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Biochemistry and Biophysics |
Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201 [R. M., H. B., S. S.], and Division of Cancer Genetics, Dana-Farber Cancer Institute, Boston, Massachusetts 02115 [R. S.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Biological studies demonstrate a tumor-suppressive role of maspin,
acting at the levels of tumor invasion and metastases. Mammary
carcinoma MDA-MB-435 cells transfected with maspin cDNA are
significantly inhibited in invasion and motility assays in
vitro and are inhibited in tumor growth and metastasis in nude
mice (1
, 5
, 7)
. It has been shown independently
that induction of maspin expression in four different breast tumor cell
lines by 
-linolenic acid dramatically reduces cell motility in
vitro (8)
. Furthermore, purified recombinant maspin
proteins produced in three different expression systems inhibit the
invasion and motility of an array of breast cancer cell lines as well
as three prostatic carcinoma cell lines in culture (5
, 9)
.
A study by Seftor et al. (10)
shows that
purified recombinant maspin provokes changes of the integrin profiles
on the MDA-MB-435 cell surface in favor of a more benign epithelial
phenotype. Recently, Zhang et al. (11)
have
shown that recombinant mouse maspin inhibits prostate tumor mitogenesis
in vitro and inhibits human prostate tumor-induced
neovascularization in a xenograft mouse model. Hence, maspin protein,
either reexpressed in carcinoma cells or as an exogenously added
purified reagent, may find novel therapeutic applications in the
intervention of both breast and prostate cancers.
The enthusiasm about the potential therapeutic value of maspin in treating human malignancy is hampered by the lack of understanding of the molecular mechanism of maspin. Central to this issue, maspin does not act as a classical serpin in cell-free conditions, i.e., it does not inhibit a series of serine proteases including tPA and uPA (12, 13, 14) , although the RSL sequence of maspin is critical for its biological activities (1 , 5 , 11) . After intensive research, tPA preactivated by fibrinogen or poly-L-lysine has been identified as a target of maspin (12) . Interestingly, detailed kinetic data suggest that recombinant maspin may use its two segregated domains to interact with the catalytic and regulatory domains of tPA, respectively (12) . The complex interaction between recombinant maspin and tPA suggests that the novel proteolytic inhibitory property of maspin depends not only on its intrinsic structural features but also on the microenvironment of the target enzyme.
We report here that rMaspin(i) competitively inhibited plasminogen activation mediated by the surface of prostate carcinoma DU145 cells. In contrast, rMaspin(i) did not inhibit purified uPA, tPA, and plasmin in cell-free conditions. Under our culture conditions, the DU145 cell surface used primarily uPA, but not tPA, to convert plasminogen to plasmin. We show that rMaspin(i) forms a stable complex with uPA in DU145 cell lysate. Furthermore, the maspin proteolytic inhibitory effect correlated quantitatively with the inhibition of cell motility in vitro. To our knowledge, this is the first evidence that maspin inhibits uPA. Moreover, our data demonstrate an important role of the prostate carcinoma cell surface in mediating the inhibitory interaction between maspin and uPA. Thus, future maspin-based therapeutic strategies may prove useful in blocking the invasion and metastasis of uPA-positive prostate carcinomas.
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MATERIALS AND METHODS |
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Chemicals and Reagents.
Polyclonal antibodies against the RSL sequence of maspin protein Abs4A
were produced and purified as described previously (1)
.
Purified recombinant PAI-1 was a generous gift from Dr. Thomas Reilly
(Dupont Merck, Wilmington, DE). Reagents of molecular biology
grade for protein gel electrophoresis, protein staining, and protein
concentration analysis were purchased from Bio-Rad. Trypan blue
solution was purchased from Flow Laboratories (McLean, VA). Proteolytic
enzymes, protease substrates, and specific protease inhibitors
purchased from American Diagnostica include recombinant single-chain
tPA, high molecular weight recombinant uPA, synthetic chromogenic
peptide substrate of uPA (Spectrozyme UK), synthetic chromogenic
peptide substrate of tPA (Spectrozyme tPA), Glu-type plasminogen,
purified plasmin, chromogenic plasmin substrate (Spectrozyme PL),
purified tPA-neutralizing monoclonal antibody (catalogue number 374B),
purified uPA-neutralizing monoclonal antibody (catalogue number
394), and purified recombinant human PAI-2. Unless otherwise specified,
all other chemicals and reagents were of the highest purity and
obtained from Sigma.
Purification of Monomeric rMaspin(i).
rMaspin(i) was first purified by a two-step chromatographic procedure
as described previously (9)
. This rMaspin(i) preparation
was further purified by a heparin column procedure using the
medium-pressure BioLogic automated chromatographic system (Bio-Rad).
Briefly, 10 mg of the initially purified rMaspin(i) were loaded onto a
heparin column. The unbound molecules were removed from the column by
an extensive washing (20 column volumes) with 20 mM
Tris-HCl (pH 7.6). The bound proteins were eluted by a NaCl gradient
(0–0.5 M) in 20 mM Tris-HCl (pH 7.6). The
fractions eluted at 0.1 M NaCl contained the purified
monomeric rMaspin(i). The purified rMaspin(i) was sterilized by
filtration through a 0.2 µm membrane for subsequent biochemical and
biological analyses.
Coupled Colorimetric Assay for Plasminogen Activation in
Conditioned Culture Medium.
Aliquots of 80 µl of reaction buffer [50 mM Tris-HCl (pH
7.5), 0.5 mM EDTA, 0.2 µg/ml leupeptin, 0.32
µM Glu-plasminogen, 0.2 mM Spectrozyme PL,
and 0.1% Triton X-100] were added to the wells of a 96-well
microplate. Glu-plasminogen was omitted in the blank reactions. The
enzyme reaction was initiated by the addition of 20 µl of the
concentrated CM of DU145 cells. The photometric absorbance of the
reaction mixtures at 405 nm was monitored at 37°C over the next 20
min using a Bio-Rad BenchMark microplate reader.
Detection of Cell Surface-associated Plasminogen Activators by
CAA.
Cells were seeded at 10,000 cells/well in 96-well plates in the
maintenance medium and allowed to reach approximately 90% confluence.
The cells were washed once with PBS and analyzed in a PBS-based
reaction buffer (100 µl/well) containing 0.8 mM
MgCl2 and 0.2 µg/ml leupeptin. The reactions
were initiated by the addition of the chromogenic amidolytic substrate
specific for either uPA (Spectrozyme UK) or tPA (Spectrozyme tPA) to a
final concentration of 0.2 mM. No amidolytic substrate was
added in the blank reactions. The photometric absorbance of the
reaction mixtures at 405 nm was monitored at 37°C over the next 30
min.
CCA-CSPA.
Cells were prepared as described for the CAA. A total of 100 µl of
the reaction mixture (PBS, 0.2 mM Spectrozyme PL, 0.32
µM Glu-plasminogen, 0.8 mM
MgCl2, 0.2 µg/ml leupeptin, and various protein
factors at the indicated final concentrations) was added to the cells
in each well. The reaction was initiated by the addition of
Glu-plasminogen, which was omitted in the blank reactions. The
photometric absorbance of the reaction mixtures at 405 nm was monitored
at 37°C over the next 20 min.
Chemical Cross-linking Assay.
One hundred µg of the total lysate of DU145 cells were incubated in
the presence or absence of 5 µg of rMaspin(i) at 37°C for 30
min. In parallel, 1 µg of purified uPA protein was incubated with 5
µg of rMaspin(i) (or BSA as a negative control) at 37°C for 30 min.
The above-mentioned reaction mixtures were incubated with 0.1%
glutaraldehyde on ice for 30 min and then quenched by glycine (pH 8.0)
at a final concentration of 100 mM. The resulting mixtures
were analyzed by Western blotting with either the polyclonal antibody
against the NH2-terminal variable sequence of
maspin [Abs3A (1)
] or the monoclonal antibody against
the A-chain of uPA (American Diagnostica).
Immunofluorescent Staining of Maspin.
Cells cultured in 8-well chamber slides (Nunc) in the maintenance
medium were washed with PBS and incubated with PBS ± 80
nM rMaspin(i) for 30 min. The cells were gently washed
twice with PBS, fixed with 4% freshly prepared paraformaldehyde/PBS
for 10 min, blotted with Abs4A (5 µg/ml), and subsequently
blotted with R-phycoerythrin-antirabbit IgG (H+L) (Zymed), as
described previously (5)
. The nuclear DNA was briefly
counterstained with Hoechst 33528 at a final concentration of 10
µg/ml. The stained cells were viewed and photographed under a Leica
fluorescent microscope.
ELISA Detection of rMaspin(i) Bound to DU145 Cells.
Cells seeded at 105 cells/well in a 96-well
microplate were incubated in the maintenance medium for 12 h. The
cells were washed with PBS, incubated overnight at 4°C in 100
µl/well SF keratinocyte growth medium containing rMaspin(i) at
various concentrations. The cells were washed gently, blocked with 1%
BSA/PBS for 1 h, blotted with 15 µg/ml Abs4A for 1 h, and
detected by the color reaction of horseradish peroxidase-conjugated
antirabbit IgG as described by Harlow and Lane (15)
.
In Vitro Motility Assays Using MICS.
Briefly, the serum-starved cells (24 h) were seeded onto the 8 µm
polycarbonate membrane insert (precoated with 50 µg/ml Matrigel) of
the MICS chamber. rMaspin(i), Abs4A, or a preincubated mixture of
rMaspin(i) and Abs4A (37°C, 30 min) was added directly to the cell
cultures at the indicated final concentrations. The MICS chamber was
incubated for 6 h at 37°C with 6.5% CO2.
Cells attached to the bottom side of the polycarbonate membrane insert
were fixed, stained, and counted under the microscope as described
previously (5)
.
Miscellaneous Procedures.
Cell staining with SRB was performed as described by Garbin et
al. (16)
. The trypan blue cell staining procedure was
performed as described by Broman et al. (17)
.
Plasminogen-dependent gelatinolytic zymogram was performed as
described previously (18)
. SDS-PAGE of protein was
performed as described by Laemmli (19)
. Nondenaturing
protein gel electrophoresis was performed as described by Lomas
et al. (20)
. Western blotting of maspin was
performed as described previously (1)
. Silver nitrate
staining of protein electrophoresis gels and protein concentration
analyses using the Bio-Rad reagents were performed according to the
manufacturer’s instructions.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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showed that the repurified rMaspin(i) was approximately
100% homogenous (Lane 3), whereas the starting material
(Lane 2) had impurities of higher and lower molecular
weights. The yield of this highly purified rMaspin(i) was estimated to
be at least 10% (w/w) of the total extractable proteins of the host
insect cells.
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was analyzed by nondenaturing protein electrophoresis
followed by silver staining. As shown in Fig. 1B
, only one
protein band was detected (Lane 2). Furthermore, the
purified rMaspin(i) remained homogeneous after being frozen and thawed
several times over the next month (Lane 3). The identity of
rMaspin(i) was further confirmed by Western blotting using the
polypeptide-derived polyclonal antibody against the RSL sequence of
maspin, Abs4A (Ref. 1
; data not shown).
Cell Surface-associate uPA as a Target of rMaspin(i).
To address the possibility that maspin may inhibit cell
surface-associated uPA, it was critical to use a homogenous cell
population that produces uPA as the predominant plasminogen activator.
For this reason, human prostate carcinoma cell line DU145 was chosen
for our study. Fig. 2A
shows the zymographic profile of the plasminogen-dependent
gelatinolytic activities of both the total cell lysate and the SF-CM of
DU145 cells. As compared with the purified tPA and uPA standards, both
the lysate and the SF-CM of DU145 cells contained primarily uPA,
although a trace amount of tPA was also detected.
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, the SF-CM
of DU145 cells was active in converting Glu-plasminogen to plasmin,
which in turn led to the cleavage of Spectrozyme PL, as shown by an
increased photometric absorbance at 405 nm (12)
. The
uPA-neutralizing antibody blocked this plasminogen activation activity
in a dose-dependent manner. In contrast, the tPA-neutralizing antibody
had no significant effect. CAA was also performed. When the chromogenic
amidolytic substrate specific for uPA or tPA was added to the monolayer
of DU145 cells, only the uPA substrate, Spectrozyme UK, was cleaved
(Fig. 2C)
. The uPA-neutralizing antibody, but not a
tPA-neutralizing antibody, blocked the cleavage of Spectrozyme UK. In
parallel experiments, the tPA-specific substrate, Spectrozyme tPA, was
not cleaved.
There appeared to be a specific interaction between rMaspin(i) and the
DU145 cell surface. DU145 cells do not produce endogenous maspin (Refs.
5
and 6
; Fig. 3A
). However, after the cells were treated with rMaspin(i) and
washed thoroughly, cell-bound rMaspin(i) was detected by
immunofluorescent staining under a nonpermeablizing condition (Fig. 3A)
. In addition, when added to the SF culture medium,
rMaspin(i) at low concentrations (<100 nM) bound
to DU145 cells in a dose-dependent manner (Fig. 3B)
. As the
concentration of rMaspin(i) exceeded 100 nM, the
amount of cell surface-bound rMaspin(i) was not further increased,
suggesting that the binding of rMaspin(i) had reached a level of
saturation.
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, rMaspin(i) at a final concentration of 90
nM exhibited a significant inhibition of DU145
cell surface-mediated plasminogen activation. In addition, purified
Abs4A blocked the inhibitory activity of rMaspin(i) in a dose-dependent
manner, whereas the preimmune rabbit IgG had no significant effect. It
is worth noting that throughout this biochemical assay, cells remained
intact, showing little or no plasma membrane permeability by trypan
blue and a full recovery of proliferation in the subsequent cell growth
assay (data not shown). The number of remaining cells in each reaction,
as quantified by the SRB staining procedure (16)
, was used
to normalize the initial velocities of plasminogen activation.
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, the uPA-neutralizing antibody inhibited
DU145 cell-mediated plasminogen activation to a similar extent as
rMaspin(i). Cells preincubated with the uPA-neutralizing antibody were
not further inhibited by rMaspin(i) in this assay. In contrast, the
tPA-neutralizing antibody neither affected the basal level
cell-mediated plasminogen activation nor altered the proteolytic
inhibitory effect of rMaspin(i). These data further support a specific
inhibitory activity of rMaspin(i) toward the cell surface-associated
uPA.
rMaspin(i) did not seem to act as a protease substrate. Fig. 4C
shows the Western blotting of rMaspin(i) before
(Lane 2) and after (Lane 3) the CCA-CSPA,
respectively. rMaspin(i) was not degraded during CCA-CSPA. Under our
assay conditions, the cleavage of the plasmin substrate Spectrozyme PL
was dependent on the exogenous supply of either Glu-plasminogen (data
not shown) or plasmin. The increased cleavage of Spectrozyme PL by
exogenously added pure plasmin, however, was not affected by rMaspin(i)
(Fig. 4B)
. Thus, it seemed unlikely that rMaspin(i) directly
inhibited plasmin.
rMaspin(i) Exerts Differential Effects on Cell Surface-associated
and Free Plasminogen Activators.
Because DU145 cells secreted both tPA and uPA into the culture media
(Fig. 2A)
, it was important to investigate whether
rMaspin(i) inhibited the unbound plasminogen activators. As shown in
Fig. 5A
, rMaspin(i) added at a final concentration of 90
nM did not inhibit the basal level activity of
plasminogen activation in the SF-CM of DU145. The exogenously added tPA
and uPA both significantly stimulated the plasminogen activation in the
CM. However, in each case, rMaspin(i) failed to inhibit the accelerated
plasminogen activation.
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. It is possible that the surface of DU145 cells was
saturated with the endogenous uPA and thus could not accommodate
additional plasminogen activators added exogenously. It has been
reported that, under an acidic condition, the cell surface-associated
uPA may be stripped and subsequently replaced by purified uPA
(21)
. However, treatment of DU145 cells by this method
caused significant damage to the cell membrane, as indicated by an
increased trypan blue permeability (data not shown), thus preventing an
accurate assessment of the binding as well as the activity of uPA on
DU145 cell surface.
To investigate whether rMaspin(i) interacts directly with uPA, chemical
cross-linking experiments were performed. As shown in Fig. 6
, the polymerization of rMaspin(i) in cell-free solution resulted in a
high molecular weight rMaspin(i) oligomer
(Mr >125,000; Fig. 6
, Lane
1). When rMaspin (i) was incubated with purified uPA, three major
polymer forms of rMaspin(i) were detected with molecular weights of
approximately 80,000, 120,000, and >125,000, respectively (Lane
3). This polymerization pattern resembles that seen with the
trypsin-cleaved maspin, a Mr 38,000
NH2-terminal fragment of maspin
(13)
. These data suggest that rMaspin(i) did not form a
complex with soluble uPA but rather was significantly degraded.
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, Lane 5).
When the same membrane containing Lanes 4 and 5
was reblotted with the uPA antibody, a uPA-containing protein of
approximately Mr 100,000 was detected
in the mixture of DU145 cell lysate and rMaspin(i) (Fig. 6
, Lane
7). In the absence of rMaspin(i), only the monomeric uPA of
Mr 55,000 was detected in DU145 cell
lysate (Fig. 6
, Lane 6). Given that the combined molecular
weight of rMaspin(i) and uPA is 97,000, it is likely that the new band
of approximately Mr 100,000 detected
by both Abs3A and the monoclonal antibody against uPA was the complex
between rMaspin(i) and uPA in DU145 cells.
The Biologically Active rMaspin(i) Acts as a Competitive Inhibitor
of the Cell Surface-mediated Plasminogen Activation.
Kinetic analyses were performed to further investigate the mode of
molecular interaction between rMaspin(i) and the DU145 cell
surface-associated uPA. The initial velocities of plasminogen
activation are presented in the Lineweaver-Burk plots as shown in Fig. 7A
. In the presence of subsaturating concentrations of
plasminogen (0–30 nM), the rate of the
plasminogen activation mediated by DU145 cell surface was dependent on
the concentration of Glu-plasminogen, [s]. The slope of the
Lineweaver-Burk plot was initially increased as the concentration of
rMaspin(i) increased from 0 to 90 nM,
demonstrating a dose-dependent inhibition by rMaspin(i). Furthermore,
these plots intersect close to the 1/v axis, suggesting a constant
maximum velocity (Vmax). Thus,
rMaspin(i) appeared to inhibit the cell surface-mediated plasminogen
activation as a competitive inhibitor. As shown in Fig. 7B
,
the replot of the slopes derived from Fig. 7A
versus the concentrations of rMaspin(i) was linear. Based on
Fig. 7B
, an apparent Ki
value of approximately 20 nM was deduced.
Interestingly, rMaspin(i) became less inhibitory as its concentration
was further increased to 240 nM (Fig. 7A)
.
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. The apparent
Ki values for PAI-1 and PAI-2 deduced
from these replots were approximately 10 and 25
nM, respectively.
We have shown previously that rMaspin(i) inhibits the invasion and
motility of three prostatic carcinoma cell lines including DU145
(5)
. To test whether repurified monomeric rMaspin(i)
retained biological activity, we performed the MICS motility assay as
described previously (5)
. As shown in Fig. 8
, the purified monomeric rMaspin(i) exhibited a biphasic dose-dependent
inhibition on the motility of DU145 cells with a nadir at 125
nM. This biphasic dose-response curve coincides with the
biphasic effects of rMaspin(i) on DU145 cell-mediated plasminogen
activation (Fig. 7A)
. In addition, preincubation with Abs4A,
but not with the preimmune rabbit IgG, abolished the inhibitory
activity of rMaspin(i). Thus, the RSL of rMaspin(i) was critical for
both its proteolytic inhibitory activity (Fig. 4A)
and its
biological activity in inhibiting tumor cell motility.
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DISCUSSION |
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The biological activity of rMaspin(i) in inhibiting cell motility and
invasion has been initially localized on the cell membrane
(5)
. Data from the current study demonstrated an important
role for the epithelial cell surface in regulating the inhibitory
interaction between rMaspin(i) and the localized plasminogen activation
system. First, rMaspin(i) bound to intact DU145 cells in a saturable
fashion (Fig. 3)
, suggesting that it may interact with a specific cell
surface-associated molecule. Second, rMaspin(i) exhibited a strong
inhibitory effect on DU145 cell surface-mediated plasminogen
activation, comparable with that of PAI-1 and PAI-2 in parallel
experiments (Fig. 7)
. In contrast, rMaspin(i) did not inhibit free uPA
(12, 13, 14)
in solution, nor did it inhibit the plasminogen
activators secreted by DU145 cells to the CM (Fig. 5)
. Moreover, we
showed that rMaspin(i) forms a complex with the DU145 cell-associated
uPA but acted as a substrate of purified uPA in solution (Fig. 6)
.
The DU145 cell surface may conceivably enhance the inhibitory interaction between rMaspin(i) and its target plasminogen activators via two mechanisms that are not mutually exclusive. On one hand, specific interactions between uPA and its associated proteins on the cell surface may render uPA prone to inhibition by rMaspin(i). It is proposed that the activation of uPA on the cell surface is mediated by a specific receptor, uPAR, which not only controls the localized plasminogen activation (22, 23, 24) but also regulates cell-matrix interaction through its close association with integrins, the transmembrane receptors of extracellular matrix proteins (25) . The elegant study of Schwartz et al. (26) demonstrates that uPAR-bound uPA and free uPA are inhibited by serpin PAI-3 via different mechanisms.
On the other hand, the cell surface microenvironment may play a
critical role in mediating the transition of rMaspin(i) from a latent
conformation to an active conformation. Based on a current paradigm,
the initial docking of the RSL of an inhibitory serpin into the
catalytic site of the target enzyme induces a massive ß sheet
rearrangement of the serpin molecule, leading to a stabilized
enzyme/inhibitor complex (27
, 28)
. Accordingly, based on
protein sequence alignments, serpins such as chick ovalbumin and
maspin, whose RSL may not undergo such conformational rearrangement
without substantial energy compensation, were predicted to be
noninhibitory (29)
. However, a study by Mellet et
al. (30)
showed that chick ovalbumin could be
converted to a potent competitive serine protease inhibitor by heat
denaturation. In fact, the activity of many inhibitory serpins such as
PAI-1 can be greatly enhanced by serpin cofactors (31
, 32)
. Central to the molecular mechanism of maspin, we show here
that the RSL of rMaspin(i) was required for its inhibitory effect on
the motility of DU145 cells (Fig. 8)
and further supports the role of
maspin as an inhibitory serpin.
Furthermore, given the earlier in vitro evidence that the Mr 38,000 NH2-terminal fragment of rMaspin(i) specifically interacts with the regulatory domain of tPA (12) , it is likely that the functional determinants of maspin are not restricted to its RSL. It is worth noting that PAI-1 uses its NH2-terminal domain to interact with heparin and vitronectin (31, 32, 33) . The latter is an extracellular matrix component and regulates cellular functions via its integrin receptor and uPAR (25) . An X-ray crystallographic study of cleaved PAI-1 revealed two additional potential regulatory sites on ß strand 3A and 5A, respectively, that may render PAI-1 inactive on additional intermolecular interaction (34) . Future study is needed to address what other functional domains of rMaspin(i) are involved in its specific interaction with the cell surface-bound plasminogen activators. In addition, because rMaspin(i) stimulates the adhesion of mammary carcinoma MDA-MB-435 cells to fibronectin and alters the integrin profiles on the cell surface (10) , it is of particular importance to investigate whether fibronectin binds directly to maspin and regulates its proteolytic inhibitory activity.
Regarding the biphasic dose effect of rMaspin(i) on DU145 cell
surface-mediated plasminogen activation (Fig. 7A)
and on
cell motility (Fig. 8)
, our chemical cross-linking evidence (Fig. 6)
supports an earlier notion that purified recombinant maspin may undergo
a concentration-dependent polymerization (35)
. Although
the intact monomeric rMaspin(i) used in this study did not undergo fast
spontaneous polymerization and was not degraded during storage (Fig. 1)
, at higher local concentrations, rMaspin(i) may polymerize and
lose its proteolytic inhibitory potency. Meanwhile, current
evidence does not exclude the possibility that the reduced biochemical
and biological activities of rMaspin(i) at higher concentrations are
caused by an increased nonspecific interaction between rMaspin(i) and
the plasminogen activation system because rMaspin(i) has been shown to
use its NH2-terminal domain to interact with the
regulatory domain of tPA (12)
. Furthermore, because the
specific receptors and other molecules associated with the plasminogen
activation system on the cell surface are of fundamental importance in
cell biology (22, 23, 24, 25, 26
, 31, 32, 33
, 36, 37, 38, 39)
, an inhibitor of
plasminogen activators such as rMaspin(i), when added at high doses,
may not only shift the balance against the enzymatic activity of its
target but may also trigger further compensating cellular
responses.
It was advantageous to use DU145 cells to investigate the effect of
maspin on cell-associated uPA because these cells do not produce
endogenous maspin but express abundant uPA and uPAR (40)
.
However, this choice may also limit our conclusion. Future studies are
needed to clarify whether maspin inhibits the uPA associated with other
cell lines and whether maspin competes with other known plasminogen
activators such as PAI-1 and PAI-2 to inhibit the cell surface-bound
uPA. On the other hand, the experimental design of this study may not
appropriately address the potential inhibitory interaction between
maspin and pericellular tPA. tPA is thought to act as a secreted
enzyme, which does not depend on the cell surface for its activity
(41, 42, 43, 44)
. To date, the role of tPA on the cell
surface-mediated plasminogen activation remains unclear. It is
important to point out that DU145 cells produce a trace amount of tPA
(Fig. 2A)
. However, the tPA activity was not detected by
either CCA-CSPA or the cell-based CAA (Fig. 2, B and C)
.
In conclusion, we provide the first evidence that rMaspin(i) acts as a potent inhibitor of cell surface-bound uPA, comparable with PAI-1 and PAI-2 in parallel experiments. We demonstrated that the DU145 cell surface played a critical role in mediating the inhibitory interaction between rMaspin(i) and uPA. Moreover, the proteolytic inhibitory activity of rMaspin(i) correlates quantitatively with its inhibition of the motility of DU145 cells in vitro. These data suggest a molecular mechanism underlying the tumor-suppressive role of maspin. It is reasonable to hypothesize that novel maspin-based therapeutic strategies may prove useful in blocking uPA-mediated tumor invasion and metastasis. To this end, it is of particular importance to note that different inhibitors of plasminogen activators may play distinct roles in tumor progression. For example, PAI-1, along with uPA and uPAR, is causatively involved in the progression of breast cancer and prostate cancer (45, 46, 47, 48) . In contrast, maspin, which is down-regulated in breast cancer and prostate cancer (1 , 5 , 6) , has been shown to have a tumor-suppressive activity. Extensive biological experiments are under way to identify the downstream molecular pathways affected by the inhibition of the cell surface-bound uPA by maspin.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Prostate Cancer Pilot Grant
CA69845 from the National Cancer Institute/Wayne State University (to
S. S.), the Ruth Sager Memorial Fund established by Dr. Arthur B.
Pardee through Wayne State University (S. S.), and NIH Grant CA61253
(to R. S.). 
3 To whom requests for reprints should be
addressed, at Department of Pathology, Wayne State University School of
Medicine, 540 East Canfield Avenue, Detroit, MI 48201. Phone:
(313) 997-8197; Fax: (313) 993-4112; E-mail: ssheng{at}med.wayne.edu
4 The abbreviations used are: PAI, plasminogen
activator inhibitor; rMaspin(i), recombinant human maspin produced in
baculovirus-infected Spodoptera frugiperda Sf9 insect
cells; tPA, tissue-type plasminogen activator; uPA, urokinase-type
plasminogen activator; uPAR, urokinase-type plasminogen activator
receptor; RSL, reactive site loop; SRB, sulforhodamine B; CM,
conditioned medium; SF, serum-free; CM, conditioned medium; CAA,
colorimetric amidolytic activity assay; CCA-CSPA, coupled colorimetric
assay for cell surface-mediated plasminogen activation; MICS, membrane
invasion culture system.
Received 2/17/00. Accepted 7/ 5/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) or the tPA-neutralizing antibody (•) at the indicated
concentrations. The data represent an average result of quadruplicate
repeats, and the error bars stand for the SDs.
C, detection of the amidolytic activity of the
plasminogen activators on the DU145 cell surface using Spectrozyme UK
(•), Spectrozyme UK plus 2.5 µg/ml uPA-neutralizing monoclonal
antibody (
), and Spectrozyme tPA plus 2.5
µg/ml tPA-neutralizing monoclonal antibody (
). Changes of
photometric absorbance at 405 nm for each reaction were normalized by
the number of remaining cells quantified by SRB staining
(A550 nm). The data represent an average of
quadruplicate repeats. The SEs are not shown for clarity.
).
B, rMaspin(i) specifically inhibits uPA on the DU145
cell surface. CCA-CSPA was performed in the presence of Glu-plasminogen
(0.32 µM), plasmin (0.1 nM), rMaspin(i) (90
nM), uPA-neutralizing antibody (2.5 µg/ml), and
tPA-neutralizing antibody (2.5 µg/ml) in the indicated combinations.
C, Western blotting of maspin. Lane 1
shows 0.5 µg of purified rMaspin(i) control with a molecular weight
of Mr 42,000. Lanes 2 and
3 are 35 µl of postassay supernatants from reaction
(
) were normalized as
100%, which represent approximately 12% of the starting cell
population. The motility data from cells treated with rMaspin(i) (
),
rMaspin(i) plus Abs4A (
) are expressed as a percentage of this control. The
data represent the average of three parallel experiments. Error
bars, SEs.