|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 939-945
Apposition-Dependent Induction of Plasminogen Activator
Inhibitor Type 1 Expression: A Mechanism for Balancing Pericellular
Proteolysis During Angiogenesis
By
Eran Bacharach,
Ahuva Itin, and
Eli Keshet
From the Department of Molecular Biology, The Hebrew University,
Hadassah Medical School, Jerusalem, Israel.
 |
ABSTRACT |
Plasminogen-activator inhibitor type I (PAI-1), the primary
inhibitor of urinary-type plasminogen activator, is thought to play an
important role in the control of stroma invasion by both endothelial
and tumor cells. Using an in vitro angiogenesis model of capillary
extension through a preformed monolayer, in conjunction with in situ
hybridization analysis, we showed that PAI-1 mRNA is specifically
induced in cells juxtaposed next to elongating sprouts. To further
establish that PAI-1 expression is induced as a consequence of a direct
contact with endothelial cells, coculture experiments were performed.
PAI-1 mRNA was induced exclusively in fibroblasts (L-cells) contacting
endothelial cell (LE-II) colonies. Reporter gene constructs driven by a
PAI-1 promoter and stably transfected into L-cells were used to
establish that both mouse and rat PAI-1 promoters mediate
apposition-dependent regulation. This mode of PAI-1 regulation is not
mediated by plasmin, as an identical spatial pattern of expression was
detected in cocultures treated with plasmin inhibitors. Because
endothelial cells may establish direct contacts with fibroblasts only
during angiogenesis, we propose that focal induction of PAI-1 at the
site of heterotypic cell contacts provides a mechanism to negate
excessive pericellular proteolysis associated with endothelial cell
invasion.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
A PROTEOLYTIC CASCADE of zymogen
activation triggered by plasminogen activation appears to be a
fundamental component in many situations of cellular invasion.
Urokinase- and tissue-type plasminogen activators (uPA, tPA) convert
plasminogen to plasmin, a serin protease capable of degrading (either
directly or indirectly through the activation of other zymogens) most
of the major components of the extracellular matrix
(ECM).1-3 The pericellular nature of uPA-triggered
proteolysis is explained by the fact that pro-uPA is converted to its
active form upon interaction with a high-affinity cell-surface
receptor.4-6 Uneven cellular distribution of uPA receptors
(uPAR) may also polarize proteolysis to particular regions of cell-cell
contacts or to the leading edge of migrating cells.7-9
Plasminogen activators (PAs) have been implicated as mediators of
extracellular proteolysis during angiogenesis. During angiogenesis, quiescent endothelial cells are induced to locally degrade their basement membrane and to form new blood vessels by sprouting into the
surrounding stroma. PAs are induced in endothelial cells upon stimulation with angiogenic factors like vascular endothelial growth
factor (VEGF) and basic fibroblast growth factor
(bFGF), and a large body of evidence implicates PAs in
angiogenesis-associated matrix degradation and acquisition by
endothelial cells of invasive properties.10-14
Excessive matrix degradation, however, is incompatible with efficient
cellular migration.15 Likewise, the maintenance of a
certain degree of ECM integrity is an essential
requirement for capillary morphogenesis.16,17 Therefore,
means must exist that will protect the stroma from adverse proteolysis
during endothelial cell invasion. Natural PA inhibitors (PAIs) are
thought to act as natural balancers of PA-mediated pericellular
proteolysis.
PA inhibitor type 1 (PAI-1) is a member of the serpin family of
protease inhibitors that reacts specifically with tPA and uPA.2,18 PAI-1 is expressed by various types of cells,
including endothelial cells.19 PAI-I may accumulate within
the tissue environment due to its sequestration and stabilization by
the ECM.20-22 Upon binding of PAI-1 to uPAR-bound uPA, the
complex (which otherwise is active on the cell surface for several
hours) is rapidly internalized and degraded.23,24 Through
the clearance of active proteolytic complexes from the surface of
invading cells, PAI-1 keeps the extent of ECM degradation in check.
Little is known about the regulation of PAI-1 in vivo. To assign a role
for PAI-1 in the restraint of pericellular proteolysis, it is necessary
to invoke that PAI-1 is locally induced at the invasion site and to
propose a mechanism how a tissue senses invasion and, in response,
upregulates PAI-1 expression.
The present study addresses these issues in the context of an in vitro
angiogenesis model and heterotypic cell cultures. It provides evidence
supporting the supposition that PAI-1 is specifically induced at the
invasion site as a result of heterotypic cell contacts.
| |
MATERIALS AND METHODS |
Cell cultures.
Cell lines used were mouse endothelial cells derived from lung
capillaries (LE-II cells)25 and mouse
fibroblasts (L-cells). Cells were grown in Dulbecco's modified
Eagle's medium (DMEM) containing 4.5 g/L glucose, 2 mmol/L glutamine,
2 µg/mL streptomycin, 20 U/mL penicillin, and 10% fetal calf serum
(FCS).
When indicated, the following materials were added to the
medium: -Amino-n-Caproic Acid (A-2504; Sigma Chemical Company, St Louis, MO), Soybean Trypsin Inhibitor (03-048; Beit Haemek - Biological Industries), Tranexamic Acid (A-6516; Sigma),
Trasylol (aprotinin; Bayer), and D-mannose 6-phosphate (M3655; Sigma).
In vitro angiogenesis in aorta explant cultures.
Explants of rat aorta rings (1 mm long) were grown in tissue-culture
chamber slides (Lab-Tek) in DMEM supplemented with 4.5 g/L glucose, 2 mmol/L glutamine, 2 mg/mL streptomycin, 20 µ/mL penicillin, and 10%
FCS, essentially as described by Diglio et al.26 Outgrowth
of aortic cells, mostly muscle cells, during the first 2 weeks was
followed by the formation of vascular-like sprouts extending from the
aorta segment. Cultures were fixed after 3 weeks and processed for
immunohistochemistry and in situ hybridization analysis.
Immunohistochemistry.
Aorta explant cultures were fixed with cold methanol and stained with
rabbit anti-human von Willebrand factor (vWF) antibody (A 082;DAKO,
Glostrup, Denmark) diluted 1:250 in phosphate-buffered saline (PBS). An
immunoperoxidase procedure was used to detect the primary antibody
using VECTASTAIN ABC kit (Vector Laboratories, Burlingame, CA)
according to the manufacturer's instructions.
In situ hybridization.
Aorta explant cultures and heterotypic cell cocultures (both seeded
directly on glass slides) were subjected to in situ hybridization as
previously described.27 DNA fragments used as templates for synthesis of specific complementary RNAs (cRNAs) were a 1.1-kb EcoRI-BglII fragment derived from the coding and
3 noncoding regions of the mouse uPA cDNA28 and a
2.4 Xho I-EcoRI fragment derived from the coding and
3 noncoding regions of the mouse PAI-1 cDNA clone
mr1.29 cDNAs were subcloned onto the polylinker of a PBS
vector (Stratagene, La Jolla, CA) and were linearized by digestion with
the appropriate restriction endonuclease to allow synthesis of a
35S-labeled cRNA in either the antisense or sense
orientation (using T3 or T7 RNA polymerase). The RNA probe was
fragmented by mild alkaline treatment before the hybridization step.
Promoter constructs and stable transfectants.
A mouse PAI-1 promoter-LacZ reporter gene was constructed by fusing a
1.3-kb fragment of mouse PAI-1 promoter (excised with HindIII
from plasmid pH 1.6 [generously provided by Dr M. Cole, Princeton,
NJ]) upstream to a LacZ-coding region (also containing a nuclear
localization signal and SV40-derived polyadenylation signal). A control
plasmid, composed of LacZ gene driven by a ubiquitous promoter, was
constructed by replacing the PAI-1 promoter with a 180-bp-long promoter
of the ribosomal protein S1630 (a kind gift of Dr O. Meyuhas, The Hebrew University). A 2.4-kb rat PAI-1
promoter31 and shorter fragments thereof were fused to a
CAT reporter gene (generously provided by Dr T.D. Gelehrter, University
of Michigan).
L-cells were cotransfected with the test plasmid and a plasmid encoding
a neomycin-resistance, using the poly-L-ornithine/dimethylsulfoxide (DMSO) method. Stably tranfected clones were selected for use in
coculture experiments with LE-II cells. Cells expressing
 -Galactosidase (-Gal) activity were identified using the
substrate X-gal (5-bromo-4-chloro-3-indolyl--galactoside). Chloramphenicol acetyltransferase (CAT) activity in cell extracts was
determined by a standard procedure using a
14C-chloramphenicol substrate, resolution of products by
silica gel chromatography, and relative quantification of reaction
products using a Fuji BAS 1000 image analyzer (Fuji).
Staining of fixed cells for -Gal activity.
Cells were fixed in 50 mmol/L phosphate buffer (pH 7.4) containing
0.2% glutaraldehyde, 2% formaldehyde, and 2 mmol/L MgCl2 at room temperature for 5 minutes. The cells were rinsed three times in
50 mmol/L phosphate buffer (pH 7.4) containing 2 mmol/L MgCl2 and 0.02% Nonidet P-40 at room temperature for 20 minutes each. Cells were stained in 50 mmol/L phosphate buffer (pH 7.4) containing 0.5 mg/mL X-gal, 5 mmol/L potassium ferocyanide, 5 mmol/L
potassium ferricyanide, and 2 mmol/L MgCl2 at 37°C.
| |
RESULTS |
In cultured cells, PAI-1 is expressed in various cell types and is
often produced by the same cells that produce uPA.13 Little
is known, however, regarding the nature of PAI-1-expressing cells in
the context of natural invasive processes. In situ analysis of natural
angiogenic processes (neovascularization of the maternal decidua and
corpus luteum) has shown that whereas uPA is produced by sprouting
endothelial cells, PAI-1 is preferentially expressed in nearby
nonendothelial cells (decidual cells and lutein cells, respectively).27 Notably, PAI-1 was not uniformly expressed in the surrounding tissue but seemed to be more abundant in the vicinity of forming capillaries, suggesting that induction of PAI-1 may
represent a tissue response to cellular invasion.
In situ analysis of natural angiogenesis, however, is short of
providing sufficient resolution to identify cellular contacts of
PAI-1-expressing cells. Therefore, we resorted to in situ analyses of
an in vitro angiogenesis model. To gain better insight into PAI-1
regulation as a function of cellular contexts, we also analyzed native
as well as genetically manipulated heterotypic cell cocultures.
PAI-1 expression is induced in cells juxtaposed to capillary sprouts
in an aorta explant system.
Rat aortic segments were maintained in culture as explants for up to 3 weeks.26,27 Following initial outgrowth of aortic cells,
mostly muscle cells,26 vascular sprouts started to grow radiating from the aorta segment and extending on and through the
established cell sheet. Figure 1A shows a
phase-contrasted image of the culture, depicting the tips of sprouts in
the process of extension through the preformed cell monolayer. Figure
1B shows a region of the aorta explant culture immunostained with the
endothelial cell-specific marker vWF, to highlight the network of
branching endothelial cell sprouts. The advantage of this system over a similar system of vascular sprouting, induced by embedding the aorta
segment in a three-dimensional fibrin gel,32 is that it allows one to examine interactions between vascular sprouts and nonendothelial cells that they contact. Specifically, it enables one to
identify changes in gene expression resulting from encounter with
invading endothelial cell sprouts.
View larger version (110K):
[in this window]
[in a new window]
| Fig 1.
Spatial relationship between uPA-expressing cells and
PAI-expressing cells during capillary sprouting in vitro. Cultured rat aortic ring explants, displaying branching capillary-like sprouts, are
shown 3 weeks after initial culture. (A) A phase-contrasted micrograph
showing the tips of sprouts and the preformed monolayer through which
sprouts extend. (B) Immunostaining with vWF antibodies. vWF-positive
cells are organized in cords, indicating that all cells seen as
surrounding the cords in (A) and (C through E) are nonendothelial
cells. (C) In situ hybridization with a uPA-specific probe. Note that
expressing cells are endothelial cells forming the cords. (D and E) In
situ hybidization with a PAI-1-specific probe. Note that strongest
hybridization signals are in cells closest to cords (highlighted by
arrows) and that fibroblasts residing more distally show much weaker
hybridization (arrowheads). Magnification: (A, C, and E) ×200
original magnification; (B) ×100 original magnification; (D) ×300
original magnification.
|
|
Aorta explant cultures were fixed and hybridized in situ with either a
uPA-specific probe or a PAI-1-specific probe. Whereas expression of
uPA was confined to the endothelial cell sprouts (Fig 1C), PAI-1
expression was mostly detected in cells adjacent to the uPA-expressing
cells (Fig 1D and E). Note that the highest level of PAI-1 expression
is detected in cells juxtaposed to the capillary sprout and that
expression drops precipitously in cells residing further away from the
capillary. These findings suggested that PAI-1 expression is
specifically upregulated in cells in close proximity, and likely also
in direct contact with invading capillaries.
PAI-1 expression in a heterotypic coculture is induced only in
fibroblasts juxtaposed to endothelial cells.
To establish that PAI-1 expression is induced as a result of
heterotypic cell contact, we used a coculture system composed of an
established endothelial cell line (LE-II) and a fibroblastic cell line
(L-cells). As shown in Fig 2A, colonies of
LE-II cells (round colonies of cobblestone-like cells) are easily
distinguishable from L-cell fibroblasts among which they are
interspersed. In situ hybridization with a PAI-1-specific probe was
used to identify cells, either fibroblasts or endothelial cells, that
have upregulated PAI-1 mRNA expression above the basal level of
expression. As shown in Fig 2A and C, expression of PAI-1 mRNA was
upregulated exclusively in fibroblasts juxtaposed to endothelial cells.
Strikingly, fibroblasts positioned only a single row away from
endothelial cells expressed only a low level of PAI-1 mRNA (L-cells are
known to express a constitutive, low level of PAI-1 mRNA and
protein,33 and the low level of expression in LE-II cells
was barely detectable due to a relatively short exposure time).
View larger version (110K):
[in this window]
[in a new window]
| Fig 2.
L-cells/LE-II cells coculture. Induction of endogenous
PAI-1 expression in fibroblasts contacting endothelial cells. (A and C)
(brightfield and darkfield, respectively) show in situ hybridization of
L-fibroblast cells (L)/ LE-II endothelial cells (E) cocultured with a
PAI-1-specific probe. (B and D) (brightfield and darkfield, respectively) show in situ hybridization of a L-fibroblast cell culture
displaying a gradient of cell density.
|
|
To rule out that induction of PAI-1 mRNA was due to the localization of
the expressing subpopulation at the edge of the fibroblastic sheet (ie,
due to fewer homotypic cell-cell contacts), the following experiment
was performed. Small coverslips were initially placed on the glass
slides before the seeding of L-cells (to preclude cell growth in
certain areas), and cells were grown to a saturation density.
Coverslips were then removed, and partial filling of cell-free areas
was allowed to take place during a further incubation for 3 days. As
shown in Fig 2B and D, no difference with respect to PAI-1 expression
could be detected between cells in confluent regions and cells residing
at the edges of the cellular sheet. These results strongly suggest that
PAI-1 is induced as a result of heterotypic cell-cell contacts.
Apposition-dependent regulation of PAI-1 expression is mediated by
PAI-1 promoter sequences.
To determine whether apposition-dependent induction of PAI-1 is
dictated by the PAI-1 promoter, reporter gene constructs were used. A
DNA fragment containing approximately 1.3 kb of the murine PAI-1
promoter sequences was ligated to a -Gal reporter gene containing a
nuclear localization signal (NLS; to improve visualization of
-Gal-expressing cells). Stably transfected L-cell colonies were
selected, transformed clones were cocultured with LE-II cells, and
confluent cocultures were examined for the distribution of -Gal-positive cells. As shown in Fig
3A, -Gal activity was confined to a subpopulation of transformed
L-cells distinguished by proximity to endothelial cells, ie, a pattern
of expression similar to that of the endogenous PAI-1 gene (Fig 2). As
a control, when expression of -Gal was driven by an irrelevant
promoter (promoter of the ribosomal protein S16), a uniform pattern of
expression in all transformed L-cells was detected (Fig 3B). This
finding indicates that apposition-dependent regulation of PAI-1
expression is mediated by PAI-1 promoter sequences.
View larger version (60K):
[in this window]
[in a new window]
| Fig 3.
PAI-1 promoter directs expression of a -Gal reporter
to fibroblasts juxtaposed to endothelial cells. (A) Fibroblasts
transfected with a PAI promoter-LacZ plasmid. (B) Fibroblasts
transfected with an L16 promoter-LacZ plasmid. See Materials and
Methods for details. L-fibroblasts (L); LE-II endothelial cells (E).
|
|
Mechanistic aspects of apposition-dependent PAI-1 regulation.
Rifkin and coworkers have shown that plasmin, generated by activated
PAs, converts latent transforming growth factor- (TGF-) to its
active form and that TGF- induces, in turn, expression of PAI-1.
Their studies further suggested that activation of latent TGF- is
achieved when two different cell types, at least one of which produces
latent TGF-, are cocultured.34,35 We therefore wished to
determine whether induction of PAI-1 at the sites of heterotypic cell
contact represents a TGF--mediated feedback response to plasmin
production.
To address this question, we performed a series of experiments similar
to those illustrated in Figs 2 and 3, except that different inhibitors
of the PA system were added and were present throughout the period of
coculture growth. Inhibitors used were the same as those used
previously to show the TGF- link34,35 and included the
following: -Amino-n-Caproic Acid (a general inhibitor of serine
proteases), Soybean Trypsin Inhibitor (a general inhibitor of serine
proteases), Tranexamic Acid (inhibits binding of plasminogen and
plasmin to the cell surface), and Trasylol (Aprotinin; inhibits plasmin, including cell-bound plasmin). In addition, we examined the
effect of mannose 6-phosphate (Man-6-P) on PAI-1 induction (exogenous
Man-6-P is known to compete with natural Man-6-P residues of latent
TGF- for binding to a cell surface receptor [of the cation-independent Man-6-P/IGFII type] and, hence, to inhibition of
its activation36). All of these treatments had no effect on
PAI-1 induction or on the size and distribution pattern of PAI-1-expressing cells. Representative examples are shown in
Fig 4 with respect to both the endogenous
PAI-1 gene and a transfected reporter gene driven by PAI-1 promoter.
These findings suggest that apposition-dependent regulation of PAI-1 is
not plasmin mediated. To further rule out a role for soluble plasmin,
experiments were also performed using a serum-free medium (devoid of
plasminogen or plasmin) and L-cells and LE-II cells adapted for growth
in a serum-free medium. An identical pattern of PAI-1 mRNA induction was observed, reinforcing the notion that plasmin activity is not
involved (data not shown).
View larger version (83K):
[in this window]
[in a new window]
| Fig 4.
Apposition-dependent regulation of PAI-1 is unaffected by
inhibitors of the PA system and effectors of latent TGF- activation. (A) In situ hybridization of L-fibroblast cells (L)/LE-II endothelial cells (E) grown in the presence of 100 mmol/L mannose-6-phosphate (brightfield, left and darkfield, right). (B) -Gal activity in fibroblasts (L) transfected with a PAI promoter-LacZ plasmid and cocultured with LE-II cells (E) in the presence of 200 mg/mL Soybean Trypsin Inhibitor.
|
|
To grossly map regulatory domains required for PAI-1 induction by
heterotypic cell contacts, 5 truncated versions of the rat PAI-1
promoter were linked to a CAT reporter gene, and clones of transfected
L-cells were obtained. CAT activity was quantified in extracts of
selected L-cell clones and was compared with the activity found in
extracts of the same L-cell clones that have been cocultured with LE-II
endothelial cells. The heterotypic/homotypic ratio of CAT activity was
taken as a measure of the extent of PAI-1 induction due to heterotypic
contacts. As shown in Fig 5, a 2.4-kb rat
PAI-1 promoter directed CAT expression in heterotypic cultures to
significantly higher levels than in homotypic cultures (also take into
account that the apparent level of stimulation is an underestimate
because CAT activity measured is averaging for the entire coculture,
whereas the fraction of L-cells contacting endothelial cells is
relatively small). Deletion of approximately 600 bp of distal promoter
sequences resulted in the loss of most of the increase in CAT activity,
and deletion of additional 600 bp completely abolished the coculture
effect. These results suggested that regulation of PAI-1 mRNA
expression by heterotypic cell contact is mediated by regulatory
elements located in a 5 distal promoter region.
View larger version (13K):
[in this window]
[in a new window]
| Fig 5.
The 5 upstream region of rat PAI-1 promoter is
required for induction in a heterotypic coculture. A series of 5
deletions of the 2.4-kb rat PAI-1 promoter31 were cloned
upstream of a CAT reporter gene and transfected into L-cells. For each
construct several transfected clones were selected and grown either
alone or after mixing with LE-II endothelial cells (in a 1:1 ratio). Cultures were grown for 5 days (ie, at least 1 day after reaching confluence) and then obtained and analyzed for CAT activity. A comparison was made between the heterotypic cocultures (Ht) and an
equal number of cells of the respective L-cell clone to which LE-II
cells were added (both obtained from individually grown cultures) (Ho).
Results are expressed as an Ht/Ho ratio and are the average of six
different clones for each construct.
|
|
To determine whether PAI-1 expression is induced in all circumstances
of heterotypic cocultures or, alternatively, that only particular
heterotypic cell contacts are conducive for PAI-1 induction, we also
examined by in situ hybridization the pattern of PAI-1 expression in
heterotypic cultures composed of L-cells fibroblast and epithelial
cells (a human carcinoma MLS line). In contrast to endothelial cells
cocultures, we could not detect elevated levels of PAI-1 expression in
fibroblasts juxtaposed to the epithelial cells (data not shown). This
finding suggests that not all heterotypic cell contacts are conducive
for PAI-1 induction.
| |
DISCUSSION |
By virtue of focusing proteolysis close to the cell surface, PAs play
an important role in clearing a path for invading cells. Because PAs
trigger a degradative cascade that is further amplified through the
activation of matrix metalloproteinases, an efficient way to limit
matrix degradation may include additional mechanism(s) to a regulated
PA expression. A tightly controlled pericellular proteolysis is
particularly important during certain processes of physiological
angiogenesis where the tissue is invaded by a large number of
endothelial cell sprouts (eg, during neovascularization of the corpus
luteum). In these highly invasive processes matrix degradation is
restricted to provide a sufficient ECM milieu around capillary sprouts
(which is necessary for proper capillary morphogenesis). PAI-1, by
virtue of its ability to clear active uPA complexes from cell
surfaces,23 is situated in an excellent position for the
role of protecting the interstitial tissue from excessive degradation.
Previous work has shown that PAI-1 mRNA and protein are produced by a
variety of cultured cell types and that high concentrations of PAI-1
are constitutively expressed in a number of mouse tissues. A widespread
expression of PAI-1 may reflect the facts that the PA system may also
play a role in cellular functions other than cellular invasion (notably
in control of fibrinolysis37), that PAI-1 might be required
to counteract both tPA and uPA, and that PAI-1 might interact with both
cell-bound and diffusible forms of PAs. Therefore, to assign a
regulatory role for PAI-1 in uPA-mediated proteolysis associated with
cellular invasion and, in particular, to examine the proposition that
PAI-1 may shield cells in the immediate surrounding of invasive
endothelium from excessive degradation, it is necessary to elucidate
the spatial relationship between uPA-producing cells and
PAI-1-producing cells in the context of natural invasive processes.
Clearly, the fact that PAI-1 is sequestered in the ECM underlying its
producer cell in a PA-accessible form9,38 is consistent
with this role.
Data presented here, showing that PAI-1 induction is confined to within
a single cell distance from endothelial cells (eg, Fig 2), strongly
suggest that a heterotypic cell-cell contact is required for PAI-1
induction. This requirement for a cell-cell contact was further
supported by findings that daily exchanges of media conditioned by
LE-II and L-cells had no effect on PAI-1 expression in either cell type
grown alone (data not shown).
Experiments using a PAI-1 promoter-reporter gene construct have shown
that a PAI-1 promoter confers an apposition-dependent expression on a
heterologous gene, and a preliminary promoter deletion analysis of the
rat PAI-1 promoter has shown that a far upstream region (from
 2.4 kb to 1.2 kb) is required for this novel mode of
PAI-1 regulation. Although this analysis is preliminary, it argues
against the notion that restricting PAI-1 induction to cells contacting
endothelial cells reflects a juxtacrine response to endothelial
cell-bound TGF-, as TGF- regulatory elements were mapped to a
more proximal promoter region.39 A more detailed analysis
is required to determine whether any of the putative regulatory
elements previously mapped to this region31 or,
alternatively, a yet unidentified regulatory element mediates the
cell contact regulation of PAI-1.
A possible mechanism for apposition-dependent PAI-1 induction is a
feedback response in which the protease plays a direct role. One
example for a protease-inducing expression of its cognate inhibitor is
the proteolytic enzyme elastase, which regulates the synthesis of its
inhibitor,  1-proteinase inhibitor.40 More relevantly, it was shown that tPA increases steady-state levels of
PAI-1 mRNA in HUVEC endothelial cells.41 Interestingly,
further studies have shown that induction of PAI-1 mRNA expression by tPA also takes place with a protease devoid of enzymatic
activity.42 Thus, the triggering molecule could be a
cell-bound PA, but not necessarily with the involvement of plasmin. The
latter possibility is consistent with our findings that induction of
PAI-1 in fibroblasts juxtaposed to endothelial cells is independent of
plasmin activity.
A fundamental difference between a quiescent endothelium and
endothelial cells engaged in angiogenesis is that, whereas the first is
enveloped in a basement membrane (BM), the latter are set free of the
BM constraints following angiogenic factor-induced matrix degradation
and, hence, are accessible to contact cells of the interstitial tissue.
We speculate that transient interactions between endothelial cells and
cells that under nonangiogenic circumstances do not contact the
endothelium, trigger a signaling pathway that culminates
in PAI-1 induction. Such a mechanism would provide an efficient way to
sense endothelial cell invasion and is consistent with the observations
that PAI-1 is induced only during endothelial cell invasion and only in
cells juxtaposed to invading endothelial cells.
| |
FOOTNOTES |
Submitted October 28, 1997;
accepted April 1, 1998.
Supported by the Cooperation Program in Cancer Research of the
Deutsches Krebsforschungszentrum (DKFZ) and Israel's Ministry of
Science and Arts.
Address reprint requests to Eli Keshet, PhD, Department of
Molecular Biology, Hebrew University, Hadassah Medical School, Jerusalem 91120, Israel.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
REFERENCES |
1.
Dano K,
Andreasen PA,
Grondahl HJ,
Kristensen P,
Nielsen LS,
Skriver L:
Plasminogen activators, tissue degradation, and cancer.
Adv Cancer Res
44:139,
1985[Medline]
[Order article via Infotrieve]
2.
Pollanen J,
Stephens RW,
Vaheri A:
Directed plasminogen activation at the surface of normal and malignant cells.
Adv Cancer Res
57:273,
1991[Medline]
[Order article via Infotrieve]
3.
Vassalli JD,
Sappino AP,
Belin D:
The plasminogen activator/plasmin system.
J Clin Invest
88:1067,
1991
4.
Pollanen J,
Vaheri A,
Tapiovaara H,
Riley E,
Bertram K,
Woodrow G,
Stephens RW:
Prourokinase activation on the surface of human rhabdomyosarcoma cells: Localization and inactivation of newly formed urokinase-type plasminogen activator by recombinant class 2 plasminogen activator inhibitor.
Proc Natl Acad Sci USA
87:2230,
1990[Abstract/Free Full Text]
5.
Roldan AL,
Cubellis MV,
Masucci MT,
Behrendt N,
Lund LR,
Dano K,
Appella E,
Blasi F:
Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis.
EMBO J
9:467,
1990[Medline]
[Order article via Infotrieve]
6.
Stephens RW,
Pollanen J,
Tapiovaara H,
Leung KC,
Sim PS,
Salonen EM,
Ronne E,
Behrendt N,
Dano K,
Vaheri A:
Activation of pro-urokinase and plasminogen on human sarcoma cells: A proteolytic system with surface-bound reactants.
J Cell Biol
108:1987,
1989[Abstract/Free Full Text]
7.
Estreicher A,
Muhlhauser J,
Carpentier JL,
Orci L,
Vassalli JD:
The receptor for urokinase type plasminogen activator polarizes expression of the protease to the leading edge of migrating monocytes and promotes degradation of enzyme inhibitor complexes.
J Cell Biol
111:783,
1990[Abstract/Free Full Text]
8.
Hebert CA,
Baker JB:
Linkage of extracellular plasminogen activator to the fibroblast cytoskeleton: Colocalization of cell surface urokinase with vinculin.
J Cell Biol
106:1241,
1988[Abstract/Free Full Text]
9.
Pollanen J,
Saksela O,
Salonen EM,
Andreasen P,
Nielsen L,
Dano K,
Vaheri A:
Distinct localizations of urokinase-type plasminogen activator and its type 1 inhibitor under cultured human fibroblasts and sarcoma cells.
J Cell Biol
104:1085,
1987[Abstract/Free Full Text]
10.
Gross JL,
Moscatelli D,
Jaffe EA,
Rifkin DB:
Plasminogen activator and collagenase production by cultured capillary endothelial cells.
J Cell Biol
95:974,
1982[Abstract/Free Full Text]
11.
Gross JL,
Moscatelli D,
Rifkin DB:
Increased capillary endothelial cell protease activity in response to angiogenic stimuli in vitro.
Proc Natl Acad Sci USA
80:2623,
1993
12.
Mignatti P,
Robbins E,
Rifkin DB:
Tumor invasion through the human amniotic membrane: Requirement for a proteinase cascade.
Cell
47:487,
1986[Medline]
[Order article via Infotrieve]
13.
Pepper MS,
Ferrara N,
Orci L,
Montesano R:
Vascular endothelial growth factor (VEGF) induces plasminogen activators and plasminogen activator inhibitor-1 in microvascular endothelial cells.
Biochem Biophys Res Commun
181:902,
1991[Medline]
[Order article via Infotrieve]
14.
Rifkin DB,
Moscatelli D,
Bizik J,
Quarto N,
Blei F,
Dennis P,
Flaumenhaft R,
Mignatti P:
Growth factor control of extracellular proteolysis.
Cell Differ Dev
32:313,
1990[Medline]
[Order article via Infotrieve]
15.
Montesano R,
Pepper MS,
Vassalli JD,
Orci L:
Phorbol ester induces cultured endothelial cells to invade a fibrin matrix in the presence of fibrinolytic inhibitors.
J Cell Physiol
132:509,
1987[Medline]
[Order article via Infotrieve]
16.
Montesano R,
Pepper MS,
Mohle SU,
Risau W,
Wagner EF,
Orci L:
Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene.
Cell
62:435,
1990[Medline]
[Order article via Infotrieve]
17.
Pepper MS,
Montesano R:
Proteolytic balance and capillary morphogenesis.
Cell Differ Dev
32:319,
1990[Medline]
[Order article via Infotrieve]
18.
Andreasen PA,
Georg B,
Lund LR,
Riccio A,
Stacey SN:
Plasminogen activator inhibitors: Hormonally regulated serpins.
Mol Cell Endocrinol
68:1,
1990[Medline]
[Order article via Infotrieve]
19.
Loskutoff DJ:
Regulation of PAI-1 gene expression.
Fibrinolysis
5:197,
1991
20.
Declerck PJ,
De MM,
Alessi MC,
Baudner S,
Paques EP,
Preissner KT,
Muller BG,
Collen D:
Purification and characterization of a plasminogen activator inhibitor 1 binding protein from human plasma. Identification as a multimeric form of S protein (vitronectin).
J Biol Chem
263:15454,
1988[Abstract/Free Full Text]
21.
Mimuro J,
Loskutoff DJ:
Purification of a protein from bovine plasma that binds to type 1 plasminogen activator inhibitor and prevents its interaction with extracellular matrix. Evidence that the protein is vitronectin.
J Biol Chem
264:936,
1989[Abstract/Free Full Text]
22.
Wun TC,
Palmier MO,
Siegel NR,
Smith CE:
Affinity purification of active plasminogen activator inhibitor-1 (PAI-1) using immobilized anhydrourokinase. Demonstration of the binding, stabilization, and activation of PAI-1 by vitronectin.
J Biol Chem
264:7862,
1989[Abstract/Free Full Text]
23.
Cubellis MV,
Wun TC,
Blasi F:
Receptor-mediated internalization and degradation of urokinase is caused by its specific inhibitor PAI-1.
EMBO J
9:1079,
1990[Medline]
[Order article via Infotrieve]
24.
Herz J,
Clouthier DE,
Hammer RE:
LDL receptor-related protein internalizes and degrades uPA-PAI-1 complexes and is essential for embryo implantation.
Cell
71:411,
1992[Medline]
[Order article via Infotrieve]
25.
Schreiber AB,
Kenney J,
Kowalski WJ,
Friesel R,
Mehlman T,
Maciag T:
Interaction of endothelial growth factor with heparin: Characterization by receptor and antibody recognition.
Proc Natl Acad Sci USA
82:6138,
1985[Abstract/Free Full Text]
26.
Diglio CA,
Grammas P,
Giacomelli F,
Wiener J:
Angiogenesis in rat aorta ring explant cultures.
Lab Invest
60:523,
1989[Medline]
[Order article via Infotrieve]
27.
Bacharach E,
Itin A,
Keshet E:
In vivo patterns of expression of urokinase and its inhibitor PAI-1 suggest a concerted role in regulating physiological angiogenesis.
Proc Natl Acad Sci USA
89:10686,
1992[Abstract/Free Full Text]
28.
Belin D,
Vassalli JD,
Combepine C,
Godeau F,
Nagamine Y,
Reich E,
Kocher HP,
Duvoisin RM:
Cloning, nucleotide sequencing and expression of cDNAs encoding mouse urokinase-type plasminogen activator.
Eur J Biochem
148:225,
1985[Medline]
[Order article via Infotrieve]
29.
Prendergast GC,
Diamond LE,
Dahl D,
Cole MD:
The c-myc-regulated gene mrl encodes plasminogen activator inhibitor 1.
Mol Cell Biol
10:1265,
1990[Abstract/Free Full Text]
30.
Levy S,
Avni D,
Hariharan N,
Perry RP,
Meyuhas O:
Oligopyrimidine tract at the 5 end of mammalian ribosomal protein mRNAs is required for their translational control.
Proc Natl Acad Sci USA
88:3319,
1991[Abstract/Free Full Text]
31.
Bruzdzinski CJ,
Riordan JM,
Nordby EC,
Suter SM,
Gelehrter TD:
Isolation and characterization of the rat plasminogen activator inhibitor-1 gene.
J Biol Chem
265:2078,
1990[Abstract/Free Full Text]
32.
Nicosia RF,
Tchao R,
Leighton J:
Histotypic angiogenesis in vitro: Light microscopic, ultrastructural, and radioautographic studies.
In Vitro
18:538,
1982[Medline]
[Order article via Infotrieve]
33.
Cajot JF,
Bamat J,
Bergonzelli GE,
Kruithof EK,
Medcalf RL,
Testuz J,
Sordat B:
Plasminogen-activator inhibitor type 1 is a potent natural inhibitor of extracellular matrix degradation by fibrosarcoma and colon carcinoma cells.
Proc Natl Acad Sci USA
87:6939,
1990[Abstract/Free Full Text]
34.
Sato Y,
Rifkin DB:
Inhibition of endothelial cell movement by pericytes and smooth muscle cells: Activation of a latent transforming growth factor-beta 1-like molecule by plasmin during co-culture.
J Cell Biol
109:309,
1989[Abstract/Free Full Text]
35.
Sato Y,
Tsuboi R,
Lyons R,
Moses H,
Rifkin DB:
Characterization of the activation of latent TGF-beta by co-cultures of endothelial cells and pericytes or smooth muscle cells: a self-regulating system.
J Cell Biol
111:757,
1990[Abstract/Free Full Text]
36.
Dennis PA,
Rifkin DB:
Cellular activation of latent transforming growth factor beta requires binding to the cation-independent mannose 6-phosphate/insulin-like growth factor type II receptor.
Proc Natl Acad Sci USA
88:580,
1991[Abstract/Free Full Text]
37.
Carmeliet P,
Schoonjans L,
Kieckens L,
Ream B,
Degen J,
Bronson R,
De Voss R,
van-den Oord JJ,
Collen D,
Mulligan RC:
Physiological consequences of loss of plasminogen activator gene function in mice.
Nature
368:419,
1994[Medline]
[Order article via Infotrieve]
38.
Salonen EM,
Vaheri A,
Pollanen J,
Stephens R,
Andreasen P,
Mayer M,
Dano K,
Gailit J,
Ruoslahti E:
Interaction of plasminogen activator inhibitor (PAI-1) with vitronectin.
J Biol Chem
264:6339,
1989[Abstract/Free Full Text]
39.
Dong G,
Schulick AH,
DeYoung MB,
Dichek DA:
Identification of a cis-acting sequence in the human plasminogen activator inhibitor type-1 gene that mediates transforming growth factor-beta 1 responsiveness in endothelium in vivo.
J Biol Chem
271:29969,
1996[Abstract/Free Full Text]
40.
Perlmutter DH,
Travis J,
Punsal PI:
Elastase regulates the synthesis of its inhibitor, alpha 1-proteinase inhibitor, and exaggerates the defect in homozygous PiZZ alpha 1 PI deficiency.
J Clin Invest
81:1774,
1988
41.
Fujii S,
Lucore CL,
Hopkins WE,
Billadello JJ,
Sobel BE:
Induction of synthesis of plasminogen activator type-1 by tissue-type plasminogen activator in human hepatic and endothelial cells.
Thromb Haemost
64:412,
1990[Medline]
[Order article via Infotrieve]
42.
Fujii S,
Sobel BE:
Determinants of induction of increased synthesis of plasminogen activator inhibitor type-1 in human endothelial cells by t-PA.
Thromb Homeost
67:233,
1992
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
H Kliem, H Welter, W D Kraetzl, M Steffl, H H D Meyer, D Schams, and B Berisha
Expression and localisation of extracellular matrix degrading proteases and their inhibitors during the oestrous cycle and after induced luteolysis in the bovine corpus luteum
Reproduction,
September 1, 2007;
134(3):
535 - 547.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. B. Saunders, K. J. Bayless, and G. E. Davis
MMP-1 activation by serine proteases and MMP-10 induces human capillary tubular network collapse and regression in 3D collagen matrices
J. Cell Sci.,
May 15, 2005;
118(10):
2325 - 2340.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. S. Pepper
Role of the Matrix Metalloproteinase and Plasminogen Activator-Plasmin Systems in Angiogenesis
Arterioscler. Thromb. Vasc. Biol.,
July 1, 2001;
21(7):
1104 - 1117.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Davis, K. Pintar Allen, R Salazar, and S. Maxwell
Matrix metalloproteinase-1 and -9 activation by plasmin regulates a novel endothelial cell-mediated mechanism of collagen gel contraction and capillary tube regression in three-dimensional collagen matrices
J. Cell Sci.,
January 3, 2001;
114(5):
917 - 930.
[Abstract]
[PDF]
|
|
|
|
|
|
|
P. E. Morange, H. R. Lijnen, M. C. Alessi, F. Kopp, D. Collen, and I. Juhan-Vague
Influence of PAI-1 on Adipose Tissue Growth and Metabolic Parameters in a Murine Model of Diet-Induced Obesity
Arterioscler. Thromb. Vasc. Biol.,
April 1, 2000;
20(4):
1150 - 1154.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Lopez, F. Peiretti, B. Bonardo, I. Juhan-Vague, and G. Nalbone
Tumor Necrosis Factor alpha Up-regulates in an Autocrine Manner the Synthesis of Plasminogen Activator Inhibitor Type-1 during Induction of Monocytic Differentiation of Human HL-60 Leukemia Cells
J. Biol. Chem.,
February 4, 2000;
275(5):
3081 - 3087.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Stefansson, E. Petitclerc, M. K. K. Wong, G. A. McMahon, P. C. Brooks, and D. A. Lawrence
Inhibition of Angiogenesis in Vivo by Plasminogen Activator Inhibitor-1
J. Biol. Chem.,
March 9, 2001;
276(11):
8135 - 8141.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract