MINIREVIEW Hepatocyte growth factor activator (HGFA): pathophysiological functions in vivo Hiroaki Kataoka and Makiko Kawaguchi Section of Oncopathology and Regenerative Biology, Faculty of Medicine, University of Miyazaki, Japan Introduction Hepatocyte growth factor (HGF), also called scatter factor (SF), is a multifunctional growth factor known to play important roles in development, tissue regeneration and tumor progression via its receptor, the tyrosine kinase MET, which is the c-met proto-oncogene product [1]. HGF ⁄ SF is secreted as an inactive proform (pro- HGF ⁄ SF), which is structurally homologous to plas- minogen. It is activated by proteolysis, generating a heterodimeric product consisting of heavy (a) and light (b) chains. Although the b-chain shows homologies with the serine protease domain of plasmin, catalytic activity is absent in HGF ⁄ SF. The activation of pro-HGF ⁄ SF is critical for the triggering of HGF⁄ SF–MET signaling [2]. Several proteases that are found in the serum or on cell membranes have been proposed to be activators of HGF ⁄ SF signaling [2,3]. These activators include HGF activator (HGFA), coagulation factors XII and XI, plasma kallikrein, urokinase-type and tissue-type plas- minogen activators, matriptase, and hepsin. Among them, HGFA, matriptase and hepsin show much more efficient processing activity than the other proteases, at least in vitro. HGFA was initially purified from bovine serum as a potent activator of HGF ⁄ SF [4]. Shortly afterwards, human HGFA was purified and its cDNA Keywords hepatocyte growth factor; hepatocyte growth factor activator; HGF activator; macrophage-stimulating protein; tissue injury Correspondence H. Kataoka, Section of Oncopathology and Regenerative Biology, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan Fax: +81 985 856003 Tel: +81 985 852809 E-mail: mejina@med.miyazaki-u.ac.jp (Received 17 November 2009, revised 21 January 2010, accepted 26 February 2010) doi:10.1111/j.1742-4658.2010.07640.x Hepatocyte growth factor activator (HGFA) is a serine protease initially identified as a potent activator of hepatocyte growth factor ⁄ scatter factor. Hepatocyte growth factor ⁄ scatter factor is known to be critically involved in tissue morphogenesis, regeneration, and tumor progression, via its recep- tor, MET. In vivo, HGFA also activates macrophage-stimulating protein, which has roles in macrophage recruitment and inflammatory processes, cellular survival and wound healing through its receptor, RON. Therefore, the pericellular activity of HGFA might be an important factor regulating the activities of these multifunctional cytokines in vivo. HGFA is secreted mainly by the liver, circulates in the plasma as a zymogen (pro-HGFA), and is activated in response to tissue injury, including tumor growth. In addition, local production of pro-HGFA by epithelial, stromal or tumor cells has been reported. Although the generation of HGFA-knockout mice revealed that the role played by HGFA in normal development and physio- logical settings can be compensated for by other protease systems, HGFA has important roles in regeneration and initial macrophage recruitment in injured tissue in vivo. Insufficient activity of HGFA results in impaired regeneration of severely damaged mucosal epithelium, and may contribute to the progression of fibrotic lung diseases. On the other hand, deregulated excess activity of HGFA may be involved in the progression of some types of cancer. Abbreviations HAI, hepatocyte growth factor activator inhibitor; HGF, hepatocyte growth factor; HGFA, hepatocyte growth factor activator; MSP, macrophage-stimulating protein; PCI, protein C inhibitor; pro-MSP, pro-macrophage-stimulating protein; SF, scatter factor. 2230 FEBS Journal 277 (2010) 2230–2237 ª 2010 The Authors Journal compilation ª 2010 FEBS was cloned [5]. HGFA is a member of the kringle serine protease superfamily, and its molecular structure resem- bles that of coagulation factor XII [2,5]. The gene encoding human HGFA consists of 14 exons spanning a genomic region of approximately 7.5 kb on chromo- some 4 [2]. Its 5¢ regulatory region lacks consensus TATA and CAAT boxes and contains multiple response elements, including sequences binding SP-1, AP-2, Ets- 1, hepatocyte nuclear factor-1, and nuclear factor-jB. It also contains a hypoxia-inducible factor-1a-binding site, which enables hypoxia-induced expression of HGFA [6]. The gene encoding mouse HGFA shows a similar genomic structure to the human gene [7]. In vivo, HGFA is synthesized primarily by hepatocytes as an inactive single-chain proform (pro-HGFA) [2,5]. There- fore, activation of pro-HGFA is a prerequisite for this activity. Once activated, the activity of HGFA is regu- lated by endogenous inhibitors, HGFA inhibitor (HAI)- 1 and HAI-2, in the pericellular microenvironment of epithelial tissues [2,8,9]. In human plasma and serum, its activity is regulated by protein C inhibitor (PCI) [10]. In spite of its efficient pro-HGF ⁄ SF-processing activity and relatively high levels in plasma, the func- tional roles of HGFA in vivo are unclear. This mini- review covers the presumed functions of HGFA in vivo that have aided our understanding of the roles of HGFA in physiological and pathological settings. Synthesis and distribution of HGFA in vivo HGFA is secreted mainly by the liver, and circulates in the plasma as a single-chain pro-HGFA, which migrates with a molecular mass of 98 or 96 kDa under reducing or nonreducing conditions, respectively [11]. The concentration of pro-HGFA in plasma of healthy individuals is around 40 nm ( 4 lgÆmL )1 ) [10], indi- cating its relative abundance as a plasma protein. The activated form of HGFA is also detectable in human plasma and serum, and the reported levels in healthy individuals are varied, possibly owing to assay condi- tions. They range from 8 pm (0.3 ngÆmL )1 ) [12] to 520 pm (18 ngÆmL )1 ) [13] in the serum, and are  140 pm ( 5ngÆmL )1 ) in the plasma [10]. Moreover, activated HGFA is reported to be complexed with PCI, and the HGFA–PCI complex has been found in the plasma of healthy individuals at a mean concentra- tion of 27 pm [10]. In addition to hepatic production of pro-HGFA, low but distinct levels of extrahepatic HGFA expres- sion have been reported in a number of tissues – gastrointestinal, renal, synovial, and central nervous system – and in hair follicles [7,14–17]. Therefore, although pro-HGFA is abundant in the plasma, local production of HGFA may also contribute to the pro- cessing of its substrate(s), such as pro-HGF ⁄ SF, in pericellular microenvironments. This might have important roles in tissue development, homeostasis and regeneration via establishment of HGF ⁄ SF–MET signaling [1]. Moreover, tumor cells can also express and secrete HGFA, suggesting a potential role of this enzyme in tumor biology [18–23]. Mechanism of pro-HGFA activation As mentioned above, HGFA is synthesized and secreted as an inactive proform (pro-HGFA) [2,5]. Therefore, activation of pro-HGFA is critical for its activity. As significant activation of pro-HGF ⁄ SF in vivo is observed exclusively in injured tissues, it is reasonable to postulate that pro-HGFA is also acti- vated in response to tissue injury [2]. In fact, the acti- vation of pro-HGF ⁄ SF in injured tissues was significantly inhibited by neutralizing antibody against HGFA [24]. In this context, thrombin is a presumed activator of pro-HGFA in vivo, as activation of the coagulation cascade occurs in injured tissues [11]. In vitro studies indicate that thrombin activates pro- HGFA through efficient cleavage of the Arg407-Ile408 bond in the presence of negatively charged substances such as dextran sulfate, heparin, and chondroitin sul- fate. This generates the two-chain HGFA (active form), consisting of a disulfide-linked 66 kDa heavy chain and a 32 kDa light chain [11]. The catalytic domain is present in the 32 kDa light chain. Plasma kallikrein may further cleave the Arg372-Val373 bond in the 66 kDa heavy chain, resulting in a final 34 kDa two-chain form (short form), which was initially puri- fied from serum [11]. This short form retains its enzy- matic activity [8,11]. There may be another activating mechanism for pro-HGFA. Human kallikrein 1-related peptidase (KLK) proteins are a family of serine proteases pro- duced and secreted by various types of tissue. Among KLKs, KLK4 and KLK5 activate pro-HGFA effi- ciently by cleaving the Arg407-Ile408 bond [25]. The activity of KLK5 is comparable with that of thrombin, and requires a negatively charged substance as well. KLK4 does not require a negatively charged substance for the activation of pro-HGFA, but its specific activ- ity is one-fifth that of KLK5 [25]. KLK5 further cleaves the Arg372-Val373 bond of the active form, eventually generating the short form. Forced expres- sion of KLK5 in the human pancreatic carcinoma cell line SUIT-2 results in enhanced processing of pro- HGF ⁄ SF, phosphorylation of MET, and enhanced H. Kataoka and M. Kawaguchi HGFA: pathophysiological functions in vivo FEBS Journal 277 (2010) 2230–2237 ª 2010 The Authors Journal compilation ª 2010 FEBS 2231 invasiveness [25]. Therefore, these pro-HGFA-activat- ing KLKs can serve as cellular activators of pro- HGFA in pericellular spaces, particularly in tumor cells concomitantly expressing both KLKs and pro-HGFA. This machinery may be important for the generation of active-form HGF ⁄ SF in tumor cell microenvironments. Once activated, HGFA can be localized to the peri- cellular microenvironment in injured tissue via its affin- ity for heparin and ⁄ or binding to HAI-1 on the cell surface [8,24]. HAI-1 is a membrane-bound Kunitz- type serine protease inhibitor that is expressed mainly on the basolateral surfaces of epithelial cells. The bind- ing of HGFA to cell surface HAI-1, a protease–inhibi- tor interaction, inhibits HGFA activity [8]. However, in vitro studies using cultured cells suggest that the HGFA–HAI-1 complexes on the cell surface can potentially be released via metalloprotease-mediated shedding of the 58 kDa low-affinity secreted HAI-1 ectodomain [8]. This regulated shedding is enhanced by inflammatory cytokines such as interleukin-1b [8]. After HGFA–HAI-1 shedding subsequent to interleu- kin-1b stimulation, HGFA can dissociate from HAI-1, resulting in considerable recovery of HGFA activity in the culture supernatant [8]. Therefore, it is possible that HAI-1 is not only an inhibitor but also a reservoir of HGFA on the cell surface in injured and inflamed tissue. Indeed, the expression of HAI-1 is enhanced in response to tissue injury [2,8]. Presumed substrates and physiological roles of HGFA in vivo The substrate specificity of HGFA appears to be very limited, and only two substrate molecules have been reported to date. One is pro-HGF ⁄ SF, a well-known substrate, as indicated by the name HGFA. Another substrate, pro-macroph age-stimulating p rotein (pro -MS P), was recently identified as a physiological substrate of HGFA in vivo [26]. Both HGF⁄ SF and macrophage- stimulating protein (MSP) are members of the kringle protein family, having four kringle domains and a serine protease-like domain in each molecule. They show significant amino acid sequence identity (45%) over all domains [26,27]. MSP was originally identi- fied as a plasma protein that promotes chemotactic responses in peritoneal resident macrophages [27]. Unlike HGF ⁄ SF, which is mainly produced by stromal cells as a paracrine factor, MSP is primarily synthesized by the liver as an inactive proform (pro- MSP), and is secreted as a plasma protein in a con- centration range of 2–5 nm (0.16–0.4 lgÆmL )1 ) [27]. The proteolytic cleavage at the Arg483-Val484 bond of pro-MSP by HGFA results in a disulfide-linked heterodimer consisting of a 60 kDa a-chain and a 30 kDa b-chain (mature MSP), which phosphorylates its specific receptor, the tyrosine kinase RON (recepteur d’origine nantais) [26]. Notably, RON is expressed not only by macrophages, but also by many types of epithelial and tumor cell [27]. There- fore, pericellular HGFA activity may influence the cellular functions through both HGF ⁄ SF–MET and MSP–RON signaling. Since the discovery of HGFA, only a limited num- ber of in vivo studies have been published describing the role of HGFA in physiological processes (Table 1). The roles of HGFA during the morphogenesis of the gastrointestinal tract and metanephric kidney have been investigated in murine developmental settings, [14,28]. The results indicated that the morphogenesis of these fetal tissues requires enhanced processing of pro-HGF ⁄ SF by HGFA. Possible roles of HGFA are also suggested in B-cell differentiation in the germinal center of the lymph node, where pro-HGF ⁄ SF and HGFA provided by dark zone follicular dendritic cells help to regulate the proliferation, survival and ⁄ or adhesion of MET-positive centroblasts [29]. Hair folli- cle elongation may also require HGFA-mediated HGF ⁄ SF activation [17]. On the other hand, our stud- ies using HGFA-knockout mice suggest that the pro- cessing of pro-HGF ⁄ SF is redundant during tissue development, as homozygous mutant HGFA-knockout mice were viable without obvious developmental abnormalities [30]. Since HGF ⁄ SF ) ⁄ ) mice are embry- onic lethal, with impaired development of the placenta and liver, it is evident that there are alternative mecha- nism(s) for pro-HGF ⁄ SF activation during tissue development. In fact, recent studies have revealed that membrane-bound serine proteases, such as matriptase and hepsin, can activate pro-HGF ⁄ SF [3]. These prote- ases may serve as cellular activators of pro-HGF ⁄ SF in local tissue environments. Thus, in spite of its rela- tively high concentration in the plasma, the precise roles of HGFA in developmental and physiological settings remain to be clarified. On the other hand, despite the apparently normal appearance of HGFA ) ⁄ ) mice, the knockout studies also confirmed that the major serum activator of pro-HGF ⁄ SF is, in fact, HGFA, as the sera from HGFA ) ⁄ ) mice were unable to process pro-HGF ⁄ SF [30]. Essential roles of HGFA in injured tissue The activation of pro-HGFA and the efficient locali- zation of mature HGFA in the desired tissue and HGFA: pathophysiological functions in vivo H. Kataoka and M. Kawaguchi 2232 FEBS Journal 277 (2010) 2230–2237 ª 2010 The Authors Journal compilation ª 2010 FEBS cells is a prerequisite for the utilization of this potent activator of HGF ⁄ SF. Considering the in vivo obser- vations that significant HGF ⁄ SF activation occurs exclusively in response to tissue injury and inflamma- tion [31], where pro-HGFA is efficiently activated by thrombin and ⁄ or KLKs, one can assume that HGFA may exercise its roles when tissues are severely dam- aged (Table 1). A critical role of HGFA in the acti- vation of pro-HGF ⁄ SF in injured tissues was initially confirmed in a rat model of liver injury induced by carbon tetrachloride [24,31]. In that study, the genera- tion of active HGF ⁄ SF in the injured liver tissue was significantly suppressed by the addition of neutralizing antibody against HGFA [24]. However, the precise roles of HGFA-mediated HGF ⁄ SF activation in liver regeneration are unclear: activation of HGF ⁄ SF was not observed in the regenerating liver tissue after par- tial hepatectomy [32]. Nonetheless, administration of exogenous HGFA accelerates liver regeneration after hepatectomy [33]. More evidence for the role of HGFA-mediated pro-HGF ⁄ SF activation in severely injured tissue was reported in HGFA-deficient mice. Although HGFA ) ⁄ ) mice showed normal develop- ment, initial regeneration after severe mucosal injury was significantly attenuated. Injured mucosa of HGFA-deficient mice showed impaired epithelial resti- tution, the first step in the regeneration of ulcerated mucosa [30]. This step appears to require HGF ⁄ SF activity, as the epithelial cells undergoing restitution on gastrointestinal ulcers show significantly enhanced phosphorylation of MET in human tissue samples [34]. Therefore, the role of HGFA in vivo may be preferentially observed in the early repair phase after tissue injury or in a tissue with persistent inflamma- tion and destruction, where transient but significant activation of pro-HGFA or low but sustained activa- tion of pro-HGFA, respectively, can be expected. Consistent with these observations, HGFA mRNA levels are upregulated in response to tissue injury and inflammation in various organs, and some consensus binding sites for several early responsive factors expressed in cases of tissue injury are present in the 5¢-flanking region of the HGFA gene [2,15,16]. Possi- ble roles of HGFA-mediated HGF ⁄ SF activation were also found in a pulmonary fibrosis model, in which the activity of HGFA appeared to be an important factor in preventing disease progression [35,36]. Table 1. Evidence for the roles of HGFA in vivo (non-neoplastic conditions). (A) Developmental and physiological processes. (B) Response to tissue injury and inflammation. (A) Species Site of action Process involved Source of HGFA Reference Rat Glandular, stomach, intestine Fetal development, morphogenesis of gastrointestinal tract Gastrointestinal epithelium 28 Mouse Kidney Ureteric bud branching, glomerulogenesis and nephrogenesis Ureteric bud 14 Human Lymph node germinal center Proliferation, survival, and ⁄ or adhesion of centroblasts Follicular dendritic cells 29 Human Hair follicle Hair follicle elongation Follicular papilla and outer root sheath cells 17 (B) Species Disorder Findings Source of HGFA Reference Rat CCl 4 -induced liver damage Critical requirement of HGFA in HGF ⁄ SF activation in liver injury Plasma 24 Mouse Experimental colitis Impaired regeneration of injured colon epithelium in HGFA ) ⁄ ) mice Plasma 30 Mouse Skin wound Reduced initial recruitment of macrophages at a site of skin wound due to impaired MSP activation in HGFA ) ⁄ ) mice Plasma 26 Mouse Bleomycin-induced pulmonary fibrosis Imbalance between HGFA and HAI-1 and defective HGF ⁄ SF activation at fibrotic stage Bronchoalveolar lavage 36 Human Idiopathic pulmonary fibrosis Reduced HGF ⁄ SF activation due to decreased HGFA production and enhanced HAI-1 and HAI-2 expression in idiopathic pulmonary fibrosis Lung fibroblasts 35 H. Kataoka and M. Kawaguchi HGFA: pathophysiological functions in vivo FEBS Journal 277 (2010) 2230–2237 ª 2010 The Authors Journal compilation ª 2010 FEBS 2233 The activation of pro-HGFA at the injured site is also an important event for the activation of pro-MSP, and HGFA may thus influence subsequent signaling through RON. The establishment of MSP-induced sig- naling has roles in macrophage recruitment and inflammatory processes, cellular survival, and wound healing [27]. Indeed, processing and activation of endogenous pro-MSP was impaired in HGFA-deficient serum, and initial infiltration of macrophages into the site of mechanical skin wounds was delayed in HGFA ) ⁄ ) mice [26]. Possible roles of HGFA in tumor progression Tissue destruction and persistent inflammation are usu- ally observed in invasive tumor tissues. Moreover, other components of the pro-HGFA-activating machinery, such as KLK4 and KLK5, are frequently upregulated in tumor cells [25]. Therefore, it is possible that enhanced activation of pro-HGFA and HGFA- mediated activation of HGF ⁄ SF and ⁄ or MSP occurs in the tumor stroma. Because ample evidence has sug- gested the roles of HGF ⁄ SF and MSP and their spe- cific receptor tyrosine kinases in invasive growth of tumor cells [1,27], HGFA-mediated activation of these factors may significantly influence tumor biology. A number of studies providing circumstantial evidence for this hypothesis have been published: (a) enhanced production and activation of HGF ⁄ SF are observed in various types of tumor tissue [1,2,6,19,37]; (b) although matriptase and hepsin may also be involved in the acti- vation of pro-HGF ⁄ SF in tumor tissues, aberrant expression of HGFA is observed in many types of tumors [18–23] – regarding the mechanism underlying enhanced HGFA expression in tumors, a recent study suggests that a hypoxic microenvironment in the tumor tissue may be responsible [6]; (c) the neutralizing anti- body against HGFA suppressed pro-HGF ⁄ SF activa- tion in colon cancer, myeloma, and diffuse large B-cell lymphoma [19–21]; and (d) overexpression of HGFA results in enhanced tumorigenicity and invasion [38]. Consequently, HAI-1 and HAI-2, both being cell surface HGFA inhibitors, suppressed tumor invasion in experimental models [39–42]. Indeed, the expression of HAI-2 is significantly downregulated by promoter hypermethylation in several tumor types: glioblastoma [42], medulloblastoma [43], hepatocellular carcinoma [44], and renal cell carcinoma [40]. Finally, clinical studies suggest that HGFA may serve as a biomarker of tumor progression. The levels of serum HGFA and active-form HGF ⁄ SF were elevated in patients with advanced prostate cancer [12,45], and the serum con- centration of activated HGFA was elevated in patients with multiple myeloma [13]. In breast cancer patients, tumor tissues from node-positive patients expressed a higher level of HGFA than those from the patients without nodal involvement [46]. Tissue injury Activation of coagulation cascade Pro-thrombin Thrombin proHGFA proMSP MSP proHGF/SF HGF/SF RON MET HGFA Matriptase KLK4, KLK5 Macrophages Epithelial cells Tumor cells Endothelial cells Plasma Tumor cells Epithelial cells Inflammatory cells Tumor cells Epithelial cells Plasma Stromal cells HAI-1 HAI-2 HAI-1 HAI-2 Protein C inhibitor Stimulation Hepsin HAI-1 HAI-2 Epithelial cells Stimulation Fig. 1. Hypothetical model for the activation of pro-HGF ⁄ SF and pro-MSP. There may be diverse pathways for the activation of these multi- functional growth factors. One pathway is mediated by HGFA that is activated in injured tissues and tumors. The second pathway is medi- ated by membrane-bound serine proteases (cell surface activators), such as matriptase and hepsin. Pericellular activities of these proteases are regulated by membrane-bound HAI-1 and HAI-2, and the serum activity of HGFA is regulated by PCI in humans. Although the activities of other pro-HGF ⁄ SF activators, such as coagulation factors XII and XI, plasma kallikrein, and plasminogen activators, are very weak, they may be stimulated by a certain microenvironment. The active forms of HGF ⁄ SF and MSP induce signaling through MET and RON, respec- tively, expressed on the surface of tumor cells, epithelial cells, and ⁄ or endothelial cells, resulting in tumor progression, epithelial restitution and regeneration, and angiogenesis. HGFA: pathophysiological functions in vivo H. Kataoka and M. Kawaguchi 2234 FEBS Journal 277 (2010) 2230–2237 ª 2010 The Authors Journal compilation ª 2010 FEBS Conclusions Considering the multifunctional aspects of HGF ⁄ SF and MSP [1,27], their processing and activation in the extracellular milieu represent a tightly controlled phe- nomenon in vivo, including both positive and negative inputs. To date, the significance of the extracellular proteolytic processing and activation of growth factors ⁄ cytokines may have been underestimated. Both HGF ⁄ SF and MSP are examples of growth factors ⁄ cytokines whose activities are regulated by the specific extracellular proteases. In this minireview, we have summarized evidence regarding the roles of HGFA in the activation of pro-HGF ⁄ SF and pro- MSP in injured tissues and tumors, and discussed its impact on subsequent tissue repair and tumor progres- sion. The structural similarities of HGFA and its sub- strates to coagulation factor XII and plasminogen, respectively, suggest that this system has a similar evo- lutionary nature to other plasma systems, such as blood coagulation and fibrinolysis. Our hypothetical model for the molecular interactions and cascades in the activation of HGF ⁄ SF and MSP is shown in Fig. 1. There is clearly a need for further studies on the pathophysiological functions of HGFA in vivo. For example, taking into account the substantial concentra- tion of pro-HGFA in human plasma, it may be possi- ble that other unknown unique substrates still exist in pathological settings in vivo. Furthermore, the clinical relevance of HGFA is an important matter to be eval- uated in the future. Efficient activation of the pre-exist- ing pericellular pro-HGF ⁄ SF and ⁄ or pro-MSP by the administration of recombinant HGFA may be benefi- cial for accelerated survival and regeneration of paren- chymal cells, and thus may have relevance in therapies for disorders with refractory wounds and ⁄ or persistent inflammation. On the other hand, the deregulated activities of HGFA and other HGF ⁄ SF-activating and MSP-activating enzymes in tumor tissue may be thera- peutic targets for some types of cancer. References 1 Matsumoto K & Nakamura T (2006) Hepatocyte growth factor and the Met system as a mediator of tumor–stromal interactions. 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Kawaguchi HGFA: pathophysiological functions in vivo FEBS Journal 277 (2010) 2230–2237 ª 2010 The Authors Journal compilation ª 2010 FEBS 2237 . cancer. Abbreviations HAI, hepatocyte growth factor activator inhibitor; HGF, hepatocyte growth factor; HGFA, hepatocyte growth factor activator; MSP, macrophage-stimulating. February 2010) doi:10.1111/j.1742-4658.2010.07640.x Hepatocyte growth factor activator (HGFA) is a serine protease initially identified as a potent activator of hepatocyte growth factor ⁄ scatter factor. Hepatocyte