Báo cáo khoa học: Hepatocyte growth factor activator (HGFA): a serine protease that links tissue injury to activation of hepatocyte growth factor pdf

MINIREVIEW Hepatocyte growth factor activator (HGFA): a serine protease that links tissue injury to activation of hepatocyte growth factor Keiji Miyazawa Department of Biochemistry, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Japan Introduction In tissues of multicellular organisms, different types of cells are intricately and precisely arranged to perform specific functions. Once such tissue architecture is destroyed, the regeneration system works to restore the structure as well as the function of the damaged tissue. Liver regeneration has long been a subject of active research, because it displays a dramatic form of organ regeneration. It also represents a good in vivo model for understanding the regulation of cell growth: hepatocytes are usually in a quiescent state, but most of them enter the cell cycle during liver regeneration. Humoral factors that trigger liver cell growth have been detected in the blood circulation of liver-injured animals, and many researchers have tried to isolate these factors. Hepatocyte growth factor (HGF) was originally identified as a potent mitogen for hepatocytes in pri- mary culture during studies of liver regeneration [1]. Studies showed that HGF was induced in the blood plasma and liver in response to liver injury. Therefore, Keywords hepatocyte growth factor; plasminogen; proteolytic activation; tissue injury Correspondence K. Miyazawa, Department of Biochemistry, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan Fax ⁄ Tel: +81 55 273 6784 E-mail: keiji-miyazawa@umin.ac.jp (Received 26 November 2009, revised 3 February 2010, accepted 26 February 2010) doi:10.1111/j.1742-4658.2010.07637.x Growth factors are a group of proteins that regulate a wide variety of cel- lular processes, including proliferation, differentiation, motility, adhesion, and apoptosis of target cells. They play crucial roles in the formation and maintenance of tissue architecture in embryonic development and adult tissue homeostasis. Because aberrations in growth factor signaling often result in pathological conditions, the activities of growth factors are tightly controlled by extracellular and intracellular regulators. Hepatocyte growth factor (HGF) is a mesenchymal cell-derived growth factor that affects various target cells, including epithelial and endothelial cells. HGF is synthesized and secreted as a latent form, and is proteolytically activated in response to tissue injury, thus participating in tissue regeneration and repair. Interestingly, HGF has a unique structural feature: it is homologous to plasminogen, a key enzyme in the fibrinolytic system. Elucidation of the regulatory mechanisms of HGF activity has revealed that a blood coagula- tion factor XII-like serine protease, hepatocyte growth factor activator, efficiently converts HGF from the latent form to the active form. Hepato- cyte growth factor activator itself is activated downstream of the blood coagulation cascade, and links tissue injury to activation of HGF. HGF thus has structural as well as functional relevance to the blood coagulation ⁄ fibrinolytic system. Abbreviations FXII, coagulation factor II; HGF, hepatocyte growth factor; HGFA, hepatocyte growth factor activator; tPA, tissue-type plasminogen activator. 2208 FEBS Journal 277 (2010) 2208–2214 ª 2010 The Author Journal compilation ª 2010 FEBS HGF was regarded as the humoral factor that triggers liver regeneration. HGF was subsequently shown to have mitogenic, motogenic and morphogenic activities on various target cells, including epithelial and endo- thelial cells. HGF is now thought to play a major role in the repair and regeneration of various tissues, including the liver, kidney, lung, and stomach [1]. A new question then arose. Why is the liver specifi- cally targeted to grow after liver injury? HGF and its receptor, MET tyrosine kinase, are widely expressed and distributed among normal as well as injured tis- sues. After liver injury, HGF is induced not only in the liver but also in blood plasma and other uninjured tissues, such as spleen [2]. Although HGF is not exclu- sively induced in the liver, its action is limited to the injured liver. Apparently, the amount of HGF is not correlated with the activity of HGF, suggesting that HGF is latent in normal states, and is activated specifi- cally at the site of tissue injury. The mechanism of localized activation of HGF, however, had remained unclear. In this minireview, I describe the discovery of a novel serine protease, HGF activator (HGFA), which has revealed the link between tissue injury and localized activation of HGF. Active and inactive forms of HGF The first step in solving this puzzle was to understand the nature of latent HGF. Several examples of latent growth factors have already been reported. Insulin-like growth factors are inactive when bound to insulin-like growth factor-binding proteins. Transforming growth factor-b is produced and secreted as a latent precursor form composed of the N-terminal ‘latency-associated peptide’ and the C-terminal mature form. It is activated after the N-terminal portion of the precursor is degraded or dissociated from the C-terminal portion. In the case of HGF, its unique primary structure pro- vided a valuable clue for solving the puzzle of HGF latency. Mature HGF consists of two polypeptide chains, a heavy a-chain (62 kDa) and a light b-chain (32–34 kDa), which are held together by a disulfide bond [3]. In 1989, cDNA of human HGF was cloned, and the primary structure was elucidated [4,5]. HGF is synthesized and secreted as a single-chain precursor [6], and is extracellularly processed to the two-chain form by proteolytic cleavage at a specific site. The heavy chain consists of four tandem repeats of a krin- gle domain, and the light chain has a structure similar to that of the catalytic domain of serine proteases (Fig. 1A) [5]. HGF, however, has no proteolytic activ- ity, because two of the conserved catalytic triad resi- dues of the serine protease domain are substituted. The domains of HGF are very similar to those of pro- teases in the blood coagulation and fibrinolytic system. HGF shows the highest similarity to plasminogen (about 40% amino acid similarity). Plasminogen is synthesized as a single-chain form that consists of five tandemly repeated kringle domains and a serine protease domain [1]. It is activated upon cleavage at a specific site between the fifth kringle Heavy chain (α-chain) Light chain (β-chain) 62 kDa 32–34 kDa Signal 1 I II III IV 2 (K)(N) (N) (K) (N) NK1 NK2 I I II (K) A B Hairpin region Kringle domain Serine protease- like domain Fig. 1. Schematic structure of HGF (A) and its variants (B). Circles denote amino acids, and lines denote disulfide bonds. Arrow- head 1 denotes the cleavage site of the signal sequence. Arrowhead 2 denotes the cleavage site for activation. Roman numbers denote kringle domain numbers. K. Miyazawa Hepatocyte growth factor activator FEBS Journal 277 (2010) 2208–2214 ª 2010 The Author Journal compilation ª 2010 FEBS 2209 domain and the serine protease domain by the action of either urokinase or tissue-type plasminogen activa- tor (tPA). After this site-specific cleavage, the protease domain changes conformation and becomes active. Plasmin then degrades fibrin clots and extracellular matrices, and activates matrix metalloproteases, thus contributing to the process of tissue remodeling. Similarly, the single-chain form of HGF was shown to be a latent form. We found that single-chain HGF was inactive in the presence of serine protease inhibi- tors in a hepatocyte proliferation assay, whereas the two-chain form was active even in the presence of the inhibitors [7]. Furthermore, an HGF mutant, in which Arg494 was replaced by glycine, and which was thus resistant to proteolytic processing, was not active at all [7,8]. HGF needs be processed to the two-chain form to exert its biological activity. Recently, proteolytic processing of HGF was shown to induce a conforma- tional change in the serine protease-like domain, which is required for functional interaction of HGF with its receptor, MET, through a region corresponding to the ‘active site’ and ‘activation domain’ of serine proteases [9,10]. HGF and serine proteases thus share a similar activation mechanism. Proteolytic activation of HGF in response to tissue injury In order to elucidate the in vivo roles of proteolytic processing of HGF, we examined the molecular forms of HGF by immunoblotting using a heavy chain- specific monoclonal antibody [11]. The antibody reacts with both the single-chain and two-chain forms of HGF, giving positive bands at 92 and 62 kDa, respec- tively. By scanning of the 92 and 62 kDa bands, the ratio of the single-chain form (inactive) to the two-chain form (active) can be quantified. We first found that HGF from various normal rat tissues (liver, kidney, lung, and spleen) was present exclusively in the inactive single-chain form. We next administered hepatotoxin or nephrotoxin to rats to induce tissue injury. HGF was extracted from injured and uninjured tissues and then analyzed. After intraga- stric administration of carbon tetrachloride, liver tissue was severely injured and the other tissues were mini- mally affected. The amount of HGF was dramatically increased in the liver and spleen tissue, but not in the kidney or lung tissue. HGF was converted to the active form only in the liver, which was injured in this experimental model. Similar results were obtained when d-galactosamine was used to induce liver injury through a different mechanism. When we injected mercuric chloride to induce renal injury in rats, HGF in the kidney increased in quantity and was activated. In contrast, HGF in the liver and spleen increased in quantity but was not activated. These findings indicated that HGF in uninjured tissue exists as the inactive form, even though it sometimes increases in amount. HGF is therefore activated exclusively in injured tissues by proteolytic processing, and thus seems to contribute to the process of tissue regenera- tion and repair [11]. The fact that the proteolytic conversion of HGF is specific to injured tissues suggests that HGF-convert- ing enzyme(s) should work exclusively in the injured tissues. We found that HGF-converting activity was induced in the injured liver but not in the normal liver tissue [11]. This activity seemed most likely to repre- sent a key enzyme regulating the action of HGF in injured tissues. We thought that identification of the enzyme would be crucial to understanding the control of HGF action in vivo. The activity of this enzyme, however, was not high enough to allow purification of the protein for identification. Fortunately, we detected strong HGF-converting activity in human serum. HGFA – one of the key enzymes of HGF activation We thus purified, from human serum, a novel serine protease of 34 kDa that activates HGF very efficiently in vitro, and designated it as HGFA [12]. The nucleo- tide sequence of HGFA cDNA showed that HGFA is derived from the C-terminal region of a precursor of 655 amino acids by proteolytic processing. Interest- ingly, the precursor consists of multiple domains, a type II fibronectin homology region, two epidermal growth factor domains, a type I fibronectin homology region, a kringle domain, and a catalytic domain (Fig. 2). These domains are homologous to those observed in blood coagulation factor XII (FXII), with an overall amino acid similarity of 39%. Analysis of genomic DNA coding for HGFA indicated a relation- ship between HGFA and FXII as well as urokinase and tPA, activators of plasminogen. These four pro- teins therefore constitute a family (the PA–FXII– HGFA family) [13]. Other proteases, such as urokinase, tPA, FXII, factor XI, plasma kallikrein, matriptase, and hepsin, were subsequently reported to activate HGF in vitro [14–18]. The first five of these are of blood plasma ori- gin, whereas the latter two are transmembrane serine proteases and may be involved in pericellular activa- tion of HGF. Among these proteases, matriptase and hepsin activate HGF with comparable efficiency to that of HGFA in vitro [17,18]. Although the activities Hepatocyte growth factor activator K. Miyazawa 2210 FEBS Journal 277 (2010) 2208–2214 ª 2010 The Author Journal compilation ª 2010 FEBS of the other proteases on HGF are very weak in vitro, they may be stimulated by a cofactor(s) or by a certain microenvironment in vivo. These proteases, as well as HGFA, are thus candidates for the HGF-converting enzyme in injured tissues. Recently, Itoh and Kataoka generated mice defi- cient in hgfa, and directly demonstrated that HGFA is the serum-derived protease responsible for activa- tion of HGF [19,20]. HGFA was also shown to be required for tissue repair in experimental colitis mod- els. Both HGF activation and tissue regeneration were markedly impaired in injured intestinal tissue of HGFA-deficient mice. The important function of HGFA during tissue repair in vivo was thus demon- strated. How is HGFA activity localized to the site of tissue injury? HGFA is produced in liver parenchymal cells, and behaves as an acute-phase protein [21]. HGFA is found in the active form in serum, but in the inactive form in plasma. This precursor form was purified and characterized [22]. The inactive form of HGFA (pro-HGFA) has a molecular mass of 96 kDa, and is converted to its mature form by thrombin and plasma kallikrein (KLKB1) [22] (Fig. 2). The process- ing by thrombin is required for activating HGFA, whereas that by plasma kallikrein is not. The role of the processing by plasma kallikrein remains to be elucidated. HGFA circulates in the bloodstream as an inactive precursor, and is activated in response to tissue injury, probably coupled with activation of the blood coagula- tion system. Immunoblotting analysis of HGFA from normal and injured tissues indicated that HGFA is activated exclusively in injured tissues [23]. Thus, injured tissue-specific activation of HGFA appears to be involved in the localized activation of HGF. HGF was originally identified as a potent mitogen for hepatocytes, but it was subsequently shown to induce angiogenesis in vivo, and to stimulate prolifera- tion ⁄ migration of vascular endothelial cells in vitro [24,25]. HGF thus appears to be linked to the blood coagulation and fibrinolytic system, not only structur- ally but also functionally (Fig. 3). The blood coagula- tion system is activated upon injury of blood vessels, leading to conversion of prothrombin to thrombin. Thrombin processes fibrinogen and coagulation fac- tor XIII (plasma transglutaminase) to form stable blood clots and prevent further hemorrhage from the injured sites. Concomitantly, thrombin induces activa- tion of HGF via HGFA. HGF then stimulates prolif- eration and migration of endothelial cells to repair blood vessels. It appears rational that the blood coagu- lation system triggers activation of a growth factor that promotes angiogenesis. Therefore, the prototypic function of HGF may be to maintain the integrity of blood vessels. Thrombin appears to be a bifurcation point for clotting and endothelial cell migration ⁄ prolif- eration, and HGFA represents the link between tissue injury and activation of HGF. Perspectives In 1995, Uehara et al. and Bladt and coworkers [26,27] reported that the embryonic lethality of HGF-knock- Kringle Type I Type II EGF EGF 1 (2) Serine protease Fig. 2. Schematic structure of HGFA. Circles denote amino acids, and lines denote disulfide bonds. The names of the domains are shown. Type I and Type II denote the type I fibronectin homology region and the type II fibronectin homology region, respectively. The arrowhead denotes the cleavage site of the signal sequence. Arrow 1 denotes the site of cleavage by thrombin, kallikrein 1- related peptidase 4, and kallikrein 1-related peptidase 5, which is required for activation of HGFA. Arrow 2 denotes the site of cleav- age by plasma kallikrein. EGF, epidermal growth factor. Cell growth, migration Pro-HGF HGF Cell FXa Pro-HGFA HGFA Thrombin FVa Prothrombin FXIII Fibrinogen fibrin FXIIIa Cross-linked fibrin clot Fig. 3. Activation of HGF in response to tissue injury. Activation of the blood coagulation cascade, through either an intrinsic or an extrinsic pathway, results in conversion of prothrombin to thrombin. Thrombin processes fibrinogen and factor XIII (FXIII; plasma trans- glutaminase) to form stable blood clots and prevent further hemor- rhage from the injured sites. Concomitantly, thrombin induces activation of HGF via HGFA, which leads to endothelial cell growth ⁄ migration and contributes to repair of the blood vessel structure. HGF also stimulates the proliferation and migration of epithelial cells to repair the tissue architecture. FVa, factor Va; FXa, factor Xa; FXIIIa, activated FXIII. K. Miyazawa Hepatocyte growth factor activator FEBS Journal 277 (2010) 2208–2214 ª 2010 The Author Journal compilation ª 2010 FEBS 2211 out mice was caused by dysfunction of the placenta and liver, indicating that HGF plays important roles during embryonic development. We can explain the activation of HGF in injured tissues in adults by the scheme illustrated in Fig. 3, but it remains unclear how HGF is activated during embryonic development in which there is no apparent tissue injury. In the Drosophila embryo, the signal transduction pathway that establishes the dorsal–ventral pattern is temporally and spatially regulated through the proteolytic cascade to activate Spa ¨ tzle, the Toll receptor ligand [28]. It is likely that a proteolytic cascade also plays an impor- tant role in the regulation of HGF activity during mammalian development. Known proteases that acti- vate HGF in vitro do not appear to be the key prote- ases activating HGF during embryonic development, because mice lacking these proteases are not embry- onic lethal. Alternatively, truncated variant forms of HGF, NK1, and NK2 (Fig. 1B), which are abundantly expressed in embryo-derived cells, might contribute to HGF activity during embryonic development. These variants exhibit weak agonistic activity, although they lack the serine protease-like domain [29,30], and do not appear to require proteolytic activation. Further progress in this field would answer this question. In chronic liver diseases, liver dysfunction often leads to disorders in blood coagulation, because the plasma levels of blood clotting enzymes, which are produced in hepatocytes, are decreased. Similarly, HGFA is produced in hepatocytes [21], and its produc- tion is impaired in a rat model of liver cirrhosis, caus- ing decreased efficiency of HGF activation [31]. Kaibori et al. [32] demonstrated that local administra- tion of HGFA promotes conversion of HGF from the inactive form to the active form, leading to accelera- tion of liver regeneration. In other cases, aberrant activation of HGF may result in overactivity of HGF, which is implicated in malignancy as well as some types of kidney disease (including polycystic disease, glomerulosclerosis, and renal tubular hyperplasia) [33]. It is important to identify HGF-activating enzymes in each pathogenic situation that is associated with aber- rant HGF function. Selective inhibitors against those enzymes, probably including HGFA, could be candi- dates for clinical drugs for treatment of such diseases with HGF overactivity. We identified HGFA as a protease that links tissue injury to activation of HGF. However, HGFA may activate HGF in other contexts in vivo. Notably, the kallikrein 1-related peptidases KLK-4 and KLK-5 were recently shown to activate HGFA [34]. These proteases cleave HGFA at the same site as thrombin. KLK-4 and KLK-5 are implicated in the activation of HGFA in tumor tissues. Further investigation of HGFA activation in vivo, as well as regulation of HGFA activity by endogenous protease inhibitors, including hepatocyte growth factor activator inhibitor-1 (HAI-1) and protein C inhibitor [35–37], will be important for understanding the pathophysiological processes regulated by the HGF–HGFA system. Acknowledgements I apologize to colleagues in the field for not citing many important papers, because of the limitation of the length of this review article. I would like to thank T. Shimomura, D. Naka and T. Kawaguchi of Mitsu- bishi Chemical Corp., A. Okajima and A. Kitamura of Kansai Medical University and N. Kitamura of Tokyo Institute of Technology for their contributions to the study of HGFA. 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