Hepatocyte growth factor activator inhibitor type 1 inhibits protease activity and proteolytic activation of human airway trypsin-like protease

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type transmembrane serine protease inhibitor initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA), a serine protease that converts pro-HGF to the active form. HAI-1 also has inhibitory activity against serine proteases such as matriptase, hepsin and prostasin. In this study, we examined effects of HAI-1 on the protease activity and proteolytic activation of human airway trypsin-like protease (HAT), a transmembrane serine protease that is expressed mainly in bronchial epithelial cells. A soluble form of HAI-1 inhibited the protease activity of HAT in vitro. HAT was proteolytically activated in cultured mammalian cells transfected with its expression vector, and a soluble form of active HAT was released into the conditioned medium. The proteolytic activation of HAT required its own serine protease activity. Co-expression of the transmembrane full-length HAI-1 inhibited the proteolytic activation of HAT. In addition, full-length HAI-1 associated with the transmembrane full-length HAT in co-expressing cells. Like other target proteases of HAI-1, HAT converted pro-HGF to the active form in vitro. These results suggest that HAI-1 functions as a physiological regulator of HAT by inhibiting its protease activity and proteolytic activation in airway epithelium.     

Functional characterization of Kunitz domains in hepatocyte growth factor activator inhibitor type 1.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type serine protease inhibitor identified as a strong inhibitor of hepatocyte growth factor (HGF) activator and matriptase. HAI-1 is first produced in a membrane-integrated form with two Kunitz domains in its extracellular region, and subsequent ectodomain shedding releases two major secreted forms, one with a single Kunitz domain and one with two Kunitz domains. To determine the roles of the Kunitz domains in the inhibitory activity of HAI-1 against serine proteases, we constructed various HAI-1 mutant proteins and examined their inhibitory activity against HGF activator and trypsin. The N-terminal Kunitz domain (Kunitz I) had potent inhibitory activity against both HGF activator and trypsin, whereas the C-terminal Kunitz domain (Kunitz II) had only very weak inhibitory activity against HGF activator, although its potency against trypsin was equivalent to that of Kunitz I. These results indicate that Kunitz I is the functional domain of HAI-1 for inhibiting the HGF-converting activity of HGF activator. Furthermore, the presence of two Kunitz domains affected the inhibitory activity of HAI-1 against HGF activator, and it showed a similar, but not additive, level of inhibitory activity against trypsin when compared with that of the individual Kunitz domains. These results suggest that serine protease binding sites of Kunitz I and Kunitz II are located close to each other and that proteolytic processing to generate HAI-1 with only one Kunitz domain regulates the activity of HAI-1.     

TMPRSS13, a type II transmembrane serine protease, is inhibited by hepatocyte growth factor activator inhibitor type 1 and activates pro-hepatocyte growth factor

Type II transmembrane serine proteases (TTSPs) are structurally defined by the presence of a transmembrane domain located near the N-terminus and a C-terminal extracellular serine protease domain. The human TTSP family consists of 17 members. Some members of the family have pivotal functions in development and homeostasis, and are involved in tumorigenesis and viral infections. The activities of TTSPs are regulated by endogenous protease inhibitors. However, protease inhibitors of most TTSPs have not yet been identified. In this study, we investigated the inhibitory effect of hepatocyte growth factor activator inhibitor type 1 (HAI-1), a Kunitz-type serine protease inhibitor, on several members of the TTSP family. We found that the protease activity of a member, TMPRSS13, was inhibited by HAI-1. A detailed analysis revealed that a soluble form of HAI-1 with one Kunitz domain (NK1) more strongly inhibited TMPRSS13 than another soluble form of HAI-1 with two Kunitz domains (NK1LK2). In addition, an in vitro protein binding assay showed that NK1 formed complexes with TMPRSS13, but NK1LK2 did not. TMPRSS13 converted single-chain pro-hepatocyte growth factor (pro-HGF) to a two-chain form in vitro, and the pro-HGF converting activity of TMPRSS13 was inhibited by NK1. The two-chain form of HGF exhibited biological activity, assessed by phosphorylation of the HGF receptor (c-Met) and extracellular signal-regulated kinase, and scattered morphology in human hepatocellular carcinoma cell line HepG2. These results suggest that TMPRSS13 functions as an HGF-converting protease, the activity of which may be regulated by HAI-1.     

Purification from human milk of matriptase complexes with secreted serpins: mechanism for inhibition of matriptase other than HAI-1

Matriptase, a type 2 transmembrane serine protease, is predominately expressed by epithelial and carcinoma cells in which hepatocyte growth factor activator inhibitor 1 (HAI-1), a membrane-bound, Kunitz-type serine protease inhibitor, is also expressed. HAI-1 plays dual roles in the regulation of matriptase, as a conventional protease inhibitor and as a factor required for zymogen activation of matriptase. As a consequence, activation of matriptase is immediately followed by HAI-1-mediated inhibition, with the activated matriptase being sequestered into HAI-1 complexes. Matriptase is also expressed by peripheral blood leukocytes, such as monocytes and macrophages; however, in contrast to epithelial cells, monocytes and macrophages were reported not to express HAI-1, suggesting that these leukocytes possess alternate, HAI-1-independent mechanisms regulating the zymogen activation and protease inhibition of matriptase. In the present study, we characterized matriptase complexes of 110 kDa in human milk, which contained no HAI-1 and resisted dissociation in boiling SDS in the absence of reducing agents. These complexes were further purified and dissociated into 80-kDa and 45-kDa fragments by treatment with reducing agents. Proteomic and immunological methods identified the 45-kDa fragment as the noncatalytic domains of matriptase and the 80-kDa fragment as the matriptase serine protease domain covalently linked to one of three different secreted serpin inhibitors: antithrombin III, 1-antitrypsin, and 2-antiplasmin. Identification of matriptase-serpin inhibitor complexes provides evidence for the first time that the proteolytic activity of matriptase, from those cells that express no or low levels of HAI-1, may be controlled by secreted serpins. protease; type 2 transmembrane serine protease; protease inhibitor; ST-14; hepatocyte growth factor activator inhibitor 1     

Potent inhibition and global co-localization implicate the transmembrane Kunitz-type serine protease inhibitor hepatocyte growth factor activator inhibitor-2 in the regulation of epithelial matriptase activity

Hepatocyte growth factor activator inhibitors (HAI)-1 and -2 are recently identified and closely related Kunitz-type transmembrane serine protease inhibitors. Whereas HAI-1 is well established as an inhibitor of the serine proteases matriptase and hepatocyte growth factor activator, the physiological targets of HAI-2 are unknown. Here we show that HAI-2 displays potent inhibitory activity toward matriptase, forms SDS-stable complexes with the serine protease, and blocks matriptase-dependent activation of its candidate physiological substrates proprostasin and cell surface-bound pro-urokinase plasminogen activator. To further explore the potential functional relationship between HAI-2 and matriptase, we generated a transgenic mouse strain with a promoterless beta-galactosidase marker gene inserted into the endogenous locus encoding HAI-2 protein and performed a global high resolution mapping of the expression of HAI-2, matriptase, and HAI-1 proteins in all adult tissues. This analysis showed striking co-localization of HAI-2 with matriptase and HAI-1 in epithelial cells of all major organ systems, thus strongly supporting a role of HAI-2 as a physiological regulator of matriptase activity, possibly acting in a redundant or partially redundant manner with HAI-1. Unlike HAI-1 and matriptase, however, HAI-2 expression was also detected in non-epithelial cells of brain and lymph nodes, suggesting that HAI-2 may also be involved in inhibition of serine proteases other than matriptase.     

Hepatocyte growth factor activator inhibitor-1 has a complex subcellular itinerary

HAI-1 [HGF (hepatocyte growth factor) activator inhibitor-1] is a Kunitz-type transmembrane serine protease inhibitor that forms inhibitor complexes with the trypsin-like serine protease, matriptase. HAI-1 is essential for mouse placental development and embryo survival and together with matriptase it is a key regulator of carcinogenesis. HAI-1 is expressed in polarized epithelial cells, which have the plasma membrane divided by tight junctions into an apical and a basolateral domain. In the present study we show that HAI-1 at steady-state is mainly located on the basolateral membrane of both Madin-Darby canine kidney cells and mammary gland epithelial cells. After biosynthesis, HAI-1 is exocytosed mainly to the basolateral plasma membrane from where 15% of the HAI-1 molecules are proteolytically cleaved and released into the basolateral medium. The remaining membrane-associated HAI-1 is endocytosed and then recycles between the basolateral plasma membrane and endosomes for hours until it is transcytosed to the apical plasma membrane. Minor amounts of HAI-1 present at the apical plasma membrane are proteolytically cleaved and released into the apical medium. Full-length membrane-bound HAI-1 has a half-life of 1.5 h and is eventually degraded in the lysosomes, whereas proteolytically released HAI-1 is more stable. HAI-1 is co-localized with its cognate protease, matriptase, at the basolateral plasma membrane. We suggest that HAI-1, in addition to its protease inhibitory function, plays a role in transporting matriptase as a matriptase-HAI-1 complex from the basolateral plama membrane to the apical plasma membrane, as matriptase is known to interact with prostasin, located at the apical plasma membrane.     

Hepsin activates pro-hepatocyte growth factor and is inhibited by hepatocyte growth factor activator inhibitor-1B (HAI-1B) and HAI-2

Hepsin, a type II transmembrane serine protease, is highly upregulated in prostate cancer and promotes tumor progression and metastasis. We generated a soluble form of hepsin comprising the entire extracellular domain to show that it efficiently converts single-chain hepatocyte growth factor (pro-HGF) into biologically active two-chain HGF. Hepsin activity was potently inhibited by soluble forms of the bi-Kunitz domain inhibitors HAI-1B (IC(50) 21.1+/-2.7 nM) and HAI-2 (IC(50) 1.3+/-0.3 nM). Enzymatic assays with HAI-1B Kunitz domain mutants (R260A and K401A) further demonstrated that inhibition was due to Kunitz domain-1. The results suggest a functional link between hepsin and the HGF/Met pathway, which may contribute to tumor progression.     

Requirement of the activity of hepatocyte growth factor activator inhibitor type 1 for the extracellular appearance of a transmembrane serine protease matriptase in monkey kidney COS-1 cells

Hepatocyte growth factor activator inhibitor type I (HAI-1) is a membrane-bound, serine protease inhibitor with two protease-inhibitory domains (Kunitz domain I and II). HAI-1 is known as a physiological inhibitor of a membrane-bound serine protease, matriptase. Paradoxically, however, HAI-1 has been found to be required for the extracellular appearance of the protease in an expression system using a monkey kidney COS-1 cell line. In the present study, we show using COS-1 cells that co-expression of recombinant variants of HAI-1 with the inhibition activity toward matriptase, including a variant consisting only of Kunitz domain I (the domain responsible for inhibition of matriptase), allowed for the appearance of this protease in the conditioned medium, whereas that of the variants without the activity did not. These findings suggest that the inhibition activity toward matriptase is critical for the extracellular appearance of protease in COS-1 cells.     

Pericellular activation of hepatocyte growth factor/scatter factor (HGF/SF) in colorectal carcinomas: roles of HGF activator (HGFA) and HGFA inhibitor type 1 (HAI-1).

Activation of hepatocyte growth factor/scatter factor (HGF/SF) is a critical limiting step in the HGF/SF-induced signaling pathway mediated by MET receptor tyrosine kinase. Although HGF/SF-MET signaling could have potentially important roles in the invasive growth of tumors and tumor angiogenesis, little is known about the regulation of HGF/SF activation in the tumor tissues. This activation occurs in the extracellular milieu caused by proteolytic cleavage at the bond between Arg194-Val195 in the single-chain HGF precursor to generate the active two-chain heterodimeric form. Here we show that activation of HGF/SF is significantly enhanced in colorectal carcinoma tissues compared with normal colorectal mucosa, and HGF activator (HGFA), a recently identified factor XII-like serine proteinase, is critically involved in this process. Furthermore, we also show that HGF activator inhibitor type 1 (HAI-1) should have an important regulatory role in the pericellular activation of HGF/SF having diverse roles acting as a cell surface specific inhibitor of active HGFA and a reservoir of this enzyme on the cell surface. The latter property might paradoxically ensure the concentrated pericellular HGFA activity in certain cellular conditions in which shedding of HAI-1/HGFA complex from the plasma membrane is upregulated.     

Roles of functional and structural domains of hepatocyte growth factor activator inhibitor type 1 in the inhibition of matriptase

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-bound, Kunitz-type serine protease inhibitor. HAI-1 inhibits serine proteases that have potent pro-hepatocyte growth factor-converting activity, such as the membrane-type serine protease, matriptase. HAI-1 comprises an N-terminal domain, followed by an internal domain, first protease inhibitory domain (Kunitz domain I), low-density lipoprotein receptor A module (LDLRA) domain, and a second Kunitz domain (Kunitz domain II) in the extracellular region. Our aim was to assess the roles of these domains in the inhibition of matriptase. Soluble forms of recombinant rat HAI-1 mutants made up with various combinations of domains were produced, and their inhibitory activities toward the hydrolysis of a chromogenic substrate were analyzed using a soluble recombinant rat matriptase. Kunitz domain I exhibited inhibitory activity against matriptase, but Kunitz domain II did not. The N-terminal domain and Kunitz domain II decreased the association rate between Kunitz domain I and matriptase, whereas the internal domain increased this rate. The LDLRA domain suppressed the dissociation of the Kunitz domain I-matriptase complex. Surprisingly, an HAI-1 mutant lacking the N-terminal domain and Kunitz domain II showed an inhibitor constant of 1.6 pm, and the inhibitory activity was 400 times higher in this HAI-1 mutant than in the mutant with all domains. These findings, together with the known occurrence of an HAI-1 species lacking the N-terminal domain and Kunitz domain II in vivo, suggest that the domain structure of HAI-1 is organized in a way that allows HAI-1 to flexibly control matriptase activity.     

Conformational lability in serine protease active sites: structures of hepatocyte growth factor activator (HGFA) alone and with the inhibitory domain from HGFA inhibitor-1B

Hepatocyte growth factor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. When activated HGF binds its cognate receptor Met, cellular signals lead to cell growth, differentiation, and migration, activities which promote tissue regeneration in liver, kidney and skin. Intervention in the conversion of HGF to its active form has the potential to provide therapeutic benefit where HGF/Met activity is associated with tumorigenesis. To help identify ways to moderate HGF/Met effects, we have determined the molecular structure of the protease domain of HGFA. The structure we determined, at 2.7 A resolution, with no pseudo-substrate or inhibitor bound is characterized by an unconventional conformation of key residues in the enzyme active site. In order to find whether this apparently non-enzymatically competent arrangement would persist in the presence of a strongly-interacting inhibitor, we also have determined, at 2.6 A resolution, the X-ray structure of HGFA complexed with the first Kunitz domain (KD1) from the physiological inhibitor hepatocyte growth factor activator inhibitor 1B (HAI-1B). In this complex we observe a rearranged substrate binding cleft that closely mirrors the cleft of other serine proteases, suggesting an extreme conformational dynamism. We also characterize the inhibition of 16 serine proteases by KD1, finding that the previously reported enzyme specificity of the intact extracellular region of HAI-1B resides in KD1 alone. We find that HGFA, matriptase, hepsin, plasma kallikrein and trypsin are potently inhibited, and use the complex structure to rationalize the structural basis of these results.     

Localization of hepatocyte growth factor activator inhibitor type 1 in Langhans' cells of human placenta

Activation of hepatocyte growth factor (HGF) is a crucial limiting step in HGF-induced signaling pathway. The HGF activator inhibitor type 1 (HAI-1) was identified as a potent inhibitor of HGF activator (HGFA), a serine proteinase that is responsible for the activation of HGF in vivo. HAI-1 is an integral membrane Kunitz-type serine proteinase inhibitor, and its mRNA has been reported to be most abundant in the placenta. In this report, specific antibody to HAI-1 was used in an immunohistochemical procedure to determine the localization of HAI-I in human placenta. HAI-1 was expressed in cytotrophoblasts (Langhans' cells) of the double-layered trophoblastic epithelium of chorionic villi tissue, and syncytiotrophoblasts were almost negative. On the other hand, extravillous trophoblasts of cytotrophoblastic columns showed markedly decreased immunoreactivity, and those infiltrating into the superficial decidua membrane of early placenta were hardly stainable. The amnionic epithelial cells were also immunostained intensely. The presence of HAI-1 mRNA was also confirmed in a cultured human cytotrophoblastic cell line. In addition to HAI-1, low but distinct expression of HGFA mRNA was observed in the placenta tissue and cultured cytotrophoblasts by using a sensitive RT-PCR method. Since HGF plays an essential role in the placenta development, expression of HAI-1 and HGFA may have an important regulatory role in the placenta. The localization of HAI-I in the proliferating trophoblastic stem cells (Langhans' cells), but not in syncytiotrophoblasts and extravillous trophoblasts, suggest a possible role of HAI-1 in the proliferation of trophoblasts.     

Upregulation of HGF activator inhibitor type 1 but not type 2 along with regeneration of intestinal mucosa

Hepatocyte growth factor (HGF) activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) are new Kunitz-type serine protease inhibitors that were recently purified and cloned from the human stomach cancer cell line MKN45 as specific inhibitors against HGF activator. Both proteins contain two Kunitz inhibitor domains and are expressed abundantly throughout the gastrointestinal tract, in addition to the placenta, pancreas, and kidney. In this study, to assess the possible roles of HAI-1 and HAI-2 in the intestinal mucosa, we examined the expression of HAI-1 and HAI-2 during regeneration of the intestinal mucosa. Immunohistochemical studies revealed that HAI-1 but not HAI-2 was detected more strongly in regenerative epithelium than in normal epithelium, although both proteins were detected throughout the human gastrointestinal tract. During the course of acetic acid-induced experimental colitis in an in vivo mouse model, HAI-1 but not HAI-2 was upregulated in the recovery phase, suggesting that HAI-1 but not HAI-2 is associated with the regeneration of damaged colonic mucosa. Upregulation of HAI-1 may serve to downregulate the proliferative response after initial activation of MET receptor by HGF/scatter factor after an injury.     

Regulation of the activity of matriptase on epithelial cell surfaces by a blood-derived factor.

Matriptase is an epithelial-derived, integral membrane, trypsin-like serine protease. We have shown previously that matriptase exists both in complexed and noncomplexed forms. We now show that the complexed matriptase is an activated, two-chain form, which is inhibited in an acid-sensitive, reversible manner through binding to its cognate, Kunitz-type inhibitor, HAI-1 (hepatocyte growth factor activator inhibitor-1). Conversely, the majority of the noncomplexed matriptase is a single-chain zymogen, which lacks binding affinity to HAI-1, suggesting that matriptase, similar to most other serine proteases, is activated by proteolytic cleavage at a canonical activation motif. We have now generated mAbs specific for the conformational changes associated with the proteolytic activation of matriptase. Using these mAbs, which specifically recognize the two-chain form of matriptase, we demonstrate that matriptase is transiently activated on 184A1N4 human mammary epithelial cell surfaces following their exposure to serum. The ability of serum to activate matriptase is highly conserved across reptilian, avian, and mammalian species. This serum-dependent activation of matriptase on epithelial cell surfaces is followed by ectodomain shedding of both matriptase and its Kunitz-type inhibitor.     

Expression of hepatocyte growth factor activator inhibitor type 1 in endothelial cells

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is an integral membrane Kunitz-type serine proteinase inhibitor initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA). HGFA is a serum proteinase that is critically involved in the activation of hepatocyte growth factor/scatter factor (HGF/SF) in injured tissue. Previous studies have shown that HAI-1 is expressed on the basolateral surface of various epithelial cells. In this study, we analyzed the expression of HAI-1 in human endothelial cells. Immunohistochemically, HAI-1 protein was observed in the endothelial cells of capillaries, venules and lymph vessels. On the other hand, arterial endothelial cells were poorly stained for HAI-1. Mesothelial cells on the serous surface were also positively immunostained. The endothelial expression of HAI-1 was also examined in cultured human endothelial cells of various origins, such as umbilical vein, microvessels and aorta. Notably, in accordance with the results of immunohistochemistry, HAI-1 mRNA and protein levels were high in the endothelial cells derived from umbilical vein and were hardly detectable in those derived from aorta. A low but distinct level of HAI-1 expression was also observed in endothelial cells from microvessels. As these HAI-1-positive endothelial cells also expressed MET tyrosine kinase, the specific receptor of HGF/SF, it is conceivable that HAI-1 might have an important regulatory role in the HGF/SF-MET signaling axis of endothelial cells, which could be involved in the process of angiogenesis.     

Simultaneous activation and hepatocyte growth factor activator inhibitor 1-mediated inhibition of matriptase induced at activation foci in human mammary epithelial cells

Activation of single-chain, latent matriptase, a type II transmembrane serine protease, depends on the weak proteolytic activity of its own zymogen as well as its cognate inhibitor, hepatocyte growth factor activator inhibitor 1 (HAI-1). Oligomerization of matriptase zymogens and HAI-1, and probably its interaction with other proteins, has been proposed to occur during matriptase activation. In the present study, we examined the cellular events associated with matriptase activation triggered either by the physiological inducer sphingosine 1-phosphate (S1P) or by a chemical inducer, the polyanionic compound suramin. S1P-induced matriptase translocation to cell-cell contacts, where it is activated, is an F-actin polymerization-dependent process. Conversely, suramin-induced matriptase accumulation and activation at vesicle-like structures is an F-actin polymerization-independent process. While matriptase activation can occur at different subcellular locations, both S1P- and suramin-induced matriptase accumulation form unique subcellular structures, termed activation foci, where oligomerization of matriptase zymogens and HAI-1 may occur, promoting matriptase activation. Furthermore, matriptase activation may be regulated by intracellular signaling, because Ro 31-8220, a bisindolylmaleimide protein kinase C inhibitor, inhibited both S1P- and suramin-induced activation. The requirement of HAI-1 for matriptase activation and the coincidence of HAI-1 and matriptase in activation foci apparently provide rapid access of HAI-1 for the inhibition of matriptase immediately after its activation. Indeed, all activated matriptase was detected in complexes with HAI-1 only 5 min after suramin stimulation. The close temporospatial coupling of matriptase activation with its inhibition suggests that the proteolytic activity of this enzyme must be well controlled and that the proteolysis of matriptase substrates may be tightly regulated by this mechanism. sphingosine 1-phosphate; suramin     

Mechanisms for the control of matriptase activity in the absence of sufficient HAI-1

Matriptase proteolytic activity must be tightly controlled for normal placental development, epidermal function, and epithelial integrity. Although hepatocyte growth factor activator inhibitor-1 (HAI-1) represents the predominant endogenous inhibitor for matriptase and the protein molar ratio of HAI-1 to matriptase is determined to be >10 in epithelial cells and the majority of carcinoma cells, an inverse HAI-1-to-matriptase ratio is seen in some ovarian and hematopoietic cancer cells. In the current study, cells with insufficient HAI-1 are investigated for the mechanisms through which the activity of matriptase is regulated. When matriptase activation is robustly induced in these cells, activated matriptase rapidly forms two complexes of 100- and 140-kDa in addition to the canonical 120-kDa matriptase-HAI-1 complex already described. Both 100- and 140-kDa complexes contain two-chain, cleaved matriptase but are devoid of gelatinolytic activity. Further biochemical characterization shows that the 140-kDa complex is a matriptase homodimer and that the 100-kDa complexes appear to contain reversible, tight binding serine protease inhibitor(s). The formation of the 140-kDa matriptase dimer is strongly associated with matriptase activation, and its levels are inversely correlated with the ratio of HAI-1 to matriptase. Given these observations and the likelihood that autoactivation requires the interaction of two matriptase molecules, it seems plausible that this activated matriptase homodimer may represent a matriptase autoactivation intermediate and that its accumulation may serve as a mechanism to control matriptase activity when protease inhibitor levels are limiting. These data suggest that matriptase activity can be rapidly inhibited by HAI-1 and other HAI-1-like protease inhibitors and "locked" in an inactive autoactivation intermediate, all of which places matriptase under very tight control.