(Received for publication, October 26, 1995)
From the
Hepatocyte growth factor (HGF) is a potent mitogen, motogen, and morphogen for epithelial cells in vitro. It appears likely that HGF participates in tissue regeneration following hepatic and renal injury in vivo. The activity of HGF is localized to the injured tissues by a proteolytic activation system; HGF remains as an inactive single-chain form in the normal state and is converted to an active heterodimeric form in response to tissue injury. A protease responsible for this conversion is induced in the injured liver, but it has not yet been identified. We have previously purified and characterized HGF activator (HGFA), a serum-derived serine protease that efficiently activates single-chain HGF in vitro. In this study, we found that the HGF-converting activity in the injured liver was inhibited by an anti-HGFA antibody. We also found that the active form of HGFA was generated exclusively in the injured tissues. Thus, it appears likely that HGFA is the key enzyme that regulates the activity of HGF in the injured tissues. We also analyzed the heparin binding properties of the precursor and mature forms of HGFA. HGFA had a weak affinity for heparin near the physiological salt concentration in its precursor form but acquired a strong affinity for heparin upon activation that is linked to blood coagulation. This property may ensure the local action of this enzyme at the site of tissue injury.
Hepatocyte growth factor (HGF), ()also known as
scatter factor, was originally described as a potent mitogen for
hepatocytes in primary culture(1, 2, 3) . It
was subsequently shown to have mitogenic, motogenic, and morphogenic
activities on various target cells, including renal tubular epithelial
cells and vascular endothelial
cells(4, 5, 6, 7, 8, 9) .
HGF is thought to play an important role in regeneration following
hepatic and renal injury(10, 11, 12) .
Mature HGF is a heterodimeric protein consisting of a heavy chain and a light chain held together by a disulfide bond(1) . The two chains are produced from a single-chain precursor by proteolytic processing(13, 14) . This processing, which is mediated by a serine protease, is required for HGF to exert both its mitogenic and motogenic activities (15, 16, 17, 18, 19) . We recently found that the biological effects of HGF in injured tissues are regulated through this proteolytic processing; HGF in normal tissue is present in the inactive single-chain form, and it is converted to the active heterodimeric form exclusively in the injured tissues(20) . We also found that this conversion was mediated by a serine protease, the activity of which was induced in the injured tissues(20) . However, the serine protease has not yet been identified.
Four proteases are reported to activate HGF in vitro. We previously purified a HGF-activating protease from bovine and human serum (21, 22) and designated it HGF activator (HGFA). Blood coagulation factor XIIa, urokinase, and tissue-type plasminogen activator (tPA) also activate HGF in vitro(16, 23, 24) . Although the action of urokinase and tPA on HGF is very weak in vitro(23, 25) , it is possible that, in vivo, the enzymatic reaction may be stimulated by a cofactor(s) or by a certain microenvironment. In fact, receptor-bound urokinase modulates the activation and receptor binding of HGF(26) . These serine proteases are thus the candidates for the HGF-converting enzyme(s) in injured tissues.
In the present study, we examined the involvement of HGFA in the activation of HGF in the injured tissues. We purified and characterized the rat counterpart of HGFA. We then analyzed the inhibitory effect of anti-human HGFA monoclonal antibodies (mAb) on rat HGFA and found that one of them inhibited rat HGFA. We demonstrated that the HGF-converting activity in the homogenate of injured rat liver was abrogated by treatment with the anti-HGFA antibody. In addition, we found that the active form of HGFA was generated exclusively in the injured tissues. Thus, we concluded that HGFA was the key enzyme regulating the activity of HGF in injured tissues. We also found that the activated HGFA acquired the capacity to interact with heparin. This property may ensure the local action of HGFA.
Figure 1: Activation of human recombinant HGF by rat HGFA (A) and the effects of monoclonal antibodies (B). A, single-chain HGF (200 µg/ml) was incubated at 37 °C for 2 h with various concentrations of rat HGFA and analyzed by SDS-PAGE (12.5% acrylamide) under reducing conditions. The gel was stained with Coomassie Brilliant Blue. Molecular mass markers are shown on the left. B, effects of monoclonal antibodies (1 µg) were analyzed by preincubation with rat HGFA (10 ng) at 37 °C for 30 min.
Figure 2: Abrogation of HGF-converting activity in the injured liver by an anti-HGFA antibody. A single-chain HGF was incubated with homogenate of injured rat liver and analyzed by immunoblotting using the P-1 mAb that was raised against the heavy chain of HGF. Lane 1, no incubation; lane 2, 37 °C for 8 h; lane 3, 37 °C for 8 h, pretreated with anti-HGFA, P1-4; lane 4, 37 °C for 8 h, pretreated with P-5, which does not react with HGFA (as a negative control).
Figure 3:
Tissue-derived HGFA after hepatic or renal
injury. Soluble proteins (40 mg) of tissue homogenates from liver,
kidney, lung, and spleen were fractionated on P1-4 mAb affinity
column and analyzed by immunoblotting. Lane 1, normal; lane 2, 12 h after CCl treatment; lane 3,
12 h after HgCl
treatment. The band of 82 kDa probably
corresponds to a degradation product of the
precursor(27) .
Figure 4: Heparin binding properties of the precursor and active forms of rat HGFA. Rat plasma (A) or serum (B) was diluted and applied to a heparin-Sepharose column. After washing was carried out, the bound proteins were eluted in a stepwise manner with buffer containing increasing concentrations of NaCl (100-800 mM) and analyzed by immunoblotting.
The biological activities of HGF have been shown to be localized to injured tissues by proteolytic processing in vivo(20) . In that study, when the rat was treated with hepatotoxin, HGF was converted to its active form in the liver but not in the kidney, lung, or spleen. Similarly, when the rat was treated with nephrotoxin, HGF was activated in the kidney but not in the liver, lung, or spleen. We found that HGF-converting activity was induced in the injured liver (20) . Since it appears likely that this activity plays a key role in regulation of HGF activities in the injured tissues, it is important to identify the protease responsible for this activity.
In the present study, we clearly demonstrated that the HGF-converting activity in the injured liver was abrogated by treatment with HGFA-specific antibody. In addition, we found that the active form of HGFA was generated in the liver and kidney after induction of hepatic and renal injury, respectively. This generation was not observed in the uninjured tissues. These results indicate that HGFA is most likely the key enzyme that is involved in the locally restricted generation of active HGF in the injured tissues.
The properties of HGFA well explain the presence of HGFA activity in the injured tissues and its absence in normal tissues. HGFA was first identified in bovine serum (21) and human serum(22) . The cDNA cloning revealed that its sequence was homologous (39%) to that of blood coagulation factor XII(22) . HGFA is a plasma protein produced in the liver. It circulates in the blood as an inactive zymogen that is converted into the active form by thrombin during blood coagulation (27) . Because tissue injury often leads to the activation of the blood coagulation pathway(31) , the zymogen form of HGFA is converted into the active enzymes to activate HGF in injured tissues. To retain the activity of HGFA in the close vicinity of the injured locus, it would be favorable if HGFA protein were not freely diffusible. The active form of HGFA appears to be associated with the cell surface. In our previous study, we detected HGFA activity in a primary culture of hepatocytes under serum-free conditions(15) . The activity was cell-bound, because the single-chain HGF was not converted to the heterodimer during incubation with the serum-free conditioned medium of the culture(32) . In the present study, we found that HGFA acquired a heparin binding ability after it was activated. These results indicate that the zymogen of HGFA is rather diffusible, whereas the activated form can associate with cell surface heparin-like molecules and is prevented from free diffusion. This property of HGFA probably ensures its localized action on HGF. The binding of HGFA with heparin-like molecules has another merit in activating HGF, because HGF is also associated with heparin-like molecules on the cell surface(16, 29) .
In conclusion, a proteolytic cascade plays a key role in the activation of HGF in response to tissue injury (Fig. 5). Recently, Uehara et al. (33) and Schmidt et al.(34) reported that disruption of the HGF gene in mice caused embryonic lethality, indicating that HGF is an essential factor for mouse development. During development, the activity of growth/differentiation factors must be spatially restricted. Thus, a proteolytic activation system for HGF appears to be required also during the development. Further study is necessary to determine whether or not HGFA also functions as a HGF-converting enzyme during mammalian development.
Figure 5: Proteolytic cascade for activation of HGF in response to tissue injury.