Genomic structure and chromosomal localization of the human hepatocyte growth factor activator inhibitor type 1 and 2 genes.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) are recently discovered Kunitz-type serine protease inhibitors which can be purified and cloned from human stomach cancer cell line MKN45 as specific inhibitors against hepatocyte growth factor activator (HGFA). HAI-2 was identical with the protein originally reported as placental bikunin. Both proteins contain two Kunitz inhibitor domains (KDs), of which the first domain (KD1) is mainly responsible for the inhibitory activity against HGFA, and are expressed ubiquitously in various tissues. In this study, we cloned the genes coding for these two structurally similar proteins by screening of human genomic bacterial artificial chromosome (BAC) library and their genomic structures were compared. HAI-1 and -2 genes consist of 11 and 8 exons spanning 12 kbp and 12.5 kbp, respectively. Three exons were inserted between KD1 and KD2 of each gene, of which the middle one was the low-density lipoprotein (LDL) receptor-like domain (HAI-1) and the testis specific exon (HAI-2). Apparently homologous regions between HAI-1 and -2 were not found in 5'-flanking region and neither TATA nor CAAT box was present. The genes were mapped to chromosome 15q15 (HAI-1) and 19q13.11 (HAI-2). These results suggested that although HAI-1 and -2 genes might be derived from same ancestor gene, they acquired distinctive in vivo roles during their evolution.     

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.     

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is required for branching morphogenesis in the chorioallantoic placenta

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-associated Kunitz-type serine proteinase inhibitor that was initially identified as a potent inhibitor of hepatocyte growth factor activator. HAI-1 is also a cognate inhibitor of matriptase, a membrane-associated serine proteinase. HAI-1 is expressed predominantly in epithelial cells in the human body. Its mRNA is also abundant in human placenta, with HAI-1 specifically expressed by villous cytotrophoblasts. In order to address the precise roles of HAI-1 in vivo, we generated HAI-1 mutant mice by homozygous recombination. Heterozygous HAI-1+/- mice underwent normal organ development. However, homozygous HAI-1-/- mice experienced embryonic lethality which became evident at embryonic day 10.5 postcoitum (E10.5). As early as E9.5, HAI-1-/- embryos showed growth retardation that did not reflect impaired cell proliferation but resulted instead from failed placental development and function. Histological analysis revealed severely impaired formation of the labyrinth layer, in contrast all other placental layers, such as the spongiotrophoblast layer and giant cell layer, which were formed. Our results indicate that mouse HAI-1 is essential for branching morphogenesis in the chorioallantoic placenta and lack of HAI-1 function may result in placental failure.     

HAI-1过表达对SW620细胞体外生长和运动能力的影响

肝细胞生长因子激活因子抑制因子1(hepatocyte growth factor activator inhibitor type 1,HAI-1)能有效抑制肝细胞生长因子激活因子(hepatocyte growth factor activator,HGFA)和丝氨酸蛋白酶Matriptase的活性,并可通过对HGFA和Matriptase活性的调控参与HGF／c—Met信号传导途径。为了解HAI-1在肿瘤细胞的生长和运动中的作用,本研究将人HAI-1基因全长cDNA克隆至pcDNA3．1( )表达载体,并转染人肠癌SW620细胞,用Western blot验证了转染细胞中HAI-1的表达情况,并分别利用生长曲线、软琼脂集落形成、穿膜运动和扩散运动测定等方法检测了HAI-1过表达对SW620细胞生长和运动能力的影响。生长曲线和软琼脂集落形成测定都显示出HAI-1转染细胞与对照组相比差异不十分明显。穿膜运动和扩散运动测定则均显示了HAI-1过表达对细胞运动能力有明显的抑制。因此,HAI-1的过表达虽然在体外对肿瘤细胞生长影响较小,但可以抑制肿瘤细胞的运动迁移能力。     

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.     

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.     

HAI-1过表达对SW620细胞体外生长和运动能力的影响

肝细胞生长因子激活因子抑制因子1(hepatocyte growth factor activator inhibitor type 1, HAI-1)能有效抑制肝细胞生长因子激活因子(hepatocyte growth factor activator,HGFA)和丝氨酸蛋白酶Matriptase的活性,并可通过对HGFA和Matriptase活性的调控参与HGF/c-Met信号传导途径。为了解HAL-1在肿瘤细胞的生长和运动中的作用,本研究将人HAI-1基因全长cDNA克隆至pcDNA3.1( )表达载体,并转染人肠癌SW620细胞,用Western blot验证了转染细胞中HAI-1的表达情况,并分别利用生长曲线、软琼脂集落形成、穿膜运动和扩散运动测定等方法检测了HAI-1 过表达对SW620细胞生长和运动能力的影响。生长曲线和软琼脂集落形成测定都显示出HAI-1 转染细胞与对照组相比差异不十分明显。穿膜运动和扩散运动测定则均显示了HAI-1过表达对细胞运动能力有明显的抑制。因此,HAI-1的过表达虽然在体外对肿瘤细胞生长影响较小,但可以抑制肿瘤细胞的运动迁移能力。     

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.     

Multiple sites of proteolytic cleavage to release soluble forms of hepatocyte growth factor activator inhibitor type 1 from a transmembrane form.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type serine protease inhibitor, which was identified as a potent inhibitor of hepatocyte growth factor (HGF) activator from the conditioned medium of a human carcinoma cell line. HGF activator is a blood coagulation factor XII-like serine protease that is responsible for proteolytic activation of the inactive single chain precursor of HGF in injured tissues. The predicted sequence of the primary translation product of HAI-1, which has a hydrophobic sequence in its COOH-terminal region, suggested that HAI-1 is first produced in a membrane-associated form. In this study, we identified a transmembrane form of HAI-1 integrated in the plasma membrane of cultured cells using a monoclonal antibody against HAI-1. We also identified several soluble forms of HAI-1 in the conditioned medium of the cells, indicating that multiple sites are present in the transmembrane form of HAI-1 at which proteolytic cleavage releases the extracellular domain. At least two proteases, one of which is a metalloprotease, appear to be responsible for the release. Further, the soluble forms of HAI-1 have different inhibitory activity against HGF activator. These findings suggest that proteolytic processing plays important roles in regulation of the inhibitory activity of HAI-1.     

Regulation of pericellular proteolysis by hepatocyte growth factor activator inhibitor type 1 (HAI-1) in trophoblast cells

Hepatocyte growth factor activator inhibitor type 1/serine protease inhibitor Kunitz type 1 (HAI-1/SPINT1) is a membrane-bound Kunitz-type serine protease inhibitor that is abundantly expressed on the surface of cytotrophoblasts, and is critically required for the formation of the placenta labyrinth in mice. HAI-1/SPINT1 regulates several membrane-associated cell surface serine proteases, with matriptase being the most cognate target. Matriptase degrades extracellular matrix protein such as laminin and activates other cell surface proteases including prostasin. This study aimed to analyze the role of HAI-1/SPINT1 in pericellular proteolysis of trophoblasts. In HAI-1/SPINT1-deficient mouse placenta, laminin immunoreactivity around trophoblasts was irregular and occasionally showed an intense punctate pattern, which differed significantly from the linear distribution along the basement membrane observed in wild-type placenta. To explore the molecular mechanism underlying this observation, we analyzed the effect of HAI-1/SPINT1 knock down (KD) on pericellular proteolysis in the human trophoblast cell line, BeWo. HAI-1/SPINT1-KD BeWo cells had increased amounts of cellular laminin protein and decreased laminin degradation activity in the culture supernatant. Subsequent analysis indicated that cell-associated matriptase was significantly decreased in KD cells whereas its mRNA level was not altered, suggesting an enhanced release and/or dislocation of matriptase in the absence of HAI-1/SPINT1. Moreover, prostasin activation and pericellular total serine protease activities were significantly suppressed by HAI-1/SPINT1 KD. These observations suggest that HAI-1/SPINT1 is critically required for the cell surface localization of matriptase in trophoblasts, and, in the absence of HAI-1/SPINT1, physiological activation of prostasin and other protease(s) initiated by cell surface matriptase may be impaired.     

Reduced Prostasin (CAP1/PRSS8) Activity Eliminates HAI-1 and HAI-2 Deficiency-Associated Developmental Defects by Preventing Matriptase Activation

Loss of either hepatocyte growth factor activator inhibitor (HAI)-1 or -2 is associated with embryonic lethality in mice, which can be rescued by the simultaneous inactivation of the membrane-anchored serine protease, matriptase, thereby demonstrating that a matriptase-dependent proteolytic pathway is a critical developmental target for both protease inhibitors. Here, we performed a genetic epistasis analysis to identify additional components of this pathway by generating mice with combined deficiency in either HAI-1 or HAI-2, along with genes encoding developmentally co-expressed candidate matriptase targets, and screening for the rescue of embryonic development. Hypomorphic mutations in Prss8, encoding the GPI-anchored serine protease, prostasin (CAP1, PRSS8), restored placentation and normal development of HAI-1-deficient embryos and prevented early embryonic lethality, mid-gestation lethality due to placental labyrinth failure, and neural tube defects in HAI-2-deficient embryos. Inactivation of genes encoding c-Met, protease-activated receptor-2 (PAR-2), or the epithelial sodium channel (ENaC) alpha subunit all failed to rescue embryonic lethality, suggesting that deregulated matriptase-prostasin activity causes developmental failure independent of aberrant c-Met and PAR-2 signaling or impaired epithelial sodium transport. Furthermore, phenotypic analysis of PAR-1 and matriptase double-deficient embryos suggests that the protease may not be critical for focal proteolytic activation of PAR-2 during neural tube closure. Paradoxically, although matriptase auto-activates and is a well-established upstream epidermal activator of prostasin, biochemical analysis of matriptase- and prostasin-deficient placental tissues revealed a requirement of prostasin for conversion of the matriptase zymogen to active matriptase, whereas prostasin zymogen activation was matriptase-independent.     

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.     

The Protease Inhibitor HAI-2, but Not HAI-1, Regulates Matriptase Activation and Shedding through Prostasin

The membrane-anchored serine proteases, matriptase and prostasin, and the membrane-anchored serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2, are critical effectors of epithelial development and postnatal epithelial homeostasis. Matriptase and prostasin form a reciprocal zymogen activation complex that results in the formation of active matriptase and prostasin that are targets for inhibition by HAI-1 and HAI-2. Conflicting data, however, have accumulated as to the existence of auxiliary functions for both HAI-1 and HAI-2 in regulating the intracellular trafficking and activation of matriptase. In this study, we, therefore, used genetically engineered mice to determine the effect of ablation of endogenous HAI-1 and endogenous HAI-2 on endogenous matriptase expression, subcellular localization, and activation in polarized intestinal epithelial cells. Whereas ablation of HAI-1 did not affect matriptase in epithelial cells of the small or large intestine, ablation of HAI-2 resulted in the loss of matriptase from both tissues. Gene silencing studies in intestinal Caco-2 cell monolayers revealed that this loss of cell-associated matriptase was mechanistically linked to accelerated activation and shedding of the protease caused by loss of prostasin regulation by HAI-2. Taken together, these data indicate that HAI-1 regulates the activity of activated matriptase, whereas HAI-2 has an essential role in regulating prostasin-dependent matriptase zymogen activation.