(Received for publication, August 19, 1994; and in revised form, October 20, 1994)
From the
Hepatocyte growth factor (HGF) is a paracrine inducer of
morphogenesis and invasive growth in epithelial and endothelial cells.
HGF is secreted by mesenchymal cells as an inactive precursor
(pro-HGF). The crucial step for HGF activation is the extracellular
hydrolysis of the Arg-Val
bond, which
converts pro-HGF into
-HGF, the high-affinity ligand for the Met receptor. We previously reported that the urokinase-type
plasminogen activator (uPA) activates pro-HGF in vitro. We now
show that this is a stoichiometric reaction, and provide evidence for
its occurrence in tissue culture.
Activation involves the formation
of a stable complex between pro-HGF and uPA. This complex was isolated
from the in vitro reaction of pure uPA with recombinant
pro-HGF, as well as from the membrane of target cells, after sequential
addition of uPA and pro-HGF. On the cell membrane, the uPAHGF
complex was bound to the Met receptor.
Monocytic cell lines, and primary monocytes after adhesion, activated efficiently pro-HGF both on their surface and in the culture medium. This activation was inhibited by anti-catalytic anti-uPA antibodies, and occurred by a stoichiometric reaction.
The stoichiometry of the activation reaction suggests that the biological effects of HGF can be titrated in vivo by the level of uPA activity. Adequate amounts of uPA can be locally provided by the macrophages, which would condition the tissue microenvironment by rendering HGF bioavailable to its target cells.
Hepatocyte growth factor (HGF), ()also known as
Scatter Factor, is a unique growth factor capable of inducing an
invasive growth pattern in its target epithelial and endothelial cells.
Among its recognized effects are: (i) dissociation of epithelial
sheets, or cell scattering (Stoker et al., 1987; Gherardi et al., 1989; Weidner et al., 1990, 1991; Naldini et al., 1991b); (ii) increased motility and chemotaxis of
dissociated cells (Morimoto et al., 1991; Bussolino et
al., 1992; Giordano et al., 1993); (iii) mitogenesis
(Michalopoulos et al., 1984; Nakamura et al., 1984;
Russel et al., 1984; Rubin et al., 1991; Kan et
al., 1991; Matsumoto et al., 1991; Igawa et al.,
1991; Bussolino et al., 1992; Halaban et al., 1992);
(iv) invasion of extracellular matrices (Weidner et al., 1990;
Naldini et al., 1991b); (v) organization of invading cells
into complex structures such as branching tubules (Montesano et
al., 1991). Coordinate execution of these events is required for
physiological processes such as morphogenesis of epithelial organs,
angiogenesis, and tissue repair, and is a feature of invasive
neoplasms. In vivo, HGF is a powerful angiogenetic factor
(Bussolino et al., 1992; Grant et al., 1993) and it
has been involved in kidney and liver regeneration (Nagaike et
al., 1991; Higuchi and Nakamura, 1991). HGF is the ligand for the
tyrosine kinase receptor encoded by the MET proto-oncogene
(Naldini et al., 1991a, 1991b; Bottaro et al., 1991).
In vivo, HGF is thought to act in a paracrine fashion: it
is produced by mesenchymal/stromal cells (Stoker et al.,
1987), and its receptor is found on epithelial cells nearby (Sonnemberg et al., 1993). HGF is secreted as a single-chain, 92-kDa
precursor devoid of biological activity (pro-HGF; Naldini et
al.(1992), Naka et al.(1992) and Mizuno et
al.(1992)). Pro-HGF binds the cell surface or the extracellular
matrix, presumably via its affinity for heparin-like glycosaminoglycans
(Naldini et al., 1991b). Pro-HGF also binds the Met receptor, but with low affinity and without triggering its kinase
activity (Naldini et al., 1992; Hartmann et al.,
1992; Lokker et al., 1992). Limited proteolysis of pro-HGF at
the Arg-Val
bond yields a disulfide-linked
heterodimer of a 60-kDa (
) and a 32-36-kDa (
) chain
(
-HGF; Nakamura et al.(1987), Gohda et
al.(1988), Zarnegar and Michalopoulos(1989), Gherardi et
al.(1989), and Weidner et al.(1990)). The
chain
consists of a putative hairpin loop and four triple-disulfide
structures called kringles (Patthy et al., 1984). The
chain has homology to the catalytic domain of serine proteases but
lacks enzymatic activity (Nakamura et al., 1989; Miyazawa et al., 1989).
-HGF binds the Met receptor
with high affinity, triggers its kinase activity and a biological
response in target cells (Naldini et al., 1991a, 1991b;
Bottaro et al., 1991).
Maturation of pro-HGF into the bioactive dimer takes place in the extracellular environment. Thus, a crucial limiting step occurs in the HGF signaling pathway after its secretion. We previously showed that the urokinase-type plasminogen activator (uPA) activates pro-HGF in vitro (Naldini et al., 1992). This finding was consistent with the high degree of structural homology between HGF and plasminogen, and with the near identity of the proteolytic cleavage site (Miyazawa et al., 1989; Nakamura et al., 1989; Sottrup-Jensen et al., 1978). Other investigators have shown that an enzyme purified from serum can also process pro-HGF in vitro (Shimomura et al., 1992). This enzyme is homologous to the blood coagulation Factor XII (Miyazawa et al., 1993), is present in plasma as an inactive zymogen, and is activated by limited proteolysis by thrombin (Shimomura et al., 1993). Whether either or both uPA and the Factor XII homolog have a role in vivo in pro-HGF activation remained unsettled.
UPA has been critically involved in tissue
remodeling, cell migration, invasion, and metastasis (reviewed in
Saksela and Rifkin(1988)). Possible mechanisms for these biological
activities are triggering of a protease cascade digesting the
extracellular matrix, and activation of latent growth factors, such as
transforming growth factor- (Sato et al., 1990) and
pro-HGF. uPA is an extracellular serine endoprotease with a
multimodular structure (reviewed in Furie and Furie (1988). It is
secreted as an inactive, single-chain protein of 55 kDa (Kasai et
al., 1985; Petersen et al., 1988), and subsequently
cleaved into an enzymatically active, two-chain molecule (Kasai et
al., 1985). The 20-kDa A chain bears an epidermal growth
factor-like domain which mediates binding to the uPA receptor on the
surface of several cell types (Appella et al., 1987), followed
by a kringle domain. The 34-kDa B chain is the catalytic moiety. The
fraction of uPA bound to its receptor on the cell surface is thought to
be the most biologically relevant. uPA activates plasminogen, present
in high concentrations in serum and extracellular fluids, to plasmin, a
fibrynolytic enzyme with broad substrate specificity. uPA is negatively
regulated by two specific inhibitors, plasminogen activator inhibitor
type-1 and type-2, and by protease nexin-1 (PN-1; reviewed in Blasi et al.(1987) and Saksela and Rifkin(1988)).
In this work, we report the unexpected finding that uPA activates pro-HGF by a stoichiometric reaction. This is due to the formation of a stable complex between HGF and uPA, both in vitro, with the pure reagents, and on the membrane of target cells, after the binding of pro-HGF and uPA to their receptors. Moreover, we provide evidence for a direct role of endogenous uPA in the activation of pro-HGF by monocytes in cell culture.
Pure human urinary, two-chain uPA
was a gift of M. L. Nolli (Dow-Lepetit, Varese, Italy). Specific
activity of uPA preparation was titrated using the chromogenic
substrate p-Glu-Gly-Arg-p-nitroanilide (Sigma). One
unit of UPA activity is defined as the amount of enzyme which yields a
2 A increase in absorbance at 405 nm with 0.6 nMp-nitroanilide in 1 min at 37 °C. uPA specific
activity was 1 unit = 0.02 µg. All the uPA concentrations
reported refer to the active portion of the enzyme present in the
preparation. Recombinant PN-1 was a gift of R. Scott (Mc Grogan et
al., 1988). Anti-human uPA monoclonal antibodies were a gift of M.
L. Nolli (Dow-Lepetit, Varese, Italy) or from American Diagnostica
(Greenwich, CT) and anti-human uPA rabbit antiserum was a gift of F.
Blasi (H. S. Raffaele, Milano, Italy). Cell lines were obtained from
ATCC, and cultured in RPMI 1640 medium containing 10% fetal calf serum
and maintained at 37 °C in a humidified atmosphere with 5%
CO.
Recombinant pro-HGF was purified by affinity chromatography on
heparin (Bio-Rad Laboratories, Hercules, CA), according to the
procedure published by Weidner et al.(1990), with some
modifications. Sf9 S. frugiperda cells were grown at 27 °C
in serum-free SF900 medium (Life Technologies, Inc. Ltd., Scotland).
Exponentially growing cultures were infected by adding the viral stock
in serum-free culture medium, and the cells were grown for 3 days. The
culture medium was collected, spun at 300 g for 15
min, to remove cellular debris, and cleared by centrifugation at 10,000
g for 1 h. The supernatant was buffered to pH 7.4 with
Tris, supplemented with a mixture of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 50 µg/ml leupeptin, 10
µg/ml aprotinin, 4 µg/ml pepstatin) and the detergent CHAPS to
a final concentration of 0.2% (w/v), filtered on a 0.45-µm pore
Tuffryn membrane filter (Gelman Sciences, Ann Arbor, MI) by vacuum
suction, cooled to 4 °C, and applied to a 5-ml heparin-agarose
column assembled in an fast protein liquid chromatography apparatus in
the cold room with a loading rate of 8 ml/h. The column was
sequentially washed with 0.15 M NaCl, 50 mM Tris-HCl,
pH 7.4, 0.2% CHAPS, and 0.5 M NaCl, 50 mM Tris-HCl,
pH 7.4, 0.2% CHAPS until the eluant absorbance returned to the
baseline. Bound materials were eluted with a linear gradient from 0.5 M to 1.8 M NaCl over 8 h in 50 mM Tris-HCl,
pH 7.4, 0.2% CHAPS, with a flow rate of 0.2 ml/min, and 2-ml fractions
were collected. The starting material, the column breakthrough and
washings, and the eluted fractions, were scored for the content of
pro-HGF by the Madin-Darby canine kidney cell scattering assay in the
presence of serum (Weidner et al., 1990; Naldini et
al., 1992). The fractions containing the peak of HGF activity,
eluting at approximately 1 M NaCl, were pooled, concentrated
with a diafiltration device with 30,000 molecular weight cut off
(Amicon Div., Grace Industrial, Switzerland), checked for biological
activity on Madin-Darby canine kidney cells, and purity by SDS-PAGE and
protein stains, and stored in aliquots in liquid nitrogen. The average
yield of the procedure was 150 µg of pro-HGF from 700 ml of culture
supernatant. The purity of pro-HGF was higher than 95%. When tested for
scattering activity on Madin-Darby canine kidney cells in the presence
of serum, pro-HGF was activated to a specific activity of 10 scatter
units/ng of protein.
We detected efficient processing of pro-HGF by nanomolar concentrations of uPA at 37 °C. Time course analysis showed saturation kinetics with a decreasing production of two-chain HGF over the hours (not shown). As expected, the initial rate of the reaction was dependent on the amount of uPA added. Surprisingly, however, the total amount of two-chain HGF produced by an overnight incubation was also dependent on the concentration of enzyme. For prolonged times, the limiting factor in product yield was found to be the concentration of enzyme added, and not, as expected from a catalytic mechanism, the initial concentration of substrate. A linear relationship was found between the total amount of HGF processed overnight and the amount of uPA, as long as the concentration of substrate was higher than that of the enzyme (Fig. 1). In these conditions, the product of the reaction accumulated to approximately one-half the molar amount of the enzyme. The uPA concentrations given refers to the catalytically active enzyme, titrated with a chromogenic substrate as described under ``Experimental Procedures.'' Control experiments showed that the specific activity of uPA was not affected by prolonged incubation in the reaction buffer (not shown). These observations pointed to a stoichiometric reaction between uPA and pro-HGF.
Figure 1:
Total yield of HGF in vitro
as a function of the amount of uPA. 5 nM
I-pro-HGF were incubated overnight with the
indicated concentrations of pure, two-chain uPA in 100 µl of
buffer. The samples were then analyzed by reducing SDS-PAGE and
autoradiography. In A, the autoradiogram is shown, and the
mobility of pro-HGF and the two chains of processed HGF are indicated.
In B, the final concentration of
-HGF, calculated
from the absorbance of the
or
chain, was plotted against
the concentration of uPA. In the inset, data are replotted for
enzyme concentrations lower than that of substrate, to show the linear
relationship between product yield and uPA. In these conditions,
-HGF accumulated to an approximately half-molar level with
uPA. A representative experiment out of three performed is
shown.
In order to
estimate the affinity of pro-HGF as substrate for uPA, we studied the
initial rate of proteolytic cleavage as a function of the substrate
concentration. We used two different approaches. In the first type of
experiment, we added increasing concentrations of I-pro-HGF to the reaction mixture, and measured the net
amount of labeled two-chain HGF produced. A fast saturation of the
reaction rate was observed with increasing concentrations of substrate.
Half-maximal rate was reached with 3.5 nM pro-HGF (Fig. 2A). In the second type of experiments,
increasing concentrations of unlabeled pro-HGF were added to a fixed
concentration of
I-labeled substrate. We then measured
the inhibition of proteolytic cleavage of
I-pro-HGF as an
indirect estimate of the saturation of the reaction. According to
theoretical predictions, the extent of proteolytic cleavage of a
labeled substrate is unaffected by the addition of unlabeled substrate
as long as their total concentration remains well below saturation of
the enzyme, the reaction rate being directly proportional to the
concentration of substrate. Approaching saturation results in the
progressive inhibition of proteolytic cleavage of the labeled
substrate. These experiments provided a similar estimate of
approximately 7 nM for the concentration of pro-HGF giving
half-maximal rate of proteolytic cleavage by uPA (Fig. 2B). As the maximal rate of the reaction was
reached with very low concentrations of substrate, a high affinity
interaction must take place between pro-HGF and uPA.
Figure 2:
Initial rate of pro-HGF activation in
vitro by uPA as a function of the substrate concentration. In A, the indicated concentrations of I-pro-HGF
were incubated with 2 units of uPA in 100 µl of buffer for 2 h at
37 °C. The samples were then analyzed by SDS-PAGE and
autoradiography. The amount of product was calculated from the net
absorbance of the band corresponding to the
or
chain of
HGF, obtained by subtracting the absorbance of a corresponding
untreated sample. In B, the indicated concentrations of
unlabeled pro-HGF were added to 0.1 nM
I-pro-HGF, and incubated with 2 units of uPA in 100
µl of buffer for 2 h at 37 °C. The samples were then analyzed
by SDS-PAGE and autoradiography. The amount of labeled product was
plotted as percentage of that obtained with the lowest concentration of
pro-HGF tested. As the concentration of unlabeled pro-HGF approached
saturation of uPA, progressive inhibition of the proteolytic cleavage
of
I-pro-HGF was observed. Both experiments provided a
similar estimate of 3.5 nM (A) and 7 nM (B) for the pro-HGF concentration giving half-maximal
rate of proteolytic cleavage by uPA. For each point, the mean and range
of duplicate determinations are given from a representative experiment
out of three performed.
Figure 3:
Isolation of a stable complex between HGF
and uPA in vitro. 1 nMI-pro-HGF was incubated
with or without 2 units of uPA for 2 h, at 37 °C in 100 µl of
buffer. The samples were diluted in stop buffer (see
``Experimental Procedures''), immunoprecipitated with
Sepharose-Protein A beads precharged with rabbit anti-uPA antibodies
(
-uPA) or normal rabbit serum (nrs), and analyzed by
reducing SDS-PAGE and autoradiography.
I-Labeled HGF was
found in immunoprecipitates of uPA only after preincubation with the
enzyme. The experiments shown are representative of four
performed.
Figure 4:
Identification of a stable complex of HGF
with uPA in vitro. 1 nMI-pro-HGF was incubated
with or without 2 units of uPA for 2 h at 37 °C in 100 µl of
buffer, then 0.1 nM DST was added for 15 min. The samples were
diluted in stop buffer (see ``Experimental Procedures''), and
analyzed by SDS-PAGE and autoradiography as such (lanes marked: total), or after immunoprecipitation with Sepharose-Protein A
beads precharged with rabbit antibodies against uPA (
-uPA) or normal rabbit serum (nrs). The low
concentration of cross-linker allowed for the resolution of several
novel labeled bands in the immunoprecipitates of uPA (indicated with a, b, and c), together with the individual
chains of processed HGF. Their M
was consistent
with the covalent addition of one or both chains of HGF to one or both
chains of uPA. Protein molecular mass markers in kDa are shown on the
right. The experiment shown is representative of three
performed.
Figure 5:
HGF and uPA form a stable complex on the
membrane of target cells, after binding to their respective receptors.
Monolayers of A549 cells were sequentially incubated to equilibrium
binding with two-chain uPA (100 units/ml) and I-pro-HGF
(0.6 nM), washed, and exposed to 1 mM of a
cross-linking agent, extracted, and analyzed by SDS-PAGE on a
5-15% polyacrylamide gradient gel under nonreducing condition and
autoradiography, as such (lane marked: total in B),
or after immunoprecipitation with anti-human Met (
-Met) or anti-human uPA (
-uPA) rabbit
antibodies, or normal rabbit serum (nrs). In panel A,
the coprecipitation of HGF both with its high-affinity Met receptor, and with uPA is shown. The gel was exposed overnight for
autoradiography. Protein molecular mass markers are shown on the left. In panel B, a 3-day exposure of the gel allowed
detection of several cross-linked species. Two major species (indicated
with a and b) correspond to previously identified
complexes of HGF with the Met receptor, and were selectively
precipitated by anti-Met antibodies. A lower molecular weight
species (indicated with c) was selectively precipitated by
anti-uPA antibodies, and was consistent with the covalent addition of
HGF to uPA. A fourth, very high molecular weight form (indicated with d) was precipitated both by anti-Met and anti-uPA
antibodies, and was consistent with the covalent addition of HGF to uPA
plus the Met receptor. The presence of even higher molecular
weight forms, which could hardly enter the gel, can be noted both in
anti-uPA and anti-Met immunoprecipitates. Protein molecular
mass markers in kDa are shown on the left. The experiments
shown are representative of five performed. In panel C, the
presence of uPA in the cross-linked adduct indicated with d,
was proved by the absence of this species in extracts of cells treated
with acid, and not exposed to uPA, prior to incubation with
I-pro-HGF. The relevant portion of the autoradiogram from
a 1-week exposure is shown.
Fig. 6shows the processing of I-pro-HGF into
the bioactive heterodimer by human monocytic cell lines. U-937 and
THP-1 cells were differentiated into a monolayer of adherent
macrophage-like cells by treatment with tetradecanoylphorbol acetate
(Genton et al., 1987). To avoid interference from serum
enzymes, the cells were cultured in the absence of serum for 24 h, and
washed several times before using in the experiments. With U-937 cells,
two-chain HGF was already detectable in the culture medium after 15 min
of incubation (Fig. 6A). Pro-HGF was also processed by
THP-1 cells, although less efficiently than U-937 cells (Fig. 6B). Correct processing of
I-pro-HGF was indicated by the M
of
the two chains and by the identical mobility of processed and
unprocessed
I-HGF in SDS-PAGE under nonreducing
conditions (not shown). With prolonged incubations, a lower M
band was also noted, apparently derived from
further processing of the
subunit. When undifferentiated U-937
and THP-1 cells were assayed, a similar but less efficient proteolytic
cleavage of
I-pro-HGF was observed (not shown).
Figure 6:
Activation of I-pro-HGF in
serum-free culture medium of human monocytic cell lines. U-937 (A) and THP-1 (B) cells were grown as a monolayer of
differentiated macrophage-like cells by treatment with 50 nM tetradecanoylphorbol acetate for 48 h, cultured in the absence of
serum for 24 h, and incubated with 0.3 nM
I-pro-HGF. After the indicated times of incubation,
the culture medium was collected and analyzed by reducing SDS-PAGE and
autoradiography. The mobility of pro-HGF, and of the two chains of
mature HGF is shown. In C, the processing activity was also
detected in the medium conditioned by incubation with the cells. 100
µl of medium conditioned by a 2-h incubation with U-937 cells were
incubated with 0.3 nM
I-pro-HGF for the
indicated times and analyzed as above. The experiments shown are
representative of four performed.
In
order to identify the enzyme(s) involved in the proteolytic cleavage of
pro-HGF, we first showed that the processing activity was also
detectable in the medium conditioned by incubation with the cells (Fig. 6C). U-937-conditioned medium was then challenged
with a panel of monoclonal antibodies against uPA and with the protease
inhibitor protease nexin 1 (PN-1), prior to incubation with I-pro-HGF. As shown in Fig. 7, anticatalytic
antibodies recognizing the B chain of uPA, and PN-1, effectively
inhibited the proteolytic cleavage of
I-pro-HGF in the
conditioned medium. Anti-uPA antibodies recognizing either the receptor
binding site in the A chain, or the B chain without hampering
catalysis, were ineffective. These results implied a direct action of
uPA on pro-HGF. A spiking test, performed by the addition of known
amounts of pure uPA to the culture medium, proved the activity of the
enzyme in these experimental conditions (not shown). Thus, subnanomolar
concentrations of pro-HGF are activated in culture by the
stoichiometric action of endogenous uPA produced by monocytic cell
lines.
Figure 7:
Activation of I-pro-HGF in
U-937-conditioned medium by the stoichiometric action of uPA. 70 µl
of U-937-conditioned medium were preincubated with 0.5 µg of the
indicated monoclonal antibodies against human uPA or with 0.5 µg of
PN-1, for 40 min at 37 °C, prior to incubation with 0.3 nM
I-pro-HGF for the indicated time. The samples were
analyzed by reducing SDS-PAGE and autoradiography. Anticatalytic
antibodies (A1 and 26A3) and PN-1, inhibited the
proteolytic cleavage of
I-pro-HGF, while antibodies
recognizing the receptor binding site in the A chain of uPA (3E4), or
the B chain without hampering catalysis (25D10), were
ineffective.
Figure 8:
Activation and cell-association of I-pro-HGF in cultures of adherent, freshly isolated,
human blood monocytes. Washed mononuclear cells, obtained by Ficoll
density gradient centrifugation of buffy coats from blood donors, were
plated on 6-well Costar plates at a concentration of about 6
10
cells/cm
, and either immediately assayed for
activation of pro-HGF (A), or incubated in endotoxin-free
medium with 10% calf serum for 90 min at 37 °C to allow adhesion,
and washed free of serum and nonadherent cells, prior to use (B). For the assay, both cell populations were incubated with
0.3 nM
I-pro-HGF in serum-free medium. After the
indicated time, the culture supernatants and the cells were separately
extracted with Laemmli buffer and analyzed by reducing SDS-PAGE and
autoradiography. After adhesion, monocytes became able to activate and
associate HGF. A representative experiment out of five performed is
shown.
Activation of pro-HGF was also detected with serum-free
conditioned medium of adherent monocytes. The same result was obtained
when monocytes were cultured in the presence of human heparinized
plasma, in place of serum, so ruling out a residual contamination from
serum components (not shown). Inhibition studies similar to those
reported above for U-937 cells, identified uPA as the enzyme
responsible for pro-HGF activation in the conditioned medium of
monocytes. Preincubation of conditioned medium with anti-catalytic
monoclonal antibodies against uPA and with the protease inhibitor
protease nexin 1, inhibited the processing of I-pro-HGF.
Anti-uPA antibodies recognizing the receptor binding site in the A
chain of uPA were ineffective (not shown). These results ruled out
aspecific proteases, released from the monocytes, from the observed
processing of
I-pro-HGF. Analyzing the relative
contribution of secreted versus cell-bound uPA to the
processing of pro-HGF by monocytes, the activity was higher in the
presence of the cell monolayer then in the conditioned medium (not
shown).
Immunohistochemistry (Zarnegar et al., 1990; Wolf et al., 1991), in situ hybridization (Sonnemberg et al., 1993), immunoassays (Tsubouchi et al., 1991),
and perfusion studies (Masumoto and Yamamoto, 1991) pointed to a wide
distribution of HGF in the tissues. Accordingly, the HGF receptor is
also expressed by several types of epithelial cells (Di Renzo et
al., 1991) and by endothelial cells (Bussolino et al.,
1992). The limiting step in the HGF signaling pathway in vivo may thus be represented by the extracellular activation of the
secreted precursor. This occurs by limited proteolysis of pro-HGF at
the Arg-Val
bond, which converts pro-HGF
into a bioactive heterodimer (Naldini et al., 1992; Naka et al., 1992; Mizuno et al., 1992). The uPA was shown
to activate pro-HGF in vitro (Naldini et al., 1992;
Mars et al., 1993).
In this work, a kinetic analysis of the reaction in vitro between purified pro-HGF and uPA indicated a surprising dependence of the total product yield from the input of enzyme assayed. The reaction did not progress further, once an amount of HGF approximately half-molar with the enzyme input had been processed. This indicates a stoichiometric reaction. In a catalytic reaction, given enough time virtually all the substrate is processed, and the total yield of product is independent from the concentration of enzyme and only limited by the initial concentration of substrate. Half-maximal rate of activation was reached with 3-7 nM pro-HGF. As this is also a measure of the affinity of pro-HGF for uPA, significant activation of pro-HGF may take place with concentrations of reactants near the physiological range.
Activation
of pro-HGF by uPA was notably different from the activation of
plasminogen. In the latter case, uPA acts as a typical catalyst with a K for the substrate in the micromolar range (Ellis et al., 1991; Franco et al., 1992). It can be noted
that the widely different affinities of uPA for its two substrates
correlate with their physiological concentrations. Thus, given the
structural homology between plasminogen and pro-HGF, a consensus for
high-affinity recognition of uPA may be built in the pro-HGF sequence.
The stoichiometric reaction of uPA with pro-HGF could be due either to
the formation of a stable complex between the reaction products or to
an irreversible modification of uPA in the course of its reaction with
pro-HGF. We isolated a complex of HGF with uPA by immunoprecipitation
of uPA after incubation with pro-HGF, and by cross-linking experiments
with the bifunctional reagent DST. The short length of the atomic
spacer in the DST molecule provides further support for a close
molecular recognition between pro-HGF and uPA. On the other hand, we
did not observe any variation in the specific activity of uPA, as
assayed by a direct chromogenic substrate, after its incubation with
pro-HGF (not shown). A similar stoichiometric mechanism was described
for the activation of plasminogen by streptokinase (Reddy and Markus,
1972).
Complex formation between HGF and uPA was also detected on the surface of target cells after equilibrium binding of both reactants to their receptors. Complexing of HGF with both uPA and the Met receptor was detected by cross-linking and immunoprecipitation experiments. The participation in the complex of other molecules such as the uPA receptor is also possible. Thus, binding of pro-HGF and uPA to their receptors in the membrane of target cells promotes complex formation, and, consequently, activation of pro-HGF. The uPA receptor may then cooperate with Met in binding HGF to the plasma membrane, and so influence the triggering of the Met receptor.
The in vitro activation of pro-HGF by uPA was slow. The
achievement of a maximal rate of reaction with low concentrations of
pro-HGF indicated that the rate-limiting step was not the diffusion of
the reactants but, more likely, the conversion of proHGF to
-HGF once bound to uPA. This may reflect a requirement for
additional cofactor(s) or for a specific surface, such as the cell
membrane, for optimal reactivity. Indeed, the activation of pro-HGF was
much more efficient in the presence of cells bearing receptors for both
reactants, such as monocytes (Vassalli et al., 1985; Galimi et al., 1993). In analogy with the activation of plasminogen
(Kirchheimer and Remold, 1989; Ellis et al., 1991), the cell
surface may facilitate activation of pro-HGF by uPA.
The finding of a stoichiometric reaction may explain the failure to observe proteolytic cleavage of pro-HGF by uPA, reported by other investigators (Mizuno et al., 1992). In fact, if uPA is added in catalytic amounts to pro-HGF in vitro, and the reaction is monitored by the quantitative processing of the substrate, only a tiny amount of product would be generated, and, most likely, go undetected.
We also provide evidence for activation of pro-HGF by endogenous uPA in the culture medium. Among the cell types tested, we detected significant activation of pro-HGF mostly by monocytic cell lines and freshly isolated, adherent blood monocytes. Activation of pro-HGF was also observed in the cell-conditioned medium, and it was inhibited by several anti-catalytic monoclonal antibodies against uPA. We obtained indications for a stoichiometric reaction similar to that observed in vitro with uPA, as the total yield of processed HGF was dependent on the input of conditioned medium. Adherent monocytes, and U-937 cells, are a rich source of enzymatically active uPA (Manchanda and Schwartz, 1990).
Monocytes activated pro-HGF only after adhesion
to a substrate, and treatment with endotoxin further increased the
extent of pro-HGF activation. ()As this treatment increases
the expression of uPA (Manchanda and Schwartz, 1990), monocytes titer
the biological activity of HGF in response to extracellular signals.
Activation of pro-HGF was more efficient in the presence of the cell
monolayer than with the cell-conditioned medium. In these conditions,
an increasing fraction of HGF became associated with the monocytes.
Association of HGF with the monocytes may represent both complexing of
HGF with receptor-bound uPA, and binding of HGF to its high-affinity Met receptor. Monocytes express the receptor for HGF, and
increase its expression upon activation (Galimi et al., 1993).
Other investigators have shown that an enzyme purified from serum can also process pro-HGF in vitro (Shimomura et al., 1992). This enzyme is homologous to the blood coagulation factor XII (Miyazawa et al., 1993), is present in plasma as an inactive zymogen, and is activated by limited proteolysis by thrombin (Shimomura et al., 1993). While no kinetic parameter was published for HGF activation, HGF activator was reported to work as a typical catalyst. Thus, if the Factor XII homolog also works in vivo, two different pathways may effect activation of HGF. The HGF activator may bring about quantitative activation of pro-HGF in response to the triggering of the blood coagulation cascade, as in the case of tissue injury. uPA may effect a more restricted activation of pro-HGF in the tissues and on the membrane of target cells (see the schematic model in Fig. 9).
Figure 9:
Schematic model of the proposed activation
pathways for pro-HGF. HGF is secreted and stored in the tissues as the
biologically inactive, single-chain precursor, pro-HGF. Pro-HGF binds
with low affinity to the Met receptor, and/or to heparin-like
sites (not shown), on the cell membranes. The unprocessed precursor
does not trigger the kinase activity of the Met receptor. The right panel shows the stoichiometric activation pathway by
uPA. uPA forms a stable complex with pro-HGF, both in the extracellular
medium and on the cell membrane, where the molecules are bound to their
receptors. Within the complex, pro-HGF is processed to the two-chain
form, -HGF, which binds with high affinity and triggers the Met receptor kinase. A biological response ensues in the
target cell. As uPA remains bound to
-HGF, the yield of
two-chain HGF is limited by the amount of active uPA available (the
observed stoichiometry predicts a complex of two molecules of uPA with
one of HGF). Adequate amounts of uPA can be produced in the tissues by
macrophages in response to activating stimuli, or possibly by other
cell types (tissue activation pathway). The left panel shows
the catalytic activation pathway by the Factor XII-like HGF activator.
As this enzyme works as a typical catalyst (Miyazawa et al.,
1993), it could effect quantitative activation of all stored pro-HGF.
The Factor XII-like HGF activator is found in the plasma as inactive
precursor and is itself activated by limited proteolysis by thrombin
(Shimomura et al., 1993). Thus, triggering of the blood
clotting cascade, as in response to tissue injury, would be a
requirement of this pathway (serum activation
pathway).
Extracellular activation by a protease has been envisaged as a critical regulatory step in the signaling mediated by morphogenic factors. It provides a mechanism for the local production of intercellular signals (reviewed in Hecht and Anderson(1992)). A stoichiometric reaction provides tight control on the extent of extracellular activation of pro-HGF. The level of bioactive HGF in a tissue microenvironment would be titrated by the extent of activity of uPA. This, in turn, is subject to strict spatial and temporal regulation by the level of expression of the protein and its interplay with activators, inhibitors and cellular receptors (reviewed in Blasi et al.(1987)). Moreover, the macrophage, by its selective ability to activate pro-HGF in the culture medium, may exert a crucial role in the conditioning of a tissue microenvironment.
Coordination of cell growth, movement, and matrix invasion occurs not only in physiological events but also in the progression of cancer cells toward malignancy. Several growth factors, including HGF (Pepper et al., 1992; Boccaccio et al., 1994), induce expression of uPA and its receptor as an early event in the course of the mitogenic response (reviewed in Saksela and Rifkin (1988)). A correlation between surface uPA activity and tumor cell invasion and metastasis has been well documented (Ossowski and Reich, 1983; Kirchheimer et al., 1987, 1989; Ossowski, 1988a, 1988b; Hearing et al., 1988; Axelrod et al., 1989; Pyke et al., 1991; Meissauer et al., 1991). It would be worth testing whether cancer cells, by enhanced expression of uPA, mimic macrophages in the activation of pro-HGF. This would trigger a positive feed-back circuit inducing growth and invasion through the HGF receptor, itself the product of a proto-oncogene often overexpressed in malignancies (Cooper et al., 1986; Giordano et al., 1989; Iyer et al., 1990; Ponzetto et al., 1991; Di Renzo et al., 1991, 1992; Liu et al., 1992; Rong et al., 1992).