©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Biological Activation of pro-HGF (Hepatocyte Growth Factor) by Urokinase Is Controlled by a Stoichiometric Reaction (*)

(Received for publication, August 19, 1994; and in revised form, October 20, 1994)

Luigi Naldini (§) Elisa Vigna (¶) Alberto Bardelli Antonia Follenzi Francesco Galimi Paolo M. Comoglio

From the Department of Biomedical Sciences and Oncology, University of Torino Medical School, corso Massimo D'Azeglio 52, 10126, Torino, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 alphabeta-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 uPAbulletHGF 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.


INTRODUCTION

Hepatocyte growth factor (HGF), (^1)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 (alpha) and a 32-36-kDa (beta) chain (alphabeta-HGF; Nakamura et al.(1987), Gohda et al.(1988), Zarnegar and Michalopoulos(1989), Gherardi et al.(1989), and Weidner et al.(1990)). The alpha chain consists of a putative hairpin loop and four triple-disulfide structures called kringles (Patthy et al., 1984). The beta chain has homology to the catalytic domain of serine proteases but lacks enzymatic activity (Nakamura et al., 1989; Miyazawa et al., 1989). alphabeta-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-beta (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.


EXPERIMENTAL PROCEDURES

Reagents and Cells

All reagents used were of analytical grade. Bovine serum albumin, crystalline, cell-culture tested, was purchased from Sigma. Staphylococcus aureus Protein A covalently coupled to Sepharose was purchased from Pharmacia LKB Biotechnology, Sweden. Reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were from Bio-Rad. [I]Iodine and ^14C-methylated molecular weight standards used in SDS-PAGE, myosin (200 kDa), phosphorylase b (97 kDa), bovine serum albumin (69 kDa), egg albumin (46 kDa), and carbonic anhydrase (30 kDa), were obtained from Amersham.

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(2).

Purification of Recombinant HGF from the Baculovirus Expression System

Full-length HGF cDNA was cloned from human liver mRNA and inserted into the baculovirus transfer vector PVL1393 (Invitrogen, San Diego, CA). The recombinant vector was cotransfected with the BsuI-digested BacPak6 viral DNA (Clontech Laboratories, Palo Alto, CA) into Spodoptera frugiperda insect cells (Sf9) by the lipofectin procedure (Life Technologies, Inc., Gaithersburg, MD). Positive clones were identified and purified by dot-blot hybridization and plaque assay. The recombinant virus was used to infect Sf9 cells with dilutions of 10, 10, 10, and 10. After 1 week, the infected cell extracts were blotted on a nylon filter and probed with radiolabeled full-length human HGF cDNA. The viruses containing the HGF cDNA gene were subsequently purified by plaque assay. Single viral clones were isolated and used for large scale infection of Sf9 cells.

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 times g for 15 min, to remove cellular debris, and cleared by centrifugation at 10,000 times 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.

In Vitro Activation of HGF by uPA

Pro-HGF (1 µg) was radiolabeled with carrier-free NaI (1 mCi) and IODO-GEN (50 µg/ml, Pierce Chemical Co.) as described previously (Naldini et al., 1991b) to a specific activity of approximately 1.8 times 10^8 cpm/µg. Trace amounts of I-pro-HGF were added to known amounts of unlabeled pro-HGF to the desired specific activity, and incubated with two-chain uPA in 100 µl of 50 mM Tris-HCl, pH 8.8, 38 mM NaCl, 0.01% (v/v) Tween 20, at 37 °C, in siliconized tubes. Proteolytic cleavage of I-pro-HGF was monitored by reducing SDS-PAGE, autoradiography, and scanning of the autoradiogram. When required, the absorbance values of the HGF bands were converted into moles by comparison with the absorbance of known amounts of untreated I-pro-HGF.

Chemical Cross-linking and Immunoprecipitation in Vitro

I-pro-HGF (1 nM) and two-chain uPA (2 units) were incubated in 100 µl of 50 mM borate, pH 8.8, 38 mM NaCl, 0.01% (v/v) Tween 20 at 37 °C, for the indicated time in siliconized tubes. When required, disuccinimidyl tartrate (DST) was then added at 0.1 mM or 1 mM and incubated for 15 min at 37 °C. The reaction was stopped by adding an equal volume of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% (v/v) Triton X-100, 2 mg/ml bovine serum albumin (stop buffer). The samples were diluted to 1 ml with the same buffer, immunoprecipitated using Sepharose-Protein A precharged either with rabbit anti-uPA antiserum (5 µl), or normal rabbit serum, washed three times with stop buffer, three times with 50 mM Tris-HCl, pH 7.4, 0.5 M LiCl, three times with 50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, and then solubilized with Laemmli buffer. Protein complexes were separated using reducing SDS-PAGE methods and visualized by autoradiography.

Chemical Cross-linking and Immunoprecipitation from Cultured Cells

Monolayers of human A549 or GTL16 cells were washed twice with RPMI supplemented with 25 mM HEPES pH 7.4 and 0.05% (w/v) bovine serum albumin (RHB), incubated with 100 units/ml two-chain uPA in RHB for 1 h at 37 °C, washed three times with RHB, and further incubated with 0.6 nMI-pro-HGF in RHB, either for 30 min at 37 °C, or for 2 h at 4 °C. After five washes with phosphate-buffered saline (PBS), the monolayers were exposed to 1 mM disuccinimidyl suberate or DST in PBS for 15 min at 4 °C. The reaction was stopped by the addition of 20 mM Tris-HCl, and the monolayers were washed once with PBS, then extracted with HEPS buffer (25 mM HEPES pH 7.4, 100 mM NaCl, 5 mM MgCl(2), 1 mM EGTA, 10% glycerol (v/v), 1.25% CHAPS (w/v)) supplemented with 10 mM iodoacetamide and 50 µg/ml leupeptin, 10 µg/ml aprotinin, 4 µg/ml pepstatin, 50 µg/ml soybean trypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride for 20 min at 4 °C. In these conditions more than 50% of the HGF receptor is solubilized. After clearing by centrifugation at 15,000 times g for 15 min at 4 °C, the extracts were immunoprecipitated with Sepharose-Protein A beads precharged either with immune or preimmune sera, for 2 h at 4 °C. After five washes with HEPS buffer, the beads were eluted with nonreducing Laemmli buffer and analyzed by SDS-PAGE and autoradiography. For control experiments, cell monolayers were washed twice with acid buffer (50 mM glycine-HCl, pH 3.0, 0.1 M NaCl) for 3 min at room temperature (Stoppelli et al., 1986), rinsed twice with RHB, and incubated with or without 100 units/ml uPA, prior to incubation with 0.6 nMI-pro-HGF for 30 min at 37 °C. After washing and cross-linking with disuccinimidyl suberate as above, antisera or control rabbit serum were added to the monolayers in RHB (10 µl/ml), on ice. After 2 h incubation on ice, the monolayers were washed three times with RHB, extracted, and immunoprecipitated with Sepharose-Protein A beads as described above.

Activation of Pro-HGF in Cell Culture

Cell monolayers, grown in 6-well Costar plates, were extensively washed with RHB, and incubated with 0.3 nMI-pro-HGF in RHB at 37 °C. Suspensions of U-937 and THP-1 cells were incubated for 48 h with or without 50 nM tetradecanoylphorbol acetate, washed, and cultured for further 18 h in serum-free medium, prior to washing and incubation with I-pro-HGF in RHB. After different incubation times, culture supernatants were collected and Laemmli buffer was added. At the same time, the cell monolayers were also solubilized with Laemmli buffer. Proteolytic cleavage of I-HGF was monitored by reducing SDS-PAGE and autoradiography.

Isolation of Human Blood Monocytes

Buffy coats from blood donors were obtained from Banca del Sangue e del Plasma (Cittá di Torino), diluted with equal volume of PBS, and separated by Ficoll (Nycomed Pharma, Norway) density gradient centrifugation. Mononuclear cells were washed three times in PBS and then plated on Petri dishes at a concentration of about 6 times 10^5 cells/cm^2 in endotoxin-free Medium 199 (M199, Sigma) supplemented with 10% endotoxin-free bovine calf serum (Hyclone Laboratories, Logan, UT) or 10% plasma from blood donors containing 50 IU/ml heparin. After incubation for 1.5 h at 37 °C, nonadherent cells were removed by washing five times with PBS. The monolayers of monocytes were then used for studying activation of I-pro-HGF. All preparations of monocytes were scored for cell viability by trypan blue exclusion, and used only if more than 98% of the cells looked viable.

Collection of Conditioned Medium and Pro-HGF Activation Assay

Monolayers of monocytes or differentiated U937 or THP-1 cells, obtained as described above, were extensively washed and incubated in serum-free RHB medium for 2 h. Cell-conditioned media were collected, centrifuged at 100 times g for 10 min, and clarified by centrifugation at 15,000 times g for 15 min. For pro-HGF activation assay, control and cell-conditioned media were incubated with 0.3 nMI-pro-HGF for increasing times. For inhibition studies, the indicated inhibitors were preincubated for 30 min at 37 °C prior to addition of I-pro-HGF.


RESULTS

Pro-HGF Is Activated in Vitro by a High-affinity Stoichiometric Interaction with uPA

The kinetics of proteolytic cleavage of pro-HGF by uPA was studied in vitro using highly purified reagents. Recombinant human pro-HGF was obtained from the Baculovirus expression system under serum-free culture conditions, and purified to near homogeneity by heparin-Sepharose chromatography. This preparation of HGF was virtually in the precursor form, and free from any possible contamination from serum components. In order to monitor proteolytic cleavage at concentrations of reactants in the physiological range, pro-HGF was radiolabeled with [I]iodine, as described previously (Naldini et al., 1991b). Two-chain human urinary uPA was purified to homogeneity and had a specific activity of 50 units/µg.

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 alphabetaHGF in vitro as a function of the amount of uPA. 5 nMI-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 alphabeta-HGF, calculated from the absorbance of the alpha or beta 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, alphabeta-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 alpha or beta 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 nMI-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.



Pro-HGF and uPA Form a Stable Complex in Vitro

In order to explain the stoichiometric reaction between uPA and HGF, we monitored the formation of a stable complex between the reaction products in vitro, by coprecipitation and covalent cross-linking experiments. I-pro-HGF was incubated with or without uPA for increasing times, and the samples were immunoprecipitated with an antiserum against uPA. The precipitation of radiolabeled molecules was then scored by SDS-PAGE and autoradiography of the immunocomplexes. Both pro-HGF and alphabeta-HGF coprecipitated with uPA after preincubation with the enzyme (Fig. 3), and the extent of coprecipitation increased with the time of preincubation (not shown). When the homobifunctional cross-linker DST was added at the end of the incubation, we also observed novel labeled M(r) species in the immunoprecipitates made with anti-uPA antibodies. Their M(r) was consistent with the covalent addition of one or both chains of HGF to one or both chains of uPA (Fig. 4). Thus, a stable complex between HGF and uPA can be isolated after their reaction.


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 (alpha-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 (alpha-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(r) 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.



Pro-HGF and uPA Form a Stable Complex on the Membrane of Target Cells

We then asked whether HGF and uPA also formed a stable complex on the surface of target cells, which express receptors for both reactants. Monolayers of human A549 cells were sequentially incubated with two-chain uPA and I-pro-HGF to saturate their surface receptors, washed, and exposed to a cross-linking agent. Then cells were extracted and immunoprecipitated either with anti-Met or anti-uPA antibodies. HGF coprecipitated both with its high-affinity Met receptor, and, to a lower extent, with uPA (Fig. 5A). Moreover, several cross-linked species were detected (Fig. 5B). Two high molecular weight species, previously identified with the cross-linked adducts of HGF with the Met receptor (indicated in the figure with a and b), were selectively precipitated by anti-Met antibodies. A third, lower molecular weight species (indicated with c), was selectively precipitated by anti-uPA antibodies. Its size was similar to that observed in the in vitro cross-linking experiments between HGF and uPA, cited above. A fourth, very high molecular weight form (indicated with d), was immunoprecipitated both by the anti-Met and anti-uPA antibodies. The precipitation pattern and the molecular size of the latter two species were consistent with the covalent addition of uPA to HGF alone, and, respectively, to HGF plus its Met receptor. The presence of uPA in these complexes was proved by control experiments in which the cell monolayers were rinsed with acidic buffer to remove most of the surface-bound uPA, prior to exposure to I-pro-HGF. In these conditions, the c and d cross-linked species were sharply decreased (Fig. 5C). The presence of very high molecular weight form(s), which could hardly enter the gel, was also noted both in anti-uPA and anti-Met immunoprecipitates. Cross-linked adducts of higher molecular weight than HGF plus the Met receptor were also seen on other cell lines, such as the GTL16 cell lines, but were notably absent from insect Sf9 cells transfected with the Met receptor (not shown). In cultures of insect cells neither uPA nor its receptor are present, as the PA/plasmin system is only found in mammals. Thus, a stable complex of HGF with uPA is formed on the membrane of target cells. This complex is bound to the Met receptor, and possibly other molecules, such as the uPA receptor.


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 (alpha-Met) or anti-human uPA (alpha-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.



Pro-HGF Is Activated by a Stoichiometric Reaction with Cell-derived uPA in Cultures of Monocytic Cells

We then asked whether pro-HGF was activated by endogenous uPA produced by cells in culture. We assayed a panel of primary and continuous cell lines for the processing of I-pro-HGF added to the culture medium in serum-free conditions. Significant activation of pro-HGF in the culture medium was a peculiar feature of a small subset of cells, mainly exhibiting monocytic features. These cells are a rich source of enzymatically active uPA (Vassalli et al., 1984).

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(r) 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(r) band was also noted, apparently derived from further processing of the alpha 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 nMI-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 nMI-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 nMI-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.



Pro-HGF Is Activated by Cell-derived uPA in Cultures of Blood Monocytes in Response to Adhesion

In order to verify the physiological occurrence of pro-HGF activation by monocytes, human peripheral blood monocytes were prepared from buffy coats from healthy donors. Freshly isolated mononuclear cells and adherent monocytes were assayed for processing of I-pro-HGF in serum-free conditions. While suspensions of circulating mononuclear cells were unable to process pro-HGF, efficient proteolytic cleavage was observed with the adherent cells. In these conditions, an increasing fraction of HGF became associated with the monocytes (Fig. 8). Thus, activation and cell-association of HGF by monocytes are induced by adhesion.


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 times 10^5 cells/cm^2, 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 nMI-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).


DISCUSSION

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(m) 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 alphabeta-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. (^2)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, alphabeta-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 alphabeta-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).


FOOTNOTES

*
This work was supported in part by grants from the Associazione Italiana Ricerche sul Cancro and the Consiglio Nazionale delle Ricerche (special project ACRO, Grant 92.02169.PF39). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biomedical Sciences and Oncology, University of Torino Medical School, corso Massimo D'Azeglio 52, 10126, Torino, Italy. Tel.: 39-11-6707739; Fax: 39-11-6509105.

Supported by the Ministero della Sanita' (Progetto AIDS).

(^1)
The abbreviations used are: HGF, hepatocyte growth factor; uPA, urokinase plasminogen activator; PN-1, protease nexin-1; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl-dimethylammonio]-1-propanesulfonic acid; DST, disuccinimidyl tartrate; PBS, phosphate-buffered saline.

(^2)
F. Galimi, M. F. Brizzi, E. Cottone, S. Giordano, L. Naldini, E. Vigna, C. Boccaccio, L. Pegoraro, and P. M. Comoglio, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank E. Cottone for preparing monocytes, and F. Blasi, M. F. Di Renzo, C. Ponzetto, and L. Tamagnone for critical reading of the manuscript.


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