Title: Hepatocyte Growth Factor Activator Inhibitor Type 1 Is a Specific Cell Surface Binding Protein of Hepatocyte Growth Factor Activator (HGFA) and Regulates HGFA Activity in the Pericellular Microenvironment
Abstract: Hepatocyte growth factor activator (HGFA) is responsible for proteolytic activation of the precursor form of hepatocyte growth factor in injured tissues. To date, two specific inhibitors of HGFA have been identified, namely HGFA inhibitor type 1 (HAI-1) and type 2 (HAI-2)/placental bikunin (PB). Both inhibitors are first synthesized as integral membrane proteins having two Kunitz domains and a transmembrane domain, and are subsequently released from cell surface by shedding. Here we show that an active form of HGFA is specifically complexed with membrane-form HAI-1, but not with HAI-2/PB, on the surface of epithelial cells expressing both inhibitors. This binding required the enzyme activity of HGFA. The selective binding of HGFA to the cell surface HAI-1 was further confirmed in an engineered system using Chinese hamster ovary cells, in which only the cells expressing HAI-1 retained exogenous HGFA. The binding of HGFA to HAI-1 was reversible, and no irreversible modifications affecting the enzyme activity occurred during the binding. Importantly, HAI-1 and the HGFA·HAI-1 complex were quickly released from the cell surface by treatment with phorbol 12-myristate 13-acetate or interleukin 1β accompanying the generation of 58-kDa fragments of HAI-1, which are less potent against HGFA, as well as significant recovery of HGFA activity in the culture supernatant. This regulated shedding was completely inhibited by BB3103, a synthetic zinc-metalloproteinase inhibitor. We conclude that HAI-1 is not only an inhibitor but also a specific acceptor of active HGFA, acting as a reservoir of this enzyme on the cell surface. The latter property appears to ensure the concentrated pericellular HGFA activity in certain cellular conditions, such as tissue injury and inflammation, via the up-regulated shedding of HGFA·HAI-1 complex. These findings shed light on a novel function of the integral membrane Kunitz-type inhibitor in the regulation of pericellular proteinase activity. Hepatocyte growth factor activator (HGFA) is responsible for proteolytic activation of the precursor form of hepatocyte growth factor in injured tissues. To date, two specific inhibitors of HGFA have been identified, namely HGFA inhibitor type 1 (HAI-1) and type 2 (HAI-2)/placental bikunin (PB). Both inhibitors are first synthesized as integral membrane proteins having two Kunitz domains and a transmembrane domain, and are subsequently released from cell surface by shedding. Here we show that an active form of HGFA is specifically complexed with membrane-form HAI-1, but not with HAI-2/PB, on the surface of epithelial cells expressing both inhibitors. This binding required the enzyme activity of HGFA. The selective binding of HGFA to the cell surface HAI-1 was further confirmed in an engineered system using Chinese hamster ovary cells, in which only the cells expressing HAI-1 retained exogenous HGFA. The binding of HGFA to HAI-1 was reversible, and no irreversible modifications affecting the enzyme activity occurred during the binding. Importantly, HAI-1 and the HGFA·HAI-1 complex were quickly released from the cell surface by treatment with phorbol 12-myristate 13-acetate or interleukin 1β accompanying the generation of 58-kDa fragments of HAI-1, which are less potent against HGFA, as well as significant recovery of HGFA activity in the culture supernatant. This regulated shedding was completely inhibited by BB3103, a synthetic zinc-metalloproteinase inhibitor. We conclude that HAI-1 is not only an inhibitor but also a specific acceptor of active HGFA, acting as a reservoir of this enzyme on the cell surface. The latter property appears to ensure the concentrated pericellular HGFA activity in certain cellular conditions, such as tissue injury and inflammation, via the up-regulated shedding of HGFA·HAI-1 complex. These findings shed light on a novel function of the integral membrane Kunitz-type inhibitor in the regulation of pericellular proteinase activity. hepatocyte growth factor HGF activator HGFA inhibitor type 1 HGFA inhibitor type 2/placental bikunin secreted form of HAI-1 single-chain HGF Chinese hamster ovary 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid phenylmethylsulfonyl fluoride phorbol 12-myristate 13-acetate disulfosuccinimidyl tartrate interleukin-1β fluorescein 5-isothiocyanate-conjugated IgG monoclonal antibody fetal bovine serum phosphate-buffered saline glyceraldehyde-3-phosphate dehydrogenase polyacrylamide gel electrophoresis tumor necrosis factor-α TNFα-converting enzyme a disintegrin and metalloproteinase low density lipoprotein bovine serum albumin base pair(s) amyloid β protein precursor Hepatocyte growth factor (HGF),1 also known as scatter factor, is a pleiotropic factor that functions as a mitogen, motogen, and/or morphogen for a variety of cells, particularly epithelial cells, bearing c-Met receptor tyrosine kinase (1Matsumoto K. Nakamura T. J. Biochem. (Tokyo ). 1996; 119: 591-600Crossref PubMed Scopus (609) Google Scholar, 2Stella M.C. Comoglio P.M. Int. J. Biochem. Cell Biol. 1999; 31: 1357-1362Crossref PubMed Scopus (136) Google Scholar). Because HGF is secreted as an inactive precursor form, proteolytic activation of the precursor form in the extracellular milieu is a critical limiting step in the HGF-induced signaling pathway. HGF activator (HGFA) is a factor XII-like serine proteinase having a critical role in the activation of HGF in injured tissue (3Shimomura T. Ochiai M. Kondo J. Morimoto Y. Cytotechnology. 1992; 8: 219-229Crossref PubMed Scopus (77) Google Scholar, 4Miyazawa K. Shimomura T. Kitamura A. Kondo J. Morimoto Y. Kitamura N. J. Biol. Chem. 1993; 268: 10024-10028Abstract Full Text PDF PubMed Google Scholar, 5Shimomura T. Kondo J. Ochiai M. Naka D. Miyazawa K. Morimoto Y. Kitamura N. J. Biol. Chem. 1993; 268: 22927-22932Abstract Full Text PDF PubMed Google Scholar, 6Miyazawa K. Shimomura T. Naka D. Kitamura N. J. Biol. Chem. 1994; 269: 8966-8970Abstract Full Text PDF PubMed Google Scholar, 7Miyazawa K. Shimomura T. Kitamura N. J. Biol. Chem. 1996; 271: 3615-3618Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Because active HGFA is not inhibited by serum proteinase inhibitors (8Shimomura T. Miyazawa K. Komiyama Y. Hiraoka H. Naka D. Morimoto Y. Kitamura N. Eur. J. Biochem. 1995; 229: 257-261Crossref PubMed Scopus (167) Google Scholar), it has been suggested that local synthesis of HGFA inhibitors could have a critical regulatory role for HGFA activity in the injured tissue. HGFA inhibitor type 1 (HAI-1) was initially identified as a potent inhibitor of HGFA present in a culture-conditioned medium of MKN45 gastric adenocarcinoma cell line (9Shimomura T. Denda K. Kitamura A. Kawaguchi T. Kito M. Kondo J. Kagaya S. Qin L. Takata H. Miyazawa K. Kitamura N. J. Biol. Chem. 1997; 272: 6370-6376Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). A second type of HGFA inhibitor (HAI-2) was subsequently identified (10Kawaguchi T. Qin L. Shimomura T. Kondo J. Matsumoto K. Denda K. Kitamura N. J. Biol. Chem. 1997; 272: 27558-27564Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). HAI-2 was also independently isolated as placental bikunin (PB) (11Marlor C.W. Delaria K.A. Davis G. Muller D.K. Greve J.M. Tamburini P.P. J. Biol. Chem. 1997; 272: 12202-12208Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 12Delaria K.A. Muller D.K. Marlor C.W. Brown J.E. Das R.C. Roczniak S.O. Tamburini P.P. J. Biol. Chem. 1997; 272: 12209-12214Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and as a protein overexpressed in pancreatic cancer (13Müller-Pillasch F. Wallrapp C. Bartels K. Varga G. Friess H. Büchler M. Adler G. Gress T.M. Biochim. Biophys. Acta. 1998; 1395: 88-95Crossref PubMed Scopus (50) Google Scholar). Both HAI-1 and HAI-2/PB have two well-defined Kunitz inhibitor domains and a presumed transmembrane domain. In addition, HAI-1 has an LDL receptor-like domain between two Kunitz domains (9Shimomura T. Denda K. Kitamura A. Kawaguchi T. Kito M. Kondo J. Kagaya S. Qin L. Takata H. Miyazawa K. Kitamura N. J. Biol. Chem. 1997; 272: 6370-6376Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 10Kawaguchi T. Qin L. Shimomura T. Kondo J. Matsumoto K. Denda K. Kitamura N. J. Biol. Chem. 1997; 272: 27558-27564Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar).HAI-1 protein is predominantly expressed on the basolateral surface of columnar epithelial cells covering the mucosal surfaces and duct lumina (14Kataoka H. Suganuma T. Shimomura T. Itoh H. Kitamura N. Nabeshima K. Koono M. J. Histochem. Cytochem. 1999; 47: 673-682Crossref PubMed Scopus (104) Google Scholar). Its localization on the cell surface as a transmembrane form integrated in the plasma membrane was also confirmed in a cultured epithelial cell line MKN45 (15Shimomura T. Denda K. Kawaguchi T. Matsumoto K. Miyazawa K. Kitamura N. J. Biochem. 1999; 126: 821-828Crossref PubMed Scopus (44) Google Scholar). HAI-1 was first purified as a 40-kDa secreted protein from the conditioned medium of MKN45 cells (9Shimomura T. Denda K. Kitamura A. Kawaguchi T. Kito M. Kondo J. Kagaya S. Qin L. Takata H. Miyazawa K. Kitamura N. J. Biol. Chem. 1997; 272: 6370-6376Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Therefore, mature membrane-form HAI-1 can be proteolytically cleaved, and the resultant truncated form with Kunitz domain(s) is released into the extracellular space as a secreted form of HAI-1 (sHAI-1). Our recent observation indicated that there are multiple sites of proteolytic cleavage to release sHAI-1 and two major secreted forms, 40 and 58 kDa in size, were identified (15Shimomura T. Denda K. Kawaguchi T. Matsumoto K. Miyazawa K. Kitamura N. J. Biochem. 1999; 126: 821-828Crossref PubMed Scopus (44) Google Scholar). The 40-kDa sHAI-1 contains only the first Kunitz domain, whereas the 58-kDa form contains both Kunitz domains. Interestingly, the 58-kDa sHAI-1 showed a markedly lower affinity for HGFA than the initially identified 40-kDa sHAI-1 (9Shimomura T. Denda K. Kitamura A. Kawaguchi T. Kito M. Kondo J. Kagaya S. Qin L. Takata H. Miyazawa K. Kitamura N. J. Biol. Chem. 1997; 272: 6370-6376Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 15Shimomura T. Denda K. Kawaguchi T. Matsumoto K. Miyazawa K. Kitamura N. J. Biochem. 1999; 126: 821-828Crossref PubMed Scopus (44) Google Scholar), although the pathophysiological significance of these differentially processed forms of sHAI-1 remains to be clarified. To date, the precise functions of HAI-1 and HAI-2/PB in vivoare undefined. However, previous studies have shown that the cellular surface expression of HAI-1 is significantly up-regulated in the epithelial cells in response to tissue injury and regeneration, suggesting a potential role of HAI-1 in the survival and regeneration of the epithelial cells in vivo (14Kataoka H. Suganuma T. Shimomura T. Itoh H. Kitamura N. Nabeshima K. Koono M. J. Histochem. Cytochem. 1999; 47: 673-682Crossref PubMed Scopus (104) Google Scholar, 16Itoh H. Kataoka H. Tomita M. Hamasuna R. Nawa Y. Kitamura N. Koono M. Am. J. Physiol. 2000; 278: G635-G643Crossref PubMed Google Scholar). In contrast, HAI-2/PB expression was not altered in the injured tissue (16Itoh H. Kataoka H. Tomita M. Hamasuna R. Nawa Y. Kitamura N. Koono M. Am. J. Physiol. 2000; 278: G635-G643Crossref PubMed Google Scholar).HGFA is produced mainly by the liver in which hepatocytes being responsible (4Miyazawa K. Shimomura T. Kitamura A. Kondo J. Morimoto Y. Kitamura N. J. Biol. Chem. 1993; 268: 10024-10028Abstract Full Text PDF PubMed Google Scholar, 17Okajima A. Miyazawa K. Naitoh Y. Inoue K. Kitamura N. Hepatology. 1997; 25: 97-102Crossref PubMed Scopus (55) Google Scholar). In addition, recent reports indicate that gastrointestinal tissues also express the HGFA gene (18Kinoshita Y. Kishi K. Asahara A. Matsushima Y. Wang H.Y. Miyazawa K. Kitamura N. Chiba T. Digestion. 1997; 58: 225-231Crossref PubMed Scopus (28) Google Scholar, 19Matsubara Y. Ichinose M. Yahagi N. Tsukada S. Oka M. Miki K. Kimura S. Omata M. Shiokawa K. Kitamura N. Kaneko Y. Fukamachi H. Biochem. Biophys. Res. Commun. 1998; 253: 477-484Crossref PubMed Scopus (44) Google Scholar, 20Itoh H. Hamasuna R. Kataoka H. Yamauchi M. Miyazawa K. Kitamura N. Koono M. Biochim. Biophys. Acta. 2000; 1491: 295-302Crossref PubMed Scopus (27) Google Scholar). HGFA is secreted as an inactive single-chain 96-kDa zymogen (pro-HGFA) and is activated by thrombin in injured tissues via a cleavage at the bond between Arg407 and Ile408 resulting in a two-chain heterodimeric form (5Shimomura T. Kondo J. Ochiai M. Naka D. Miyazawa K. Morimoto Y. Kitamura N. J. Biol. Chem. 1993; 268: 22927-22932Abstract Full Text PDF PubMed Google Scholar, 6Miyazawa K. Shimomura T. Naka D. Kitamura N. J. Biol. Chem. 1994; 269: 8966-8970Abstract Full Text PDF PubMed Google Scholar, 7Miyazawa K. Shimomura T. Kitamura N. J. Biol. Chem. 1996; 271: 3615-3618Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). The active form of HGFA efficiently activates the single-chain precursor form of HGF bound to the extracellular matrix (6Miyazawa K. Shimomura T. Naka D. Kitamura N. J. Biol. Chem. 1994; 269: 8966-8970Abstract Full Text PDF PubMed Google Scholar, 7Miyazawa K. Shimomura T. Kitamura N. J. Biol. Chem. 1996; 271: 3615-3618Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 21Naka D. Ishii T,. Yoshiyama Y. Miyazawa K. Hara H. Hishida T. Kitamura N. J. Biol. Chem. 1992; 267: 20114-20119Abstract Full Text PDF PubMed Google Scholar), and the activated HGF in turn facilitates the repair and regeneration of a variety of injured tissues through binding to the c-Met receptor expressed in epithelial and endothelial cells (1Matsumoto K. Nakamura T. J. Biochem. (Tokyo ). 1996; 119: 591-600Crossref PubMed Scopus (609) Google Scholar, 2Stella M.C. Comoglio P.M. Int. J. Biochem. Cell Biol. 1999; 31: 1357-1362Crossref PubMed Scopus (136) Google Scholar, 22Kawaida K. Matsumoto K. Shimazu H. Nakamura T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4357-4361Crossref PubMed Scopus (382) Google Scholar, 23Ohmichi H. Matsumoto K. Nakamura T. Am. J. Physiol. 1996; 270: L1031-L1039PubMed Google Scholar, 24Schmassmann A. Stettler C. Poulsom R. Tarasova N. Hirschi C. Flogerzi B. Matsumoto K. Nakamura T. Halter F. Gastroenterology. 1997; 113: 1858-1872Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Thus, it is important to investigate the regulation of HGF activation in the injured tissue in which HGFA and HAIs could be critically involved. Based on the circumstantial evidence that HAI-1, but not HAI-2/PB, is significantly up-regulated in injured and regenerating tissues (14Kataoka H. Suganuma T. Shimomura T. Itoh H. Kitamura N. Nabeshima K. Koono M. J. Histochem. Cytochem. 1999; 47: 673-682Crossref PubMed Scopus (104) Google Scholar, 16Itoh H. Kataoka H. Tomita M. Hamasuna R. Nawa Y. Kitamura N. Koono M. Am. J. Physiol. 2000; 278: G635-G643Crossref PubMed Google Scholar), it can be hypothesized that, despite their similar molecular structures, the roles of HAI-1 and HAI-2/PB may be distinct from each other, and HAI-1 may have an undefined, selective, and important role in the injured tissue. It also remains to be determined whether membrane forms of HAI-1 and HAI-2/PB in fact interact with HGFA on the cellular surface.In this report, we describe evidence showing that active HGFA selectively binds to the membrane-form HAI-1, but not to HAI-2/PB, of epithelial cells, and that this binding is reversible. Further investigation revealed that membrane-form HAI-1 acts not only as a cellular surface inhibitor of HGFA but also as a reservoir for this enzyme. The latter function of HAI-1 paradoxically contributed to ensure the pericellular HGFA activity in certain cellular conditions via up-regulated shedding of the HGFA·HAI-1 complex from the cell surface followed by the recovery of HGFA activity. These results propose a novel important role of the membrane-type Kunitz inhibitor in the regulation of pericellular proteolysis.DISCUSSIONIn the present study, we showed that only an active form of HGFA was bound to the epithelial cell surface. This binding was mediated by membrane-form HAI-1 and was considered to be a proteinase-proteinase inhibitor interaction, because the pretreatment of active HGFA with low molecular weight synthetic serine proteinase inhibitors abolished the binding completely. Therefore, it can be postulated that the membrane-form HAI-1 is a specific inhibitor of active HGFA acting on the epithelial cell surface. Rather surprisingly, although the molecular structure of HAI-2/PB is similar to HAI-1 having a transmembrane domain and two Kunitz domains, active HGFA was not complexed with the cellular HAI-2/PB. Thus, the membrane-form HAI-2/PB does not act as an HGFA inhibitor on the cell surface, although the secreted form of HAI-2/PB shows potent anti-HGFA activity in vitro (10Kawaguchi T. Qin L. Shimomura T. Kondo J. Matsumoto K. Denda K. Kitamura N. J. Biol. Chem. 1997; 272: 27558-27564Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 28Qin L. Denda K. Shimomura T. Kawaguchi T. Kitamura N. FEBS Lett. 1998; 436: 111-114Crossref PubMed Scopus (36) Google Scholar). These observations support a hypothesis that HAI-1 and HAI-2/PB acquire distinct biological roles in vivoduring their evolution, although they might be derived from the same ancestor gene (29Itoh H. Yamauchi M. Kataoka H. Hamasuna R. Kitamura N. Koono M. Eur. J. Biochem. 2000; 267: 3351-3359Crossref PubMed Scopus (25) Google Scholar). HAI-1 has an LDL receptor-like domain that is absent in HAI-2/PB between two Kunitz inhibitor domains. Thus, it may be that the LDL receptor-like domain is somehow involved in the accessibility of active HGFA to the membrane-form HAI-1. Another possibility is that a Kunitz-containing part of HAI-2/PB is not exposed to the extracellular milieu but is exposed to a lumen of a vesicular compartment depending on its post-Golgi trafficking pathway. Although convincing evidence supporting this possibility is lacking at present, our previous immunohistochemical study showed that HAI-2/PB was preferentially stained intracellularly in the epithelial cells, whereas HAI-1 was stained on the basolateral cellular surface (14Kataoka H. Suganuma T. Shimomura T. Itoh H. Kitamura N. Nabeshima K. Koono M. J. Histochem. Cytochem. 1999; 47: 673-682Crossref PubMed Scopus (104) Google Scholar, 15Shimomura T. Denda K. Kawaguchi T. Matsumoto K. Miyazawa K. Kitamura N. J. Biochem. 1999; 126: 821-828Crossref PubMed Scopus (44) Google Scholar, 25Kataoka H. Itoh H. Uchino H. Hamasuna R. Kitamura N. Nabeshima K. Koono M. Cancer Lett. 2000; 148: 127-134Crossref PubMed Scopus (26) Google Scholar, 26Kataoka H. Uchino H. Denda K. Kitamura N. Itoh H. Tsubouchi H. Nabeshima K. Koono M. Cancer Lett. 1998; 128: 219-227Crossref PubMed Scopus (25) Google Scholar). Alternatively, HAI-2/PB may be very quickly released from the cell surface.The most important aspect of this study is the demonstration that HGFA proteins complexed with membrane-form HAI-1 were released into the extracellular milieu by regulated shedding of the membrane-form HAI-1 followed by a considerable recovery of HGFA activity. This shedding was significantly enhanced by PMA or IL-1β in a metalloproteinase-dependent manner, accompanying the generation of 58-kDa sHAI-1 fragments. Previous study has revealed that this 58-kDa sHAI-1 shows a significantly lower affinity for HGFA than the 40-kDa one that was initially identified as an HGFA inhibitor in a conditioned medium of MKN45 cells (9Shimomura T. Denda K. Kitamura A. Kawaguchi T. Kito M. Kondo J. Kagaya S. Qin L. Takata H. Miyazawa K. Kitamura N. J. Biol. Chem. 1997; 272: 6370-6376Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 15Shimomura T. Denda K. Kawaguchi T. Matsumoto K. Miyazawa K. Kitamura N. J. Biochem. 1999; 126: 821-828Crossref PubMed Scopus (44) Google Scholar). Indeed, immunoprecipitation study indicated that only a minor portion of HGFA proteins was tightly complexed with 58-kDa sHAI-1 in the PMA-treated culture supernatant. Therefore, it is conceivable that, after regulated shedding of the HGFA·HAI-1 complex, an equation among the inhibitor (58-kDa sHAI-1), enzyme (active HGFA), and substrate (single-chain HGF) shifts in favor of the enzyme·substrate complex, eventually resulting in an efficient generation of an active two-chain form of HGF. Currently, the precise biochemical mechanism underlying the decreased affinity of 58-kDa sHAI-1 to HGFA is undefined. Because the NH2-terminal amino acid sequence of the 58-kDa form is identical to those of membrane-form HAI-1 and 40-kDa sHAI-1 (15Shimomura T. Denda K. Kawaguchi T. Matsumoto K. Miyazawa K. Kitamura N. J. Biochem. 1999; 126: 821-828Crossref PubMed Scopus (44) Google Scholar), the 58-kDa form appears to be generated by the cleavage at the COOH-terminal region of the extracellular part of membrane-form HAI-1 (15Shimomura T. Denda K. Kawaguchi T. Matsumoto K. Miyazawa K. Kitamura N. J. Biochem. 1999; 126: 821-828Crossref PubMed Scopus (44) Google Scholar). The similar immunoreactivity of 58-kDa sHAI-1 to C76-18 mAb and 1N7 mAb, recognized around the first and second Kunitz domains, respectively, confirmed the existence of both Kunitz domains in this form. The presence of the second Kunitz domain and free COOH terminus may somehow interfere with the binding of HGFA to the first domain that is responsible to the inhibition of HGFA (9Shimomura T. Denda K. Kitamura A. Kawaguchi T. Kito M. Kondo J. Kagaya S. Qin L. Takata H. Miyazawa K. Kitamura N. J. Biol. Chem. 1997; 272: 6370-6376Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) in a soluble phase. For instance, bikunin (the light chain of inter-α-trypsin inhibitor) also has two Kunitz domains, and proteinase binding to the second domain is affected by the presence of the first domain (30Morishita H. Yamakawa T. Matsusue T. Kusuyama T. Sameshima-Aruga R. Hirose J. Nii A. Miura T. Isaji M. Horisawa-Nakano R. Nagase Y. Kanamori T. Nobuhara M. Tanaka R. Koyama S. Nakatsuka M. Thromb. Res. 1994; 73: 193-204Abstract Full Text PDF PubMed Scopus (22) Google Scholar, 31Xu Y. Carr P.D. Guss J.M. Ollis D.L. J. Mol. Biol. 1998; 276: 955-966Crossref PubMed Scopus (60) Google Scholar). Although the molecular mechanism involved in the dissociation of 58-kDa sHAI-1 and HGFA after regulated shedding remains to be clarified, these observations propose a novel and unexpected mechanism for the regulation of serine proteinase activity in the pericellular microenvironment, as well as on the cell surface, mediated by an integral membrane Kunitz-type serine proteinase inhibitor. In Fig. 10, we propose a hypothetical model for the regulation of pericellular HGFA activity by HAI-1 in which the membrane-form HAI-1 is acting not only as a cell-surface inhibitor of active HGFA but also as a reservoir for this enzyme protecting the active enzyme from other possibly co-existing inhibitor(s) of HGFA and concentrating the enzyme on the cell surface. This reservoir function would be terminated by the regulated shedding of the 58-kDa low affinity form of sHAI-1 followed by dissociation of active HGFA from 58-kDa sHAI-1 in the extracellular space, resulting in efficient activation of HGF in the pericellular microenvironment. In this model, the epithelial cell can utilize a single molecule (HAI-1) as a negative or positive regulator of pericellular proteinase activity depending on the cellular conditions, these being the levels of protein kinase C activation and presence or absence of cytokine stimulation. Importantly, HAI-1 inhibits not only HGFA but also other serine proteinases such as plasmin 2H. Kataoka, R. Hamasuna, H. Itoh, and M. Koono, unpublished observation.and matriptase, a recently identified matrix-degrading serine proteinase (32Lin C.-Y. Anders J. Johnson M. Sang Q.A. Dickson R.B. J. Biol. Chem. 1999; 274: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 33Lin C.-Y. Anders J. Johnson M. Dickson R.B. J. Biol. Chem. 1999; 274: 18237-18242Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Therefore, it is of interest to test the possibility of whether the membrane-form HAI-1 can also serve as a reservoir for these enzymes and recruit the enzyme activity in the pericellular microenvironment. It is also interesting to know whether another integral membrane Kunitz-type inhibitor also exhibits the diverse functions similar to HAI-1 against its target proteinase. To date, three Kunitz-type serine proteinase inhibitors having a transmembrane domain have been reported in human species in addition to HAI-1. These include amyloid β protein precursor (APP), APP-like protein-2, and HAI-2/PB. All are type-I integral membrane proteins having one (APP and APP-like protein-2) or two (HAI-1 and HAI-2/PB) Kunitz domains, and their physiological functions have been poorly understood.A significant body of evidence is accumulating in favor of the existence of a general regulated shedding machinery acting on diverse groups of transmembrane proteins (34Hooper N.E. Karran E.C. Turner A.J. Biochem. J. 1997; 321: 265-279Crossref PubMed Scopus (558) Google Scholar). It has been shown that the proteinase responsible for the shedding of tumor necrosis factor-α (TNFα) is also involved in the shedding of various other proteins such as APP, L-selectin, and transforming growth factor-α (35Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. Science. 1998; 282: 1281-1284Crossref PubMed Scopus (1355) Google Scholar, 36Buxbaum J.D. Liu K.-N. Luo Y. Slack J.S. Stocking K.L. Peschon J.J. Johnson R.S. Castner B.J. Cerretti D.P. Black R.A. J. Biol. Chem. 1998; 273: 27765-27767Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar). This TNFα-converting enzyme (TACE) is a family member of the cell surface zinc-metalloproteinase called ADAM (a disintegrin and metalloproteinase) (37Werb Z. Yan Y. Science. 1998; 282: 1279-1280Crossref PubMed Google Scholar), and the shedding mediated by TACE has been shown to be significantly enhanced by PMA (35Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. 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For example, the numbers of amino acids from the predicted transmembrane domain to the cleavage site are 20, 12, 11, and 9 in the shedding of TNFα, APP, L-selectin, and transforming growth factor-α, respectively (34Hooper N.E. Karran E.C. Turner A.J. Biochem. J. 1997; 321: 265-279Crossref PubMed Scopus (558) Google Scholar). Although we tried to determine the COOH-terminal amino acid sequence of 58-kDa sHAI-1, it was not successful. However, from this evidence, it seems likely that the distance of cleavage site for the generation of 58-kDa sHAI-1 from the predicted transmembrane domain may be around 10–20 amino acids. The identification of the precise cleavage site for the shedding of 58-kDa sHAI-1 would be necessary to clarify the mechanism of decreased affinity of this secreted form for active HGFA.Like APP (39Buxbaum J.D. Oishi M. Chen H.I. Pinkas-Kramarski R. Jaffe E.A. Gandy S.E. Greengard P. Proc. Natl. Acad. Sci. U. S. 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