Báo cáo khoa học: Hepatocyte growth factor activator is a serum activator of single-chain precursor macrophage-stimulating protein potx

Hepatocyte growth factor activator is a serum activator of single-chain precursor macrophage-stimulating protein Makiko Kawaguchi, Hiroshi Orikawa, Takashi Baba, Tsuyoshi Fukushima and Hiroaki Kataoka Section of Oncopathology and Regenerative Biology, Department of Pathology, Faculty of Medicine, University of Miyazaki, Japan Macrophage-stimulating protein (MSP) was originally identified as a plasma protein that promotes chemotac- tic responses in peritoneal resident macrophages [1–3]. Mature MSP is a disulfide-linked heterodimer with a relative molecular mass of 80–95 kDa, consisting of an a chain of approximately 60 kDa and a b chain of approximately 30 kDa, that autophosphorylates its specific receptor tyrosine kinase RON (recepteur d’origine nantais) [4,5]. MSP is a member of the krin- gle proteins, which contain multiple copies of a highly conserved triple disulfide loop structure (kringle domain). The a chain contains an N-terminal hairpin loop, followed by four kringle domains, and the b chain has a serine protease-like domain [5]. MSP is synthesized and secreted by hepatocytes [6] and circu- lates in plasma as a single-chain precursor (pro-MSP) Keywords activation; hepatocyte growth factor activator; macrophage-stimulating protein; recepteur d’origine nantais (RON); serum Correspondence H. Kataoka, Section of Oncopathology and Regenerative Biology, Department of Pathology, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan Fax: +81 985 85 6003 Tel: +81 985 85 2809 E-mail: mejina@med.miyazaki-u.ac.jp (Received 14 February 2009, revised 7 April 2009, accepted 22 April 2009) doi:10.1111/j.1742-4658.2009.07070.x Macrophage-stimulating protein (MSP) is a plasma protein that circu- lates as a single-chain proform. It acquires biological activity after prote- olytic cleavage at the Arg483–Val484 bond, a process in which serum and cell surface serine proteinases have been implicated. In this article, we report that hepatocyte growth factor activator (HGFA), a serum proteinase which activates hepatocyte growth factor in response to tissue injury, may have a critical role in the activation of pro-MSP. In vitro analysis has revealed that human HGFA efficiently cleaves human pro- MSP at the physiological activation site without further degradation, resulting in biologically active MSP, as measured by the chemotactic response and MSP-induced morphological change of peritoneal macro- phages. The processing of pro-MSP by HGFA is 10-fold more efficient than processing by factor XIa. To search for a role of HGFA in pro- MSP activation, we analyzed the processing of mouse pro-MSP in sera from HGFA-knockout (HGFA )/) ) mice. The proform of MSP was the predominant molecular form in the plasma of both wild-type and HGFA )/) mice. In wild-type sera, endogenous pro-MSP was progres- sively converted to the mature two-chain form during incubation at 37 °C. However, this conversion was significantly impaired in sera from HGFA )/) mice. The addition of recombinant HGFA to HGFA-deficient serum restored pro-MSP convertase activity in a dose-dependent manner, and a neutralizing antibody to HGFA significantly reduced the conver- sion of pro-MSP in wild-type serum. Moreover, initial infiltration of macrophages into the site of mechanical skin injury was delayed in HGFA )/) mice. We suggest that HGFA is a major serum activator of pro-MSP. Abbreviations CHO, Chinese hamster ovary; HGF/SF, hepatocyte growth factor/scatter factor; HGFA, hepatocyte growth factor activator; LPS, lipopolysaccharide; MSP, macrophage-stimulating protein; PC50%, processing concentration 50%; PCI, protein C inhibitor; RON, recepteur d’origine nantais. FEBS Journal 276 (2009) 3481–3490 ª 2009 The Authors Journal compilation ª 2009 FEBS 3481 that has no biological activity until the protein is cleaved into a and b chains at the Arg483–Val484 bond [5]. Several proteinases have been identified as candidate convertases in the processing of pro-MSP to mature MSP. Of interest is the observation that pro- MSP activation occurs in the presence of fetal bovine serum in vitro, suggesting the existence of a pro-MSP convertase in serum [7]. However, this conversion was not observed in freshly prepared human serum [8]. As pro-MSP is abundant in plasma (2–5 nm) [5], activation of pro-MSP by the serum convertase may be an important physiological response to tissue injury. Previous studies have suggested that the proteinases involved in the coagulation cascade and inflammation, such as factor XIa, factor XIIa and serum kallikrein, are responsible for pro-MSP convertase activity in serum [7]. However, the physiological serum activator of pro-MSP remains to be determined. Membrane-bound serine proteinases are also important [8]. Matriptase/ST14 may be an important cellular activator of pro-MSP in the pericellular microenviron- ment [9]. Other potential activators of pro-MSP include mouse epidermal growth factor-binding protein and nerve growth factor c (kallikrein 1-related peptidase b3), both of which have serine proteinase activity [10]. Hepatocyte growth factor/scatter factor (HGF/SF) is also a member of the kringle protein family and shows significant sequence homology to MSP (45% amino acid sequence identity) [2,3,11]. Like MSP, HGF/SF is secreted as an inactive single-chain precur- sor (pro-HGF/SF), and the cleavage between Arg494 and Val495 by an extracellular proteinase is critical for signal transduction via its specific cell surface receptor tyrosine kinase, MET, the protein product of the c-met proto-oncogene [12]. The serine proteinase hepatocyte growth factor activator (HGFA) is a very efficient pro- cessor of pro-HGF/SF [12,13]. HGFA is synthesized by the liver and circulates as an inactive zymogen (pro-HGFA) at a concentration of approximately 40 nm [14]. It is activated in response to tissue injury via cleavage of the bond between Arg407 and Ile408, resulting in a two-chain heterodimeric active form [12,15]. This cleavage is assumed to be mediated by thrombin in the serum and by kallikrein 1-related peptidases, such as KLK4 and KLK5, in the pericellu- lar microenvironment [16,17]. Activated serum HGFA retains sufficient activity in bovine serum and also in mouse serum [12,18]. However, in human serum, its activity is inhibited by protein C inhibitor (PCI) [14]. The activity of HGFA is also regulated by a cell surface inhibitor, namely HGFA inhibitor, in local tissues [19]. Considering the significant structural similarity of MSP to HGF/SF, we hypothesized that HGFA, a serum activator of pro-HGF/SF, may be an important candidate for the serum pro-MSP convertase in vivo. 188 188 62 49 38 38 28 49 62 28 L ys Leu Arg V a l V a l Gly Gly His Pro 483 484 anti-MSP anti-His tag kDa kDa 0 0.005 0.05 0.5 5 10 200 0.005 0.05 0.5 5 pro-MSP α chain HGFA (nM) Factor XIa (nM) 0 5 10 30 60 120 0 5 10 30 60 120 pro-MSP α chain HGFA (0.5 nM) Factor XIa (0.5 nM) Incubation time (min) 0 0.05 0.5 5 0 0.05 0.5 5 0 0.05 0.5 5 10 2001020 50 100 150 50 150 HGFA (nM) Factor XIa (nM) pro-MSP α chain NaCl concentration (mM) A B C D Fig. 1. Processing of pro-MSP by HGFA. (A) Immunoblot analysis of proteolytic cleavage of a His-tagged human pro-MSP recombi- nant protein by human HGFA. Pro-MSP at a concentration of 5 n M was incubated with 0.5 nM of HGFA in 20 mM Tris buffer (pH 7.6), 150 m M NaCl and 0.05% Chaps for 8 h at 37 °C. Anti-MSP IgG rec- ognized the a chain of MSP and the anti-His tag IgG recognized the poly-His tag at the C-terminus of MSP. The N-terminal amino acid sequence of a product of approximately 30 kDa is indicated. (B) Effects of NaCl concentration on the processing of pro-MSP. Pro-MSP at a concentration of 5 n M was incubated with various concentrations of HGFA or factor XIa in 20 m M Tris buffer (pH 7.6) and 0.05% Chaps, with 50–150 m M of NaCl, for 4 h at 37 °C. The processed products were analyzed by immunoblot. (C) Dose-depen- dent processing of pro-MSP (5 n M) by HGFA or factor XIa in Tris buffer (pH 7.6), 50 m M NaCl and 0.05% Chaps. The reaction mix- tures were incubated for 4 h at 37 °C. (D) Time-dependent process- ing of pro-MSP (5 n M) by HGFA (0.5 nM) or factor XIa (0.5 nM)in Tris buffer (pH 7.6), 50 m M NaCl and 0.05% Chaps. Activation of pro-MSP by HGFA M. Kawaguchi et al. 3482 FEBS Journal 276 (2009) 3481–3490 ª 2009 The Authors Journal compilation ª 2009 FEBS In this study, we found that recombinant human HGFA efficiently converts human pro-MSP to its active form in vitro. Subsequent experiments using an HGFA-deficient mouse model [18] revealed that HGFA is a major serum activator of pro-MSP. Results Processing of pro-MSP by HGFA The effect of recombinant human HGFA was tested on the processing of recombinant human pro-MSP. Incubation of pro-MSP with different concentrations of HGFA at 37 °C led to the processing of pro-MSP in a dose-dependent manner. Immunoblot analysis using an anti-MSP IgG revealed a band of approxi- mately 60 kDa, presumably the a chain of mature MSP (Fig. 1A). Generation of a band of approxi- mately 30 kDa, presumably the b chain, was also detected by an anti-His tag IgG (Fig. 1A). Cleavage site analysis was performed after separating the products of HGFA cleavage by SDS–PAGE under reducing conditions. The N-terminal amino acid sequence of the 30 kDa product was Val-Val-Gly-Gly- His. Therefore, this 30 kDa band was in fact the b chain of mature MSP, and HGFA cleaved pro-MSP at the normal processing site, Arg483–Val484 (Fig. 1A). The processing was suppressed at higher concentra- tions of NaCl, and this tendency was also observed for factor XIa, a known serum activator of pro-MSP (Fig. 1B). The concentration of HGFA required to acti- vate 50% of 5 nm pro-MSP (PC50%) after 4 h at 37 °C was 0.05 nm, whereas that of factor XIa was 0.5 nm. Therefore, HGFA was a 10-fold more potent convertase of pro-MSP than factor XIa (Fig. 1C). Further degra- dation of mature MSP was not observed by HGFA. We also examined the time course of pro-MSP processing by HGFA (Fig. 1D). More than 50% of pro-MSP (5 nm) was processed within 30 min by 0.5 nm of HGFA, again showing superior efficiency to factor XIa. Biological activity of MSP processed by HGFA The biological activity of MSP after HGFA processing was determined using macrophage chemotaxis assays. Pro-MSP could not efficiently induce the chemotactic migration of macrophages. However, after incubation of pro-MSP with HGFA, the processed products showed a significant induction of macrophage migra- tion, and the activity was comparable with that of commercially available recombinant mature human MSP a/b heterodimer (Fig. 2A). Macrophages derived from HGFA )/) mice also responded to the recombi- nant mature MSP a/b heterodimer (data not shown). HGFA alone did not detectably induce the chemotac- tic response. We also examined the effect of HGFA processing on the culture morphology of mouse perito- neal macrophages. The MSP processed by HGFA induced an elongated, migratory morphology of macrophages within 1 h, showing an effect similar to +–+ –– 0 20 80 AB +–+–– –– +–– pro-MSP HGFA MSP Migrated cells/field 40 60 * * No treatment + MSP + pro-MSP + HGFA-treated pro-MSP Control pro-MSP pro-MSP + HGFA Fig. 2. Biological activity of MSP processed by HGFA. (A) Results of chemotaxis assays. Murine peritoneal resident macrophages (1 · 10 5 cells) were placed in the upper well of Chemotaxicells and incubated for 3.5 h at 37 °C. The bottom well contained pro-MSP (1.25 n M) with or without HGFA (0.125 nM) pretreatment (2 h) or recombinant active MSP (1.25 nM). Values are the mean number ± stan- dard deviation of migrated cells per high-power field in triplicate experiments. *P < 0.01 compared with control (pro-MSP only, HGFA only or no addition) (Mann–Whitney U-test). Representative photographs of migrating cells are also shown. (B) Morphology of macrophages in the presence of pro-MSP (1.25 n M), MSP (1.25 nM) or pro-MSP (1.25 nM) pretreated with HGFA (0.125 nM). After 1 h in culture, the cells were observed by phase-contrast microscopy. M. Kawaguchi et al. Activation of pro-MSP by HGFA FEBS Journal 276 (2009) 3481–3490 ª 2009 The Authors Journal compilation ª 2009 FEBS 3483 that of the recombinant mature MSP a/b heterodimer (Fig. 2B). Impaired processing of endogenous pro-MSP in serum from HGFA -/- mice In order to study further the role of HGFA in the acti- vation of pro-MSP, we used HGFA )/) mice. After incubation at 37 °C for 2 h, most of the endogenous MSP proteins in the plasma from both wild-type and HGFA )/) mice were the 90 kDa single-chain pro- forms (Fig. 3A). In contrast, pro-MSP was apparently processed in the sera from wild-type mice after a 2 h incubation at 37 °C (Fig. 3B), indicating the presence of pro-MSP convertase in the wild-type serum, as pre- viously observed in bovine serum [7]. However, the processing of pro-MSP was reduced significantly in the sera from HGFA )/) mice (Fig. 3B). A subsequent time course study also confirmed the significantly reduced processing activity of pro-MSP in HGFA-deficient sera relative to that in wild-type sera (n = 5 for each group) (Fig. 3C). Although the processing of endo- genous pro-MSP was apparent within 15 min of incu- bation and had reached 20% at 30 min in wild-type serum, there was less than 10% processing even after 120 min of incubation in HGFA-deficient serum (Fig. 3C). Therefore, the absence of HGFA resulted in a markedly delayed and reduced processing of pro- MSP in mouse serum. Indeed, the addition of recombi- nant HGFA to the sera of HGFA )/) mice restored the pro-MSP processing activity (Fig. 4). Inhibition of pro-MSP processing activity in serum by anti-HGFA neutralizing IgG The efficient pro-MSP activating activity of human HGFA in vitro and the significantly reduced processing of endogenous pro-MSP in HGFA-deficient mouse serum suggest that HGFA is a major serum activator of pro-MSP in vivo. Therefore, we examined the effect of a neutralizing antibody raised against HGFA (P1-4) on the pro-MSP convertase activity of wild-type mouse serum. The P1-4 antibody suppressed significantly the processing of pro-MSP in sera obtained from wild-type mice (Fig. 5). We concluded that HGFA is a major activator of pro-MSP in mouse serum. Delayed infiltration of macrophages in HGFA -/- mice at a site of tissue injury To test the physiological role of HGFA-mediated acti- vation of pro-MSP, we compared the recruitment of macrophages in injured tissues, in which the activation of pro-HGFA was anticipated by thrombin and/or 0 10 20 30 40 50 60 70 01530 60 120 pro-MSP pro-MSP pro-MSP α-chain α-chain α-chain 1212 1212 120 min 120 min 60 min Incubation time Incubation time PlasmaA B C Serum Wild HGFA –/– HGFA –/– Wild % converted pro-MSP Incubation time (min) Wild-type serum HGFA-deficient serum * Fig. 3. Impaired processing of endogenous pro-MSP in HGFA-defi- cient serum. (A) Processing of endogenous pro-MSP in plasma from wild-type and HGFA )/) mice. Plasma was incubated at 37 °C and the processing of endogenous pro-MSP was analyzed by immunoblot using anti-MSP antibody. (B) Processing of endo- genous pro-MSP in sera from wild-type and HGFA )/) mice. Serum was incubated at 37 °C and the processing of endogenous pro- MSP was analyzed by immunoblot. (C) Time course of pro-MSP processing in serum. Values are the mean processing rate ± stan- dard deviation (n = 5). *P < 0.001, Mann–Whitney U-test. 0 0.018 0.18 1.8 18 180 I ncu b at i on: 120 m i n HGFA (n M) % converted pro-MSP pro-MSP MSP α chain 56 85 > 90% > 90 > 90 Fig. 4. Reversion of pro-MSP convertase activity in serum from HGFA )/) mice by recombinant HGFA. HGFA-deficient serum was incubated with recombinant human HGFA (0–180 n M) for 2 h, and the processing of endogenous pro-MSP was analyzed by immunoblot. Activation of pro-MSP by HGFA M. Kawaguchi et al. 3484 FEBS Journal 276 (2009) 3481–3490 ª 2009 The Authors Journal compilation ª 2009 FEBS KLKs, between wild-type mice and HGFA )/) mice. We generated a small mechanical wound in the dorsal skin of mice and examined the infiltration of macro- phages by measuring the CD68 mRNA level. One day after injury, the levels of CD68 mRNA in the wounds of HGFA )/) mice were significantly lower than those of wild-type mice (Fig. 6A). The level of pro-MSP pro- cessing was also low in HGFA )/) wounds (Fig. 6B). However, at the fifth day of injury, the CD68 mRNA level was comparable between HGFA )/) wounds and wild-type wounds (Fig. 6A). These results suggest that serum HGFA is required for the early-phase recruit- ment of macrophages at the injured tissue, possibly via its efficient pro-MSP processing activity. Discussion Pro-MSP is primarily produced by the liver [6] and cir- culates in blood with a concentration of 2–5 nm [5]. It is converted to its mature active form during blood coagu- lation and local inflammation [5,7]. Wound fluids also contain pro-MSP convertase activity, and a cellular sur- face proteinase is also an important convertase [5,8,9]. This activation step of pro-MSP might serve as a critical regulatory mechanism in MSP-induced physiological and pathophysiological tissue responses. After proteo- lytic cleavage, it stimulates resident macrophages via its specific receptor tyrosine kinase, RON [5,11]. Epithelial cells and neoplastic cells also frequently express RON [11,20,21]. The establishment of RON-induced signaling appears to have an important role in inflammatory pro- cesses [1,5,22–24], cellular survival and wound healing [25,26]. It is also important in the progression and meta- static spread of various types of tumor [11,27,28]. In this study, we have shown that human HGFA efficiently activates human pro-MSP in vitro. In mice, serum HGFA represents the major pro-MSP convertase activ- ity of the serum. Indeed, the conversion of endogenous pro-MSP to its mature form was impaired in sera from HGFA )/) mice and the convertase activity in wild-type sera was significantly attenuated by the addition of anti- HGFA neutralizing IgG. Moreover, initial infiltration of macrophages into the site of mechanical skin injury was delayed in HGFA )/) mice. Together with the fact that matriptase, a cell surface activator of pro-MSP [9], is also a potent activator of pro-HGF/SF [29], we sug- gest that pro-MSP might share its activation machiner- ies with pro-HGF/SF (Fig. 7). The identification of HGFA as a major serum activa- tor of pro-MSP may explain why the serum convertase activity of pro-MSP is different between species. Although bovine serum [7] and mouse serum showed significant processing activity for endogenous pro-MSP, the processing activity of human serum was very weak and the molecular form of MSP in human serum was mostly proforms (data not shown), as described previ- ously [8]. HGFA is resistant to major serum proteinase inhibitors [12] and is active in mouse serum [18]. How- ever, it can be inhibited by PCI, a serpin-type protein- ase inhibitor present in human plasma [14,30]. However, mouse plasma does not contain PCI [30]. Therefore, HGFA-mediated conversion of pro-MSP may be tightly regulated by PCI in human serum, whereas HGFA remains active and easily converts pro- MSP in mouse serum because of the absence of PCI. HGFA is present in plasma as an inactive zymogen at a concentration of approximately 40 nm in humans [14]. During tissue injury, pro-HGFA is converted to the active heterodimeric form by thrombin [12,15]. Human kallikrein 1-related peptidases, KLK4 and KLK5, are also candidate activators of pro-HGFA in the local tissue environment [17]. After conversion, mature HGFA very efficiently activates pro-HGF/SF at the site of injury [16], which might have important roles in survival, repair and regeneration of the injured tissue [12,19]. The activity of HGFA is tightly regu- lated by PCI in human serum and also by HGFA inhibitor type 1 and type 2 on the epithelial cell surface [12,14,19]. Nonetheless, the activity of HGFA is detectable in injured human tissues, such as invasive tumors, accompanying the activation of pro-HGF/SF [31]. To date, the possible involvement of HGFA in tissue repair and cancer progression has been discussed primarily in the context of its presumed capability to activate pro-HGF/SF and the subsequent MET signal- ing cascade [11,12,18]. This study indicates that MSP- induced RON signaling can be initiated by HGFA activity and may contribute to the role of HGFA in tissue repair and cancer progression. Furthermore, the activation of pro-MSP by HGFA prompts the consid- eration of the possible role of HGFA in inflammation via modulation of macrophage function. IgG1 P1-4 IgG1 P1-4 IgG1 P1-4 0 min 30 min 60 min Incubation time Antibody pro-MSP MSP α chain — — — 35 38 14 52 61 29% % converted pro-MSP Fig. 5. Inhibition of pro-MSP processing activity in serum by anti- HGFA IgG. Serum from wild-type mice was incubated for the indicated time periods at 37 °C without or with 400 lgÆmL )1 of anti-HGFA neutralizing IgG (P1-4) or nonspecific mouse IgG1. The processing of endogenous pro-MSP was analyzed by immunoblot. M. Kawaguchi et al. Activation of pro-MSP by HGFA FEBS Journal 276 (2009) 3481–3490 ª 2009 The Authors Journal compilation ª 2009 FEBS 3485 Evidence suggests that MSP exerts a dual function, both stimulatory and inhibitory, on macrophages [5]. Stimulatory functions include its ability to induce mac- rophage spreading, migration, phagocytosis and the production of cytokines [1,5,32]. However, MSP inhib- its lipopolysaccharide (LPS)-induced production of inflammatory mediators and, consequently, RON-defi- cient mice show increased inflammatory responses and susceptibility to LPS-induced septic death [5,22–24]. Therefore, MSP is also required to attenuate an exces- sive inflammatory response to LPS stimulation, and thus may have an important regulatory role in septic Wild-type HGFA KO 1.11 ± 0.22 1.08 ± 0.16 CD68 β -actin Wild-type A B HGFA KO 0 day 0.33 ± 0.08 CD68/actin 0.30 ± 0.06 Wild-type HGFA KO 1.25 ± 0.05 0.79 ± 0.15 * 1 day 5 day Wild KO Wild KO Incubated Serum 1 day 0 day pro-MSP MSP α chain % converted pro-MSP 44 33 — < 5 18 48 84 < 5 — — — Fig. 6. Delayed infiltration of macrophages in cutaneous wounds of HGFA )/) mice. (A) Infiltration of macrophages in wounded skin tissue was evaluated by CD68 mRNA level. *P < 0.05, Mann–Whitney U-test (n = 4). (B) Processing level of pro-MSP in injured tissues (1 day after injury). Skin tissues without injury (0 day) were also examined. For positive control, wild-type serum after incubation for the processing of endogenous pro-MSP (incubated serum) was also applied. Wild, wild-type mice; KO, HGFA )/) mice. Tissue injury Activation of coagulation cascade pro-thrombin Thrombin pro-HGFA pro-MSP MSP pro-HGF/SF HGF/SF RON MET HGFA EGF-BP, NGF-γ Matriptase KLK4, KLK5 Cell surface proteinase(s) Macrophages Epithelial cells Tumour cells Endothelial cells Factor XIa Factor XIIa Fig. 7. Hypothetical model for the activation of pro-MSP. There may be diverse pathways for the activation of pro-MSP, and pro-MSP might share the activation machinery with its homologous protein, pro-HGF/SF. One pathway is mediated by membrane-bound serine proteinases (cell surface activator), such as matriptase [9]. Matriptase is also a potent activator of pro-HGF/SF [12,30]. The second pathway is mediated by humoral activators that are generated in injured tissues. The activation of the coagulation cascade by tissue injury eventually results in the active form of HGFA that efficiently activates both pro-MSP and pro-HGF/SF. Other coagulation proteinases, such as factor XIa and fac- tor XIIa, may also mediate the activation of pro-MSP [7] and pro-HGF/SF [12]. Wound fluids in the injured tissues contain other pro-MSP acti- vators, such as epidermal growth factor-binding protein (EGF-BP) and nerve growth factor c (NGF-c) [10]. The effects of EGF-BP and NGF-c on pro-HGF/SF are unknown. Activation of pro-MSP by HGFA M. Kawaguchi et al. 3486 FEBS Journal 276 (2009) 3481–3490 ª 2009 The Authors Journal compilation ª 2009 FEBS inflammation. In the septic condition, intravascular hypercoagulation occurs, which might result in the conversion of pro-HGFA to its active form. The HGFA-mediated activation of pro-MSP may be an important process in the regulation of macrophage functions in septic inflammatory responses. To test this hypothesis, future studies of HGFA )/) mice under vari- ous inflammatory stimuli, including LPS stimulation, will be required. Moreover, the difference in serum HGFA activity between human and mouse may have implications in the different susceptibility to LPS-induced septic death between these species. In summary, we have demonstrated for the first time that HGFA is a potent activator of pro-MSP. Although the activation of pro-MSP is a redundant system which can be mediated by various proteinases (Fig. 7) [7–10], the major pro-MSP convertase in serum is HGFA. As pro-HGFA is activated in response to tissue injury, we suggest that HGFA-mediated activation may play an important role in the regulation of MSP/RON signaling involved in inflammation, wound healing and cancer progression. Further experiments of tissue injury and inflammation using genetically engineered mouse mod- els of the HGFA and MSP genes are needed to explore the in vivo significance of HGFA in MSP/RON signal- ing. However, our study also indicates that caution should be exercised when interpreting the function of MSP/RON signaling using a mouse model in vivo,as HGFA activity would be much higher in mouse serum than in human serum because of the absence of circulat- ing PCI in mice [30]. Experimental procedures Antibodies Anti-human MSP goat polyclonal IgG and the recombinant active form of human MSP were purchased from R&D Sys- tems (Minneapolis, MN, USA). Recombinant human factor XIa was obtained from Haematologic Technologies, Inc. (Essex Junction, VT, USA). Anti-mouse MSP goat polyclonal IgG (T-19) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The preparation of the recombinant active form of human HGFA and anti- human HGFA mouse monoclonal neutralizing IgG P1-4, which is also cross-reactive to mouse HGFA, has been described previously [15]. Anti-His tag rabbit polyclonal IgG was purchased from MBL (Nagoya, Japan). Preparation of recombinant proteins The preparation of the recombinant active form of HGFA has been described previously [19]. To obtain a recombinant pro-MSP protein, the entire coding region of the MSP gene was subcloned into the pcDNA3.1/myc-HisA expression plasmid (Invitrogen, Carlsbad, CA, USA) and transfected into Chinese hamster ovary (CHO) cells using Lipofectamine 2000 reagent (Invitrogen). After transfection, the cells were cultured in DMEM containing 10% fetal bovine serum and gradually changed to serum-free medium (CHO-S-SFMII; Invitrogen) containing 250 lgÆmL )1 G418 (Sigma-Aldrich, St Louis, MO, USA). To prevent the cleavage of pro-MSP by cellular and fetal bovine serum-derived proteases, cells were cultured in the presence of 50 lm nafamostat mesilate (Torii Pharmaceutical Co., Tokyo, Japan). G418-resistant colonies were selected and screened for the expression and production of pro-MSP. Supernatants were collected from the serum-free cultures every day and 0.1% Chaps (Sigma- Aldrich) was added. Recombinant pro-MSP in the condi- tioned medium was affinity purified with TALON His-Tag Purification Resins (Clontech Laboratories, Mountain View, CA, USA) according to the manufacturer’s instructions. Activation of pro-MSP Recombinant pro-MSP (final concentration, 5 nm) was incubated with various concentrations of HGFA or factor XIa in 20 lL reactions containing 20 mm Tris/HCl, 50–150 mm NaCl and 0.05% Chaps (pH 7.6) for the indi- cated time periods at 37 °C. The processing of pro-MSP was determined by immunoblot analysis under reducing conditions, and the extent of processing was verified using photoshop software (Adobe Systems, San Jose, CA, USA). The specific activity for pro-MSP processing was expressed as the enzyme concentration required for the conversion of 50% of 5 nm pro-MSP to its mature form, and was desig- nated as the processing concentration 50% (PC50%). To assess the time course of cleavage by HGFA or factor XIa, pro-MSP (5 nm) was incubated with 0.5 nm of each proteinase at 37 °C for various time periods (0–120 min). Immunoblot analysis Each sample was mixed with SDS–PAGE sample buffer and heated for 15 min at 70 °C. SDS–PAGE was per- formed under reducing conditions using 4–12% gradient gels. After electrophoresis, samples were transferred to Immobilon poly(vinylidene difluoride) membranes (Milli- pore, Bedford, MA, USA). After blocking with 3% BSA in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBS-T), the membranes were incubated with primary antibody at 4 °C overnight, followed by washing in TBS-T and incuba- tion with a horseradish peroxidase-conjugated rabbit anti- goat IgG (DAKO, Glostrup, Denmark) diluted in TBS-T with 1% BSA for 1 h at room temperature. The labeled proteins were visualized with a chemiluminescence reagent (PerkinElmer Life Science, Boston, MA, USA). M. Kawaguchi et al. Activation of pro-MSP by HGFA FEBS Journal 276 (2009) 3481–3490 ª 2009 The Authors Journal compilation ª 2009 FEBS 3487 N-terminal amino acid sequencing of cleaved pro-MSP Pro-MSP (final concentration, 416 nm) was incubated with 97 nm HGFA in a 40 lL reaction containing 20 mm Tris/ HCl, 150 mm NaCl and 0.05% Chaps (pH 7.6) at 37 °C for 11 h. The reaction mixture was subjected to SDS–PAGE, after which the proteins were transferred to an Immobilon membrane and stained with 0.1% Coomassie Brilliant Blue in a water–methanol–acetic acid solution (4.5 : 4.5 : 1, v/v). The cleaved MSP protein band was cut and processed for N-terminal amino acid sequencing by automated Edman degradation using the Procise 494 HT Protein Sequencing System (Applied Biosystems, Foster City, CA, USA). Preparation of peritoneal macrophages and bioassays Murine peritoneal resident macrophages were obtained from C57BL/6 mice by washing the peritoneal cavity with 3 mL per mouse of serum-free RPMI-1640 medium. Cells were washed and resuspended in RPMI-1640 medium con- taining 25 mm Hepes at a concentration of 1 · 10 6 cell- sÆmL )1 . The macrophage chemotaxis assay was performed using a polycarbonate membrane with a pore size of 5 lm (Chemotaxicells; Kurabo, Osaka, Japan). One hundred microliters of the cell suspension (i.e. 10 5 macrophages) were added to the upper wells of the Chemotaxicells. The bottom wells were filled with RPMI-1640 medium contain- ing purified pro-MSP treated or not with HGFA at 37 °C for 2 h. The recombinant active form of human MSP (R&D Systems) was used as a positive control. After incu- bation at 37 °C for 3.5 h, the cells on the upper surface of the membrane were wiped off with a cotton swab and the membranes were fixed with 3.7% formaldehyde in NaCl/P i and stained with hematoxylin. Migration was quantified by counting the cells on the lower surface in 10 randomly selected high-power fields (200-fold magnification). To test the effect of MSP on the morphological changes of macro- phages, murine peritoneal resident macrophages (1 · 10 6 cellsÆmL )1 ) were cultured in serum-free RPMI-1640 medium overnight. After incubation, nonadherent cells were removed and pro-MSP (1.25 nm), pretreated or not with HGFA, was added to the culture medium. After an addi- tional incubation at 37 °C for 1 h, morphological changes of the macrophages were observed by phase-contrast microscopy. Analysis of molecular forms of MSP in wild-type and HGFA-deficient mice The generation of HGFA knockout (HGFA )/) ) mice by the targeting of gene disruption has been reported previ- ously [18]. Sera and EDTA-treated plasma were obtained from C57BL/6 wild-type (HGFA +/+ ) and HGFA )/) mice, and diluted 10-fold with phosphate buffer (pH 7.4). Molecular forms of endogenous MSP in the plasma and serum were analyzed by immunoblots. To test the effect of complementation of HGFA activity on serum pro- MSP convertase activity, the diluted serum from an HGFA )/) mouse was incubated with varying concentra- tions of recombinant HGFA at 37 °C for 2 h, and ana- lyzed by immunoblot. For a neutralizing study, the diluted serum from a C57BL/6 mouse was incubated with or without 400 lgÆmL )1 of anti-HGFA neutralizing anti- body at 37 °C for the indicated time periods. The molec- ular forms of endogenous MSP were analyzed by immunoblot. Skin injury model Eight-week-old male wild-type and HGFA )/) mice were deeply anesthetized by intraperitoneal administration of ketamine hydrochloride [100 lgÆ(g body weight) )1 ; Sankyo, Tokyo, Japan] and xylazine [10 lgÆ(g body weight) )1 ; Bayer, Tokyo, Japan]. After shaving the dorsal hair and cleaning with 70% ethanol, two full-thickness excisional skin wounds (5 mm in diameter) were made. Mice were sac- rificed at 1 or 5 days after the generation of wounds. The wounded tissues were excised and used for RT–PCR, immunoblot analysis for pro-MSP processing and routine histological analysis with hematoxylin and eosin staining. For control, normal skin tissues were also biopsied (0 day). For RT-PCR, total RNA was prepared with TRIzol (Invi- trogen Japan, Tokyo, Japan) followed by DNase I (Takara Bio, Shiga, Japan) treatment. Three micrograms of total RNA were reverse transcribed with a mixture of oligo (dT) 12)18 (Invitrogen Japan) and random primers (6-mer) (Takara Bio) using 200 units of ReverTraAceÔ (TOYOBO, Osaka, Japan), and 1/30 of the resultant cDNA was pro- cessed for each PCR with 0.1 lm of both forward and reverse primers and 2.5 units of HotStarÔ Taq DNA poly- merase (Qiagen, Tokyo, Japan). The following primers were used: b-actin: forward, 5¢-TGACAGGATGCAGAAGGA GA; reverse, 5¢-GCTGGAAGGTGGACAGTGAG; CD68: forward, 5¢-TCTACCTGGACTACATGGCGGTGG; reverse, 5¢-ACATGGCCCGAAGTGTCCCTTGTC. For immuno- blot, tissues were homogenized on ice in lysis buffer (CelLyticÔ-MT; Sigma-Aldrich) supplemented with prote- ase inhibitor cocktail (Sigma-Aldrich). The extracts were centrifuged at 20 000 g for 20 min at 4 °C, and the result- ing supernatants were used for immunoblot. 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Hepatocyte growth factor activator is a serum activator of single-chain precursor macrophage-stimulating protein Makiko Kawaguchi, Hiroshi Orikawa, Takashi Baba, Tsuyoshi Fukushima and Hiroaki. zymogen of hepatocyte growth factor activator by thrombin. J Biol Chem 268, 22927–22932. 17 Mukai S, Fukushima T, Naka D, Tanaka H, Osada Y & Kataoka H (2008) Activation of hepatocyte growth factor. proteinases (cell surface activator) , such as matriptase [9]. Matriptase is also a potent activator of pro-HGF/SF [12,30]. The second pathway is mediated by humoral activators that are generated