electron microscopy was carried out as described previously (23) using gelatinase B and TIMP1
antibodies (1:50 dilution) or nonimmune IgG antibody. Thin sections
were examined with the Joel 100-S electron microscope.
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RESULTS |
Cellular Localization of Gelatinase B by Flow
Cytometry--
Previous workers have shown that cultured human
endothelial cells secrete gelatinase B into the conditioned medium in
response to PMA (4, 5). To determine whether gelatinase B was also localized to the endothelial cell, we performed flow cytometry using an
antibody directed against the latent and active forms of gelatinase B. Results are shown in Fig. 1. Under basal
conditions, FSE showed a 2.1-fold increase in gelatinase B expression
compared with the nonimmune control. Stimulation of FSE with PMA
resulted in a 9.6-fold increase in the expression of gelatinase B
compared with unstimulated cells. In contrast, HUVE expressed markedly lower levels of gelatinase B.

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Fig. 1.
Localization of gelatinase B by flow
cytometry. Confluent endothelial cell monolayers were stimulated
with 100 ng/ml PMA (dark shading) or no test agent
(light shading) for 24 h. After fixation and
permeabilization of the cells, expression of gelatinase B was assessed
by flow cytometry using an antibody to gelatinase B. Nonimmune IgG
(no shading) was used as a negative control.
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Gelatinase B Present in Endothelial Cell Conditioned Medium and
Cell Fractions--
To determine whether gelatinase B was located on
the cell membrane or intracellularly, the membrane and cytosolic
fractions from endothelial cells were separated using Triton extraction as described under "Experimental Procedures." Gelatinase B levels were measured in these fractions as well as in the conditioned medium
using zymography. Results are shown in Fig.
2. Under basal conditions, gelatinase B
was not detected in the conditioned medium or membrane fraction of
endothelial cells. However, endothelial cells expressed gelatinase B in
the cytosolic fraction as three distinct bands at 88, 82, and 74 kDa,
the latter two bands representing the active forms of gelatinase B. PMA
markedly increased gelatinase B expression in FSE (7.1-fold compared
with basal using scanning densitometry; mean, 3 cell lines) but not in
HUVE. PMA also stimulated both the latent and active forms of
gelatinase B in the membrane fraction of FSE. However, only the latent
form was present in the conditioned medium of FSE and to a lesser
extent in HUVE.

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Fig. 2.
Latent and active forms of gelatinase B in
conditioned medium and cytosolic and membrane fractions of FSE and
HUVE. Confluent endothelial monolayers were preincubated in basal
medium (Biorich plus 1% normal pooled serum) for 6 h followed by
incubation for 24 h in fresh basal medium in the presence of 100 ng/ml PMA or no test agent (Basal). The conditioned medium
(Medium) was collected and the cytosolic fraction
(Cytosol) and membrane fraction (Membrane) were
extracted using Triton X-114 as described under "Experimental
Procedures." Zymography was used to assess gelatinase activity in the
conditioned medium and the cytosolic and membrane fractions of FSE and
HUVE. These experiments were performed on three different FSE and HUVE
cell lines, each with similar results.
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To assess whether the activation of gelatinase B in the cytosolic and
membrane fractions was caused by the Triton extraction procedure, the
conditioned medium from PMA-stimulated FSE, which contained only the
latent enzyme, was incubated with the detergent for 16 h at
37 °C. This treatment did not activate the latent gelatinase B
present in the conditioned medium, thus indicating that gelatinase B
was not activated by the extraction technique (Fig.
3).

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Fig. 3.
Effect of Triton X-114 extraction on the
activation of gelatinase B. Confluent FSE were preincubated in
basal medium (Biorich plus 1% normal pooled serum) for 6 h
followed by incubation for 24 h in fresh basal medium in the
presence of 100 ng/ml PMA. Conditioned medium was then incubated with
or without 1.5% Triton X-114 in Tris-buffered saline for 16 h at
37 °C. Control represents conditioned media treated with Triton but
not incubated. The samples were then assessed for gelatinase activity
by zymography.
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In addition to gelatinase B there was a prominent band of gelatinolytic
activity at 66 kDa in the conditioned medium and the cytosolic fraction
of both FSE and HUVE, representing the latent form of gelatinase A
(Fig. 2). Two additional bands at 62 and 59 kDa, being the active
species of gelatinase A, were also present. The treatment of both FSE
and HUVE with PMA for 24 h resulted in the further activation of
gelatinase A. The total amount of latent and active forms of gelatinase
A were increased in the membrane fraction by PMA (Fig. 2).
We next examined the time-course expression of gelatinases by FSE after
2, 8, and 16 h of PMA treatment (Fig.
4). In the conditioned medium, latent
gelatinase B only became detectable after 16 h of treatment with
PMA. In the cytosol, latent gelatinase B was first detected after
2 h, whereas the active forms of gelatinase B were detected after
8 h of PMA treatment, the levels of which were substantially
higher at 16 h. Gelatinase A first appeared in the cytosol after
2 h but, in contrast to gelatinase B, the levels remained constant
over the 16-h incubation period. This suggested that gelatinase A was
secreted from the cell, whereas gelatinase B was stored in the
cell.

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Fig. 4.
Time course of gelatinase B expression by
FSE. Confluent FSE were preincubated in basal medium (Biorich plus
1% normal pooled serum) for 6 h followed by incubation for
24 h in fresh basal medium in the presence of 100 ng/ml PMA for 2, 8, or 16 h. The conditioned medium (Medium) and
cytosolic fraction (Cytosol) were assessed for gelatinase
activity by zymography.
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Western blotting disclosed the presence of TIMP1 in the cytosol, which
was markedly elevated in FSE but not HUVE after treatment with PMA
(Fig. 5). Stromelysin-1 was also detected
in the FSE cytosol under basal conditions, the levels of which were
increased after treatment with PMA (data not shown).

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Fig. 5.
Western blot analysis of TIMP1.
Confluent endothelial monolayers were preincubated in basal medium
(Biorich plus 1% normal pooled serum) for 6 h followed by
incubation for 24 h in fresh basal medium in the presence of 100 ng/ml PMA or no test agent (Basal). TIMP1 antigen was
measured in the cytosolic fraction of FSE and HUVE using an antibody to
TIMP1.
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Cytosolic Gelatinase B Is TIMP-free, whereas Secreted Gelatinase B
Is Bound to TIMP1--
Previous workers have reported that the
aminophenylmercuric acetate (APMA) activation of the purified
gelatinase B-TIMP complex results in the loss of an 8-10-kDa
NH2-terminal domain of the enzyme, generating a final
product of 82 kDa (24, 25). In contrast, activation of TIMP-free
gelatinase B with APMA results in the formation of not only the
intermediate 82-kDa species, but also the active species of 67 kDa and
40-50 kDa (24-26). To determine whether gelatinase B in the cytosolic
fraction was complexed with TIMP1, the conditioned medium and the
cytosolic fraction of PMA-stimulated FSE were incubated with 2 mm
APMA at 37 °C. Results are shown in Fig.
6. Incubation of the conditioned medium with APMA for 1 h resulted in the partial conversion of the latent gelatinase B to an 82-kDa species. No further activation occurred after
18 h. Treatment of the cytosolic fraction with APMA produced 82-, 74-, and 67-kDa species and diffusely resolved bands at 40-50 kDa
similar to those described by Senior et al. (26). Western analysis confirmed these results (Fig. 6b). These
observations indicate that gelatinase B present in the cytosolic
fraction is free of TIMP1, whereas the latent species present in the
conditioned medium is complexed with TIMP1.

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Fig. 6.
Activation and processing of gelatinase B by
APMA. Confluent FSE were preincubated in basal medium (Biorich
plus 1% normal pooled serum) for 6 h followed by incubation for
24 h in fresh basal medium in the presence of 100 ng/ml PMA or no
test agent (Basal). The conditioned medium
(Medium) or cytosolic fraction (Cytosol) of
PMA-stimulated FSE were incubated with 2 mM APMA for 0, 1, or 18 h at 37 °C and then analyzed for gelatinase B by
zymography (a) and Western blotting (b) using an
antibody to gelatinase B.
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Gelatinase B Is Localized to Intracellular Vesicles--
Because
both gelatinase B and TIMP1 were present in the cytosol yet were not
complexed, we used immunogold electron microscopy to investigate
whether TIMP1 and gelatinase B were separately compartmentalized within
the cell. FSE were grown on type I collagen gel to provide support for
maintaining cell integrity during processing. Immunogold electron
microscopy confirmed the presence of gelatinase B and TIMP1 in the
cytosol of FSE which had been treated with PMA for 24 h.
Gelatinase B gold colloid particles were mainly localized in
membrane-bound secretory vesicles (Fig.
7a). These vesicles were
slightly more electron dense than the surrounding cytoplasm and were
encapsulated by a clearly defined plasma membrane. In quiescent cells,
the vesicles were usually found in close proximity to the cell membrane
facing the collagen gel. Interestingly, the vesicles were more abundant
in pseudopod extensions from the cells and, in some instances, the
immunogold labeling showed gelatinase B being secreted from the tips of
pseudopodia which were invading the collagen gel (Fig. 7c).
Using double immunogold labeling, we found that TIMP1 was spread more
diffusely throughout the cytoplasm (Fig. 7d). Although TIMP
was occasionally present in small vesicles (Fig. 7d), it was
never found in the gelatinase B-containing vesicles.

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Fig. 7.
Immunogold labeling of gelatinase B. Electron micrographs of immunogold labeling for intracellular
gelatinase B and TIMP1 in FSE grown on a type I collagen gel
(Coll) in the presence of PMA (100 ng/ml) for 24 h.
Protein A20-nm colloidal gold particles were used to localize
gelatinase B. a, gelatinase B (G) localized in
membrane-bound vesicles (× 60,000). b, negative control.
When the antibody to gelatinase B was replaced by a nonimmune IgG there was minimal nonspecific binding of gold particles (× 25,000). c, a pseudopod which was invading the collagen gel contained
many gelatinase vesicles. One vesicle (S) at the tip of the
pseudopod is releasing gelatinase B into the matrix (× 96,000).
d, double labeling. The protein A20-nm and protein A10-nm
colloidal gold particles were used to localize gelatinase B
(G) and TIMP1 (T), respectively. TIMP1 was
located throughout the intracellular matrix, occasionally in vesicles
(Tv), but was not present within the gelatinase B-containing
vesicles (× 58,000).
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DISCUSSION |
We have demonstrated that gelatinase B accumulates in human
microvascular endothelial cells and that the enzyme is present in both
its latent and active forms. Previous to this report, only
stromelysin-3 and MT1-MMP were known to be processed to their enzymatically active forms intracellularly (27, 28). The activation of
stromelysin-3 and MT1-MMP is regulated by the presence of an unusual
10-amino acid insert sandwiched between the propeptide and the
catalytic domains of the enzyme, containing an amino acid sequence
Arg-Arg-Lys-Arg which is recognized and activated by the
Golgi-associated proteinase furin. However, it is not clear how the
activation of gelatinase B, which does not have this amino acid insert,
occurs intracellularly. Recently, Baramova et al. (29) have
shown that the plasminogen activator-plasmin system may be involved in
gelatinase B activation. Free gelatinase B, but not gelatinase B bound
to TIMP, can also be activated by active stromelysin-1 (6, 14, 15) and
by the 62- and 45-kDa active forms of gelatinase A (30).
Stromelysin-activated TIMP-free gelatinase B generates the intermediate
82-kDa form, the active 67-kDa species, and the inactive 50-kDa species
(6, 24), whereas gelatinase A generates an 82-kDa active species as the final product. The active species of gelatinase B present in the cytosolic fraction of our study are similar to those generated by
stromelysin, suggesting that this may be the activating agent. Further
evidence for this was provided by our finding that stromelysin-1 was
also present in the cytosol of FSE. Whether stromelysin-1 is
responsible for the activation of intracellular gelatinase B needs
further investigation.
Gelatinase B is unique among the MMPs in that it strongly interacts
with TIMP1 in its latent form via the C-terminal domain (6). Murphy
et al. (3) have proposed that gelatinase B-TIMP1 complexes
are formed during intracellular protein folding. It is generally
believed that the gelatinase B-TIMP1 complexes are then secreted from
cells (24). Our study has verified that gelatinase B secreted into the
conditioned medium of endothelial cells is bound to TIMP1; however, the
gelatinase B expressed in the cytosolic fraction of FSE can exist
uncomplexed. Thus, the formation of the gelatinase B-TIMP1 complex
occurs after (or while) these molecules are secreted from microvascular
endothelial cells. Further evidence for this was obtained using
immunogold electron microscopy, which revealed that gelatinase B was
separately compartmentalized from TIMP1 within the endothelial cell by
being localized in secretory vesicles (Fig. 7).
The presence of gelatinase A on the cell membrane of human umbilical
vein endothelial cells has been previously reported (19). Our
experiments confirmed these findings and also found that both gelatinase A and gelatinase B were expressed in the membrane fraction of FSE in response to PMA. The association of gelatinase B with the
plasma membrane has been previously shown in bone metastatic tissue
(31) and in basal cell carcinomas (32), although the binding mechanism
is not clear. In contrast, the binding and activation of gelatinase A
to the cell membrane via TIMP2 and MT1-MMP are well documented (12,
33). Brooks et al. (34) have also found that the C terminus
of active gelatinase A is associated with the
v
3 integrin receptor on the cell surface
of angiogenic blood vessels and melanoma tumors. Similar interactions
may exist for the binding of gelatinase B to the membrane.
In this report, we have demonstrated that FSE have the ability to
accumulate and activate gelatinase B intracellularly, especially in the
presence of the tumor-promoting chemical PMA. We and others (4, 5) have
previously shown that PMA induces the synthesis and secretion of
gelatinase B by FSE. To date, there have been few reports on the
effects of cytokines or angiogenic/growth factors on gelatinase B
induction by human microvascular endothelial cells. Hanemaaijer
et al. (4) have shown that tumor necrosis factor-
can
enhance the effect of PMA, but it does not stimulate gelatinase B
synthesis by FSE when used alone (4, 5). Future studies need to
investigate the interplay of physiological agents that can induce
gelatinase B accumulation in microvascular endothelial cells.
We have demonstrated that gelatinase B can exist as a free active
enzyme in the cytoplasm and to a lesser extent on the cell membrane of
FSE. These findings are likely to be relevant in angiogenesis, a
phenomenon which only occurs in microvascular endothelial cells. It is
feasible that as endothelial cells migrate during angiogenesis, the
active forms of gelatinase B are secreted from the cell in short bursts
to locally degrade the basement membrane. They are then rapidly
inhibited by TIMP and/or degraded in the extracellular milieu (3),
rendering them inactive. This inhibitory mechanism is important, as it
prevents uncontrolled proteolysis. Pepper et al. (35) have
shown that if proteolysis goes uninterrupted the dissolution of the
matrix prevents endothelial cells from migrating and forming tube-like
structures due to the absence of a scaffold. The ability of
microvascular endothelial cells to accumulate active gelatinase B in
secretory vesicles, ready for release, in addition to the presence of
active species of gelatinase A and gelatinase B on the cell membrane,
would enable microvascular endothelial cells to focalize proteolytic
activity to the pericellular environment and thus be very effective in the process of cell migration.
We thank Dr. Ross Davey, Assoc. Prof. Leslie
Schrieber, Prof. Philip Sambrook, and Kate Gibbons for helpful
discussions and review of the manuscript; Peter Jameison (Gore Hill
Research Laboratories) and Anne Simpson-Gomes (University of Sydney
Electron Microscope Unit) for expert electron microscope assistance;
Dr. Malcolm King for performing flow cytometry; and Eddie Jozefiak
for photography.