1 Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel
2 InSight Ltd., Rehovot, Israel
3 Weizmann Institute of Science, Rehovot, Israel
4 Hadassah-Hebrew University Hospital, Jerusalem, Israel
* Author for correspondence (e-mail: sbkatz{at}netvision.net.il )
Accepted 12 December 2001
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Summary |
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Key words: Endocytosis, Heparan sulfate proteoglycans, Heparanase, Processing
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Introduction |
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Transfection experiments indicated that the major heparanase form produced
by insect and mammalian cells is the non-processed, inactive 65 kDa precursor
(Vlodavsky et al., 1999a). In
fact, attempts to express the truncated 50 kDa
(Lys158-Ile543) protein failed to yield an active
enzyme, suggesting that the region starting at the N-terminus to
Lys158 plays a role in mediating expression and/or function of
heparanase (Fairbanks et al.,
1999
; Hulett et al.,
1999
; Kussie et al.,
1999
; Toyoshima et al., 1999;
Vlodavsky et al., 1999a
). In
the present study we investigated the interactions of the extracellular
inactive 65 kDa heparanase with primary human fibroblasts devoid of endogenous
heparanase. We found that human fibroblasts readily convert the precursor form
of heparanase into its highly active proteolytically processed form. During
this process, heparanase binds to the cells, and following cleavage the
processed form of the enzyme is endocytosed. Heparanase endocytosis is
inhibited in the presence of excess heparin and requires integrity of the
actin cytoskeleton. Endocytosis of the 50 kDa, processed form is significantly
enhanced compared with the non-processed proenzyme. Following endocytosis, the
truncated active enzyme is stored within the cytoplasm for a prolonged period
of time (at least 16 hours). This pattern of events provides a novel
regulatory mechanism by which extracellular heparanase, an important source of
heparanase activity, is activated and later on may enhance the invasive
behavior of malignant cells.
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Materials and Methods |
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Heparanase activity
Cell-associated heparanase activity was determined as previously described
(Vlodavsky et al., 1992;
Vlodavsky et al., 1994
;
Vlodavsky et al., 1999a
).
Briefly, metabolically sulfate
(Na2[35S]O4)-labeled ECM was prepared as
described previously (Vlodavsky,
1999b
) and incubated (4 hours, 37°C) with the cells at pH 6.6.
In order to evaluate the occurrence of heparan sulfate degradation products,
the incubation medium was collected and applied for gel filtration on
Sepharose 6B columns. Degradation fragments of HS side chains were eluted at
0.5<kav<0.8, peak II. Nearly intact HSPGs were eluted next to
the V0, (kav<0.2, peak I)
(Vlodavsky et al., 1992
;
Vlodavsky et al., 1994
;
Vlodavsky et al., 1999a
). Each
experiment was performed at least three times, and the variation of elution
position (kav values) did not exceed ±15%.
Indirect immunofluorescence
Cultured primary human foreskin fibroblasts were stained by indirect
immunofluorescence, as previously described
(Katz et al., 1999). Cells
were fixed with either acetone (-20°C, 5 minutes) or 4% formaldehyde in
PBS for 20 minutes and permeabilized with 0.5% Triton X-100 in PBS for 5
minutes. The cells were then incubated with the indicated first (45 minutes)
and second (45 minutes) antibodies in PBS. Anti-heparanase antibodies were
used at 10 µg/ml followed by staining with secondary Alexa- or
Cy3-conjugated goat anti-mouse antiserum diluted 1:200.
Labeling of intracellular organelles
HFF were incubated with 50 nM Lyso TrackerTM DNA99,
Texas-red-conjugated dextran or TRITC-conjugated ConA (Molecular Probes,
Eugene, OR) in growth medium containing recombinant heparanase for 2 hours at
37°C. The cells were then fixed and permeabilized with acetone (-20°C,
5 minutes), followed by immunofluorescent staining for heparanase, as
described above.
Digital fluorescence imaging analysis of heparanase subcellular
distribution
Quantitative fluorescence microscopy was performed using the DeltaVision
system (Applied Precision Inc., Issaqua, WA) attached to an inverted Zeiss
Axiovert microscope using a 100x1.4 Plan-APOCHROMAT objective (Zeiss,
Oberkochen, Germany). The image processing and analysis methods used here were
described in details previously (Levkowitz
et al., 1998; Zamir et al.,
2000
). Briefly, these methods include: (a) image filtration;
original images of fluorescently labeled cells were subjected to high-pass
filtration, which subtracts the local average intensity surrounding each
vesicle, using the Priism software as described elsewhere
(Levkowitz et al., 1998
;
Zamir et al., 2000
); (b)
segmentation and quantitation; vesicles in fluorescently labeled cells were
segmented using the `water' software
(Levkowitz et al., 1998
;
Zamir et al., 2000
) in order
to measure the area and the average fluorescence intensity of each individual
vesicle. The parameters of the water software were adjusted to the dimensions
of the vesicles as described (Levkowitz et
al., 1998
; Zamir et al.,
2000
); (c) `Spectral' presentation of fluorescence intensities: in
order to compare fluorescence intensities visually, filtered images were
presented using a blue-to-red linear spectrum scale; (d) fluorescence ratio
imaging; to analyze the relationships between heparanase-labeled structures
and LysoTracker- or TRIT-ConA-labeled vesicles, ratio images
(heparanase:LysoTracker or heparanase:TRIT-ConA) were calculated as described
previously (Levkowitz et al.,
1998
; Zamir et al.,
2000
) and presented in a spectral, log scale color look-up table
that ranged from blue for low heparanase:fluorescent label ratios (
0.1) to
red for high heparanase:fluorescent label ratios (
10). To utilize this
two-order of magnitude range optimally and to compensate for the differences
in photon yields of different fluorescent labels, all the ratios were
normalized linearly by a constant that shifted their average ratio toward a
value around 1.
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Results |
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Although untreated HFF do not express the heparanase protein (Fig. 2A), western blot analysis revealed that the 65 kDa heparanase precursor became associated with the cells and was already partially converted to a 50 kDa form after 30 minutes (Fig. 2A). Analysis of longer incubation time points indicated that although the amount of heparanase precursor associated with the cells remained constant, the amount of 50 kDa processed form increased significantly (Fig. 2A).
|
To investigate whether the enzyme is associated with the cells, and/or undergoes internalization, the subcellular localization of heparanase was determined by immunofluorescent staining. For this purpose, HFF incubated for 30, 60 and 120 minutes with the heparanase precursor were fixed, permeabilized and stained with anti-heparanase mAb 130. Intracellular accumulation of heparanase was clearly observed in a granular pattern after 60 minutes of incubation and was significantly increased after 120 minutes (Fig. 2B-D). In contrast, non-permeabilized cells exhibited only low levels of diffuse heparanase staining at the different time points (data not shown), confirming internalization of cell-associated heparanase and accumulation in the cytoplasm. Interestingly, only weak immunofluorescent staining of the cells was observed at the same time points (30-120 minutes) when a mAb directed against the N-terminus of heparanase, which is present only in the precursor protein, was utilized (not shown). These findings indicate that the heparanase precursor is proteolytically cleaved, followed by internalization and cytoplasmic accumulation of the processed enzyme. Heparanase-containing cytoplasmic granules were observed even after 16 hours following internalization, suggesting a prolonged cytoplasmic storage (not shown).
The subcellular heparanase-containing granules were characterized using
either LysoTracker (an acidic granules marker) or TRITC-conjugated ConA that
marks endosomes (Barzu et al.,
1996). These dyes were applied to living HFF, which were
simultaneously incubated with the 65 kDa heparanase precursor for 120 minutes.
Following staining with anti-heparanase mAb 130, we observed colocalization of
heparanase-containing granules with the endosomal
(Fig. 3, left panels) but not
lysosomal (Fig. 3, right
panels) labels.
|
In order to further characterize the heparanase-containing granules, we
performed fluorescent labeling of cells incubated for two hours with
extracellular heparanase using antibodies directed against either EEA1 (an
early endosome resident molecule), dextran (an endosomal marker), Lamp-1 (a
protein associated with lysosomes) or cathepsin D (a proteolytic enzyme
located in lysosomes) (Nagamatsu et al.,
2001; Thomsen et al.,
2000
; Reddy et al.,
2001
; Storrie and Desjardins,
1996
). As shown in Fig.
4, the heparanase-containing granules were negative for EEA1
staining. In a similar manner, heparanase-containing granules did not contain
Lamp-1 or cathepsin D, both molecules found in lysosomes
(Table 1). In contrast, the
endosomal marker dextran accumulated in the heparanase-containing granules
(Table 1), again indicating
that the enzyme is stored primarily in the endosomal compartment.
|
|
The most common endocytosis process is relatively rapid, depends on the
integrity of the actin cytoskeleton and results in the formation of either
clathrin-coated or non-coated membrane pits
(Corvera et al., 1999). Another
major endocytosis pathway is mediated through caveolae, specialized membrane
invaginations previously shown to be involved in HSPGs-mediated uptake
(Williams and Fuki, 1997
). To
test the possibility that heparanase is endocytosed via caveolae we have
double-labeled cells for heparanase using mAb 130 and for caveolae using
anti-caveolin-1 polyclonal antibodies, and we found no colocalization
(Table 1). Incubation with
cytochalasin D, which destroys the actin cytoskeleton, completely inhibited
heparanase endocytosis (Fig.
5). In contrast, nocodazole, which inhibits microtubules
integrity, had no effect on heparanase endocytosis
(Fig. 5).
|
As described above, heparanase-containing endosomes were observed
relatively late, only after 60 minutes of incubation of the cells with the
heparanase precursor enzyme. Since heparanase processing apparently involves
at least two distinct events proteolytic activation and endocytosis
we examined whether cleavage of the heparanase N-terminus affects the
cytoplasmic accumulation rate of the enzyme. For this purpose, HFF were
incubated with the 50 kDa truncated heparanase form for 30, 60 and 120
minutes, followed by staining with anti-heparanase mAb 130, as described
above. Accumulation of heparanase-containing granules was already observed
after 30 minutes, indicating that uptake of the processed enzyme is
significantly more efficient then that of the non-processed precursor
(Fig. 6). Unlike the latent
enzyme, endocytosis of processed heparanase reaches saturation after 30
minutes, with respect to the number and size of endosomes, as well as their
heparanase content (Fig. 6). Taken together, the data indicate that proteolytic activation of the enzyme on
the cell surface is a rate-limiting step in heparanase endocytosis. Also, the
experiment demonstrates that uptake of the enzyme does not involve the
N-terminal portion of the protein but rather recognizes the C-terminal region,
containing both the heparinbinding domain and the active site of heparanase
(Hulett et al., 2000).
|
Previous studies have shown that at a neutral pH heparanase can bind but
not degrade HSPGs (Gilat et al.,
1995). Thus, cell-surface HSPGs might function as receptors that
mediate the uptake of extracellular heparanase. In fact, cell-surface HSPGs
were shown to facilitate the binding and endocytosis of a variety of
extracellular ligands, including growth factors (bFGF), lipoproteins (LDL) and
various pathogens (Colin et al.,
1999
; David et al., 1993; Datta
et al., 2000
; Fuki et al.,
2000
; Ji et al.,
1998
; Sperinde and Nugent,
2000
; Shukla et al.,
1999
; Summerford et al.,
1999
). We therefore examined whether cell association with
heparanase and its subsequent endocytosis depend on cell surface HSPGs. For
this purpose, the 65 kDa heparanase precursor was pre-incubated for 30 minutes
at 37°C with various concentrations of heparin (0.5-200 µg/ml). The
pretreated enzyme was then incubated with the cells for 120 minutes in the
continuous presence of heparin, followed by immunofluorescent staining of
heparanase. As shown in Fig.
7A-C, heparanase endocytosis was completely blocked in the
presence of 10 µg/ml heparin. Similarly, pretreatment of the cells with
bacterial heparinase III inhibited heparanase endocytosis (not shown).
Previous studies indicated that mannose-6-phosphate may mediate heparanase
association with cells (Bartlett et al.,
1995
). We did not observe any inhibition of heparanase endocytosis
in the presence of mannose-6-phosphate (not shown).
|
Heparanase endocytosis is accompanied by its processing
(Fig. 2). We examined whether
association of the 65 kDa heparanase with the cells and/or its processing are
inhibited by heparin. As shown in Fig.
7D (lane 2), a significant reduction ( 50% as evaluated by
densitometry) in the amount of heparanase associated with the cells was
observed when the cells were incubated for 120 minutes with the enzyme in the
presence of 1 µg/ml heparin, compared with control cells that were
incubated with the enzyme in the absence of heparin
(Fig. 7D, lane 1). However, a
significant amount of heparanase was still associated with the cells following
heparin treatment (Fig. 7D,
lane 2). A similar partial inhibition of binding was observed when the cells
were incubated with heparanase at 4°C
(Fig. 7D, lane 3) rather then
37°C. Both heparin (Fig.
7A-C) and incubation at 4°C (not shown) almost totally
inhibited endocytosis but did not affect the cleavage of the latent heparanase
into an active form. The ratio of cell-associated heparanase precursor to the
processed form was similar in control cells, cells incubated with heparin or
cells kept at 4°C (Fig.
7D). In a similar manner, cytochalasin D, which totally inhibited
heparanase endocytosis (Fig.
5), had no effect on its extracellular processing (not shown).
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Discussion |
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We have demonstrated that extracellular heparanase is both activated and endocytosed by primary human fibroblasts. The amount of heparanase precursor associated with the cells reaches saturation within 30 minutes and remains steady, indicating a relatively limited precursor-binding capacity. On the other hand, the cellular content of the processed enzyme significantly increases during 120 minutes of incubation, apparently because of cytoplasmic accumulation of heparanase in endosomes.
Heparanase endocytosis seems to share some of the diverse properties of
HSPG-mediated endocytosis pathways
(Bernfield et al., 1999; David
et al., 1993). Heparanase internalization occurs through an endocytosis
pathway, involving the actin cytoskeleton and is not mediated by caveolae.
This internalization mechanism differs from that previously suggested for
HSPG-mediated uptake of bFGF or LDL via caveolae (Williams et al., 1997) or
the perlecan HSPG-mediated endocytosis that is not inhibited by cytochalasin D
(Fuki et al., 2000
). Moreover,
endocytosed heparanase did not translocate into the lysosomal compartment,
even after an extended incubation period. This finding is in agreement with a
previous study indicating a relative late entry of bFGF into lysosomes during
HSPG-mediated endocytosis (Gleizes et al.,
1995
). Also, heparanase endocytosis was not blocked by
mannose-6-phosphate, which may facilitate lysosomal targeting. These results
indicate that any heparanase activity exerted by the cells during the 2 hour
incubation period originates either from the cell surface or endosomes.
While pre-incubation of heparanase with heparin completely abolished
endocytosis, binding of heparanase to the cells was only partially inhibited,
suggesting an involvement of non-heparan-sulfate binding sites to heparanase
on the cell surface. Conversion of heparanase from the latent precursor form
to an active enzyme was not inhibited by heparin. Since heparanase proteolytic
processing took place even when its endocytosis was completely blocked, we
conclude that proteolytic activation of the enzyme is a cell surface or
juxta-surface event. Heparanase processing may be essential, but not
sufficient to induce its endocytosis, which apparently depends on a proper
interaction with cell-surface HSPGs (Fig.
8). Previous studies showed that cell-surface HSPGs serve as
co-receptors, partially responsible for surface binding of various
heparin-binding extracellular molecules
(Colin et al., 1999;
Datta et al., 2000
;
Fuki et al., 2000
;
Ji et al., 1998
;
Sperinde and Nugent, 2000
).
Moreover, HSPGs may also be critical for the internalization of these
molecules into the cells. For example, HSPGs mediate the uptake and
degradation of LDL (Fuki et al.,
2000
) and bFGF by fibroblasts. Also, HSPGs serve as a primary
attachment receptor for herpes simplex type 1 virus and adeno-associated virus
type 2 (Shukla et al., 1999
;
Summerford et al., 1999
).
|
Endocytosis of the processed enzyme was found to be significantly more efficient then that of its precursor protein, already reaching saturation after 30 minutes. This may stem from the fact that the precursor enzyme is proteolytically cleaved outside the cell, which may be the rate-limiting step. Another possible explanation is that binding of the processed enzyme is more efficient in terms of affinity and/or capacity, in other words, more enzyme can bind to HSPGs or other putative receptor(s). Once endocytosed, heparanase can accumulate within the cytoplasm for a prolonged period of time (at least 16 hours).
Our results indicate that the presence of active heparanase in cells is not
necessarily regulated at the gene expression level. Thus, cells that lack
endogenous heparanase can acquire heparanase activity by surface binding,
activation and internalization of exogenous heparanase. Previous studies have
shown that heparanase is excreted by activated platelets and cells of the
immune system (Parish et al.,
2001; Vlodavsky et al.,
1992
) and by highly metastatic tumor cells
(Dempsey et al., 2000a
;
Irimura et al., 1986
;
Parish et al., 2001
;
Vlodavsky et al., 1990
;
Vlodavsky et al., 1994
;
Vlodavsky and Friedman, 2001
).
Interestingly, cell surface binding, activation and endocytosis of exogenous
heparanase appears to alter the metastatic potential of malignant cells from a
low metastatic into a highly metastatic phenotype (O.Y.-Z., unpublished).
Thus, cancer cells may acquire a highly invasive phenotype by endocytosis and
storage of heparanase secreted from stromal cells
(Marchetti et al., 2000
).
Heparanase endocytosis by normal cells may regulate the course of
physiological processes such as wound healing and angiogenesis. During the
initial stages of tissue remodeling and repair, the damaged tissue is
populated by platelets, neutrophils and macrophages, which release lysosomal
enzymes. In these inflammatory, slightly acidic conditions, the extracellular
latent heparanase is readily converted to the active form. We show here that
heparanase may also be activated on the surface of primary human cells and not
necessarily under inflammatory conditions. The active enzyme degrades heparan
sulfate and thereby may contribute to the inflammatory process by enabling
neutrophils and lymphocytes to migrate from the vasculature into the target
tissue (Parish et al., 2001;
Vlodavsky et al., 1992
).
However, at later stages, migration of fibroblasts and endothelial cells
occurs, followed by deposition of ECM macromolecules
(Eckes et al., 2000
).
Heparanase activity may therefore constrain this later response (e.g. ECM
deposition and assembly) via degradation of the ECM scaffold. Thus, an
efficient uptake of the active form of heparanase (and potentially other
ECM-degrading enzymes) may accelerate the late stages of wound healing by
enabling tissue remodeling and reorganization to occur. In agreement with this
concept, we found that endocytosis of processed, highly active heparanase by
primary human skin fibroblasts is significantly more efficient than that of
the non-processed precursor.
Unlike matrix metalloproteases involved in tumor cell metastasis and
angiogenesis, heparanase is a well conserved protein that exists as a single
functional endoglycosidase, utilized by normal and malignant cells to degrade
HSPGs (Dempsey et al., 2000a;
Fairbanks et al., 1999
;
Hulett et al., 1999
;
Kussie et al., 1999
;
Parish et al., 2001
; Toyoshima
et al., 1999; Vlodavsky et al.,
1999a
; Vlodavsky and Friedman,
2001
). Thus, heparanase represents an attractive target for the
development of anti-tumor and anti-inflammatory drugs. Understanding the
intracellular localization of heparanase and its endocytotic mechanisms under
normal and pathological conditions may thus provide novel therapeutic
approaches. In fact, heparanase inhibitors (e.g. non-anticoagulant species of
heparin, suramine, castanospermine and phosphomannopenpaose sulfate) were
previously shown to inhibit autoimmune disorders (i.e. EAE and adjuvant
arthritis) and cancer metastasis in animal models
(Bartlett et al., 1995
;
Parish et al., 1999
;
Parish et al., 2001
;
Vlodavsky et al., 1992
;
Vlodavsky et al., 1994
;
Vlodavsky and Friedman, 2001
).
Our study suggests that modulators of heparanase uptake and/or its proteolytic
activation should be looked for as such compounds may be utilized as
regulators of tissue repair, vascularization and immune surveillance, as well
as for pathological conditions such as autoimmunity and metastasis.
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Footnotes |
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References |
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---|
Bartlett, M. R., Cowden, W. B. and Parish, C. R. (1995). Differential effects of the anti-inflammatory compounds heparin, mannose-6-phosphate, and castanospermine on degradation of the vascular basement membrane by leukocytes, endothelial cells, and platelets. J. Leukoc. Biol. 57,207 -213.[Abstract]
Barzu, T., Pascal, M., Maman, M., Roque, C., Lafont, F. and Rousselet, A. (1996). Entry and distribution of fluorescent antiproliferative heparin derivatives into rat vascular smooth muscle cells: comparison between heparin-sensitive and heparin-resistant cultures. J. Cell. Physiol. 167,8 -21.[Medline]
Bernfield, M., Gotte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J. and Zako, M. (1999). Functions of cell surface heparan sulfate proteoglycans. Annu. Rev. Biochem. 68,729 -777.[Medline]
Colin, S., Jeanny, J. C., Mascaelli, F., Vienet, R., Al-Mahmood,
S., Courtois, Y. and Labarre, J. (1999). In vivo involvement
of heparan sulfate proteoglycan in the bioavailability, internalization, and
catabolism of exogenous basic fibroblast growth factor. Mol.
Pharmacol. 55,74
-82.
Corvera, S., D'Arrigo, A. and Stenmark, H. (1999). Phosphoinositides in membrane traffic. Curr. Opin. Cell Biol. 11,460 -465.[Medline]
David, G. (1993). Integral membrane heparan
sulfate proteoglycans. FASEB J.
7,1023
-1030.
Dempsey, L. A., Brunn, G. J. and Platt J. L. (2000a). Heparanase, a potent regulator of cell matrix interactions. Trends Biochem. Sci. 25,349 -355.[Medline]
Dempsey, L. A., Plummer, T. B., Coombes, S. L. and Platt, J.
L. (2000b). Heparanase expression in invasive trophoblasts
and acute vascular damage. Glycobiol.
10,467
-475.
Datta, G., Chaddha, M., Garber, D. W., Chung, B. H., Tytler, E. M., Dashti, N., Bradley, W. A., Gianturco, S. H. and Anantharamaiah, G. M. (2000). The receptor binding domain of apolipoprotein E, linked to a model class A amphipathic helix, enhances internalization and degradation of LDL by fibroblasts. Biochemistry 39,213 -220.[Medline]
Eckes, B., Zigrino, P., Kessler, D., Holtkotter, O., Shephard, P., Mauch, C. and Krieg, T. (2000). Fibroblast-matrix interactions in wound healing and fibrosis. Matrix Biol. 19,325 -332.[Medline]
Fairbanks, M. B., Mildner, A. M., Leone, J. W., Cavey, G. S.,
Mathews, W. R., Drong, R. F., Slightom, J. L., Bienkowski, M. J., Smith, C.
W., Bannow, C. A. and Heinrikson, R. L. (1999). Processing of
the human heparanase precursor and evidence that the active enzyme is a
heterodimer. J. Biol. Chem.
274,29587
-29590.
Friedmann, Y., Vlodavsky, I., Aingorn, H., Aviv, A., Peretz, T.,
Pecker, I. and Pappo, O. (2000). Expression of heparanase in
normal, dysplastic and neoplastic human colon mucosa and stroma.
Am. J. Pathol. 157,1167
-1175.
Fuki, I. V., Iozzo, R. V. and Williams, K. J.
(2000). Perlecan heparan sulfate proteoglycan. A novel receptor
that mediates a distinct pathway for ligand catabolism. J. Biol.
Chem. 275,25742
-25750.
Gilat, D., Heshkoviz, R., Goldkorn, I., Cahalon, L., Korner, G., Vlodavsky, I. and Lider, O. (1995). Molecular behavior adapts to context: heparanase functions as an extracellular matrix-degrading enzyme or as a T cell adhesion molecule, depending on the local pH. J. Exp. Med. 181,1929 -1934.[Abstract]
Gleizes, P. E., Noaillac-Depeyere, J., Amalric, F. and Gas, N. (1995). Basic fibroblast growth factor (FGF-2) internalization through heparan sulfate proteoglycans-mediated pathway: an alternative ultrastructural approach. Eur. J. Cell Biol. 66,47 -59.[Medline]
Hulett, M. D., Freeman, C., Hamdorf, B. J., Baker, R. T., Harris, M. J. and Parish, C. R. (1999). Cloning of mammalian heparanase, an important enzyme in tumor invasion and metastasis. Nat. Med. 5,803 -809.[Medline]
Hulett, M. D., Hornby, J. R., Ohms, S. J., Zuegg, J., Freeman, C., Gready, J. E. and Parish, C. R. (2000). Identification of active-site of the prometastatic endoglycisidase heparanase. Biochemistry 39,15659 -15667.[Medline]
Iozzo, R. V. (1998). Matrix proteoglycans: from molecular design to cellular function. Annu. Rev. Biochem. 67,609 -652.[Medline]
Irimura, T., Nakajima, M. and Nicolson, G. L. (1986). Heparanases and tumor metastasis. Biochemistry 25,5322 -5328.[Medline]
Ji, Z. S., Pitas, R. E. and Mahley, R. W.
(1998). Differential cellular accumulation/retention of
apolipoprotein E mediated by cell surface heparan sulfate proteoglycans.
Apolipoproteins E3 and E2 greater than e4. J. Biol.
Chem. 273,13452
-13460.
Katz, B.-Z., Zamir, E., Bershadsky, A., Kam, Z., Yamada, K. M.
and Geiger, B. (1999). Physical state of the extracellular
matrix regulates the structure and molecular composition of cell-matrix
adhesions. Mol. Biol. Cell
11,1047
-1060.
Kjellen, L. and Lindahl, U. (1991). Proteoglycans: structures and interactions. Annu. Rev. Biochem. 60,443 -475.[Medline]
Kussie, P. H., Hulmes, J. D., Ludwig, D. L., Patel, S., Navarro, E. C. Seddon, A. P., Giorgio, N. A. and Bohlen, P. (1999). Cloning and functional expression of a human heparanase gene. Biochem. Biophys. Res. Commun. 261,183 -187.[Medline]
Levkowitz, G., Waterman, H., Zamir, E., Kam, Z., Oved, S.,
Langdon, W. Y., Beguinot, L., Geiger, B. and Yarden, Y.
(1998). c-Cbl/Sli-1 regulates endocytic sorting and
ubiquitination of the epidermal growth factor receptor. Genes
Dev. 12,3663
-3674.
Marchetti, D., Li, J. and Shen, R. (2000).
Astrocytes contribute to the brainmetastatic specificity of melanoma cells by
producing heparanase. Cancer Res.
60,4767
-4770.
McKenzie, E., Tyson, K., Stamps, A., Smith, P., Turner, P., Barry, R., Hircock, M., Patel, S., Barry, E., Stubberfield, C. et al. (2000). Cloning and expression profiling of hpa2, a novel mammalian heparanase family member. Biochem. Biophys. Res. Commun. 276,1170 -1177.[Medline]
Nagamatsu, S., Nakamichi, Y., Watanabe, T., Matsushima, S.,
Yamaguchi, S., Ni, J., Itagaki, E. and Ishida, H. (2001).
Localization of cellubrevin-related peptide, endobrevin, in the early endosome
in pancreatic beta cells and its physiological function in exo-endocytosis of
secretory granules. J. Cell Sci.
114,219
-227.
Parish, C. R., Freeman, C., Brown, K. J., Francis, D. J. and
Cowden, W. B. (1999). Identification of sulfated
oligosaccharide-based inhibitors of tumor growth and metastasis using novel in
vitro assays for angiogenesis and heparanase activity. Cancer
Res. 59,3433
-3441.
Parish, C. R., Freeman, C. and Hulett, M. D. (2001). Heparanase: a key enzyme involved in cell invasion. Biochem. Biophys. Acta 1471,M99 -M108.[Medline]
Reddy, A., Caler, E. V. and Andrews, N. W. (2001). Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes. Cell 106,157 -169.[Medline]
Shukla, D., Liu, J., Blaiklock, P., Shworak, N. W., Bai, X., Esko, J. D., Cohen, G. H., Eisenberg, R. J., Rosenberg, R. D. and Spear, P. G. (1999). A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry. Cell 99, 13-22.[Medline]
Sperinde, G. V. and Nugent, M. A. (2000). Mechanisms of fibroblast growth factor 2 intracellular processing: a kinetic analysis of the role of heparan sulfate proteoglycans. Biochemistry 39,3788 -3796.[Medline]
Storrie, B. and Desjardins, M. (1996). The biogenesis of lysosomes: is it a kiss and run, contiuous fusion and fission process? BioEssays 18,895 -903.[Medline]
Summerford, C., Bartlett, J. S. and Samulski, R. J. (1999). Alpha Vbeta5 integrin: a co-receptor for adeno-associated virus type 2 infection. Nat. Med. 5, 78-82.[Medline]
Thomsen, P., van Deurs, B., Norrild, B. and Kayser, L. (2000). The HPV16 E5 oncogene inhibits endocytic trafficking. Oncogene 19,6023 -6032.[Medline]
Toyoshima, M. T. and Nakajima, M. (1999). Human
heparanase. Purification, characterization, cloning, and expression.
J. Biol. Chem. 274,24153
-24160.
Vlodavsky, I., Korner, G., Ishai-Michaeli, R., Bashkin, P., Bar-Shavit, R. and Fuks Z. (1990). Extracellular matrix-resident growth factors and enzymes: Possible involvement in tumor metastasis and angiogenesis. Cancer Metastasis Rev. 9, 203-226.[Medline]
Vlodavsky, I., Eldor, A., Haimovitz-Friedman, A., Matzner, Y., Ishai-Michaeli, R., Lider, O., Naparstek, Y., Cohen, I. R. and Fuks, Z. (1992). Expression of heparanase by platelets and circulating cells of the immune system: possible involvement in diapedesis and extravasation. Invasion Metastasis 12,112 -127.[Medline]
Vlodavsky, I., Bar-Shavit, R., Korner, G. and Fuks, Z. (1993). Extracellular matrix-bound growth factors, enzymes and plasma proteins. In Basement membranes: Cellular and molecular aspects (eds D. H. Rohrbach and R. Timpl), pp.327 -343. Orlando: Academic Press.
Vlodavsky, I., Mohsen, M., Lider, O., Svahn, C. M., Ekre, H. P., Vigoda, M., Ishai-Michaeli, R. and Peretz, T. (1994). Inhibition of tumor metastasis by heparanase inhibiting species of heparin. Invasion Metastasis 14,290 -302.[Medline]
Vlodavsky, I., Miao, H.-Q., Medalion, B., Danagher, P. and Ron, D. (1996). Involvement of heparan sulfate and related molecules in sequestration and growth promoting activity of fibroblast growth factor. Cancer Metastasis Rev. 15,177 -186.[Medline]
Vlodavsky, I., Friedmann, Y., Elkin, M., Aingorn, H., Atzmon, R., Ishai-Michaeli, R., Bitan, M., Pappo, O., Peretz, T., Michal, I., Spector, L. and Pecker, I. (1999a). Mammalian heparanase: gene cloning, expression and function in tumor progression and metastasis. Nat. Med. 5,793 -802.[Medline]
Vlodavsky, I. (1999b). Preparation of extracellular matrices produced by cultured corneal endothelial and PF-HR9 cells. In Current Protocols in Cell Biology (eds J. S. Bonifacino, M. Dasso, J. B. Harford, J. Lippincott-Schwartz and K. M. Yamada), pp. 10.4.1-10.4.14. New York: John Wiley and Sons.
Vlodavsky, I. and Friedman, Y. (2001).
Molecular properties and involvement of heparanase in cancer metastasis and
angiogenesis. J. Clin. Invest.
108,341
-347.
Wight, T. N., Kinsella, M. G. and Qwarnstromn, E. E. (1992). The role of proteoglycans in cell adhesion, migration and proliferation. Curr. Opin. Cell Biol. 4, 793-801.[Medline]
Williams, K. J. and Fuki, I. V. (1997). Cell-surface heparan sulfate proteoglycans: dynamic molecules mediating ligand catabolism. Curr. Opin. Lipidol. 8, 253-262.[Medline]
Zamir, E., Katz, M., Posan, Y., Erez, N., Yamada, K. M., Katz, B.-Z., Lin, S., Lin, C. D., Bershadsky, A., Kam, Z. and Geiger, B. (2000) Dynamics and segregation of cell-matrix adhesions in cultured fibroblasts. Nat. Cell Biol. 2, 191-196.[Medline]